alphaT3-1 Cells Research Articles

MRG1 Binds to the LIM Domain of Lhx2 and May Function as a Coactivator to Stimulate Glycoprotein Hormone α-Subunit Gene Expression

Tissue-specific expression of the alpha-subunit gene of glycoprotein hormones involves an enhancer element designated the pituitary glycoprotein basal element, which interacts with the LIM homeodomain transcription factor, Lhx2. In the present studies we have explored the function of the LIM domain of Lhx2 in stimulating alpha-subunit transcription. When fused to the GAL4 DNA-binding domain, the LIM domain of Lhx2 was shown to contain a transcriptional activation domain. Furthermore, in the context of an alpha-subunit reporter gene in which a GAL4-binding site replaced the pituitary glycoprotein basal element, the LIM domain enhanced both basal and Ras-mediated transcription. In addition, a synergistic response to Ras activation was observed when the Lhx2 LIM domain and the transactivation domain of Elk1 are directed to a minimal reporter gene. A yeast two-hybrid screen identified the recently described melanocyte-specific gene-related gene 1 (MRG1) as an Lhx2 LIM-interacting protein. MRG1 was shown to bind Lhx2 in vitro, and a co-immunoprecipitation assay provided evidence that endogenous MRG1 forms a complex with Lhx2 in alphaT3-1 cells. Expression of MRG1 in alphaT3-1 cells enhanced alpha-subunit reporter gene activity. MRG1 was also shown to bind in vitro to the TATA-binding protein and the transcriptional coactivator, p300. These data suggest a model in which the Lhx2 LIM domain activates transcription through interaction with MRG1 leading to recruitment of p300/CBP and the TATA-binding protein.

Calcium Influx through L-type Channels Is Required for Selective Activation of Extracellular Signal-regulated Kinase by Gonadotropin-releasing Hormone

The hypothalamic decapeptide gonadotropin-releasing hormone stimulates mobilization of two discrete pools of calcium in clonal (alphaT3-1) and primary pituitary gonadotropes. A multidisciplinary approach was implemented to investigate the effects of discrete calcium fluctuations on the signaling pathways linking the gonadotropin-releasing hormone receptor to activation of mitogen-activated protein kinases and immediate early genes. Blockade of calcium influx through nifedipine-sensitive voltage-gated calcium channels reduced buserelin-induced activation of extracellular signal-regulated kinase (ERK) and c-Fos while activation of c-Jun N-terminal kinase and c-Jun was unaffected. Inhibition of buserelin-stimulated ERK activity by nifedipine was also observed in rat pituitary cells in primary culture. Direct activation of alphaT3-1 cell L-type calcium channels with the agonist Bay-K 8644 resulted in phosphorylation of ERK and induction of c-Fos. However, simple voltage-induced channel activation did not produce a sufficient calcium signal, since depolarization with 35 mM KCl failed to induce activation of ERK. Depletion of intracellular calcium stores with thapsigargin did not affect buserelin-induced ERK activation. An inhibitor of protein kinase C decreased calcium influx through nifedipine-sensitive calcium channels and phosphorylation of ERK induced by buserelin. Pharmacological inhibition of protein kinase C did not block Bay-K 8644-induced ERK activation. These observations suggest that calcium influx through L-type channels is required for GnRH-induced activation of ERK and c-Fos and that the influence of calcium lies downstream of protein kinase C.

The equine luteinizing hormone beta-subunit promoter contains two functional steroidogenic factor-1 response elements.

The requirements for basal expression of the LH beta-subunit promoter in pituitary gonadotropes are largely unknown. We have used the equine (e) LHbeta subunit promoter as a model to unravel the combinatorial code required for gonadotrope expression. Through the use of 5'-deletion mutagenesis, a region between -185 and -100 of the eLHbeta promoter was shown to play a critical role in maintaining basal promoter activity in alphaT3-1 and LbetaT2 cells. This region encompasses the steroidogenic factor-1 (SF-1) binding site that has been reported to have a functional role in expression of the LHbeta promoter in other species. We have also identified an additional SF-1 site at -55 to -48. Binding of SF-1 to both sites was confirmed by electrophoretic mobility shift assays. Mutations within these sites, either individually or in combination, did not attenuate basal activity of the eLHbeta promoter in alphaT3-1 cells, but did diminish promoter activity in LbetaT2 cells. Interestingly, cotransfection with an expression vector encoding SF-1 induced eLHbeta promoter activity, and this induction was abrogated by mutations within the SF-1 sites in alphaT3-1 cells. Block replacement mutagenesis was performed on the -185/-100 region of the eLHbeta promoter to identify DNA response elements responsible for maintaining basal promoter activity. From this analysis, two regions emerged as being important: a distal 31-bp segment (-181 to -150) and an element located immediately 3' to the distal SF-1 site (-119 to -106). It is hypothesized that these two regions as well as the SF-1 sites represent regulatory elements that contribute to a combinatorial code involved in targeting expression of the eLHbeta promoter to gonadotropes.

