Download Biosynthesis of estradiol. Cloning and characterization of rodent

BIOSYNTHESIS OF ESTRADIOL
Cloning and characterization of rodent 17β-hydroxysteroid
dehydrogenase/17-ketosteroid reductase types 1 and 7
PA SI
NOKE LAINE N
Biocenter Oulu and
WHO Collaborating Centre for
Research on Reproductive Health
OULU 2000
PASI NOKELAINEN
BIOSYNTHESIS OF ESTRADIOL
Cloning and characterization of rodent
17β-hydroxysteroid dehydrogenase/17-ketosteroid
reductase types 1 and 7
Academic Dissertation to be presented with the assent
of the Faculty of Medicine, University of Oulu, for public
discussion in Auditorium 9 of the University Hospital of
Oulu, on September 29th, 2000, at 12 noon.
O U L U N YL I O PI ST O , O U L U 2 0 0 0
Copyright © 2000
Oulu University Library, 2000
Manuscript received 16 August 2000
Accepted 21 August 2000
Communicated by
Professor Heikki Ruskoaho
Professor Timo Ylikomi
ISBN 951-42-5751-0
ALSO AVAILABLE IN PRINTED FORMAT
ISBN 951-42-5750-2
ISSN 0355-3221
(URL: http://herkules.oulu.fi/issn03553221/)
OULU UNIVERSITY LIBRARY
OULU 2000
Nokelainen, Pasi, Biosynthesis of estradiol. Cloning and characterization of rodent
17β
β-hydroxysteroid dehydrogenase/17-ketosteroid reductase types 1 and 7
Biocenter Oulu and WHO Collaborating Centre for Research on Reproductive
Health, P.O.Box 5000, FIN-90014 University of Oulu, Finland
2000
Oulu, Finland
(Manuscript received 16 August 2000)
Abstract
17β-Hydroxysteroid dehydrogenases (17HSDs)/17-ketosteroid reductases (17KSRs) modulate the
biological activity of certain estrogens and androgens by catalyzing dehydrogenase and reductase
reactions between 17β-hydroxy and 17-ketosteroids.
In the present study, cDNAs encoding mouse and rat 17HSD/KSR1 were cloned in order to
study the role of rodent type 1 enzyme in ovarian estradiol (E2) biosynthesis and its enzymatic
characteristics. Both rat and mouse 17HSD/KSR1 were expressed in granulosa cells of developing
follicles, where diethylstilbestrol and follicle-stimulating hormone stimulated follicular maturation
and up-regulated the expression of 17HSD/KSR1, whereas human chorionic gonadotropin caused
luteinization of follicles and down-regulation of the enzyme. In line with this, the rodent type 1
enzymes are not expressed in the corpus luteum (CL). Mouse 17HSD/KSR1 showed substrate
specificity different from that of the human counterpart. The mouse type 1 enzyme catalyzed the
reaction from androstenedione to testosterone at least as efficiently as estrone (E1) to E2, while
human 17HSD/KSR1 clearly preferred the E1 to E2 reaction.
A mouse mammary epithelial cell line was found to possess strong estrogenic 17KSR activity.
A novel type of 17HSD/KSR responsible for this activity was expression-cloned on the basis of its
ability to convert E1 to E2 and it was chronologically named 17HSD/KSR7. Interestingly, it showed
89 % identity with a rat protein called prolactin receptor-associated protein (PRAP), which is
expressed in the CL. Enzymatic characterization showed that both mouse 17HSD/KSR7 and PRAP
efficiently catalyzed the reaction from E1 to E2. The mouse type 7 enzyme was most abundantly
expressed in the ovary and placenta. Similar primary structure, enzymatic characteristics, and tissue
distribution of mouse 17HSD/KSR7 and PRAP suggest that PRAP is rat 17HSD/KSR7.
Further studies showed that in rat ovaries 17HSD/KSR7 is primarily expressed in the middle
and second half of pregnancy, in parallel with E2 secretion from the CL. Using in situ hybridization,
cell-specific expression of 17HSD/KSR7 was studied in the mouse ovary, uterus and placenta. In
the mouse ovary, the enzyme was expressed exclusively in the CL. In the uterus on day 5½ post
coitum (p.c.), the type 7 enzyme was expressed in the decidua, mostly in the inner zone of
antimesometrial decidua. Between day 8 and 9½ p.c. the enzyme was abundant in decidua
capsularis of the developing placenta, after which expression moved to the basal zone. On days
12½ and 14½ p.c., mouse type 7 enzyme was abundantly expressed in the spongiotrophoblasts,
where expression decreased towards parturition. Altogether, rodent 17HSD/KSR7 is a new
17HSD/KSR which is involved in the biosynthesis of E2 in the ovaries. In addition, E2 produced
locally in the decidua and placenta by the type 7 enzyme may have a role in decidualization and/or
implantation and placentation.
Keywords: steroidogenesis, estrogen, ovary, placenta
Acknowledgements
The present study was carried out at the World Health Organization Collaborating Centre
(WHOCCR) for Research on Reproductive Health, University of Oulu, Finland.
I wish to express my deepest gratitude to my supervisors, first to the President of the
Academy of Finland, Professor Reijo Vihko, M.D., Ph.D., who introduced me to this
interesting field of molecular endocrinology and provided excellent research facilities,
and thereafter to Professor Pirkko Vihko, M.D., Ph.D., who after Reijo had left his
position has shared her knowledge and given support and encouragement. I also owe my
gratitude to my additional supervisor, Docent Hellevi Peltoketo, Ph.D., for her devoted
and patient tutoring and contribution to my project, for her friendship and for valuable
comments on the manuscript of this thesis. In addition, I would like to extend my
appreciation to Mauri Orava, Ph.D., who introduced me to the secrets of clonings at the
beginning of this study.
I am grateful to Professor Heikki Ruskoaho, M.D., Ph.D., and Professor Timo
Ylikomi, M.D., Ph.D., for their constructive criticism on the manuscript of this thesis. I
also wish to thank Docent Nicholas Bolton, Ph.D. for his careful revision of the language
of the manuscript and the original articles. I would like to extend my thanks to Docent
Veli Isomaa, Ph.D., for his support in completing this thesis and for help in all practical
arrangements needed to attain the Ph.D. degree.
I am also indebted to all my co-authors Professor Matti Poutanen, Ph.D., Professor
Hannu Rajaniemi, M.D., Ph.D., Docent Helena Autio-Harmainen, M.D., Ph.D., Sergio
Ghersevich, Ph.D., Mika Mustonen, Ph.D., and Terhi Puranen, Ph.D., for their valuable
contribution to this work.
I wish to extend my sincere thanks to all my co-workers at WHOCCR for sharing their
knowledge and expertise. Especially I am grateful to Ms. Minna Eskelinen, whose
outstanding and invaluable technical assistance and sincere friendship have been of great
importance. The skillful technical assistance of Ms. Eeva Holopainen, Ms. Liisa Kaarela
and Ms. Helmi Konola is also gratefully acknowledged. I am also grateful to Anja
Raatikainen and Sirpa Annanperä for their skillful secretarial assistance and help in many
daily problems. In addition, special thanks are reserved for Juhani and Jaakko Heikkilä
for their invaluable help with computers.
I wish to express my warmest thanks to my mother, my parents- and sister-in-law and
all my friends, who have given solid support and encouragement throughout these years
and shared the interesting life outside science.
Finally, I owe my sincerest appreciation to my wife, Minna, for her love, unfailing
support and patience during these years. I am also grateful for her efficiency in finalizing
her doctoral thesis and the practical arrangements, which gave me a special
encouragement to keep going toward my dissertation.
This study was mainly supported by Biocenter Oulu, Research Council for Health of
the Academy of Finland, the Cancer Foundation of Northern Finland and the Emil
Aaltonen Foundation. The WHOCCR is supported by the Ministries of Education, Social
Affairs and Health, and Foreign Affairs, Finland.
Oulu, August 2000
Pasi Nokelainen
Abbreviations
Hormones and steroids:
16α-OH-DHEAS
17-OH-P
17-hydroxypregnenolone
20-OH-P
3α-diol
5α-A-dione
A-diol
A-dione
ADT
apigenin
coumestrol
DES
DHEA
DHEAS
DHT
E1
E2
E3
FSH
genistein
hCG
LH
P
PLP-B
pregnenolone
PRL
T
16α-hydroxydehydroepiandrosterone sulfate
17α-hydroxy-4-pregnene-3,20-dione
3β,17α-dihydroxy-5-pregnen-20-one
20α-hydroxyprogesterone, 20α-hydroxy-4-pregnen-3-one
5α-androstane-3α,17β-diol
5α-androstane-3,17-dione
androstenediol, 5-androstene-3β,17β-diol
androstenedione, 4-androstene-3,17-dione
androsterone, 3α-hydroxy-5α-androstan-17-one
4,5,7-trihydroxyflavone
2-(2,4-dihydroxyphenyl)-6-hydroxy-3-benzofurancarboxylic
acid δ-lactone
diethylstilbestrol
dehydroepiandrosterone, 3β-hydroxy-5-androsten-17-one
dehydroepiandrosterone sulfate
dihydrotestosterone, 17β-hydroxy-5α-androstan-3-one
estrone, 3-hydroxy-1,3,5(10)-estratriene-17-one
estradiol, 1,3,5(10)-estratriene-3,17β-diol
estriol, 1,3,5(10)-estratriene-3,16α,17β-triol
follicle-stimulating hormone
4,5,7-trihydroxyisoflavone
human chorionic gonadotropin
luteinizing hormone
progesterone, 4-pregnene-3,20-dione
PRL-like protein-B
3β-hydroxy-5-pregnen-20-one
prolactin
testosterone, 17β-hydroxy-4-androsten-3-one
Others:
17HSD/KSR
17HSD/KSR1–8
20α-HSD
3α-HSD
3β-HSD
AF
AKR
ATP
BSA
cAMP
cDNA
CMV
ER
ERE
ERKO
GAPDH
HEK
HPLC
HSD17B1–5
HSD17BP1
hsd17b1
kb
kDa
Ke6
Km
MFP
mRNA
+
NAD
+
NADP
P450arom
P450c17
P450scc
PBS
p.c.
PKA
PKC
PR
PRAP
RACE
RT-PCR
SDR
SHBG
Vmax
X
17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase
17HSD/KSR type 1–type 8
20α-hydroxysteroid dehydrogenase
3α-hydroxysteroid dehydrogenase
5
4
3β-hydroxysteroid dehydrogenase/∆ -∆ isomerase
transcriptional activation function
aldoketoreductase
adenosine-5’-triphosphate
bovine serum albumin
cyclic adenosine-3’,5’-monophosphate
complementary DNA
cytomegalovirus
estrogen receptor
estrogen-responsive element
estrogen receptor knockout
glyceraldehyde-3-phosphate dehydrogenase
human embryonic kidney
high performance liquid chromatography
gene for 17HSD/KSR type 1–type 5
17HSD/KSR1 pseudogene
rat 17HSD/KSR1 gene
kilobase
kilodalton
mouse 17HSD/KSR8 gene
Michaelis-Menten constant
multifunctional protein
messenger RNA
nicotinamide-adenine dinucleotide
nicotinamide-adenine dinucleotide phosphate
cytochrome P450 aromatase
17α-hydroxylase/C17-20 lyase
cholesterol side chain cleavage enzyme
phosphate-buffered saline
post coitum
protein kinase A
protein kinase C
progesterone receptor
PRL receptor-associated protein
rapid amplification of cDNA ends
reverse transcriptase PCR
short-chain dehydrogenase/reductase
sex hormone binding globulin
maximal velocity
any amino acid
List of original publications
This thesis is based on following articles, which are referred to in the text by their Roman
numerals:
I
Ghersevich S, Nokelainen P, Poutanen M, Orava M, Autio-Harmainen H,
Rajaniemi H & Vihko R (1994) Rat 17β-hydroxysteroid dehydrogenase
type 1: Primary structure and regulation of enzyme expression in rat ovary
by diethylstilbestrol and gonadotropins in vivo. Endocrinology 135: 14771487.
II
Nokelainen P, Puranen T, Peltoketo H, Orava M, Vihko P & Vihko R (1996)
Molecular cloning of mouse 17β-hydroxysteroid dehydrogenase type 1 and
characterization of enzyme activity. Eur J Biochem 236: 482-490.
III
Nokelainen P, Peltoketo H, Vihko R & Vihko P (1998) Expression cloning
of a novel estrogenic mouse 17β-hydroxysteroid dehydrogenase/17ketosteroid reductase (m17HSD7), previously described as a prolactin
receptor associated protein (PRAP) in rat. Mol Endrocrinol 12: 1048-1059.
IV
Nokelainen P, Peltoketo H, Mustonen M & Vihko P (2000) Expression of
mouse 17β-hydroxysteroid dehydrogenase/17-ketosteroid reductase type 7
in the ovary, uterus and placenta: localization from implantation to late
pregnancy. Endocrinology 141: 772-778.
Contents
Abstract
Acknowledgements
Abbreviations
List of original publications
Contents
1 Introduction................................................................................................................... 13
2 Review of the literature................................................................................................. 15
2.1 Physiological roles of estrogens ............................................................................. 15
2.2 Principles of estrogen action................................................................................... 16
2.2.1 Estrogen receptors ......................................................................................... 16
2.2.1.1 Domain structure of steroid hormone receptors ................................ 16
2.2.1.2 Estrogen receptors α and β................................................................ 17
2.2.2 Molecular mechanisms of estrogen action..................................................... 18
2.3 Pathway of estrogen biosynthesis ........................................................................... 19
2.4 Ovarian physiology................................................................................................. 21
2.4.1 Follicle........................................................................................................... 21
2.4.1.1 Follicular maturation and luteinization.............................................. 21
2.4.1.2 Estradiol biosynthesis in the follicle and its role during
folliculogenesis ................................................................................. 22
2.4.2 Corpus luteum ............................................................................................... 23
2.4.2.1 Steroidogenesis and role of estradiol in the corpus luteum ............... 23
2.5 The fetoplacental unit as a sex hormone source ..................................................... 25
2.6 17β-Hydroxysteroid dehydrogenases (17HSDs)/17-ketosteroid reductases
(17KSRs)................................................................................................................ 26
2.6.1 The short-chain dehydrogenases/reductases family....................................... 29
2.6.2 17HSD/KSR type 1 ....................................................................................... 29
2.6.2.1 Structural and functional properties of human 17HSD/KSR1........... 29
2.6.2.2 Tissue distribution and role of human 17HSD/KSR1 in estradiol
biosynthesis....................................................................................... 31
2.6.2.3 Human 17HSD/KSR1 in normal mammary gland and in
breast cancer...................................................................................... 32
2.6.2.4 Structure of the human 17HSD/KSR1 gene ...................................... 32
2.6.2.5 Rat 17HSD/KSR1.............................................................................. 33
2.6.2.6 Regulation of 17HSD/KSR1 ............................................................. 34
2.6.3 17HSD/KSR type 2 ....................................................................................... 35
2.6.3.1 Human 17HSD/KSR2 ....................................................................... 35
2.6.3.2 Rodent 17HSD/KSR2........................................................................ 36
2.6.4 17HSD/KSR type 3 ....................................................................................... 37
2.6.5 17HSD/KSR type 4 ....................................................................................... 38
2.6.6 17HSD/KSR type 5 ....................................................................................... 40
2.6.7 17HSD/KSR type 6 ....................................................................................... 41
2.6.8 17HSD/KSR type 8 ....................................................................................... 41
2.6.9 Other 17HSD/KSRs....................................................................................... 42
3 Outlines of the present study......................................................................................... 44
4 Materials and methods .................................................................................................. 45
4.1 Isolation of RNAs (I-IV) ........................................................................................ 45
4.2 Cloning of rat and mouse 17HSD/KSR1 cDNAs (I, II) ......................................... 45
4.3 Cloning of mouse and rat 17HSD/KSR7 cDNAs (III) ........................................... 46
4.4 Sequencing and bioinformatics (I-IV) .................................................................... 46
4.5 Measurement of 17HSD/KSR activity of human and mouse 17HSD/KSR1 (II) ... 47
4.6 Measurement of 17HSD/KSR activity of mouse and rat 17HSD/KSR7 (III)......... 47
4.7 Northern blot analysis (I-III) .................................................................................. 48
4.8 Reverse transcriptase polymerase chain reaction (II, III) ....................................... 48
4.9 Animal treatments (I, II) ......................................................................................... 49
4.10 Tissue samples (I-IV) ........................................................................................... 49
4.11 Immunohistochemistry (I, II)................................................................................ 49
4.12 In situ hybridization (I, IV)................................................................................... 50
5 Results........................................................................................................................... 51
5.1 Cloning and characterization of rat and mouse 17HSD/KSR1 cDNAs (I, II) ........ 51
5.2 Expression of 17HSD/KSR1 in rat ovary and regulation by gonadotropins (I)...... 52
5.3 Characterization of 17HSD/KSR activity of mouse 17HSD/KSR1 and
comparison with the human enzyme (II)................................................................ 52
5.4 Tissue distribution of mouse 17HSD/KSR1 (II)..................................................... 53
5.5 Cloning and characterization of mouse 17HSD/KSR7 cDNA (III)........................ 53
5.6 Characterization of the enzymatic properties of mouse and rat 17HSD/KSR7 (III) .. 54
5.7 Tissue distribution of 17HSD/KSR7 and its expression pattern in the ovary
during gestation (III, IV) ........................................................................................ 54
5.8 Expression of mouse 17HSD/KSR7 in the uterus and placenta (IV)...................... 55
6 Discussion ..................................................................................................................... 56
6.1 Cloning and characterization of mouse and rat 17HSD/KSR1 ............................... 56
6.2 Expression and regulation of rat 17HSD/KSR1 in granulosa cells......................... 57
6.3 Cloning and characterization of mouse and rat 17HSD/KSR7, and its expression
pattern in the ovary................................................................................................. 57
6.4 17HSD/KSR7 in the mouse uterus and placenta .................................................... 59
7 Summary and conclusions............................................................................................. 60
8 References..................................................................................................................... 61
1 Introduction
Estrogens, female sex steroid hormones, not only affect reproduction but also participate
in a large number of different functions outside the female reproductive system, such as
bone and lipid metabolism and functions in the cardiovascular system. Estrogen action is
transmitted through two nuclear estrogen receptors, estrogen receptor α (ERα) and ERβ,
which are ligand-inducible transcription factors (Tsai & O'Malley 1994, Couse & Korach
1999). They interact with transcription machinery to initiate transcription of target genes
of estrogen action. Of natural human estrogens, estradiol (E2) is biologically the most
active sex hormone, whereas estrone (E1) and estriol (E3) are less active, as a result of
their lower affinity to ERs and decreased affinity of E1-ER and E3-ER complexes to
estrogen-responsive elements (EREs) in target genes (Hisaw 1959, Martucci & Fishman
1979, Kuiper et al. 1997, Melamed et al. 1997). In humans, E2 biosynthesis occurs at a
number of tissue sites. The principal sites are granulosa cells of the ovary in
premenopausal women and syncytiotrophoblasts of the placenta in pregnant women. In
addition, estrogens are produced in peripheral tissues such as the mammary gland and
adipose tissue, especially in postmenopausal women (Labrie 1991, Simpson et al. 1997,
Peltoketo et al. 1999a). In rodents, E2 biosynthesis is thought not to take place in the
placenta but in the corpus luteum (CL) (Gibori et al. 1988 and references therein).
In the traditional endocrine system, estrogens are synthesized in the ovaries, released
to the blood circulation and transported to target tissues (Speroff et al. 1994). In the
blood, estrogens are bound with high affinity to sex hormone-binding globulin (SHBG)
and with low affinity to albumin, as a consequence of which only 2 % of total E2 is free
(Siiteri et al. 1982, Goldfien & Monroe 1991). Estrogens are released from their binding
proteins before they diffuse into the target cells. In addition to endocrine signaling, E2
produced in peripheral tissues can locally act in an autocrine, paracrine or intracrine
manner (Labrie 1991).
In the pathway of E2 biosynthesis, the last two reactions are catalyzed by cytochrome
P450 aromatase (P450arom) and 17β-hydroxysteroid dehydrogenase/17-ketosteroid
reductase (17HSD/KSR) enzymes. P450arom catalyzes conversion of androgens to
estrogens (reviewed by Simpson et al. 1997), whereas 17HSD/KSRs catalyze
interconversion between E1 and E2, and androstenedione (A-dione) and testosterone (T),
for example (reviewed by Peltoketo et al. 1999a). When this work began, two
14
17HSD/KSRs were known, type 1 and type 2. Human 17HSD/KSR1 catalyzes the
reaction from E1 to E2, while 17HSD/KSR2 mainly catalyzes reactions from E2 to E1, and
T to A-dione. The type 2 enzyme is mostly expressed in the placenta, endometrium, liver,
kidney and gastrointestinal tract, where it has been suggested to have a protective role,
inactivating excess E2 (Miettinen et al. 1996a, Moghrabi et al. 1997, Mustonen et al.
1998a, Mustonen et al. 1998b). Originally 17HSD/KSR1 was known to be expressed in
the placenta, ovary, mammary gland and endometrium, both normal and malignant.
However, in the ovaries as well as in peripheral tissues, P450arom was considered to be
the important enzyme involved in E2 biosynthesis, while 17HSD/KSR1, though needed in
E2 biosynthesis, was assumed to be of lesser importance and an enzyme whose expression
is not regulated.
The present study focuses on 17HSD/KSR enzymes participating in E2 biosynthesis.
Role of 17HSD/KSR1 in E2 biosynthesis was studied by cloning rat and mouse
17HSD/KSR1 cDNAs and characterizing expression and regulation of the enzyme in
ovaries. Enzymatic properties of the rodent 17HSD/KSR1 were determined using mouse
17HSD/KSR1 expressed in insect cells and the results compared with the human enzyme.
In further studies, a mouse mammary epithelial cell line was found to possess strong
estrogenic 17KSR activity, profile of which did not match with mouse 17HSD/KSRs
known at that time. Thereby, expression cloning and characterization of this novel type of
17HSD/KSR, type 7, and its cell-specific expression in the placenta and uterus came an
important part of this thesis.
2 Review of the literatu re
2.1 Physiological roles of estrogens
Sexual maturation and function of the female reproductive tract require estrogen action
even though initial development and differentiation of the tract appear to be estrogenindependent (Couse & Korach 1999 and references therein). Estrogens, together with
other hormones, particularly gonadotropins, coordinate the menstrual cycle and stimulate
proliferation of granulosa cells and growth of follicles (Goldfien & Monroe 1991, Adashi
1996a, Robker & Richards 1998). Estrogens also induce and maintain female secondary
sexual characteristics, enhance stromal development and ductal growth in the mammary
gland, and stimulate development of the endometrial lining (Goldfien & Monroe 1991).
More specifically, in the endometrium E2 stimulates proliferation of luminal and
glandular epithelium, and increases expression of progesterone receptor (PR) (Couse &
Korach 1999). In addition, estrogens together with progesterone (P) are necessary for
maintaining pregnancy (Pepe & Albrecht 1995, Albrecht et al. 2000).
In addition to these effects in the reproductive system, estrogens are involved in a wide
variety of other functions in the human body, such as bone and lipid metabolism and
functions in the cardiovascular system. Estrogens play an important role in maintaining
bone mass in adult women by suppressing bone remodeling and maintaining a balance
between osteoblastic and osteoclastic activity (Turner et al. 1994). Recent studies have
also shown the importance of estrogens in bone development and mineralization in men
(Smith et al. 1994, Simpson 1998). Estrogens have cardiovascular-protective effects that
are reported to be mediated indirectly by an effect on lipoprotein metabolism and directly
by an effect on the vessel wall itself (Farhat et al. 1996, Mendelsohn & Karas 1999). The
central nervous system is also a target of estrogen action, since estrogens affect certain
types of memory, mood and behavior and they may even have neuroprotective effects in
the brain as regards Alzheimer’s disease, for example (Panay & Studd 1998, McEwen &
Alves 1999). In addition, estrogens have been suggested to influence the immune system
(Olsen & Kovacs 1996).
16
At the molecular level, E2 is a mitogenic factor that in several target tissues stimulates
cell proliferation by regulating the expression of enzymes, proteins and growth factors
involved in cell replication and nucleic acid synthesis (Cullen & Lippman 1989, Robker
& Richards 1998). Hence, estrogens can also act as tumor-promoting agents by
stimulating the growth of malignant cells transformed by other mechanisms. The best
known estrogen-dependent malignancies are breast cancer and endometrial cancer (Benz
& Lewis 1991). Peripheral or local estrogen biosynthesis, which is significant after
menopause, in particular (Labrie 1991), was connected to breast cancer when it was
realized that E2 accumulates in breast cancer tissue (Fishman et al. 1977, McNeill et al.
1986, Vermeulen et al. 1986, Thijssen & Blankenstein 1994). In line with this, enzymes
catalyzing the last steps of E2 biosynthesis (17HSD/KSR1 and P450arom) are expressed
in peripheral tissues.
2.2 Principles of estrogen action
2.2.1 Estrogen receptors
2.2.1.1 Domain structure of steroid hormone receptors
The mediators of estrogen action, ERs, belong to the steroid/thyroid hormone receptor
superfamily, represented by a large number of genes encoding these ligand-activated
transcription factors. In addition to estrogen and thyroid hormone, the family includes
receptors for androgens, progesterone, glucocorticoids, mineralocorticoids, vitamin D and
retinoic acids. Furthermore, a number of orphan receptors, for which ligands have not yet
been identified, have been cloned (Tsai & O'Malley 1994, Mangelsdorf et al. 1995).
These nuclear receptors can be aligned on the basis of their primary structure homology,
which shows six distinct domains, named A to F (Green & Chambon 1988). The Nterminal region (A/B domain) is of variable length and composition and is poorly
conserved between different receptors. This domain contains a hormone-independent
transcriptional activation function, (AF)-1 (Lees et al. 1989, Tora et al. 1989, Tsai &
O'Malley 1994). The C-domain, containing two zinc-fingers, is a DNA-binding region,
which is the most conserved region in the receptor family. It is also involved in receptor
dimerization (Kumar & Chambon 1988, Tsai & O'Malley 1994). The D-domain, also
called the hinge region, often contains a nuclear localization signal (Picard et al. 1990,
Tsai & O'Malley 1994). The ligand-binding domain (E-domain) is relatively large and is
functionally complex. It not only harbors regions important for ligand binding but also
regions involved in receptor dimerization, nuclear localization and interaction with
transcriptional coactivators and corepressors (Tsai & O'Malley 1994, Horwitz et al.
