Download Molecular Involvement of the Pit-2 Gene in Anterior Pituitary Cell

Molecular Involvement of the Pit-2 Gene in
Anterior Pituitary Cell Commitment'
Simon J. Rhodes* and Michael G. Rosenfeld?
*Department of Biology, Indiana University-Purdue University
a t Indianapolis, Indianapolis, IN 46202-5132
and +Department of Medicine and Howard Hughes Medical Institute,
University of California San Diego, La Jolla, CA 92093-0648
ABSTRACT
growth hormone and are dwarfed. Two distinct
populations of thyrotrope cells arise during pituitary
development: a Pit-l-independent group of thyrotropes
is found transiently at the rostral tip of the gland
during early embryogenesis, whereas a second population in the caudomedial region is detectable following
activation of the pzt-l gene and persists into the
mature animal. The pit-1 gene is subject to autoregulation by its own product, and regulatory regions of
the gene contain response elements conferring activation by retinoic acid, vitamin D3 derivatives, cyclic
AMP signaling pathways, and pituitary-restricted
factors. The p i t - l gene retinoic acid response element
is entirely dependent on Pit-1 for function: this
synergistic interaction suggests a general mechanism
by which signaling molecules might achieve cellspecific effects.
Hormones secreted by the mammalian pituitary gland regulate growth, lactation,
sexual function, and homeostasis. The anterior
pituitary-specific transcription factor Pit-1 directly
activates the growth hormone, prolactin, and thyroidstimulating hormone beta genes. Dwarf animals with
defective p i t - l genes lack the thyrotrope, somatotrope,
and lactotrope cell types that express these three
genes. Similar pituitary dysfunction and short stature
are also observed in humans with mutations a t the
p i t - l locus. Pit-1 therefore is required for both the
transcriptional activation of pituitary target genes and
for survival of these three cell lineages. Pit-l-defective
animals do not express the receptor for growth
hormone- releasing factor, a key regulator of somatotrope proliferation. Mice harboring defects in the
receptor gene express significantly reduced levels of
Key Words: Growth Hormone, Prolactin, Thyroid-stimulating Hormone,
Somatotrope, Lactotrope, Thyrotrope
J. h i m . Sci. 1996. 74(Suppl. 2):94-106
Introduction
each cell type (Figure 1 ) . The five cell types ( a n d
their respective hormone products) are corticotropes
(producing adrenocorticotropin [ACTH] by proteolytic
processing of the product of the proopiomelanocortin
[POMC] gene); gonadotropes (follicle-stimulating
hormone FSH] and luteinizing hormone [LHI ); thyrotropes (thyroid-stimulating hormone [TSHI1; somatotropes (growth hormone [GHl ); and lactotropes
(prolactin [PRL]) , Follicle-stimulating hormone, LH,
and TSH are polypeptide heterodimers consisting of a
common subunit, alpha glycoprotein ( arGSU), and a
distinct 0 subunit ( FSW, LH@,TSH@).The development of the five cell types in a precise temporal order
from a common origin (Figure 1) and the physiological importance of the pituitary gland make the
anterior pituitary a n attractive system with which t o
analyze the mechanisms that regulate the commitment and subsequent differentiation of specific phenotypes during mammalian organogenesis.
The anterior pituitary arises from Rathke's Pouch,
an outfolding of embryonic ectoderm first observed a t
about embryonic d 9 of mouse development, and the
mature gland secretes polypeptide trophic hormones
that regulate growth, adrenal function, sexual activity, thyroid gland function, and lactation (Schwind,
1928; Dubois and Hemming, 1991; Voss and Rosenfeld, 1992; Andersen and Rosenfeld, 1994; Rhodes e t
al., 1994). The hormones are released from five
distinct cell types and serve as specific markers for
'We are grateful to Susan Martin and Kathleen Scully for their
assistance. SJR is a Special Fellow of the Leukemia Society of
America. MGR is an Investigator with the Howard Hughes Medical
Research Institute. Studies performed in the Rosenfeld laboratory
were supported by grants from the NIH to MGR and the Human
Growth Foundation to SJR.
94
95
PIT-1 A N D ANTERIOR PITUITARY DEVELOPMENT
e9
e12.
