Download Identification of Progesterone Receptor in Human Subcutaneous Adipose Tissue

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Journal of Clinical Endocrinology and Metabolism
Copyright © 1998 by The Endocrine Society
Vol. 83, No. 2
Printed in U.S.A.
Identification of Progesterone Receptor in Human
Subcutaneous Adipose Tissue
SUSAN N. O’BRIEN, BRENDA H. WELTER, KIMBERLY A. MANTZKE,
THOMAS M. PRICE
AND
Greenville Hospital System/Clemson University Biomedical Cooperative Research Program, Clemson
University, Clemson, South Carolina 29634
tissue of premenopausal women. These preliminary studies revealed
that PR messenger RNA levels are higher in the stromal-vascular
fraction as opposed to the adipocyte fraction. Western blot analysis
demonstrates both PR protein isoforms (human PR-A and human
PR-B) in human subcutaneous adipose tissue. Using an enzymelinked immunosorbent assay, total PR could be quantitated. These
studies substantiate that sex steroid receptors are present in human
adipose tissue, thereby providing a direct route for regulation of adipose tissue by female sex steroids. (J Clin Endocrinol Metab 83:
509 –513, 1998)
S
Subjects and Methods
EX steroids are postulated to influence adipose tissue
regulation and distribution, because changes in fat
distribution coincide with onset of ovarian production of
estrogen and progesterone during puberty and with cessation of hormone production during menopause (1).
Women typically demonstrate a gynoid or lower body fat
distribution, whereas women with excessive androgens
and glucocorticoids manifest an android or upper body fat
distribution (2). Android adipose tissue distribution in
women has been associated with an increased risk of diseases, including coronary artery disease (3), adult-onset
diabetes mellitus (4), and endometrial cancer (5). Although
clinical observations suggest that sex steroids influence
adipose distribution, controversy exists as to whether sex
steroids mediate these effects directly or indirectly in human adipose tissue. The dispute has been fueled by technical difficulty in detecting sex steroid hormone receptors
in human adipose tissue (6, 7).
Estrogen receptor (ER) messenger RNA (mRNA) and protein have been identified in human subcutaneous adipose
tissue (8, 9). We have reported the presence of higher ER
mRNA levels in the adipocyte fraction of adipose tissue
compared with ER mRNA levels in the stromal-vascular
portion (8). In this study, we demonstrated the presence of
progesterone receptor (PR) mRNA and protein in subcutaneous adipose tissue of premenopausal women and
examined the distribution of PR message in the two cell
fractions.
Subjects
Subcutaneous adipose tissue was obtained from five premenopausal
women undergoing elective adominoplasty with an average age of 43 6
4 yr and mean body mass index of 26.8 6 5.5 kg/m2. The included
subjects were not taking exogenous hormones and were free of neoplastic, inflammatory, or autoimmune disease. All subjects signed an
Institutional Review Committee-approved consent form (Greenville
Hospital System) before surgery for use of tissue in research.
Tissue acquisition and processing
A portion of tissue was frozen immediately in liquid nitrogen for
subsequent Western blot analyses and enzyme-linked immunosorbent
assay (ELISA). Fresh adipose tissue was processed into adipocyte and
stromal-vascular fractions as previously described (8). Both fractions
were frozen in liquid nitrogen for Northern blot analyses.
RNA isolation and Northern blot analysis
Total RNA was isolated from whole abdominal adipose and myometrium using an RNeasy kit (Qiagen, Santa Clarita, CA) according to
the manufacturer’s directions. Five micrograms of RNA was electrophoresed on a 1.2% denaturing agarose gel. The integrity of RNA was
assessed by ethidium bromide staining of the gel. RNA was transferred
to nylon by capillary blotting and fixed by ultraviolet cross-linking and
vacuum baking. The filter was hybridized overnight at 65 C using a 32P
random-prime labeled complementary DNA (cDNA) for human PR (10)
added to prehybridization solution (7% SDS, 0.25 m sodium phosphate
buffer, pH 7.2). Northern blots were washed at room temperature with
23 SSC and 0.1% SDS for 15 min followed by 23 SSC and 0.1% SDS for
15 min at 65 C and then exposed to film.
