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Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208
Address all correspondence and requests for reprints to: Kelly E. Mayo, Department of Biochemistry, Northwestern University, Evanston, Illinois 60208. E-mail: k-mayo{at}nwu.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
ßA, and ßB). Inhibin A and inhibin B are
formed by heterodimerization of the -subunit and one of the two
ß-subunits, whereas the three forms of activin are formed by
dimerization of the highly related ß-subunits. The molecular cloning
of the , ßA, and ßB complementary DNAs
(cDNAs) has revealed that each subunit is encoded by a separate gene
and is derived from the carboxyl-terminus of a larger precursor protein
(5, 6, 7, 8).
In the rodent ovary, messenger RNAs (mRNAs) for the -,
ßA-, and ßB-subunit genes are expressed
predominantly in the granulosa cells of healthy follicles, and their
abundance increases during follicular maturation and decreases in
preovulatory follicles following the proestrous LH surge (9, 10). In
the adult rat, FSH induces the expression of the -,
ßA-, and ßB-subunit genes in preovulatory
follicles (11, 12). FSH also stimulates inhibin secretion and -and
ß-subunit mRNA accumulation in cultured primary granulosa cells (9, 13). Forskolin (FSK), a pharmacological agent that mimics the activity
of FSH by activating adenylyl cyclase and increasing intracellular
levels of cAMP, also stimulates inhibin gene expression and secretion
in granulosa cells, suggesting a role for cAMP in the regulation of
- and ß-subunit gene expression (11, 14). To explore the role of
cAMP in the regulation of inhibin and activin gene expression, we and
others reported earlier the isolation and structural characterization
of the rat inhibin -subunit gene. We identified a cAMP response
element (CRE) in the promoter of the -subunit gene that is necessary
for mediating gonadotropin and cAMP regulation of the gene in rat
granulosa cells (15, 16, 17). We, and others, also characterized the rat
inhibin and activin ßB-subunit gene and found that, while
it is structurally similar to the -subunit gene, it is not regulated
by cAMP in transfected gonadal cells (14, 18). We have now extended
those studies and report the isolation of the rat inhibin and activin
ßA-subunit gene and characterization of its promoter
region. To assess the regulation of this gene, we use a recently
characterized immortalized granulosa cell line, GRMO2, that retains
many of the characteristics of ovarian granulosa cells. Using transient
transfection assays, we identify a variant CRE element as being
critical for regulation of this gene by both cAMP and phorbol esters,
and demonstrate that AP-1 family proteins play an important role in the
regulation of ßA-subunit gene transcription.
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Materials and Methods |
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Charon 4A, the other with HTC hepatoma cell DNA in -Fix, Stratagene
(La Jolla, CA) (19) were screened with probes from the rat
ßA-subunit cDNA clone rINB-5 (8). The 5' probe was a
240-bp EcoRI/BamHI fragment that contained
untranslated sequences and the first 24 amino acids of the
ßA prohormone. The 3' probe was a 510-bp
PstI/EcoRI fragment spanning the mature
ßA-subunit sequences. No plaques that hybridized to both
probes were found; however, a 5'-positive phage was purified from the
-Fix library, and a 3'-positive phage was purified from the Charon
4A library. Southern blotting of these two clones identified a 4.3
BamHI fragment and a 6.1-kb EcoRI fragment that
hybridized to the 5' and 3' probes, respectively. These two fragments,
as well as the 0.5 kb BamHI fragment immediately 3' of the
4.3-kb BamHI fragment, were subcloned into pGEM vectors.
Subsequent Southern DNA blotting, subcloning, dideoxynucleotide chain
termination DNA sequencing (Sequenase, U.S. Biochemical Corp.,
Cleveland, OH) and primer extension and S1 nuclease analysis (described
below) revealed that these three genomic DNA clones, in combination,
contain three exons comprising the ßA-subunit mRNA as
well as approximately 1.5 kb of 5' flanking sequences. To construct the
initial fusion gene, a 609-bp NaeI fragment extending from
-571 to +38 bp was ligated into a SmaI site adjacent to the
luciferase coding region of the vector pA3-Luc (kindly provided by Dr.
