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Receptors coupled to heterotrimeric G proteins are linked to activation of mitogen-activated protein kinases (MAPKs) via receptor-and cell-specific mechanisms. We have demonstrated recently that gonadotropin-releasing hormone (GnRH) receptor occupancy results in activation of extracellular signal-regulated kinase (ERK) through a mechanism requiring calcium influx through L-type calcium channels in ␣T3-1 cells and primary rat gonadotropes. Further studies were undertaken to explore the signaling mechanisms by which the GnRH receptor is coupled to activation of another member of the MAPK family, c-Jun N-terminal kinase (JNK). GnRH induces activation of the JNK cascade in a dose-, time-, and receptor-dependent manner in clonal ␣T3-1 cells and primary rat pituitary gonadotrophs. Coexpression of dominant negative Cdc42 and kinase-defective p21-activated kinase 1 and MAPK kinase 7 with JNK and ERK indicated that specific activation of JNK by GnRH appears to involve these signaling molecules. Unlike ERK activation, GnRH-stimulated JNK activity does not require activation of protein kinase C and is not blocked after chelation of extracellular calcium with EGTA. GnRH-induced JNK activity was reduced after treatment with the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester), whereas activation of ERK was not affected. Chelation of intracellular calcium also reduced GnRH-induced activation of JNK in rat pituitary cells in primary culture. GnRH-induced induction and activation of the JNK target c-Jun was inhibited after chelation of intracellular calcium, whereas induction of c-Fos, a known target of ERK, was unaffected. Therefore, although activation of ERK by GnRH requires a specific influx of calcium through L-type calcium channels, JNK activation is independent of extracellular calcium but sensitive to chelation of intracellular calcium. Our results provide novel evidence that GnRH activates two MAPK superfamily members via strikingly divergent signaling pathways with differential sensitivity to activation of protein kinase C and mobilization of discrete pools of calcium.

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.

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The aim of these studies was to elucidate a role for epidermal growth factor (EGF) signaling in the transcriptional regulation of the glycoprotein hormone ␣ subunit gene, a subunit of chorionic gonadotropin. Studies examined the effects of EGF and the adenylate cyclase activator forskolin on the expression of a transfected ␣ subunit reporter gene in a human choriocarcinoma cell line (JEG3). At maximal doses, administration of EGF resulted in a 50% increase in a subunit reporter activity; forskolin administration induced a fivefold activation; the combined actions of EGF and forskolin resulted in synergistic activation (greater than eightfold) of the ␣ subunit reporter. Mutagenesis studies revealed that the cyclic AMP response elements (CRE) were required and sufficient to mediate EGF-forskolin-induced synergistic activation. Chorionic gonadotropin (CG) is a heterodimeric glycoprotein hormone consisting of an ␣ subunit common to other glycoprotein hormone family members noncovalently linked to a CG-specific ␤ subunit (58). CG is synthesized and secreted by placental syncytiotrophoblast cells during the first trimester of pregnancy in women and nonhuman primates. CG is a luteotropin required for maintenance of progesterone production by the ovarian corpus luteum in early gestation. An important factor in the establishment of early pregnancy appears to be the timing and rate of increase in the secretion of CG that is highly correlated with a rise in progesterone levels in peripheral circulation (42). Insufficient progesterone production during early pregnancy is correlated with the potential for early or recurrent pregnancy loss in women (6, 7, 27, 47). Thus, endocrine mechanisms that potentiate the synthesis of CG subunits and CG secretion are essential for the establishment of pregnancy. Despite the clear importance of CG to early pregnancy, the specific ligands and signaling mechanisms that regulate the expression of CG subunit genes in placental cells have not been fully elucidated.The ␣ subunit of the glycoprotein hormones is a unique and useful transcriptional model for the study of tissue-specific gene expression because the ␣ subunit gene is expressed in placental and pituitary cells, albeit by various mechanisms. Analysis of the architecture of the ␣ subunit promoter revealed the presence of multiple promoter elements that are required for transcriptional regulation. These include the pituitary glycoprotein hormone basal element, which binds members of the LIM class of homeobox-containing proteins (67,71,72); the ␣ basal element (32); the gonadotrope-specific element, which binds steroidogenic factor 1 (9, 36); the upstream regulatory element (URE) (12,26,38,59); the GATA element, which binds several GATA factors (75); the dual tandem cyclic AMP (cAMP) response elements (CREs), which bind CRE binding protein (CREB) (5,10,12,13,22,25,32,35,52,59,74); the junctional regulatory element (JRE) (4, 12); and a unique CAAT box (12,40). Extensive mutagenesis studies have begun to unravel the specific requirements f...