1996). It also contains AF-2, which in contrast to AF-1, is ligand-dependent (Webster et
17
al. 1988, Lees et al. 1989). The F-domain has been suggested to modulate
agonist/antagonist effects of ligands (Montano et al. 1995).
2.2.1.2 Estrogen receptors α and β
Two estrogen receptors, ERα and ERβ, have been cloned to date. They are similar only in
the C and E regions. Human ERβ shows a high degree of identity (96 %) in the DNAbinding domain and moderate identity (58 %) in the ligand-binding domain compared
with human ERα, whereas other regions are weakly conserved (Mosselman et al. 1996).
Both receptor types bind E2 with high affinity, although ERβ has a slightly lower affinity
(Kd = 0.5–0.6 nM) compared with ERα (Kd = 0.2 nM) (Kuiper et al. 1996, Tremblay et al.
1997). The binding affinity of the two ERs towards other ligands is similar overall,
although there are certain differences, e.g. regarding phytoestrogens (Kuiper et al.
1997). In addition to their capacity to bind to DNA as homodimers, ERα and ERβ can
form heterodimers which bind to the ERE with an affinity similar to ERα homodimer,
and they all are able to stimulate transcription (Cowley et al. 1997, Pettersson et al.
1997). In addition, several ERβ isoforms have been cloned from rat and human tissues
(Warner et al. 1999 and references therein), but wether these isoforms have significant
biological and physiological roles remains to be investigated.
ERs are rather widely distributed in mammals. Regarding reproduction, important
tissues in which ERα is expressed include uterus, mammary gland, ovary, pituitary and
hypothalamus, whereas ERβ is mostly expressed in the granulosa cells of developing
follicles (Couse et al. 1997, Enmark et al. 1997, Kuiper et al. 1997). The biological role
of ERs has been elucidated by studying ER null mice: estrogen receptor α knockout
(αERKO) and βERKO (reviewed by Couse & Korach 1999). Even though the uterus
undergoes normal pre- and neonatal development in ER null mice, in αERKO adults it is
hypoplastic and estrogen-insensitive. In addition, the mammary glands of αERKO mice
exhibit impaired development. Female αERKO mice are unovulatory and thus infertile.
The sterile phenotype is most probably due to defects in the negative feedback loop of E2
on the hypothalamic-pituitary unit, which leads to chronic exposure to abnormally high
concentrations of LH, as a result of which follicles either prematurely luteinize or become
atretic. Adult βERKO mice are subfertile, having reduced litter size due to a decline in
completed folliculogenesis and therefore they scarcely ovulate (Couse & Korach 1999). It
is of interest that the βERKO ovarian phenotype has incomplete penetrance when taking
into account that ERβ is specifically expressed in the granulosa cells (Byers et al. 1997,
Fitzpatrick et al. 1999). It has been suggested that follicle-stimulating hormone (FSH) is
able to correct the defect to some extent since it stimulates proliferation of granulosa cells
(Couse & Korach 1999).
18
2.2.2 Molecular mechanisms of estrogen action
In the general estrogen action mode (Fig. 1), estrogens as lipophilic molecules passively
diffuse into cells (MacLusky 1996), where they either bind to their specific receptors or
are metabolized. According to the current concept, ERs when not occupied by ligand,
form large heterocomplexes that contain heat shock proteins and immunophilins, which
have been suggested to have chaperone functions (Pratt & Toft 1997 and references
therein). In the case of steroid receptors, heat shock proteins have been suggested to
facilitate folding of the hormone-binding domain of the receptors into a high-affinity
steroid-binding conformation (Pratt & Toft 1997). There is also contradictory but
convincing data suggesting that at least in the case of PR and heat shock protein-90,
which is a major constituent of heterocomplexes of steroid receptors, heterooligomerization of the two is an artifact taking place only in vitro (reviewed by Ylikomi
et al. 1998).
Fig. 1. Simplified mechanism of classical estrogen action. In the blood circulation estrogens
(E) are mostly bound to SHBG and albumin (= 98 %), from which they are released before
diffusing into a target cell. Most ERs are present in the nucleus, where they are complexed
with several proteins such as heat shock proteins (Hsp). When estrogen binds to ER,
receptors are released from heterocomplexes, phosphorylated and dimerized. Activated ERdimer is then able to bind basal transcriptional machinery and coactivators in a way that
initiates transcription of an estrogen target gene.
19
Similarly to PR, ER is mainly localized in the nucleus (King & Greene 1984,
Guiochon-Mantel et al. 1996), where E2 then binds to ER. This is believed to cause a
conformational change in the receptor, as a result of which the receptor dissociates from
the heterocomplex, dimerizes and binds to the palindromic ERE sequence (Pratt & Toft
1997). Binding of the receptor dimer to DNA takes place via the zinc-fingers of the DNAbinding domain (Tsai & O'Malley 1994). Upon binding to ERE the agonist-bound
receptor interacts directly or indirectly with basal transcription machinery to enhance
transcription (Tsai & O'Malley 1994, Horwitz et al. 1996, Shibata et al. 1997). During
this binding and activation process, ER is also phosphorylated, which is believed to
stimulate the binding of ER to DNA and/or enhance the gene transactivation capacity of
ER (Tsai & O'Malley 1994, MacLusky 1996, Weigel 1996). The efficiency of
transcription is additionally modulated by coactivators and/or corepressors that interact
with nuclear receptors to enhance or suppress, respectively, the transcription rate at
estrogen target genes (Horwitz et al. 1996, McKenna et al. 1998).
ERs also mediate gene transcription from an AP1 response element via binding of
AP1 transcription factors Fos and Jun (Gaub et al. 1990, Umayahara et al. 1994).
Interestingly, at AP1 sites E2 mediates transcription only with ERα, whereas with ERβ it
inhibits transcription. Antiestrogens mediate transcription at AP1 sites with both ERα
and ERβ (Paech et al. 1997). Similarly to the AP1 element, ER mediates transactivation
via binding to Sp1 transcription factor, which binds to a GC-box and an ERE half-site
(Batistuzzo de Medeiros et al. 1997, Porter et al. 1997). ER can also be activated in the
absence of E2 by agents such as growth factors, dopamine, cyclic adenosine-3’,5’monophosphate (cAMP) and cyclins (Cenni & Picard 1999 and references therein).
In addition to the genomic action of steroids directly on target genes, estrogens can
exert their effects in a nonclassic or nongenomic way through receptors on the cell
surface (Revelli et al. 1998). For instance, E2 has been shown to signal by way of the
SHBG receptor (Nakhla et al. 1997, Rosner et al. 1999). Furthermore, a fraction of the
population of both ERα and ERβ has recently been shown to reside on the cell membrane
and activate G-proteins and cell proliferation (Razandi et al. 1999). Most nongenomic
2+
actions of steroids so far described involve Ca as a second messenger, preceded by an
2+
increase in intracellular Ca concentration which takes place via release of intracellular
2+
calcium stores or stimulation of Ca influx (Revelli et al. 1998). In addition, there is most
probably cross-talk between the genomic and nongenomic actions of estrogens (Revelli et
al. 1998).
2.3 Pathway of estrogen biosynthesis
Biosynthesis of sex steroids starts from cholesterol, which is a precursor of all steroid
hormones (Fig. 2). Cholesterol can be synthesized from acetate in most steroidogenic
tissues, but the favored route is to obtain it from circulating LDL cholesterol or HDL
cholesterol, at least in rats (Albrecht & Pepe 1990, Gibori 1993, Adashi 1996a, Miller
1999). Cholesterol resides in LDL as esters which are hydrolyzed and transported to the
outer membrane of mitochondria by a process not completely clear but which most
20
probably involves sterol carrier protein 2 (Miller 1995). The movement of cholesterol
from the outer to inner mitochondrial membranes is facilitated by the steroidogenic acute
regulatory protein (Stocco 1999). Thereafter, cholesterol is converted to 3β-hydroxy-5pregnen-20-one (pregnenolone) in three sequential steps, 20α-hydroxylation, 22hydroxylation and C20,22-bond scission catalyzed by cholesterol side-chain cleavage
enzyme (P450scc) at a single active site (Duque et al. 1978). This reaction has been
considered to be the rate-limiting step in steroidogenesis but the availability of cholesterol
in the inner mitochondrial membrane is actually the limiting factor in vivo (Miller 1995,
Stocco 1999).
Fig. 2. Pathway of E2 biosynthesis.
In the endoplasmic reticulum, pregnenolone is then converted either to 3β,17αdihydroxy-5-pregnen-20-one (17-hydroxypregnenolone) by 17α-hydroxylase activity of
17α-hydroxylase/C17-20 lyase (P450c17) (Conley & Bird 1997, Miller et al. 1997) or to
5
4
P by 3β-hydroxysteroid dehydrogenase/∆ -∆ isomerase (3β-HSD) (Simard et al. 1996).
Similarly to pregnenolone, P can be converted to a corresponding 17α-hydroxysteroid,
17α-hydroxy-4-pregnene-3,20-dione (17-OH-P) by P450c17. Thereafter, 17hydroxypregnenolone and 17-OH-P can be catalyzed to dehydroepiandrosterone (DHEA)
and A-dione, respectively, by the 17,20-lyase activity of P450c17 (Conley & Bird 1997,
Miller et al. 1997).
5
Reactions from pregnenolone to DHEA, and further to A-dione, are those of the ∆ 4
pathway, whereas reactions from pregnenolone to A-dione via P are those of the ∆ pathway (Conley & Bird 1997). While rat P450c17 is able to utilize both pathways, the
5
human enzyme almost exclusively uses the ∆ -pathway (Conley & Bird 1997). Whether
or not a human endocrine cell synthesizes DHEA or P is thought to depend on the 3βHSD to P450c17 ratio, in addition to which DHEA synthesis can be controlled by
regulating the 17,20-lyase activity of P450c17 by phosphorylation (Conley & Bird 1997,
Miller et al. 1997).
21
DHEA, a general precursor of both androgens and estrogens, is converted to A-dione
by 3β-HSD (Simard et al. 1996). A-dione is then converted to T by 17HSD/KSR3
(Geissler et al. 1994) or by 17HSD/KSR5 (Dufort et al. 1999, Pelletier et al. 1999). The
final steps of E2 biosynthesis are catalyzed by P450arom, which catalyzes aromatization
of androgens to estrogens (Simpson et al. 1997), and by 17HSD/KSR1, which converts E1
to E2 (Peltoketo et al. 1999a and references therein).
2.4 Ovarian physiology
2.4.1 Follicle
The ovaries are responsible for the maturation and release of fertilizable ova, and for the
biosynthesis of steroid hormones, mainly E2, A-dione and P (Adashi 1996a). Ovarian
functions are controlled by complex feedback mechanisms that involve gonadotropinreleasing hormone from the hypothalamus, gonadotropins from the pituitary, and steroid
hormones and protein hormones secreted locally in the ovary (Adashi 1996a, Ferin 1996).
E2 is synthesized in the granulosa cells of follicles, where its synthesis is in parallel with
follicular maturation, so that preovulatory follicles are the most abundant E2 source
(Gougeon 1996).
2.4.1.1 Follicular maturation and luteinization
Follicular maturation starts from primordial follicles, where a single layer of granulosa
cells surrounding the oocyte exit the G0 phase of the cell cycle and start slow proliferation
under circumstances that are still unclear (Gougeon 1996, Robker & Richards 1998).
Once follicles have begun to grow, basal concentrations of the gonadotropins, FSH and
LH, maintain growth from primary follicles to the small antral stage (Richards 1994,
Gougeon 1996). During this maturation process the follicles acquire more gonadotropin
receptors, the expression of which is stimulated by FSH and E2. Follicles thereby gain
enhanced responsiveness to FSH and LH, and begin producing E2, which leads to
maturation of the follicles to the antral stage (Adashi 1996a, Adashi 1996b, Gougeon
1996). At the final phase, exposure to gonadotropins and estrogen triggers a rapid burst of
proliferation that results in the formation of large preovulatory follicles (Adashi 1996b,
Gougeon 1996, Robker & Richards 1998). The mitogenic effect of FSH and E2 on the
growth of granulosa cells is transduced via increasing expression of cyclin D2 and E,
which are regulatory proteins of the cell cycle (Robker & Richards 1998). It is
characteristic of primate folliculogenesis that usually only one follicle reaches the
preovulatory stage, while the rest of the follicles become atretic through the process of
apoptosis (Adashi 1996b, Ferin 1996, Gougeon 1996). Granulosa cells of preovulatory
22
follicles are not only highly proliferative but also differentiating and they acquire LH
receptors, whose expression is stimulated by LH itself (Richards 1994, Adashi 1996a,
Gougeon 1996, Robker & Richards 1998). At the end of the follicular phase, the serum
concentration of E2 rises to a level able to stimulate the gonadotropin surge, an LH peak
particularly, that triggers changes leading to ovulation (Ferin 1996). During this process
granulosa cells exit the cell cycle, cease to divide and terminally differentiate, i.e.
luteinize to form mature CL (Robker & Richards 1998 and references therein). Upon
luteinization, the expression of cyclin D2 and E is down-regulated, whereas expression of
KIP1
the negative regulatory protein of the cell cycle, p27 , is enhanced (Robker & Richards
1998). Other proteins whose expression is changed during luteinization include
P450arom, P450c17 and ERβ. Their expression is down-regulated, whereas expression of
PR, P450scc, proteolytic enzymes and prostaglandin endoperoxide synthase-2 is upregulated (Richards 1994, Tsafriri & Chun 1996, Byers et al. 1997, Richards et al. 1998,
Fitzpatrick et al. 1999).
2.4.1.2 Estradiol biosynthesis in the follicle and its role during
folliculogenesis
The pathway of E2 biosynthesis in follicles is compartmentalized, which is known as the
two-cell, two-gonadotropin model (Hillier et al. 1994, Adashi 1996a). LH stimulates
biosynthesis of androgens, mainly A-dione (Bergh et al. 1993), a precursor of E2
biosynthesis, in theca cells, which express LH receptor. One of the target genes of LH
action in theca cells is that for P450c17. From theca cells, androgens traverse the
basement membrane to the neighboring granulosa cells, which express FSH receptor. In
granulosa cells A-dione is first catalyzed to E1 by P450arom and then to E2 by
17HSD/KSR1, the expression of both of these enzymes being stimulated by FSH
(Ghersevich et al. 1994a, Hillier et al. 1994, Adashi 1996a, Kaminski et al. 1997,
Peltoketo et al. 1999a and references therein). In other words, theca cells do not
synthesize estrogens since they do not express P450arom or 17HSD/KSR1 (Hillier et al.
1994, Sawetawan et al. 1994). Granulosa cells in turn are not able to produce androgens
since they do not express P450c17. However, both cell types express P450scc and 3βHSD (Hillier et al. 1994, Richards 1994).
In addition to its multiple systemic effects, E2 exerts a variety of critical actions at the
level of the ovary, where it affects the growth and development of ovarian follicles by
stimulating the proliferation of granulosa cells (Robker & Richards 1998), stimulating
antrum formation, increasing, together with FSH, gonadotropin receptor levels in the
granulosa cells, enhancing gonadotropin-stimulated P450arom activity, and stimulating
gap junction formation among granulosa cells (Adashi 1996a, Shoham & Schachter 1996
and references therein,). Furthermore, E2 plays a major role in feedback communication
between the ovary and the hypothalamic-pituitary unit. The first of two feedback loops is
the negative loop, whereby E2 feeds back to adjust gonadotropin-releasing hormone
and/or gonadotropin secretion (Ferin 1996). Positive feedback occurs as a stimulus to
ovulation, as described in section 2.4.1.1. In line with these estrogen effects, both ER
23
types are expressed in the ovary, ERβ being the dominant form in granulosa cells (Byers
et al. 1997, Enmark et al. 1997, Fitzpatrick et al. 1999). In addition, recent studies with
ERKO mice suggests that both ERα and ERβ are involved in the negative feedback loop
of E2 on the hypothalamic-pituitary unit, suppressing gonadotropin secretion (Couse &
Korach 1999, Couse et al. 1999).
2.4.2 Corpus luteum
As a result of gonadotropin surge-stimulated follicular rupture, ovulation, the follicle
becomes the CL. The main role of the CL is the production of P, in addition to which it
secretes estrogens, and in humans, peptide/protein hormones such as relaxin, oxytocin
and inhibin-like factors. The physiological role of P is related to preparation of the
reproductive tract and gametes for fertilization, implantation, and the maintenance of the
uterine environment during gestation (Gibori 1993, Pfeifer & Strauss 1996). The human
CL is an active endocrine gland during the luteal phase, while in rodents, secretion of P
after ovulation is short-lived and therefore the CL of the cycle is not believed to have a
clearly defined role. In both humans and rodents, the CL is a transient organ if not
hormonally stimulated as a result of pregnancy, or as a result of mating in rodents. The
human CL is rescued by human chorionic gonadotropin (hCG), while in rodents PRL is
needed for this purpose. In rodents, function of the CL is indispensable throughout
gestation, whereas in humans, the placenta replaces luteal functions in early pregnancy
(luteal-placental shift), after which the CL is luteolyzed (Taya & Greenwald 1981, Gibori
et al. 1988, Gibori 1993, Stouffer 1996).
2.4.2.1 Steroidogenesis and role of estradiol in the corpus luteum
Although there is a transient decline in androgen and estrogen synthesis around the time
of ovulation, P450c17 and P450arom mRNAs and activity remain elevated in human
luteal tissue throughout the luteal phase as well as in the CL of pregnancy until the lutealplacental shift (Stouffer 1996). In rat CL of pregnancy, P450scc and 3β-HSD are
expressed more or less in a constitutive manner (Gibori 1993), whereas expression of
P450c17 and P450arom are regulated. Expression of P450c17 is stimulated by LH until
mid-pregnancy, when LH levels drop (Morishige et al. 1973, Khan et al. 1987), which
results in a decline of androgen production. At this stage, the rodent placenta becomes the
primary source of androgen (Fig. 3) which is necessary for maximal ovarian E2
biosynthesis (Warshaw et al. 1986, Gibori et al. 1988). The amount of P450arom is low
in the first half of pregnancy, it increases from mid-pregnancy, and declines at the end of
pregnancy (Hickey et al. 1988). In contrast to granulosa cells, P450arom expression in
the rat CL is not affected by FSH, LH or cAMP but is regulated by PRL, placental
lactogen and luteal E2 itself, for example (Gibori 1993). No specific 17HSD/KSR has
24
been demonstrated in the CL of rodents, whereas in humans, 17HSD/KSR1 is expressed
at least to some extent in the CL (Sawetawan et al. 1994).
Fig. 3. Estrogen biosynthesis during pregnancy: primates vs. rodents. In primates estrogens
are predominantly synthesized from fetal and maternal precursors in the placenta, whereas
in rodents synthesis mainly takes place in the CL. Estrogen biosynthesis in the luteal cells of
rodent CL relies on luteal androgen precursors in the first half of gestation, while in the
second half of pregnancy the placenta supplies the CL with androgen precursors. In rodents,
cooperation between the CL and placenta is also evident in the source of P. The placenta
utilizes luteal P as an additional source for androgen biosynthesis since P production is
limited in the rodent placenta (Gibori et al. 1988, Gibori 1993, Kuss 1994, Wilson & Parson
1996).
In rodents, E2 acts by an intracrine mechanism to affect steroidogenesis,
vascularization and protein synthesis of the CL (Gibori et al. 1988, Gibori 1993). During
the first week of pregnancy, PRL secreted by the pituitary is able to sustain basal levels of
P, but E2 is necessary for maximal P production (Gibori & Keyes 1980). In the second
week, E2 becomes essential for normal production of P. Between days 12-21, E2 is
25
responsible together with placental lactogen, a PRL-related protein, for the remarkable
increase in CL weight, vascularization and steroidogenic capacity (Gibori et al. 1988,
Gibori 1993). In line with estrogen’s local effects in the CL, luteal cells express both ERα
and ERβ (Telleria et al. 1998, Misao et al. 1999). A prerequisite for E2 action is previous
exposure of the CL to PRL and PRL-related hormones, an important role of which is to
sustain high levels of ER (Gibori & Keyes 1980, Gibori et al. 1988). In fact, expression of
both ERs has been shown to be up-regulated by PRL and placental lactogens (Telleria et
al. 1998). The main action of E2 in steroidogenesis is to increase the supply of cholesterol
substrate by mobilizing cholesterol storage, stimulating cholesterol synthesis and
enhancing luteal cell content of lipoprotein receptors and thus uptake of circulating
cholesterol. It also stimulates the transport of cholesterol to mitochondrial P450scc. In
addition, E2 enhances luteal cell hypertrophy by increasing protein content through
stimulation of expression of elongation factor 2. E2 stimulation of luteal cell hypertrophy
is accompanied by a remarkable proliferation of vascular endothelial cells (Gibori et al.
1988, Gibori 1993).
2.5 The fetoplacental unit as a sex hormone source
The placenta has several important functions during pregnancy: it physically anchors the
fetus to the uterus, it transports nutrients from the maternal circulation to the fetus, it
excretes fetal metabolites into the maternal compartment, it has an immunomodulatory
role in the maternal acceptance of the fetus, and it produces a wide range of peptide and
steroid hormones (Strauss et al. 1996, Wilson & Parson 1996). Estrogens (E1, E2 and E3)
and P are the principal steroid hormones produced by the placenta during primate
pregnancy (Albrecht & Pepe 1990, Miller 1999). At the beginning of human pregnancy
these hormones are produced by the CL, whose functional life span is only ten weeks.
From approximately the eighth week, the placenta acts as a significant sex hormone
source (Albrecht & Pepe 1990, Strauss et al. 1996).
The main role of estrogen during pregnancy is to stimulate key steps of P biosynthesis
in syncytiotrophoblasts by enhancing cholesterol formation in the fetal liver, LDL uptake
and P450scc activity (Pepe & Albrecht 1995). In addition, E2 enhances uteroplacental
blood flow and possibly placental neovascularization to provide optimal gas exchange
and nutrients, and it is also involved in the regulation of mammary gland development
during pregnancy. The key role of P is to maintain pregnancy by promoting uterine
myometrial quiescence, in addition to which it has a suppressive action on the immune
system to prevent rejection of the developing fetus and placenta (Pepe & Albrecht 1995,
Wilson & Parson 1996).
Human placental villi consist of two types of trophoblast cells: an inner layer of
cytotrophoblasts, which are mitotically active mononuclear cells prominent early in
pregnancy, and an outer layer of syncytiotrophoblasts, which are fused nondividing
multinuclear cells that become predominant later in pregnancy. Cytotrophoblasts and
syncytiotrophoblasts produce peptide and protein hormones, whereas the
syncytiotrophoblasts produce all of the steroid hormones (Ringler & Strauss 1990,
26
Wilson & Parson 1996). Biosynthesis of P starts from the uptake of LDL cholesterol,
since de novo synthesis of cholesterol is believed to be limited in the placenta (Albrecht
& Pepe 1990). Because human placenta does not express P450c17 (Albrecht & Pepe
1990, Strauss et al. 1996), estrogen biosynthesis depends on fetal and maternal adrenal
precursors, DHEA and dehydroepiandrosterone sulfate (DHEAS), and 16αhydroxydehydroepiandrosterone sulfate (16α-OH-DHEAS) produced by the fetal liver
(Fig. 3) (Albrecht & Pepe 1990, Miller 1999). In the placenta, these steroids are taken up
by syncytiotrophoblasts, where they are desulfonated by steroid sulfatase (Salido et al.
1990) and thereafter converted to E1, E2 and E3 by sequential reactions catalyzed by 3βHSD type 1, P450arom and 17HSD/KSR1 (Albrecht & Pepe 1990 and references therein,
Miller 1999, Peltoketo et al. 1999a). The normal ratio of E1, E2 and E3 in term cord blood
is 14:5:81 (Miller 1999).
The rodent chorioallantoic placenta is structurally different from the primate placenta
and consists of four types of differentiated trophoblast cells: trophoblast giant cells,
spongiotrophoblasts, glycogen cells and syncytial trophoblast cells (Soares et al. 1996).
They contribute to the formation of two morphologically and functionally distinct regions
of the chorioallantoic placenta, the junctional zone and the labyrinth zone. Trophoblast
giant cells and spongiotrophoblasts exhibit endocrine activities by producing a variety of
peptide hormones and steroid hormones, whereas syncytial trophoblast cells in the
labyrinth zone carry out transport functions and the glycogen cells serve as a potential
energy reserve (Sherman 1983, Soares et al. 1996). The biosynthesis of sex steroids
represents another major difference between the human and rodent placenta. In contrast
to human placenta, rodent placenta is believed to be unable to synthesize E2 due to its lack
of P450arom (Townsend & Ryan 1970, Rembiesa et al. 1971). In addition, only a limited
amount of P is secreted in the rodent placenta (Matt & MacDonald 1984). An unique
feature of the rodent placenta is synthesis of androgen precursors (Fig. 3), which are then
used in E2 biosynthesis in the CL (Warshaw et al. 1986, Gibori et al. 1988).
2.6 17β
β-Hydroxysteroid dehydrogenases (17HSDs)/17-ketosteroid
reductases (17KSRs)
The 17HSD/KSRs are nicotinamide adenine dinucleotide [NAD(H)]- and/or its phosphate
form [NADP(H)]-dependent enzymes that in a positional and stereospecific manner
catalyze hydride transfer from NAD(P)H to 17-ketosteroids (17KSR activity) or from
+
17β-hydroxysteroids to NAD(P) (17HSD activity) (Peltoketo et al. 1999a). Since both
estrogens and androgens have their highest activity in the 17β-form, 17HSD/KSRs
regulate the biological activity of these sex steroids. The 17HSD/KSRs primarily convert
the relatively inactive sex steroids E1, A-dione and 5α-androstane-3,17-dione (5α-Adione) to their more potent forms: E2, T and dihydrotestosterone (DHT), and vice versa.
