Mechanism of Mitogen-Activated Protein Kinase Activation by Gonadotropin-Releasing Hormone in the Pituitary αT3–1 Cell Line: Differential Roles of Calcium and Protein Kinase C*

Abstract

The mechanism of mitogen-activated protein kinase (MAPK, ERK) stimulation by the GnRH analog [D-Trp6]GnRH (GnRH-a) was investigated in the gonadotroph-derived alphaT3-1 cell line. GnRH-a as well as the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated a sustained response of MAPK activity, whereas epidermal growth factor (EGF) stimulated a transient response. MAPK kinase (MEK) is also activated by GnRH-a, but in a transient manner. GnRH-a and TPA apparently activated mainly the MAPK isoform ERK1, as revealed by Mono-Q fast protein liquid chromatography followed by Western blotting as well as by gel kinase assay. GnRH-a and TPA stimulated the tyrosine phosphorylation of several proteins, and this effect as well as the stimulation of MAPK activity were inhibited by the PKC inhibitor GF 109203X. Similarly, down-regulation of TPA-sensitive PKC subspecies nearly abolished the effect of GnRH-a and TPA on MAPK activity. Furthermore, the protein tyrosine kinase (PTK) inhibitor genistein inhibited protein tyrosine phosphorylation and reduced GnRH-a-stimulated MAPK activity by 50%, suggesting the participation of genistein-sensitive and insensitive pathways in GnRH-a action. Although Ca2+ ionophores have only a marginal stimulatory effect, the removal of Ca2+ markedly reduced MAPK activation by GnRH-a and TPA, but had no effect on GnRH-a and TPA stimulation of protein tyrosine phosphorylation. Interestingly, the removal of Ca2+ also partly inhibited the activation of MAPK by EGF and vanadate/H2O2. Thus, a calcium-dependent component(s) downstream of PKC and PTK might also participate in MAPK activation. Elevation of cAMP by forskolin exerted partial inhibition on EGF, but not on TPA or GnRH-a action, suggesting that MEK activators other than Raf-1 might be involved in GnRH action. We conclude that Ca2+, PTK, and PKC participate in the activation of MAPK by GnRH-a, with Ca2+ being necessary downstream to PKC and PTK.

Constitutive Localization of the Gonadotropin-releasing Hormone (GnRH) Receptor to Low Density Membrane Microdomains Is Necessary for GnRH Signaling to ERK

“…The use of PMA in these studies would, presumably, directly activate PKC isozymes thus effectively bypassing the GnRHR, G␣ q/11 , and phospholipase C. Consistent with earlier studies (43)(44)(45)(46), both GnRH and PMA induced ERK phosphorylation in control cells (Fig. 10A).…”

c-Src Is Activated by the Epidermal Growth Factor Receptor in a Pathway That Mediates JNK and ERK Activation by Gonadotropin-releasing Hormone in COS7 Cells

“…Here we studied the signaling processes that lead from GnRHR to ERK and JNK in GnRHR-transfected COS7 cells. The pathways that we have identified in these cells differ substantially from those observed in the pituitary-derived ␣T3-1 cells (32,33,35). Nevertheless, the signaling mechanisms observed here are important for several reasons: (i) the unique mechanism of JNK and ERK activation by GnRH reported here can serve as a model for other GPCRs activation of the MAPK cascades; (ii) a second GnRHR has been recently described (55,56), and therefore it is important to characterize signaling mechanisms specific for type 1 GnRHR; and (iii) aside from its pituitary functions, GnRH exerts also extrapituitary effects upon the gonads and gonadal steroid-dependent tumor cells (42) where the GnRHR may couple to different signaling components.…”

“…Thus, activation of ERK in these cells involves simultaneous activation of a pathway that involves activation of Raf-1 by PKC (33), together with a supportive pathway that includes dynamin, c-Src, and Ras (33). In addition, Ca 2ϩ from various pools has been implicated in the differential activation of ERK and JNK by GnRH in ␣T3-1 cells (32,37). The three pathways do not seem to contradict, but rather to complement each other to form a signaling network, which is essential to achieve the full GnRH effect on MAPKs.…”

“…9 and 31). Studies from our laboratories have shown that the signaling of GnRH in ␣T3-1 cells involves a direct activation of Raf-1 by PKC, and this step is partially dependent on a second pathway consisting of Ras activation downstream of dynamin and c-Src (32)(33)(34). The activation of JNK in these cells is also mediated primarily by PKC that further induces the sequential activation of c-Src and CDC42/RAC (35).…”

Dependence of Gonadotropin-releasing Hormone-induced Neuronal MAPK Signaling on Epidermal Growth Factor Receptor Transactivation

“…The major signal transduction pathways in cells expressing GnRH receptors are initiated by activation of phospholipase C. The consequent calcium (Ca 2ϩ ) mobilization and activation of protein kinase C (PKC) by GnRH are key elements in the hypothalamic control of gonadotropin secretion from the anterior pituitary gland (1,3,5). Activation of PKC and Ca 2ϩ mobilization during GnRH receptor stimulation are also responsible for mediating downstream signals leading to activation of extracellularly regulated mitogen-activated protein kinases (ERK1/2 MAPKs) that transmit signals from the cell surface to the nucleus to regulate transcriptional and other processes (7)(8)(9)(10)(11)(12)(13). However, the specific PKC isoforms that are involved in GnRH-induced ERK1/2 activation in GT1-7 cells are not known.…”