Follicle-Stimulating Hormone Induction of Ovarian Insulin-Like Growth Factor-Binding Protein-3 Transcription Requires a TATA Box-Binding Protein and the Protein Kinase A and Phosphatidylinositol-3 Kinase Pathways

Elimelda Moige Ongeri, Michael F. Verderame and James M. Hammond

Pennsylvania State University, College of Medicine, Department of Medicine, Hershey Medical Center, Hershey, Pennsylvania 17033

Address all correspondence and requests for reprints to: James M. Hammond, Pennsylvania State University, College of Medicine, Hershey Medical Center, 500 University Drive, Hershey, Pennsylvania 17033. E-mail: jhammond{at}psu.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The current study was done to elucidate the mechanism of the FSH stimulation of IGF-binding protein 3 (IGFBP-3) expression and map the FSH response element on the pig IGFBP-3 promoter. Forskolin induced IGFBP-3 reporter activity in transiently transfected granulosa cells. The protein kinase A (PKA) inhibitor [N-[2-(p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl] (and cotransfection with a PKA inhibitor expression vector), the phosphatidylinositol-3 kinase inhibitor [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], and the ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene], all blocked FSH stimulation. Use of serial deletion constructs and site-directed mutagenesis show that a TATA box-binding protein site is required for FSH stimulation and that a specific protein 1 (Sp1) site is required for basal transcription. Gel shift assays of nuclear protein with a –61/–25 probe detected four protein-DNA complexes, with bands I and II having significantly higher intensities in FSH-treated cells than in controls. Mutation of the Sp1 site prevented formation of bands I and II whereas mutation of the TATA box-binding protein site prevented formation of band IV. Use of specific antibodies showed that Sp1 participates in formation of band I, Sp3 band II, and p300 in both I and II. Band III was nonspecifically competed out. We conclude that FSH stimulation of IGFBP-3 transcription is mediated by cAMP via the PKA pathway and requires the P1–3 kinase and likely the MAPK pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IGF-BINDING PROTEIN-3 (IGFBP-3), the most abundant IGFBP in the circulation and in ovarian follicular fluid, has been shown to regulate cell growth and differentiation in the ovary and other tissues. IGFBP-3 acts via modulation of IGF bioavailability and through IGF-independent pathways. Factors that modulate IGFBP-3 transcription and translation include IGF-I (1) and cAMP (1, 2, 3). In mammary epithelial cells transfected to overexpress IGFBP-3, responsiveness to IGF-I stimulation was enhanced and shown to involve the phosphatidylinositol-3 (PI-3) kinase pathway (4, 5). In rat C6 glioma cells, cAMP reduced IGFBP-3 expression in a dose-dependent manner (3). However, in a Madin bovine kidney cell line, cAMP and agents that increase intracellular cAMP levels (such as forskolin) stimulated IGFBP-3 mRNA via protein kinase A (PKA) (2). Collectively, these results suggest a species and/or cell line-specific response. Little is known about the regulation of IGFBP-3 in ovarian cells. Our studies have shown that FSH, the dominant gonadotropin regulator of ovarian granulosa cells, controls expression of this protein in a biphasic, time-dependent fashion (6). Luciferase assays with serial deletion constructs of the IGFBP-3 promoter showed that a 13-bp nucleotide sequence between –61 and –48 of the IGFBP-3 promoter was required for basal and FSH-induced transcriptional activity. The mechanism underlying FSH stimulation of IGFBP-3 gene activity and the transcription factors involved in this induction are not known. The objective of the current study was to elucidate the mechanism of FSH stimulation of IGFBP-3 gene activity in porcine ovarian granulosa cells and to map more precisely the FSH response element on the IGFBP-3 promoter. Because the 13-bp sequence that was required for basal and FSH-stimulated gene activity had a putative binding site for the specific protein 1 (Sp1) transcription factor, we sought to determine whether Sp1 plays a role in FSH regulation of the IGFBP-3 gene.

