Steroidogenic Factor-1 and The Gonadotrope-Specific Element Enhance Basal and Pituitary Adenylate Cyclase-Activating Polypeptide-Stimulated Transcription of the Human Glycoprotein Hormone {alpha}-Subunit Gene in Gonadotropes

Robert C. Fowkes, Marion Desclozeaux, Mayur V. Patel, Simon J. B. Aylwin, Peter King, Holly A. Ingraham and Jacky M. Burrin

Department of Endocrinology, Barts and Royal London School of Medicine and Dentistry (R.C.F., M.V.P., S.J.B.A., P.K., J.M.B.), West Smithfield, London, United Kingdom EC1A 7BE; and Department of Physiology, University of California, San Francisco, California 94143-0444

Address all correspondence and requests for reprints to: Dr. R. C. Fowkes, Department of Physiology, S-1479, 513 Parnassus Avenue, University of California, San Francisco, California 94143-0444. E-mail: rfow0187{at}itsa.ucsf.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the anterior pituitary, expression of the common glycoprotein hormone {alpha}-subunit ({alpha}GSU) is mediated in part by multiple response elements residing in the distal promoter (-435 bp). One such site is the gonadotrope-specific element (GSE), which is bound by the orphan nuclear receptor steroidogenic factor-1 (SF-1) and confers pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated {alpha}GSU expression. Here we investigated the functional importance of the GSE and SF-1 phosphorylation in both basal and stimulated {alpha}GSU transcription. Mutation of the GSE reduced basal and PACAP-stimulated {alpha}GSU promoter activity in the {alpha}T3-1 gonadotrope cell line. Overexpression of wild-type SF-1, but not an S203A mutant form of SF-1, enhanced basal and PACAP-stimulated {alpha}GSU promoter activity. The effect of PACAP on {alpha}GSU promoter activity was inhibited after overexpression of MAPK phosphatase. Helix assembly of the SF-1 ligand-binding domain was stimulated by PACAP in vitro via a MAPK-dependent pathway, as determined using a mammalian two-hybrid assay. PACAP quickly activated MAPK (within 5 min) and also resulted in elevated levels of phospho-cAMP response element-binding protein and phospho-SF-1, as judged by a specific antiphospho-S203 antibody; this effect was blocked by the MAPK kinase inhibitor, UO126. Collectively, these data demonstrate that SF-1 binds to the GSE and activates both basal and PACAP-stimulated {alpha}GSU transcription, which is further increased by phosphorylation at Ser203 via MAPK. These data suggest strongly that the induction of {alpha}GSU gene expression by peptide hormone signaling is coupled directly to the posttranslational status of SF-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE SYNTHESIS OF the pituitary glycoprotein hormones LH, FSH, and TSH is essential for the regulation of normal reproductive function, growth, and metabolism. Each of these hormones is composed of a common glycoprotein hormone {alpha}- subunit ({alpha}GSU) and a unique ß-subunit that conveys specificity (1). Pituitary-specific expression of the human {alpha}GSU has been attributed to multiple response elements that reside within the first 435 bp of the promoter (2, 3, 4). Transfection studies in the clonal gonadotrope-derived {alpha}T3-1 cell line have identified elements important for gonadotrope- specific expression, including a pituitary glycoprotein basal element (5), a gonadotrope-specific element (GSE), the cAMP response elements (CREs), and the upstream regulatory element (2, 3).

The consensus GSE sequence (TGACCTTGT) in the human {alpha}GSU promoter interacts with the transcription factor steroidogenic factor-1 (SF-1) (6, 7) and {alpha}GSU reporter constructs that contain the GSE are expressed at higher levels in cell lines that contain endogenous SF-1 than cells that lack SF-1, consistent with a role for SF-1 in cell-specific transcriptional activation of the {alpha}GSU gene (2, 7). SF-1 is a nuclear transcription factor that has well defined roles in adrenal and gonadal development and steroidogenesis (8) and has also been identified in the pituitary, where it is localized to the gonadotrope (6). SF-1 has been shown to increase basal transcriptional activation of heterologous promoter constructs containing GSE consensus sites (7) and regulate LHß-subunit promoter activity (9), suggesting that SF-1 can function as a regulator of basal gonadotropin subunit transcriptional activity. Furthermore, SF-1 has been shown to interact (both physically and functionally) with several other transcription factors and coactivators/repressors in a range of tissues (8). In particular, SF-1 is involved in the cAMP-regulated expression of various genes, including the bovine Cyp17 gene and the rat inhibin {alpha} gene (10, 11). The role of SF-1 in cAMP responsiveness of the human {alpha}GSU in gonadotropes has yet to be reported.

The regulation of SF-1 activation has focused recently on the phosphorylation status of this orphan nuclear receptor. The phosphorylation status of SF-1 has been shown to be altered by cAMP/protein kinase A (PKA) in several tissues, a posttranslational modification that can modify SF-1-mediated gene transcription. PKA activation increases SF-1 phosphorylation in vitro, an effect that apparently regulates SF-1- enhanced basal and cAMP-stimulated transcription of the rat p450c17 gene (12). In contrast, cAMP stimulation causes the recruitment of protein phosphatases and subsequent dephosphorylation of SF-1 in human adrenal H295R cells, resulting in an increase in SF-1-mediated Cyp17 expression (13, 14). Other investigators have reported that PKA failed to phosphorylate SF-1 in H295R cells at a putative PKA phosphorylation site (Ser430), but acknowledged the potential for serine/threonine protein kinases to phosphorylate SF-1 (15). More compelling observations revealed a consensus MAPK phosphorylation site that resides in the hinge region of SF-1, N-terminally located to the ligand-binding domain, at Ser203. Epidermal growth factor stimulation of SF-1-transfected HEK293 cells resulted in an increase in SF-1 phosphorylation at this residue, an enhancement of SF-1 target gene transcription, and recruitment of SF-1 coactivators (16). Overexpression of MAPK phosphatase-1 (MKP-1) or mutation of this serine residue to alanine blocked these effects of SF-1, but did not alter the binding characteristics of SF-1 to its consensus response elements (16). Most recently, the Ser203 residue has been shown to act as a surrogate ligand for SF-1 by altering the protein conformation to mimic ligand binding to a nuclear receptor (17). However, the phosphorylation status of SF-1 in gonadotropes under basal or hormone-stimulated conditions remains to be elucidated.

Almost all proposed SF-1 target genes are responsive to peptide hormones that transduce signals through G protein-coupled receptors. One such peptide hormone is pituitary adenylate cyclase-activating polypeptide (PACAP), a putative hypophysiotropic factor that acts via G protein-coupled receptors as a potent stimulator of {alpha}GSU transcription in primary pituitary cells (18) and in {alpha}T3-1 cells (19). The signal transduction pathways that mediate these transcriptional effects are still not fully characterized. PACAP has been shown to stimulate cAMP production and inositol phosphate turnover, increase the cytosolic free Ca2+ concentration (20), and activate a member of the MAPK family, ERK1/2, in {alpha}T3-1 cells (21), acting predominantly via splice variants of the PAC1-R receptor. Activation of all of these intracellular signaling pathways has been shown to increase {alpha}GSU gene expression and transcription (18, 19, 22, 23, 24, 25). The effects of PACAP on {alpha}GSU transcription are potent and rapid, with increased {alpha}GSU promoter activity occurring within 2 h (19). Previous work from our laboratory has suggested that full transcriptional activation of the {alpha}GSU by PACAP requires an intact CRE, but also involves sequences between -244 and -195 bp of the human {alpha}GSU promoter, the region that contains the consensus GSE sequence (19). We have also shown this region to regulate basal promoter activity and contribute to protein kinase C (PKC)/ERK-mediated {alpha}GSU transcription in the LßT2 gonadotrope cell line (25).

