Activation of the p 38 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 Nterminal 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 aT3–1 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 aT3–1 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 a and e. 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 a-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 GnRHinduced c-jun or glycoprotein hormone a-subunit promoter activity. These studies provide evidence that the GnRH signaling pathway in aT3–1 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. (Endocrinology 140: 1310–1318, 1999) G IS REQUIRED for normal function of the hypothalamo-pituitary-gonadal axis in mammals. The GnRH receptor is a serpentine, G protein-coupled receptor. Binding of GnRH to its receptor results in Gaq/11-mediated activation of phospholipase C, leading to the production of inositol 1,4,5-trisphosphate and diacylglycerol, increased intracellular calcium, and activation of protein kinase C (PKC) (1–12). The consequences of GnRH receptor occupancy include increased synthesis and secretion of the gonadotropic hormones, LH and FSH. Ablation of GnRH stimulation of the anterior pituitary results in decreased glycoprotein hormone subunit messenger RNA concentrations and diminished secretion of the gonadotropic hormones (13, 14). In addition to the glycoprotein hormone subunit genes, GnRH stimulates increased c-fos (15, 16), c-jun (15), mitogen-activated protein kinase phosphatase 2 messenger RNA and transcriptional activation of the ternary complex factor Elk-1 (17). GnRH-dependent intracellular signaling events downstream of PKC have only recently been characterized. Several groups have demonstrated that GnRH receptor occupancy results in activation of extracellular signal regulated protein kinases (ERK) (17–20) and c-jun N-terminal protein kinase (21) (Mulvaney, J. M., T. Zhang, and M. S. Roberson, in preparation), members of the mitogen-activated protein kinase (MAPK) superfamily. Acute phorbol ester administration can mimic GnRH action on ERKs (17–19). Chronic phorbol ester treatment to deplete PKC isozymes reduces GnRHinduced ERK activity, suggesting a requirement for PKC in GnRH activation of the ERK pathway (18, 19). GnRH activation of the JNK cascade is dependent on the low mol wt GTP-binding protein, Cdc42 (21), and appears to be activated independently of diacylglycerol-dependent PKC isozymes, which differentiates GnRH modulation of the ERK and JNK cascades (Mulvaney, J. M. , T. Zhang, and M. S. Roberson, in