Extracellular Signal-Regulated Kinase and c-Src, But Not Jun N-Terminal Kinase, Are Involved in Basal and Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone α-Subunit Promoter

Abstract

Addition of a GnRH agonist (GnRH-A) to alphaT3-1 cells stimulates different MAPK cascades: ERK, Jun N-terminal kinase (JNK), and p38. Activation of JNK, ERK, and p38 shows a unique fold activation ratio of 25:12:2, which might encode signal specificity. ERK is translocated to the nucleus within 20 min with a peak at 120 min of GnRH-A stimulation. We used the human alpha-subunit promoter linked to chloramphenicol acetyl transferase (alphaCAT) to examine the role of ERK, JNK, and c-Src, which is implicated in MAPK activation, in basal and GnRH-stimulated alphaCAT. Addition of GnRH-A resulted in a 3-fold increase in alphaCAT, whereas the Ca(2+) ionophore ionomycin and the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect. Addition of GnRH-A and TPA, but not GnRH-A and ionomycin, produced a synergistic response, whereas removal of Ca(2+), but not down-regulation of TPA-sensitive PKCs, abolished GnRH-A-stimulated alphaCAT. Thus, regulation of alpha-promoter activity by GnRH is Ca(2+) dependent and is further augmented by PKC. Cotransfection of alphaCAT and constitutively active or dominant negative plasmids of ERK and JNK cascade members, or the use of the ERK inhibitor PD98059, revealed that ERK, but not JNK, is involved in basal and GnRH-A-stimulated alphaCAT. Because c-Src participates in MAPK activation by GnRH, we also studied its role. Cotransfection of alphaCAT and the dominant negative form of c-Src or incubation with the c-Src inhibitor PP1 reduced GnRH-A-stimulated alphaCAT. The 5'-deletion analysis revealed that the -846/-420 region participated in basal alpha-transcription. In addition, the -346/-156 region containing the pituitary glycoprotein hormone basal element, alpha-basal elements, glycoprotein-specific element, and upstream response element is involved in basal and GnRH-A-stimulated alphaCAT. ERK contribution to GnRH maps to -346/-280 containing the pituitary glycoprotein hormone basal element and alpha-basal elements 1/2. Surprisingly, although c-Src is involved in GnRH-A-stimulated ERK, its involvement is mapped to another region (-280/-180) containing the glycoprotein-specific element. Thus, ERK and c-Src but not JNK are involved in basal and GnRH-A-stimulated-alphaCAT, whereas c-Src contribution is independent of ERK activation.

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

“…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).…”

Frequency-Dependent Regulation of Follicle-Stimulating Hormone β by Pulsatile Gonadotropin-Releasing Hormone Is Mediated by Functional Antagonism of bZIP Transcription Factors

“…The answer may rest in either the differential activation of signaling systems within the gonadotrope and/or synthesis, modification, and degradation of either transcription factors or regulatory proteins. It is well established that members of the mitogen activated protein kinase (MAPK) family of signaling proteins are stimulated by GnRH in vivo (50), in cultured rat pituitary fragments (22) and in L␤T2 cells (9,31) to contribute to regulation of gonadotropin subunit gene expression. In particular, activation of phosphorylated extracellular signal-regulated kinases (pERK) 1/2 is more rapid and more sustained in L␤T2 cells perifused with low, rather than high, GnRH pulse frequencies, suggesting that pERK is important in the preferential increase of FSH␤ transcription observed at low GnRH pulse frequency (27).…”

Arrestin-mediated ERK Activation by Gonadotropin-releasing Hormone Receptors

“…Thus, the endogenous mouse GnRHRs of gonadotrope lineage ␣T3-1 cells act primarily via activation of PKC and entry of Ca 2ϩ to cause a Raf-1-dependent activation of ERK (40,41). In these cells, EGF receptor trans-activation is not required (42,43), whereas ERK activation by mouse GnRHRs stably transfected into COS7 cells is dependent upon EGF receptor trans-activation (44). In the HeLa cell model used here, both receptors increase pp-ERK1/2 (as measured by Western blotting or immunohistochemistry) (Figs.…”