In this study we have investigated the functional importance of both the GSE and SF-1 phosphorylation in mediating basal and PACAP-stimulated {alpha}GSU transcription in gonadotrope cells. Our data support a model in which posttranslational modification of SF-1 at Ser203, mediated by PACAP activation of the MAPK signaling pathway, contributes to {alpha}GSU gene expression via the GSE in {alpha}T3-1 gonadotropes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutation of the SF-1-Binding Site Reduces PACAP and cAMP-Stimulated Increases in {alpha}GSU Promoter Activity
The region between -219 and -211 of the human {alpha}GSU promoter contains a consensus SF-1-binding site (TGACCTTGT) known as the GSE. Deletion or mutation of the GSE has demonstrated its importance in basal {alpha}GSU promoter activity in gonadotropes, but not in thyrotropes or placental cells (2, 3). To delineate the role of the GSE in mediating the human {alpha}GSU promoter, a 2-bp mutation was introduced into the GSE (from CC to TT) of the -517{alpha}LUC promoter construct to form -517{alpha}MUT (Fig. 1AGo). We examined this 2-bp mutation in the GSE, which resulted in loss of DNA binding by SF-1, as demonstrated by EMSA using gonadotrope {alpha}T3-1 cells and adrenocortical Y1 cells (Fig. 1BGo). This mutation substantially decreased basal promoter activity to 39.6 ± 5.0% of the wild-type transcription (P < 0.001; Fig. 1CGo). These studies confirm a role for the GSE in regulating basal human {alpha}GSU promoter activity in {alpha}T3-1 cells.



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Fig. 1. Mutation of the GSE Blocks SF-1 Binding and Reduces Basal and PACAP-Stimulated {alpha}GSU Promoter Activity in {alpha}T3-1 Gonadotropes

A, Schematic depiction of the proximal region of the wild-type (-517{alpha}LUC) and GSE mutant (-517{alpha}MUT) human {alpha}GSU promoter constructs. PGBE, Pituitary glycoprotein hormone basal element. B, EMSAs were performed with {alpha}T3-1 and Y1 nuclear extracts and 32P-labeled oligonucleotides encoding the wild-type or mutant GSE. C, Transient transfection of {alpha}T3-1 cells with -517{alpha}LUC or -517{alpha}MUT. Cells were incubated for 8 h after transfection before harvesting. LUC activity is expressed as arbitrary light units (ALU) normalized for ß-galactosidase activity. The mean fold increase over -517{alpha}MUT is indicated above the bar. Each bar represents the mean ± SEM of at least three independent experiments, each performed in triplicate. ***, P < 0.001 compared with the ALU for -517{alpha}MUT. D, Transiently transfected {alpha}T3-1 cells were untreated or treated with 100 nM PACAP or 0.5 mM 8-Br-cAMP and harvested 8 h after transfection. LUC activity is expressed as ALU normalized for ß-galactosidase activity. The data are normalized to the basal expression of each promoter construct (dotted line) to account for differences in basal activity. The mean fold increase over basal is indicated above the bar. Each bar represents the mean ± SEM of at least three independent experiments, each performed in triplicate. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (significantly different from relevant basal, except where indicated by brackets).

 
To assess the effect of the mutant GSE on PACAP-stimulated {alpha}GSU promoter activity, {alpha}T3-1 cells transfected with either -517{alpha}LUC or -517{alpha}MUT were stimulated for 8 h with PACAP (100 nM) or 8-bromo-cAMP (8-Br-cAMP; 0.5 mM; Fig. 1DGo). The enhancements of basal promoter activity seen with PACAP (7.3 ± 0.7-fold; P < 0.001) and 8-Br-cAMP (4.9 ± 1.1-fold; P < 0.001) were significantly reduced to 50.0 ± 11.0% and 45.4 ± 5.0% of wild-type responses (for PACAP and 8-Br-cAMP, respectively, P < 0.01 and P < 0.05; Fig. 1DGo). However, a significant 3.1 ± 0.7-fold stimulation above basal promoter activity was still evident after PACAP stimulation of the -517{alpha}MUT promoter (P < 0.05), which suggests that the PACAP effect may be mediated through multiple response elements. These data support our original findings that the region between -244 and -195 bp of the {alpha}GSU promoter contributes to the PACAP effect on {alpha}GSU transcription and for the first time demonstrate that the GSE within this region is involved. By implication, these findings also suggest that SF-1 can mediate basal and PACAP-stimulated {alpha}GSU promoter activity in {alpha}T3-1 cells.

PACAP-Stimulated {alpha}GSU Promoter Activity Requires an Intact Ser203 Residue in SF-1 and MAPK Activity
Having shown the role of the GSE in mediating part of the PACAP effect on {alpha}GSU promoter activity, we next investigated the role of SF-1. {alpha}T3-1 cells express high levels of endogenous SF-1, similar to those seen in Y1 adrenal cortical cells (Fowkes, R. C., and P. King, unpublished observations). We cotransfected {alpha}T3-1 cells with -517{alpha}LUC and increasing concentrations of either wild-type or S203A mutant SF-1. The Ser203 residue in SF-1 is a consensus MAPK phosphorylation site and is required for full transcriptional activity and coactivator recruitment (16, 17). Overexpression of wild-type SF-1 significantly enhanced {alpha}GSU promoter activity by up to 7.4 ± 1.5-fold over basal (P < 0.01), whereas expression of the S203A mutant SF-1 failed to significantly alter basal {alpha}GSU promoter activity (Fig. 2AGo). To determine whether the S203A mutation affected PACAP stimulation of -517{alpha}LUC activity, the cotransfected cells were stimulated with 0 or 100 nM PACAP for 8 h. As shown (Fig. 2BGo), the presence of the S203A mutant SF-1 consistently attenuated the PACAP effect by at least 40% (to 51.1 ± 11.0% and 61.0 ± 3.2% of the response seen in wild-type transfected cells; P < 0.05). These data suggest that the Ser203 phosphorylation site in SF-1 is important for both basal and PACAP-stimulated {alpha}GSU promoter activity in {alpha}T3-1 cells.



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Fig. 2. Role of Ser203 in SF-1 and MAPK Activity in Mediating PACAP-Stimulated {alpha}GSU Promoter Activity in {alpha}T3-1 Gonadotropes

A, {alpha}T3-1 cells were cotransfected with -517{alpha}LUC (200 ng), wild-type SF-1 or S203A-SF-1 (100 and 200 ng), and BosßGAL (100 ng) and harvested 24 h after transfection. The data shown are representative of two such experiments, each performed in quadruplicate (mean ± SEM), normalized to the basal promoter activity, and expressed as fold increases. **, P < 0.01; *, P < 0.05 (significantly different from basal). B, {alpha}T3-1 cells were transfected as described above, stimulated with 100 nM PACAP for 8 h, and harvested 24 h after transfection. The data shown are representative of two such experiments, each performed in quadruplicate (mean ± SEM), and normalized to the control PACAP response, expressed as 100%. *, P < 0.05 (significantly different from wild-type SF-1). C, {alpha}T3-1 cells were transiently transfected with -517{alpha}LUC (200 ng), BosßGAL (100 ng), and increasing concentrations of MKP-1 (0–125 ng) and were stimulated for the last 8 h with 0 or 10 nM PACAP, 24 h after transfection. The data are normalized to basal -517{alpha}LUC promoter activity. Each bar represents the mean ± SEM of at least two independent experiments, each performed in quadruplicate. ***, P < 0.001; *, P < 0.05 [significantly different from control (0 ng MKP-1)].