Therefore, reductive 17HSD/KSRs are indispensable for E2 and T biosynthesis in the
ovary and testis, respectively. In addition to the gonads, these 17HSD/KSRs in primates
and rodents are also expressed in certain peripheral tissues (Martel et al. 1992, Labrie et
al. 1997), where they participate in regulation of local steroid hormone concentrations. In
27
turn, oxidative 17HSD/KSRs, preferring inactivation of sex hormones by
dehydrogenation, are generally more widely expressed and they are likely to protect
tissues from excessive hormone action (Moghrabi et al. 1997, Mustonen et al. 1998b,
Peltoketo et al. 1999a, Peltoketo et al. 1999b).
At least eight different 17HSD/KSRs have been cloned to date, and they have been
named chronologically types 1–8 (Peltoketo et al. 1999b and references therein). Despite
the same reaction type catalyzed, they share an amino acid sequence identity of less than
30 %. In addition to their distinct primary structures, they are dissimilarly distributed,
they differ in substrate and cofactor specificities, and they consequently have apparently
separate physiological functions. In particular, 17HSD/KSR types 1, 3 and 5 mainly
catalyze reductive reactions of estrogens and androgens (Poutanen et al. 1993, Geissler et
al. 1994, Miettinen et al. 1996a, Dufort et al. 1999), whereas types 2, 4, 6 and 8 can be
considered as oxidative enzymes (Wu et al. 1993, Adamski et al. 1995, Biswas & Russell
1997, Fomitcheva et al. 1998). Several 17HSD/KSR enzymes, even counteracting ones,
may coexist in the same tissue and even in the same cell type (Miettinen et al. 1996a),
where steady-state concentrations of sex steroids are a result of the sum of action of
different 17HSD/KSRs.
In addition, some of the enzymes named 17HSD/KSR have also been reported to
catalyze several other reactions, such as 20α-HSD, 3α-HSD and 3β-HSD reactions, βoxidation of fatty acids, oxidation of xenobiotics and reactions between retinoic acids
(Wu et al. 1993, Deyashiki et al. 1995, Dieuaide-Noubhani et al. 1996, Leenders et al.
1996, Qin et al. 1997a, Dufort et al. 1999, Su et al. 1999). The properties of different
17HSD/KSRs are summarized in Table 1.
Human
Mouse
8/SDR
Microsomal
(Ando et al. 1996)
(Aziz et al. 1993)
(Biswas & Russell 1997)
(Deyashiki et al. 1995)
Membranebound?
Unknown
(Geissler et al. 1994)
Microsomal
(Sha et al. 1997)
(Adamski et al. 1995)
Peroxisomal
(Corton et al. 1996),
(Dieuaide-Noubhani et al. 1996),
(Qin et al. 1997a)
(Normand et al. 1995)
(Leenders et al. 1994)
(Kobayashi et al. 1997)
(Caira et al. 1998)
(Lin et al. 1997),
Cytosolic
(Dufort et al. 1999)
(Wajima et al. 1999)
(Wu et al. 1993)
(Akinola et al. 1996)
(Mustonen et al. 1997a)
Subcellular
localization
Cytosolic
Adapted and modified from a review by Peltoketo et al. (1999b).
Rat
Mouse
Mouse
Porcine
Chicken
Guinea Pig
Human
Human
Mouse
Human
Rat
6/SDR
5/AKR
4/SDR
3/SDR
2/SDR
Chicken
Human
Rat
Mouse
Type/protein Species
References regarding
family
cloned from cloning
1/SDR
Human
(Peltoketo et al. 1988),
(Luu The et al. 1989)
Table 1. Properties of 17HSD/KSR enzymes
Liver, kidney
Prostate, liver
Liver, prostate,
CL, testis, uterus,
mammary gland
Liver, kidney,
testis
Widely
distributed
Reductase
T production
Dehydrogenase E2 & T inactivation,
(P production)
Type of activity Putative
function
Reductase
E2 production
Estrogens,
androgens
Androgens,
estrogens,
(xenobiotics)
Androgens
Androgens,
progestins
T production
Dehydrogenase DHT inactivation pathway
Dehydrogenase E2 & androgen
inactivation
Reductase
Fatty acyl-CoA, Dehydrogenase β-oxidation of
(estrogens)
fatty acids, (E2
inactivation)
Mainly expressed Substrate
specificity
Ovary, placenta, Estrogens
(mammary gland,
endometrium)
Ovary
Placenta, liver,
Estrogens,
gastrointestinal
androgens
tract,
(progestins)
endometrium (in
humans)
Testis
Androgens
28
29
2.6.1 The short-chain dehydrogenases/reductases family
Apart from 17HSD/KSR5, all 17HSD/KSRs cloned so far belong to a large protein
family of short-chain dehydrogenases/reductases (SDRs) which are also called shortchain alcohol dehydrogenases. 17HSD/KSR5 belongs to the aldoketoreductase (AKR)
protein family. The SDR family includes enzymes from humans, animals, plants, insects
and bacteria that use steroids, sugars, prostaglandins, antibiotics, aromatic hydrocarbons
and compounds involved in nitrogen metabolism as substrates (Krozowski 1992,
Krozowski 1994, Jörnvall et al. 1995). These enzymes have an average length of 250
amino acids and they act independently of metal cofactors, in contrast to medium-chain
dehydrogenases/reductases that need zinc (Persson et al. 1991). Even though SDRs share
a low overall amino acid identity (15–30 %), their three-dimensional folding and
hydrophobic core are similar, and most SDRs are dimers or tetramers (Jörnvall et al.
1995). In addition, overall comparisons of SDR enzymes suggest the possibility of related
reaction mechanisms and domain properties within the family (Persson et al. 1991). Of
the family, bacterial 3α,20β-hydroxysteroid dehydrogenase was the first SDR whose
tertiary structure was determined using crystallographic analyses (Ghosh et al. 1991,
Ghosh et al. 1994).
The only amino acid residue totally conserved in the entire family is the tyrosine
residue in the highly conserved Tyr-X-X-X-Lys motif (X, any amino acid) (Jörnvall et al.
1995), which forms part of a catalytic triad involved in the transfer of hydride between
cofactor and substrate (Ghosh et al. 1995, Jörnvall et al. 1995). A largely conserved
serine residue, 13 residues upstream from the tyrosine motif (Jörnvall et al. 1995) is also
part of the catalytic triad (Ghosh et al. 1995). An additional conserved region is a
glycine-rich motif (Gly-X-X-X-Gly-X-Gly) in the N-terminal part of the molecule that
binds cofactor, NAD(H) or NADP(H) (Krozowski 1992, Jörnvall et al. 1995). As a
phylogenetic study has shown, 17HSD/KSRs do not form a subclass within the SDR
family but are in different branches (Baker 1995). It is not known, however, if different
17HSD/KSRs are a result of gene duplication followed by sequence divergence or
functional convergence (Baker 1995).
2.6.2 17HSD/KSR type 1
2.6.2.1 Structural and functional properties of human 17HSD/KSR1
Human 17HSD/KSR1 was partially purified from human placenta as early as in 1958
(Langer & Engel 1958). The complete amino acid sequence based on cloned cDNA was
described 30 years later by Peltoketo et al. (1988) and Luu-The et al. (1989). The years
between the 60s and the late 80s were the time of development of purification methods
and analyses of enzymatic properties. Human 17HSD/KSR1 is a cytosolic enzyme that
was initially found to catalyze mainly the interconversion between E1 and E2 using
30
NAD(H) or NADP(H) as a cofactor (Jarabak 1969, Jarabak & Sack 1969, Karavolas &
Engel 1971, Jin & Lin 1999). Purified native 17HSD/KSR1 and recombinant type 1
enzyme, produced in Sf9 cells, catalyze more efficiently the reaction from E1 to E2 than
the opposite reaction (Puranen et al. 1994, Puranen et al. 1997a, Jin & Lin 1999). In
addition, when the activity of 17HSD/KSR1 is measured in intact cultured cells, which is
believed to correspond well to conditions in vivo, the enzyme almost exclusively
catalyzes the reaction from E1 to E2 (Poutanen et al. 1993). Furthermore, there is a
correlation between E1 to E2 17KSR activity and type 1 enzyme expression in several cell
lines (Miettinen et al. 1996a).
In addition to estrogenic reactions, 17HSD/KSR1 has been detected to catalyze
interconversions between A-dione and T, DHEA and androstenediol (A-diol), 5α-Adione and DHT, and between P and 20α-hydroxyprogesterone (20-OH-P) (Purdy et al.
1964, Jarabak & Sack 1969, Strickler et al. 1981, Mendoza-Hernández et al. 1984,
Puranen et al. 1997a). However, the results of kinetic studies have shown that human
17HSD/KSR1 prefers C18 steroids over C19 and C20 steroids as substrates (Langer et al.
1959, Adams et al. 1962, Purdy et al. 1964, Jarabak & Sack 1969, Mendoza-Hernández
et al. 1984, Puranen et al. 1997a).
Native human 17HSD/KSR1 is a dimer composed of two identical subunits which are
non-covalently bound, but strongly associated with each other (Burns et al. 1971, Burns
et al. 1972, Lin et al. 1992, Puranen et al. 1997b). According to the results of
polyacrylamide gel electrophoresis the size of a subunit is approximately 34 kilodaltons
(kDa), which corresponds well with the calculated molecular mass (34.9 kDa) based on
the cDNA (Peltoketo et al. 1988, Luu The et al. 1989). The native enzyme has been
reported to show microheterogeneity, with several isoelectric points (Engel & Groman
1974) that could be due to post-translational modifications. The absence of carbohydrates
in the purified protein (Burns et al. 1972) and the absence of N-linked glycosylation sites
in the primary structure (Peltoketo et al. 1988) rules out this source of heterogeneity.
Recent studies have shown that the enzyme possesses a serine residue, Ser134, which is
phosphorylated by protein kinase A (PKA) in vitro (Barbieri et al. 1994, Puranen et al.
1997b). Phosphorylation, however, has no significant effect on the catalytic properties or
microheterogeneity of the enzyme (Puranen et al. 1997b).
Using X-ray crystallographic techniques, the three-dimensional structure of human
17HSD/KSR1 has recently been resolved by Ghosh et al. (1995), utilizing the structure of
3α,20β-hydroxysteroid dehydrogenase. The structure and reaction mechanism have been
further resolved in subsequent studies (Gibori et al. 1988, Azzi et al. 1996). Human
17HSD/KSR1 has a secondary structure with alternating α-helices and β-strands
resulting in a similar overall fold as in other SDRs (Ghosh et al. 1995). At the catalytic
site, Tyr155 is essential for catalysis (Puranen et al. 1994, Puranen et al. 1997b). This
tyrosine residue is a proton donor in the hydride transfer chain. According to the current
model, the hydroxyl groups at Tyr155 and Ser142 and the steroid O17 atom form a triangular
hydrogen bond network enabling hydride transfer and reduction of E1 to E2. Ser142 has
been suggested to stabilize the intermediate transition states. In addition, a third amino
acid, Lys159, has been suggested to be directly involved in catalysis by forming hydrogen
bonds with the cofactor and apparently lowering the pKa of the hydroxyl proton in Tyr155
to facilitate proton transfer (Ghosh et al. 1995, Azzi et al. 1996, Breton et al. 1996,
Peltoketo et al. 1998). The difference in substrate specificity between human and rat
31
17HSD/KSR1 (see section 2.6.2.5.) has been suggested to be due to Asn152, Pro187 and
amino acids between residues 190–230, and membrane association near the active site of
the enzyme (Puranen et al. 1997b, Peltoketo et al. 1998). Detailed knowledge of the
structure of 17HSD/KSR1 is being utilized to design and generate specific and potent
inhibitors of 17HSD/KSR1 for clinical use.
2.6.2.2 Tissue distribution and role of human 17HSD/KSR1
in estradiol biosynthesis
A 1.3-kilobase (kb) mRNA encoding human 17HSD/KSR1 is mainly expressed in the
ovary and placenta, in addition to which it is also expressed in the mammary gland,
endometrium, and certain breast cancer and choriocarcinoma cell lines (Luu-The et al.
1990, Poutanen et al. 1990, Miettinen et al. 1996a, Zeitoun et al. 1998, Miettinen et al.
1999). In line with this, 17HSD/KSR1 protein is expressed in the ovary, placenta, in
choriocarcinoma cell lines, normal endometrium (Mäentausta et al. 1990, Mäentausta et
al. 1991, Sawetawan et al. 1994, Miettinen et al. 1996a), endometriotic lesions (Zeitoun
et al. 1998) and in around 50 % of endometrial adenocarcinoma specimens (Mäentausta
et al. 1992). The protein has also been detected in some samples of normal mammary
gland (Söderqvist et al. 1998), and in about 70 % of benign and 50 % of malignant breast
specimens, where it is present in the epithelial cells, although in highly variable amounts
(Poutanen et al. 1992a, Sasano et al. 1996).
In the ovary 17HSD/KSR1 is expressed in the granulosa cells of developing follicles,
ranging from primary to tertiary follicles (Sawetawan et al. 1994). The association
between 17HSD/KSR1 expression and the maturation stage of follicles (Ghersevich et al.
1994a, Sawetawan et al. 1994) is related to their capacity to produce E2 (Gougeon 1996).
The correlation of 17HSD/KSR1 expression with 17KSR activity and with E2 production
in granulosa cells further confirms that the type 1 enzyme is the major 17HSD/KSR
involved in E2 biosynthesis during follicle maturation (Ghersevich et al. 1994a). Human
17HSD/KSR1 is also expressed in the granulosa-luteal cells of the CL (Ghersevich et al.
1994a, Sawetawan et al. 1994).
In the placenta 17HSD/KSR1 is expressed in the syncytiotrophoblasts (FournetDulguerov et al. 1987, Mäentausta et al. 1991, Sawetawan et al. 1994, Mustonen et al.
1998a, Takeyama et al. 1998), which have a high capacity to produce E2 from androgen
precursors of fetal and maternal origin, since the cells also abundantly express P450arom
(Fournet-Dulguerov et al. 1987, Simpson et al. 1995). In the endometrium the type 1
enzyme is present in glandular and surface epithelial cells (Mäentausta et al. 1991),
where its concentration is highest during the early to mid-secretory phase (Mäentausta et
al. 1990, Mäentausta et al. 1991). E2 stimulates proliferation of the endometrial
epithelium in the proliferative phase, but the role of 17HSD/KSR1 and E2 biosynthesis in
secretory endometrium is obscure since ER concentrations decrease in the secretory
phase (Fleming et al. 1980) and it is oxidative 17HSD/KSR2 that predominates in the
endometrium (Casey et al. 1994).
32
2.6.2.3 Human 17HSD/KSR1 in normal mammary gland and
in breast cancer
During adolescence the duct system of the mammary gland proliferates extensively, and
this is under the control of estrogen and growth hormones (Couse & Korach 1999 and
references therein). It is thus not surprising that estrogen-induced proliferation is assumed
to play a central role in the promotion of breast cancer and tumor growth (Cullen &
Lippman 1989 and references therein). Estrogen metabolism in the mammary gland and
its influence on the risk of breast cancer have been subjects of interest lately (Labrie
1991, Santen 1996, Bulun et al. 1997), since it was realized that E2 accumulates in breast
cancer tissue (Fishman et al. 1977, McNeill et al. 1986, Vermeulen et al. 1986, Thijssen
& Blankenstein 1994), and 17HSD/KSR1, P450arom and steroid sulfatase are expressed
in the mammary gland (Santner et al. 1984, Sasano et al. 1996, Bulun et al. 1997). These
enzymes are needed to synthesize E2 in situ from the circulating precursors A-dione, E1
and E1-sulfate. After menopause, in particular, peripheral estrogen biosynthesis plays a
major role in estrogen action (Labrie 1991).
The role of human 17HSD/KSR1 in local E2 biosynthesis and its impact on
proliferation have been demonstrated in studies where the enzyme was expressed in
cultured breast cancer cells to the same degree as in certain breast tumors. E1 increased
the growth of cells in a similar manner as those treated with E2, showing the activity and
growth-promoting role of 17HSD/KSR1. A growth response was not achieved with E1 in
breast cancer cells not expressing 17HSD/KSR1 (Miettinen et al. 1996b). Hence,
inhibitors of the type 1 enzyme could be feasible as regards additional breast cancer
treatment, since some breast cancer patients initially treated with antiestrogens relapse,
probably due to enhanced estrogen sensitivity, indicating that inhibitors that block
estrogen biosynthesis completely are needed (Santen et al. 1990, Masamura et al. 1995).
Antiestrogens and inhibitors of E2 biosynthesis, mainly inhibitors of P450arom, have
already been utilized in the treatment of breast cancer for over two decades (Santen et al.
1990, Miller 1996). Designing of inhibitors of 17HSD/KSR1 and steroid sulfatase is also
in progress (Penning 1996, Reed et al. 1996). Interestingly, several phytoestrogens,
especially apigenin, coumestrol and genistein, have inhibitory effects on 17HSD/KSR1
activity in vitro and in cultured breast cancer cells (Mäkelä et al. 1995, Mäkelä et al.
1998). Flavonoids are considered to be the least estrogenic of phytoestrogens. In line with
this, higher concentrations of the flavonoid apigenin are needed to bring about estrogen
action than to inhibit 17HSD/KSR1 (Mäkelä et al. 1995, Mäkelä et al. 1998).
2.6.2.4 Structure of the human 17HSD/KSR1 gene
The gene encoding human 17HSD/KSR1, HSD17B1 (previously also called EDH17B2)
is located in tandem with the HSD17BP1 gene in chromosome region 17q12-21
(Winqvist et al. 1990, Luu-The et al. 1990, Peltoketo et al. 1992, Simard et al. 1993).
HSD17B1 is a small gene consisting of six exons and five short introns. It shares 89 %
identity with HSD17BP1, which is organized in a similar manner. HSD17BP1 is
33
considered to be a pseudogene, since it contains a premature in-frame stop codon at the
position coding for amino acid 214 (Luu-The et al. 1990). In addition, there is a
nucleotide change (A→C) at the putative TATA box of HSD17BP1 which significantly
decreases promoter activity in vitro (Peltoketo et al. 1994). However, Touitou et al.
(1994) reported that by using the reverse transcriptase polymerase chain reaction (RTPCR) a transcript from HSD17BP1 can be detected in several cancer cell lines and biopsy
samples from the mammary gland, uterus and placenta.
Human HSD17B1 is transcribed into two mRNAs, 1.3 and 2.3 kb in size, using two
major transcription start sites located 9–10 and 971 nucleotides upstream of the
translation initiation codon (Luu The et al. 1989, Luu-The et al. 1990). The shorter
transcript is expressed in cells containing 17HSD/KSR1 enzyme, and its amount
correlates well with the concentration of the protein and 17KSR activity (Miettinen et al.
1996a). So far, three elements regulating the efficiency of transcription of the 1.3-kb
mRNA have been identified in the 5’-region of human HSD17B1. First, the fragment
between -78 to +9, with respect to the cap site for the 1.3-kb transcript, has been
characterized as a proximal promoter. Second, the region from -661 to -392 is a cellspecific enhancer and third, the fragment between these two elements, from -392 to -78,
acts as a silencer (Piao et al. 1995, Peltoketo et al. 1999a). The role of the 2.3-kb
transcript is less clear than that of the shorter type, since 2.3-kb mRNA is constitutively
expressed in several tissues, and its concentration does not correlate with 17HSD/KSR1
protein levels or 17KSR activity (Miettinen et al. 1996a). In addition, no typical promoter
elements proximal to the transcription start site of the 2.3-kb mRNA have been detected
(Peltoketo et al. 1992).
Fourteen single base allelic variations characterized as polymorphisms have been
detected in human HSD17B1 (Normand et al. 1993, Simard et al. 1993, Mannermaa et al.
1994, Peltoketo et al. 1994). Of these, only two resulted in a change of amino acid,
namely Ala237→Val and Ser312→Gly. However, these amino acid changes do not affect the
activity of 17HSD/KSR1 (Puranen et al. 1994). Interestingly, one single base allelic
variation located in the putative TATA box decreases promoter activity by 45 % in vitro
(Peltoketo et al. 1994). However, its frequency is the same among breast cancer patients
and control individuals.
2.6.2.5 Rat 17HSD/KSR1
The rat hsd17b1 gene is transcribed into two mRNAs, 1.4 and 1.7 kb in size, as a result of
alternative polyadenylation (Akinola et al. 1998). Cis-acting elements known to modulate
expression of the human HSD17B1 gene are not conserved in the rat gene, indicating that
the two genes are partly differentially regulated (Akinola et al. 1998). The rat
17HSD/KSR1 mRNAs are almost exclusively expressed in the ovary, and only very low
amounts of the transcripts have been found in the kidney (Akinola et al. 1996). In
addition, using human 17HSD/KSR1 antibody, type 1 enzyme has been detected in rat
brain (Pelletier et al. 1995).
34
Unlike the human enzyme, rat 17HSD/KSR1 catalyzes the reaction from A-dione to T
as efficiently as that of E1 to E2, both in cultured cells and in vitro (Akinola et al. 1996,
Puranen et al. 1997a). Of the estrogens, the rat enzyme strongly prefers E1 over E2 as a
substrate (Puranen et al. 1997a). In contrast to human type 1 enzyme, which is a cytosolic
protein, rat 17HSD/KSR1 is inactive in cell homogenates without a nonionic detergent.
Therefore, membrane interaction has been suggested to be important in maintaining its
full activity (Puranen et al. 1997a).
Rat 17HSD/KSR1 is expressed throughout the rat estrous cycle in the granulosa cells
of developing follicles of different stages. The expression of rat type 1 enzyme is
constitutive in whole ovaries in different phases of the estrous cycle (Akinola et al. 1997).
However, studies by Ghersevich et al. (1994b) have shown that treatment with pregnant
mare serum gonadotropin for 1 day up-regulates the expression of 17HSD/KSR1 in the
follicles of rat ovaries, whereas after 2-day treatment the expression is down-regulated.
The expression is further decreased by 1-day treatment with hCG (Ghersevich et al.
1994b). Therefore, the expression of rat 17HSD/KSR1 in granulosa cells is suggested to
be related to the maturation and luteinization stage of the follicles (Ghersevich et al.
1994b, Akinola et al. 1997). In addition, rat type 1 enzyme is more or less constitutively
expressed in whole ovaries during gestation, though a slight increase in the mRNA level
has been detected from day 5 to day 15 (Akinola et al. 1997).
2.6.2.6 Regulation of 17HSD/KSR1
Regulation of 17HSD/KSR1 is best characterized in the ovaries and in cells of placental
origin. Pituitary gonadotropins (FSH and LH) are the main regulators of the maturation of
follicles and E2 secretion, and this process includes regulation of P450arom and
17HSD/KSR1 (Hickey et al. 1988, Fitzpatrick & Richards 1991, Peltoketo et al. 1999a
and references therein). As mentioned above, in vivo studies using pregnant mare serum
gonadotropin-treatment showed that 17HSD/KSR1 expression is related to the maturation
and luteinization of follicles, suggesting that FSH may up-regulate expression of the
enzyme whereas LH may down-regulate it, at least in rodents (Ghersevich et al. 1994b).
The effect of FSH is supported by the results of in vitro studies showing that FSH
partially prevents the decrease in expression of 17HSD/KSR1 in cultured rat granulosa
cells (Ghersevich et al. 1994c). The stimulatory effect of FSH appears to be mediated by
a cAMP-dependent pathway, since a cAMP analog and forskolin mimic the affect of FSH
(Ghersevich et al. 1994c). The PKA-dependent up-regulation of 17HSD/KSR1
expression is further modulated by protein kinase C (PKC)-dependent inhibition,
estrogens, androgens and autocrine/paracrine growth factors present in the ovary
(Ghersevich et al. 1994c, Kaminski et al. 1997).
While granulosa cells have a unigue gonadotropin-dependent system for regulation of
17HSD/KSR1, the synergy of retinoic acid action with the influence of epidermal growth
factor and PKA and PKC activators results in an increase of 17HSD/KSR1 concentration
in human trophoblast-like cells (Piao et al. 1997, Peltoketo et al. 1999a and references
therein). In T-47D breast cancer cells, epidermal growth factor and PKA and PKC
35
activators decrease or lack influence on retinoic acid-stimulated 17HSD/KSR1
expression, further pointing to tissue-specific regulation of 17HSD/KSR1 expression
(Piao et al. 1997). According to Poutanen et al. (1990, 1992b), expression of the type 1
enzyme in the same cell line is up-regulated by progestins and the effect is mediated
through PR. Similarly to the breast cancer cell line, expression of 17HSD/KSR1 in the
endometrium has been suggested to be up-regulated by P, since the concentration of the
enzyme is higher in the secretory than in the proliferative phase (Mäentausta et al. 1990,
Mäentausta et al. 1991). This is supported by the results of a study showing that an
antiprogestin, mifepristone, prevented expression of 17HSD/KSR1 in the endometrium,
in 8 out of 10 subjects (Mäentausta et al. 1993).
2.6.3 17HSD/KSR type 2
2.6.3.1 Human 17HSD/KSR2
The human HSD17B2 gene has been mapped to chromosome region 16q24 (Casey et al.
1994) and it encodes two alternatively spliced mRNAs. The mRNAs give rise to active
human 17HSD/KSR2, a protein of 387 amino acids, as well as to a related 291-amino
acid protein of unknown function (Labrie et al. 1995). Human 17HSD/KSR2 is an
+
oxidative enzyme that prefers NAD as a cofactor and it catalyzes C18 and C19 steroids
equally well. More specifically, the type 2 enzyme converts E2, T, DHT and A-diol to E1,
A-dione, 5α-A-dione and DHEA, respectively. In addition, it catalyzes reduction of 20OH-P to P, but less efficiently than the other substrates (Wu et al. 1993, Puranen et al.
1999). 17HSD/KSR2 protein is bound to the microsomal fraction in vitro (Wu et al.
1993). In line with this, the primary structure of human 17HSD/KSR2 has been found to
contain a putative N-terminal signal sequence followed by a hydrophobic region that is
apparently a transmembrane domain. The C-terminus contains a putative motif for
endoplasmic reticulum retention (Wu et al. 1993). In addition, the protein has been
localized in the endoplasmic reticulum, by using a double immunofluorescence labeling
technique (Puranen et al. 1999). The study also confirmed that the putative N-terminal
signal sequence is not cleaved.
Human 17HSD/KSR2 mRNA, 1.5 kb in size, is mostly expressed in the placenta,
endometrium, liver and small intestine. Using Northern blot, faint to moderate signals for
17HSD/KSR2 have also been detected in the kidney, pancreas, colon, normal prostate,
benign prostatic hyperplasia and in prostatic carcinoma (Casey et al. 1994, Elo et al.