Gonadotropin-Releasing Hormone Activates the Equine Luteinizing Hormone β Promoter Through a Protein Kinase C/Mitogen-Activated Protein Kinase Pathway1

GnRH regulation of LH secretion is well understood and involves Ca(2+) mobilization. However, the mechanism by which GnRH activates transcription of the LHbeta gene is controversial. GnRH is known to elevate intracellular calcium and activate the protein kinase C (PKC) pathway. The present study evaluated the pathway(s) involved in GnRH induction of LHbeta transcription. We have previously reported that the equine LHbeta (eLHbeta -448/+60) promoter is active in alphaT3-1 cells. Therefore, we created a clonal, stably transfected alphaT3-1 gonadotroph cell line harboring the eLHbeta promoter (-448/+60) fused to the luciferase reporter gene. Administration of a GnRH agonist resulted in induction of promoter activity that was completely inhibited by the antagonist antide. Various calcium-affecting drugs had no effect on the promoter. Administration of phorbol 12-myristate 13-acetate (PMA) elicited an activation similar to, albeit lower than, that with GnRH. Down-regulation or pharmacological inhibition of PKC completely blocked PMA's induction of the promoter, while GnRH induction was only partly attenuated. Treatment with the mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059, completely inhibited the activation of eLHbeta by PMA but only partly diminished GnRH's induction. Expression of the transcription factor, early growth response protein 1 (Egr1), correlated completely with activation of MAPK, suggesting that Egr1 is the factor through which PKC/MAPK acts. Our data suggest that GnRH induces activity of the eLHbeta promoter by activating a signal transduction cascade involving PKC-MAPK-Egr1 but that has no significant requirement for calcium.

Steroidogenic factor-1 interacts with a gonadotrope-specific element within the first exon of the human gonadotropin-releasing hormone receptor gene to mediate gonadotrope-specific expression.

GnRH plays a pivotal role in regulating human reproductive functions. This hypothalamic peptide interacts with its receptor (GnRHR) on the pituitary gonadotropes to trigger the secretion of gonadotropins, which, in turn, regulates the release of sex steroids from the gonads. In light of the importance of GnRHR, the molecular mechanisms underlying the transcriptional regulation of the human GnRHR (hGnRHR) gene become a key issue in understanding human reproduction. In this report, the possible involvement of steriodogenic factor-1 (SF-1) as a key cell-specific regulator for hGnRHR gene expression was examined. By the transient luciferase reporter gene assays, the wild-type promoter, containing 2.3 kb ofthe hGnRHR gene 5'-flanking region relative to the ATG codon, was able to drive a 3.6 +/- 0.2-fold (P < 0.05) increase in luciferase activity in the mouse alphaT3-1 gonadotropes. Subsequent deletion analysis indicated that the most proximal 173 bp within the first exon of the gene, although not a promoter itself, contains a critical regulatory element(s) essential for the basal expression of the hGnRHR gene. The functional roles of the putative gonadotrope-specific elements (GSE; consensus 5'-CTG(A)/(T)CCTTG-3') residing at positions -5, -134, and -396 were studied by site-directed mutagenesis, and it was found that only the mutation at position -134 significantly reduced the promoter activity (80% reduction; P < 0.05). The attenuation effect of this GSE mutant was cell specific, as it was restricted to alphaT3-1 cells, but not to COS-7 and human ovarian adenocarcinoma (SKOV-3) cells. Competitive mobility shift assays using either alphaT3-1 nuclear extract or recombinant SF-1 protein clearly indicated that SF-1 is able to interact specifically with this GSE element positioned at -134. Using a SF-1 antibody that completely abrogated complex formation in the gel shift assays, the involvement of endogenous nuclear SF-1 was further evidenced. By competitive gel shift assays using oligoprimers with 2-bp scanning mutations, the sequences essential for the interaction with SF-1 were identified (5'-TTG(A)/(T)CCCTG-3', underlined sequences were important). To study the in vivo function of SF-1, vector directing expression of sense or antisense SF-1 messenger RNA (mRNA) was cotransfected with the hGnRHR promoter-luciferase construct into alphaT3-1, SKOV-3, and COS-7 cells. Overexpression of the SF-1 mRNA was able to enhance promoter activities in all of the cells tested. On the contrary, expression of the antisense SF-1 mRNA reduced the hGnRHR promoter activity only in alphaT3-1 cells, not in COS-7 or SKOV-3 cells. In summary, the data reported here provide conclusive evidence that SF-1 interacts with the GSE motif at position -134 within the first exon of the hGnRHR gene to mediate its cell-specific expression.