 
We next investigated the role of MAPK in mediating PACAP-stimulated {alpha}GSU transcription. Our previous investigation of the role of PACAP-stimulated MAPK activation revealed that UO126 [a specific MAPK kinase (MEK) inhibitor] pretreatment alone was unable to significantly inhibit the potent PACAP effect on {alpha}GSU promoter activity (21). Other researchers have reported the inhibition of the PACAP effect on the mouse {alpha}GSU promoter using the PKA inhibitor H-89 (26), but the concentration required for inhibition was over 200-fold greater than the Ki for H-89 on PKA. At such high concentrations, H-89 is known to inhibit other protein kinases (27). Using a wide range of pharmacological inhibitors of PKA (H-89 and PKi), PKC (GF109203X), PI3-K (wortmannin), Ca2+ entry (nifedipine), or calmodulin K (W-7) at their established Ki values, we have been unable to inhibit the potent effect of PACAP on the human {alpha}GSU promoter in {alpha}T3-1 cells (21) (data not shown). Therefore, we cotransfected {alpha}T3-1 cells with -517{alpha}LUC and increasing concentrations of the MKP-1 expression vector (0–125 ng), then stimulated them with 10 nM PACAP for 8 h (Fig. 2CGo). Overexpression of MKP-1 significantly attenuated the PACAP-stimulated {alpha}GSU promoter activity in a concentration-dependent manner (to 76.3 ± 8.9%, 57.8 ± 1.8%, and 33.7 ± 1.0% of the control response; P < 0.05, P < 0.01, and P < 0.01, respectively). Collectively, these data strongly suggest that PACAP stimulates {alpha}GSU promoter activity via a mechanism that involves MAPK activation and implies that phosphorylation of Ser203 in SF-1 is required.

Role of MAPK Activation in PACAP Stimulation of SF-1 Ligand-Binding Domain (LBD) Activity and {alpha}GSU Transcription in {alpha}T3-1 Cells
Previous studies in our laboratory have established that SF-1 adopts an active conformation upon MAPK-dependent phosphorylation at Ser203 (17), in a novel mechanism that mimics ligand binding. In addition, phosphorylation at Ser203 increases the recruitment of the truncated LBD (helix 2–12) by the hinge-helix 1 portion of SF-1 (helix assembly assay using the two-hybrid system; see Fig. 3AGo). To determine whether PACAP treatment of {alpha}T3-1 gonadotropes could cause similar changes in SF-1 LBD activity, we investigated the interaction between the hinge-helix 1 region of SF-1 (which contains the Ser203 residue) with helixes 2–12 of SF-1, using GAL4 expression plasmids in a mammalian two-hybrid assembly assay (17). {alpha}T3-1 cells were cotransfected with wild-type pGAL4-SF-1-hinge-helix 1, helixes 2–12 of SF-1 fused to the activation domain of VP16, a GAL4 response element-luciferase (LUC) reporter and the MKP-1 expression vector, before stimulation with 0 or 100 nM PACAP for 8 h (Fig. 3BGo). PACAP significantly enhanced SF-1 helix assembly by 46.5 ± 5.3-fold over pGAL4 (P < 0.001). After overexpression of MKP-1, the PACAP effect on SF-1 LBD helix assembly (Fig. 3BGo) was significantly attenuated to 64.2 ± 4.3% (P < 0.001). MKP-1 overexpression failed to alter basal helix assembly (P > 0.5).



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Fig. 3. Role of MAPK in SF-1 Helix Assembly in Response to PACAP

A, Schematic representation of the mammalian two-hybrid assay. B, {alpha}T3-1 cells were cotransfected with pGAL4 or GAL4SF-1 hinge-helix-1, VP-16SF-1, GAL4-RE-LUC, BosßGAL, and MKP-1 (125 ng) before stimulation with 0 or 100 nM PACAP for 8 h. The data shown are normalized to basal pGAL4 activity and expressed as the fold increase. Each bar represents the mean ± SEM of at least three independent experiments, each performed in triplicate. ***, P < 0.01 (significantly different as indicated). C, {alpha}T3-1 cells were cotransfected with pGAL4 (100 ng) or GAL4SF-1 hinge-helix-1 (100 ng), VP-16SF-1 (100 ng), BosßGAL (100 ng), and GAL4-RE-LUC (200 ng) and pretreated with 0 or 1 µM UO126 for 30 min before stimulation with 0 or 100 nM PACAP for 8 h. The data shown are normalized to basal pGAL4 activity, and expressed as the fold increase. Each bar represents the mean ± SEM of at least three independent experiments, each performed in triplicate. *, P < 0.05 (significantly different as indicated). D, {alpha}T3-1 cells were cotransfected with pGAL4 (100 ng), GAL4SF-1 hinge-helix-1 (100 ng), or S203A-GAL4SF-1 hinge-helix-1 (100 ng), VP-16SF-1 (100 ng), BosßGAL (100 ng), and GAL4-RE-LUC (200 ng) and stimulated with 0 or 100 nM PACAP for 8 h. The data shown are normalized to basal pGAL4 activity and expressed as the fold increase. Each bar represents the mean ± SEM of at least two independent experiments, each performed in quadruplicate. ***, P < 0.001 (significantly different as indicated).

 
To establish whether the UO126 could elicit effects similar to MKP-1 overexpression, {alpha}T3-1 cells were cotransfected with wild-type pGAL4-SF-1-hinge-helix 1, helixes 2–12 of SF-1 fused to the activation domain of VP16, and a GAL4 response element-LUC reporter and pretreated with 0 or 1 µM UO126 for 30 min before stimulation with 0 or 100 nM PACAP for 8 h. Pretreatment with UO126 significantly reduced PACAP-stimulated helix assembly to 53.5 ± 9.0% (P < 0.05) without significantly altering the effect on basal helix assembly (Fig. 3CGo).

The inhibitory effects of MKP-1 and UO126 on PACAP-stimulated SF-1 helix assembly strongly support a role for MAPK in the regulation of SF-1 LBD activity. To confirm this, we performed similar mammalian two-hybrid assays comparing the activities of wild-type pGAL4-SF-1-hinge-helix 1 and S203A pGAL4-SF-1-hinge-helix 1 after stimulation with 0 or 100 nM PACAP. As shown (Fig. 3DGo), the S203A mutation significantly attenuated PACAP-stimulated SF-1 helix assembly (to 16.0 ± 7.6% of the wild-type response; P < 0.001), although basal helix assembly was not significantly affected (P > 0.5). Collectively, these data show that PACAP enhances SF-1 helix assembly via a MAPK and Ser203-dependent mechanism, suggesting that PACAP may enhance SF-1 phosphorylation at Ser203.

PACAP Enhances Phosphorylation of Multiple Downstream Signaling Targets in {alpha}T3-1 Cells
To establish the potential signaling cascade initiated by PACAP in {alpha}T3-1 cells, we performed Western blotting for known cAMP target proteins. Total cell lysates were made from {alpha}T3-1 cells stimulated with or without 100 nM PACAP for up to 8 h, mimicking the time course performed for the transient transfection analysis. Using phospho-specific primary antibodies, we found that PACAP enhanced CRE-binding protein (CREB) phosphorylation within 5 min, an effect that lasted for at least 30 min (Fig. 4AGo, upper panel). As SF-1 is phosphorylated by MAPK on Ser203, we sought to confirm our previous observations that PACAP could activate the ERK1/2 pathway in {alpha}T3-1 cells (21). As expected, PACAP transiently increased ERK1/2 phosphorylation at 5 min (Fig. 4BGo, middle panel). As new cAMP-activated signaling pathways have recently been elucidated, we examined whether PACAP could activate the PKB/Akt pathway in {alpha}T3-1 cells. Phosphorylated Akt was detected in untreated and PACAP-stimulated protein extracts at all time points, but PACAP failed to enhance the level of phosphorylation, suggesting that the PACAP signaling pathway in {alpha}T3-1 cells does not activate the PKB/Akt cascade (Fig. 4AGo, bottom panel).