William Wood, University of Colorado Health Sciences Center, Denver,
CO) (20). 5' deletions extending to an AccI site at -362 bp
and a HinfI site at -110 were made, and these three
constructs were designated (-571)rßA-Luc,
(-362)rßA-Luc, and (-110)rßA-Luc.
Analysis of the ßA-subunit mRNA structure
The ßA gene transcriptional start site was
determined by combining S1 nuclease mapping and primer extension
analysis. For S1 nuclease mapping, probes S1–1 and C were
3'-end-labeled with [
32P]ATP (DuPont NEN, Boston, MA)
and T4 polynucleotide kinase. Approximately 1 x
105 cpm/ng probe was hybridized to 5 µg of
polyA+ ovarian RNA (or liver RNA as a control) in 20 µl
of formamide hybridization buffer (80% formamide, 40 mM
PIPES (piperazine-N,N'-bis[2-ethane-sulfonic acid), pH 6.8,
400 mM NaCl, and 1 mM EDTA. Hybridization was
initially at 80 C for 5 min, then at 35–55 C for 12–16 h. Three
hundred microliters of 1500 U/ml S1 nuclease (Gibco BRL, Gaithersburg,
MD) in S1 salts buffer (0.28 M NaCl, 50 mM
NaOAc (pH 4.5), 4.5 mM ZnSO4, 20 µg/ml yeast transfer
RNA) were added, and S1 nuclease digestion was allowed to proceed for
1.5 h at 15–22 C. Samples were extracted with an equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1), ethanol precipitated, and
resuspended in formamide loading buffer. Protected fragments were
resolved on 6% polyacrylamide/50% urea denaturing gels and compared
with adjacent sequencing ladders to establish their size. Primer
extension analysis was performed using a synthetic 30-bp
oligonucleotide (5'-CCAAAGCAAGGGCATCCTGGCAGCAAAAGT-3') complimentary to
exon 2 sequences. The oligonucleotide was kinased as described above
and 1 x 105 cpm/ng primer was hybridized to 5–20
µg of polyA+ ovarian RNA (or liver, placenta, testis RNA
as a control) in 20 µl of 100 mM KCl. Hybridization was
performed at 80 C for 5 min followed by 12–16 h at 55 C. The extended
product was synthesized by adding 20 µl of 2x extension mix (100
mM Tris base (pH 8.0), 20 mM MgCl, 20
mM dithiothreitol (DTT), 10 mM each of dGTP,
dATP, dTTP, and dCTP and 1 U RNasin/ml (Promega, Madison, WI) and 30 U
of AMV reverse transcriptase (Promega) and then incubated at 42 C for
1.5 h. One milliliter of 0.5 M EDTA was added to stop
the reaction. Samples were extracted with phenol/chloroform/isoamyl
alcohol, ethanol precipitated, resuspended in 2 µl of 0.1
M NaOH, and combined with 4 µl of formamide loading
buffer. Gel electrophoresis was performed as described above, and
parallel sequencing reactions were used to size the extended
products.
GRMO2 cell culture, transfections, and luciferase assays
GRMO2 cells (kindly provided by Innogenetics N.V., Ghent,
Belgium), were maintained in Ham’s F-12/DMEM with 2% FCS and
supplemented with 10 µg/ml transferrin, 5 µg/ml insulin, 30
nM sodium selenite, 3 µg/ml BSA, and antibiotics as
described previously (21). Transfection of GRMO2 cells was performed at
50–70% cell confluency using a modified lipofectin-mediated method
and 1.25 µg of the relevant DNAs (22). After 6 h at 37 C, cells
were washed and incubated overnight. Cells were treated with FSK (10
µM) and/or TPA (30 nM) for 24 h before
harvest and assayed for luciferase reporter gene activity as described
previously (23).
Site-directed mutagenesis
To construct plasmids containing promoter point mutations,
oligonucleotide-directed mutagenesis was performed (24). An
NaeI/KpnI fragment (-571 bp to -62 bp) from the
ßA-subunit promoter was subcloned into pGEM3Zf(-)
(Promega) and transformed into the dut-ung-- Escherichia coli strain RZ1032.