Rathke's
pouch
Corticotrope
Rostral
Pit- 1
detected -.)
e15.
absent in Snell dwarf mice
1
Caudomedial
Thyrotrope
somatotrope
Birth
AC
H
FSH
LH
TSH
GH
PRL
Figure 1. Commitment and differentiation of the five anterior pituitary hormone-secreting cell types from Rathke's
pouch. The approximate times (e = mouse embryonic day) at which markers of individual cell types are first
observed are shown on the left. Transcription factors and the products of trophic peptide hormone genes that
distinguish each cell lineage are shown. A transient population of thyrotrope cells is detected in the rostral tip of the
gland during embryogenesis. A second population that is dependent on Pit-1 and absent in the Snell dwarf appears
later in the caudomedial portion of the gland and is maintained into adulthood. NEct = neuroectoderm, Ect =
ectoderm.
Identification and Cloning of Pit-1
Analyses of the GH and PRL gene promoters
indicated that a common factor bound t o and activated
these genes (Nelson et al., 1986, 1988; Bodner and
Karin, 1987; Cao et al., 1987; Gutierrez-Hartmann et
al., 1987; Lufkin and Bancroft, 1987; Ye and Samuels,
1987). The subsequent cloning of this factor (known
as Pit-1/GHF-1) revealed that the protein was a novel
homeodomain-containing transcription factor (Bodner
et al., 1988; Ingraham et al., 1988). Pit-1 was
demonstrated to trans-activate both GH and PRL
genes and to bind to multiple elements in the
regulatory regions of these genes (Ingraham et al.,
1988, 1990a; Mangalam et al., 1989; Elsholtz et al.,
1990). Pit-1 has subsequently been shown to activate
other anterior pituitary genes, including the pit-1 gene
(Chen et al., 1990; McCormick et al., 1990; Rhodes et
al., 19931, the growth hormone-releasing factor
(GRF)receptor gene (Lin, et al., 1992, 19931, the
TSHO gene (Steinfelder et al., 1991, 1992a,b; Haugen
et al., 1993; Gordon et al., 1993; Mason et al., 1993;
96
RHODES AND ROSENFELD
Lin et al., 19941, and the thyroid hormone receptor
beta type 2 pituitary promoter (Wood et al., 1994).
Pit-l-related factors have also been implicated in
regulation of the renin gene ( S u n e t al., 1993, 1994;
Borensztein et al., 1994; Catanzaro et al., 1994;
Gilbert et al., 1994). Pit-1 recognizes an A/"-rich DNA
sequence related to the octamer motif bound by Oct-1
and Oct-2 (Elsholtz e t al., 1990). Figure 2 shows a n
alignment of Pit-1 binding sites giving a consensus
sequence with a core structure of APT T/A T/A
TATNCAT. The regulatory regions recognized by Pit-1
within the PRL, GH, and pzt-l genes have been
demonstrated t o be required for appropriate, pituitaryspecific expression of these genes in both cultured cell
lines and transgenic animals (Lira et al., 1988, 1993;
Crenshaw et al., 1989). In addition, the widelyseparated proximal and distal regulatory regions of
the rat PRL gene, which both contain multiple binding
sites for Pit-1, are juxtaposed in chromatin (Cullen et
al., 1993). Phosphorylation of certain Pit-1 amino acid
residues can modulate binding to specific target
sequences (Kapiloff et al., 1991), but the basal
transactivation function of Pit-1 may be independent
of the phosphorylation status of the protein a t these
residues (Fischberg et al., 1994; Howard et al., 1994;
Okimura et al., 1994).
Pit-1 Protein Structure a n d
Alternate Splice Variants
Pit-1 is a member of the POU domain family of
developmental regulatory proteins. The POU domain
class of proteins was named for its original members:
Pit-1, Oct-l/2 and the C. elegans factor unc-86. Each of
these proteins contains a structurally related DNA
binding domain, the POU domain (Herr et al., 1988).
The POU domain is a bipartite DNA-binding structure
consisting of the POU-specific domain ( POUS) joined
by a linker region to a homeodomain, the POU
homeodomain ( POUm) (Figure 3). POU domain
factors play important roles in the regulation of
developmental decisions in many organs and tissues
and are found in a wide range of organisms (Rosenfeld, 1991; Ruvkun and Finney, 1991; Scholer, 1991,
Wegner et al., 1993). Pit-1 and Oct-1 can interact and
form heterodimers in activation of Pit-1 target genes
(Voss et al., 1991a). Both the POUs and POUHD
domains of Pit-1 are required for high affinity binding
of Pit-1 dimers t o DNA sites (Ingraham et al., 1990b).