Functional uncoupling between intracellular calcium dynamics and secretion in the alphaT3-1 gonadotropic cell line.

Gonadotropin releasing hormone (GnRH) stimulates both transcription and secretion of the alpha subunit of the gonadotropins in a Ca2+-dependent fashion. In this study, we examined the role of Ca2+ as the signal coupling agonist occupancy of GnRH receptors to hormone secretion using the gonadotropic cell line alphaT3-1. Treatment of alphaT3-1 cells for 60 min with GnRH (0.1-100 nM), veratridine (50 microM) or high K+ (56 mM) was completely ineffective in stimulating secretion. The lack of effect occurred in spite of a robust, specific, and dose-dependent biphasic [Ca2+]i response consisting of a rapid peak sensitive to thapsigargin (200 nM) followed by a smaller plateau sensitive to the extracellular application of EGTA (5 mM). On the other hand, treatment of alphaT3-1 cells with the Ca2+ ionophore ionomycin resulted in a significant dose-dependent stimulation of secretion and [Ca2+]i responses comparable to those elicited by GnRH. Binding assays revealed the presence of Ins(1,4,5)P3 receptors (Kd = 3.2 nM, Bmax = 50.5 fmol/mg protein) but not ryanodine receptors in alphaT3-1 cell membranes. Together, these results show a functional uncoupling between the [Ca2+]i response and secretion in this cell line, suggesting that the increase in [Ca2+]i triggered by GnRH and depolarization may be necessary but not sufficient to stimulate exocytosis.

Temperature-dependent conformational changes in a voltage-gated potassium channel.

Temperature was used as a biophysical tool to investigate the energy changes associated with conformational change during the gating of a non-inactivating voltage-gated K+ channel present in the membrane of alpha T3-1 cells, a gonadotroph cell line. The time course of the current activation was described by a single exponential function at three temperatures: 15, 25 and 35 degrees C. The Q10 values were between 1.5 to 1.9 and in agreement with the activation energy determined from Arrhenius plots of the forward and backward rate constants associated with channel opening. The Gibb's free energy change associated with channel opening and closing at various membrane potentials estimated by two approaches yield similar values. The changes in Gibb's free energy (delta G degree) with depolarization potential is a quadratic and more prominent at 15 than at 25 or 35 degrees C. The results suggest that increase in temperature favours movement of voltage sensing segments, and reduces the restraint on them brought about by other parts of the channel molecule.

Early growth response protein 1 binds to the luteinizing hormone-beta promoter and mediates gonadotropin-releasing hormone-stimulated gene expression.

The hypothalamic neuropeptide, GnRH, regulates the synthesis and secretion of LH from pituitary gonadotropes. Furthermore, it has been shown that the LH beta-subunit gene is regulated by the transcription factors steroidogenic factor-1 (SF-1) and early growth response protein 1 (Egr1) in vitro and in vivo. The present study investigated the roles played by Egr1 and SF-1 in regulating activity of the equine LH beta-subunit promoter in the gonadotrope cell line, alpha T3-1, and the importance of these factors and cis-acting elements in regulation of the promoter by GnRH. All four members of the Egr family were found to induce activity of the equine promoter. The region responsible for induction by Egr was localized to the proximal 185 bp of the promoter, which contained two Egr response elements. Coexpression of Egr1 and SF-1 led to a synergistic activation of the equine (e)LH beta promoter. Mutation of any of the Egr or SF-1 response elements attenuated this synergism. Endogenous expression of Egr1 in alpha T3-1 cells was not detectable under basal conditions, but was rapidly induced after GnRH stimulation. Reexamination of the promoter constructs harboring mutant Egr or SF-1 sites indicated that these sites were required for GnRH induction. In fact, mutation of both Egr sites within the eLH beta promoter completely attenuated its induction by GnRH. Thus, GnRH induces expression of Egr1, which subsequently activates the eLH beta promoter. Finally, GnRH not only induced expression of Egr1, but also its corepressor, NGFI-A (Egr1) binding protein (Nab1), which can repress Egr1- induced transcription of the eLH beta promoter.