1996, Miettinen et al. 1996a). In addition, human 17HSD/KSR2 has been detected in
sebaceous glands (Thiboutot et al. 1998). In endometrium, expression of the type 2
enzyme has been localized to the glandular epithelium (Casey et al. 1994, Mustonen et
al. 1998a), and in the intestine, to the surface epithelium of the villi (Mustonen et al.
1998a). In the placenta, it is expressed in cytotrophoblasts (Mustonen et al. 1998a), or,
according to a contradictory report, in endothelial cells of certain blood vessels
36
(Takeyama et al. 1998). In the liver, it has been reported to be expressed in hepatocytes
(Takeyama et al. 1998).
The 17HSD/KSR2 enzyme may primarily be involved in decreasing the stimulatory
effects of highly active 17β-hydroxysteroids, even in tissues not classified as traditional
sex steroid target tissues. The presence of the type 2 enzyme in the placenta may serve to
inactivate both E2 and T in order to protect the fetus from excess estrogen and androgen
(Moghrabi et al. 1997, Mustonen et al. 1997b, Mustonen et al. 1998a). The capability of
17HSD/KSR2 to convert 20α-OH-P to P, and the role of this activity in the maintenance
of pregnancy and P concentrations in the maternal circulation remain to be studied.
In the endometrium the expression of 17HSD/KSR2 mRNA is regulated according to
the menstrual cycle. The concentration of the mRNA is greater in the secretory than in the
proliferative phase and is highest during the mid- to late secretory phase, when plasma P
levels are at a maximum, suggesting that the expression is stimulated by P (Casey et al.
1994). The predominant oxidative 17HSD activity in the endometrium (Tseng 1980) and
lower expression of 17HSD/KSR1 than of 17HSD/KSR2 indicate that the type 2 enzyme
is the principal 17HSD/KSR in the endometrium (Casey et al. 1994). Thus,
17HSD/KSR2 together with decreased ER concentrations (Fleming et al. 1980) and
increased steroid sulfotransferase activity, may participate in the progestin-induced downregulation of estrogen action during the secretory phase (Tseng 1980 and references
therein). Interestingly, inactivation of E2 has been assumed to be impaired in
endometriotic tissues due to deficient expression of 17HSD/KSR2 (Zeitoun et al. 1998).
Greater expression of 17HSD/KSR2 in benign prostatic hyperplasia than in prostatic
carcinoma also indicates a protective role of the enzyme (Elo et al. 1996). Moreover, the
wide expression of the type 2 enzyme in the gastrointestinal and urinary tracts (see
section 2.6.3.2) indicates that the protein may be one of the enzymes in the intestine and
liver that is involved in the rapid degradation and excretion of steroids and steroid-like
compounds originating from nutriments, for example (Mustonen et al. 1998b, Peltoketo
et al. 1999a).
2.6.3.2 Rodent 17HSD/KSR2
Mouse and rat 17HSD/KSR2, consisting of 381 amino acids and having a molecular mass
of approximately 42.0 kDa, have been cloned from liver and placenta, respectively
(Akinola et al. 1996, Mustonen et al. 1997a). In contrast to the human type 2 enzyme, the
primary structure of rodent 17HSD/KSR2 lacks an N-terminal lysine-rich putative signal
sequence and a C-terminal lysine-rich endoplasmic reticulum retention signal (Akinola et
al. 1996, Mustonen et al. 1997a). However, both rodent and human type 2 enzymes have
an arginine-rich motif in the C-terminus which is believed to play a role as a retrieval
signal for endoplasmic reticulum-resident membrane proteins (Mustonen et al. 1997a).
The activity of rodent 17HSD/KSR2 has been characterized in cultured cells, where it
catalyzes oxidation of E2 and T with an efficacy similar to that of the human enzyme
(Akinola et al. 1996, Mustonen et al. 1997a).
37
The expression pattern of 17HSD/KSR2 in rodent tissues is similar to that in human
tissues, being highest in the placenta, small intestine and liver. An exception is the uterus,
where the rodent enzyme has not been detected (Akinola et al. 1996, Mustonen et al.
1997a, Mustonen et al. 1998b). Expression of the type 2 enzyme in the liver and small
intestine is constitutive throughout the life span of rats, including gestation. In the
placenta, the enzyme is modestly expressed in early and mid-pregnancy but it is upregulated toward the end of pregnancy (Akinola et al. 1997).
Cell-specific expression of 17HSD/KSR2 has been studied in more detail in mice,
where the enzyme is expressed in epithelial cells of various types, such as stratified
squamous epithelium of the esophagus, the surface epithelial cells of the stomach, small
intestine, and colon, and the epithelium of the urinary bladder (Mustonen et al. 1998b).
Mouse type 2 enzyme is also abundantly expressed in the hepatocytes of the liver and
thick limbs of the loops of Henle in the kidneys. In addition, the transcript can be
detected in the seminiferous tubules, and in the sebaceous glands of the skin (Mustonen et
al. 1998b). Another cell-specific expression study has been conducted with the mouse
fetus and developing placenta (Mustonen et al. 1997b). In the choriovitelline placenta, the
enzyme is expressed both in mural and polar giant cells. In mid-gestation it is present at
the junctional zone, whereas towards the end of pregnancy the expression shifts mainly to
the labyrinth region of the placenta. In addition, the transcript can be found in the visceral
yolk sac throughout pregnancy. In the fetus the expression is congruent with that in adult
mice. It is expressed in the liver from day 11, in epithelial cell layers of the esophagus
and intestine from day 12, in the stomach and kidney from day 15, and in the oropharynx,
nasopharynx and tongue from day 16. In conclusion, the expression pattern of mouse
17HSD/KSR2 in the developing placenta and fetus suggests a role for the enzyme in
maintaining a barrier to the highly active 17β-hydroxy forms of sex steroids between
fetus and mother. This in turn could be vital for the normal development of the fetus as
well as for the normal progression of pregnancy (Mustonen et al. 1997b).
2.6.4 17HSD/KSR type 3
Human 17HSD/KSR3 is a 310-amino acid microsomal enzyme which catalyzes most
efficiently the reaction from A-dione to T (Geissler et al. 1994). In addition, it is to a
lesser extent able to catalyze reduction of E1 and DHEA to E2 and A-diol, respectively. As
a cofactor the enzyme prefers NADP(H) over NAD(H). The human HSD17B3 gene,
consisting of 11 exons, is located at chromosome region 9q22. A 1.3-kb transcript of the
type 3 enzyme is almost exclusively expressed in the testis (Geissler et al. 1994,
Miettinen et al. 1996a). Using RT-PCR, 17HSD/KSR3 mRNA has been found in a
Sertoli-Leydig cell tumor adjacent theca lutein ovarian tissue (Barbieri & Gao 1997) and
in adipose tissue (Corbould et al. 1998). Expression of the type 3 enzyme has not been
found in other tissues having androgenic 17HSD/KSR activity such as the placenta and
liver (Miettinen et al. 1996a, Labrie et al. 1997).
The physiological role of 17HSD/KSR3 is biosynthesis of T (Geissler et al. 1994),
which is needed with Müllerian inhibiting substance and DHT to induce male sexual
38
differentiation during fetal development (Conte & Grumbach 1991, Wilson et al. 1993).
This is evidenced by mutations found in the HSD17B3 gene which lead to decreased T
formation in the fetal testis and consequently to a human intersex disorder termed male
pseudohermaphroditism (Geissler et al. 1994). The disorder can also be caused by other
defects in androgen action, mainly mutations in the androgen receptor gene (McPhaul et
al. 1993) and in the steroid 5α-reductase 2 gene (Wilson et al. 1993), which encodes an
enzyme responsible for the conversion of T to DHT in the urogenital tract. 17HSD/KSR3
deficiency is an autosomal and recessive disorder affecting males, whereas females are
asymptomatic (Rösler et al. 1996, Mendonca et al. 1999).
The characteristic phenotype of pseudohermaphroditism is a 46,XY individual with
testes and male Wolffian duct-derived urogenital structures such as epididymides, vas
deferens, and seminal vesicles, but with female external genitalia (Conte & Grumbach
1991, Geissler et al. 1994). At puberty, these individuals often start to show signs of
virilization. At that time the concentration of A-dione in the plasma is characteristically
tenfold elevated. The plasma T levels are higher than in 46,XX women but may be in the
normal male range. Interestingly, virilization at puberty and almost normal development
of Wolffian ducts suggests residual A-dione to T activity, possibly catalyzed by another
17HSD/KSR (Andersson et al. 1996 and references therein). In studies of 17HSD/KSR3
deficiency, several mutations have been found in the 17HSD/KSR3 gene. These include
missense mutations and mutations causing a splicing error which inactivate
17HSD/KSR3 either severely or completely (Geissler et al. 1994, Moghrabi & Andersson
1998).
17HSD/KSR3 has been cloned from mouse (Sha et al. 1997) and rat tissues (TsaiMorris et al. 1999). In mice, the type 3 enzyme is expressed in fetal, postnatal and adult
testes, as shown by RT-PCR analysis (Sha et al. 1996). In the adult testis, mouse
17HSD/KSR3 transcripts have been localized in the interstitial tissue, most likely in
Leydig cells (Baker et al. 1997). Using RT-PCR, mouse 17HSD/KSR3 has also been
detected in many other tissues, such as the ovary and prostate (Sha et al. 1997). Similar to
the human enzyme, rat 17HSD/KSR3 is almost exclusively expressed in the testis. The rat
enzyme catalyzes most efficiently the reaction from A-dione to T, and to a lesser extent,
DHEA to A-diol (Tsai-Morris et al. 1999). In contrast to the human enzyme, the rat type
3 enzyme does not possess estrogenic 17KSR activity. Interestingly, 17KSR activity of rat
17HSD/KSR3 is increased by glucose and its expression is down-regulated by hCG in
Leydig cells (Tsai-Morris et al. 1999). It has been shown that activation of the glycolytic
pathway is needed to meet ATP requirements for maximal A-dione to T 17KSR activity
(Khanum et al. 1997).
2.6.5 17HSD/KSR type 4
17HSD/KSR4 is peroxisomal multifunctional protein II (MFP-II), with several different
names (see review de Launoit & Adamski 1999). It was first purified and cloned from
porcine tissues (Adamski et al. 1992, Leenders et al. 1994). 17HSD/KSR4/MFP-II is
primarily translated as an 80-kDa protein from a 3-kb transcript (Leenders et al. 1994).
39
The protein is later cleaved to approximately 32-kDa and 45-kDa fragments having
distinct activities (Adamski et al. 1992, Dieuaide-Noubhani et al. 1996, DieuaideNoubhani et al. 1997). In addition to porcine tissues, this multifunctional and widely
expressed protein has been cloned from human (Adamski et al. 1995), mouse (Normand
et al. 1995) and rat tissues (Corton et al. 1996, Dieuaide-Noubhani et al. 1996, Qin et al.
1997a), for example. The similarity of the protein between different species is very high,
around 80 %, and they all have the same multidomain structure (de Launoit & Adamski
1999).
17HSD/KSR4/MFP-II has been found to be expressed in almost all tissues examined,
being highest in the liver and kidney. In addition, its 3-kb mRNA is expressed in several
cancer cell lines (Adamski et al. 1995, Normand et al. 1995, Qin et al. 1997a, Möller et
al. 1999). Expression of the rat enzyme has been shown to be induced by peroxisomal
proliferator chemicals in the liver and kidney and this induction was dependent on
peroxisome proliferator-activated receptor α (Corton et al. 1996, Fan et al. 1998). In
porcine endometrium, the enzyme has been suggested to be up-regulated by P (Kaufmann
et al. 1995, Markus et al. 1995).
Based on homology and the results of functional studies, the 17HSD/KSR4/MFP-II
polypeptide can be divided into three different regions. The N-terminal domain, including
the first 300 amino acids, is similar to that of several members of the SDR family,
especially two dehydrogenase domains of a yeast enzyme participating in peroxisomal βoxidation of fatty acids (Adamski et al. 1995, Normand et al. 1995, Leenders et al. 1996,
Qin et al. 1997a). In line with sequence identity, the N-terminal domain of
17HSD/KSR4/MFP-II catalyzes dehydrogenase reactions of fatty acids, 3-hydroxyacylCoA esters. Therefore, a role in the β-oxidation of fatty acids has been suggested for
MFP-II (Dieuaide-Noubhani et al. 1996, Leenders et al. 1996, Qin et al. 1997a). The 3hydroxyacyl-CoA dehydrogenase activity of 17HSD/KSR4/MFP-II is D-specific while
that of MFP-I is L-specific (Qin et al. 1997a). The central region, corresponding to amino
acids 343–607 in the human protein, shows moderate identity with the C-terminal
hydratase domain of the same yeast protein (Adamski et al. 1995, Normand et al. 1995,
Leenders et al. 1996, Qin et al. 1997a). In agreement with this, the central/2-enoyl-CoA
hydratase domain catalyzes hydration of intermediates of bile acid synthesis (Qin et al.
1997b). The least characterized domain is the C-terminal region of 17HSD/KSR4/MFP-II
(residues 596–736 in the human protein). It shares 39 % identity with human sterol
carrier protein 2 which is assumed to participate in the intracellular transport of sterols
and lipids (Adamski et al. 1995, Normand et al. 1995, Leenders et al. 1996, Qin et al.
1997a). The C-terminal domain of the porcine protein has been shown in vitro to transfer
7-dehydrocholesterol and phosphatidylcholine between membranes, similarly to human
sterol carrier protein 2 (Leenders et al. 1996).
The 17HSD activity of 17HSD/KSR4/MFP-II is an interesting enigma.
17HSD/KSR4/MFP-II has been shown to be able to convert E2 to E1 to some extent
(Adamski et al. 1995, Carstensen et al. 1996), but studies with the rat protein have shown
that 17HSD activity is remarkably lower than 3-hydroxyacyl-CoA dehydrogenase activity
(Dieuaide-Noubhani et al. 1996, Qin et al. 1997a). In spite of that, the Michaelis-Menten
constant (Km) for E2 is similar to those which other 17HSD/KSRs have for their preferred
substrates (de Launoit & Adamski 1999). However, in a recent study no correlation
between 17HSD/KSR4/MFP-II expression and 17HSD activity (E2→E1) was seen in
40
various cell lines (Miettinen et al. 1999). Additional studies are needed to find out if
17HSD/KSR4/MFP-II has any role in estrogen metabolism.
Direct evidence concerning the role of 17HSD/KSR4/MFP-II in peroxisomal βoxidation has come from the discovery that mutations in the HSD17B4 gene encoding
MFP-II cause serious peroxisomal disorders. Mutations have been found both in Dhydroxyacyl-CoA dehydrogenase (van Grunsven et al. 1998, van Grunsven et al. 1999a)
and enoyl-CoA hydratase regions (Suzuki et al. 1997, van Grunsven et al. 1999b).
Mutations lead to accumulation of intermediates of bile acid metabolism and/or verylong-chain fatty acid metabolism which influence renal function and neuronal
development. A D-hydroxyacyl-CoA dehydrogenase deficiency patient has a Gly16→Ser
+
mutation which resides in an important loop of Rossman fold forming the NAD -binding
site (van Grunsven et al. 1998). It is thus not surprising that the mutation also inactivates
17HSD activity of the protein (Möller et al. 1999).
2.6.6 17HSD/KSR type 5
Human 17HSD/KSR5 belongs to the aldoketoreductase family and is highly homologous
with type 1 (84 %) and type 3 (86 %) 3α-HSD as well as with 20α-HSD (88 %) (Dufort
et al. 1999). 17HSD/KSR5 has also been cloned from mouse tissues (Deyashiki et al.
1995). The human HSD17B5 gene is located at chromosome region 10p14-15 and the
corresponding mouse gene at chromosome 13, region A2 (Rheault et al. 1999a). The
human enzyme has been cloned and characterized as 3α-HSD type 2 (Khanna et al. 1995,
Lin et al. 1997), but activity measurements in intact cells showed that both mouse and
human 17HSD/KSR5 more efficiently catalyze conversion of A-dione to T than
conversion of DHT to 5α-androstane-3α,17β-diol (3α-diol), which represents 3α-HSD
activity (Dufort et al. 1999). The sequence of the human type 5 enzyme reported by
Khanna et al. (1995) shows a two-amino acid difference from the one cloned by Dufort et
al. (1999). However, change of these two amino acids did not significantly affect
17HSD/KSR activity (Dufort et al. 1999). The 17HSD/KSR activity of the human type 5
enzyme, but not that of the mouse counterpart, is very labile, since the activity in cell
homogenates is only 10 % of that observed in intact cells. However, 3α-HSD activity
does not decrease that much upon homogenization. Therefore, if enzymatic properties of
17HSD/KSR5 were measured only in vitro, the protein would erratically be considered as
3α-HSD and not 17HSD/KSR (Dufort et al. 1999).
Human and mouse 17HSD/KSR5 show differences in their substrate specificity
patterns. The mouse type 5 enzyme was originally reported to catalyze the reaction from
E2 to E1 (Deyashiki et al. 1995), but in a recent study using cultured cells it was shown
that transformation of E2 to E1 represents only 1 % of the rate of conversion of A-dione to
T, which is the highest activity (Dufort et al. 1999, Rheault et al. 1999b). The mouse
enzyme is also able to catalyze conversions of E1, 5α-A-dione, androsterone (ADT),
DHEA and P to E2, DHT, 3α-diol, A-diol and 20-OH-P respectively (Dufort et al. 1999,
Rheault et al. 1999b). In addition, mouse 17HSD/KSR5 has been reported to be active
toward xenobiotic substrates in vitro (Deyashiki et al. 1995). Human 17HSD/KSR5 also
41
efficiently converts A-dione to T, but most efficiently it catalyzes P to 20-OH-P. In
addition, it is able to catalyze conversion of T to A-dione and interconversion between
ADT and 3α-diol (Dufort et al. 1999). Furthermore, it has 3α-HSD activity towards ADT
and 3α-diol (Lin et al. 1997, Dufort et al. 1999).
Results of RNase protection assays show that human 17HSD/KSR5 is abundantly
expressed in the liver, prostatic cancer cells and osteocarcinoma cells, in addition to
which it is modestly expressed in normal prostate and in the adrenal (Dufort et al. 1999).
Mouse 17HSD/KSR5 is also abundantly expressed in the liver (Deyashiki et al. 1995). In
the human prostate 17HSD/KSR5 is mainly expressed in the basal cells of glandular
epithelium, in stromal fibroblasts and in endothelial cells (El-Alfy et al. 1999). An
immunohistochemical study has shown that human 17HSD/KSR5 is also expressed in the
CL, in the epithelial cells of the endometrium and mammary gland, in Leydig cells and
occasionally in Sertoli and germ cells (Pelletier et al. 1999). This relatively wide tissue
distribution is in line with a previous report showing that several human and rhesus
monkey tissues have androgenic 17KSR activity (Labrie et al. 1997).
2.6.7 17HSD/KSR type 6
17HSD/KSR6 cDNA has so far been cloned only from rat prostate (Biswas & Russell
1997). Rat 17HSD/KSR6 is a protein of 327 or 317 amino acids depending on which
AUG codon is used to encode for the first methionine. In vitro, the enzyme most
efficiently catalyzes oxidation of 3α-diol to ADT, which is part of the inactivation path of
DHT. DHT is first reduced at the 3-keto group by 3α-HSD to yield 3α-diol which is then
oxidized at the 17β-hydroxy group to ADT by 17HSD/KSR6. As a cofactor the enzyme
+
uses NAD . The enzyme is mainly expressed in the ventral prostate and liver, in addition
to which a 1.5-kb transcript is modestly expressed in the kidney. In castrated rats, the
level of 17HSD/KSR6 mRNA was decreased. 17HSD/KSR6 belongs to the SDR family
and has its greatest sequence identity (65 %) with retinol dehydrogenase 1. Retinoic acids
have been found to inhibit the reaction from 3α-diol to ADT, suggesting that they could
be potential substrates. However, the concentrations of retinoic acids used in the
experiment were above physiological levels, i.e. µM vs. nM (Biswas & Russell 1997).
2.6.8 17HSD/KSR type 8
17HSD/KSR8 cDNA was cloned from a mouse kidney cDNA library and initially given
the name ke 6 (Aziz et al. 1993). Five years later ke 6 was reported to be a 17HSD/KSR,
with the highest identity (35 %) to 17HSD/KSR4 (Fomitcheva et al. 1998). 17HSD/
KSR8 is slightly smaller than other 17HSD/KSRs, consisting of 260 amino acids and
having a molecular mass of 34 kDa (Aziz et al. 1993, Aziz et al. 1996, Fomitcheva et al.
1998). In vitro characterization of the enzymatic nature of 17HSD/KSR8 has shown that
it catalyzes oxidation of E2, T and DHT, and reduction of E1, using NAD(H) as a cofactor
42
(Fomitcheva et al. 1998). The enzyme has the highest affinity for E2 (Km = 0.11 µM). The
results of kinetic studies thus suggest that E2 inactivation may be the main physiological
function of the enzyme (Fomitcheva et al. 1998).
The Ke6 gene encoding mouse 17HSD/KSR8 consists of nine exons and it resides in
mouse chromosome 17, among genes for a major histocompatibility complex (Maxwell
et al. 1995). The gene is transcribed into two mRNAs, 1.0 kb and 1.2 kb in size. The 1.0kb transcript is most abundantly expressed in the kidney and liver, in addition to which it
is moderately expressed in the ovary, testis, spleen, brain, heart and intestine (Aziz et al.
1993, Maxwell et al. 1995, Fomitcheva et al. 1998). An immunohistochemical study has
shown that in the ovary 17HSD/KSR8 is expressed in the cumulus cells and in the
endothelial cells of blood vessels (Fomitcheva et al. 1998). The 1.2-kb transcript is
expressed in the spleen (Aziz et al. 1993, Maxwell et al. 1995). This second transcript is
a splicing variant, where exon 9 is replaced by intron 8, leading to an altered amino acid
sequence in the C-terminus of the protein (Maxwell et al. 1995). The biological impact of
the 1.2-kb transcript is unknown. It is of interest that compared with normal mice there
are decreased 17HSD/KSR8 transcript and protein concentrations in the kidneys of mice
having various cystic kidney diseases which are considered to be models of human
heritable polycystic kidney disease (Aziz et al. 1993, Maxwell et al. 1995, Aziz et al.
1996). Down-regulation of 17HSD/KSR8 expression has recently been suggested to be
due to impaired gene regulation and not because of mutation in the Ke6 gene (Ramirez et
al. 1998).
Human 17HSD/KSR8 cDNA has also been cloned from kidney tissue (Ando et al.
1996). Its gene consists of eight exons and it is located at chromosome region 6p21
(Kikuti et al. 1997), where the gene for human autosomal recessive polycystic kidney
disease has been mapped. Characterization of the enzymatic nature of human
17HSD/KSR8 has not yet been reported. A 1-kb transcript encoding human type 8
enzyme is well expressed in the liver and pancreas, and it is expressed at moderate levels
in the kidney and skeletal muscle. The enzyme is also weakly expressed in the heart,
placenta and lung (Kikuti et al. 1997).
The physiological role of 17HSD/KSR8 is unknown, but a protective role as a
regulator of sex steroid levels, especially in the kidney, which is a highly steroid-sensitive
organ, has been suggested (Fomitcheva et al. 1998). This is supported by the results of a
study showing that antisense oligonucleotide-mediated inhibition of expression of the
enzyme caused formation of renal cysts in embryonic kidney organ cultures (Aziz et al.
1995).
2.6.9 Other 17HSD/KSRs
Recently, 17β/3α-hydroxysteroid/retinoid short chain dehydrogenase/reductase, a novel
protein having 17HSD/KSR activity, has been cloned from mouse tissues (Su et al. 1999).
It has greatest identity (88 %) with rat 17HSD/KSR6, but the two proteins differ in
+
substrate specificity. Using NAD as a cofactor the enzyme most efficiently catalyzes the
reactions from E2 to E1 and 3α-diol to ADT, and then ADT to 5α-A-dione, thus showing
43
3α-HSD activity. In addition, the enzyme is able to convert all-trans-retinol to all-transretinal. The enzyme, however, is not believed to function in the pathway of retinoic acid
biosynthesis, while its physiological role has been suggested to be inactivation of
estrogen and a variety of androgens in the liver, where it has been reported to be
exclusively expressed (Su et al. 1999).
Other examples of proteins having 17HSD/KSR activity are a protein from fungus
(Lanisnik Rizner et al. 1999) and human brain short chain L-3-hydroxyacyl coenzyme A
dehydrogenase, which has been shown to convert E2 to E1 (He et al. 1999). Whether E2 is
the physiological substrate for these enzymes is not yet known.
3 Outlines of the prese nt study
17HSD/KSR1 catalyzes conversion of E1 to E2, which is biologically the most active
natural estrogen and which is mainly synthesized in the ovary and placenta. E2 has an
important role in female reproduction, including folliculogenesis. This study was aimed
to characterize enzymatic properties of rodent 17HSD/KSR1 and its physiological role
and regulation in ovaries. In addition, an expression cloning strategy was used to
demonstrate that rodents possess a novel type of 17HSD/KSR, which is involved in E2
biosynthesis in ovaries as well as in other tissues, as determined by studying cell-specific
expression of the enzyme. Specific aims of the study were:
1. to clone rat 17HSD/KSR1 cDNA and use it in studies concerning expression and
regulation of the rat type 1 enzyme in the ovary,
2. to clone mouse 17HSD/KSR1 cDNA, and characterize the mouse type 1 enzyme and
its tissue distribution,
3. to expression-clone a novel type of mouse 17HSD/KSR cDNA and study the
catalytic properties of the enzyme encoded by the cloned cDNA, named
17HSD/KSR7, and
4. to characterize the tissue distribution and cell-specific expression of 17HSD/KSR7 in
rodents and thereby elucidate the role of the type 7 enzyme in estrogen biosynthesis.
4 Materials and metho ds
Detailed descriptions of the materials and methods are presented in the original articles, IIV.
4.1 Isolation of RNAs (I-IV)
Total RNA was isolated by homogenization of samples in a guanidine-isothiocyanate
buffer, followed by CsCl density ultracentrifugation (Davis et al. 1986). For RT-PCR,
total RNA was alternatively isolated using an RNeasy total RNA isolation kit (Qiagen).