The amino terminus of the Pit-1 protein contains the
major transferable trans-activation domain of the
molecule (Ingraham et al., 1990b), but the POUs
domain also seems to contribute to Pit-1 transactivation function (Voss et al., 1993).
Three-dimensional nuclear magnetic resonance and
x-ray crystallographic studies have revealed that the
Oct-1 POU-specific domain adopts a structure similar
to those of the well-characterized helix-turn-helix
domains of the lambda and 434 repressor molecules
and that the POU homeodomain has a tertiary
structure related to the Drosophila Engrailed and
Antennapedia homeodomain proteins (Assa-Munt et
al., 1993; Dekker e t al., 1993; Klemm et al., 1993).
These structures will be useful in modeling interactions of Pit-1 with its binding site until the structure
of a Pit-UDNA complex is determined. Using other
approaches, distinct classes of POU domain proteins
have been found to recognize DNA binding motifs of
specific spacing and orientation and t o adopt different
conformations when bound to DNA ( L i et al., 1993).
The POU-specific sub-domains of Pit-1 and Oct-1 seem
to adopt an apparent opposite orientation on DNA
response elements, with respect to their POU homeodomains, to that noted for the brain POU factors Brn2 and Brn-3.0.
The primary amino acid sequence of Pit-1 has been
well conserved throughout evolution and Pit-1 cDNAs
have been sequenced from mice, rats, humans, turkeys, cattle, swine, and fish (Bodner et al., 1988;
Ingraham et al., 1988; Lew and Elshotz, 1991;
Elsholtz et al., 1992; Ohta et al., 1992b; Ono and
Takayama, 1992; Tatsumi et al., 199213; Wong e t al.,
1992; Pernasetti et al., 1993; Tuggle et al., 1993;
Yamada et al., 1993; Yu et al., 1994). Pit-1 function is
apparently maintained within endocrine systems
producing GH, TSH, and PRL, and Pit-1 may also play
a role in the expression of somatolactin, a fish
hormone of unknown function (Ono et al., 1994).
Alternative translation initiation site usage results in
two forms of Pit-1 of 31 and 33 kDa (Voss et al.,
1991b).
Alternative splice forms of Pit-1, expressed a t low
levels in pituitary cells, have been reported (Figure
3). One variant, named Pit-l@/GHF-2/Pit-la,contains
a n insertion of 26 amino acids in the amino terminal
trans-activation domain of the molecule (Konzak and
Moore, 1992; Morris et al., 1992; Theill et al., 1992;
Delhase et al., 1995). The Pit-lP isoform is only
moderately active in trans-activation of the GH
promoter and is strongly impaired in induction of the
PRL and p i t - l gene promoters. A second Pit-1 splice
product (Pit-1T) results from the insertion of 14
amino acids a t the same position as the Pit-10
insertion. Pit-1T is restricted to cells of the thyrotrope
lineage and increases transcription of the TSHP
promoter, but not the GH and PRL promoters, when
co-transfected with wild-type Pit-1 (Haugen et al.,
1993, 1994). The third class of Pit-1 isoform results
from a splice choice that excludes exon 4 information,
removing most of the POUs domain (Voss et al., 1993;
Day and Day, 1994). This molecule (A4Pit-1)
provided a natural mutagenesis with which to examine the role of the POUs domain in Pit-1 function
(Voss e t al., 1993). DNA binding, site selection, and
trans-activation studies revealed that A4Pit-1 is a
transcriptionally inert protein with a limited range of
high-affinity binding sites. It was concluded that the
PIT-1 A N D ANTERIOR PITUITARY DEVELOPMENT
rPrl-1P
rPrl-2P
rPrl-3P
rPrl-4P
rPrl-1D
rPrl-2D
r P r 1- 3D
rPrl-4D
rGHla
rGHlb
rGH2
rPitBl
rPitB2
mP i t - 1a
mPit-lb
mPit-lc
mP i t - I d
mPit-le
mTSHl
mTSH2
hTSH3
mTB2-1
mTB2-2
mTB2-3
SSLl
sSL2
sSL3
sSL4
sSL5a
sSL5b
97
Nelson et al., 1989
Chen et al., 1991
Rhodes et al., 1993
Lin et al., 1993
Steinfelder et al., 1992
Wood et al., 1994
Yamada et al., 1993
tGHl
tGH2
tGH3
tGH4a
tGH4b
tGH4c
/
Consensus: N N
a t
Pit-1 domains:
\
AT AT A1 aT A T Nt C A T N N N Na
POU-homeodomain
domain
P.
a,
POU Domain
Figure 2. Alignment of established and predicted Pit-1 binding sites. Pit-1 recognition motifs from the prolactin
(Prl), growth hormone (GH), pit-1 (Pit), thyroid-stimulating hormone beta (TSH), thyroid hormone receptor beta 2
(TB2), and somatolactin (SL) genes. r = rat, m = mouse, h = human, s = salmon, t = rainbow trout. A consensus
sequence is given: uppercase letters represent strong preferences at a position; lowercase letters represent a weaker
preference. N = any nucleotide. The regions of the DNA binding site recognized by the subdomains of the Pit-1 POU
domain are indicated.