For comparison of mRNA from the stromal-vascular and adipocyte
cell fractions, total RNA was isolated as described above. Equal amounts
of RNA from each fraction were denatured and applied to a nylon
membrane housed in a slot-blot manifold. RNA was fixed and hybridized as described above. The autoradiograph was quantitated by scanning two-dimensional densitometry. The filter was then stripped and
rehybridized with a 1.076-kilobase (kb) fragment of the mouse cytoplasmic b-actin gene (Ambion, Austin, TX). Following densitometry, PR
values were normalized with b-actin values. The difference between the
Received June 2, 1997. Revision received October 17, 1997. Accepted
October 28, 1997.
Address all correspondence and requests for reprints to: Susan N.
O’Brien, Greenville Hospital System/Clemson University Biomedical
Cooperative, Clemson University, 124 Long Hall, Clemson, South Carolina 29634-5112.
509
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ABSTRACT
Sex steroids are postulated to play a role in adipose tissue regulation and distribution, because the amount and location of adipose
tissue changes during puberty and menopause. Because of the nature
of adipose tissue, receptors for the female sex steroids have been
difficult to demonstrate. To date, estrogen receptor messenger RNA
and protein have been identified in human subcutaneous adipose
tissue, but the presence of progesterone receptor (PR) has not been
reported. In this study, we demonstrate PR message by Northern blot
analysis in RNA isolated from the abdominal subcutaneous adipose
510
O’BRIEN ET AL.
JCE & M • 1998
Vol 83 • No 2
ratio of PR to b-actin densitometry values for adipocytes compared with
stromal-vascular cells in the five subjects were compared with a paired
sample t test.
Ammonium sulfate precipitation and Western blot analysis
FIG. 1. Western blot analysis for hPR protein after ammonium sulfate precipitation. In myometrium (lane 1, 40 mg total protein), hPR-B
isoform was identified at 116 kDa, whereas hPR-A isoform was primarily seen at 83 kDa, with a less intense band at 92 kDa. In adipose
tissue (lane 2, 2 mg total protein), hPR-B isoform was identified at 116
kDa, whereas hPR-A isoform was primarily seen at 92 kDa. Control
lane (lane 3, 2 mg adipose total protein) showed no specific bands
when rabbit serum was used in place of primary antibody. A control
lane with myometrial total protein also showing no specific binding
is not shown.
ELISA
Frozen tissue was prepared in the same manner as for Western
analyses. Protein was quantitated using Bradford analysis (BioRad). A
96-well plate was coated with a mixture of PR antibodies from NeoMarkers, PR antibodies 3, 4, 5, and 7 (Labvision, Freemont, CA) diluted
to 1.5 mg/mL in 0.1 m sodium carbonate buffer (pH 9.3) at 100 mL/well.
All subsequent volumes were 100 mL. The plate was incubated overnight
at 4 C and then washed with PBS with 0.1% BSA and 0.05% Tween-20
(PBS-BSA-T) and blocked with 3% BSA in PBS. PR standard (Hormone
Receptor Laboratory, Louisville, KY) at concentrations of 0, 3.75, 7.5, and
15 fmol/mg protein or 1 mg sample diluted with TEMMG were added
to quadruplicate wells and incubated overnight at 4 C. After washing,
a rabbit polyclonal PR antibody, C19, diluted to 0.5 mg/mL in PBS-BSA-T
was added to triplicate wells. For a blank, one well included only
PBS-BSA-T, without the primary C19 antibody. The plate was incubated
overnight at 4 C. Biotinylated goat antirabbit IgG (Kirkegaard & Perry,
Gaithersburg, MD) diluted 1:5000 in PBS-BSA-T was added to each well
and incubated 1 h at room temperature. ImmunoPure Ultra-Sensitive
ABC (Pierce, Rockford, IL) reagents were prepared according to the
manufacturer’s directions, added to each well, and incubated 30 min at
room temperature. Color developer (Horseradish Peroxidase Substrate
Kit, BioRad) was added, and color development was measured after 1 h
at 405 nm. Each sample was assayed in triplicate in three separate assays.