Our data show that FSH induction of the IGFBP-3 gene is mediated by cAMP via the PKA pathway but requires the PI-3 kinase pathway. Although the 13-bp sequence with the Sp1 binding site is necessary, our data show that it is not sufficient for FSH induction of the IGFBP-3 gene. We further show a requirement for a TATA box-binding protein (TBP) binding site in FSH stimulation of the IGFBP-3 gene in granulosa cells. Although a significant amount of literature exists on the role of TBP in basal transcription, little or nothing is known about its role in hormone-regulated cell function. To our knowledge, this is the first study to show that TBP participates in hormone-induced transcription.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FSH Stimulation of IGFBP-3 Is Mediated by cAMP via the PKA Pathway but Requires PI-3 Kinase and ERK
We used granulosa cells transiently transfected with a –191/+47 IGFBP-3 promoter construct to evaluate the signaling pathways involved in FSH induction of the IGFBP-3 gene. To determine whether the cAMP pathway plays a role in IGFBP-3 gene induction, transfected granulosa cells were treated with 10 µl forskolin, an agent that activates adenylyl cyclase. Forskolin caused a significant induction of IGFBP-3 reporter activity (Fig. 1Go). However, the level of stimulation by forskolin was lower than that achieved by FSH treatment of cells, suggesting that, in addition to activating adenylyl cyclase and the PKA pathway, FSH stimulation activates other signaling pathway(s). However, combining forskolin with FSH did not have an additive effect. To confirm that the PKA pathway is involved, we preincubated transiently transfected cells with the PKA inhibitor H89 (10 µM). The luciferase activity of cells preincubated with H89 was comparable to control cells treated with dimethylsulfoxide (DMSO) and significantly lower than cells treated with FSH (Fig. 2AGo). Treatment of cells with either H89 or LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one] on their own did not have any effect on the basal luciferase activity of transfected cells. Because the specificity of H89 has been questioned in some cell systems (7), we confirmed the role of PKA by cotransfecting granulosa cells with a PKA inhibitor expression vector (8). Granulosa cells cotransfected with the PKA inhibitor expression vector were not responsive to FSH treatment (Fig. 2BGo). In addition to blocking FSH responsiveness, the PKA inhibitor expression vector significantly decreased the basal transcription activity of the cells.



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Fig. 1. Luciferase Activity of Granulosa Cells Transfected with a –191/+47 Pig IGFBP-3 Promoter Construct

Cells were cultured in 10% FBS for 18 h and transfected with the IGFBP-3 reporter construct using the Lipofectin method for 6 h in serum-free medium. Cell culture was continued in 10% FBS to 95% confluence. Cells were then serum starved overnight before being treated with either 100 ng/ml FSH, 10 µM of the adenylyl cyclase activator, forskolin, or a combination of both FSH and forskolin for 3 h. Control cells were treated with vehicle (PBS). ß-Galactosidase plasmid was used as an internal control. Results are mean ± SEM of three different experiments, each done in triplicate with different batches of granulosa cells. Different superscripts represent significant differences.

 


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Fig. 2. Luciferase Activity of Granulosa Cells Transiently Transfected with a –191/+47 IGFBP-3 Promoter Construct

Cells were seeded in 10% FBS for 18 h, transfected in serum-free medium using the Lipofectin method for 6 h, and subsequently cultured in 10% FBS to 95% confluency. The cells were then serum starved overnight and preincubated in 10 µM of either the PKA inhibitor H89 or the PI-3 kinase inhibitor LY294002 (LY) for 30 min before being exposed to 100 ng/ml FSH for 3 h (panel A). Control cells were treated with equivalent volumes of vehicle. Blockage of the PKA signaling pathway was confirmed by cotransfection with a PKA inhibitor expression vector (PKI; panel B).

 
Because signaling via the FSH receptor (FSHR) has been reported to branch upstream and downstream of PKA (9), we evaluated other pathways that may play a role. To determine whether the P1–3 kinase pathway is involved in FSH-induced transcription of the IGFBP-3 gene, granulosa cells transiently transfected with IGFBP-3 promoter constructs were incubated for 30 min with 10 µM LY294002, a specific inhibitor of the PI-3 kinase pathway, before being treated with FSH for 3 h. Preincubation with LY294002 blocked the FSH stimulation of IGFBP-3 transcription activity (Fig. 2AGo). Preincubation with the ERK inhibitor, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene], significantly reduced the FSH response of IGFBP-3 (Fig. 3Go). To evaluate whether FSH stimulates ERK activation, Western blot analysis with a phospho-ERK-specific antibody was used. Our data show that FSH induced ERK phosphorylation (Fig. 3BGo). This induction was seen at 3 h and was blocked by the ERK inhibitor U0126. FSH did not significantly affect the level of total ERK.



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Fig. 3. Luciferase Activity of Granulosa Cells Transiently Transfected with a –191/+47 IGFBP-3 Promoter Construct and Treated with FSH

Cells were cultured and transfected as described in Fig. 2Go and preincubation in 5 µM of the ERK inhibitor U0126 for 30 min before being treated with 100 ng/ml FSH. In panel B, granulosa cells were cultured to 95% confluence, serum starved overnight, and treated with FSH for time points shown. Total protein was extracted, and Western blot analysis with a phospho-ERK-specific antibody was used to evaluate ERK activation. The membrane was stripped and probed for total ERK. In panel C, cells were preincubated in 0.5–5.0 µM U0126 for 30 min before FSH exposure. Control cells were treated with equivalent volumes of vehicle (lower panel). The blots are representative of three different experiments. P-ERK, Phosphorylated ERK.

