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T3-1 Cells
Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523
Address all correspondence and requests for reprints to: Colin M. Clay, Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Foothills Campus, Colorado State University, Fort Collins, Colorado 80523. E-mail: cclay{at}cvmbs.colostate.edu.
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Abstract |
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T3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH3). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in T3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components. |
Introduction |
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-subunit, the unique LHß- and FSHß-subunits, and the GnRH receptor (GnRHR). Furthermore, expression of each of these genes is tightly regulated by a variety of different stimulatory and inhibitory endocrine inputs. Thus, any substantive physiological response of gonadotropes represents a complex integration of multiple signals impacting at multiple sites in multiple genes. As such, the gonadotrope represents an excellent model for studying the basic biology of mechanisms underlying the integration of multiple signaling systems in a single endocrine cell type. Of the different hormones that directly mediate gonadotrope function, none are more fundamental than the hypothalamic decapeptide GnRH (1, 2, 3, 4). Quite simply, in the absence of GnRH input, gonadotropin secretion and, consequently, gonadal function ceases. Thus, the GnRHR represents the site that ultimately receives and mediates the primary stimulatory input to gonadotropes. Accordingly, much effort has been expended toward understanding the biological events evoked by GnRH on binding to the GnRHR. In this regard, it is clear that GnRH activation of the GnRHR leads to increased expression of the genes that encode for not only the gonadotropin subunits but also the GnRHR gene itself (5, 6, 7, 8, 9, 10, 11, 12, 13). Thus, the GnRHR gene is a target for homologous up-regulation by GnRH, an event that likely contributes to the high rates of LH secretion necessary for ovulation in mammals. At issue is the identity of the signaling cascades, regulatory proteins, and DNA elements that underlie the sensitivity of the gonadotropin subunit genes and the GnRHR gene to GnRH stimulation.
Lacking an intracellular carboxyl terminus, the mammalian GnRHR is an unusual member of the rhodopsin-like family of G protein-coupled receptors (14, 15, 16). GnRH-induced signal transduction partially occurs via coupling of the receptor to members of the Gq/G11 family of G proteins leading to stimulation of multiple phospholipase activities, formation of inositol 1,4,5-trisphosphate and diacylglycerol, elevation of intracellular free calcium concentrations, and activation of one or more isoforms of protein kinase C (PKC; Refs. 17, 18, 19, 20). In the gonadotrope-derived T3-1 cell line, GnRH has been shown to activate multiple MAPK signaling cascades including ERK (7, 8, 21), c-Jun N-terminal kinase (JNK) (22, 23) and p38 MAPK (24).
Consistent with GnRH regulation of the endogenous GnRHR gene, we have found that approximately 1900 bp of proximal 5' flanking region of the GnRHR gene is sufficient to confer GnRH responsiveness on a heterologous reporter gene in transgenic mice (25). We and others have found that mutation of a canonical activator protein-1 (AP-1) element located within 500 bp of proximal promoter leads to a loss of GnRH responsiveness in the gonadotrope-derived T3-1 cell line (12, 13). In addition to AP-1, sequence underlying regulation of GnRH-1 (SURG-1), an element located upstream of the AP-1 site, appears to partially contribute to GnRH responsiveness in T3-1 cells (13). Finally, in contrast to T3-1 cells, a noncanonical cAMP response element appears to be necessary for GnRH responsiveness of the GnRHR gene in GH3 cells stably expressing the rat GnRHR (GGH3; Refs. 26 and 27). Thus, multiple elements have been implicated in mediating the GnRH response of the GnRHR gene; however, the physiological relevance of these elements has not been established. Herein, we report that GnRH responsiveness of the GnRHR gene promoter in transgenic mice requires an intact AP-1 element. Furthermore, we suggest that GnRH activation of JNK but not ERK is necessary for GnRH responsiveness of the GnRHR gene promoter.