Poly(A)-enriched RNA was extracted from total RNA using oligo-dT-cellulose
chromatography (Sambrook et al. 1989). For a cDNA expression library, poly(A)enriched RNA was isolated using a FastTrack 2.0 kit (Invitrogen).
4.2 Cloning of rat and mouse 17HSD/KSR1 cDNAs (I, II)
A cDNA library, constructed from FSH- and E2-stimulated ovaries of Sprague-Dawley
32
rats (Clontech), was screened with a [ P]-labeled fragment (nucleotides 1–969) of human
17HSD/KSR1 cDNA (Peltoketo et al. 1988) and a rat 17HSD/KSR1 cDNA fragment
(nucleotides 374–767) was obtained. To clone cDNA containing the whole coding region,
the same cDNA library was screened with the rat 17HSD/KSR1 cDNA fragment as a
probe. The cDNA was then cloned into pBluescript KS+ vector (Stratagene) and
sequenced.
To clone mouse 17HSD/KSR1 cDNA, a cDNA library was constructed from poly(A)enriched RNA extracted from the ovaries of diethylstilbestrol- (DES-) treated mice, using
32
a TimeSaver cDNA synthesis kit (Pharmacia). The library was screened with a [ P]labeled fragment (nucleotides 374–767) of rat 17HSD/KSR1 cDNA to obtain a first
cDNA fragment (nucleotides 167–1317). The missing 5’-fragment was cloned using a 5’-
46
AmpliFINDER RACE kit (Clontech), from the same RNA fraction. Mouse 17HSD/KSR1
cDNA containing the whole coding region was cloned into pBluescript KS+ vector by
ligating the two sequenced cDNA fragments simultaneously.
4.3 Cloning of mouse and rat 17HSD/KSR7 cDNAs (III)
Mouse 17HSD/KSR7 cDNA was cloned by expression cloning. A cDNA expression
library was generated by Invitrogen in cDNA3.1 plasmids containing cytomegalovirus
(CMV) promoter. Poly(A)-enriched RNA extracted from the HC11 cell line (Ball et al.
1988) was used for generation of the library. The cDNA library was divided into pools of
2500 clones/colonies from which plasmid DNAs were isolated and transfected into
human embryonic kidney (HEK) 293 cells grown on six-well plates. After transfection
3
the medium was replaced with one containing 100 nM unlabeled E1 + 200000 cpm of Hlabeled E1/ml. After 18 h the media were collected, and the substrates and products were
separated in a Symmetry C18 reverse-phase chromatography column connected to a high
performance liquid chromatography (HPLC) system (Waters) as described by Puranen et
al. (1997b). Radioactivity of steroids was measured by an on-line β-counter and pools
showing conversion of E1 to E2 were identified. Thereafter, plasmids from positive pools
were retransformed to generate 20 pools of 1000 transformants on the second round of
screening and 20 pools of 100 transformants on the third round of screening. Plasmids
from new pools were transfected into HEK-293 cells to identify a pool where the positive
clone was enriched. At the final stage, positive plasmid pools were retransformed, and
100 single colonies were picked up for plasmid isolation on a miniprep scale. After
transfection of these 100 minipreps, a pure enriched plasmid encoding 17HSD/KSR
activity was identified and sequenced.
Rat 17HSD/KSR7 cDNA was cloned by RT-PCR on the basis of the sequence
published as a PRL receptor-associated protein (PRAP) cDNA at that time (Duan et al.
1996). A 1013 bp fragment (nucleotides 9–1021, containing the whole coding region)
was reverse-transcribed and amplified from total RNA isolated from the ovaries of
pregnant rats (12-day p.c.) using SuperScriptII RT (GIBCO BRL) and Pyrococcus
furiosus DNA polymerase (Stratagene). The cDNA fragment was then cloned into
Bluescript KS+ and CMV6 plasmid vectors and sequenced.
4.4 Sequencing and bioinformatics (I-IV)
Rat and mouse 17HSD/KSR1 cDNAs were sequenced from both their strands by the
T7
dideoxy termination method described by Sanger et al. (1977), using a Sequencing kit
T7
and Deaza G/A Sequencing mixes (Pharmacia). Other sequencings thereafter were
performed using an automatic DNA sequencer (ABI Prism 377 DNA sequencer, PerkinElmer). Sequence analyses were carried out with a GCG package (Genetics Computer
47
Group, Inc.) on a Unix computer at CSC (Center for Scientific Computing, Espoo) run
through the internet.
4.5 Measurement of 17HSD/KSR activity of human and mouse
17HSD/KSR1 (II)
17HSD/KSR activity in cultured Sf9 cells was measured in six-well plates. The cells
were infected with baculoviruses (AcNPVs) containing a fragment coding for
17HSD/KSR1. After 50 h infection, both reductive (E1→E2, A-dione→T) and oxidative
(E2→E1, T→A-dione) 17HSD/KSR activities were measured by adding 2 ml serum-free
3
TNF-FH medium, containing 10 µM unlabeled and 300000 cpm H-labeled substrate. At
four time points (10, 20, 30, 40 min), the media were collected, and the substrates and
products were separated in a Sephasil C18 reverse-phase chromatography column
TM
connected to a SMART System (Pharmacia) (Jantus Lewintre et al. 1994), and the
amount of substrate converted was calculated.
In addition, substrate specificities of mouse and human 17HSD/KSR1 were further
compared in the conditions described above. In these experiments, the medium contained
either 10 µM E1 and different amounts (5–100 µM) of A-dione or 10 µM A-dione and
different amounts (5–100 µM) of E1.
4.6 Measurement of 17HSD/KSR activity of mouse and rat
17HSD/KSR7 (III)
The 17HSD/KSR activity of mouse and rat 17HSD/KSR7 was measured under CMVpromoter after transient transfection in HEK-293 cells grown on six-well plates. After
transfection the medium was replaced with one containing 100 nM or 1 nM unlabeled
3
substrate + 200000 cpm of H-labeled substrate/ml. After incubation (2–24 h), the media
were collected, and the substrates and products were separated as described in section
4.3, and the amount of substrate converted was calculated.
For activity measurements in vitro, transfected HEK-293 cells were lysed in 10 mM
potassium phosphate buffer, pH 8, containing 1 mM EDTA, 20 % glycerol, 0.08 % N,Nbis-(3-D-gluconamidopropyl)cholamide
(Big
Chap,
Calbiochem-Novabiochem
International), 0.05 % bovine serum albumin (BSA), 0.02 % NaN3 and
phenylmethylsulfonyl fluoride (87 ng/ml). For determination of Km and maximal velocity
(Vmax) values, cell extracts were diluted in 10 mM potassium phosphate buffer, pH 8,
containing 1 mM EDTA and 0.05 % BSA, and the samples were then mixed with
different amounts of substrate. The enzymatic reactions were started by adding a cofactor
+
(NADP /NADPH, Boehringer Mannheim) to a final concentration of 1 mmol/l, and the
samples were incubated for an appropriate time at 37 °C. The reactions were stopped by
freezing the reaction mixture quickly in an ethanol-dry ice bath. Steroids were extracted,
48
separated from each other (see section 4.3) and the amounts of substrate formed per min
and per total amount of protein were calculated. Kinetic parameters (Km and Vmax) were
calculated by using a GraFit program (Erithacus Software Ltd). The program fits data to
the Michaelis-Menten equation using nonlinear regression analysis.
4.7 Northern blot analysis (I-III)
Samples of RNA were resolved by 1 % gel electrophoresis and transferred overnight to
nylon membranes (Hybond N, Amersham) by capillary force. The RNAs were fixed to
the membrane by UV cross-linking. The membranes were prehybridized for 2–4 h in a
solution containing 5 x SSPE [0.75 M NaCl, 50 mM sodium phosphate (pH 7.4), 5 mM
EDTA], 50 % formamide, 0.1 % BSA, 0.1 % Ficoll, 0.1 % polyvinyl pyrrolidone, 0.5 %
SDS and 20 µg or 100 µg salmon sperm DNA/ml at the same temperature as used in
hybridization. The membranes were then hybridized overnight with one of the following
32
[ P]-labeled cDNA probes: rat 17HSD/KSR1 (nucleotides 374–767) at 48 °C, rat
P450arom at 45 °C, mouse 17HSD/KSR1 (nucleotides 1–1059) at 50 °C, mouse
17HSD/KSR7 (nucleotides 33–849) at 42 °C, or rat 17HSD/KSR7 (nucleotides 9–897) at
42 °C. After hybridization, the membranes were washed in decreasing salt concentrations
and exposed to Kodak XAR films. In order to estimate the amount and quality of RNA
applied to the gels, the membranes were hybridized at 42 °C with a glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA probe.
4.8 Reverse transcriptase polymerase chain reaction (II, III)
RT-PCR was carried out on 250 ng of total RNA using a Thermostable recombinant
Thermus thermophilus (rTth) Reverse Transcriptase RNA PCR kit (Perkin Elmer).
Amplified fragments corresponded to mouse 17HSD/KSR1 cDNA (nucleotides 45–492)
or mouse 17HSD/KSR7 cDNA (nucleotides 351–849). A fraction of each PCRs was
resolved electrophoretically and transferred to nylon membranes. The membranes were
hybridized with mouse 17HSD/KSR1 or 17HSD/KSR7 cDNA probes as described in
section 4.7. The quality of the RNA used was tested by checking the integrity of
ribosomal bands after electrophoresis and/or by performing RT-PCR with mouse GAPDH
primers.
49
4.9 Animal treatments (I, II)
For preparation of a mouse ovarian cDNA library, 75 immature BALB/c mice were
implanted subcutaneously with DES pellets (Innovative Research of America), which for
5 days released 0.25 mg diethylstilbestrol/day.
To study regulation of 17HSD/KSR1 in ovaries, female Sprague-Dawley rats, 26-28
days old, were used 2 days after hypophysectomy. One group of rats was implanted
subcutaneously with DES pellets (Innovative Research of America) releasing 1.2 mg
DES/day. Rats in this group were then further treated subcutaneously with placebo, FSH,
FSH followed by hCG, or hCG alone. Another group of animals were not primed with
DES but were injected with vehicle alone [1 % BSA in phosphate-buffered saline (PBS)]
or gonadotropins as above.
4.10 Tissue samples (I-IV)
Tissue specimens for RNA isolation were frozen in liquid nitrogen and stored at -70 °C
until processed. For immunohistochemistry, tissues excised from animals were
immediately fixed in 4 % paraformaldehyde-0.5 % glutaraldehyde-PBS at 4 °C for 3 h, in
4 % paraformaldehyde-5 % sucrose-PBS at 4 °C for 6 h and in 20 % sucrose-PBS
overnight. Samples were thoroughly washed with PBS after each treatment. The fixed
samples were then frozen in isopentane cooled with liquid nitrogen. Sections of 7 µm
were cut and mounted on polylysine-coated glass slides.
For in situ hybridization using rat 17HSD/KSR1 riboprobe, 8–10 µm cryosections
were cut and mounted on slides coated with aminopropyltriethoxysilane and fixed with
4 % paraformaldehyde (Autio-Harmainen et al. 1992). For in situ hybridization using
other probes, tissues excised were immediately fixed overnight in 4 % paraformaldehydePBS solution at 4 °C. Next day they were dehydrated with ethanol, and embedded in
paraffin (Merck) overnight at 58 °C. Thereafter, 7 µm sections were cut and collected on
Super Frost+ glass slides (Menzel-Gläser).
4.11 Immunohistochemistry (I, II)
Non-specific binding of antibodies was blocked by incubating the slides in 10 % goat
serum-PBS at room temperature for 30 min. Immunoaffinity-purified human
17HSD/KSR1 antibodies (Mäentausta et al. 1990) in 10 % goat serum-PBS were added
to the tissue sections and incubated at 4 °C overnight. Thereafter, all steps were
performed at room temperature. The slides were washed 3 x 5 min with PBS, and
incubated for 2 h in a 1:50 dilution of biotinylated anti-rabbit antibody (Dakopatts) in
10 % goat serum-PBS. After washing, the slides were further treated for 1 h in a 1:50
dilution of streptavidin-conjugated FITC (Dakopatts) in 10 % goat serum-PBS. The
50
preparations were then washed 5 x 5 min with PBS, and mounted with a drop of 5 %
(w/w) n-propylgallate-glycerol.
4.12 In situ hybridization (I, IV)
A 394-bp fragment (nucleotides 374–767) of rat 17HSD/KSR1 was cloned into pGEM4Z vector (Promega) and a 499-bp fragment (nucleotides 351–849) of mouse
17HSD/KSR7 was cloned into pBluescript KS+ vector. In addition, a 509-bp fragment
(nucleotides 185–693) of prolactin-like protein B (PLP-B) cDNA (Müller et al. 1998)
was cloned from poly(A)-enriched RNA from placentas of pregnant mice (day 11–13) by
RT-PCR and was cloned into pBluescript KS+. The rat 17HSD/KSR1 fragment in
pGEM-4Z and the mouse 17HSD/KSR7 fragment in pBluescript KS+, the mouse PLP-B
cDNA fragment in pBluescript KS+, a 737-bp fragment (nucleotides 584–1320) of mouse
17HSD/KSR2 in SP72 plasmid (Mustonen et al. 1998b) and a 403-bp fragment
(nucleotides 1–403) of mouse 17HSD/KSR1 in pBluescript KS+ were used as templates
to transcribe rat 17HSD/KSR1, mouse 17HSD/KSR7, mouse PLP-B, mouse
35
17HSD/KSR2 and mouse 17HSD/KSR1 [α- S]-labeled riboprobes, respectively.
In situ hybridization using rat 17HSD/KSR1 riboprobe was performed in cryosections
as previously described (Autio-Harmainen et al. 1992). The in situ hybridization protocol
using other probes and paraffin sections was based on that described by Quéva et al.
(1992) with minor modifications described by Mustonen et al. (1998b). Each section pair
was hybridized with an antisense and a sense probe. To visualize cells, sections were
stained with Hoechst 33258 or hematoxylin and eosin.
5 Results
5.1 Cloning and characterization of rat and mouse 17HSD/KSR1
cDNAs (I, II)
A cDNA encoding rat 17HSD/KSR1 was cloned from a cDNA library which was
constructed from FSH- and E2-stimulated rat ovaries. The cloned cDNA, 1.2 kb in size,
encodes a protein of 344 amino acids with a calculated molecular mass of 37.0 kDa.
Similarly to rat ovary, DES was found to increase the number of secondary follicles and
granulosa cells expressing 17HSD/KSR1 in the mouse ovary. Therefore, to clone mouse
17HSD/KSR1 cDNA, a library was prepared from mRNA extracted from the ovaries of
mice treated with DES. A 1.2-kb cDNA clone for mouse 17HSD/KSR1 obtained from the
library was not full-length since it lacked the region coding 45 N-terminal amino acids.
The missing region was cloned using the RACE technique. The whole cDNA, 1317 bp in
size, similarly to the rat, encoded a protein of 344 amino acids with a calculated
molecular mass of 36.8 kDa.
The overall amino acid identity between rat and human 17HSD/KSR1 was 68 %. The
identity of rat 17HSD/KSR1 was further proved by comparing hydropathy plots of the
human and rat enzymes. These were very similar apart from the C-terminal region.
Between the mouse and rat enzyme, the identity was 93 % and between the three
proteins, 63 %. The lowest identity was localized to the C-terminus, which was 17 amino
acids longer in the rodent enzymes than in the human counterpart. Like human
17HSD/KSR1, both rodent enzymes belong to the SDR family and they contain five
conserved regions (A-E) typical of the SDRs characterized at that time (Krozowski 1992,
Krozowski 1994) and the consensus sequence of SDR family members in the PROSITE
data bank. The consensus sequence includes a strictly conserved Tyr-X-X-X-Lys
segment, in addition to which there is a Gly-X-X-X-Gly-X-Gly segment near the Nterminus, also typical of the SDRs. The former segment is part of the catalytic center,
while the latter binds cofactor. Furthermore, a potential cAMP-dependent
phosphorylation site, Ser134, shown to be phosphorylated in the human enzyme (Barbieri
et al. 1994, Puranen et al. 1997b), is also present in mouse and rat 17HSD/KSR1. Like
52
rat 17HSD/KSR1 cDNA, the mouse cDNA contains an Alu-like or B1-like repetitive
sequence in the antisense strand of the 3’-noncoding region.
5.2 Expression of 17HSD/KSR1 in rat ovary and regulation by
gonadotropins (I)
Expression and regulation of 17HSD/KSR1 in the ovaries of immature rats were analyzed
using immunohistochemistry, Northern blots and in situ hybridization. In the ovaries of
hypophysectomized but otherwise untreated animals, 17HSD/KSR1 was modestly but
exclusively expressed in the granulosa cells of a few small antral follicles. One to two
days of FSH treatment clearly induced 17HSD/KSR1 expression in the granulosa cells of
growing antral follicles, whereas one-day treatment of FSH-primed animals with hCG
caused luteinization of follicles and a lower expression level of 17HSD/KSR1. Similarly
to FSH, DES strongly up-regulated expression of the type 1 enzyme. In contrast to
hypophysectomized but otherwise untreated animals, in DES-primed rats FSH caused
luteinization of follicles and a decrease of enzyme expression, starting from the basal
layer of granulosa cells. In line with this, 17HSD/KSR1 was not expressed in the rat CL.
In addition, 17HSD/KSR1 was not detected in either theca cells or stromal tissues.
Expression of P450arom in the ovaries was also followed. Similar to the effect on
17HSD/KSR1, FSH stimulated the expression of P450arom in hypophysectomized
immature rats. However, in contrast to the down-regulation of 17HSD/KSR1 by hCG,
expression of P450arom was strongly increased after one-day treatment with hCG. DES
alone did not stimulate P450arom expression but it enhanced the effect of FSH on
P450arom expression.
5.3 Characterization of 17HSD/KSR activity of mouse 17HSD/KSR1
and comparison with the human enzyme (II)
Mouse and human recombinant 17HSD/KSR1 in cultured Sf9 insect cells mainly
catalyzed reductive reactions from E1 to E2 and from A-dione to T. Both enzymes
catalyzed E1 to E2 similarly (Fig. 4). However, mouse 17HSD/KSR1 converted A-dione to
T at least as efficiently as E1 to E2, while the production rate of T from A-dione catalyzed
by the human enzyme was less than 10 % of that catalyzed by the mouse enzyme under
the conditions used. Oxidative activities (E2→E1 and T→A-dione) were minor with both
enzymes. The difference in substrate specificity towards androgens was further confirmed
by inhibiting conversion of E1 to E2 with increasing amounts of A-dione and, vice versa.
For example, a 10-fold excess of A-dione did not decrease E2 formation catalyzed by
human 17HSD/KSR1, while E2 formation catalyzed by the mouse enzyme was reduced
by 43 % with a fivefold excess of A-dione. In line with this, the minor formation of T
catalyzed by the human enzyme was decreased close to baseline level by E1 at a
53
concentration of only 50 % that of A-dione. In contrast, the androgenic activity of mouse
17HSD/KSR1 was inhibited by 43 % with a fivefold excess of E1.
Fig. 4. Comparison of substrate specificity of mouse and human 17HSD/KSR1 in cultured Sf9
cells. Medium containing 10 µM E1 or A-dione was applied to the cells grown on six-well
plates after infection with baculoviruses containing a fragment coding mouse or human type
1 enzyme. After 10 min, the media were collected and the amount of product formed was
measured.
5.4 Tissue distribution of mouse 17HSD/KSR1 (II)
The expression pattern of mouse 17HSD/KSR1 was studied using Northern blot and RTPCR analyses. In Northern blots, the probe used recognized in ovarian samples a 1.4-kb
mRNA as the main transcript and in addition, low amounts of 2.0-kb and 2.3-kb
transcripts were detected. A very faint signal for the 1.4-kb mRNA was also observed in
samples from the uterus and adrenal gland. No expression was detected in other tissues
studied, e.g. the placenta and mammary gland. Expression of mouse 17HSD/KSR1 in the
uterus and adrenal gland was confirmed by RT-PCR, which showed a moderate level of
amplification in RNA samples from the two tissues. In addition, modest amplification
took place in samples from male liver, testis and female brain.
5.5 Cloning and characterization of mouse 17HSD/KSR7 cDNA (III)
The HC11 cell line, originating from the epithelial cells of the mammary gland of a
pregnant mouse, was found to possess strong estrogenic 17KSR activity. E1 was
efficiently converted to E2 in cultured cells, whereas A-dione was poorly converted to T.
Because the activity profile did not match with mouse 17HSD/KSRs known at that time,
54
and because mouse 17HSD/KSR probes did not recognize any transcripts, the cell line
was assumed to express a novel type of 17HSD/KSR. To clone a corresponding cDNA,
an expression cDNA library was prepared from HC11 cells. Based on the ability of
clones to convert E1 to E2 after transfection into cultured HEK-293 cells, two cDNA
clones encoding mouse 17HSD/KSR7 were obtained. The longer cDNA, 1756 bp in size,
encodes a protein of 334 amino acids with a predicted molecular mass of 37.3 kDa. The
deduced amino acid sequence of the protein was compared with those in a sequence
database, which showed that the protein had a 89 % identity to a rat protein called PRL
receptor-associated protein, PRAP. Analysis using the PROSITE databank showed that
mouse 17HSD/KSR7 and PRAP belong to the SDR family and they possess the
conserved glycine pattern for cofactor-binding and the three most important catalytic
amino acids within the SDR consensus sequence (Ser180, Tyr193 and Lys197).
5.6 Characterization of the enzymatic properties of mouse and rat
17HSD/KSR7 (III)
To characterize the enzymatic activities of mouse 17HSD/KSR7, the cDNA was
expressed under the CMV promoter in cultured HEK-293 cells. The mouse enzyme
efficiently catalyzed only the reductive reaction from E1 to E2. No significant
17HSD/KSR or 20α-HSD activity was detected when E2, A-dione, T, P or 20-OH-P was
used as a substrate. Further characterization of the activity was carried out in vitro using a
homogenate of transfected HEK-293 cells. Kinetic parameters (Km and Vmax) were
determined only for E1 and E2, since activity with A-dione, T, DHEA, A-diol, P or 20OH-P was almost negligible. Vmax values for E1 and E2 were 556.5 and 616.3 pmol
product/min/mg total protein, and Km values were 2.28 µM and 12.87 µM, respectively.
The calculated catalytic efficiency (Vmax/Km) for E1 was 244 whereas for E2 it was 48. The
catalytic efficiency values and Km clearly showed that E1 is the preferred substrate for the
enzyme. In addition, it was shown that the mouse type 7 enzyme preferred NADPH over
NADH as a cofactor. In order to characterize the enzymatic activity of rat
17HSD/KSR7/PRAP, its cDNA was cloned by RT-PCR from the ovaries of pregnant rats
and expressed under the CMV promoter in cultured HEK-293 cells. Similarly to the
mouse enzyme, the rat protein catalyzed only the reaction from E1 to E2 and no activity
was detected when E2, A-dione, T, P or 20-OH-P was used as a substrate.
5.7 Tissue distribution of 17HSD/KSR7 and its expression pattern in
the ovary during gestation (III, IV)
In Northern blot analysis, the mouse 17HSD/KSR7 probe recognized several transcripts,
a 4.6-kb mRNA being the dominant form, and others (3.6, 1.7 and 1.2 kb) being weakly
detected. The 4.6-kb transcript was most abundantly expressed in the ovaries of pregnant
55
mice and in the placenta. 17HSD/KSR7 was weakly expressed in the ovaries of
nonpregnant mice, in the mammary gland, liver, kidney, testis and in the HC11 cell line.
Because of the remarkable size difference between the major mRNA and the cloned
cDNA, and because of the fact that the size of the mRNA was about the same as that of
28S ribosomal RNA (4.7 kb), the authenticity of the weak signals in the Northern blot
was confirmed by RT-PCR, which showed that the enzyme was expressed in the tissues
identified in the Northern blot.
Expression of 17HSD/KSR7 in the ovaries during pregnancy was followed in Northern
blots, using RNA samples from rat ovaries. The rat 17HSD/KSR7/PRAP probe
recognized two main transcripts, 4.6 kb and 1.8 kb in size, of which the longer transcript
was the dominant form. 17HSD/KSR7/PRAP was expressed throughout pregnancy but
was remarkably up-regulated at day 8 p.c. and strongly decreased close to parturition, on
day 21 p.c.
Expression of the type 7 enzyme in mouse ovary was studied in more detail using in
situ hybridization, which showed that the enzyme was exclusively and highly expressed
in the CLs of both nonpregnant and pregnant animals. The 17HSD/KSR7 transcript was
present only in fresh CLs, i.e. in those of the cycle and those of the pregnancy, but not in
those of previous cycle(s), which were regressing. In agreement with the Northern blot
results, the expression level of the type 7 enzyme in the CLs was decreased close to
parturition, when they showed clear signs of luteolysis.
5.8 Expression of mouse 17HSD/KSR7 in the uterus and placenta (IV)
Using in situ hybridization, expression of the mouse type 7 enzyme was studied in the
uterus and placenta, where cell-specific expression was followed from implantation to
late pregnancy. In the uterus, 17HSD/KSR7 was first detected on day 5½ p.c., when
expression surrounded the implantation site on the antimesometrial side. Expression was
more abundant in the inner zone than in the outer zone of the decidua. Expression of
mouse 17HSD/KSR7 was congruent with that of PLP-B, which was used as a marker of a
decidual reaction. PLP-B was expressed in the inner zone of the decidua on the
antimesometrial side, but not in the outer zone. As gestation progressed, the type 7
enzyme was abundantly expressed in the decidua capsularis on day 8 and 9½ p.c., and
disappeared thereafter from declining antimesometrial decidua. On day 9 p.c. onward,
expression of 17HSD/KSR7 was observed in the basal layer of the developing
chorioallantoic placenta. On days 12½ and 14½ p.c., the enzyme was abundantly
expressed in the spongiotrophoblasts, where expression gradually declined toward
parturition. Similarly, PLP-B, a marker of spongiotrophoblasts, was abundantly expressed
in the spongy layer on days 12½ and 14½ p.c., after which its expression declined
somewhat faster than that of 17HSD/KSR7, being almost undetectable on day 17½ p.c.
No specific signals for mouse 17HSD/KSR7 or PLP-B were detected in the giant cells at
any stage of pregnancy.