 
An Sp1-Binding Site Is Required for IGFBP-3 Basal Transcription Activity
Because the sequence from –61 to –48 of the IGFBP-3 promoter required for basal and FSH induction contained an Sp1 binding site, we evaluated the role of Sp1 in FSH stimulation of the IGFBP-3 gene. To determine whether this sequence was sufficient for FSH induction of the IGFBP-3 gene, we first generated and cloned a tetramer construct of the 13-bp sequence into the pLUC vector. Transient transfection of granulosa cells with this construct resulted in a significant restoration of basal transcription activity. The level of luciferase activity was about 50% of that observed for cells transfected with the wild-type –61/+9 IGFBP-3 promoter construct. However, there was no significant FSH induction in granulosa cells transiently transfected with the tetramer construct (Fig. 4Go). These data suggested that this sequence was necessary, but not sufficient, for the FSH response. This outcome was confirmed by data from IGFBP-3 promoter constructs with 1, 2, 3, and 4 base substitution mutations in the Sp1 binding site. Although all four mutations caused a large and statistically significant (P < 0.0001) reduction in basal luciferase activity compared with the wild-type promoter construct, these cells retained FSH responsiveness (Fig. 5Go).



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Fig. 4. Luciferase Activity of Granulosa Cells Transfected with Various IGFBP-3 Promoter Constructs

Granulosa cells were cultured in 10% FBS for 18 h and transfected with IGFBP-3 promoter constructs using the Lipofectin method. AT 95% confluence, cells were treated with FSH for 3 h. Control cells were treated with PBS. A ß-galactosidase plasmid was used as an internal control. In the bottom set of bars, sequence between –47 and +9 of the IGFBP-3 promoter was cloned upstream of the 13 base tetramer.

 


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Fig. 5. Luciferase Activity of Granulosa Cells Transfected with a –191/+47 IGFBP-3 Promoter Construct and Four Mutants with One-, Two-, Three-, and Four-Nucleotide Substitution Mutations in the Sp1-Binding Site Found between –58 and –52

The IGFBP-3 mutant constructs were generated using site-directed mutagenesis. Cell culture, transfection, FSH treatment, and luciferase assays were done as described in Fig. 2Go. Control cells were treated with PBS. WT, Wild type; Mut, mutation.

 
Because the major FSH response elements are retained in a truncated sequence between –61 and +9, and the –61/–48 tetramer was not FSH responsive, these results pointed to the presence of an enhancer site downstream of –48 required for FSH induction. To test this hypothesis we inserted the –47/+9 sequence upstream of the –61/–48 tetramer construct. This rearranged construct was not FSH responsive (Fig. 4Go), suggesting either that the FSH responsive element is elsewhere or that it is position dependent, and requires that the sequence between –61 and +9 be intact. These issues were resolved by the experiments described below.

A TATA Box-Binding Protein Site Is Required for FSH stimulation of IGFBP-3 Transcription
To determine minimal sequence elements crucial for FSH induction of IGFBP-3, we generated serial deletion constructs with a common distal end, all of which included the 13 bp that appeared essential for the core promoter. Initially we compared luciferase activity of three constructs: –106/+9, –75/+9, and –61/+ 9. There was no difference in either basal or FSH-induced transcriptional activity of the –75/+9 or –61/+9 constructs. Although the –106 construct gave a higher basal activity, the fold change in transcription activity following FSH treatment was the same as the other two constructs. This is in agreement with previous studies showing that upstream elements enhance, but do not mediate, FSH effects (Ref. 6 and Fig. 4Go). Based on this outcome, we made four constructs with a –106 common distal end. Data from cells transfected with these constructs indicate a requirement for sequence between –39 and –25 for FSH induction of the IGFBP-3 gene (Fig. 6AGo). A similar result was found for constructs with a common –61 distal end (Fig. 6BGo). Deletion of sequence between –25 and +9 significantly reduced the basal luciferase activity. However, the fold change in FSH response was not affected, suggesting that elements in this region play an important role in basal transcription activity. Site-directed substitution mutations in the –39 to –25 region mapped the FSH response region to between –30 and –25 (Fig. 7Go). Three base substitution mutations of the –30 to –28 and the –27 to –25 sequence regions both significantly reduced the FSH response. The sequence between –30 and –25 is the predicted TATA box and a putative binding site for the TBP.



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Fig. 6. Luciferase activity of Granulosa Cells Transfected with IGFBP-3 Promoter Constructs with a Common –106 Distal End (A)

Serial deletion constructs were generated by PCR using the –191/+47 construct as template. Granulosa cells were cultured, transfected, and treated with FSH as described in Fig. 2Go. In panel B, deletion constructs with a common –61 distal end were generated to evaluate the FSH response of the sequence that contains the critical basal and FSH response elements on the IGFBP-3 promoter.

 


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Fig. 7. Luciferase Activity of Granulosa Cells Transfected with –191/+47 IGFBP-3 Promoter Constructs with 3 Base Substitution Mutations in the –39 to –25 Sequence Region of the Pig IGFBP-3 Promoter, Which Has a Putative Binding Site for the TBP

Mutant constructs were generated by site-directed mutagenesis in the context of the –191/+47 construct and validated by sequence analysis. Cells were cultured, transfected, and treated with FSH for 3 h as described in Fig. 1Go. Control cells were treated with PBS.