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Plasmids
The plasmid pMGR-600LUC consisted of 600 bp of 5' flanking region from the murine GnRHR gene fused to the cDNA-encoding luciferase in the pGL3 basic vector (Promega Corp., Madison, WI) (28). The control vector used to test for transfection efficiency in all experiments contained the Rous sarcoma virus promoter linked to the cDNA-encoding ß-galactosidase (RSV-ßgal). The Gal4-c-Fos (pFA-c-Fos), Gal4-c-Jun (pFA2-c-Jun), and pBK-CMV vectors were purchased from Stratagene (La Jolla, CA). The Gal4-FosB (pBind-FosB) and Gal4-JunD (pBind-JunD) vectors were generously provided by Dr. G. T. Bowden (University of Arizona Health Sciences Center, Tucson, AZ), and the Gal4-LUC vector and Raf-CAAX overexpression vector were kindly provided by Dr. M. S. Roberson (Cornell University, Ithaca, NY). The dominant negative SEK1K-R (pEGB) plasmid that was used to create the stable cell lines was kindly supplied by Dr. L. I. Zon (Children’s Hospital, Boston, MA).
Generation and screening of transgenic mice
A total of 1900 bp of the 5' flanking region from the mGnRHR gene (-1900wt) or this same region containing a recognition site for EcoRI in place of the AP-1 element (-1900µAP1) was subcloned upstream of the cDNA encoding firefly luciferase. The fusion genes were released from pMGR-1900LUC by digestion with SacI and BamHI and purified by electroelution. After extraction with phenol-chloroform-isoamyl alcohol (24:24:1), the DNA was precipitated by the addition of 0.1 vol 3 M sodium acetate and 2 vol ice-cold ethanol. The precipitated DNA was resuspended in 10 mM Tris-HCl (pH 7.5) and 0.2 mM EDTA to a final concentration of 5 ng/µl. The linearized fusion gene was microinjected with continuous positive flow into pronuclei of fertilized mouse oocytes. Injected embryos were reimplanted into oviducts of recipient females and allowed to develop to term (29). Genomic DNA was extracted from tail biopsies and analyzed for the presence of the transgene by slot blot hybridization (30) or PCR amplification using primers specific to luciferase. Animals were maintained under a 14-h light, 10-h dark cycle and received food and water ad libitum. All experiments were performed under veterinary supervision with approval from the Colorado State University Animal Care and Use Committee and in accordance with the NIH Animal Care and Use Guidelines. All experiments were conducted using animals older than 6 wk of age.
Luciferase assays
Tissue extracts were prepared by homogenization in 200 µl cold lysis buffer (25 mM glycyl-glycine, pH 7.8; 1.0% Triton X-100; 10 mM MgSO4; and 1.0 mM dithiothreitol). Cellular debris was pelleted by microcentrifugation at 16,000 x g for 5 min at 4 C. Cellular lysates were immediately assayed for luciferase activity by adding 20 µl lysate to 100 µl luciferin substrate (Promega Corp.) and measuring luminescence with a Turner model TD-20E luminometer set for a 5-sec delay and 10-sec integration. Total protein was precipitated from lysates with 10% trichloroacetic acid and then dissolved in 0.1 N NaOH. Protein concentrations were determined using bicinchoninic acid (BCA assay, Pierce Chemical Co., Rockford, IL). Luciferase activity was adjusted for protein content by dividing the arbitrary light units by the protein content in milligrams.
Animal treatments
Experiment 1: basal expression of -1900wt, -1900µAP1:line1, and -1900µAP1:line2.
Male and female transgenic mice were killed and pituitary, brain, ovaries, testes, liver, heart, pancreas, kidney, lung, and spleen were harvested and assayed for luciferase activity.
Experiment 2: GnRH regulation of -1900wt, -1900µAP1:line1, and -1900µAP1:line2.
Female transgenic mice (-1900wt, -1900µAP1:line1, -1900µAP1:line2) were ovariectomized. After 4 d, half of the -1900wt and half of the -1900µAP1 mice received a single ip injection of 300 µl GnRH antiserum (AS). After 48 h of treatment with AS, animals were killed and pituitary glands were harvested for luciferase activity. Trunk blood was collected for analysis of serum concentrations of LH (31). Each treatment group contained eight animals.