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Fig. 4. Activation of Multiple Signaling Components by PACAP in {alpha}T3-1 Cells

A, Total proteins were extracted from {alpha}T3-1 cells serum-starved for 2 h before stimulation with 0 or 100 nM PACAP for the indicated time periods. Western blotting was performed for phospho-CREB, total-CREB, phospho-ERK, total-ERK, phospho-Akt, and total-Akt. The gels shown are representative of three such experiments. B, Total proteins were extracted from {alpha}T3-1 cells serum-starved for 2 h before stimulation with 0 or 100 nM PACAP for the indicated time period. Western blotting was performed for phospho-SF-1, total SF-1, and ß-actin (as a loading control). The gels shown are representative of three such experiments. C, Total proteins were extracted from {alpha}T3-1 cells serum-starved for 2 h before incubation with 0 or 1 µM UO126 or 1 µM GF109203X for 30 min before stimulation with 0 or 100 nM PACAP for 5 min. Western blotting was performed for phospho-SF-1, phospho-Elk-1, and ß-actin. The gels shown are representative of two such experiments.

 
We previously failed to observe any changes in SF-1 mRNA (by real-time RT-PCR), SF-1 promoter activity (reporter gene assays), or SF-1 protein levels (by Western blotting) after PACAP treatment of {alpha}T3-1 cells (28). Therefore, we next examined whether posttranslational modification of SF-1 occurred in {alpha}T3-1 gonadotropes. SF-1 is phosphorylated by MAPK on Ser203 in vitro (16, 17). Using a specific antibody directed against the phosphorylated Ser203 residue of SF-1 (17), we performed Western blotting for phospho-SF-1 and found that PACAP stimulation of {alpha}T3-1 cells resulted in an enhancement of SF-1 phosphorylation within 5 min that was maintained at 15 min (Fig. 4BGo). Levels of total SF-1 and ß-actin (Fig. 4BGo, lower panel) remained the same. These data suggest that increased phosphorylation of SF-1 after PACAP stimulation may contribute to the PACAP effects on {alpha}GSU promoter activity via the GSE in {alpha}T3-1 cells.

To establish which of these pathways are involved in PACAP-induced SF-1 phosphorylation, we produced total protein extracts from {alpha}T3-1 cells pretreated with pharmacological inhibitors of MEK (UO126, 1 µM) and PKC (GF109203X, 1 µM) and then stimulated cells with 100 nM PACAP for 5 min before Western blotting for SF-1 phosphorylation and ERK activity (as determined by Elk-1 phosphorylation). PACAP-stimulated SF-1 phosphorylation and ERK activation were both blocked by UO126 and GF109203X, implying that the PACAP-stimulated enhancement of SF-1 phosphorylation is MAPK dependent in {alpha}T3-1 cells (Fig. 4CGo). Similar experiments using inhibitors of PKA (KT5720 and H-89, both at 100 nM) failed to significantly reduce SF-1 phosphorylation at Ser203 (data not shown). These data are consistent with the activation of multiple signaling pathways by PACAP and demonstrate that Ser203 in SF-1 is a MAPK phosphorylation site in {alpha}T3-1 cells, as shown previously in transfected JEG-3 cells (16) and Y1 adrenal cells (17).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human {alpha}GSU promoter contains a single SF-1 binding site and two CREs. Both elements are thought to be important for basal and regulated expression of the {alpha}GSU gene in pituitary gonadotropes. We have shown that mutating the consensus GSE sequence is sufficient to markedly blunt basal and PACAP-stimulated {alpha}GSU promoter activity in gonadotropes. We also provide evidence that these effects involve phosphorylation of SF-1 at Ser203 rather than via changes in SF-1 expression.

Previous studies of the human {alpha}GSU promoter have demonstrated that the GSE is important for promoter activity in the pituitary gonadotrope cell line {alpha}T3-1 (2, 3). Horn et al. (2) mutated the GSE element in the -224 truncated human {alpha}GSU promoter by the substitution of two T nucleotides for two C nucleotides. When the activity of this mutated construct was compared with the activity of the wild-type -224 truncated construct in {alpha}T3-1 cells, they found a 50% decrease in expression, a reduction of the same magnitude as that found using a truncated construct that eliminated the GSE. We found a similar 2-fold decrease in the activity of a construct containing an identical 2-bp mutation in the GSE compared with a wild-type -517{alpha}LUC promoter. Studies using a block mutation of the GSE within the context of the naturally occurring -1500 bp promoter sequence have also shown a similar reduction in expression to 41% of full-length promoter activity (3). Additionally, we have recently shown that using the same CC to TT mutation obliterates basal {alpha}GSU promoter activity in transfected LßT2 gonadotropes (25). Thus, the presence of a GSE in the human {alpha}GSU promoter consistently enhances basal expression in pituitary gonadotropes.

The role of the GSE in hormone-regulated expression of the {alpha}GSU gene is less well defined. Previous studies have shown that cAMP analogs or peptides such as PACAP, which activate the cAMP signaling pathway, stimulate human {alpha}GSU promoter activity in primary rat pituitary cells (29) and {alpha}T3-1 cells (19). In the latter study, we demonstrated that full transcriptional activation of the {alpha}GSU by PACAP, while requiring an intact CRE, also involved the GSE-containing region of the promoter. PACAP is able to stimulate cAMP production, increase inositol phospholipid turnover, and increase the cytosolic free Ca2+ concentration in {alpha}T3-1 cells (20, 23), whereas we and others have also shown PACAP to transiently activate the ERK1/2 pathway in {alpha}T3-1 cells (21) and nonfunctioning pituitary adenomas of gonadotrope origin (30). However, pharmacological inhibition of the PKC/Ca2+/ERK pathway appears to have little impact alone on the {alpha}GSU transcriptional response to PACAP (19, 21, 31), and its transcriptional effects on the {alpha}GSU promoter may occur through stimulation of the cAMP/PKA pathway (19) or, indeed, alternative and as yet unidentified signaling pathways that may impinge on the PKC/Ca2+/ERK pathways. Dissection of the signaling pathways employed by PACAP to stimulate {alpha}GSU gene transcription is complicated due to activation of the multiple signaling pathways described above. All of these pathways have been shown to contribute to {alpha}GSU transcription (18, 19, 22, 24) in response to GnRH, PACAP, or pharmacological activators of PKA and PKC. This complexity is further compounded by the existence of cross-talk between the many pathways, as has been demonstrated between GnRH and PACAP (19, 32). Therefore, it is possible that some degree of redundancy exists within this system. Nevertheless, using overexpression of MKP-1, we have demonstrated a partial requirement for MAPK activation to mediate the PACAP effect on the human {alpha}GSU promoter. The failure to inhibit the potent transcriptional effect of PACAP on the {alpha}GSU promoter using pharmacological inhibitors supports the possibility that numerous signaling pathways are involved in mediating the PACAP response, and that compensatory mechanisms exist to overcome the effects of these inhibitors.