 
FSH-Stimulated Transcription of IGFBP-3 Involves Formation of Multiple Protein-DNA Complexes That Include Sp1, Sp3, TBP, and p300
Using a 32P-labeled –61/–25 IGFBP-3 promoter sequence probe and nuclear protein extracted from FSH-treated granulosa cells and controls without FSH, we ran gel shift assays. This assay detected four protein-DNA complexes (herein referred to as bands I, II, III, and IV) in both groups of cells (Fig. 8Go). These complexes were not detected in two negative control lanes, one without protein and the second loaded with an equivalent amount of BSA. The intensity of bands I and II was significantly higher (P < 0.02 and P < 0.04, respectively) in nuclear protein from cells treated with FSH compared with controls without FSH. Subsequent studies (see below) disclosed that band III was nonspecific. Preincubation with a 20-fold excess of the cold probe competed out all four bands. To evaluate the role of the Sp1 site between –58 and –52 (GGGGCGT) and the TBP site between –30 and –25 (TATATA) of the IGFBP-3 promoter, we generated and labeled four additional DNA probes in the –61/–25 sequence region. The first had a four base substitution mutation of the Sp1 binding site (GGAATAT). Two TBP mutant probes had three base substitution mutations in the –30/28 (CGCATA) and –27/–25 (TATGCG) regions, respectively, and the third had mutations in all 6 bases (–30/–25; AAAAAA). Bands I and II were not detected when the Sp1 site on the probe was mutated. In contrast, band IV did not appear when mutations were made in the TBP site. Band III was detected even when the mutant probes were used. To evaluate the transcription factors that bind to the –65/–25 DNA sequence, we preincubated the nuclear protein with antibodies against Sp1, Sp3, p300, and TBP (Fig. 9BGo). Antibodies against Sp1 partially shifted band I whereas preincubation with antibodies against Sp3 shifted band II. Antibodies against TBP did not have any effect. Antibodies against p300 significantly reduced the intensities of bands I and II (P < 0.002). The formation of band III was blocked in a nonspecific manner when the nuclear protein was preincubated for 15 min before adding the 32P-labeled probe, even in the absence of cold probes or antibodies (Fig. 9BGo). This was true even when preincubations were done on ice. Due to the nonspecificity and the lack of FSH responsiveness, we have not further characterized this protein-DNA interaction. Taken together, these data suggest that the Sp1 binding site participates in the formation of two protein-DNA complexes (bands I and II). Although mutation of the TBP site prevented formation of band IV, direct binding of TBP to this site could not be shown with the available antibodies. These antibodies [to rabbit transcription factor IID (TFIID] recognized a porcine form in Western blots (data not shown). However, it is possible that they may not work in gel shifts or that another of the many TBP forms that bind to this site are active in this system.



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Fig. 8. A Representative Gel Shift Assay Blot of Nuclear Protein Extracted from Cultured Granulosa Cells Treated with FSH and Vehicle-Treated Controls

Granulosa cells were cultured to 95% confluence, serum starved overnight, and treated with FSH or PBS for 3 h in serum free medium. Protein was extracted and fractionated. Nuclear protein was then probed with a 32P-labeled –61/–25 IGFBP-3 promoter DNA oligonucleotide. Lane 1 is a negative control without any protein whereas lane 2 was loaded with an equivalent amount of BSA. Protein in lane 5 was preincubated with a 20-fold excess cold probe before addition of the labeled probe. The assay was repeated four times with protein from different batches of granulosa cells.

 


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Fig. 9. A Representative Gel Shift Assays of Nuclear Protein Extracted from Granulosa Cells Treated with FSH and Probed with 32P-Labeled Mutants of the –61/–25 Probe