Is gonadotrope expression of the gonadotropin releasing hormone receptor gene mediated by autocrine/paracrine stimulation of an activin response element?

Expression of the FSHbeta subunit and GnRH receptor (GnRHR) genes in gonadotropes is stimulated by activin. We sought to identify the cis-acting element(s) in the murine GnRHR gene promoter which confer activin responsiveness. We established that 600 bp of 5'flanking sequence from the murine GnRHR gene were sufficient to confer activin responsiveness in the gonadotrope-derived alphaT3-1 cell line. Since alphaT3-1 cells, like gonadotropes, secrete activin, we examined the ability of follistatin, an activin binding protein, to block the activin response. Increasing concentrations of follistatin from 0 to 100 ng/ml resulted in a dose dependent decrease in activity of the -600 promoter. Contained within this region are three elements important for expression in alphaT3-1 cells: a Steroidogenic Factor-1 binding site (SF-1), an Activator Protein-1(AP-1) element, and an element termed the GnRH receptor activating sequence or GRAS. A block mutation of GRAS inhibited the ability of the promoter to respond to follistatin. A more refined analysis using a series of two-bp mutations which scan GRAS and flanking sequence revealed exact convergence of GRAS with activin/follistatin responsiveness. Finally, a construct consisting of 3 copies of GRAS placed upstream of a heterologous minimal promoter (3xGRAS-PRL-LUC) was responsive to both activin stimulation and follistatin inhibition in alphaT3-1 cells. Thus, autocrine/paracrine stimulation of gonadotropes by activin illustrates a unique mechanism for cell-specific gene expression.

Homologous regulation of the gonadotropin-releasing hormone receptor gene is partially mediated by protein kinase C activation of an activator protein-1 element.

Homologous regulation of GnRH receptor (GnRHR) gene expression is an established mechanism for controlling the sensitivity of gonadotropes to GnRH. We have found that expression of the GnRHR gene in the gonadotrope-derived alpha T3-1 cell line is mediated by a tripartite enhancer that includes a consensus activator protein-1 (AP-1) element, a binding site for SF-1 (steroidogenic factor-1), and an element we have termed GRAS (GnRHR-activating sequence). Further, in transgenic mice, approximately 1900 b.p. of the murine GnRHR gene promoter are sufficient for tissue-specific expression and GnRH responsiveness. The present studies were designed to further delineate the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. Vectors containing 600 bp of the murine GnRHR gene promoter linked to luciferase (LUC) were transiently transfected into alpha T3-1 cells and exposed to treatments for 4 or 6 h. A GnRH-induced, dose-dependent increase in LUC expression of the -600 promoter was observed with maximal induction of LUC noted at 100 nM GnRH. We next tested the ability of GnRH to stimulate expression of vectors containing mutations in each of the components of the tripartite enhancer. GnRH responsiveness was lost in vectors containing mutations in AP-1. Gel mobility shift data revealed binding of fos/jun family members to the AP-1 element of the murine GnRHR promoter. Treatment with GnRH or phorbol-12-myristate-13-acetate (PMA) (100 nM), but not forskolin (10 microM), increased LUC expression, which was blocked by the protein kinase C (PKC) inhibitor, GF109203X (100 nM), and PKC down-regulation (10 nM PMA for 20 h). In addition, a specific MEK1/MEK2 inhibitor, PD98059 (60 microM), reduced the GnRH and PMA responses whereas the L-type voltage-gated calcium channel agonist, +/- BayK 8644 (5 microM), and antagonist, nimodipine (250 nM), had no effect on GnRH responsiveness. Furthermore, treatment of alpha T3-1 cells with 100 nM GnRH stimulated phosphorylation of both p42 and p44 forms of extracellular signal-regulated kinase (ERK), which was completely blocked with 60 microM PD98059. We suggest that GnRH regulation of the GnRHR gene is partially mediated by an ERK-dependent activation of a canonical AP-1 site located in the proximal promoter of the GnRHR gene.