Uracil-enriched single-stranded phagemid was produced by growing a
single colony in Terrific Broth supplemented with 5 mg/ml uridine and
100 µg/ml ampicillin for 3 h, infecting with 100 ml R408 helper
phage (1 x 1011 pfu/ml), and continuing growth for
6 h. Collected phagemid particles were resuspended in TE, and the
phagemid DNA was obtained after phenol/chloroform extraction. The
oligonucleotide primer used for constructing
(mCRE)rßA-Luc had the sequence
(5'-AATCAGCATGATCT-CAGCAGATGA-3'). The oligonucleotide primer used to
generate the (cCRE)rßA-Luc, had the sequence
(5'-AATCAGCATGACGTCAGCAGATGA-3'). The primers were phosphorylated with
T4 polynucleotide kinase and annealed to phagemid DNA in annealing
buffer (40 mM Tris base, pH 7.5, 0.1 M NaCl, 20
mM MgCl2 and 5 mM DTT). Primer
extension and ligation were performed using Klenow and T4
DNA ligase in extension buffer (20 mM Tris base, pH 7.5, 10
mM MgCl2, 5 mM DTT, 1
mM each of dGTP, dATP, dTTP, and dCTP, and 0.5
mM ATP). The reaction proceeded on ice for 30 min, at room
temperature for 4 h, and then was used to transform
Escherichia coli strain JM109. Several colonies were
screened by DNA sequencing, and the correct mutation was subcloned as
an AccI/KpnI fragment into
ßANae-Luc to generate the (cCRE)rßA-Luc and
(mCRE) rßA-Luc mutant plasmid constructs.
Electrophoretic mobility shift assays
An oligonucleotide probe corresponding to the -106 bp to
-135-bp region in the ßA-subunit promoter
(5'-GAGTCATCTGCTGATGTCATGCTGATTCTA-3') was end-labeled using
T4 kinase and [32P]ATP. The probe was
heated to 95 C for 5 min in the presence of a complementary
oligonucleotide and annealed gradually by cooling to room temperature
in 0.1 M NaCl solution. Oligonucleotides used in
competition assays were similarly annealed to their complementary
strands to form double-stranded probes. Binding reactions were carried
out in the presence (100x) or absence of competitors (wild-type,
mutant oligonucleotides, or oligonucleotides containing consensus TRE
or CRE sites). The labeled probes (104 cpm, approximately
105 cpm/ng) were incubated with 10 µg of nuclear extract
and 1 µg of poly (dI-dC) in a buffer containing (final concentration)
10 mM HEPES (pH 7.8), 50 mM KCl, 5
mM MgCl2, 0.5 mM DTT, 1
mM EDTA, and 10% glycerol. The reaction mixture, in a
volume of 20 µl, was incubated on ice for 20 min, and DNA-protein
complexes were separated from unbound probe on a 5% polyacrylamide gel
electrophoresed in 0.5x TBE (90 mM Tris borate, pH 8.2,
2.5 mM EDTA at 4 C for 2 h at 200 V. Antibody
incubations with 1 µg of anti-CREB (kindly provided by Dr. Joel F.
Habener, Harvard Medical School, Boston, MA), anti-ATF-1, anti-ATF-2,
anti-jun-B, anti-jun-D, anti-c-jun, anti-c-fos, anti-fos-B or
anti-fra-2 (Santa Cruz Biotechnology, Santa Cruz, CA), were performed
before binding for 45 min at room temperature.
Statistical analysis
Data for transfection and cotransfection studies were expressed
as the mean ± SEM; n = 3. Statistical analyses were
performed using unpaired student’s t test. A two-tailed
probability of less than 5% (i.e. P < 0.05) was
considered statistically significant.
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depicts the structure of the rat
ßA gene.