98
RHODES AND ROSENFELD
A. Pit-1 Gene
r v v
m
I
VI
VII
3'
5'
FJit-1p
A4F'it- 1
Pit-1T
Pit-la
B. Pit-1 proteins
A147-202
A4Pit-1
1
5
236
+14aa
Pit-1
Pit-1T
Pit-lb 1
1
1
305
\
+26aa
317
291
I
I
POU-specificdomain
Major transactivation
domain
8
1
POU-homeodomain
I
I
POU Domain
(DNA bindin&/transactivation)
Figure 3. The mouse/rat pit-1 gene encodes the major Pit-1 protein isoform (Pit-la) and three splice variants
(A4Pit-1, Pit-1T. Pit-lo). (A) Organization of the pit-1 gene. White boxes indicate noncoding exons, black boxes
indicate coding exons, and shaded/hatched boxes indicate extensions of exon 2 resulting from alternative splicing.
The various splice choices that result in alternate forms of the Pit-1 protein are indicated. Exons are given in roman
numerals. (B) Structure of Pit-1 protein isoforms. The amino terminus trans-activation domain and the POU-specific
and POU-homeo subdomains of the DNA-binding POU domain are denoted. aa = amino acid.
POUs domain is a modular DNA-binding structure
and a non-modular contributor to the trans-activation
function of Pit-1. In a second study, A4Pit-1 was
identified in transplanted pituitary cells and was
proposed to serve a dominant negative function in
regulation of the PRL gene (Day and Day, 1994). The
physiological importance of the Pit- 1 alternate splice
forms is not clear due to their low levels of expression
and weak trans-activation capabilities. Should they be
demonstrated to exhibit patterns of expression during
pituitary development or activities that are distinct
from the major Pit-1 isoform then their importance to
anterior pituitary function will be better established.
Pit-1 is Required for the Thyrotrope,
Somatotrope, and Lactotrope Lineages
The pit-1 gene is expressed in a pituitary-specific
fashion: Pit-1 transcripts are first detected in the
developing pituitary on mouse embryonic d 14.5, after
the first detection of TSHP cells but prior to the
activation of the GH and PRL genes in somatotrope
and lactotrope cells (Doll6 et al., 1990; Simmons et al.,
1990). Pit-1 protein is restricted to the thyrotrope,
somatotrope, and lactotrope cell types. Expression of
Pit-1 in non-pituitary tissues, such as the placenta,
has been reported (Bamberger et al., 19951, but the
importance of very low levels of Pit-1 in these tissues
has yet to be established.
The h e l l ( d w ) and Jackson ( d w J ) strains of dwarf
mice carry heritable recessive defects that segregate
with chromosome 16 and phenotypically lack the
thyrotrope, somatotrope, and lactotrope each cell types
and therefore their hormone products (Snell, 1929;
Table 1). The mapping of the p i t - l gene t o mouse
chromosome 16 and the subsequent demonstration
that the pit-1 genes of these mice are defective
confirmed the importance of Pit-1 in both pituitary
target gene activation and the proliferation and
maintenance of pituitary cell phenotypes (Li et al.,
1990). The Snell mouse pit-1 gene contains a single
point mutation that alters a conserved amino acid
residue in the POU homeodomain of the POU DNA
binding domain, whereas the Jackson pit-1 gene is
rearranged. A further strain of dwarf mouse, the Ames
dwarf ( d f ) , is phenotypically similar t o the Jackson
and Snell mice and also displays a n absence of the
99
PIT-1 AND ANTERIOR PITUITARY DEVELOPMENT
Table 1. Defects in anterior pituitary-specific genes underlying dwarf phenotypes. The characteristics
of four strains of dwarf mice exhibiting pituitary dysfunction are given
Dwarf mouse
Snell
Jackson
Ames
Little
Symbol
Chromosome
Phenotype
Affected gene
dw
dw
df
lit
16
16
11
6
TSHP, GH, PRL absent
TSHP, GH, PRL absent
TSHP, GH, PRL absent
GH, PRL reduced
pLt-l
pit-1
three Pit-l-dependent pituitary cell types (Gage et al.,
1995 and references therein; Table 1). The Ames
allele is, however, located on chromosome 11 and
mutations of Pit-1 are therefore not responsible for
this phenotype. Determination of the nature of the
Ames defect will be important to our understanding of
pituitary cell development and the regulation of Pit-1
function.