Activation of the p38 mitogen-activated protein kinase pathway by gonadotropin-releasing hormone.

Previous studies have shown that interaction of GnRH with its serpentine, G protein-coupled receptor results in activation of the extracellular signal regulated protein kinase (ERK) and the Jun N-terminal protein kinase (JNK) pathways in pituitary gonadotropes. In the present study, we examined GnRH-stimulated activation of an additional member of the mitogen-activated protein kinase (MAPK) superfamily, p38 MAPK GnRH treatment of alphaT3-1 cells resulted in tyrosine phosphorylation of several intracellular proteins. Separation of phosphorylated proteins by ion exchange chromatography suggested that GnRH receptor stimulation can activate the p38 MAPK pathway. Immunoprecipitation studies using a phospho-tyrosine antibody resulted in increased amounts of immunoprecipitable p38 MAPK from alphaT3-1 cells treated with GnRH. Immunoblot analysis of whole cell lysates using a phospho-specific antibody directed against dual phosphorylated p38 kinase revealed that GnRH-induced phosphorylation of p38 kinase was dose and time dependent and was correlated with increased p38 kinase activity in vitro. Activation of p38 kinase was blocked by chronic phorbol ester treatment, which depletes protein kinase C isozymes alpha and epsilon. Overexpression of p38 MAPK and an activated form of MAPK kinase 6 resulted in activation of c-jun and c-fos reporter genes, but did not alter the expression of the glycoprotein hormone alpha-subunit reporter. Inhibition of p38 activity with SB203580 resulted in attenuation of GnRH-induced c-fos reporter gene expression, but was not sufficient to reduce GnRH-induced c-jun or glycoprotein hormone alpha-subunit promoter activity. These studies provide evidence that the GnRH signaling pathway in alphaT3-1 cells includes protein kinase C-dependent activation of the p38 MAPK pathway. GnRH integration of c-fos promoter activity may include regulation by p38 MAPK.

Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion.

The GnRH receptor (GnRHR) is a G protein-coupled receptor expressed by gonadotropes in the anterior pituitary gland. In the past several years, much has been learned about the structure-function relationships that exist in this receptor with regard to ligand binding and signal transduction. However, the lack of specific antibodies has precluded any analyses of the behavior of the unbound form of this receptor. We have constructed a functional GnRHR in which enhanced green fluorescent protein is fused to the carboxyl-terminus of the murine GnRHR. This fusion receptor was expressed diffusely throughout the cell, with approximately 38% of the fusion receptors colocalized with a plasma membrane marker in the gonadotrope-derived alphaT3 cell line, and approximately 82% of the fusion receptors colocalized with a membrane marker in Chinese hamster ovary cells. Furthermore, the fusion receptor displayed a Kd of 0.8 nM for iodinated des-Gly10,D-Ala-6-GnRH N-ethyl amide in Chinese hamster ovary cells, which was similar to the Kd of the native GnRHR expressed in alphaT3 cells. The surface mobility of the fusion protein was examined by fluorescence photobleaching recovery methods. In the unbound state the majority of the receptors were laterally mobile and displayed a lateral diffusion rate of 1.2-1.6 x 10(-9) cm2/sec. Binding of GnRH reduced the rate of lateral diffusion over 3-fold and reduced the fraction of mobile receptors from approximately 76-91% to 44-61%. Like GnRH, the competitive GnRH antagonist antide slowed the rate of receptor diffusion approximately 3-fold. In contrast to GnRH, antide had no effect on the fraction of mobile receptors. Thus, an intrinsically fluorescent GnRHR is trafficked to the plasma membrane of mammalian cells, is capable of ligand binding and signal transduction, and allows direct observation of the GnRHR in the nonligand-bound state. Furthermore, fluorescence photobleaching recovery analysis of the GnRHR-green fluorescent protein fusion reveals fundamental differences in the membrane dynamics of the GnRHR induced by the binding of an agonist vs. that induced by the binding of an antagonist.