Granulosa cells were cultured to 95% confluence, serum starved overnight, and treated with FSH in serum-free medium for 3 h. Protein was then extracted and fractionated, and nuclear protein was probed with 32P-labeled oligonucelotide probes. In lane 1 the wild-type probe was used as a positive control. In lane 2, four bases of the Sp1 site were mutated by substituting A for G and T for C and vice versa (wild-type Sp1 site: GGGGCGT; mutant Sp1 site: GGAATAT). Substitution mutations were similarly made on the TBP site (wild-type TBP site: TATATA). Three base mutations were generated for lanes 3 (–30/–28; CGCATA) and 4 (–27/–25; TATGCG) and a six base mutation for lane 5 (–30/–25; AAAAAA). In panel B antibody interference was evaluated by preincubating the nuclear protein with antibodies against Sp1 (lane 2), Sp3 (lane 3), TBP (lane 4), and p300 (lane 5) before addition of the labeled probe. Lane 1 of panel B is the control in which nuclear protein was preincubated only in binding buffer. The experiment was repeated three times with protein from different batches of granulosa cells.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The signaling mechanism for FSH appears to be gene specific. Although the cAMP/PKA pathway is most widely accepted, other pathways seem essential for some responses. A novel finding in this study is the requirement of a TBP site for FSH-induced transcription of IGFBP-3 in pig ovarian granulosa cells. Insertion of a TBP-binding site upstream of the core promoter elements restored basal, but not FSH-activated, transcription, suggesting that the TBP plays a dual role, with mediation of FSH stimulation being position dependent. To our knowledge, this is the first time the TBP [or a TBP-associated factor (TAF)] has been shown to mediate hormonal regulation of a gene. TBP is better known as a component of the basal transcription factor TFIID, which is part of a general transcription complex that, together with polymerase II, is required for basal transcription of genes. However, TAFs have been shown to enhance recruitment of RNA polymerase II by transcription factors (10). Recently cell type-specific TAFs have been identified. The first, TAFII105, first identified in human B cell-derived TFIID complexes (11), was shown to be expressed most highly in the testis and ovary (12). In the ovary, TAFII105 was expressed exclusively in granulosa cells. Follow-up studies showed that TAFII105 null mice exhibited normal B lymphocyte development, suggesting a redundant role for TAFII105 in the regulation of B cell-specific genes (13). In contrast, female mice lacking TAFII105 are infertile due to defects in folliculogenesis (13), suggesting that TAFII105 plays a crucial role in the regulation of granulosa cell genes required for follicular development. Recently, expression of a testis-specific form of TBP-related factor 2 mRNA has been reported during mouse spermatogenesis (14). Accordingly, we hypothesize that the TBP/TAF system plays a key role in ovarian granulosa cell function.

Upstream of recruited transcription factors, the effects of FSH are initiated via FSHRs, which are solely expressed on the surface of ovarian granulosa cells and Sertoli cells in the testis. The FSHR is a member of the seven-transmembrane protein family and is coupled to adenylyl cylase via heterodimeric G proteins. In classical cAMP-mediated responses, cAMP activates the cAMP response element binding protein (CREB), which then activates cAMP-driven genes [see review (7)]. However, cAMP action has divergent outcomes, and this pathway has been shown to be more complex. In granulosa cells, there is evidence for branching of the signaling pathway upstream of CREB with cAMP being coupled to other upstream signaling pathways such as PI-3 kinase and MAPK (9, 15, 16). Based on data from the present study, it appears that the induction of IGFBP-3 represents another case of branching of the cAMP-driven signal to incorporate the PI-3 kinase and MAPK pathways. The PI-3 kinase pathway may also play an important role in phosphorylation of CREB via intermediate protein kinases. A component of the PI-3 kinase pathway, protein kinase B (PKB), was found to be essential for FSH stimulation of the aromatase gene in rat granulosa cells (17), and CREB is a regulatory target of PKB/Akt (18). Furthermore, FSH increases phosphorylation of PKB/Akt (9) and ERK (19, 20) in a cAMP- and PKA-dependent manner. The p38 MAPK, part of a kinase cascade distinct from PI-3 kinase and the Raf-MAPK kinase-ERK, is also activated by FSH, although evidence for involvement of PKA in this activation is inconclusive (9, 21). FSH activation of ERK promotes phosphorylation of a 100-kDa phosphotyrosine phosphatase, causing it to dissociate from ERK, and allows ERK to translocate to the nucleus (22). Data from the present study suggest that ERK participates in FSH stimulation of the IGFBP-3 gene in pig granulosa cells. We further show that FSH induces ERK phosphorylation.

In the present study, the minimal pig promoter sequence mediating FSH responsiveness does not have a CREB-binding site. Thus, the most obvious pathway from cAMP to gene activation is through p300, which may be an essential part of the transcription complex. Our data show that p300 participates in a critical protein complex that binds to the IGFBP-3 promoter. Studies in our laboratory have shown that FSH stimulates phosphorylation of CREB in pig granulosa cells (Qin, Z., M. A. Cunningham, and J. M. Hammond, unpublished data), and phosphorylated CREB has been shown to recruit p300 (23). This postulated pathway is shown in Fig. 10Go whereby p300 mediates CREB activity, possibly by forming complexes with Sp1, Sp3, and a gonadal TAF. The N-terminal transcriptional activation domain of the p300 homolog CBP has been shown to bind TBP (24). In addition, p300 interacts with Sp1 and Sp3 in stimulation of the p21waf1/cip1 promoter by a histone deacetylase inhibitor (25).



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Fig. 10. A Summary of the Proposed Mechanism of FSH Induction of the IGFBP-3 Gene

Binding of FSH to its receptor, FSHR, activates adenylyl cyclase via coupling with the heterodimeric G proteins. The resulting increase in cAMP activates both PKA and the PI-3 kinase. Activation of PKA causes dissociation of the catalytic subunit, which enters the nucleus and phosphorylates transcription factors such as CREB. The phosphorylated CREB then recruits the CREB binding protein homolog p300. Phospho-CREB-p300 associates with Sp1, Sp3, and TBP (or a gonadal TAF) to form a complex that stimulates IGFBP-3 transcription. The PI-3 kinase pathway may be important in the phosphorylation of the transcription factors recruited. Activation of the PI-3 kinase pathway may be partly mediated via the PKA pathway. Intermediate signaling molecules involved are yet to be determined but include ERK. Solid lines represent pathways that have been documented in granulosa cells, whereas dashed lines represent pathways suggested by our data. AC, Adenylyl cyclase; gdTAF, gonadal TAF.