The studies reported here suggest for the first time that in pituitary gonadotropes, PACAP responsiveness of the {alpha}GSU promoter involves the GSE. We have shown that transfection of an {alpha}GSU promoter construct containing a mutated GSE into {alpha}T3-1 cells significantly reduced the transcriptional response to both PACAP and the cAMP analog, 8-Br-cAMP, although significant stimulation above basal promoter activity was still evident in response to PACAP. This observation implicates additional response elements and/or signaling pathways in the regulation of PACAP-stimulated {alpha}GSU promoter activity and mimics the effect of a GSE mutation on GnRH- and PKC-stimulated {alpha}GSU promoter activity in LßT2 cells (25). Our current data also reveal the novel finding that PACAP can induce CREB phosphorylation in {alpha}T3-1 cells, but does not alter the phosphorylation status of PKB/Akt in these cells. As there is some degree of cross-reactivity between phospho-CREB and phospho-activating transcription factor (phospho-ATF) proteins with the antibody employed for this study, we were also able to observe that PACAP enhanced ATF phosphorylation in {alpha}T3-1 cells as well (data not shown). Therefore, there are at least two additional candidates that may act as PACAP-responsive transcriptional regulators in {alpha}T3-1 cells. Interestingly, both have been shown to physically interact with SF-1 in vitro (11, 33).

From the results of the present study it is apparent that an intact GSE is necessary, but not sufficient, for full PACAP responsiveness, as mutations within the GSE reduced, but did not eliminate, PACAP-stimulated LUC activity compared with that in the wild-type construct when transfected in to {alpha}T3-1 cells. Classically, transcriptional activation in response to increases in intracellular cAMP has been found to be regulated by CREs that bind trans-activating factors of the CREB/ATF family. We have observed that PACAP and forskolin can potently stimulate the transcriptional activity of a GAL4-CREB expression vector in {alpha}T3-1 cells (34). It is likely that both the GSE and the CREs in the human {alpha}GSU promoter are required for transcriptional responsiveness to PACAP. Double-mutational analysis of these sites might delineate the relative contributions of these two sites, but given that individual mutations in either the GSE or the CREs results in a 50% inhibition of basal and hormone-stimulated promoter activity (current observations and Refs. 3 ,19 , and 25), it is likely that such a combined mutation would result in an unresponsive promoter construct.

Having demonstrated that an intact GSE specifically enhances basal and PACAP- and 8-Br-cAMP-stimulated {alpha}GSU transcription in {alpha}T3-1 cells, we next sought to determine whether SF-1 was involved in conferring PACAP responsiveness or augmenting the expression of the human {alpha}GSU promoter. {alpha}T3-1 cells express relatively high levels of endogenous SF-1, yet we have still shown an enhancement in basal and PACAP-stimulated {alpha}GSU promoter activity in {alpha}T3-1 cells after overexpression of wild-type SF-1, an effect that was absent when the S203A-SF-1 vector was used. We have confirmed these observations in an SF-1-negative pituitary cell line, GH3, in which transfected SF-1 increased basal and forskolin-stimulated promoter activity, effects that were blocked (basal) or reduced by at least 50% (forskolin-stimulated) in the presence of S203A-SF-1 (Fowkes, R. C., M. Desclozeaux, H. A. Ingraham, and J. M. Burrin, manuscript in preparation). This mutant SF-1 cannot be phosphorylated by MAPK, and this results in a failure to recruit coactivators in vitro (16). The actions of SF-1 are known to be dependent on cofactor binding (16) and may therefore be cell type specific. This is supported by a report by Ito et al. (11) that transfection of SF-1 into kidney cells did not activate {alpha}GSU activity. It is likely, therefore, that the observed actions involve coactivators that are expressed in a restricted cell type-specific pattern.

The effects of SF-1 are thought to be mediated by phosphorylation in response to MAPK activation enhancing coactivator recruitment (16), increasing the SF-1 DNA-binding capability (35) or phosphorylation by cAMP/PKA (12). Our current studies reveal that SF-1 phosphorylation is enhanced after PACAP stimulation, an effect apparently mediated via ERK activation. This would support previous observations of the mechanism of SF-1 activation (16). In the absence of a functional GSE, phosphorylated SF-1 could still recruit coactivators, thus limiting their ability to bind to phosphorylated CREB/ATF, for example, and abrogating the responses to PACAP. However, recent studies have suggested that cAMP can dephosphorylate SF-1 in H295R cells (13, 14) to recruit the coactivators p54neb/NonO. In this study, treatment with cAMP analogs for 12 h enhanced SF-1 binding to its response element within the human Cyp17 promoter and simultaneously decreased 32P labeling of SF-1. In contrast, in {alpha}T3-1 cells we observed strong binding of phosphorylated SF-1 to the GSE under basal conditions and failed to see any alteration in complex formation upon treatment with PACAP (data not shown). Furthermore, in Y-1 adrenal cortical cells, forskolin treatment enhanced SF-1 phosphorylation and binding to consensus sites in the steroidogenic acute regulatory promoter in a MAPK-dependent manner (35). Thus, it is clear that the characteristics of SF-1 binding are both cell type specific and dependent upon the context of the response element within each promoter sequence.

Previous studies have shown cAMP can regulate SF-1 expression in equine granulosa cells, thecal cells, and immortalized human granulosa cells (36, 37). Using real-time RT-PCR, reporter gene assays, and Western blotting, we failed to see changes in the expression of SF-1 after PACAP treatment in {alpha}T3-1 cells (28), suggesting that the control of SF-1 expression is cell type specific. As changes in SF-1 expression were unlikely to explain the effect of PACAP on SF-1 activity, we were interested to observe the up-regulation in SF-1 phosphorylation after PACAP treatment and sought to determine the functional consequence of such an event. Traditional nuclear receptors that have established ligands, such as the T3 receptors (e.g. retinoid X receptor), undergo conformational changes upon ligand binding, which stabilizes the receptor and allows interactions with coactivators and corepressors (38). Recently, we observed similar effects after phosphorylation of SF-1 at Ser203, suggesting that phosphorylation might act as a surrogate ligand-binding event for orphan nuclear receptors (17). Using similar experimental approaches, we observed that PACAP potently enhanced the assembly of the helical structure of SF-1 in {alpha}T3-1 cells, and that S203A mutation, treatment with the MEK inhibitor UO126, or overexpression of MKP-1 resulted in a significant attenuation (by at least 50%) of this effect. Similar experiments performed in the presence of the PKA inhibitor H-89 failed to inhibit PACAP-stimulated SF-1 helical assembly, but did inhibit PACAP-stimulated GAL4CREB activation (data not shown). This suggests that the downstream biological consequences of PACAP stimulation of gonadotropes are sensitive to specific components of the multifaceted signaling pathways activated by the PAC1-R in these cells. However, it remains to be seen whether the Ser203 residue of SF-1 is important to {alpha}GSU transcription and PACAP responsiveness in vivo.

There is increasing evidence for a number of genes in which hormone responsiveness maps to SF-1-binding elements (12, 25, 39). However, few studies have addressed the role of SF-1 in mediating this response. Our studies show that PACAP responsiveness maps to the GSE in the human {alpha}GSU promoter, and we clearly show an increase in PACAP responsiveness in SF-1-overexpressing {alpha}T3-1 cells compared with S203A-SF-1-expressing {alpha}T3-1 cells. Our data suggest that both SF-1 and its cognate binding site are involved in the mediation of this response, with phosphorylation of SF-1 being involved in the mechanism for ligand-dependent gene expression. The activation of multiple signaling pathways and transcription factors by PACAP in {alpha}T3-1 gonadotropes suggests that complex interaction exists between these different components to regulate transcription of the human {alpha}GSU gene (Fig. 5Go). This may underlie a degree of redundancy within the control of this key component of reproductive hormone synthesis.