Cell culture and transient transfections
Cultures of T3-1 cells were maintained at 37 C in a humidified 5% CO2 in air atmosphere. Cells were cultured before transfection in high-glucose DMEM containing 2 mM glutamine, 5% fetal bovine serum, 5% horse serum, 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate (Mediatech, Herndon, VA). Following transfection, the cells were cultured in the same medium without fetal bovine serum. Transient transfections were carried out using a calcium phosphate/DNA coprecipitation method as previously described (32). Briefly, the day before transfection 2 x 106 cells were plated in 100-mm tissue culture dishes. Complete media were removed and calcium phosphate/DNA precipitates in a total volume of 1 ml were added to the plates. At 30 min, posttransfection media was added and cells were treated for 6 h with either GnRH or the treatment as indicated. In Fig. 6
, the mitogen-activated ERK kinase (MEK1/MEK2) inhibitor PD98059 was added 15 min before GnRH and again at 3 h post treatment. Within each assay, treatments were performed in triplicate and different plasmid preparations were used for each assay. After 6 h of treatment, cells were washed twice with ice-cold PBS and lysed in 400 µl 25 mM glycyl-glycine (pH 7.8), 15 mM MgSO4, 1% Triton X-100, and 1 mM dithiothreitol. Lysates were cleared by centrifugation at 16,000 x g for 2 min. Lysates (40 and 100 µl for LUC and ß-gal, respectively) were assayed according to the manufacturer’s instructions for luciferase (Promega Corp., Madison, WI) and ß-gal (Tropix, Bedford, MA) activity using a Turner 20D luminometer (Turner Designs, Sunnyvale, CA). Luciferase values were divided by ß-gal activity to normalize for transfection efficiency (33).
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T3-1 cells were prepared according to Hagenbuchle and Wellauer (34). Gel-shift assays were conducted as previously described (28). Briefly, nuclei (1.5 x 106) were incubated for 15 min at 4 C in 18 ml Dignam buffer D (20 mM HEPES, pH 7.9; 20% vol/vol glycerol; 0.1 M KCl; 0.2 mM EDTA; 0.5 mM dithiothreitol) containing 2 mg poly(deoxyinosine-deoxycytidine) (Pharmacia Biotech, Piscataway, NJ), sonicated for 5 min in an ice slurry, and centrifuged for 1 min at 1310 x g. The supernatant was incubated, where indicated, with 2 µg rabbit polyclonal antibodies directed against either the amino terminus of human c-Jun p39, the amino terminus of murine JunB p39, the carboxy terminus of murine JunD p39, the amino terminus of human c-Fos p62, the central domain of murine FosB, the amino terminus of murine Fra-1, the carboxy terminus of human Fra-2, or an equal mass of rabbit IgG. The activity of these antibodies in Western blotting and gel-mobility shift assays has been described (35). For antibodies against the Jun family members, the radiolabeled probe (100,000 cpm) was added after incubation. Conversely, the antibodies against the Fos family members were incubated following addition of the radiolabeled probe. Reactions were incubated at room temperature for 30 min, and free probe was separated from bound probe by electrophoresis for 1–2 h at 40 mA in 6% polyacrylamide gels that were prerun at 100 V for 30 min in 25 mM Tris, 190 mM glycine, and 1 mM EDTA (pH 8). Gels were transferred to blotting paper, dried, and exposed to Hyperfilm MP (Amersham, Arlington Heights, IL) for approximately 6 h at -70 C with DuPont Cronex intensifying screens (DuPont, Boston, MA). Radiolabeled probes were prepared by labeling the antisense strand with [
-32P]ATP (4500 Ci/mmol; ICN, Irvine, CA) and T4 poly-nucleotide kinase followed by annealing to the complementary strand. Double-stranded DNA probes were purified by centrifugation on a G-25 Microspin column (Pharmacia Biotech).
Immunoblot analysis
The T3-1 cells were grown to approximately 70% confluence and serum starved for 2 h before drug treatment and lysis. Cells were treated with 100 nM GnRH for 0, 1, or 6 h. In Fig. 10, cells were treated with 100 nM GnRH and 10 µM forskolin for either 1 or 6 h. In addition, control vehicle (dimethyl sulfoxide) was applied to the cells receiving no drug treatment. Following treatment, cells were washed with ice-cold buffer containing 150 mM NaCl and 10 mM HEPES (pH 7.5) and lysed in radioimmunoprecipitation assay (RIPA) buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.5% deoxycholate, 2 mM EDTA, 5 mM sodium vanadate, 5 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride on ice. The cell lysates were collected and debris cleared by centrifugation. Proteins were resolved using denaturing PAGE followed by transfer to nitrocellulose membrane (Osmonics, Westborough, MA) by electroblotting. Samples were analyzed for c-Fos, FosB, and JunD total protein by Western blotting using antibodies obtained from Santa Cruz Biotechnology, Inc. Membranes were blocked at room temperature in TBS-T (140 mM NaCl, 10 mM Tris, 0.1% Tween 20, pH 7.4) plus 5% dry, nonfat milk for 30 min.