The intraassay coefficient of variation (COV), calculated from values for
the 7.5 fmol/mg protein standard was 4.5%. The interassay COV (11%)
was calculated from the same standard values.
Results
Figure 1 shows the PR protein by Western blot analysis
after ammonium sulfate precipitation in myometrium and
abdominal adipose tissue. The human PR-B (hPR-B) isoform
appeared at approximately 116 kilodaltons (kDa) and was
identical in size to that found in the myometrium. The hPR-A
isoform was located at approximately 92 kDa in adipose
tissue, which was slightly greater than the predominant
hPR-A band found at 83 kDa in the myometrium. A less
intense third band in the myometrium was seen at 92 kDa
FIG. 2. Northern blot analysis of hPR mRNA in adipose tissue (adip)
and myometrium (uterus). Five micrograms total RNA was separated
by denaturing gel electrophoresis, applied to nylon, and probed with
a randomly primed labeled hPR cDNA. Bands of identical sizes were
predominantly seen at 11.4, 6.1, 4.5, and 2.9 kb.
corresponding to the hPR-A of the adipose tissue. Bands of
these sizes were not detected in negative controls.
In Fig. 2, Northern blot analysis of total RNA isolated from
abdominal adipose tissue demonstrating the same size transcripts as seen in RNA isolated from myometrium is shown.
The most prominent bands corresponded to sizes of 11.4, 6.1,
4.5, and 2.9 kb.
Figure 3 shows the levels of PR protein in abdominal
adipose tissue from five subjects and the level in myometrium from one subject as determined by ELISA. PR protein
levels in adipose tissue varied from approximately 3– 8
fmol/mg total protein and were approximately 100-fold less
than those found in myometrium. The insert shows a typical
standard curve used for the PR ELISA.
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Frozen abdominal adipose tissue was ground with mortar and pestle
in liquid nitrogen, added to TEMMG buffer (10 mm Tris-HCl, 1.5 mm
EDTA, 12 mm monothioglycerol, 10 mm sodium molybdate, 10% glycerol) with protease inhibitors (10 mg/mL leupeptin, 1 mm phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin, 1 mg/mL pepstatin), and homogenized on ice. As a positive control, uterine tissue was prepared in
the same manner as adipose tissue. Following centrifugation for 20 min
at 16,000 3 g, the layer between the floating lipid and pellet was removed. Ammonium sulfate was added to an aliquot of the cytosol to 30%
saturation and rotated overnight at 4 C. The mixture was centrifuged at
3000 3 g for 30 min. Precipitates were resuspended in TEMMG plus SDS
sample buffer and boiled for 2 min before loading.
Samples were electrophoresed on a 7.5% polyacrylamide gel and
transferred to polyvinylidene difluoride (Bio-Rad Labs., Hercules, CA).
The membrane was incubated with rabbit antihuman PR (C19, Santa
Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C. As a second
control, duplicate lanes were incubated with rabbit sera in place of the
primary antibody. The membrane was washed and incubated with
secondary antibody, peroxidase-conjugated goat antirabbit IgG (Cappel,
ICN Pharmaceuticals, Costa Mesa, CA). Proteins were detected using an
Enhanced Chemi Luminescense Western blotting detection system (Amersham, Buckinghamshire, England) according to the manufacturer’s
directions.
PR IN HUMAN ADIPOSE TISSUE
511
Figure 4 shows PR mRNA levels in adipocytes compared
with stromal-vascular cells from abdominal adipose tissue.
Northern blot analysis (Fig. 4A) for PR, performed with total
RNA after denaturing gel electrophoresis, demonstrated
greater PR mRNA levels in adipose stromal cells compared
with adipocytes. Slot-blot analysis (Fig. 4B) was performed
to semiquantitate the difference in PR mRNA levels between
the two cell fractions in five subjects. Northern blot analysis
of b-actin was used to control for differences in loading of
total RNA. Densitometry values for the slots were expressed
as the ratio of the value for the PR and the corresponding
value for b-actin. Stromal-vascular cells contained approximately 2.5-fold greater levels of PR mRNA compared with
adipocytes.