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Fig. 5. Proposed Model of PACAP Transcriptional Regulation of the Human {alpha}GSU Promoter via SF-1 in Gonadotropes

PACAP binding to its cell surface GPCR activates specific G proteins to stimulate adenylyl cyclase (AC) activity, cAMP production, PKA or PKC activity, and MEK and MAPK phosphorylation. Induction of these signaling pathways may, cooperatively or independently, enhance the phosphorylation of SF-1, CREB, and ATF and the subsequent recruitment of coactivators (CoA) or corepressors (CoR), such as CREB-binding protein (CBP)/p300 and dosage-sensitive sex reversal adrenal hypoplasia congenita, X chromosome (DAX-1), to regulate the transcriptional activity of the human {alpha}GSU promoter.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
All chemicals were purchased from Sigma-Aldrich Corp. (Poole, UK) unless otherwise stated. PACAP was purchased from CN Biosciences (Nottingham, UK). PACAP and 8-Br-cAMP were diluted in sterile ddH2O to form stock solutions of 1 mM (PACAP) or 100 mM (8-Br-cAMP) and were stored at -20 C before dilution in culture medium. GF109203X (PKC inhibitor) and UO126 (MEK inhibitor) were purchased from CN Biosciences and stored as stock solutions (10 or 1 mM) at -20 C before dilution in culture medium. Working concentrations of inhibitors were those predetermined (21, 25) or the established 50% inhibitory concentrations.

Plasmids Used in Transfection Studies
An expression vector encoding mouse SF-1 was prepared by subcloning the mouse full-length SF-1 cDNA sequence (from Prof. K. L. Parker, University of Texas Southwestern, Dallas, TX) into pCIneo (Promega Corp., Southampton, UK), an expression vector containing a cytomegalovirus promoter for high levels of constitutive expression and imparting neomycin resistance. Restriction endonuclease digests verified correct orientation of the SF-1-coding sequence, and the vector was termed pCI-SF-1neo. The reporter construct -517{alpha}LUC contains 517 bp of the 5'-flanking sequence and 44 bp of exon 1 of the human {alpha}GSU gene, linked to the LUC reporter gene in the plasmid pA3LUC (40). The promoterless LUC expression vector pA3LUC was used as an internal control plasmid for basal LUC expression. The internal control plasmid BosßGAL contains the promoter of the human elongation factor 1 gene driving the expression of ß-galactosidase (41) and was used as an internal control to normalize transfection efficiencies. The expression vectors encoding the mutant SF-1, S203A-SF-1 in pCIneo, SF-1 hinge-helix 1, S203A-SF-1 hinge-helix 1, VP16-SF-1, pGAL4, and MKP-1 plasmids have been described previously (16, 17). All constructs were verified for orientation and correct sequence by restriction endonuclease digests and the dideoxy-DNA sequencing method. Large-scale preparation and purification of plasmids were performed by alkaline lysis and resin purification (Qiagen, Dorking, UK).

Site-Directed Mutagenesis
The -517{alpha}LUC construct was subcloned in to the HindIII site in pBluescript, and site-directed mutagenesis using the QuikChange kit (Stratagene, Cambridge, UK) was conducted to introduce a 2-bp mutation within the GSE region. Mutagenesis was performed according to manufacturer’s instructions and using the following primers: forward, 5'-CTCTCTTTTCATGGGCTGATTTTGTCGTCACCATCACCTG-3'; and reverse, 5'-AGGTGATGGTGACGACAAAATCAGCCCATGAAAAGAGAG-3', with the mutation underlined. After sequence analysis, the mutated -517{alpha}LUC (named -517{alpha}MUT) was cloned back into the HindIII site of pA3LUC.

Cell Culture
{alpha}T3-1 cells (provided by Prof. P. Mellon, University of California, San Diego, CA), were maintained in monolayer culture in high glucose (4500 mg/liter) DMEM containing 10% (vol/vol) fetal calf serum, penicillin (100 IU/ml), streptomycin (100 µg/ml), and fungizone (125 mg/liter; Invitrogen, Paisley, UK). Cells were passaged twice weekly and were grown at 37 C in a humidified 5% (vol/vol) CO2/95% (vol/vol) air incubator.

Transient Expression Assays
{alpha}T3-1 cells were transfected by the calcium phosphate method as described previously (42). The cells were plated at 1 x 106 cells/well in six-well plates and left overnight to attach. After a change of medium, the cells were transfected with 10 µg/well -517{alpha}LUC,-517{alpha}MUT, and pA3LUC and with 5 µg/well BosßGAL. The cells were stimulated for 8 h with {alpha}T3-1 growth medium without (control) or with 100 nM PACAP or 0.5 mM 8-Br-cAMP. The cells were harvested, and cellular extracts were assayed for LUC and ß-galactosidase activity (24).

For the mammalian two-hybrid assembly assays and to determine the effect of MKP-1 on -517{alpha}LUC activity, {alpha}T3-1 cells (2.5 x 105 cells/well) were transfected with 100 ng/well pGAL4, SF-1 hinge-helix 1 or S203A-SF-1 hinge-helix 1; 100 ng/well VP-16SF-1, 100 ng/well BosßGAL, and 200 ng GAL4-LUC (for 2-hybrid); or 200 ng/well -517{alpha}LUC, 100 ng/well BosßGAL, and increasing concentrations of MKP-1 expression vector (25–125 ng/well; for MKP-1 experiments) using Fugene-6 (Roche). Cells were left for 24 h before stimulation with the indicated concentration of PACAP for 8 h.

Nuclear Protein Extraction and Western Blotting
Nuclear protein extracts were prepared from {alpha}T3-1cells using a modification of a method described previously (43). Briefly, 5 x 106 cells were cultured in T25 flasks overnight in serum-free DMEM, before replacement of medium with 0 or 100 nM PACAP for up to 24 h. The medium was removed, and the cells were washed with PBS and scraped into 2 ml ice-cold PBS. After centrifugation (5 min, 1500 x g, 4 C), the cells were resuspended in 400 µl ice-cold buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonylfluoride] and transferred to a cold microfuge tube. Having left the cells to swell on ice for 15 min, 25 µl of a 10% solution of Nonidet P-40 (made in buffer A) were added to each sample, followed by vortexing for 10 sec. After microcentrifugation (10,000 x g, 30 sec), the pellets were resuspended in 300 µl buffer B [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonylfluoride] before vigorous rocking on a shaking platform at 4 C for 15 min. The nuclear extract was centrifuged (10,000 x g, 5 min) and stored at -70 C before protein determination, using the Bradford reagent (Bio-Rad Laboratories, Hertfordshire, UK). Normalized concentrations of nuclear extract (typically 10–15 µg/well) were loaded onto a 10% SDS-PAGE stacking gel and electrophoresed at 200 V. Briefly, samples were boiled in an equal volume of 2x sample buffer to cell lysate, electrophoresed, and transferred to Hybond-ECL nitrocellulose membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). The membrane was blocked with 5% nonfat milk and incubated overnight at 4 C with agitation using 1:1,000 dilutions of rabbit anti-SF-1 (Upstate Biotechnology, Inc., Lake Placid, NY). The membrane was then washed three times with PBS containing 0.05% Tween and subsequently incubated with 1:1,000 goat antirabbit IgG coupled to horseradish peroxidase (DAKO, Glostrup, Denmark) for 2 h at room temperature, again with agitation. The membrane was washed as before, and bound antibody was detected using enhanced chemiluminescence (Amersham Pharmacia Biotech).