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Blotting for phospho-JunD was performed similarly with the following exceptions: Primary antibodies were incubated with membranes overnight in TBS-T plus 5% BSA at 4 C and then washed six times with TBS-T before incubating for 1 h at room temperature in TBS-T plus 5% BSA with secondary antibody.
JNK activation assays were performed in a similar manner, except for the following differences: 20 µl lysate was loaded onto gels, antibodies were obtained from Cell Signaling Technology, and primary antibodies were incubated with membranes in TBS-T plus 5% BSA at 4 C overnight.
Dominant negative SEK-1 cell line
Using a Superfect protocol (QIAGEN, Valencia, CA), T3-1 cells were transfected with 1.8 µg dominant negative SEK1K-R plasmid (36) and 200 ng pBK-CMV. Cells were grown in T3-1 culture media plus 600 mg/liter G418. Surviving colonies were isolated and expanded under selection. Colonies were then assayed for JNK activity according to the Western blot protocol discussed previously. A positive clone was identified by its lack of phosphorylated JNK but presence of total JNK protein following GnRH treatment. GnRH responsiveness of this cell line was then tested using the calcium phosphate/DNA coprecipitation method described previously with one exception. The pH-1500LUC was transfected using the lipofectamine procedure (Life Technologies, Inc., Gaithersburg, MD) as previously described (33).
Statistical analysis
Data were analyzed using SAS (37). Tissues expressing luciferase activity at levels above the mean + 2 SD of values in tissues from nontransgenic animals were considered positive for expression of the transgene. Differences in luciferase activity and serum concentrations of LH were determined by one-way ANOVA. Means for Fig. 2 were separated using t test and Tukey’s method of multiple comparisons. Means for GnRH-treated cells were expressed as fold increases over nontreated cells. Means for luciferase activity in Figs. 6–8
were logarithmically transformed because of nonnormality and then analyzed. Means for luciferase activity were analyzed by ANOVA and compared with control values with Dunnett’s two-tailed t test.
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T3-1 cells (12, 13). Here we sought to establish the relevance of this element in mediating GnRH responsiveness of the GnRHR gene in vivo. Toward this end, we constructed two independent lines of transgenic mice harboring 1900 bp of proximal promoter from the murine GnRHR gene in which the AP-1 element has been replaced with the recognition site for EcoRI (-1900µAP1:line1 and -1900µAP1:line2). We have already established tissue-specific expression and GnRH regulation of the wild-type -1900-bp promoter in transgenic mice (25); however, as a contemporary control, we constructed a second line of mice harboring the identical wild-type promoter fragment (-1900wt). F1 progeny were used to assess expression of the transgenes by assaying for luciferase activity in pituitary gland, brain, gonads, lung, heart, kidney, spleen, liver, and pancreas (25, 38). Transgene expression was confined to pituitary, brain, and gonads in both -1900wt and -1900µAP1 mice (Fig. 1). Thus, the tissue-specific properties of the GnRHR promoter are retained despite the absence of the canonical AP-1 element located between -336 and -330. These data are, in fact, consistent with in vitro analysis of promoter function in which mutation of the AP-1 site reduces but does not eliminate promoter activity in gonadotrope-derived cell lines (33).
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As expected, immunoneutralization of GnRH reduced pituitary expression of luciferase by approximately 80% in mice harboring the -1900 wild-type promoter (Fig. 2). In contrast, pituitary expression of luciferase in both lines of -1900µAP1 mice was unaffected by GnRH immunoneutralization (Fig. 2). Based on reduced LH levels, GnRH immunoneutralization was equally efficacious in all lines of mice.