 
We further show that the Sp1 family of transcription factors (and the closely related Sp3) plays a role in FSH induction of the IGFBP-3 gene. Sp1 response elements have been shown to play a crucial role in the binding of protein-DNA complexes such as Sp1, Sp3, histone deacetylase 1, ZBP-89, and the histone acetyltransferase CREB-binding protein (CBP/p300) (25, 26, 27, 28, 29). Gel shift assays in the present study demonstrate a requirement for an Sp1-binding site between –58 and –52 of the IGFBP-3 promoter for formation of two protein-DNA complexes that have a higher intensity in FSH-treated cells than in controls. The fact that antibodies against Sp1 and Sp3 shifted different protein-DNA bands in the gel shift assays suggests that both Sp1 and Sp3 may bind to this site. In rat granulosa cells Sp1 and Sp3 have been shown to have a role in the FSH induction of the Sgk gene promoter (30), another induction that requires cAMP and PI-3 kinase. The PI-3 kinase pathway may also be important for phosphorylation of Sp1/Sp3. In hepatoma cells, activation of transcription of the IGFBP-3 promoter by a histone deacetylase inhibitor, trichostatin A, was shown to require Sp1 phosphorylation and formation of an Sp1/Sp3/ histone deacetylase 1 multiprotein complex (29). In breast cancer cells and the human prostate cancer cell line PC-3, Sp1 sites within the IGFBP-3 promoter were shown to be critical in the regulation of IGFBP-3 by a histone deacetylase inhibitor, sodium butyrate (28, 31).

The present study also indicates that a component of the TFIID complex is required for FSH stimulation of the IGFBP-3 gene. In particular, the TBP site is crucial for FSH action. Thus, deletion mutations mapped this site as a crucial locus for FSH action, and mutation of this site blocked FSH effects. ERK has been shown to phosphorylate TBP, potentially enhancing its binding to the TATA box and to transcription factors (32). FSH could thus enhance the binding of TBP to the IGFBP-3 TATA box via ERK. Our studies do not exclude this possibility. However, there was no difference in the intensity of the protein-DNA complex associated with the TBP site in FSH-treated cells and controls without FSH. Further, antibodies against TBP did not shift band IV, the protein-DNA complex linked to FSH by TBP site mutations. This could reflect the fact that the antibodies to TFIID used did not recognize the TBP form binding to the porcine promoter. An alternative mechanism is that FSH stimulation enhances recruitment of a presynthesized TBP component, e.g. a TAF.

One such component, TAFII105, is highly expressed in granulosa cells (12), and TAFII105 null mice have defects in follicular development (13). Accordingly, we postulate that the required TFIID component is TAFII105 or a related protein. Although the TATA box has not been shown previously to modulate hormonal regulation of genes, the IMD2 gene requires the TATA box for activation in response to guanylic nucleoside (33). TAFII55 is also important in transcriptional activation of the AP-1 gene by c-Jun (34). In the cyclin D1 gene, another TAF, TAFI, serves two independent functions, one at the core promoter to enhance recruitment of RNA polymerase II by transcription factors, and another at an upstream activating Sp1 site (35). In the fruit fly, TAFIIcan, a homolog of TAFII80 has been shown to be a critical regulator of the gene expression program that directs gametogenesis (36). In his review, Peter (37), suggests that a granulosa cell-specific TFIID complex containing TAFII105 may be selectively recruited by a cell- and stage-specific activator. FSH is one activator that fits this hypothesis. We were not able to establish whether TAFII105 is required in this system due to unavailability of antibodies that cross-react with the pig. We are currently developing peptides and antibodies to examine this possibility. The –61/–25 segment of the IGFBP-3 promoter, which has the elements essential for FSH induction, is very similar in the human, bovine, pig (6, 38, 39), and a mouse cDNA clone (Oliver, K., 2002, GenBank accession no. AL607124). Furthermore, the Sp1 and TBP sites are identical in all three promoters, suggesting that a common mechanism of FSH/cAMP regulation of IGFBP-3 may be employed in these species.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
FSH was purchased from Dr. Albert Parlow at the National Hormone and Peptide Program (Harbor-University of California-Los Angeles Medical Center, Torrance, CA). Forskolin, LY294002, H89, U0126, and okadoic acid were purchased from EMD Biosciences (San Diego, CA). Both H89 and forskolin were constituted in DMSO to a concentration of 10 mM and used at final concentrations of 10 µM, whereas LY294002 was supplied as a 10 mM solution and used at a final concentration of 10 µM. U0126 was constituted in DMSO and used at a final concentration of 0.5–5.0 µM. 32P-labeled deoxy-GTP was purchased from NEN Life Science Products (Boston, MA). A luciferase assay kit and reporter lysis buffer were purchased from Promega Corp. (Madison WI) whereas the ß-galactosidase assay kit was from BD Biosciences (Palo Alto, CA). The following reagents were purchased from Invitrogen Life Technologies, Inc. (Gaithersburg, MD): fetal bovine serum, lipofectin, gentamicin, and OPT-MEM 1 medium. Cell culture medium was supplied by the Department of Microbiology and Immunology, Pennsylvania State University, College of Medicine (Hershey, PA). Polyclonal antibodies against Sp1, Sp3 and TBP, ERK1, and a monoclonal mouse phospho-ERK antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and a monoclonal p300 antibody was purchased from Abcam (Cambridge, UK). DNA oligonucleotides were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). The pLUC vector used for subcloning was a gift from Dr. Rich Day (University of Virginia, Charlottesville, VA). Falcon brand culture dishes were purchased from BD Biosciences (Franklin Park, NJ). Leupeptin, aprotinin, antipain, and general laboratory reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Granulosa Cell Culture
Granulosa cells were recovered from slaughterhouse pig ovaries using the dissection method previously described (6, 40, 41, 42). Primary granulosa cells were cultured in 10% horse serum to confluence, trypsinized, and frozen in liquid nitrogen until used. These passaged cells were then thawed and cultured in 10% fetal bovine serum and treated as described in each experiment.