Total Protein Preparation
Total proteins were extracted from {alpha}T3-1 cells using the commercially available CytoBuster reagent (CN Biosciences) with added protein phosphatase inhibitor cocktails I and II (Sigma-Aldrich Corp.; referred to as extraction buffer). Briefly, 1 x 106 {alpha}T3-1 cells/well were plated in six-well plates and left to adhere overnight. After a 2-h serum starvation, the cells were incubated with the indicated stimuli and inhibitors for various time points, washed briefly with ice-cold PBS before adding 150 µl/well extraction buffer, and sonicated for 5 sec before storage at -70 C to await analysis by Western blotting (as above) using a 1:1000 dilution of rabbit antiphospho-CREB, rabbit anti-CREB (Upstate Biotechnology, Inc.), rabbit antiphospho-ERK, rabbit anti-ERK (Promega), rabbit antiphospho-Akt, rabbit anti-Akt (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), a 1:5000 dilution of mouse anti-ß-actin (AbCam, Cambridge, UK), or a 1:2500 dilution of rabbit antiphospho-SF-1 (17).

EMSA
EMSA was performed using 1–3 µg nuclear protein extract/reaction. Probes were created by filling in the 5'-AGCT overhangs of the annealed GSE or mutant GSE oligonucleotides with Klenow polymerase using a mixture of deoxy (d)-ATP, dGTP, dTTP, and [{alpha}-32P]dCTP (ICN, Hampshire, UK) (GSE: forward, 5'-AGCTGCTGACCTTGTCGTCAC-3'; reverse, 5'-AGCTGTGACGACAAGGTCAGC-3'; GSE-MUT: forward, 5'-AGCTGCTGATTTTGTCGTCAC-3'; reverse, 5'-AGCTGTGACGACGGGGTCAGC-3'). Three microliters of nuclear extracts were incubated at room temperature for 5 min in a 20-µl vol of 20 mM Tris (pH 8.0), 60 mM KCl, 2 mM MgCl2, 1.2 mM dithiothreitol, 12% glycerol, 2.5 µg poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech). In some instances, 1 µl antiphospho-SF-1 was added to the GSE reaction to compete specific binding, and these samples were incubated on ice for 60 min. The reactions were then incubated for 15 min at 30 C in the presence of 1 ng probe. Complexes were electrophoresed on a 5% native acrylamide gel, dried, and visualized by autoradiography.

Statistical Analysis
All graphical data were prepared using GraphPad PRISM 3.0 (GraphPad, San Diego, CA) and analyzed using preprogrammed analysis equations within PRISM. Transfection data are presented as normalized data pooled from multiple experiments (each in triplicate and performed at least twice). Where appropriate, an ANOVA was performed on data, followed by t test or Tukey’s multiple comparisons test, accepting P < 0.05 as significant.


    ACKNOWLEDGMENTS
 
We thank Prof. P. L. Mellon (University of California, San Francisco, CA) for the gift of the {alpha}T3-1 cells, Prof. K. L. Parker (University of Texas Southwestern, Dallas, TX) for the mouse full-length SF-1 cDNA expression vector, Dr. D. Bryan and Ms. E. Volyanik (Barts and Royal London School of Medicine and Dentistry, London, UK) for practical assistance, and Mrs. N. S. Fowkes-Gajan for assistance in preparing this manuscript.


    FOOTNOTES
 
This work was supported by a Wellcome Trust project grant (to J.M.B.).

Abbreviations: ATF, Activating transcription factor; 8-Br-cAMP, 8-bromo-cAMP; CRE, cAMP response element; CREB, CRE-binding protein; d, deoxy; GSE, gonadotrope-specific element; {alpha}GSU, glycoprotein hormone {alpha}-subunit; LBD, ligand-binding domain; LUC, luciferase; MEK, MAPK kinase; MKP-1, MAPK phosphatase; PACAP, pituitary adenylate cyclase-activating polypeptide; PKA, protein kinase A; PKC, protein kinase C; SF-1, steroidogenic factor-1.