GnRH treatment leads to increased binding of JunD, FosB, and cFos at the murine GnRHR AP-1 element
An intact AP-1 element appears to be necessary for GnRH activation of the GnRHR gene promoter in both T3-1 cells and transgenic mice. Previously we used broadly cross-reactive antibodies in gel-mobility shift assays to establish the binding of both Jun and Fos family members to the GnRHR AP-1 site (12). Here we ask whether GnRH activation might be revealed as an increase in AP-1-binding activity. Consistent with this notion, total AP-1 binding activity in T3-1 nuclei appears to be enhanced with increasing duration of GnRH treatment (Fig. 3A). To identify the specific binding components, nuclei isolated from T3-1 cells following 1 or 6 h of GnRH treatment were preincubated with specific antibodies directed against c-Jun, JunD, or JunB or an equal mass of nonimmune IgG and then analyzed by gel-mobility shift assays using the murine GnRHR AP-1 site as the radioactive probe. Inclusion of the anti-JunD antibody resulted in a supershifted complex at time zero, and this binding activity appeared to increase with increasing duration of GnRH treatment (Fig. 3B). In a similar paradigm, we examined the Fos-binding component. In contrast to Jun in which a single family member was detected at AP-1, GnRH appeared to induce increased binding activity of both FosB and, to a lesser extent, cFos (Fig. 3B). Although Fra2 may also represent a minor binding component, its binding activity did not appear to be altered by GnRH treatment.
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, T3-1 cellular proteins were subjected to Western analysis using antibodies directed against total JunD, c-Fos, and FosB. Consistent with others (23, 24, 39), we found a marked induction of c-Fos expression associated with GnRH treatment. In contrast, immunodetectable levels of total JunD and FosB were unaffected by GnRH treatment. Thus, whereas enhanced binding of c-Fos to the murine GnRHR gene likely reflects GnRH induction of c-Fos synthesis, JunD and FosB regulation appears to be largely posttranslational. Consistent with this possibility, GnRH-induced phosphorylation of JunD was evident in Western blots conducted using an anti-phospho-JunD antibody (Fig. 4A). Because of the lack of phospho-specific antibodies for FosB we tested the ability of GnRH to activate both FosB and JunD expressed as fusion proteins with the Gal4 DNA-binding domain. GnRH treatment (100 nM for 6 h) led to a significant induction of a Gal4-LUC reporter in T3-1 cells expressing the Gal4-JunD and Gal4-FosB fusion proteins (Fig. 4B). There was no effect of GnRH on Gal4-LUC expression in the absence of the Jun and Fos fusion proteins (data not shown). Thus, both JunD and FosB can serve as targets for posttranslational regulation by GnRH.
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). As a positive control in this experiment, we used a fusion protein consisting of the Gal4 DNA-binding domain fused to the transactivation domain of Elk cotransfected with a Gal4-LUC reporter construct (35). Coexpression of Raf-CAAX with Gal4-Elk led to an approximately 6-fold induction of the Gal4-LUC reporter in T3-1 cells (Fig. 5). In contrast, activity of the GnRHR -600 promoter was not affected by coexpression with Raf-CAAX. Thus, ERK activation is either not necessary or not sufficient for transcriptional induction of the murine GnRHR gene promoter. With regard to the second possibility, several groups have established that JNK, like ERK, is activated in response to GnRH stimulation. Given the involvement of both Jun and Fos in mediating GnRH regulation of the GnRHR gene, we sought to determine whether both ERK and JNK activation is required for GnRH responsiveness.
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, GnRH activation of ERK and JNK was evaluated by immunoblot using antibodies specific for the phosphorylated forms of the enzymes. GnRH-induced phosphorylation of ERK and JNK was clearly evident (Fig. 6). Furthermore, consistent with inhibition of MEK1/2, relatively low doses of PD98059 (10 µM) were effective in blocking GnRH activation of ERK. It is notable, however, that higher doses of PD98059 also interrupted GnRH-induced phosphorylation of JNK. What is striking is that GnRH responsiveness of the GnRHR promoter is only compromised at the highest doses of PD98059. Thus, doses of PD98059 that effectively eliminated GnRH activation of ERK had little or no effect on the GnRH response of the GnRHR gene promoter. Rather, only the highest doses of the MEK1/2 inhibitor were effective in attenuating and ultimately eliminating GnRH responsiveness. Assuming a predictive relationship between the patterns of ERK and JNK activation following 30 min of GnRH exposure and the functional response of the promoter, it would appear that the loss of GnRH regulation of the GnRHR gene promoter was more closely associated with the loss of JNK rather than ERK activation.