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. Comparison of the sizes of the
extended product (193 nt, Fig. 2B) and S1-protected fragment (189 nt,
data not shown) revealed the existence of an additional intron (between
nucleotides 52 and 53 of rINB-5) and untranslated exon in the
ßA gene. To establish if this exon was present in clone
rINBgen-5', an EcoRI/NIaIV fragment consisting of the first
40 bp of rINB-5 was used to probe an rINBgen-5' DNA blot (data not
shown). A presumptive first exon was found to be in rINBgen-5'
approximately 2.5 kb upstream of the first protein-coding exon (Fig. 1
). A second S1 nuclease experiment (Fig. 2C), performed with a probe
specific for the untranslated exon (C in Fig. 2A), mapped the
transcriptional start site to the same A nucleotide as the previous
primer extension analysis (Fig. 2A and Fig. 3A). These results indicate that the
newly identified 5' exon is 66 bp in length. The identification of this
small additional exon containing only 5' noncoding sequences of the
inhibin and activin ßA-subunit gene was reported
previously in preliminary form by our laboratory (25), and recent
studies on the human ßA gene indicate that it has an
analogous noncoding exon (26).
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shows the DNA
sequences of the 5' flanking region of the rat ßA gene
and identifies a number of consensus and near-consensus binding sites
for selected transcription factors. A TATA box and a CCAAT box are
located at -30 bp and -62 bp with respect to the transcriptional
start site. Within the 571 bp promoter region of the rat
ßA gene, a variant CRE (cAMP-response element) site at
-120 bp, two variant TREs (TPA-responsive elements) at -150 bp and
-197 bp and a consensus TRE site at -537 bp are identified with
respect to the transcriptional start site. Figure 3B shows the
schematic representation of the ßA promoter region and
the plasmid constructs containing point mutations at the variant CRE
site (to be discussed in the following section).
Activity of the rat inhibin and activin ßA promoter
in GRMO2 cells
To analyze the promoter activity of the ßA-subunit
gene 5'-flanking sequences and delineate elements mediating regulation
of the gene, luciferase fusion constructs containing 5'-flanking
sequences were generated and used to transiently transfect GRMO2
immortalized mouse granulosa cells that expresses the endogenous
ßA-subunit gene (21). Because potential TRE and CRE sites
were found in the 5'-flanking region and the endogenous
ßA-subunit gene has been shown to be regulated by phorbol
esters and cAMP (27, 28), we tested responses to the phorbol ester TPA
(12-O-tetradecanoylphorbol-13-acetate) and to the adenylate
cyclase activator FSK. Figure 4 shows a
significant activation of the reporter gene in response to TPA or FSK
treatment when ßA promoter regions to -571 bp and -362
bp were used. Although the (-362)rßA-Luc reporter
construct does not include the consensus TRE at -537 bp, it remained
fully responsive to TPA. We also examined the effects of FSK and TPA in
combination on ßA-subunit promoter in GRMO2 cells. As
shown in Fig. 4, the two agents in combination give a response that is
larger than that observed with either FSK or TPA alone but is less than
additive. When a ßA promoter region deleted to -110 bp
was tested, a dramatic and significant decrease in both basal and
inducible expression was observed. Because deletion construct
(-110)rßA-Luc removes a variant CRE at position -120 bp
and a nonconsensus CRE at a similar position in the -subunit gene is
critical for cAMP regulation, we generated two constructs bearing
mutations in the CRE region in the context of the -571 bp promoter.
These include a mutation that was expected to disrupt CRE function
(TGATGTCA
TGAGATCA) and a mutation that converted the
variant CRE into a consensus, palindromic CRE
(TGATGTCATGACGTCA). Figure 5 demonstrates that in GRMO2 cells, the
basal activity and FSK or TPA-inducibility of the
(mCRE)rßA-Luc construct was significantly reduced
compared with the wild-type -571-bp promoter. Surprisingly, a similar
decrease in activity was observed with the (cCRE)rßA-Luc
construct that has a consensus CRE. These results indicate that the
variant CRE site in the rat inhibin and activin ßA gene
promoter is essential for mediating both FSK and TPA responses in GRMO2
cells. In addition, the finding that conversion to a consensus CRE
impairs activity suggests that this element is not acting like a
classical CREB-binding CRE.