Targeted expression of toxic genes directed by pituitary hormone promoters: a potential strategy for adenovirus-mediated gene therapy of pituitary tumors.

Pituitary adenomas cause clinical manifestations because of mass effects and excess hormone production. This group of tumors represents a tractable target for gene therapy because they are rarely metastatic and because reductions in tumor size and function, in addition to those achieved after surgery, may be of clinical benefit. In this report we describe a strategy for targeting the expression of toxic genes to pituitary cells using adenoviral vectors. Pituitary hormone promoters (human GH or glycoprotein hormone alpha-subunit) were used to express either a marker gene [beta-galactosidase (beta-gal)] or a toxic gene [herpes simplex virus thymidine kinase (TK)]. In GH-producing GH3 cells and in alpha-subunit-producing pituitary tumor cell lines, recombinant adenoviruses containing either the alpha-subunit promoter (Ad alpha Gal; AdaTK) or the GH promoter (AdGHGal; AdGHTK) were expressed at-high levels. Using histological studies and assays for beta-gal activity, expression was shown to persist for at least 21 days, and it was relatively selective for pituitary cell lines. Cytotoxicity studies were performed using the TK-containing vectors and treatment with ganciclovir. Both AdGHTK and AdaTK caused greater than 95% cytotoxicity of GH3 and alphaT3 cells, respectively, at a viral dose (multiplicity of infection, 5 plaque-forming units/cell) that induced minimal toxicity using control viruses. Little cellular toxicity was seen using a nonpituitary cell line (T47D breast tumor cells). The AdGHTK virus also caused marked reduction in the size of GH3 cell tumors that were propagated in nude mice. These studies suggest that adenoviral vectors carrying human pituitary gland specific promoters may be useful for developing gene therapy strategies for the treatment of pituitary adenomas.

Protein kinase C isoforms in pituitary cells displaying differential sensitivity to phorbol ester.

Investigations with protein kinase C (PKC) isoform-specific antisera, revealed distinct profiles of PKC isoform content amongst pituitary tissues. Western analysis revealed the alpha, beta, delta, epsilon, zeta and theta isoforms of PKC are present in rat anterior and posterior pituitary tissue as well as in the GH3 somatomammotrophic cell line. AtT-20/D16-V corticotrophic and alphaT3-1 gonadotrophic murine cell lines contained no PKC-delta. The gamma or eta isoforms were undetected in any pituitary tissue. PKC activity measurements revealed Ca2+-independent PKCs in alphaT3-1 and GH3 cells which were more sensitive to activation by phorbol-dibutyrate (PDBu) than the corresponding PKC activity found in COS cells. However, Ca2+-dependent PKC activities were of similar sensitivity to PDBu in GH5, alphaT3-1 and COS cells, indicating that functional differences observed in PDBu-sensitivity in these cells may be due to differential activation of Ca2+-independent PKC isoforms. Moreover, substrate-specificity of these PKCs were also compared indicating that the amount of Ca2+-dependency of the observed PKC activity from the same pituitary tissue is dependent upon the substrate utilized by the PKC isotypes present. These findings explain differential sensitivities of PKC-mediated actions that have previously been observed in a range of pituitary cells.

Identification and Characterization of the Gonadotropin-releasing Hormone Response Elements in the Mouse Gonadotropin-releasing Hormone Receptor Gene

The response of the pituitary gonadotrope to gonadotropin-releasing hormone (GnRH) correlates directly with the concentration of GnRH receptors (GnRHR) on the cell surface, which is mediated in part at the level of GnRHR gene expression. Several hormones have been implicated in this regulation, most notably GnRH itself. Despite these observations and the central role that GnRH is known to play in reproductive development and function, the molecular mechanism(s) by which GnRH regulates transcription of the GnRHR gene has not been well elucidated. Previous studies in this laboratory have identified and partially characterized the promoter region of the mouse GnRHR gene and demonstrated that the regulatory elements for tissue-specific expression as well as for GnRH regulation are present within the 1.2-kilobase 5'-flanking sequence. By using deletion and mutational analysis as well as functional transfection studies in the murine gonadotrope-derived alphaT3-1 cell line, we have localized GnRH responsiveness of the mouse GnRHR gene to two DNA sequences at -276/-269 (designated Sequence Underlying Responsiveness to GnRH-2 (SURG-2), which contains the consensus sequence for the activating protein-1-binding site) and -292/-285 (a novel element designated SURG-1), and demonstrated that this response is mediated via protein kinase C. By using the electrophoretic mobility shift assay, we further demonstrate that a member(s) of the Fos/Jun heterodimer superfamily is responsible in part for the DNA-protein complexes formed on SURG-2, using alphaT3-1 nuclear extracts. These data define a bipartite GnRH response element in the mouse GnRHR 5'-flanking sequence and suggest that the activating protein-1 complex plays a central role in conferring GnRH responsiveness to the murine GnRHR gene.