Generation of IGFBP-3 Promoter Constructs
IGFBP-3 promoter constructs were generated by PCR employing our published sequence (GenBank accession no. AY464121) and cloned into a luciferase vector, pLUC, as previously described (6). Presence of insert in each construct was verified by agarose gel electrophoresis and confirmed by sequence analysis. In a previous study, we demonstrated that the FSH response required a 13-bp nucleotide sequence between –61 and –48 of the IGFBP-3 gene promoter (6). We used a –191/+47 IGFBP-3 promoter construct as template to generate mutant plasmid constructs in this 13-bp sequence segment. In previous experiments, the –191/+47 IGFBP-3 promoter construct was shown to have maximal reporter activity in response to FSH treatment. A QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) was used in accordance with the manufacturer’s instructions. Because of the high GC content of the template, 1.3 M betaine (Sigma-Aldrich, St. Louis, MO) was added to the PCR mix and the annealing temperature was raised to 62 C. Sequence for the promoter constructs was confirmed by sequence analysis using an ABI PRISM 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA) at the Hershey Medical Center Core Facility. In the mutants, nucleotide A was substituted for G, T was substituted for C, and vice versa. A total of four mutants with 1, 2, 3, and 4 base substitutions, respectively, were generated in this region (Fig. 3Go). Plasmid DNA from these constructs was used for transient transfection of granulosa cells. The wild-type –191/+47 IGFBP-3 promoter construct was used as a positive control, and the promoterless pLUC vector was used as a negative control. A second set of 3 base substitutions in the –40 to –25 promoter sequence was similarly generated and cloned into the pLUC vector.

Granulosa Cell Transfection and Reporter Assays
Granulosa cells were seeded at densities of 1 x 10–5 in 60-mm Falcon brand culture dishes in 10% serum and incubated at 37 C in a CO2 incubator to 60% confluence (usually 18 h). Transient transfection was done using the Lipofectin method according to the manufacturer’s instructions (Invitrogen Life Technologies, Inc., Carlsbad, CA). Briefly, cells were incubated in serum free OPTI MEM 1 medium for 2 h. A mixture of IGFBP-3 promoter construct plasmid (1 µg per dish), ß-galactosidase plasmid (50 ng per dish), and Lipofectin, all in OPTMEM I medium, was then added and the cells were incubated for another 6 h. Cell culture was continued in 10% serum to 90–95% confluence. The cells were then serum starved overnight and FSH was added at a concentration of 100 ng/ml in serum free medium for 3 h. Cells were preincubated with the signaling pathway inhibitors, where applicable, for 30 min before addition of FSH. Control cells were treated with PBS or DMSO, as appropriate. The cells were then lysed in reporter lysis buffer, and assays were done using Promega’s luciferase assay kit and BD Biosciences’ ß-galactosidase assay systems. Both luciferase and ß galactosidase activity were read in a Monolight luminometer (BD Pharmingen, San Diego, CA).