Defects in the human p i t - l gene also lead to
pituitary dysfunction (reviewed by Parks et al., 1993;
Table 2). A mutation of the POUs domain that has
only moderate effects on DNA binding, but that
impairs the trans-activation abilities of Pit-1, was
reported in patients with multiple pituitary hormone
deficiency (Pfaffle et al., 1992). A different mutation
in the POU homeodomain of Pit-1 in patients with
combined pituitary hormone deficiency may act as a
dominant negative; patients with one affected allele
ofien display a diseased phenotype (Radovick et al.,
1992; Okamoto et al., 1994; Cohen et al., 1995). Other
pit-1 mutations affecting different regions of the
molecule or causing premature termination of Pit-1
translation have been reported in patients with
pituitary hormone deficiency and its associated symptoms (Ohta et al., 1992a; Tatsumi et al., 1992a). Pit1 transcripts have been detected a t high levels in
pituitary adenomas in both humans and rodents (e.g.,
Delhase et al., 1993; Lloyd et al., 1993; Sanno et al.,
1994, 1995, and references therein), but the role of
Pit-1 in proliferation of cells within these adenomas is
as yet unclear.
?
GRF receptor
the appearance of the thyrotrope lineage (Simmons et
al., 1990). A detailed analysis of TSHP gene expression during pituitary development, however, in both
wild type and Pit-l-defective Snell mice revealed that
there are two spatially and temporally distinct populations of thyrotrope cells (Figure 1; Lin et al., 1994).
The first population is found in the rostral tip of the
developing pituitary and is independent of Pit-1
activity. The rostral thyrotropes are first detected in
both normal and Pit-l-defective animals on about
embryonic d 12.5 and are present for only a period of
several days, disappearing at around the time of birth.
The second thyrotrope population is detected in the
central caudomedial region of the gland. These cells
co-localize with Pit-l-positive cells and appear at the
time of pzt-1 gene activation. The caudomedial thyrotropes are absent in the Pit-l-defective Snell mouse
pituitary, demonstrating their dependence on Pit- 1.
The caudomedial thyrotropes are maintained following embryogenesis, forming the thyrotropes of the
adult animal. Determination of the mechanisms that
control the initial appearance of these two populations
of cells and the pathways that dictate their different
fates will be important in our understanding of
pituitary organogenesis. Thyrotroph embryonic factor
(TEF,Drolet et al., 1991), a transcription factor of
the leucine zipper class of DNA binding proteins, is a
potential candidate for involvement in the initial
activation of the rostral tip thyrotropes (Figure 1).
Thyrotroph embryonic factor is present a t the appropriate time in pituitary development and has been
shown t o activate the mouse TSHP gene in transfection assays.
Pit-1-Dependent and Pit-1-Independent Events
During Thyrotrope Development
A GRF Receptor Defect in the Little Dwarf Mouse
The observation that the Pit-l-defective Snell dwarf
mouse lacked thyrotrope cells suggested that functional Pit-1 was required for the proliferation and(or)
maintenance of this cell type (Li et al., 1990). In
addition, Pit-1 has been shown to transcriptionally
activate the TSHP gene, and modulators of TSHP gene
activity have been demonstrated to act through Pitl-binding sites within the gene (Steinfelder et al.,
1991, 1992a,b; Gordon et al., 1993; Haugen et al.,
1993; Kim et al., 1993; Mason et al., 1993; Lin et al.,
1994). The detection of TSHP transcripts in the
pituitary prior to pit-1 gene activation indicated that
Pit-1 was not required for the initial events controlling
The anterior pituitary is regulated by feedback
pathways and also by signaling molecules delivered
from the hypothalamus by a portal blood supply.