To further address the potential requirement for JNK in mediating the GnRH response of the GnRHR gene, we next sought a pharmacological paradigm that would clearly extinguish GnRH activation of ERK yet retain activated JNK. Based on work from Cornell (39), we tested the effects of increasing doses of the L-type voltage-gated calcium channel (VGCC) inhibitor nimodipine. As evident in Fig. 7, ERK activation in T3-1 cells is highly sensitive to blockade of VGCC such that at even the lowest doses of nimodipine GnRH-induced activation of ERK is lost. In contrast, there was little effect of nimodipine on GnRH activation of JNK. Of particular interest, however, is that despite the complete loss of activated ERK, the functional response of the GnRHR gene promoter to GnRH stimulation was attenuated but clearly retained.
GnRH responsiveness of the GnRHR promoter is lost in T3-1 cells stably expressing dominant-negative SEK1
Because of the lack of pharmacological agents that are specific for intermediates in the JNK-signaling cascade, we sought to determine whether a dominant-negative form of a signaling intermediate in the JNK cascade, SEK-1 (36) was able to inhibit GnRH responsiveness of pMGR-600LUC. To address this issue, we constructed an T3-1 cell line that stably expressed the SEK-1 dominant-negative (SEK1K-R) (36). Although ERK activation was retained in this cell line, GnRH activation of JNK and the GnRHR promoter was lost (Fig. 8). In contrast to GnRH, phorbol ester (PMA) treatment of these cells did elicit JNK activation and reconstituted a functional transcriptional response of the GnRHR promoter. To verify that GnRH signaling in the SEK1K-R cell line was retained through an ERK-dependent pathway, we examined the GnRH response of the proximal promoter from the human glycoprotein hormone -subunit gene (pH-1500LUC) (12). Consistent with the retention of ERK activation, the GnRH response of the human -promoter was also retained (Fig. 8).
GnRH activation of the GnRHR promoter and JNK is inhibited by forskolin and cAMP
In contrast to studies conducted with the GGH3 cell line (26, 27), activation of protein kinase A via forskolin or membrane-permeable cAMP analogs does not activate but rather inhibits GnRH responsiveness of the GnRHR promoter in T3-1 cells (12). Here we asked whether the ability of forskolin to inhibit the GnRH response of the GnRHR promoter might be manifested as a loss of JNK activation. Increasing concentrations of forskolin did not inhibit GnRH activation of ERK (Fig. 9). In contrast, GnRH-stimulated JNK activation was lost on addition of 1 and 10 µM forskolin. These same doses of forskolin also effectively blocked GnRH activation of the GnRHR promoter. The effects of forskolin presumably reflect increased intracellular concentrations of cAMP. Consistent with this notion, the addition of 8-Br-cAMP led to a similar loss in GnRH activation of JNK and responsiveness of the GnRHR gene promoter (data not shown).
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T3-1 cells that had been treated with 100 nM GnRH for 0, 1, or 6 h in the presence or absence of 10 µM forskolin. Forskolin clearly attenuated GnRH induction of AP-1 at both 1 and 6 h (Fig. 10A). Furthermore, this attenuation is reflected as both an inhibition of GnRH-induced phosphorylation of JunD (Fig. 10B) and loss of GnRH activation of Gal4-JunD and Gal4-FosB (Fig. 10C). In contrast, forskolin appeared to have little effect on GnRH induction of cFos expression, a result consistent with ERK-dependent activation of cFos gene expression. |
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-subunit, LHß subunit, and GnRHR itself (7, 8, 10, 12, 13). GnRH signaling to the -subunit gene appears to be mediated by PKC and ERK-dependent activation of an Ets-binding site (7, 9, 40). GnRH induction of LHß gene expression has been shown to require ERK activation of Egr-1 (41, 42); however, a role for JNK in GnRH signaling to the LHß gene has also been suggested (43, 44). Using the gonadotrope-derived T3-1 cell line, we and others have shown that GnRH stimulation of the GnRHR gene requires a canonical AP-1 element (12, 13). These results, however, stand in contrast to work conducted using a heterologous cell line (GGH3) in which GnRH responsiveness was mapped to a cAMP response-like element (26, 27). To more clearly define a role for AP-1, we sought to determine the contribution of this element to GnRH regulation of the GnRHR promoter in