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shows formation of a predominant
protein-DNA complex (lane 2) that is specifically competed by a
100-fold excess of the unlabeled wild-type ßA variant CRE
(lane 6) but not by a nonspecific oligonucleotide (lane 5). An
oligonucleotide containing the mutant ßA variant CRE
partially competed the formation of this complex (lane 7), although
much less effectively than the wild-type ßA CRE. The
sequence of the ßA variant CRE is very similar to both
CRE and TRE consensus sequences. We therefore used oligonucleotides
containing CRE and TRE consensus sequences as competitors in an EMSA.
As shown in lanes 3 and 4 of Fig. 6, both CRE and TRE consensus
sequences abolished the binding of the protein complex to the
ßA variant CRE. These data indicate that the
ßA gene variant CRE protein-DNA complex may contain
proteins of both the CREB/ATF and AP-1 family of transcription factors,
or at least includes proteins able to bind to either the CRE or TRE
site.
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shows that the CREB antibody did not affect the protein complex binding
the variant CRE site (lane 3). Similarly, no significant interference
with the protein complex was observed in the presence of the ATF-2
antibody, as shown in lane 5. However, in the presence of the ATF-1
antibody, the intensity of the protein-DNA complex is slightly reduced
relative to the other bands (lane 4). Among the antibodies used to
identify potential members of the AP-1 family in the protein complex
binding the ßA CRE, only the antibody to jun-B was
observed to supershift the protein-DNA complex (lane 8), whereas the
antibody to fos-B interfered with formation of the protein-DNA complex
(lane 12) as shown in Fig. 7B. No significant change in formation of
the protein complex on the ßA CRE was observed in
presence of antibodies to other members of the AP-1 family including,
jun-D, c-jun, c-fos, fos-B, and fra-2 (lanes 9, 10, 11, and 13,
respectively). Thus the variant CRE element appears to bind
predominantly members of the AP-1 family of transcription factors,
rather than CREB/ATF-1 family proteins, in GRMO2 cells.
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, jun-B alone had little effect on
promoter activity; however, fos-B significantly activated the -572-bp
promoter. This activation was substantially higher when both jun-B and
fos-B were coexpressed in GRMO2 cells. The (mCRE)rßA-Luc
was also activated by fos-B and jun-B, but to a lesser extent than the
wild-type promoter, whereas the (cCRE)rßA-Luc was not
activated at all in response to fos-B/jun-B overexpression. These
experiments indicate that the AP-1 family proteins jun-B and fos-B can
transactivate the ßA-subunit gene through the variant CRE
sequence.
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and ßB-subunit genes, which contain only two
exons (6, 16, 18, 30), in that the ßA gene includes a
small 5'-nontranslated exon. Using S1 nuclease mapping and primer
extension, we have defined a unique transcriptional start site of the
ßA-subunit gene, and have sequenced 761 bp of 5' flanking
DNA upstream of this start site. This led to the identification of
potential binding sites for trans-acting factors known to
mediate cAMP or phorbol ester-stimulated gene transcription. Three
potential TRE or AP-1 binding sites were found between -140 bp and
-450 bp in the inhibin and activin ßA-subunit promoter.
A CRE-like sequence was found in the 5'-flanking region at -120 bp,
which is similar in position to the cAMP-responsive element in the
promoter of the rat inhibin gene (16). A track of TG repeats was
found to extend from -691 bp to -732 bp of the ßA
promoter. We previously reported that a similar TG repeat found in the
rat inhibin gene affected basal promoter activity but did not
affect FSK-responsiveness (16). We have not yet tested the role of the
TG repeat in ßA-subunit gene expression. Recently, the genomic structure of the human inhibin and activin ßA-subunit gene has been reported (26), and comparison of the 5'-flanking regions to -275 bp of the two genes shows a greater than 90% sequence identity. Furthermore, the human ßA gene promoter has also been shown to be activated in response to 8-Br-cAMP and TPA (26). The strong conservation of potential transcription factor binding sites, including the TRE and variant CRE sites discussed here, in the 5'-flanking regions of the rat and human ßA-subunit genes suggests that these sites are likely to play an important role in the regulation of ßA-subunit gene expression.