6 Discussion
6.1 Cloning and characterization of mouse and rat 17HSD/KSR1
17HSD/KSR1 was cloned from mouse and rat tissues, and as expected, rodent type 1
enzymes have similar amino acid sequences and they belong to the SDR family,
containing the glycine pattern for cofactor-binding in the N-terminus, and the most
conserved and important catalytic amino acids. The greatest identity between rodent and
human 17HSD/KSR1 is in the first 200 amino acids (81 %), which also is typical of
SDRs (Jörnvall et al. 1995). The most striking difference between the human and rodent
type 1 enzymes is their substrate specificity. Human 17HSD/KSR1 mainly catalyzes
conversion of E1 to E2, while mouse 17HSD/KSR1 converts A-dione to T as efficiently as
E1 to E2. In line with the mouse enzyme, rat 17HSD/KSR1 equally well catalyzes
conversion of both E1 and A-dione (Puranen et al. 1997a). Low homology (32 %) in the
amino acid region 197–230 between human and rodent 17HSD/KSR1 has been suggested
to partly explain why the rodent type 1 enzyme has broader substrate specificity (Puranen
et al. 1997a). The biological significance of this broader substrate acceptance is not
known, but at least it shows that two routes are possible for E2 biosynthesis in the rodent
ovary: 1) from A-dione to E1 and then to E2, and/or 2) from A-dione to T and then to E2.
In the human ovary, the favored route is the former pathway, since theca cells mainly
produce A-dione (Bergh et al. 1993) and P450arom catalyzes A-dione as efficiently as T
(Kellis & Vickery 1987).
Rodent 17HSD/KSR1 is most abundantly expressed in the granulosa cells of
developing follicles (present study, Akinola et al. 1996). According to the Northern blot
and RT-PCR analyses, the mouse type 1 enzyme is modestly expressed in the uterus and
adrenals. The human type 1 enzyme is also expressed in the endometrium (Mäentausta et
al. 1991, Miettinen et al. 1996a, Zeitoun et al. 1998), whereas expression in the adrenals
has not been reported. In contrast to humans, mouse 17HSD/KSR1 is not expressed in the
mammary gland or placenta, where the human counterpart is expressed (Mäentausta et al.
1990, Miettinen et al. 1996a, Söderqvist et al. 1998). Furthermore, using RT-PCR, mouse
17HSD/KSR1 was found to be modestly expressed in the liver, testis and brain, which,
excluding the liver, has been confirmed by Sha et al., who also used RT-PCR (1997). In
57
addition, Sha et al. (1996, 1997) detected the transcript in several other tissues such as the
prostate, fetal and young postnatal testes, heart and kidney, and female kidney and male
spleen.
On the basis of these studies and other studies using rodent and human samples
(reviewed in Peltoketo et al. 1999a), it can be concluded that in the mouse, 17HSD/KSR1
is the main 17HSD/KSR which in the granulosa cells is involved in biosynthesis of E2.
The physiological significance, if any, of the low expression of 17HSD/KSR1 in other
mouse tissues, as detected by RT-PCR, remains open.
6.2 Expression and regulation of rat 17HSD/KSR1 in granulosa cells
Rat 17HSD/KSR1 is exclusively expressed in the granulosa cells of growing follicles,
where FSH stimulates expression of the enzyme, as is the case for P450arom (Adashi
1996a, Fitzpatrick et al. 1997). Thus, FSH stimulation of E2 biosynthesis occurs via
enhancement of both enzymes catalyzing the last two reactions of E2 biosynthesis. In
addition, the maturation stage of granulosa cells most likely affects the response of the
cells to gonadotropins, since in hypophysectomized but otherwise untreated animals FSH
stimulates expression of 17HSD/KSR1 and P450arom, while in DES-primed animals it
causes down-regulation of the type 1 enzyme. hCG, in turn, stimulated luteinization in
FSH-treated granulosa cells, while in DES-treated animals it caused follicular atresia.
Strong up-regulation of 17HSD/KSR1 expression by DES suggests an autocrine role
for estrogens, establishing a short positive feedback loop that could maintain increasing
E2 biosynthesis during folliculogenesis. Interestingly, DES has been found to be
ineffective as regards 17HSD/KSR1 expression in granulosa cells in vitro, which suggests
that the estrogen effect may be indirect and may require a paracrine factor or factors
missing in cell culture conditions (Ghersevich et al. 1994c). Unlike the expression of
17HSD/KSR1, that of P450arom is not induced by estrogen alone (Hickey et al. 1988,
Fitzpatrick & Richards 1991, present study), but estrogen is able to enhance the FSHinduced expression of P450arom (Fitzpatrick & Richards 1991, present study).
In agreement with decreased E2 biosynthesis taking place upon luteinization, the
results indicate that the ovulatory surge of LH down-regulates expression of
17HSD/KSR1 in granulosa cells.
6.3 Cloning and characterization of mouse and rat 17HSD/KSR7, and
its expression pattern in the ovary
Mouse 17HSD/KSR7 shows a 89 % identity with rat PRAP, which has been
demonstrated to be associated with a short form of PRL receptor (Duan et al. 1996).
Sequence analyses indicated that both proteins belong to the SDR family and contain
catalytic amino acids needed in the 17KSR reaction (present study). Both mouse
58
17HSD/KSR7 and rat PRAP catalyze the reaction from E1 to E2 in vitro and in intact
cultured cells. The enzyme was also able to catalyze the reverse reaction in vitro, which is
not surprising, since as in the case of human 17HSD/KSR1 (Puranen et al. 1997a), the
direction of reaction may be reversed by excess of cofactor. Kinetic parameters showed
that the enzyme prefers E1 over E2 as a substrate. As suggested to be typical of reductive
enzymes (Luu-The et al. 1995), mouse 17HSD/KSR7 also more efficiently utilizes the
phosphorylated forms of cofactor, NADP(H), than the nonphosphorylated
forms. Altogether, similar primary structures, enzymatic characteristics, and the tissue
distribution of mouse 17HSD/KSR7 and rat PRAP suggest that PRAP is rat
17HSD/KSR7.
Recently, a cDNA encoding human 17HSD/KSR7 and its gene located at chromosome
region 10p11.2 have been cloned (Krazeisen et al. 1999). This uncharacterized protein is
reported to be clearly expressed in the placenta and a liver cell line. In addition, it is
expressed in the ovary, mammary gland and testis. Furthermore, based on Expressed
Sequence Tag clones in the GenBank, the protein is assumed to be expressed in several
additional tissues such as fetal liver and spleen (Krazeisen et al. 1999).
Similarly to mouse 17HSD/KSR7, a cDNA probe for rat type 7 enzyme recognized
two transcripts, 1.8 kb and 4.3 kb in size, and in both species the longer mRNA is the
dominant form. In addition to these two transcripts, a third rat 17HSD/KSR7 mRNA, 5.5
kb in size, has been detected by Duan et al. (1997). At least in the case of the mouse, the
long transcript is mostly comprised of a long 3’-noncoding region, probably a result of
alternative polyadenylation (Nokelainen, P., unpublished data). Nevertheless, the results
of functional studies, and an in-frame stop codon in the 5’-noncoding region, have
confirmed that the cDNA contains the whole coding area.
In agreement with the results of recent studies on rats (Parmer et al. 1992, Duan et al.
1997), this study (III, IV) shows that 17HSD/KSR7 is most abundantly expressed in the
ovaries, more specifically in the CL of both pregnant and nonpregnant animals. A marked
increase of 17HSD/KSR7 expression around gestation day 8 p.c., and sustained abundant
expression until close to parturition coincide with enhanced androgen secretion from the
placenta (Warshaw et al. 1986), expression of P450arom in the CL (Hickey et al. 1988)
and elevated serum E2 concentrations (Taya & Greenwald 1981). This indicates that
17HSD/KSR7, not 17HSD/KSR1, is the 17HSD/KSR which is necessary for E2
biosynthesis in the rodent CL. However, an additional role for 17HSD/KSR7 in PRL
signaling cannot be excluded, since rat 17HSD/KSR7 has been found to be associated
with a short form of PRL receptor, and PRL up-regulates expression of
17HSD/KSR7/PRAP (Duan et al. 1997). Furthermore, E2 has been reported to increase
expression of 17HSD/KSR7 in hypophysectomized rats in vivo (Duan et al. 1997) and
PRL up-regulates expression of ERs in the CL (Telleria et al. 1998). It could therefore be
possible that there is cross-talk between E2 and PRL signaling via 17HSD/KSR7.
59
6.4 17HSD/KSR7 in the mouse uterus and placenta
In addition to the CL, mouse 17HSD/KSR7 is expressed in the decidua, more abundantly
in the inner zone of the antimesometrial layer of the decidua. Estrogen is known to play a
crucial role in implantation, particularly in rodents (Cross et al. 1994). Together with P, it
primes the uterus for implantation, nidatory estrogen triggers implantation, and in
addition it augments several effects of P on the decidual cell layer after implantation.
Since ovariectomy prevents implantation (McLaren 1971, Paria et al. 1993), local E2
biosynthesis possibly occurring in the uterus is not able to replace the ablation of ovarian
estrogen. However, E2 produced in the uterus may have a role in stromal proliferation and
differentiation into decidual cells when E2 produced in the ovaries is not maximal. Indeed,
E2-induced cell proliferation is increased on the antimesometrial side of the implantation
site in particular (Yamada & Nagata 1992, Weitlauf 1994), concurrently with the
expression of 17HSD/KSR7. In decidual cells, E2 together with P is known to modulate
the expression of several genes suggested to have a role in decidualization and mural
trophoblast giant cell invasion (Schatz et al. 1994, Schatz et al. 1995, Das et al. 1999).
Expression of the enzyme in the placenta is interesting, since rodent placenta, in
contrast to the human placenta, has been assumed to be unable to synthesize E2 as a result
of its lack of P450arom (Rembiesa et al. 1971) and 17HSD/KSR1 (present study). The
mouse placenta is rich in 17HSD/KSR2, which is suggested to prevent the transfer of
active 17β-hydroxy forms of sex steroids between the fetus and the maternal circulation,
by converting E2 to E1 (Mustonen et al. 1997a, Mustonen et al. 1997b). However, it has
also previously been proposed that mouse placenta may express multiple forms of
17HSD/KSR (Blomquist et al. 1993). It is therefore possible that 17HSD/KSR7 catalyzes
conversion of circulating E1 to E2 in the placenta for local needs. Accordingly, mouse
placenta may not secrete estrogens in the classical endocrine manner, since 17HSD/KSR2
can limit the entrance of E2 to the circulation (Mustonen et al. 1997b).
As shown in this study, E2 synthesized by 17HSD/KSR7 in situ, may exert paracrine,
autocrine or even intracrine effects in the placenta. This is supported by a report showing
that mouse trophoblasts contain estrogen receptors (Iguchi et al. 1993). In addition,
treatment of pregnant rats on day 9 to 11 p.c. with an antiestrogen, tamoxifen, has been
demonstrated to cause severe impairment of decidual development, which is associated
with altered placental bed vascularization, deteriorated fetoplacental development and
increased incidences of growth-retarded fetuses and fetal death (Sadek & Bell 1996).
Hence, estrogen action could be needed to carry rodent placentation successfully to
completion, and 17HSD/KSR2 and 17HSD/KSR7, which show precise cell-specific
expression, may be needed in the placenta for appropriate regulation of estrogen action.
Altogether, the results have shown cell-specific expression of 17HSD/KSR7 in the
ovaries, placenta and uterus. In rodent ovaries, 17HSD/KSR1 and 17HSD/KSR7 are
expressed in the follicles and CLs, respectively. In the placenta and uterus, 17HSD/KSR7
and 17HSD/KSR2 are expressed in adjacent cells. This may reflect part of a system by
which estrogen action is cell-specifically regulated in these tissues.
7 Summary and conclu sions
Overall, 17HSD/KSR1 and -7 were cloned from mouse and rat. Both enzymes catalyze
the reaction from E1 to E2 and are important enzymes in E2 biosynthesis taking place in
the ovary. In detail, the findings of the present study are summarized as follows:
1. This study showed that both rat and mouse 17HSD/KSR1 are expressed in the
granulosa cells of developing follicles, where the expression and thereby E2
biosynthesis increases along with follicular maturation. Estrogen and FSH upregulate both the maturation of follicles and the expression of 17HSD/KSR1 in them,
whereas LH causes luteinization of follicles and down-regulation of the enzyme.
Consequently, the rodent type 1 enzyme is not expressed in the CL, indicating that
another 17HSD/KSR catalyzes the E1 to E2 reaction in luteal cells.
2. A comparison of the enzymatic properties of mouse and human 17HSD/KSR1
showed that the mouse type 1 enzyme catalyzes the reaction from A-dione to T at
least equally efficiently as the reaction E1 to E2, while human 17HSD/KSR1 clearly
prefers the E1 to E2 reaction, suggesting that in rodent ovaries E2 can be synthesized
equally well from E1 and T.
3. A novel type of 17HSD/KSR was expression-cloned from a mouse mammary
epithelial cell line and was chronologically named 17HSD/KSR7. Both mouse and
rat 17HSD/KSR7 efficiently catalyze the reaction from E1 to E2. 17HSD/KSR7 is
most abundantly expressed in the CL, where the expression parallels E2 secretion
from the CL. Therefore, it is 17HSD/KSR7 and not 17HSD/KSR1 that participates in
E2 biosynthesis in the rodent CL.
4. A cell-specific expression study showed that the mouse type 7 enzyme is also
expressed in the decidua surrounding the implantation site, in the decidua capsularis
of the developing placenta and in the spongiotrophoblasts of the mature placenta,
where the expression was particularly high on days 12½ and 14½ p.c. E2 synthesized
locally by 17HSD/KSR7 is thus suggested to have a role in decidualization and/or
implantation and placentation.
8 References
Adams JA, Jarabak J & Talalay P (1962) The steroid specificity of the 17β-hydroxysteroid
dehydrogenase of human placenta. J Biol Chem 237: 3069-3073.
Adamski J, Husen B, Marks F & Jungblut PW (1992) Purification and properties of estradiol 17βdehydrogenase extracted from cytoplasmic vesicles of porcine endometrial cells. Biochem J
288: 375-381.
Adamski J, Normand T, Leenders F, Monté D, Begue A, Stéhelin D, Jungblut PW & de Launoit Y
(1995) Molecular cloning of a novel widely expressed human 80 kDa 17β-hydroxysteroid
dehydrogenase IV. Biochem J 311: 437-443.
Adashi EY (1996a) The ovarian follicle: life cycle of a pelvic clock. In: Adashi EY, Rock JA, &
Rosenwaks Z (eds) Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven
Publishers, Philadelphia, p 211-234.
Adashi EY (1996b) The ovarian follicular apparatus. In: Adashi EY, Rock JA, & Rosenwaks Z
(eds) Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven Publishers,
Philadelphia, p 17-40.
Akinola LA, Poutanen M, Peltoketo H, Vihko R & Vihko P (1998) Characterization of rat 17βhydroxysteroid dehydrogenase type 1 gene and mRNA transcripts. Gene 208: 229-238.
Akinola LA, Poutanen M & Vihko R (1996) Cloning of rat 17β-hydroxysteroid dehydrogenase
type 2 and characterization of tissue distribution and catalytic activity of rat type 1 and type 2
enzymes. Endocrinology 137: 1572-1579.
Akinola LA, Poutanen M, Vihko R & Vihko P (1997) Expression of 17β-hydroxysteroid
dehydrogenase type 1 and type 2, P450 aromatase, and 20α-hydroxysteroid dehydrogenase
enzymes in immature, mature, and pregnant rats. Endocrinology 138: 2886-2892.
Albrecht ED, Aberdeen GW & Pepe GJ (2000) The role of estrogen in the maintenance of primate
pregnancy. Am J Obstet Gynecol 182: 432-438.
Albrecht ED & Pepe GJ (1990) Placental steroid hormone biosynthesis in primate pregnancy.
Endocr Rev 11: 124-150.
Andersson S, Russell DW & Wilson JD (1996) 17β-Hydroxysteroid dehydrogenase 3 deficiency.
Trends Endrocrinol Metab 7: 121-126.
Ando A, Kikuti YY, Shigenari A, Kawata H, Okamoto N, Shiina T, Chen, L, Ikemura T, Abe K,
Kimura M & Inoko H (1996) cDNA cloning of the human homologues of the mouse Ke4 and
Ke6 genes at the centromeric end of the human MHC region. Genomics 35: 600-602.
Autio-Harmainen H, Hurskainen T, Niskasaari K, Höyhtyä M & Tryggvason K (1992)
Simultaneous expression of 70 kilodalton type IV collagenase and type IV collagen α1 (IV)
chain genes by cells of early human placenta and gestational endometrium. Lab Invest 67: 191200.
62
Aziz N, Brown D, Lee W-S & Naray-Fejes-Toth A (1996) Aberrant 11β-hydroxysteroid
dehydrogenase-1 activity in the cpk mouse: implications for regulation by the Ke 6 gene.
Endocrinology 137: 5581-5588.
Aziz N, Maxwell MM, St.-Jacques B & Brenner BM (1993) Downregulation of Ke 6, a novel gene
encoded within the major histocompatibility complex, in murine polycystic kidney disease. Mol
Cell Biol 13: 1847-1853.
Aziz N, Nearing J, Kriedberg J & Donovan M (1995) Antisense oligonucleotide mediated
inhibition of ke 6 gene expression induces cysts in embryonic kidneys in organ cultures
(abstract). J Am Soc Nephrol 6: 690.
Azzi A, Rehse PH, Zhu D-W, Campbell RL, Labrie F & Lin S-X (1996) Crystal structure of human
estrogenic 17β-hydroxysteroid dehydrogenase complexed with 17β-estradiol. Nat Struct Biol 3:
665-668.
Baker ME (1995) Unusual evolution of 11β- and 17β-hydroxysteroid and retinol dehydrogenases.
Bioessays 18: 63-70.
Baker PJ, Sha JH & O'Shaughnessy PJ (1997) Localisation and regulation of 17β-hydroxysteroid
dehydrogenase type 3 mRNA during development in the mouse testis. Mol Cell Endocrinol 133:
127-133.
Ball RK, Friis RR, Schoenenberger CA, Doppler W & Groner B (1988) Prolactin regulation of βcasein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial
cell line. EMBO J 7: 2089-2095.
Barbieri RL & Gao X (1997) Presence of 17β-hydroxysteroid dehydrogenase type 3 messenger
ribonucleic acid transcript in an ovarian Sertoli-Leydig cell tumor. Fertil Steril 68: 534-537.
Barbieri RL, Gao X & Frost RA (1994) Phosphorylation of 17β-hydroxysteroid dehydrogenase in
BeWo choriocarcinoma cells. Am J Obstet Gynecol 171: 223-230.
Batistuzzo de Medeiros SR, Krey G, Hihi AK & Wahli W (1997) Functional interactions between
the estrogen receptor and the transcription activator Sp1 regulate the estrogen-dependent
transcriptional activity of the vitellogenin A1 io promoter. J Biol Chem 272: 18250-18260.
Benz CC & Lewis BJ (1991) Hormones & Cancer. In: Greenspan FS (ed) Basic and Clinical
Endocrinology. Prentice-Hall International Inc., East Norwalk, USA, p 701-714.
Bergh C, Olsson J-H, Selleskog U & Hillensjö T (1993) Steroid production in cultured thecal cells
obtained from human ovarian follicles. Hum Reprod 8: 519-524.
Biswas MG & Russell DW (1997) Expression cloning and characterization of oxidative 17β- and
3α-hydroxysteroid dehydrogenases from rat and human prostate. J Biol Chem 272: 1595915966.
Blomquist CH, Hensleigh HC, Block DL & Feeney LA (1993) Placental 17β-hydroxysteroid
oxidoreductase, lactate dehydrogenase and malate dehydrogenase during the latter half of
pregnancy in the mouse. J Steroid Biochem Mol Biol 46: 61-67.
Breton R, Housset D, Mazza C & Fontecilla-Camps JC (1996) The structure of a complex of
+
human 17β-hydroxysteroid dehydrogenase with estradiol and NADP identifies two principal
targets for the design of inhibitors. Structure 4: 905-915.
Bulun SE, Noble LS, Takayama K, Michael MD, Agarwal V, Fisher C, Zhao Y, Hinshelwood MM,
Ito Y & Simpson ER (1997) Endocrine disorders associated with inappropriately high aromatase
expression. J Steroid Biochem Mol Biol 61: 133-139.
Burns DJW, Engel LL & Bethune JL (1971) The subunit structure of human placental 17βestradiol dehydrogenase. Biochem Biophys Res Commun 44: 786-792.
Burns DJW, Engel LL & Bethune JL (1972) Amino acid composition and subunit structure. Human
placental 17β-estradiol dehydrogenase. Biochemistry 11: 2699-2703.
Byers M, Kuiper GGJM, Gustafsson J-Å & Park-Sarge O-K (1997) Estrogen receptor-β mRNA
expression in rat ovary: down-regulation by gonadotropins. Mol Endocrinol 11: 172-182.
Caira F, Clémencet M-C, Cherkaoui-Malki M, Dieuaide-Noubhani M, Pacot, C, van Veldhoven PP
& Latruffe N (1998) Differential regulation by a peroxisome proliferator of the different
multifunctional proteins in guinea pig: cDNA cloning of the guinea pig D-specific
multifunctional protein 2. Biochem J 330: 1361-1368.
63
Carstensen JF, Tesdorpf JG, Kaufmann M, Markus MM, Husen B, Leenders F, Jakob F, de Launoit
Y & Adamski J (1996) Characterization of 17β-hydroxysteroid dehydrogenase IV. J Endocrinol
150: S3-S12.
Casey ML, MacDonald PC & Andersson S (1994) 17β-Hydroxysteroid dehydrogenase type 2:
chromosomal assignment and progestin regulation of gene expression in human endometrium. J
Clin Invest 94: 2135-2141.
Cenni B & Picard D (1999) Ligand-independent activation of steroid receptors: new roles for old
players. Trends Endrocrinol Metab 10: 41-46.
Conley AJ & Bird IM (1997) The role of cytochrome P450 17α-hydroxylase and 3βhydroxysteroid dehydrogenase in the integration of gonadal and adrenal steroidogenesis via the
∆5 and ∆4 pathways of steroidogenesis in mammals. Biol Reprod 56: 789-799.
Conte FA & Grumbach MM (1991) Abnormalities of sexual differentiation. In: Greenspan FS (ed)
Basic and Clinical Endocrinology. Prentice-Hall International Inc., East Norwalk, USA, p 491518.
Corbould AM, Judd SJ & Rodgers RJ (1998) Expression of types 1, 2, and 3 17β-hydroxysteroid
dehydrogenase in subcutaneous abdominal and intra-abdominal adipose tissue of women. J Clin
Endocrinol Metab 83: 187-194.
Corton JC, Bocos C, Moreno ES, Merritt A, Marsman DS, Sausen PJ, Cattley RC & Gustafsson JÅ (1996) Rat 17β-hydroxysteroid dehydrogenase type IV is a novel peroxisome proliferatorinducible gene. Mol Pharmacol 50: 1157-1166.
Couse JF, Hewitt SC, Bunch DO, Sar M, Walker VR, Davis BJ & Korach KS (1999) Postnatal sex
reversal of the ovaries in mice lacking estrogen receptors α and β. Science 286: 2328-2331.
Couse JF & Korach KS (1999) Estrogen receptor null mice: what have we learned and where will
they lead us? Endocr Rev 20: 358-417.
Couse JF, Lindzey J, Grandien K, Gustafsson J-Å & Korach KS (1997) Tissue distribution and
quantitative analysis of estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) messenger
ribonucleic acid in the wild-type and ERα-knockout mouse. Endocrinology 138: 4613-4621.
Cowley SM, Hoare S, Mosselman S & Parker MG (1997) Estrogen receptors α and β form
heterodimers on DNA. J Biol Chem 272: 19858-19862.
Cross JC, Werb Z & Fisher SJ (1994) Implantation and the placenta: key pieces of the development
puzzle. Science 266: 1508-1518.
Cullen KJ & Lippman ME (1989) Estrogen regulation of protein synthesis and cell growth in
human breast cancer. Vitam Horm 45: 127-172.
Das SK, Lim H, Paria BC & Dey SK (1999) Cyclin D3 in the mouse uterus is associated with the
decidualization process during early pregnancy. J Mol Endocrinol 22: 91-101.
Davis LG, Dibner MD & Battey JF (1986) Preparation and analysis of RNA from eukaryotic cells.
In: Basic Methods in Molecular Biology. Elsevier, New York, p 130-135.
Deyashiki Y, Ohshima K, Nakanishi M, Sato K, Matsuura K & Hara A (1995) Molecular cloning
and characterization of mouse estradiol 17β-dehydrogenase (A-specific), a member of the
aldoketoreductase family. J Biol Chem 270: 10461-10467.
Dieuaide-Noubhani M, Novikov D, Baumgart E, Vanhooren JCT, Fransen M, Goethals M,
Vandekerckhove J, van Veldhoven PP & Mannaerts GP (1996) Further characterization of the
peroxisomal 3-hydroxyacyl-CoA dehydrogenases from rat liver. Relationship between the
different dehydrogenases and evidence that fatty acids and the C27 bile acids di- and trihydroxycoprostanic acids are metabolized by separate multifunctional proteins. Eur J Biochem
240: 660-666.
Dieuaide-Noubhani M, Novikov D, Vandekerckhove J, van Veldhoven PP & Mannaerts GP (1997)
Identification and characterization of the 2-enoyl-CoA hydratases involved in peroxisomal βoxidation in rat liver. Biochem J 321: 253-259.
Duan WR, Linzer DIH & Gibori G (1996) Cloning and characterization of an ovarian-specific
protein that associates with the short form of the prolactin receptor. J Biol Chem 271: 1560215607.
Duan WR, Parmer TG, Albarracin CT, Zhong L & Gibori G (1997) PRAP, a prolactin receptor
associated protein: its gene expression and regulation in the corpus luteum. Endocrinology 138:
3216-3221.
64
Dufort I, Rheault P, Huang X-F, Soucy P & Luu-The V (1999) Characteristics of a highly labile
human type 5 17β-hydroxysteroid dehydrogenase. Endocrinology 140: 568-574.