Protein Extraction and Fractionation
For protein extraction, granulosa cells were seeded in 100-mm Falcon brand dishes and cultured in 10% serum to 90–95% confluence. The cells were serum starved overnight and treated with 100 ng FSH/ml for 3 h. To recover cytoplasmic protein, cells were washed with PBS and lysed in a hypotonic buffer [10 mM HEPES, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; 0.5 mM dithiothreitol; 1 mM phenylmethylsulfonylfluoride (PMSF); 1 mM Na3VO4; 1 mM NaF3; 2 µg/µl leupeptin; 2 µg/µl aprotinin]. The lysate was allowed to sit on ice for 20 min and centrifuged at 4 C, 14,000 x g, for 10 min. To extract nuclear protein, the pellet was resuspended in a high-salt buffer (20 mM HEPES, pH 7.9; 420 mM NaCl; 1.5 mM MgCl2; 0.5 mM dithiothreitol; 0.2 mM EDTA; 1 mM PMSF; 1 mM Na3VO4; 1 mM NaF3; 2 µg/µl leupeptin; 2 µg/µl aprotinin; and 25% glycerol) and allowed to sit on ice for another 20 min. It was then centrifuged at 14,000 x g, 4 C for 10 min, and the supernatant containing the nuclear extract protein was frozen at –80 C until used. Protein levels were quantified using a protein assay reagent from Bio-Rad Laboratories, Inc. (Hercules, CA) and BSA protein standards, with the optical density being read at 595{lambda}. Previous studies with this technique have indicated that nuclear protein is minimally contaminated with cytoplasmic protein (16).

EMSAs
Nuclear protein extracted from cells treated with FSH and from controls without FSH was used. A DNA oligonucleotide, (36 bases long; –61 to –25 on the pig IGFBP-3 promoter) was labeled with 32P using Klenow (Promega Corp.) and used as a probe. In the binding reaction, 50,000 cpm of labeled DNA oligonucleotide was added to 10 µg protein in a buffer consisting of 50 mM HEPES (pH 7.6), 200 mM NaCl, 5 mM EDTA, 20% glycerol, and 0.2 µg/µl poly-(dI-dC). The reaction was allowed to sit for 15 min at room temperature and resolved on a 5% polyacrylamide gel. For supershift reactions, the protein was first preincubated with 2 µg antibody (Sp1, Sp3, TBP, or P300) for 15 min before addition of the labeled probe. For competition assays, the protein was preincubated with a 20-fold excess of cold oligonucleotide before addition of the labeled probe. Electrophoresis was carried out at 120 V for 3 h at room temperature. The gel was then dried, exposed to x-ray film, and subjected to densitometric analysis.

Western Blot Analysis
Granulosa cells were cultured as described to 95% confluence, serum starved overnight, and treated with 100 ng/ml FSH for 3 h. Preincubation in 0.5–5.0 µM U0126 was done for 30 min before the FSH was added where appropriate. The cells were then scrapped in 300 µl lysis buffer (20 mM HEPES, pH 7.9; 150 mM NaCl; 1% Nonidet P-40; 0.1% sodium dodecyl sulfate; 1 mM Na3OV4; 1 mM NaF; 10 mM PMSF; 20 µg/µl aprotinin; 2 µg/µl leupeptin; 0.1 µM okadoic acid; and 2 µg/ml antipain). The lysate was allowed to sit on ice for 30 min and centrifuged at 14,000 x g for 10 min at 4 C. Protein concentrations in the supernatant were determined using a protein assay reagent (Bio-Rad) and BSA protein standards. Electrophoresis was carried out in NuPage 4–12% Bis-Tris gels (Invitrogen Life Technologies, Inc.) at 200 V. Protein was transferred to nitrocellulose membranes at 100 V for 2 h, blocked in 5% fat-free milk for 1 h, and incubated with primary antibody (1:5000 dilution) for 1.5 h at room temperature or overnight at 4 C. After three 10-min washes, the membranes were incubated with secondary antibody (1:3000 dilution) for 1.5 h at room temperature (or overnight at 4 C) and washed for 10 min three times. Antigen-antibody complexes were visualized using chemiluminescence (Amersham Pharmacia, Piscataway, NJ) following exposure to Biomax light film (Eastman Kodak, Rochester, NY). The membrane was stripped and probed with antibody against total ERK.

Statistical Analysis
Transfections were done in triplicate and repeated at least three times with different batches of granulosa cells. Luciferase activity was normalized by the ß-galactosidase activity. Gel shift assays were also performed at least three times with protein from different batches of granulosa cells. Data were analyzed using ANOVA (GraphPad, Inc, San Diego, CA) with P ≤ 0.05 being considered significant. Values reported are mean ± SEM.


    ACKNOWLEDGMENTS
 
We thank Dr. Qin Zhu of the Endocrinology Division, Hershey Medical Center, for valuable critique of our data.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grant HD24564 and The Pennsylvania State University College of Medicine Dean’s Feasibility Grant.

First Published Online February 17, 2005

Abbreviations: CBP, CREB-binding protein; CREB, cAMP response element-binding protein; DMSO, dimethyl sulfoxide; FSHR, FSH receptor; H89, N-[2-(p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl; IGFBP-3, insulin-like growth factor binding protein-3; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PI-3 kinase, phosphatidyl inositol-3 kinase; PKA, protein kinase A; PKB, protein kinase B; PMSF, phenylmethylsulfonylfluoride; Sp1, specific protein 1; Sp3, specific protein 3; TAF, TBP associated factor; TBP, TATA box-binding protein; TFIID, transcription factor IID; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene.

Received for publication December 3, 2004. Accepted for publication February 8, 2005.


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