The expression patterns of the -, ßA-, and
ßB-subunit genes in granulosa cells during the estrous
cycle (11) and in response to a variety of other hormones, growth
factors and steroids (10, 29) have been well documented. However, very
little is known about the signaling pathways and the intracellular
effector molecules regulating the expression of these genes in response
to physiological signals that change rapidly during the reproductive
cycle. This has been due in part to the unavailability of appropriate
granulosa cell lines. Recently, a mouse granulosa cell line designated
GRMO2 was generated (21) that exhibits many features characteristic of
primary granulosa cells in culture, including the expression and
secretion of inhibin and activin (31). In this study, we found that
GRMO2 cells express the transfected ßA-subunit gene
promoter and regulate its activity in response to cAMP and TPA
treatment. While the studies reported here use the GRMO2 granulosa cell
line, we have found the ßA-subunit gene promoter to be
similarly regulated by cAMP in transfected primary rat granulosa cells
(Romanelli, J. C. D., and K. E. Mayo, unpublished
results).
The major signaling pathway mediating the regulation of the inhibin and
activin subunit genes by pituitary gonadotropins in ovarian granulosa
cells is likely to be the cAMP-PKA pathway (29). We reported previously
that a CRE-like element in the rat inhibin -subunit gene promoter
mediates the cAMP response in primary granulosa cells (16). In the
present study, we have shown that the variant CRE in the
ßA-subunit gene is capable of mediating cAMP induction of
transcription in GRMO2 granulosa cells. Although cAMP regulation was
first associated with the presence of a palindromic consensus CRE in
gene promoters, in the past few years it has become increasingly
evident that nonconsensus CREs are also able to mediate PKA
responsiveness in a number of genes (32, 33, 34, 35, 36). In addition, the flanking
sequence context of CRE and CRE-related elements is reported to be
important for their activity (37, 38). It has been suggested that the
weaker contribution of a variant CRE can be compensated for by the
activity of sequences surrounding the CRE (37). Our results indicate
that the conversion of the variant CRE to a consensus CRE does not
enhance promoter activity, as has been reported to occur with the
glycoprotein hormone -subunit gene promoter in a trophoblast cell
line (39). In addition, the ßA variant CRE was found to
be responsive to TPA, an agent known to activate the PKC signaling
pathway and induce effector molecules that classically act through a
TRE consensus sequence, TGAC/GTCA. Taken together, our findings suggest
that the unique sequence of the variant CRE found in the rat
ßA-subunit gene promoter is essential for regulation of
the gene and that this variant CRE does not function as a classical
CREB-binding element.
The protein kinase A and protein kinase C signaling pathways appear to
be major regulators of ßA-subunit gene expression in a
variety of cell types. An elevation of ßA mRNA species in
human fibrosarcoma HT 1080 cell lines in response to 8-bromo-cAMP and
TPA has been reported (27, 40). In human adrenal cells, ACTH, cAMP, and
TPA stimulate ßA-subunit transcripts (15). Recently,
and ßA mRNA levels were shown to be induced by
8-bromo-cAMP and TPA in human granulosa-luteal cells in culture (28).
The data presented here demonstrate that the variant CRE in the
promoter of the ßA-subunit gene is likely to mediate
induction by both these signaling pathways. Although CRE-like elements
have been shown to mediate responsiveness to the PKC signaling pathway
in other genes (34, 35), the ability of the ßA-subunit
gene variant CRE to respond strongly to both signaling pathways appears
to be unique. In cultured rat granulosa cells, stimulation of the PKA
or PKC signaling pathways has been demonstrated to differentially favor
the formation of inhibin or activin, respectively (41). Recently the
- and ßA-subunit genes in human granulosa-luteal cells
were reported to be differentially expressed in response to treatment
with cAMP or phorbol esters (28), in that the cAMP was found to have a
predominant effect on -subunit expression, whereas TPA was a potent
stimulator of ßA-subunit gene expression but did not
affect -subunit mRNA levels. We have made similar observations in
rat granulosa cells (Pei, L., and K. E. Mayo, unpublished
results). Taken together, these observations indicate that the level of
induction of the - and ßA-subunit genes in response to
these two signaling pathways may be critical regulatory step for the
formation of inhibins vs. activins in the granulosa
cell.