Duque C, Morisaki M & Ikekawa N (1978) The enzyme activity of bovine adrenocortical
cytochrome P-450 producing pregnenolone from cholesterol: kinetic and electrophoretic studies
on the reactivity of hydroxycholesterol intermediates. Biochem Biophys Res Commun 82: 179187.
El-Alfy M, Luu-The V, Huang X-F, Berger L, Labrie F & Pelletier G (1999) Localization of type 5
17β-hydroxysteroid dehydrogenase, 3β-hydroxysteroid dehydrogenase, and androgen receptor
in the human prostate by in situ hybridization and immunocytochemistry. Endocrinology 140:
1481-1491.
Elo JP, Akinola LA, Poutanen M, Vihko P, Kyllönen AP, Lukkarinen O & Vihko R (1996)
Characterization of 17β-hydroxysteroid dehydrogenase isoenzyme expression in benign and
malignant human prostate. Int J Cancer 66: 37-41.
Engel LL & Groman EV (1974) Human placental 17β-estradiol dehydrogenase: characterization
and structural studies. Recent Prog Horm Res 30: 139-169.
Enmark E, Pelto-Huikko M, Grandien K, Lagercrantz S, Lagercrantz J, Fried G, Nordenskjöld M &
Gustafsson J-Å (1997) Human estrogen receptor β-gene structure, chromosomal localization,
and expression pattern. J Clin Endocrinol Metab 82: 4258-4265.
Fan L-Q, Cattley RC & Corton JC (1998) Tissue-specific induction of 17β-hydroxysteroid
dehydrogenase type IV by peroxisome proliferator chemicals is dependent on the peroxisome
proliferator-activated receptor α. J Endocrinol 158: 237-246.
Farhat MY, Lavigne MC & Ramwell PW (1996) The vascular protective effects of estrogen.
FASEB J 10: 615-624.
Ferin MJ (1996) The menstrual cycle: an integrative view. In: Adashi EY, Rock JA, & Rosenwaks
Z (eds) Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven Publishers,
Philadelphia, p 103-121.
Fishman J, Nisselbaum JS, Menendez-Botet CJ & Schwartz MK (1977) Estrone and estradiol
content in human breast tumors: relationship to estradiol receptors. J Steroid Biochem 8: 893896.
Fitzpatrick SL, Carlone DL, Robker RL & Richards JS (1997) Expression of aromatase in the
ovary: down-regulation of mRNA by the ovulatory luteinizing hormone surge. Steroids 62: 197206.
Fitzpatrick SL, Funkhouser JM, Sindoni DM, Stevis PE, Deecher DC, Bapat AR, Merchenthaler I
& Frail DE (1999) Expression of estrogen receptor-β protein in rodent ovary. Endocrinology
140: 2581-2591.
Fitzpatrick SL & Richards JS (1991) Regulation of cytochrome P450 aromatase messenger
ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology
129: 1452-1462.
Fleming H, Namit C & Gurpide E (1980) Estrogen receptors in epithelial and stromal cells of
human endometrium in culture. J Steroid Biochem 12: 169-174.
Fomitcheva J, Baker ME, Anderson E, Lee GY & Aziz N (1998) Characterization of Ke 6, a new
17β-hydroxysteroid dehydrogenase, and its expression in gonadal tissues. J Biol Chem 273:
22664-22671.
Fournet-Dulguerov N, MacLusky NJ, Leranth CZ, Todd R, Mendelson CR, Simpson ER &
Naftolin F (1987) Immunohistochemical localization of aromatase cytochrome P-450 and
estradiol dehydrogenase in the syncytiotrophoblast of the human placenta. J Clin Endocrinol
Metab 65: 757-764.
Gaub M-P, Bellard M, Scheuer I, Chambon P & Sassone-Corsi P (1990) Activation of the
ovalbumin gene by the estrogen receptor involves the fos-jun complex. Cell 63: 1267-1276.
Geissler WM, Davis DL, Wu L, Bradshaw KD, Patel S, Mendonca BB, Elliston KO, Wilson JD,
Russell DW & Andersson S (1994) Male pseudohermaphroditism caused by mutations of
testicular 17β-hydroxysteroid dehydrogenase 3. Nat Genet 7: 34-39.
Ghersevich SA, Poutanen MH, Martikainen HK & Vihko RK (1994a) Expression of 17βhydroxysteroid dehydrogenase in human granulosa cells: correlation with follicular size,
cytochrome P450 aromatase activity and oestradiol production. J Endocrinol 143: 139-150.
65
Ghersevich SA, Poutanen MH, Rajaniemi HJ & Vihko RK (1994b) Expression of 17βhydroxysteroid dehydrogenase in the rat ovary during follicular development and luteinization
induced with pregnant mare serum gonadotrophin and human chorionic gonadotrophin. J
Endocrinol 140: 409-417.
Ghersevich S, Poutanen M, Tapanainen J & Vihko R (1994c) Hormonal regulation of rat 17βhydroxysteroid dehydrogenase type 1 in cultured rat granulosa cells: effects of recombinant
follicle-stimulating hormone, estrogens, androgens, and epidermal growth factor. Endocrinology
135: 1963-1971.
Ghosh D, Pletnev VZ, Zhu D-W, Wawrzak Z, Duax WL, Pangborn W, Labrie F & Lin S-X (1995)
Structure of human estrogenic 17β-hydroxysteroid dehydrogenase at 2.20 Å resolution.
Structure 3: 503-513.
Ghosh D, Wawrzak Z, Weeks CM, Duax WL & Erman M (1994) The refined three-dimensional
structure of 3α,20β-hydroxysteroid dehydrogenase and possible roles of the residues conserved
in short-chain dehydrogenases. Structure 2: 629-640.
Ghosh D, Weeks CM, Grochulski P, Duax WL, Erman M, Rimsay RL & Orr JC (1991) Threedimensional structure of holo 3α,20β-hydroxysteroid dehydrogenase: a member of a short-chain
dehydrogenase family. Proc Natl Acad Sci USA 88: 10064-10068.
Gibori G (1993) The corpus luteum of pregnancy. In: Adashi EY & Leung PCK (eds) The Ovary.
Raven Press, New York, p 261-317.
Gibori G & Keyes PL (1980) Luteotropic role of estrogen in early pregnancy in the rat.
Endocrinology 106: 1584-1588.
Gibori G, Khan I, Warshaw ML, McLean MP, Puryear TK, Nelson S, Durkee TJ, Azhar S,
Steinschneider A & Rao MC (1988) Placental-derived regulators and the complex control of
luteal cell function. Recent Prog Horm Res 44: 377-429.
Goldfien AG & Monroe SE (1991) Ovaries. In: Greenspan FS (ed) Basic and Clinical
Endocrinology. Prentice-Hall International Inc., East Norwalk, USA, p 442-490.
Gougeon A (1996) Regulation of ovarian follicular development in primates: facts and hypotheses.
Endocr Rev 17: 121-155.
Green S & Chambon P (1988) Nuclear receptors enhance our understanding of transcription
regulation. Trends Genet 4: 309-314.
van Grunsven EG, van Berkel E, Ijlst L, Vreken P, de Klerk JBC, Adamski J, Lemonde H, Clayton
PT, Cuebas DA & Wanders RJA (1998) Peroxisomal D-hydroxyacyl-CoA dehydrogenase
deficiency: resolution of the enzyme defect and its molecular basis in bifunctional protein
deficiency. Proc Natl Acad Sci USA 95: 2128-2133.
van Grunsven EG, van Berkel E, Mooijer PA, Watkins PA, Moser HW, Suzuki Y, Jiang LL,
Hashimoto T, Hoefler G, Adamski J & Wanders RJ (1999a) Peroxisomal bifunctional protein
deficiency revisited: resolution of its true enzymatic and molecular basis. Am J Hum Genet 64:
99-107.
van Grunsven EG, Mooijer PA, Aubourg P & Wanders RJ (1999b) Enoyl-CoA hydratase
deficiency: identification of a new type of D-bifunctional protein deficiency. Hum Mol Genet 8:
1509-1516.
Guiochon-Mantel A, Delabre K, Lescop P & Milgrom E (1996) Intracellular traffic of steroid
hormone receptors. J Steroid Biochem Mol Biol 56: 3-9.
He X-Y, Merz G, Mehta P, Schulz H & Yang S-Y (1999) Human brain short chain L-3hydroxyacyl coenzyme A dehydrogenase is a single-domain multifunctional enzyme.
Characterization of a novel 17β-hydroxysteroid dehydrogenase. J Biol Chem 274: 15014-15019.
Hickey GJ, Chen S, Besman MJ, Shively JE, Hall PF, Gaddy-Kurten D & Richards JS (1988)
Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger
ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: relationship to estradiol
biosynthesis. Endocrinology 122: 1426-1436.
Hillier SG, Whitelaw PF & Smyth CD (1994) Follicular oestrogen synthesis: the 'two-cell, twogonadotropin' model revisited. Mol Cell Endocrinol 100: 51-54.
Hisaw FL (1959) Comparative effectiveness of estrogens on fluid imbibition and growth of the rat's
uterus. Endocrinology 64: 276-289.
66
Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS & Tung L (1996) Nuclear receptor
coactivators and corepressors. Mol Endocrinol 10: 1167-1177.
Iguchi T, Tani N, Sato T, Fukatsu N & Ohta Y (1993) Developmental changes in mouse placental
cells from several stages of pregnancy in vivo and in vitro. Biol Reprod 48: 188-196.
Jantus Lewintre E, Orava M, Peltoketo H & Vihko R (1994) Characterization of 17βhydroxysteroid dehydrogenase type 1 in choriocarcinoma cells: regulation by basic fibroblast
growth factor. Mol Cell Endocrinol 104: 1-9.
Jarabak J (1969) Soluble 17β-hydroxysteroid dehydrogenase of human placenta. In: Methods
Enzymol. Academic Press, New York-London, p 746-752.
Jarabak J & Sack GH (1969) A soluble 17β-hydroxysteroid dehydrogenase from human placenta.
The binding of pyridine nucleotides and steroids. Biochemistry 8: 2203-2212.
Jin J-Z & Lin S-X (1999) Human estrogenic 17β-hydroxysteroid dehydrogenase: predominance of
estrone reduction and its induction by NADPH. Biochem Biophys Res Commun 259: 489-493.
Jörnvall H, Persson B, Krook M, Atrian S, Gonzàlez-Duarte R, Jeffery J & Ghosh D (1995) Shortchain dehydrogenases/reductases (SDR). Biochemistry 34: 6003-6013.
Kaminski T, Akinola L, Poutanen M, Vihko R & Vihko P (1997) Growth factors and phorbol-12myristate-13-acetate modulate the follicle-stimulating hormone- and cyclic adenosine-3',5'monophosphate-dependent regulation of 17β-hydroxysteroid dehydrogenase type 1 expression
in rat granulosa cells. Mol Cell Endocrinol 136: 47-56.
Karavolas HJ & Engel LL (1971) Human placental 17β-estradiol dehydrogenase. VI. Substrate
specificity of the diphosphopyridine nucleotide (triphosphopyridine nucleotide)-linked enzyme.
Endocrinology 88: 1165-1169.
Kaufmann M, Carstensen J, Husen B & Adamski J (1995) The tissue distribution of porcine 17βestradiol dehydrogenase and its induction by progesterone. J Steroid Biochem Mol Biol 55: 535539.
Kellis JT, & Vickery LE (1987) Purification and characterization of human placental aromatase
cytochrome P-450. J Biol Chem 262: 4413-4420.
Khan I, Sridaran R, Johnson DC & Gibori G (1987) Selective stimulation of luteal androgen
biosynthesis by luteinizing hormone: comparison of hormonal regulation of P45017α activity in
corpora lutea and follicles. Endocrinology 121: 1312-1319.
Khanna M, Qin K-N, Wang RW & Cheng K-C (1995) Substrate specificity, gene structure, and
tissue-specific distribution of multiple human 3α-hydroxysteroid dehydrogenases. J Biol Chem
270: 20162-20168.
Khanum A, Buczko E & Dufau ML (1997) Essential role of adenosine triphosphate in activation of
17β-hydroxysteroid dehydrogenase in the rat Leydig cell. Endocrinology 138: 1612-1620.
Kikuti YY, Tamiya G, Ando A, Chen L, Kimura M, Ferreira E, Tsuji K, Trowsdale J & Inoko H
(1997) Physical mapping 220 kb centromeric of the human MHC and DNA sequence analysis
of the 43-kb segment including the RING1, HKE6, and HKE4 genes. Genomics 42: 422-435.
King WJ & Greene GL (1984) Monoclonal antibodies localize oestrogen receptor in the nuclei of
target cells. Nature 307: 745-747.
Kobayashi K, Kobayashi H, Ueda M & Honda Y (1997) Expression of 17β-hydroxysteroid
dehydrogenase type IV in chick retinal pigment epithelium. Exp Eye Res 64: 719-726.
Krazeisen A, Breitling R, Imai K, Fritz S, Möller G & Adamski J (1999) Determination of cDNA,
gene structure and chromosomal localization of the novel human 17β-hydroxysteroid
dehydrogenase type 7. FEBS Lett 460: 373-379.
Krozowski Z (1992) At the cutting edge: 11β-hydroxysteroid dehydrogenase and the short-chain
alcohol dehydrogenase (SCAD) superfamily. Mol Cell Endocrinol 84: C25-C31.
Krozowski Z (1994) The short-chain alcohol dehydrogenase superfamily: variations on a common
theme. J Steroid Biochem Mol Biol 51: 125-130.
Kuiper GGJM, Carlsson B, Grandien K, Enmark E, Häggblad J, Nilsson S & Gustafsson J-Å
(1997) Comparison of the ligand binding specificity and transcript tissue distribution of estrogen
receptors α and β. Endocrinology 138: 863-870.
Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S & Gustafsson J-Å (1996) Cloning of a novel
estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93: 5925-5930.
67
Kumar V & Chambon P (1988) The estrogen receptor binds tightly to its responsive element as a
ligand-inducer homodimer. Cell 55: 145-156.
Kuss E (1994) The fetoplacental unit of primates. Exp Clin Endocrinol 102: 135-165.
Labrie F (1991) Intracrinology. Mol Cell Endocrinol 78: C113-C118.
Labrie F, Luu-The V, Lin S-X, Labrie C, Simard J, Breton R & Bélanger A (1997) The key role of
17β-hydroxysteroid dehydrogenases in sex steroid biology. Steroids 62: 148-158.
Labrie Y, Durocher F, Lachance Y, Turgeon C, Simard J, Labrie C & Labrie F (1995) The human
type II 17β-hydroxysteroid dehydrogenase gene encodes two alternatively spliced mRNA
species. DNA Cell Biol 14: 849-861.
Langer LJ, Alexander JA & Engel LL (1959) Human placental estradiol-17β dehydrogenase.
Kinetics and substrate specificities. J Biol Chem 234: 2609-2614.
Langer LJ & Engel LL (1958) Human placental estradiol-17β dehydrogenase: concentration,
characterization and assay. J Biol Chem 233: 583-588.
Lanisnik Rizner T, Moeller G, Thole HH, Zakelj-Mavric M & Adamski J (1999) A novel 17βhydroxysteroid dehydrogenase in the fungus Cochliobolus lunatus: new insights into the
evolution of steroid-hormone signalling. Biochem J 337: 425-431.
de Launoit Y & Adamski J (1999) Unique multifunctional HSD17B4 gene product: 17βhydroxysteroid dehydrogenase 4 and D-3-hydroxyacyl-coenzyme A dehydrogenase/hydratase
involved in Zellweger syndrome. J Mol Endocrinol 22: 227-240.
Leenders F, Adamski J, Husen B, Thole HH & Jungblut PW (1994) Molecular cloning and amino
acid sequence of the porcine 17β-estradiol dehydrogenase. Eur J Biochem 222: 221-227.
Leenders F, Tesdorpf JG, Markus M, Engel T, Seedorf U & Adamski J (1996) Porcine 80-kDa
protein reveals intrinsic 17β-hydroxysteroid dehydrogenase, fatty acyl-CoA-hydratase/
dehydrogenase, and sterol transfer activities. J Biol Chem 271: 5438-5442.
Lees JA, Fawell SE & Parker MG (1989) Identification of constitutive and steroid-dependent
transactivation domains in the mouse estrogen receptor. J Steroid Biochem 34: 33-39.
Lin H-K, Jez JM, Schlegel BP, Peehl DM, Pachter JA & Penning TM (1997) Expression and
characterization of recombinant type 2 3α-hydroxysteroid dehydrogenase (HSD) from human
prostate: demonstration of bifunctional 3α/17β-HSD activity and cellular distribution. Mol
Endocrinol 11: 1971-1984.
Lin S-X, Yang F, Jin J-Z, Breton R, Zhu D-W, Luu-The V & Labrie F (1992) Subunit identity of
the dimeric 17β-hydroxysteroid dehydrogenase from human placenta. J Biol Chem 267: 1618216187.
Luu The V, Labrie C, Zhao HF, Couët J, Lachance Y, Simard J, Leblanc G, Côté J, Bérubé D,
Gagné R & Labrie F (1989) Characterization of cDNAs for human estradiol 17β-dehydrogenase
and assignment of the gene to chromosome 17: evidence of two mRNA species with distinct 5'termini in human placenta. Mol Endocrinol 3: 1301-1309.
Luu-The V, Labrie C, Simard J, Lachance Y, Zhao H-F, Couët J, Leblanc G & Labrie F (1990)
Structure of two in tandem human 17β-hydroxysteroid dehydrogenase genes. Mol Endocrinol 4:
268-275.
Luu-The V, Zhang Y, Poirier D & Labrie F (1995) Characteristics of human types 1, 2 and 3 17βhydroxysteroid dehydrogenase activities: oxidation/reduction and inhibition. J Steroid Biochem
Mol Biol 55: 581-587.
MacLusky NJ (1996) Sex steroid receptors. In: Adashi EY, Rock JA, & Rosenwaks Z (eds)
Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven Publishers,
Philadelphia, p 627-663.
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, Blumberg B, Kastner P,
Mark M, Chambon P & Evans RM (1995) The nuclear receptor superfamily: the second decade.
Cell 83: 835-839.
Mannermaa A, Peltoketo H, Winqvist R, Ponder BAJ, Kiviniemi H, Easton DF, Poutanen M,
Isomaa V & Vihko R (1994) Human familial and sporadic breast cancer: analysis of the coding
regions of the 17β-hydroxysteroid dehydrogenase 2 gene (EDH17B2) using a single-strand
conformation polymorphism assay. Hum Genet 93: 319-324.
Markus M, Husen B & Adamski J (1995) The subcellular localization of 17β-hydroxysteroid
dehydrogenase type 4 and its interaction with actin. J Steroid Biochem Mol Biol 55: 617-621.
68
Martel C, Rhéaume E, Takahashi M, Trudel C, Couët J, Luu-The V, Simard J & Labrie F (1992)
Distribution of 17β-hydroxysteroid dehydrogenase gene expression and activity in rat and
human tissues. J Steroid Biochem Mol Biol 41: 597-603.
Martucci CP & Fishman J (1979) Impact of continuously administered catechol estrogens on
uterine growth and luteinizing hormone secretion. Endocrinology 105: 1288-1292.
Masamura S, Santner SJ, Heitjan DF & Santen RJ (1995) Estrogen deprivation causes estradiol
hypersensitivity in human breast cancer cells. J Clin Endocrinol Metab 80: 2918-2925.
Matt DW & MacDonald GJ (1984) In vitro progesterone and testosterone production by the rat
placenta during pregnancy. Endocrinology 115: 741-747.
Maxwell MM, Nearing J & Aziz N (1995) Ke 6 gene: sequence and organization and aberrant
regulation in murine polycystic kidney disease. J Biol Chem 270: 25213-25219.
McEwen BS & Alves SE (1999) Estrogen actions in the central nervous system. Endocr Rev 20:
279-307.
McKenna NJ, Lanz RB & O'Malley BW (1998) Nuclear receptor coregulators: cellular and
molecular biology. Endocr Rev 20: 321-344.
McLaren A (1971) Blastocysts in the mouse uterus: the effect of ovariectomy, progesterone and
oestrogen. J Endocrinol 50: 515-526.
McNeill JM, Reed MJ, Beranek PA, Bonney RC, Ghilchik MW, Robinson DJ & James VHT
3
3
(1986) A comparison of the in vivo uptake and metabolism of H-oestrone and H-oestradiol by
normal breast and breast tumour tissues in post-menopausal women. Int J Cancer 38: 193-196.
McPhaul MJ, Marcelli M, Zoppi S, Griffin JE & Wilson JD (1993) Genetic basis of endocrine
disease 4. The spectrum of mutations in the androgen receptor gene that causes androgen
resistance. J Clin Endocrinol Metab 76: 17-23.
Melamed M, Castaño E, Notides AC & Sasson S (1997) Molecular and kinetic basis for the mixed
agonist/antagonist activity of estriol. Mol Endocrinol 11: 1868-1878.
Mendelsohn ME & Karas RH (1999) The protective effects of estrogen on the cardiovascular
system. N Engl J Med 340: 1801-1811.
Mendonca BB, Arnhold IJP, Bloise W, Andersson S, Russell DW & Wilson JD (1999) 17βhydroxysteroid dehydrogenase 3 deficiency in women. J Clin Endocrinol Metab 84: 802-804.
Mendoza-Hernández G, Calcagno M, Sánchez-Nuncio HR & Diaz-Zagoya JC (1984)
Dehydroepiandrosterone is a substrate for estradiol 17β-dehydrogenase from human placenta.
Biochem Biophys Res Commun 119: 83-87.
Miettinen M, Mustonen M, Poutanen M, Isomaa V, Wickman M, Söderqvist G, Vihko R & Vihko
P (1999) 17β-hydroxysteroid dehydrogenases in normal human mammary epithelial cells and
breast tissue. Breast Cancer Res Treat 57: 175-182.
Miettinen MM, Mustonen MVJ, Poutanen MH, Isomaa VV & Vihko RK (1996a) Human 17βhydroxysteroid dehydrogenase type 1 and type 2 isoenzymes have opposite activities in cultured
cells and characteristic cell- and tissue-specific expression. Biochem J 314: 839-845.
Miettinen MM, Poutanen MH & Vihko RK (1996b) Characterization of estrogen-dependent growth
of cultured MCF-7 human breast-cancer cells expressing 17β-hydroxysteroid dehydrogenase
type 1. Int J Cancer 68: 600-604.
Miller WL (1995) Mitochondrial specificity of the early steps in steroidogenesis. J Steroid Biochem
Mol Biol 55: 607-616.
Miller WL (1999) Steroid hormone biosynthesis and actions in the materno-feto-placental unit.
Clin Perinatol 25: 799-817.
Miller WL, Auchus RJ & Geller DH (1997) The regulation of 17,20 lyase activity. Steroids 62:
133-142.
Miller WR (1996) Aromatase inhibitors. Endocr-Relat Cancer 3: 65-79.
Misao R, Nakanishi Y, Sun WS, Fujimoto J, Iwagaki S, Hirose R & Tamaya T (1999) Expression
of oestrogen receptor α and β mRNA in corpus luteum of human subjects. Mol Hum Reprod 5:
17-21.
Moghrabi N & Andersson S (1998) 17β-Hydroxysteroid dehydrogenases: physiological roles in
health and disease. Trends Endrocrinol Metab 9: 265-270.
69
Moghrabi N, Head JR & Andersson S (1997) Cell type-specific expression of 17β-hydroxysteroid
dehydrogenase type 2 in human placenta and fetal liver. J Clin Endocrinol Metab 82: 38723878.
Montano MM, Müller V, Trobaugh A & Katzenellenbogen BS (1995) The carboxy-terminal F
domain of the human estrogen receptor: role in the transcriptional activity of the receptor and
the effectiveness of antiestrogens as estrogen antagonists. Mol Endocrinol 9: 814-825.
Morishige WK, Pepe GJ & Rothchild I (1973) Serum luteinizing hormone, prolactin and
progesterone levels during pregnancy in the rat. Endocrinology 92: 1527-1530.
Mosselman S, Polman J & Dijkema R (1996) ERβ: Identification and characterization of a novel
human estrogen receptor. FEBS Lett 392: 49-53.
Mustonen M, Poutanen M, Chotteau-Lelievre A, de Launoit Y, Isomaa V, Vainio S, Vihko R &
Vihko P (1997b) Ontogeny of 17β-hydroxysteroid dehydrogenase type 2 mRNA expression in
the developing mouse placenta and fetus. Mol Cell Endocrinol 134: 33-40.
Mustonen MV, Isomaa VV, Vaskivuo T, Tapanainen J, Poutanen MH, Stenbäck F, Vihko RK &
Vihko PT (1998a) Human 17β-hydroxysteroid dehydrogenase type 2 messenger ribonucleic
acid expression and localization in term placenta and in endometrium during the menstrual
cycle. J Clin Endocrinol Metab 83: 1319-1324.
Mustonen MVJ, Poutanen MH, Isomaa VV, Vihko PT & Vihko RK (1997a) Cloning of mouse
17β-hydroxysteroid dehydrogenase type 2, and analysing expression of the mRNAs for types 1,
2, 3, 4 and 5 in mouse embryos and adult tissues. Biochem J 325: 199-205.
Mustonen MVJ, Poutanen MH, Kellokumpu S, de Launoit Y, Isomaa VV, Vihko RK & Vihko PT
(1998b) Mouse 17β-hydroxysteroid dehydrogenase type 2 mRNA is predominantly expressed in
hepatocytes and in surface epithelial cells of the gastrointestinal and urinary tracts. J Mol
Endocrinol 20: 67-74.
Müller H, Ishimura R, Orwig KE, Liu B & Soares MJ (1998) Homologues for prolactin-like
proteins A and B are present in the mouse. Biol Reprod 58: 45-51.
Mäentausta O, Boman K, Isomaa V, Stendahl U, Bäckström T & Vihko R (1992)
Immunohistochemical study of the human 17β-hydroxysteroid dehydrogenase and steroid
receptors in endometrial adenocarcinoma. Cancer 70: 1551-1555.
Mäentausta O, Peltoketo H, Isomaa V, Jouppila P & Vihko R (1990) Immunological measurement
of human 17β-hydroxysteroid dehydrogenase. J Steroid Biochem 36: 673-680.
Mäentausta O, Sormunen R, Isomaa V, Lehto V-P, Jouppila P & Vihko R (1991)
Immunohistochemical localization of 17β-hydroxysteroid dehydrogenase in the human
endometrium during the menstrual cycle. Lab Invest 65: 582-587.