Stimulation of Jun N-terminal kinase (JNK) by gonadotropin-releasing hormone in pituitary alpha T3-1 cell line is mediated by protein kinase C, c-Src, and CDC42.

The signaling of ligands operating via heterotrimeric G proteins is mediated by a complex network that involves sequential phosphorylation events. Signaling by the G protein-coupled receptor GnRH was shown to include elevation of Ca2+ and activation of phospholipases, protein kinase C (PKC) and extra-cellular signal-regulated kinase (ERK). In this study, GnRH was shown to activate Jun N-Terminal Kinase (JNK)/SAPK in alpha T3-1 cells in a PKC- and tyrosine kinase-dependent manner. GnRH as well as tumor-promoting agent (TPA) also increased c-Src activity, which peaked at 2 min after GnRH stimulation and was sensitive both to PKC and to tyrosine kinase inhibitors. Coexpression of Csk, which serves as a Src-dominant interfering kinase, and constitutively active forms of Src, together with JNK, confirmed the involvement of c-Src downstream of PKC in the GnRH-JNK pathway. Coexpression of dominant negative and constitutively active forms of CDC42, Rac1, Ras, MEKK1, and MEK1 with JNK indicated that JNK activation by GnRH and TPA is mediated by CDC42 and MEKK1. Ras and MEK1, which are involved in a related mitogen-activated protein kinase (MAPK) pathway, did not affect JNK activation in alpha T3-1 cells. Taken together, our results suggest that GnRH stimulation of JNK activity is mediated by a unique pathway that includes sequential activation of PKC, c-Src, CDC42, and probably also MEKK1.

Luteinizing hormone-releasing hormone-signal transduction and stathmin phosphorylation in the gonadotrope alphaT3-1 cell line.

We have investigated the effects of GnRH (LHRH) and of the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate on stathmin phosphorylation in the gonadotrope alphaT3-1 cell line. Stathmin expression and its phosphorylation were maximal during the exponential phase of cell growth. LHRH stimulated stathmin phosphorylation through a specific receptor in a dose- and time-dependent manner, and TPA induced a similar extensive stathmin phosphorylation. Their effects were inhibited either in PKC-depleted alphaT3-1 cells, or by the PKC inhibitor staurosporine. In the context of the known implication of PKC in LHRH-induced signal transduction, our results show that stathmin phosphorylation is involved in LHRH transduction, either as a result of direct activation of specific PKC isoforms or through a pathway involving kinases downstream to PKC activation.

The pan-pituitary activator of transcription, Ptx1 (pituitary homeobox 1), acts in synergy with SF-1 and Pit1 and is an upstream regulator of the Lim-homeodomain gene Lim3/Lhx3.

The Ptx1 (pituitary homeobox 1) homeobox transcription factor was isolated as a transcription factor of the pituitary POMC gene. In corticotrope cells that express POMC, cell-specific transcription is conferred in part by the synergistic action of Ptx1 with the basic helix-loop-helix factor NeuroD1. Since Ptx1 expression precedes pituitary development and differentiation, we investigated its expression and function in other pituitary lineages. Ptx1 is expressed in most pituitary-derived cell lines and as is the related Ptx2 (Rieger) gene. However, Ptx1 appears to be the only Ptx protein in corticotropes and the predominant one in gonadotrope cells. Most pituitary hormone-coding gene promoters are activated by Ptx1. Thus, Ptx1 appears to be a general regulator of pituitary-specific transcription. In addition, Ptx1 action is synergized by cell-restricted transcription factors to confer promoter-specific expression. Indeed, in the somatolactotrope lineage, synergism between Ptx1 and Pit1 is observed on the PRL promoter, and strong synergism between Ptx1 and SF-1 is observed in gonadotrope cells on the betaLH promoter but not on the alphaGSU (glycoprotein hormone alpha-subunit gene) and betaFSH promoters. Synergism between these two classes of factors is reminiscent of the interaction between the products of the Drosophila genes Ftz (fushi tarazu) and Ftz-F1. Antisense RNA experiments performed in alphaT3-1 cells that express the alphaGSU gene showed that expression of endogenous alphaGSU is highly dependent on Ptx1 whereas many other genes are not affected. Interestingly, the only other gene found to be highly dependent on Ptx1 for expression was the gene for the Lim3/Lhx3 transcription factor. Thus, these experiments place Ptx1 upstream of Lim3/Lhx3 in a cascade of regulators that appear to work in a combinatorial code to direct pituitary-, lineage-, and promoter-specific transcription.