The regulation of gene expression by the PKA and PKC signaling pathways is often mediated by two related regulatory elements, the CRE (TGACGTCA) and TRE (TGAC/GTCA), respectively (42, 43). Generally, CRE and TRE elements are thought to bind distinct sets of transcription factors, the CREB/ATF and Fos/Jun family of proteins, respectively, both of which belong to the bZip (basic region leucine-zipper) family of proteins (43). However, an interaction between these two inducing pathways at the transcriptional level is an increasingly common finding. For example, AP-1 family proteins can bind a CRE site (44, 45), and the nuclear factors CREB and CREM can bind to AP-1 sites (46). Although cAMP and TPA are classical inducers of the PKA and PKC signaling pathways, respectively, cross-talk between these two pathways has been reported. For example, FSH (an inducer of the PKA pathway) is reported to stimulate Jun-B and c-Fos expression in rat Sertoli cells (47), and hCG stimulates c-Fos expression in rat ovarian granulosa cells (48). Conversely, phorbol esters (TPA) have been shown to induce adenylate cyclase phosphorylation (49), affecting production of the cAMP second messenger, and to stimulate CREB phosphorylation (50).
We reported previously that the ßA-subunit variant CRE
binds recombinant CREB protein, as established using DNA footprinting
analysis (51). However, in comparison with a consensus CRE, the binding
affinity is substantially reduced, consistent with strict sequence
requirements for high affinity CREB binding (38). Recently, a variant
CRE in the promoter of the glycoprotein hormone -subunit gene,
identical in sequence to the variant CRE in the promoter of the
ßA-subunit gene, has been shown to bind a protein complex
that cross-reacts with antibodies against AP-1 family transcription
factors in gonadotrope and trophoblast cells (37). Based on our
results, it appears that mediation of the effects of the PKA and/or PKC
signaling pathways in GRMO2 granulosa cells occurs predominantly
through AP-1 family proteins. However, we cannot rule out the
possibility of hetero-dimerization between members of the CREB/ATF and
AP-1 family, which has been previously reported (52), and it will be
important to establish whether such heterodimeric protein complexes
form on the ßA-subunit variant CRE in response to
stimulants that can activate both the PKA and PKC signaling pathways in
granulosa cells.
In summary, our results demonstrate a direct transcriptional regulation of the inhibin and activin ßA-subunit gene in response to cAMP and TPA, show that a variant CRE in the promoter of the ßA-subunit gene is critical for this regulation, and identify AP-1 family proteins as playing an important role in mediating this regulation. It will be important in future studies to analyze in greater detail the sequence requirements for activity of this novel CRE-like element, to establish more completely the repertoire of proteins able to mediate regulation through this element, and to investigate ways in which signals from the PKA and PKC pathways are integrated to appropriately regulate inhibin and activin subunit gene expression in the ovarian granulosa cell.
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Acknowledgments |
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Footnotes |
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2 Supported in part by NIH Training Grant HD-07068.
Received February 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-subunit
complementary deoxyribonucleic acids and expression in the ovary. Mol
Endocrinol 1:561–568, ßA, and
ßB subunits in various tissues predicts diverse
functions. Proc Natl Acad Sci USA 85:247–251 inhibin gene by cyclic adenosine
3',5'-monophosphate after transfection into rat granulosa cells. Mol
Endocrinol 5:521–534- and
ßA-subunit and follistatin mRNAs by cyclic AMP and
phorbol ester in cultured human granulosa-luteal cells. Mol Cell
Endocrinol 121:1–10[CrossRef][Medline]
subunit gene bind similar proteins in
trophoblasts and gonadotropes but have distinct functional sequence
requirements. J Biol Chem 271:31650–31656-subunit gene in the placenta
requires a functional cyclic AMP response element, whereas a different
cis-acting element mediates pituitary-specific expression.