Discussion
A substantial amount of clinical evidence indicates that sex
steroids may play a role in fat tissue regulation and distribution. Prepubertal boys and girls do not differ significantly
in adipose tissue percentage or distribution. However, with
puberty, females experience an increase in fat percentage as
well as selective adipose tissue deposition in the breast and
gluteo-femoral regions. This gynoid fat distribution commonly exists until menopause in normal nonobese women.
Women with disorders of androgen excess often display an
upper body or android type of fat distribution characterized
by abdominal adipose deposition and a higher waist-to-hip
ratio (2). During menopause, at which time ovarian production of estrogen and progesterone ceases, the female phenotype becomes more android (11, 12). Because upper body fat
distribution is strongly correlated with pathophysiology
(13), the understanding of sex steroid regulation of adipose
tissue distribution may be important in ultimately preventing this process and decreasing disease risk.
FIG. 4. A, Northern blot analysis comparison of PR mRNA levels in
adipocytes vs. stromal-vascular cells. Five micrograms total RNA was
separated by denaturing gel electrophoresis, applied to nylon, and
probed with a randomly primed labeled hPR cDNA. Insert, 28S and
18S ribosomal RNA bands of ethidium bromide gel. B, Slot-blot analysis was used to semiquantitate PR mRNA levels in adipocytes (Adip)
vs. stromal-vascular (SV) cells of samples from five subjects. Five
micrograms total RNA was applied to nylon and probed with a randomly primed labeled hPR cDNA. The membrane was then stripped
and reprobed with a labeled b-actin cDNA. Slot-blots were quantitated with laser densitometry, and results expressed as ratio of PR to
b-actin. For each subject, samples were run in triplicate, with SD bars
showing variance. A paired t test for all patients showed a significant
difference between SV and Adip, (P # 0.001). A representative slotblot is shown.
Detection of sex steroid receptors has been technically
difficult in human adipose tissue because of the inherent
properties of the tissue. In past studies, ERs could not be
demonstrated with routine binding assays because of the
high nonspecific binding of estrogens stored in adipose tissue (14). With the development of more sensitive assays, ER
mRNA (by PCR and Northern blot analysis) and ER protein
(8, 9) have been demonstrated in human adipose tissue. To
date, there have been no reports of the presence of PR mRNA
or protein (7, 15) in human adipose tissue, although specific
progestin binding has been demonstrated in the adipose
tissue of rat (16) and sheep (17). In addition to traditional
target tissues, PR has been identified in such nonclassical
human tissues as osteoclasts (18), the peripheral veins (19),
and prostate stromal cells (20).
In this study, we were able to demonstrate PR mRNA by
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FIG. 3. Quantitation of PR protein levels by ELISA in adipose tissue
of five subjects and from one myometrium sample. SD bars represent
variation for each subject within three separate assays. Insert, A
typical standard curve using PR standards as described in Subjects
and Methods.
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JCE & M • 1998
Vol 83 • No 2
O’BRIEN ET AL.
to investigate the target genes for these sex steroids in this
tissue. Ultimately, these studies will help clarify the role of
sex steroids in the regulation and distribution of adipose
tissue.
Acknowledgments
We thank Dr. Geoffrey Greene for his kind gift of human PR cDNA.
We gratefully acknowledge the technical assistance of Stephanie Weathers (Greenville Hospital System/Clemson University Biomedical Cooperative) and Dawn Blackhurst (Greenville Hospital System, Department of Medical Research).
References
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Northern blot analysis and PR protein by Western blot analysis in the abdominal subcutaneous adipose tissue of premenopausal women. Discrete transcripts of sizes identical to
those present in uterine tissue can be detected in Northern
blot analyses of total RNA isolated from human adipose
tissue, suggesting that regulation of transcription for PR is as
complicated in adipose tissue as in reproductive tissues (21).
These multiple transcripts are obtained from two promoters,
yielding hPR-A- and hPR-B- specific transcripts (22, 23), the
identities of which have not been determined.