Differential gonadotropin-releasing hormone stimulation of rat luteinizing hormone subunit gene transcription by calcium influx and mitogen-activated protein kinase-signaling pathways.

Gonadotropin secretion and gene expression are differentially regulated by hypothalamic GnRH pulses by unknown mechanisms. GnRH stimulates calcium influx through L-type voltage-gated channels and activates phospholipase C, leading to increased protein kinase C (PKC) and mitogen-activated protein kinase activity. We found differential contributions of these pathways to GnRH-stimulated rat LH subunit transcription in pituitary gonadotropes and cell lines. Endogenous transcription of the alpha- and LHbeta-subunits in rat pituitary cells was stimulated by GnRH. Independent PKC activation by phorbol myristate acid stimulated only the alpha-subunit gene. In contrast, an L-channel antagonist (nimodipine) inhibited only LHbeta stimulation by GnRH, and an L-channel agonist (BayK 8644) stimulated only basal LHbeta transcription. GnRH induction of a rat alpha-subunit promoter construct in alphaT3 cells was unaffected by nimodipine or elimination of external calcium, while both treatments eliminated the LHbeta response. Application of a mitogen-activated kinase kinase (MEK) inhibitor (PD098059) decreased basal and GnRH-stimulated alpha-subunit promoter activity and had no effect on LHbeta promoter activity. In pituitary cells from mice bearing an LHbeta promoter-luciferase reporter transgene, GnRH stimulation was inhibited by nimodipine but not by PD098059. Thus, GnRH induction and basal control of the alpha-subunit gene seem to occur through the PKC/mitogen-activated protein kinase pathway, while induction of the LHbeta gene is dependent on calcium influx. Differential signaling from the same receptor may be a mechanism for preferential regulation of transcription.

Characterization of the equine glycoprotein hormone alpha-subunit gene reveals divergence in the mechanism of pituitary and placental expression.

The equine glycoprotein hormone alpha-subunit gene is expressed in both pituitary and placenta, unlike that of all other nonprimate mammals studied, in which expression is limited to pituitary. Previous studies of the 5'-flanking region of the equine alpha-subunit promoter have revealed unique characteristics as well as similarities with the human alpha-subunit promoter, which demonstrates a similar pattern of tissue-specific expression. We have cloned and sequenced the equine alpha-subunit gene and have used tissue culture systems and transgenic mice to characterize its expression. Unlike the human promoter, the cloned equine alpha-subunit promoter failed to direct trophoblast-specific expression in either tissue culture or transgenic mouse models, suggesting an entirely different mechanism for expression. In contrast, the equine alpha-subunit promoter was able to direct gonadotroph expression in both tissue culture and transgenic mouse models. In alphaT3-1 cells, 550 base pair (bp) was sufficient for expression. This expression involves promoter elements identified in other species as playing a role in gonadotroph expression, but mutation of these elements reveals differences in their relative contributions to promoter activity. In mice, 2800 bp of 5'-flanking sequence allowed specific expression in gonadotrophs but not in thyrotrophs or placenta. The pattern of estrogen regulation observed in transgenic mice matched neither the repression that has been observed with human and bovine alpha-subunit promoters in transgenic mice nor the stimulation in mRNA levels reported in mares, suggesting a unique mechanism that is not recapitulated in the transgenic model. Thus the equine alpha-subunit promoter uses a combination of conserved and unique features of gene regulation to direct its pattern of tissue-specific expression.