Growth hormone-releasing factor ( GRF 1 stimulates
somatotrope proliferation and GH gene expression and
secretion by increasing cellular cyclic AMP levels. The
Pit-l-dependent GRF receptor ( GRFR) gene encodes
a seven transmembrane helix receptor molecule with
similarity to the receptors for other signaling
molecules such as parathyroid hormone, vasoactive
intestinal peptide, and calcitonin (Lin et al., 1992;
Mayo, 1992). The Little (lit) mouse exhibits a dwarf
100
RHODES AND ROSENFELD
Table 2. Point mutations of Pit-1 associated with pituitary dysfunction in mice and humans
Organism
Mutation
Affected domain
Reference
Snell mouse
Human
Human
Human
Human
Human
Trp261 -+ Cys
Pro24 -+ Leu
Arg143 -+ Gln
Ala158 -+ Pro
Arg172 4 Stop
Arg271 --f Trp
POU-homeodomain
Amino terminus
POU-specific domain
POU-specific domain
Truncated protein
POU-homeodomain
Li et al., 1990
Ohta et al.. 1992a
Ohta et al.. 1992a
Pfaffle et al., 1992
Tatsumi et al., 1992a
Radovick et al., 1992
Ohta et al., 1992a
Okamoto et al., 1994
Cohen et al., 1995
phenotype and has highly reduced levels of GH and
PRL (Table 1 ) . It has recently been demonstrated
that the mouse GRFR gene co-localizes with the Zit
allele on chromosome 6 and that Eitllit mice express a
defective GRFR with a point mutation in a conserved
residue within the ligand-binding amino terminus of
the molecule (Godfrey et al., 1993; Lin et al., 1993).
In situ hybridization studies during embryonic development of the little mouse suggested the presence
of distinct zones of proliferating growth-hormoneproducing cells that are regulated by different trophic
signaling pathways (Lin et al., 1993). By this model,
precursor cells (pre-somatotrope stem cells) for the
somatotrope and lactotrope lineages develop in a GRFindependent fashion, whereas differentiated somatotropes in the caudomedial portion of the pituitary are
dependent on GRF ( a n d therefore CAMP) signaling
pathways.
Transcriptional Regulation of the pit-2 Gene
The lack of thyrotrope, somatotrope, and lactotrope
cell types in the Pit-1-defective Snell and Jackson
dwarf mice demonstrates the importance of Pit-1 in
the commitment and differentiation of these pituitary
lineages. Determination of the molecular mechanisms
involved in the initial activation, maintenance, and
regulation of pit-1 gene activity are therefore required
to understand early events controlling pituitary organogenesis. The rat pit-1 promoter is weakly active
in pituitary cells and contains binding sites for Pit-1
protein, allowing autoregulation by Pit-1 of its own
gene (Figure 4A) (Chen et al., 1990; McCormick et
al., 1990). One Pit-1 site positively regulates promoter
activity; the other lies downstream of the TATA box
and confers negative regulation (Chen et al., 1990;
Smith and Sharp, 1991). In addition, the promoter
contains elements that confer response to cyclic AMP
and other signaling pathways (Chen et al., 1990;
McCormick et al., 1990; Jong e t al., 1994). The rat pit1 promoter Pit-1 binding sites are conserved in both
the mouse and human pit-1 promoters, whereas the
CREB sites are less well positionally conserved
(Pfaffle et al., 1992). It has also been reported that
the TATA box region of the rat pit-1 promoter is
recognized by factors that contribute to the cellspecific expression of the gene (McCormick et al.,
1991).
The weak activity of the pit-1 promoter suggested
that the gene n i g h t contain additional regulatory
regions. T w o approaches have been used to identify
such regions. I n one study, 15 kb of rat pit-1 gene 5’
flanking sequence was used to guide the expression of
the SV40 T-antigen gene in transgenic animals (Lew
et al., 1993). Some of the resulting transgenic mice
were dwarfed and developed pituitary tumors, indicating targeting of the T-antigen oncogene t o the
pituitary. A cell line (GHFT-1) that expressed high
levels of T-antigen and Pit-1, but not other markers of
differentiated pituitary cells such as ctGSU, TSHP,
GH, or PRL, was derived from one tumor. This cell
line was therefore proposed t o be a progenitor cell
representing a stage following initial pit-1 gene
activation, but prior t o GH or PRL gene expression. A
2-kb region of the pit-1 gene was identified as having
higher activity in this cell line than in pituitary cells
considered to represent later stages of pituitary
differentiation.