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 J. L. Kipp, S. M. Kilen, S. Bristol-Gould, T. K. Woodruff, and K. E. Mayo Neonatal Exposure to Estrogens Suppresses Activin Expression and Signaling in the Mouse Ovary Endocrinology, May 1, 2007; 148(5): 1968 - 1976. [Abstract] [Full Text] [PDF]
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 Y Okuma, A E O'Connor, T Hayashi, K L Loveland, D M de Kretser, and M P Hedger Regulated production of activin A and inhibin B throughout the cycle of the seminiferous epithelium in the rat. J. Endocrinol., August 1, 2006; 190(2): 331 - 340. [Abstract] [Full Text] [PDF]
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S. E. Harris, A. L. Chand, I. M. Winship, K. Gersak, Y. Nishi, T. Yanase, H. Nawata, and A. N. Shelling INHA promoter polymorphisms are associated with premature ovarian failure Mol. Hum. Reprod., November 1, 2005; 11(11): 779 - 784. [Abstract] [Full Text] [PDF]
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Y Okuma, A E O'Connor, J A Muir, P G Stanton, D M de Kretser, and M P Hedger Regulation of activin A and inhibin B secretion by inflammatory mediators in adult rat Sertoli cell cultures J. Endocrinol., October 1, 2005; 187(1): 125 - 134. [Abstract] [Full Text] [PDF]
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Y Okuma, K Saito, A E O'Connor, D J Phillips, D M de Kretser, and M P Hedger Reciprocal regulation of activin A and inhibin B by interleukin-1 (IL-1) and follicle-stimulating hormone (FSH) in rat Sertoli cells in vitro J. Endocrinol., April 1, 2005; 185(1): 99 - 110. [Abstract] [Full Text] [PDF]
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Y. Wang and W. Ge Involvement of Cyclic Adenosine 3',5'-Monophosphate in the Differential Regulation of Activin {beta}A and {beta}B Expression by Gonadotropin in the Zebrafish Ovarian Follicle Cells Endocrinology, February 1, 2003; 144(2): 491 - 499. [Abstract] [Full Text] [PDF]
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S. R. Cunha and K. E. Mayo Ghrelin and Growth Hormone (GH) Secretagogues Potentiate GH-Releasing Hormone (GHRH)-Induced Cyclic Adenosine 3',5'-Monophosphate Production in Cells Expressing Transfected GHRH and GH Secretagogue Receptors Endocrinology, December 1, 2002; 143(12): 4570 - 4582. [Abstract] [Full Text] [PDF]
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 C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752. [Abstract] [Full Text] [PDF]
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W. E. Thompson, A. Branch, J. A. Whittaker, D. Lyn, M. Zilberstein, K. E. Mayo, and K. Thomas Characterization of Prohibitin in a Newly Established Rat Ovarian Granulosa Cell Line Endocrinology, September 1, 2001; 142(9): 4076 - 4085. [Abstract] [Full Text] [PDF]
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 C. K. Welt and A. L. Schneyer Differential Regulation of Inhibin B and Inhibin A by Follicle-Stimulating Hormone and Local Growth Factors in Human Granulosa Cells from Small Antral Follicles J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 330 - 336. [Abstract] [Full Text]
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 M. Ito, Y. Park, J. Weck, K. E. Mayo, and J. L. Jameson Synergistic Activation of the Inhibin {alpha}-Promoter by Steroidogenic Factor-1 and Cyclic Adenosine 3',5'-Monophosphate Mol. Endocrinol., January 1, 2000; 14(1): 66 - 81. [Abstract] [Full Text]
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 S. C. Sharma and J. S. Richards Regulation of AP1 (Jun/Fos) Factor Expression and Activation in Ovarian Granulosa Cells. RELATION OF JunD AND Fra2 TO TERMINAL DIFFERENTIATION J. Biol. Chem., October 20, 2000; 275(43): 33718 - 33728. [Abstract] [Full Text] [PDF]
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