transgenic mice, perhaps the most robust test for the potential contribution of a regulatory element to promoter activity. Consistent with earlier work (25), we found that 1900 bp of proximal promoter from the GnRHR promoter is sufficient to confer both tissue-specific expression and GnRH responsiveness on a heterologous reporter gene. In contrast, removal of the AP-1 element in the context of the same 1900-bp promoter region leads to a complete loss of GnRH responsiveness of this promoter in independent lines of mice. Thus, whereas we do not preclude potential contributions of other regulatory sites, an intact AP-1 element appears to be necessary for GnRH responsiveness of the GnRHR gene in vivo. Activation at an AP-1 site typically requires an increase in either the amount or activity of one or more members of the Jun and Fos families of DNA-binding proteins (45, 46). Interaction of these proteins yields the functionally dimeric AP-1 complex (45, 46). Consistent with a previous report (13), we found that GnRH enhances Jun and Fos binding activity, a response that appears to involve both transcriptional and posttranslational mechanisms. In regard to the latter, we found that both JunD and FosB are targets for posttranslational regulation by GnRH. It is interesting to note that a functional interaction of JunD and FosB is characteristic of AP-1 activation by the tumor promoter okadaic acid (35). Furthermore, GnRH has been shown to induce JunD DNA binding in ovarian cancer cells (47). Thus, GnRH regulation of JunD may have implications in affecting gene expression in both normal and transformed cell types. Although JunD and FosB represent the primary binding components, GnRH also appears to modestly enhance cFos binding at AP-1; however, consistent with others (23, 24, 39), GnRH regulation of cFos was mediated at the level of gene expression and synthesis of new protein.
Based on the ability of a 60-µM dose of PD98059 to block GnRH responsiveness of the GnRHR gene promoter, we had previously suggested that ERK is essential in transducing the GnRH signal to the GnRHR gene (12). Thus, we were somewhat surprised that overexpression of a constitutively active form of Raf-kinase (Raf-CAAX) was ineffective in activating the GnRHR promoter. In fact, this observation served as the primary impetus to reexamine the relative importance of ERK and JNK in GnRH activation of the GnRHR gene. Based on several independent experimental paradigms, we feel that the cumulative body of data supports the interpretation that JNK, more so than ERK, is the essential MAPK underlying the transcriptional response of the GnRHR promoter to GnRH stimulation. First, doses of PD98059 that eliminated GnRH activation of ERK did not affect GnRH regulation of GnRHR promoter activity. Rather, a loss of GnRH responsiveness of the promoter was evident only at doses of PD98059 that effectively eliminated both ERK and JNK activation. Second, pharmacological blockade of L-type calcium channels with nimodipine completely abrogated GnRH activation of ERK. However, both JNK activation and the GnRH response of the GnRHR promoter were retained. In contrast, GnRH induced JNK activation and GnRH responsiveness of the GnRHR promoter were both lost in T3-1 cells stably expressing a dominant-negative form of SEK1 (36). Finally, we should note that, whereas GnRH responsiveness of the GnRHR promoter was retained in the presence of nimodipine, the magnitude of the response was attenuated. Thus, we cannot reject the possibility that ERK partially contributes to GnRH regulation of the GnRHR promoter. Alternatively, the contribution of calcium influx via L-type VGCC to the GnRH response of the GnRHR promoter may be independent of MAPK activation.
It is interesting to note that, whereas GnRH activation of JNK as well as the functional response of the GnRHR promoter was lost in the SEK1 dominant-negative cell line, both responses were fully retained by treatment with PMA. Thus, PMA would appear to activate JNK through a PKC-dependent JNKK isoform that is unaffected by the SEK1 dominant-negative. A similar phenomenon has been reported in studies examining the role of JNK in Fas-mediated apoptosis (48). A member of the TNF/nerve growth factor receptor superfamily, Fas plays a key role in transducing apoptotic signals (49). As in the present studies, overexpression of a dominant-negative SEK-1 inhibited JNK activation by Fas but did not affect PMA-mediated JNK activation (48).