A second approach examined the pit-1 gene for the
presence of regions that enhanced activity of the pit-1
promoter in both cultured pituitary cell lines and in
transgenic animals (Rhodes et al., 1993). 0-galactosidase and human growth hormone reporter transgenes
containing a region encompassing the 14.8 kb upstream of the mouse pit-1 gene were specifically
expressed in the pituitaries of transgenic mice. In
contrast, reporter genes containing only the pit-1
promoter were inactive. DNA fragments covering the
entire mouse pit-1 locus were examined for their
ability to enhance activity of the minimal promoter in
pituitary GC cells. A 700-bp enhancer was identified
that was located a t -10 kb, within the region required
to appropriately target pit-1 gene expression in
transgenic animals (Figure 4A). This enhancer ( a n d
500-bp and 390-bp sub-fragments) activated pit-1
promoter reporter genes 50- to 100-fold in transient
transfection assays using pituitary cells (Figure 4B).
The enhancer was demonstrated to work in a
pituitary-specific and orientation-independent fashion.
Three Pit-1 binding sites were shown to be function-
PIT-1 A N D ANTERIOR PITUITARY DEVELOPMENT
A.
-10 kb Enhancer
I
Promoter
I
-5OObp
B.
101
pit-I
Luciferase
Genomic
fragment
GC Cells
Ptomotcr
PromoternOObp enhancer
Promoter/SOObp enhancer
PrmoterD9Obp enhancer
0 2 b 4 0 B o 8 0 1
Luciferase Activity (xlO-3)
Figure 4.Transcriptional regulation of the mouse pit-1 gene. (A) Regulatory regions of the pit-1 gene. The -10-kb
enhancer with the retinoic acid/vitamin D response element (RDE), the pituitary-specific element (PSE), and the Pit1-dependent retinoic acid response element (PRE) are depicted. Binding sites for Pit-1 (P), and CREB (C) within the
enhancer and promoter are indicated (B) The -10-kb pit-1 enhancer strongly increases the activity of pit-1 promoter/
luciferase reporter genes following transient transfection of pituitary GC cells. The luciferase activity of various
promoterjenhancer (and control promoter alone) constructs is indicated. Luciferase activity is the mean observed
light units from triplicate assays. Error bars denote SEM.
ally important for enhancer activity. The pit-1 gene is
therefore autoregulated through Pit-1 binding sites in
the promoter and within this distal enhancer.
However, an examination of pit-1 gene expression by
in situ hybridization revealed that the pit-1 gene is
activated for several days during pituitary development in the Pit-1-defective Snell dwarf mouse, indicating that functional Pit-1 is not required for the initial
activation and early stages of gene activation. In
addition to Pit-1 binding sites, the 390-bp minimal
RDE element, which primarily confers response to
vitamin D, consists of a direct repeat of a core half-site
sequence spaced by four nucleotides. This element
therefore provides an exception to the “3-4-5 rule” for
nuclear receptor recognition motifs, in which vitamin
D receptorhetinoid X receptor ( RXR) heterodimers
are considered to prefer direct repeats spaced by three
base pairs ( N a a r et al., 1991; Umesono et al., 1991;
Yu et a]., 1991). The PRE retinoic acid response
element is dependent on Pit-1 for activity and
PIT-1 A N D ANTERIOR PITUITARY DEVELOPMENT
It is now important to define the components of the
signaling pathways that translate extracellular sigactivity within the
nals into changes in
(see Bradford et al., 1995; Lew and Elsholtz, 1995).
Further, elucidation of early events in anterior
pituitary cell commitment and differentiation will be
achieved by characterization of the mechanism of
Of the defect in the
activation Of the pit-1 gene,
Ames dwarf mouse, and investigation- -of the
. _.primary
103
Cullen, K. E., M. P. Kladde, and M. A. Seyfred. 1993. Interaction
between transcription regulatory regions of prolactin chromatin. Science (Wash., DC) 261:203.
Day R. N., and K. H. Day. 1994. An alternatively spliced form of Pit1 represses prolactin gene expression. Mol. Endocrinol. 8:374.
Day R. N., S. Kiike, H. Sakai, M. Muramatsu, and R. A. Maurer.
1990. Both Pit-1 and estrogen receptor are required for estrogen responsiveness of the rat prolactin gene. Mol. Endocrinol.
12:1964.
Defier, N,, M. Cox, R,Boelens, C, P. Verrijzer, P. c van der Wiet,
and R. Kaptein. 1993. Solution structure of the POU-specific
102
RHODES AND ROSENFELD
A.