Received for publication November 22, 2002. Accepted for publication August 7, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Pierce J, Parson TF 1981 Glycoprotein hormones: structure and function. Annu Rev Biochem 50:465–495[CrossRef][Medline]
  2. Horn F, Windle JJ, Barnhart KM, Mellon PL 1992 Tissue-specific gene expression in the pituitary: the glycoprotein hormone {alpha}-subunit gene is regulated by a gonadotrope-specific protein. Mol Cell Biol 12:2143–2153[Abstract]
  3. Heckert LL, Schultz K, Nilson JH 1995 Different composite regulatory elements direct expression of the human {alpha}-subunit gene to pituitary and placenta. J Biol Chem 270:26497–26504[Abstract/Free Full Text]
  4. Aylwin SJB, Burrin JM 1995 The role of transcriptional factors in the pituitary expression of the glycoprotein hormone {alpha} subunit. J Mol Endocrinol 15:221–231[Medline]
  5. Schoderbek WE, Kim KE, Ridgway EC, Mellon PL, Maurer RA 1992 Analysis of DNA sequences required for pituitary-specific expression of the glycoprotein hormone alpha-subunit gene. Mol Endocrinol 6:893–903[Abstract]
  6. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen W-H, Nachtigal MW, Abbud R, Nilson JH, Parker KL 1994 The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Gene Dev 8:2302–2312[Abstract]
  7. Barnhart KM, Mellon PL 1994 The orphan nuclear receptor, steroidogenic factor-1, regulates the glycoprotein hormone {alpha}-subunit gene in pituitary gonadotropes. Mol Endocrinol 8:878–885[Abstract]
  8. Morohashi K 1999 Gonadal and extragonadal functions of Ad4BP/SF-1: developmental aspects. Trends Endocrinol Metab 10:169–173[CrossRef][Medline]
  9. Halvorson LM, Kaiser UB, Chin WW 1996 Stimulation of LHß gene promoter activity by the orphan nuclear receptor, steroidogenic factor-1. J Biol Chem 271:6645–6650[Abstract/Free Full Text]
  10. Bakke M, Lund J 1995 Mutually exclusive interactions of two nuclear orphan receptors determine activity of a cyclic adenosine 3',5'-monophosphate-responsive sequence in the bovine CYP17 gene. Mol Endocrinol 9:327–339[Abstract]
  11. Ito M, Park Y, Weck J, Mayo KE, Jameson JL 2000 Synergistic activation of the inhibin {alpha}-promoter by steroidogenic factor-1 and cyclic adenosine 3',5'-monophosphate. Mol Endocrinol 14:66–81[Abstract/Free Full Text]
  12. Zhang P, Mellon SH 1996 The orphan nuclear receptor steroidogenic factor-1 regulates the cyclic adenosine 3',5'-monophosphate-mediated transcriptional activation of rat cytochrome p450c17 (17{alpha}-hydroxylase/c17–20 lyase). Mol Endocrinol 10:147–158[Abstract]
  13. Sewer MB, Nguyen VQ, Huang C-J, Tucker PW, Kagawa N, Waterman MR 2002 Transcriptional activation of human Cyp17 in H295R adrenocortical cells depends on complex formation among p54nrb/NonO, protein-associated splicing factor, and SF-1, a complex that also participates in repression of transcription. Endocrinology 143:1280–1290[Abstract/Free Full Text]
  14. Sewer MB, Waterman MR 2002 Adrenocorticotropin/cyclic adenosine 3',5'-monophosphate-mediated transcription of the human Cyp17 gene in the adrenal cortex is dependent on phosphatase activity. Endocrinology 143:1769–1777[Abstract/Free Full Text]
  15. Æsøy R, Mellgren G, Morohashi K, Lund J 2002 Activation of cAMP-Dependent protein kinase increases the protein level of steroidogenic factor-1. Endocrinology 143:295–303[Abstract/Free Full Text]
  16. Hammer GD, Krylova I, Zhang Y, Darimont BD, Simpson K, Weigel NL, Ingraham HA 1999 Phosphorylation of the nuclear receptor SF-1 modulates cofactor recruitment: integration of hormone signaling in reproduction and stress. Mol Cell 3:521–526[Medline]
  17. Desclozeaux M, Krylova IN, Horn F, Fletterick RJ, Ingraham HA 2002 Phosphorylation and intramolecular stabilization of the ligand binding domain in the nuclear receptor steroidogenic factor 1. Mol Cell Biol 22:7193–7203[Abstract/Free Full Text]
  18. Tsuji T, Ishizaka K, Winters SJ 1994 Effects of pituitary adenylate cyclase-activating polypeptide on gonadotropin secretion and subunit messenger ribonucleic acids in perifused rat pituitary. Endocrinology 135:826–833[Abstract]
  19. Burrin JM, Aylwin SJB, Holdstock JG, Sahye U 1998 Mechanism of action of pituitary adenylate cyclase-activating polypeptide on human glycoprotein hormone {alpha}-subunit transcription in {alpha}T3-1 gonadotropes. Endocrinology 139:1731–1737[Abstract/Free Full Text]
  20. Rawlings SR, Hezarah M 1996 Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP/vasoactive intestinal polypeptide receptors: actions on the anterior pituitary gland. Endocr Rev 17:4–28[Medline]
  21. Fowkes RC, Burch J, Burrin JM 2001 Stimulation of extracellular signal-regulated kinase by pituitary adenylate cyclase-activating polypeptide in {alpha}T3-1 gonadotrophs. J Endocrinol 171:R5–R10
  22. Roberson MS, Misra-Press A, Laurance ME, Stork PJ, Maurer RA 1995 A role for mitogen-activated protein kinase in mediating activation of the glycoprotein hormone {alpha}-subunit promoter by gonadotropin-releasing hormone. Mol Cell Biol 15:3531–3539[Abstract]
  23. Schomerus E, Poch A, Bunting R, Mason WT, McArdle CA 1994 Effects of pituitary adenylate cyclase-activating polypeptide in the pituitary: activation of two signal transduction pathways in the gonadotrope-derived {alpha}T3-1 cell line. Endocrinology 134:315–323[Abstract]
  24. Holdstock JG, Aylwin SJB, Burrin JM 1995 Calcium and glycoprotein hormone {alpha}-subunit gene expression and secretion in {alpha}T3-1 gonadotropes. Mol Endocrinol 10:1308–1317
  25. Fowkes RC, King P, Burrin JM 2002 Regulation of human glycoprotein hormone {alpha}-subunit gene transcription in Lß T2 gonadotropes by protein kinase C and extracellular signal-regulated kinase 1/2. Biol Reprod 67:725–734[Abstract/Free Full Text]
  26. Attardi B, Winters SJ 1998 Transcriptional regulation of the glycoprotein hormone {alpha}-subunit gene by pituitary adenylate cyclase-activating polypeptide (PACAP) in {alpha}T3-1 cells. Mol Cell Endocrinol 137:97–107[CrossRef][Medline]
  27. Mulvaney JM, Zhang T, Fewtrell C, Roberson MS 1999 Calcium influx through L-type channels is required for selective activation of extracellular signal-regulated kinase by gonadotropin-releasing hormone. J Biol Chem 274:29796–29804[Abstract/Free Full Text]
  28. Fowkes RC, Gyselman VG, McKay IA, Bustin SA, JM Burrin, Expression of SF1 and DAX-1 in mouse gonadotroph-derived {alpha}T3-1 cells. 11th International Congress of Endocrinology, Sydney, Australia, 2000 (Abstract P1255)
  29. Burrin JM, Jameson JL 1989 Regulation of transfected glycoprotein hormone {alpha}-gene expression in primary pituitary cells. Mol Endocrinol 3:1643–1651[Abstract]
  30. Lania A, Filopanti M, Corbetta S, Losa M, Ballare E, Beck-Peccoz P, Spada A 2003 Effects of hypothalamic neuropeptides on extracellular signal-regulated kinase (ERK1 and ERK2) cascade in human tumoral pituitary cells. J Clin Endocrinol Metab 88:1692–1696[Abstract/Free Full Text]
  31. Tsuji T, Attardi B, Winters SJ 1995 Regulation of {alpha}- subunit mRNA transcripts by pituitary adenylate cyclase-activating polypeptide (PACAP) in pituitary cell cultures and {alpha}T3-1 cells. Mol Cell Endocrinol 113:123–130[CrossRef][Medline]
  32. McArdle CA, Counis R 1996 GnRH and PACAP action in gonadotropes: cross-talk between phosphoinositidase C and adenylyl cyclase mediated signaling pathways. Trends Endocrinol Metab 7:168–175[CrossRef]
  33. Jorgensen JS, Nilson JH 2001 AR suppresses transcription of the LHß subunit by interacting with steroidogenic factor-1. Mol Endocrinol 15:1505–1516[Abstract/Free Full Text]
  34. Fowkes RC, Sidhu KK, Sosabowski JK, King P, Burrin JM, Absence of pituitary adenylate cyclase activating polypeptide (PACAP)-stimulated transcription of the human glycoprotein {alpha}-subunit ({alpha}GSU) gene in Lß T2 gonadotrophs reveals disrupted cyclic 3'5'-adenosine monophosphate (cAMP)-mediated gene transcription. J Mol Endocrinol, in press
  35. Gyles SL, Burns CJ, Whitehouse BJ, Sugden D, Marsh PJ, Persaud SJ, Jones PM 2001 ERKs regulate cyclic AMP-induced steroid synthesis through transcription of the steroidogenic acute regulatory (StAR) gene. J Biol Chem 276:34888–34895[Abstract/Free Full Text]
  36. Boerboom D, Pilon N, Behdjani R, Silversides DW, Sirois J 2000 Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process. Endocrinology 141:4647–4656[Abstract/Free Full Text]
  37. Hosokawa K, Dantes A, Schere-Levy C, Barash A, Yoshida Y, Kotsuji F, Vlodavsky I, Amsterdam A 1998 Induction of Ad4BP/SF-1, steroidogenic acute regulatory protein, and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines. Endocrinology 139:4679–4687[Abstract/Free Full Text]
  38. Renaud JP, Moras D 2000 Structural studies on nuclear receptors. Cell Mol Life Sci 57:1748–1769[Medline]
  39. Michael MD, Kilgore MW, Morohashi K, Simpson ER 1995 Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase p450 (CYP19) gene in the ovary. J Biol Chem 270:13561–13566[Abstract/Free Full Text]
  40. Maxwell IH, Harrison GS, Wood WM, Maxwell F 1989 A DNA cassette containing a trimerised SV40 polyadenylation signal which efficiently blocks spurious plasmid initiated transcription. Biotechniques 7:276–280[Medline]
  41. Mizushima S, Nagata S 1990 pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res 18:5322–5326[Medline]
  42. Graham FL, van der Eb AJ 1973 Transformation of rat cells by DNA of human adenovirus 5. Virology 52:456–467[Medline]
  43. Schreiber E, Matthias P, Muller MM, Schaffner W 1989 Rapid detection of octamer binding proteins with ‘mini-extracts,’ prepared from a small number of cells. Nucleic Acids Res 17:6419[Medline]