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.
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ABSTRACT |
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INTRODUCTION |
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The consensus GSE sequence (TGACCTTGT) in the human GSU promoter interacts with the transcription factor steroidogenic factor-1 (SF-1) (6, 7) and
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
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
gene (10, 11). The role of SF-1 in cAMP responsiveness of the human
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 GSU transcription in primary pituitary cells (18) and in
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
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
GSU gene expression and transcription (18, 19, 22, 23, 24, 25). The effects of PACAP on
GSU transcription are potent and rapid, with increased
GSU promoter activity occurring within 2 h (19). Previous work from our laboratory has suggested that full transcriptional activation of the
GSU by PACAP requires an intact CRE, but also involves sequences between -244 and -195 bp of the human
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
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 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
GSU gene expression via the GSE in
T3-1 gonadotropes.
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RESULTS |
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PACAP-Stimulated 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 GSU promoter activity, we next investigated the role of SF-1.
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
T3-1 cells with -517
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
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
GSU promoter activity (Fig. 2A
). To determine whether the S203A mutation affected PACAP stimulation of -517
LUC activity, the cotransfected cells were stimulated with 0 or 100 nM PACAP for 8 h. As shown (Fig. 2B
), 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
GSU promoter activity in
T3-1 cells.
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Role of MAPK Activation in PACAP Stimulation of SF-1 Ligand-Binding Domain (LBD) Activity and GSU Transcription in
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 212) by the hinge-helix 1 portion of SF-1 (helix assembly assay using the two-hybrid system; see Fig. 3A). To determine whether PACAP treatment of
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 212 of SF-1, using GAL4 expression plasmids in a mammalian two-hybrid assembly assay (17).
T3-1 cells were cotransfected with wild-type pGAL4-SF-1-hinge-helix 1, helixes 212 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. 3B
). 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. 3B
) 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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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. 3D), 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 T3-1 Cells
To establish the potential signaling cascade initiated by PACAP in T3-1 cells, we performed Western blotting for known cAMP target proteins. Total cell lysates were made from
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. 4A
, 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
T3-1 cells (21). As expected, PACAP transiently increased ERK1/2 phosphorylation at 5 min (Fig. 4B
, middle panel). As new cAMP-activated signaling pathways have recently been elucidated, we examined whether PACAP could activate the PKB/Akt pathway in
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
T3-1 cells does not activate the PKB/Akt cascade (Fig. 4A
, bottom panel).
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To establish which of these pathways are involved in PACAP-induced SF-1 phosphorylation, we produced total protein extracts from 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
T3-1 cells (Fig. 4C
). 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
T3-1 cells, as shown previously in transfected JEG-3 cells (16) and Y1 adrenal cells (17).
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DISCUSSION |
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Previous studies of the human GSU promoter have demonstrated that the GSE is important for promoter activity in the pituitary gonadotrope cell line
T3-1 (2, 3). Horn et al. (2) mutated the GSE element in the -224 truncated human
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
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
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
GSU promoter activity in transfected LßT2 gonadotropes (25). Thus, the presence of a GSE in the human
GSU promoter consistently enhances basal expression in pituitary gonadotropes.
The role of the GSE in hormone-regulated expression of the 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
GSU promoter activity in primary rat pituitary cells (29) and
T3-1 cells (19). In the latter study, we demonstrated that full transcriptional activation of the
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
T3-1 cells (20, 23), whereas we and others have also shown PACAP to transiently activate the ERK1/2 pathway in
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
GSU transcriptional response to PACAP (19, 21, 31), and its transcriptional effects on the
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
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
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
GSU promoter. The failure to inhibit the potent transcriptional effect of PACAP on the
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 GSU promoter involves the GSE. We have shown that transfection of an
GSU promoter construct containing a mutated GSE into
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
GSU promoter activity and mimics the effect of a GSE mutation on GnRH- and PKC-stimulated
GSU promoter activity in LßT2 cells (25). Our current data also reveal the novel finding that PACAP can induce CREB phosphorylation in
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
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
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 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
T3-1 cells (34). It is likely that both the GSE and the CREs in the human
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 GSU transcription in
T3-1 cells, we next sought to determine whether SF-1 was involved in conferring PACAP responsiveness or augmenting the expression of the human
GSU promoter.
T3-1 cells express relatively high levels of endogenous SF-1, yet we have still shown an enhancement in basal and PACAP-stimulated
GSU promoter activity in
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
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 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 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
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
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 GSU promoter, and we clearly show an increase in PACAP responsiveness in SF-1-overexpressing
T3-1 cells compared with S203A-SF-1-expressing
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
T3-1 gonadotropes suggests that complex interaction exists between these different components to regulate transcription of the human
GSU gene (Fig. 5
). This may underlie a degree of redundancy within the control of this key component of reproductive hormone synthesis.
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MATERIALS AND METHODS |
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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 -517LUC contains 517 bp of the 5'-flanking sequence and 44 bp of exon 1 of the human
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 -517LUC 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 manufacturers 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
LUC (named -517
MUT) was cloned back into the HindIII site of pA3LUC.
Cell Culture
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
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
LUC,-517
MUT, and pA3LUC and with 5 µg/well BosßGAL. The cells were stimulated for 8 h with
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 -517LUC activity,
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
LUC, 100 ng/well BosßGAL, and increasing concentrations of MKP-1 expression vector (25125 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 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 1015 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 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
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 13 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 [-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 Tukeys multiple comparisons test, accepting P < 0.05 as significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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; GSU, glycoprotein hormone
-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.
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REFERENCES |
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