Lucifenrsc
should be tested in both wild-type and Pit-l-defective
animals, such as the Jackson and Snell dwarf mice, in
order to separate autoregulatory and Pit-l-independent signaling pathways.
An
PRE
ENector:
cv-1cells
Synergism Between Pit-1 a n d Nuclear Receptors
1
2 0 4 0 6 0
80
Fold Induction by Rctinoic Acid
100
Synergism
n
B.
.
--L * *
pit-1
- .
PRE
C.
prolactin
growth hormone
Figure 5. Synergistic interactions of Pit-1. (A)
Oligonucleotides representing the Pit-l-dependent
retinoic acid response element (PRE) from the mouse
pit-1 gene enhancer were cloned adjacent to the
thymidine kinase (TK) promoter in a luciferase reporter
gene. The reporter gene was transiently transfected into
heterologous monkey CV-1 cells seeded in charcoalstripped medium with expression vectors for the
indicated effector molecules. RAR = retinoic acid
receptor alpha. The fold induction (k SEM) following
treatment with 5 x
M all-trans retinoic acid are
depicted. (B) Model of Pit-1/RAR synergism on the PRE.
Arrows indicate the left (L) and right (R) half-sites of the
imperfect direct repeat motif and the Pit-1 binding site.
RA = retinoic acid. (C) Synergistic interactions of Pit-1
with other factors in regulation of anterior pituitary
gene activity. E2 = estrogen, ER = estrogen receptor, T3 =
thyroid hormone, T3R = thyroid hormone receptor.
An oligonucleotide containing the pit-1 gene retinoic
acid response element ( P R E ) transfers retinoic acid
response to the heterologous thymidine kinase
promoter. This reporter gene is only active when
expression vectors for Pit-1 and the retinoic acid
receptor ( RAR) are co-transfected into heterologous
cells (Figure 5A). The PRE consists of a n imperfect
direct repeat motif that is bound by RAR/RxR
heterodimers in in vitro binding assays and a Pit-1
binding site located on the antisense strand (Figure
5B). Pit-1 has also been observed to synergize with
additional signaling receptors in activation of other
pituitary genes (Figure 5C). Synergism has been
reported between the thyroid hormone receptor ( T3R)
and Pit-1 in the rat GH gene promoter (Schaufele et
al., 1992; Tansey et al., 1993; Force and Spindler,
1994) and between the estrogen receptor (ER)and
Pit-1 in regulation of the r a t PRL gene distal enhancer
(Day e t al., 1990; Simmons et al., 1990; Nowakowski
and Maurer, 1994; Smith et al, 1995). Pit-l/"3R
synergism has also been reported a t a site within the
human placental lactogen gene (Voz et al., 1992).
Interactions such as these may play a part in the
restriction of GH, PRL ( a n d TSHP) gene expression t o
their respective cell types, because Pit-1 is a common
activator of all three genes and is found in each cell
type. Synergistic interaction between Pit-1 and other
classes of transcription factors, such as the zinc-finger
protein Zn-15, which binds a site within the GH gene
promoter (Lipkin et al., 1993), and the LIM-homeodomain factor P-LidmLIM3Lhx3 (Seidah et al., 1994;
Bach et al., 1995; Zhadanov et al., 1995) (Figure 5 C )
may also contribute to this process. Determination of
the mechanism by which interactions between Pit-1
and its partners in these synergistic activities are
translated into responses by the transcriptional
machinery will add to our understanding of pathways
of pituitary cell commitment.
Conclusion
Pit-1 is a prototype member of the POU homeodomain class of developmental regulatory proteins. It is
expressed only in specialized cells of the anterior
pituitary, where it directly regulates transcriptional
control from multiple pituitary-specific genes. In
addition, mutations a t the pit-1 gene locus underlie
pituitary disease in animals and humans and reveal
the importance of Pit-1 to the survival of the
somatotrope, thyrotrope, and lactotrope cell lineages.
PIT-I AND ANTERIOR PITUITARY DEVELOPMENT
It is now important to define the components of the
signaling pathways that translate extracellular signals into changes in Pit-1 activity within the nucleus
(see Bradford et al., 1995; Lew and Elsholtz, 1995).
Further, elucidation of early events in anterior
pituitary cell commitment and differentiation will be
achieved by characterization of the mechanism of
activation of the p i t - 1 gene, cloning of the defect in the
Ames dwarf mouse, and investigation of the primary
events that regulate the formation of Rathke’s pouch
during embryogenesis.
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