In contrast to GGH3 cells in which GnRH activation of the GnRHR gene proceeds through a cAMP-dependent mechanism (26, 27), elevation of intracellular levels of cAMP in T3-1 cells inhibits GnRH responsiveness of the GnRHR gene (12). Here we found that one plausible explanation for this inhibition is a cAMP-mediated blockade of JNK activation and the subsequent loss of posttranslational regulation of JunD and FosB by GnRH. Clearly such a mechanism presupposes a regulatory role for JNK in phosphorylation of JunD and FosB, the primary binding components at the GnRHR AP-1 site. In this regard, JunD has been shown to serve as a direct target for JNK phosphorylation (50). Similarly there is recent evidence supporting JNK mediated phosphorylation of Fos family members (51). Consistent with cFos gene induction proceeding through an ERK rather than JNK-dependent mechanism (7, 40), we find little effect of forskolin on GnRH-induced cFos gene expression.
In the current studies, we have not sought to determine at what level cAMP may be affecting the JNK regulatory cascade; however, it is interesting to note that an inhibitory effect of both cAMP and forskolin on JNK activation has been reported in other systems including smooth muscle (52), thyroid cells (53), and osteoblasts (54). As a final point, the physiological relevance of the biochemical cross-talk between cAMP and GnRH activation of JNK is difficult to establish. Partially at issue is the identity of a physiological ligand that would activate an increase in intracellular levels of cAMP in gonadotropes. Certainly one candidate is pituitary adenylate cyclase activating polypeptide (PACAP). So named based on its ability to activate adenylate cyclase and increase intracellular concentrations of cAMP, PACAP regulates multiple endocrine cell types in the anterior pituitary gland (55). As such, PACAP receptors are found on both gonadotrope and T3-1 cells (56, 57). With regard to the latter, we found that PACAP, like cAMP and forskolin, also inhibits GnRH activation of JNK and the GnRHR gene promoter (data not shown).
The pulsatile discharge of GnRH from the hypothalamus not only stimulates but is obligatory for synthesis and secretion of LH. These stimulatory effects of GnRH are manifested at multiple levels including expression of genes encoding for the common - and unique ß-subunits of LH and enhanced pituitary sensitivity to GnRH resulting from increased expression of GnRH receptors. With the advent of permanent cell lines of gonadotrope origin, much progress has been made in elucidating the signaling pathways, protein, and DNA regulatory elements that underlie GnRH activation of the gonadotropin subunit genes. We found that the mechanisms underlying GnRH regulation of the GnRHR gene possess features both common to and distinct from those that have been defined for the LH subunit genes. As to the former, it is becoming increasingly clear that GnRH activation of multiple MAPK including ERK and JNK underlies GnRH signaling to the genes encoding the gonadotropin subunits and the GnRHR; however, the DNA elements and proteins impacted by these pathways differ. Based on functional assays in both T3-1 cells and transgenic mice, we suggest that the AP-1 element located in the proximal promoter of the GnRHR gene is critical for GnRH responsiveness. Furthermore, we suggest that GnRH regulation at this site reflects enhanced binding of JunD and FosB and requires an intact JNK signaling cascade.
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Acknowledgments |
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T3-1 cells were a generous gift from Dr. Pamela Mellon (Salk Institute, La Jolla, CA). The SEK1-DN plasmid was generously provided by Dr. Leonard I. Zon. The authors would like to thank Dr. Richard Bowen, Dr. Dawn Duval, Dr. Todd Farmerie, Amy Farris, Meredith Holtzen, Dr. Scott Nelson, and Michelle Simms for their time and effort toward completion of this study. |
Footnotes |
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1 B.S.E. and B.R.W. contributed equally to this work. 
2 New address: Department of Animal Science, A224j Animal Science Building, University of Nebraska, Lincoln, Nebraska 68583-0908.
Abbreviations: AP-1, Activator protein-1; AS, antiserum; GnRHR, GnRH receptor; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; MEK, mitogen-activated ERK kinase; PKC, protein kinase C; PACAP, pituitary adenylate cyclase activating polypeptide; PMA, phorbol ester; RIPA, radioimmunoprecipitation assay; RSV-ßgal, Rous sarcoma virus promoter linked to the cDNA-encoding ß-galactosidase; TBS-T, NaCl, Tris, and Tween 20; VGCC, voltage-gated calcium channel.
Received July 31, 2002.
Accepted for publication November 27, 2002.
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