The physiological response to progesterone is conveyed by
two discrete forms of the PR (hPR-A and hPR-B) in target
tissues (24). The B isoform is a 933-amino acid protein that
acts primarily as an activator of transcription. hPR-A (768
amino acids) is an N-terminally truncated form of the larger
B protein lacking the first 164 amino acids and functions as
a repressor of hPR-B activity (25). In addition, hPR-A can
repress transcriptional activation of glucocorticoid, mineralocorticoid, and ERs, suggesting that PR may play a pivotal
role in the regulation of other steroid hormones (26, 27).
Both the A and B isoforms of the PR appear to be present
in human adipose tissue. The B isoform was of similar size
to that of uterine tissue at approximately 116 kDa. The A
isoform in adipose tissue was slightly greater in size (92 kDa)
than the predominant A isoform in uterine tissue used in this
study as a positive control (83 kDa). This may be because of
differences in the degree of phosphorylation of the protein,
which has been described in other tissues (28). The ratio of
the two PR isoforms has been shown to vary in chick oviduct
tissue according to estrogen status (29). Whether the ratio of
the isoforms will vary in adipose tissue with changes in
endogenous estrogen levels remains to be investigated.
Further analysis of the PR involved quantitation of the
protein levels by a multiple antibody ELISA. The values of
the PR in human abdominal adipose tissue of premenopausal
women are similar to those reported by Watson et al. (14) in
sheep and are much lower than the uterine control, as
expected.
Differences in the cellular content of PR in human adipose
tissue are also evident. In this study, we demonstrated higher
levels of PR mRNA in the stromal-vascular cells compared
with adipocytes. Previous studies have shown ER mRNA
levels to be higher in adipocytes compared with stromalvascular cells (8). In addition, cytochrome P450 aromatase,
the enzyme responsible for conversion of C19 steroids into
C18 estrogens, has higher activity and mRNA levels in stromal-vascular cells compared with adipocytes (8, 30). These
observations suggest a differential type of regulation by sex
steroids of the cells in human adipose tissue. Estrogen of
ovarian origin or estrogen produced by aromatization in
adipose stromal cells may transcriptionally regulate adipocytes. In contrast, progesterone of ovarian origin may primarily regulate adipose stromal cells. This selective distribution of ER and PR levels in different cells of a given tissue
has also been reported with endometrium of the rabbit uterus
(31), where PR levels are higher in endometrial stromal cells
compared with glandular cells, but ER levels are higher in
glandular cells.
Now that the presence of the ER and PR in human adipose
tissue has been established, further studies will be possible
PR IN HUMAN ADIPOSE TISSUE
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forms is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol. 7:1244 –1255.
26. McDonnell DP, Shahbbaz MM, Vegeto E, Goldman ME. 1994 The human
progesterone receptor A-form functions as a transcriptional modulator of
mineralocorticoid receptor transcriptional activity. J Steroid Biochem Mol Biol.
48:425– 432.
27. McDonnell DP, Goldman ME. 1994 RU486 exerts antiestrogenic activities
through a novel progesterone receptor A form-mediated mechanism. J Biol
Chem. 269:11945–11949.
28. Clarke CL, Feil PD, Satyaswaroop PG. 1987 Progesterone regulation by 17b-
513
estradiol in human endometrial carcinoma grown in nude mice. Endocrinology. 121:1642–1648.
29. Syvala H, Vienonen A, Ylikomi T, et al. 1997 Expression of the chicken
progesterone receptor forms A and B is differentially regulated by estrogen in
vivo. Biochem Biophys Res Commun. 231:573–576.
30. Cleland WH, Mendelson CR, Simpson ER. 1983 Aromatase activity of membrane fractions of human adipose tissue, stromal cells, and adipocytes. J Endocrinol. 113:2155–2157.
31. Zaino RJ, Clarke CL, Feil PD, Satyaswaroop PG. 1989 Differential distribution
of estrogen and progesterone receptors in rabbit uterus detected by dual
immunofluorescence. Endocrinology. 125:2728 –2734.
The National Institute on Aging announces the annual Summer Institute on Aging Research, a week-long
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