Activation of MAPK Cascades by GnRH: ERK and Jun N-Terminal Kinase Are Involved in Basal and GnRH-Stimulated Activity of the Glycoprotein Hormone LH -Subunit Promoter

Abstract

The role of ERK and Jun N-terminal kinase (JNK) in basal- and GnRH-stimulated LHβ-promoter activity was examined in the gonadotroph cell line LβT-2. GnRH agonist (GnRH-A) stimulates the MAPK cascades ERK, JNK, and p38MAPK, with a peak at 7 min for ERK and at 60 min for JNK and p38MAPK. The rat glycoprotein hormone LHβ-subunit promoter, linked to the chloramphenicol acetyl transferase (CAT) reporter gene, was used to follow its activation. Addition of GnRH-A (10 nm) to LβT-2 cells resulted in a 6-fold increase in LHβ-CAT activity at 8 h, which was markedly reduced by a GnRH antagonist. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca²⁺ ionophore ionomycin, stimulated LHβ-CAT activity. Addition of GnRH-A and TPA together did not produce an additive response. Down-regulation of PKC, but not removal of Ca²⁺, abolished the GnRH-A and the TPA response. Cotransfection of the LHβ-promoter and the constitutively active form of Raf-1 stimulated basal and GnRH-A-induced LHβ-CAT activity. The dominant negative forms of the ERK cascade members Ras, Raf-1, and MAPK/ERK kinase (MEK) markedly reduced basal and GnRH-A-induced LHβ-CAT activity, Similar results were obtained with the MEK inhibitor PD 098059. Cotransfection of the LHβ-promoter and the constitutively active CDC42 stimulated basal and GnRH-A-induced LHβ-CAT activity. The dominant negative forms of the JNK cascade members Rac, CDC42, and SEK markedly diminished basal and GnRH-A-induced LHβ-CAT activity. Interestingly, the constitutively active form of c-Src stimulated the basal and the GnRH-A response, whereas the dominant negative form of c-Src, or the c-Src inhibitor PP1 diminished basal and the GnRH-A response. We conclude that ERK and JNK are involved in basal and GnRH-A stimulation of LHβ-CAT activity. c-Src participates also in LHβ-promoter activation by a mechanism which might be linked to ERK and JNK activation.