Mäentausta O, Svalander P, Danielsson KG, Bygdeman M & Vihko R (1993) The effects of an
antiprogestin, mifepristone, and an antiestrogen, tamoxifen, on endometrial 17β-hydroxysteroid
dehydrogenase and progestin and estrogen receptors during the luteal phase of the menstrual
cycle: an immunohistochemical study. J Clin Endocrinol Metab 77: 913-918.
Mäkelä S, Poutanen M, Kostian ML, Lehtimäki N, Strauss L, Santti R & Vihko R (1998) Inhibition
of 17β-hydroxysteroid oxidoreductase by flavonoids in breast and prostate cancer cells. Proc
Soc Exp Biol Med 217: 310-316.
Mäkelä S, Poutanen M, Lehtimäki J, Kostian M-L, Santti R & Vihko R (1995) Estrogen-specific
17β-hydroxysteroid oxidoreductase type 1 (E.C. 1.1.1.62) as a possible target for the action of
phytoestrogens. Proc Soc Exp Biol Med 208: 51-59.
Möller G, Leenders F, van Grunsven EG, Dolez V, Qualmann B, Kessels MM, Markus M,
Krazeisen A, Husen B, Wanders RJA, de Launoit Y & Adamski J (1999) Characterization of the
HSD17B4 gene: D-specific multifunctional protein 2/17β-hydroxysteroid dehydrogenase IV. J
Steroid Biochem Mol Biol 69: 441-446.
Nakhla AM, Romas NA & Rosner W (1997) Estradiol activates the prostate androgen receptor and
prostate-specific antigen secretion through the intermediacy of sex hormone-binding globulin. J
Biol Chem 272: 6838-6841.
Normand T, Husen B, Leenders F, Pelczar H, Baert J-L, Begue A, Flourens A-C, Adamski J & de
Launoit Y (1995) Molecular characterization of mouse 17β-hydroxysteroid dehydrogenase IV. J
Steroid Biochem Mol Biol 55: 541-548.
70
Normand T, Narod S, Labrie F & Simard J (1993) Detection of polymorphisms in the estradiol
17β-hydroxysteroid dehydrogenase II gene at the EDH17B2 locus on 17q11-q21. Hum Mol
Genet 2: 479-483.
Olsen NJ & Kovacs WJ (1996) Gonadal steroids and immunity. Endocr Rev 17: 369-384.
Paech K, Webb P, Kuiper GGJM, Nilsson S, Gustafsson J-Å, Kushner PJ & Scanlan TS (1997)
Differential ligand activation of estrogen receptors ERα and ERβ at AP1 sites. Science 277:
1508-1510.
Panay N & Studd JWW (1998) The psychotherapeutic effects of estrogens. Gynecol Endocrinol 12:
353-365.
Paria BC, Huet-Hudson YM & Dey SK (1993) Blastocyst's state of activity determines the
"window" of implantation in the receptive mouse uterus. Proc Natl Acad Sci USA 90: 1015910162.
Parmer TG, McLean MP, Duan WR, Nelson SE, Albarracin CT, Khan I & Gibori G (1992)
Hormonal and immunological characterization of the 32 kilodalton ovarian-specific protein.
Endocrinology 131: 2213-2221.
Pelletier G, Luu-The V & Labrie F (1995) Immunocytochemical localization of type I 17βhydroxysteroid dehydrogenase in the rat brain. Brain Res 704: 233-239.
Pelletier G, Luu-The V, Têtu B & Labrie F (1999) Immunocytochemical localization of type 5 17βhydroxysteroid dehydrogenase in human reproductive tissues. J Histochem Cytochem 47: 731737.
Peltoketo H, Ghosh D & Vihko P (1998) 17β-hydroxysteroid dehydrogenase type 1: towards
structure and function-based drug design. Curr Top Ster Res 1: 111-117.
Peltoketo H, Isomaa V, Mäentausta O & Vihko R (1988) Complete amino acid sequence of human
placental 17β-hydroxysteroid dehydrogenase deduced from cDNA. FEBS Lett 239: 73-77.
Peltoketo H, Isomaa V & Vihko R (1992) Genomic organization and DNA sequences of human
17β-hydroxysteroid dehydrogenase genes and flanking regions. Localization of multiple Alu
sequences and putative cis-acting elements. Eur J Biochem 209: 459-466.
Peltoketo H, Luu-The V, Simard J & Adamski J (1999b) 17β-Hydroxysteroid dehydrogenase
(HSD)/17-ketosteroid reductase (KSR) family; nomenclature and main characteristics of the
17HSD/KSR enzymes. J Mol Endocrinol 23: 1-11.
Peltoketo H, Piao Y, Mannermaa A, Ponder BAJ, Isomaa V, Poutanen M, Winqvist R & Vihko R
(1994) A point mutation in the putative TATA box, detected in nondiseased individuals and
patients with hereditary breast cancer, decreases promoter activity of the 17β-hydroxysteroid
dehydrogenase type 1 gene 2 (EDH17B2) in vitro. Genomics 23: 250-252.
Peltoketo H, Vihko P & Vihko R (1999a) Regulation of estrogen action: role of 17βhydroxysteroid dehydrogenases. Vitam Horm 55: 353-398.
Penning TM (1996) 17β-Hydroxysteroid dehydrogenase: inhibitors and inhibitor design. EndocrRelat Cancer 3: 41-56.
Pepe GJ & Albrecht ED (1995) Actions of placental and fetal adrenal steroid hormones in primate
pregnancy. Endocr Rev 16: 608-648.
Persson B, Krook M & Jörnvall H (1991) Characteristics of short-chain alcohol dehydrogenases
and related enzymes. Eur J Biochem 200: 537-543.
Pettersson K, Grandien K, Kuiper GGJM & Gustafsson J-Å (1997) Mouse estrogen receptor β
forms estrogen response element-binding heterodimers with estrogen receptor α. Mol
Endocrinol 11: 1486-1496.
Pfeifer SM & Strauss JF (1996) Progestins. In: Adashi EY, Rock JA, & Rosenwaks Z (eds)
Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven Publishers,
Philadelphia, p 493-504.
Piao Y-S, Peltoketo H, Jouppila A & Vihko R (1997) Retinoic acids increase 17β-hydroxysteroid
dehydrogenase type 1 expression in JEG-3 and T47D cells, but the stimulation is potentiated by
epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, and cyclic adenosine 3',5'monophosphate only in JEG-3 cells. Endocrinology 138: 898-904.
Piao Y-S, Peltoketo H, Oikarinen J & Vihko R (1995) Coordination of transcription of the human
17β-hydroxysteroid dehydrogenase type 1 gene (EDH17B2) by a cell-specific enhancer and a
silencer: identification of a retinoic acid response element. Mol Endocrinol 9: 1633-1644.
71
Picard D, Kumar V, Chambon P & Yamamoto KR (1990) Signal transduction by steroid hormones:
nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell
Regul 1: 291-299.
Porter W, Saville B, Hoivik D & Safe S (1997) Functional synergy between the transcription factor
Sp1 and the estrogen receptor. Mol Endocrinol 11: 1569-1580.
Poutanen M, Isomaa V, Kainulainen K & Vihko R (1990) Progestin induction of 17βhydroxysteroid dehydrogenase enzyme protein in the T-47D human breast-cancer cell line. Int J
Cancer 46: 897-901.
Poutanen M, Isomaa V, Lehto V-P & Vihko R (1992a) Immunological analysis of 17βhydroxysteroid dehydrogenase in benign and malignant human breast tissue. Int J Cancer 50:
386-390.
Poutanen M, Miettinen M & Vihko R (1993) Differential estrogen substrate specificities for
transiently expressed human placental 17β-hydroxysteroid dehydrogenase and an endogenous
enzyme expressed in cultured COS-m6 cells. Endocrinology 133: 2639-2644.
Poutanen M, Moncharmont B & Vihko R (1992b) 17β-hydroxysteroid dehydrogenase gene
expression in human breast cancer cells: regulation of expression by a progestin. Cancer Res 52:
290-294.
Pratt WB & Toft DO (1997) Steroid receptor interactions with heat shock protein and
immunophilin chaperones. Endocr Rev 18: 306-360.
Puranen T, Poutanen M, Ghosh D, Vihko R & Vihko P (1997a) Origin of substrate specificity of
human and rat 17β-hydroxysteroid dehydrogenase type 1, using chimeric enzymes and sitedirected substitutions. Endocrinology 138: 3532-3539.
Puranen T, Poutanen M, Ghosh D, Vihko P & Vihko R (1997b) Characterization of structural and
functional properties of human 17β-hydroxysteroid dehydrogenase type 1 using recombinant
enzymes and site-directed mutagenesis. Mol Endocrinol 11: 77-86.
Puranen TJ, Kurkela RM, Lakkakorpi JT, Poutanen MH, Itäranta PV, Melis JP, Ghosh D, Vihko
RK & Vihko PT (1999) Characterization of molecular and catalytic properties of intact and
truncated human 17β-hydroxysteroid dehydrogenase type 2 enzymes: intracellular localization
of the wild-type enzyme in the endoplasmic reticulum. Endocrinology 140: 3334-3341.
Puranen TJ, Poutanen MH, Peltoketo HE, Vihko PT & Vihko RK (1994) Site-directed mutagenesis
of the putative active site of human 17β-hydroxysteroid dehydrogenase type 1. Biochem J 304:
289-293.
Purdy RH, Halla M & Little B (1964) 20α-Hydroxysteroid dehydrogenase activity a function of
human placental 17β-hydroxysteroid dehydrogenase. Biochim Biophys Acta 89: 557-560.
Qin Y-M, Haapalainen AM, Conry D, Cuebas DA, Hiltunen JK & Novikov DK (1997b)
Recombinant 2-enoyl-CoA hydratase derived from rat peroxisomal multifunctional enzyme 2:
role of the hydratase reaction in bile acid synthesis. Biochem J 328: 377-382.
Qin Y-M, Poutanen MH, Helander HM, Kvist A-P, Siivari KM, Schmitz W, Conzelmann E,
Hellman U & Hiltunen JK (1997a) Peroxisomal multifunctional enzyme of β-oxidation
metabolizing D-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and
characterization. Biochem J 321: 21-28.
Quéva C, Ness SA, Grässer FA, Graf T, Vandenbunder B & Stéhelin D (1992) Expression patterns
of c-myb and of v-myb induced myeloid-1 (mim-1) gene during the development of the chick
embryo. Development 114: 125-133.
Ramirez S, Fomitcheva I & Aziz N (1998) Abnormal regulation of the Ke 6 gene, a new 17βhydroxysteroid dehydrogenase in the cpk mouse kidney. Mol Cell Endocrinol 143: 9-22.
Razandi M, Pedram A, Greene GL & Levin ER (1999) Cell membrane and nuclear estrogen
receptors (ERs) originate from a single transcript: studies of ERα and ERβ expressed in Chinese
hamster ovary cells. Mol Endocrinol 13: 307-319.
Reed MJ, Purohit A, Woo LWL & Potter BVL (1996) The development of steroid sulphatase
inhibitors. Endocr-Relat Cancer 3: 9-23.
Rembiesa R, Marchut M & Warchol A (1971) Formation and metabolism of progesterone by the
mouse placenta in vitro. J Steroid Biochem 2: 111-119.
Revelli A, Massobrio M & Tesarik J (1998) Nongenomic actions of steroid hormones in
reproductive tissues. Endocr Rev 19: 3-17.
72
Rheault P, Charbonneau A & Luu-The V (1999b) Structure and activity of the murine type 5 17βhydroxysteroid dehydrogenase gene. Biochim Biophys Acta 1447: 17-24.
Rheault P, Dufort I, Soucy P & Luu-The V (1999a) Assignment of HSD17B5 encoding type 5
17beta-hydroxysteroid dehydrogenase to human chromosome bands 10p15–>p14 and mouse
chromosome 13 region A2 by in situ hybridization: identification of a new syntenic relationship.
Cytogenet Cell Genet 84: 241-242.
Richards JS (1994) Hormonal control of gene expression in the ovary. Endocr Rev 15: 725-751.
Richards JS, Russell DL, Robker RL, Dajee M & Alliston TN (1998) Molecular mechanisms of
ovulation and luteinization. Mol Cell Endocrinol 145: 47-54.
Ringler GE & Strauss JF (1990) In vitro systems for the study of human placental endocrine
function. Endocr Rev 11: 105-123.
Robker RL & Richards JS (1998) Hormonal control of the cell cycle in ovarian cells: proliferation
versus differentiation. Biol Reprod 59: 476-482.
Rosner W, Hryb DJ, Khan MS, Nakhla AM & Romas NA (1999) Sex hormone-binding globulin
mediates steroid hormone signal transduction at the plasma membrane. J Steroid Biochem Mol
Biol 69: 481-485.
Rösler A, Silverstein S & Abeliovich D (1996) A (R80Q) mutation in 17β-hydroxysteroid
dehydrogenase type 3 gene among Arabs of Israel is associated with pseudohermaphroditism in
males and normal asymptomatic females. J Clin Endocrinol Metab 81: 1827-1831.
Sadek S & Bell SC (1996) The effects of the antihormones RU486 and tamoxifen on fetoplacental
development and placental bed vascularisation in the rat: a model for intrauterine fetal growth
retardation. Br J Obstet Gynaecol 103: 630-641.
Salido EC, Yen PH, Barajas L & Shapiro LJ (1990) Steroid sulfatase expression in human placenta:
immunocytochemistry and in situ hybridization study. J Clin Endocrinol Metab 70: 1564-1567.
Sambrook J, Fritsch EF & Maniatis T (1989) Extraction, purification, and analysis of messenger
RNA from eukaryotic cells. In: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, p 7.1-7.84.
Sanger F, Nicklen S & Coulson AR (1977) DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci USA 74: 5463-5467.
Santen RJ (1996) Estrogen synthesis inhibitors for breast cancer: an introductory overview.
Endocr-Relat Cancer 3: 1-8.
Santen RJ, Manni A, Harvey H & Redmond C (1990) Endocrine treatment of breast cancer in
women. Endocr Rev 11: 221-265.
Santner SJ, Feil PD & Santen RJ (1984) In situ estrogen production via the estrone sulfatase
pathway in breast tumors: relative importance versus the aromatase pathway. J Clin Endocrinol
Metab 59: 29-33.
Sasano H, Frost AR, Saitoh R, Harada N, Poutanen M, Vihko R, Bulun SE, Silverberg SG &
Nagura H (1996) Aromatase and 17β-hydroxysteroid dehydrogenase type 1 in human breast
carcinoma. J Clin Endocrinol Metab 81: 4042-4046.
Sawetawan C, Milewich L, Word RA, Carr BR & Rainey WE (1994) Compartmentalization of
type I 17β-hydroxysteroid oxidoreductase in the human ovary. Mol Cell Endocrinol 99: 161168.
Schatz F, Aigner S, Papp C, Toth-Pal E, Hausknecht V & Lockwood CJ (1995) Plasminogen
activator activity during decidualization of human endometrial stromal cells is regulated by
plasminogen activator inhibitor 1. J Clin Endocrinol Metab 80: 2504-2510.
Schatz F, Papp C, Toth-Pal E & Lockwood CJ (1994) Ovarian steroid-modulated stromelysin-1
expression in human endometrial stromal and decidual cells. J Clin Endocrinol Metab 78: 14671472.
Sha J, Baker P & O'Shaughnessy PJ (1996) Both reductive forms of 17β-hydroxysteroid
dehydrogenase (types 1 and 3) are expressed during development in the mouse testis. Biochem
Biophys Res Commun 222: 90-94.
Sha JA, Dudley K, Rajapaksha WRAKJS & O'Shaughnessy PJ (1997) Sequence of mouse 17βhydroxysteroid dehydrogenase type 3 cDNA and tissue distribution of the type 1 and type 3
isoform mRNAs. J Steroid Biochem Mol Biol 60: 19-24.
73
Sherman MI (1983) Endocrinology of rodent trophoblast cells. In: Loke Y & Whyte A (eds)
Biology of Trophoblast. Elsevier, Amsterdam, p 401-467.
Shibata H, Spencer TE, Oñate SA, Jenster G, Tsai SY, Tsai M-J & O'Malley BW (1997) Role of
co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Prog
Horm Res 52: 141-165.
Shoham Z & Schachter M (1996) Estrogen biosynthesis – regulation, action, remote effects, and
value of monitoring in ovarian stimulation cycles. Fertil Steril 65: 687-701.
Siiteri PK, Murai JT, Hammond GL, Nisker JA, Raymoure WJ & Kuhn RW (1982) The serum
transport of steroid hormones. Recent Prog Horm Res 38: 457-510.
Simard J, Durocher F, Mébarki F, Turgeon C, Sanchez R, Labrie Y, Couet J, Trudel C, Rhéaume E,
Morel Y, Luu-The V & Labrie F (1996) Molecular biology and genetics of the 3βhydroxysteroid dehydrogenase/∆5-∆4 isomerase gene family. J Endocrinol 150: S189-S207.
Simard J, Feunteun J, Lenoir G, Tonin P, Normand T, Luu The V, Vivier A, Lasko D, Morgan K,
Rouleau GA, Lynch H, Labrie F & Narod SA (1993) Genetic mapping of the breast-ovarian
cancer syndrome to a small interval on chromosome 17q12-21: exclusion of candidate genes
EDH17B2 and RARA. Hum Mol Genet 2: 1193-1199.
Simpson ER (1998) Genetic mutations resulting in estrogen insufficiency in the male. Mol Cell
Endocrinol 145: 55-59.
Simpson ER, Michael MD, Agarwal VR, Hinshelwood MM, Bulun SE & Zhao Y (1997)
Expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage.
FASEB J 11: 29-36.
Simpson ER, Zhao Y, Agarwal VR, Michael MD, Bulun SE, Hinshelwood MM, Graham-Lorence
S, Sun T, Fisher CR, Qin K & Mendelson CR (1995) Aromatase expression in health and
disease. Recent Prog Horm Res 52: 185-214.
Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB &
Korach KS (1994) Estrogen resistance caused by a mutation in the estrogen-receptor gene in a
man. N Engl J Med 331: 1056-1061.
Soares MJ, Chapman BM, Rasmussen CA, Dai G, Kamei T & Orwig KE (1996) Differentiation of
trophoblast endocrine cells. Placenta 17: 277-289.
Speroff L, Glass RH & Kase NG (1994) Hormone biosynthesis, metabolism, and mechanism of
action. In: Clinical Gynecologic Endocrinology and Infertility. Williams & Wilkins, Baltimore,
USA, p 31-107.
Stocco DM (1999) Steroidogenic acute regulatory (StAR) protein: what's new. Bioessays 21: 768775.
Stouffer RL (1996) Corpus luteum formation and demise. In: Adashi EY, Rock JA, & Rosenwaks
Z (eds) Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven Publishers,
Philadelphia, p 251-269.
Strauss JF, Martinez F & Kiriakidou M (1996) Placental steroid hormone synthesis: unique features
and unanswered questions. Biol Reprod 54: 303-311.
Strickler RC, Tobias B & Covey DF (1981) Human placental 17β-estradiol dehydrogenase and
20α-hydroxysteroid dehydrogenase. J Biol Chem 256: 316-321.
Su J, Lin M & Napoli JL (1999) Complementary deoxyribonucleic acid cloning and enzymatic
characterization of a novel 17β/3α-hydroxysteroid/retinoid short chain dehydrogenase/
reductase. Endocrinology 140: 5275-5284.
Suzuki Y, Jiang LL, Souri M, Miyazawa S, Fukuda S, Zhang Z, Une M, Shimozawa N, Kondo N,
Orii T & Hashimoto T (1997) D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA
dehydrogenase bifunctional protein deficiency: a newly identified peroxisomal disorder. Am J
Hum Genet 61: 1153-1162.
Söderqvist G, Poutanen M, Wickman M, von Schoultz B, Skoog L, Vihko & R. (1998) 17βhydroxysteroid dehydrogenase type 1 in normal breast tissue during the menstrual cycle and
hormonal contraception. J Clin Endocrinol Metab 83: 1190-1193.
Takeyama J, Sasano H, Suzuki T, Iinuma K, Nagura H & Andersson S (1998) 17β-hydroxysteroid
dehydrogenase types 1 and 2 in human placenta: an immunohistochemical study with
correlation to placental development. J Clin Endocrinol Metab 83: 3710-3715.
74
Taya K & Greenwald GS (1981) In vivo and in vitro ovarian steroidogenesis in the pregnant rat.
Biol Reprod 25: 683-691.
Telleria CM, Zhong L, Deb S, Srivastava RK, Park KS, Sugino N, Park-Sarge O-K & Gibori G
(1998) Differential expression of the estrogen receptors α and β in the rat corpus luteum of
pregnancy: regulation by prolactin and placental lactogens. Endocrinology 139: 2432-2442.
Thiboutot D, Martin P, Volikos L & Gilliland K (1998) Oxidative activity of the type 2 isozyme of
17β-hydroxysteroid dehydrogenase (17β-HSD) predominates in human sebaceous glands. J
Invest Dermatol 111: 390-395.
Thijssen JHH & Blankenstein MA (1994) Steroid hormones in the human breast. Endocr-Relat
Cancer 2: 43-48.
Tora L, White J, Brou C, Tasset D, Webster N, Scheer E & Chambon P (1989) The human estrogen
receptor has two independent nonacidic transcriptional activation functions. Cell 59: 477-487.
Touitou I, Cai QQ & Rochefort H (1994) 17 beta hydroxysteroid dehydrogenase 1 "pseudogene" is
differentially transcribed: still a candidate for the breast-ovarian cancer susceptibility gene
(BRCA1). Biochem Biophys Res Commun 201: 1327-1332.
3
14
Townsend L & Ryan KJ (1970) In vitro metabolism of pregnenolone-7α- H, progesterone-4- C
14
and androstenedione-4- C by rat placental tissue. Endocrinology 87: 151-155.
Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie, F & Giguère V (1997)
Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor β.
Mol Endocrinol 11: 353-365.
Tsafriri A & Chun S-Y (1996) Ovulation. In: Adashi EY, Rock JA, & Rosenwaks Z (eds)
Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven Publishers,
Philadelphia, p 235-249.
Tsai M-J & O'Malley BW (1994) Molecular mechanisms of action of steroid/thyroid receptor
superfamily members. Annu Rev Biochem 63: 451-486.
Tsai-Morris CH, Khanum A, Tang P-Z & Dufau ML (1999) The rat 17β-hydroxysteroid
dehydrogenase type III: molecular cloning and gonadotropin regulation. Endocrinology 140:
3534-3542.
Tseng L (1980) Hormonal regulation of steroid metabolic enzymes in human endometrium. In:
Thomas JA & Singhal L (eds) Advances in Sex Steroid Hormone Research. Urban &
Schwarzenberg, Baltimore and Munich, p 329-361.
Turner RT, Riggs BL & Spelsberg TC (1994) Skeletal effects of estrogen. Endocr Rev 15: 275-300.
Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N, Morishima T, Yamasaki Y, Kajimoto
Y & Kamada T (1994) Estrogen regulation of the insulin-like growth factor I gene transcription
involves an AP-1 enhancer. J Biol Chem 269: 16433-16442.
Vermeulen A, Deslypere JP, Paridaens R, Leclercq G, Roy F & Heuson JC (1986) Aromatase, 17βhydroxysteroid dehydrogenase and intratissular sex hormone concentrations in cancerous and
normal glandular breast tissue in postmenopausal women. Eur J Cancer Clin Oncol 22: 515-525.
Wajima Y, Furusawa T, Kawauchi S, Wakabayashi N, Nakabayashi O, Nishimori K & Mizuno S
(1999) The cDNA cloning and transient expression of an ovary-specific 17β-hydroxysteroid
dehydrogenase of chickens. Gene 233: 75-82.
Warner M, Nilsson S & Gustafsson J-Å (1999) The estrogen receptor family. Curr Opin Obstet
Gynecol 11: 249-254.
Warshaw ML, Johnson DC, Khan I, Eckstein B & Gibori G (1986) Placental secretion of
androgens in the rat. Endocrinology 119: 2642-2648.
Webster NJG, Green S, Jin JR & Chambon P (1988) The hormone-binding domains of the estrogen
and glucocorticoid receptors contain an inducible transcription activation function. Cell 54: 199207.
Weigel NL (1996) Steroid hormone receptors and their regulation by phosphorylation. Biochem J
319: 657-667.
Weitlauf HM (1994) Biology of implantation. In: Knobil E & Neill JD (eds) The Physiology of
Reproduction. Raven Press, New York, p 391-440.
Wilson J & Parson M (1996) Endocrinology of human gestation. In: Adashi EY, Rock JA, &
Rosenwaks Z (eds) Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven
Publishers, Philadelphia, p 451-475.
75
Wilson JD, Griffin JE & Russell DW (1993) Steroid 5α-reductase 2 deficiency. Endocr Rev 14:
577-593.
Winqvist R, Peltoketo H, Isomaa V, Grzeschik K-H, Mannermaa A & Vihko R (1990) The gene for
17β-hydroxysteroid dehydrogenase maps to human chromosome 17, bands q12-q21, and shows
an RFLP with ScaI. Hum Genet 85: 473-476.
Wu L, Einstein M, Geissler WM, Chan HK, Elliston KO & Andersson S (1993) Expression cloning
and characterization of human 17β-hydroxysteroid dehydrogenase type 2, a microsomal enzyme
possessing 20α-hydroxysteroid dehydrogenase activity. J Biol Chem 268: 12964-12969.
Yamada AT & Nagata T (1992) Light and electron microscopic radioautography of DNA synthesis
in the endometria of pregnant-ovariectomized mice during activation of implantation window.
Cell Mol Biol 38: 763-774.
Ylikomi T, Wurtz J-M, Syvälä H, Passinen S, Pekki A, Haverinen M, Bläuer M, Tuohimaa P &
Gronemeyer H (1998) Reappraisal of the role of heat shock proteins as regulators of steroid
receptor activity. Crit Rev Biochem Mol 33: 437-466.
Zeitoun K, Takayama K, Sasano H, Suzuki T, Moghrabi N, Andersson S, Johns A, Meng L,
Putman M, Carr B & Bulun SE (1998) Deficient 17β-hydroxysteroid dehydrogenase type 2
expression in endometriosis: failure to metabolize 17β-estradiol. J Clin Endocrinol Metab 83:
4474-4480.