HORMONE-DEPENDENT POST TRANSLATIONAL REGULATION OF MAP KINASE PHOSPHATASE-1 (MKP-1) IN STEROIDOGENIC CELLS

BRION L; GOROSTIZAGA A; GARCÍA MMS; GÓMEZ N; MACIEL FC; MALOBERTI P; MÉNDEZ CF; PODESTÁ EJ; PAZ C

Rio de Janeiro, Brasil

Congreso; 13th International Congress of Endocrinology; 2008

International Society of Endocrinology

Steroid production in Leydig and adrenocortical cells is controlled by LH and ACTH respectively by a mechanism that involves PKA and ERK activation. In these cells hormone-dependent ERK1/2 activation is a key step in Steroidogenic Acute Regulatory (StAR) protein induction and steroidogenesis. LH and ACTH signaling pathway also includes the activation of MAP kinase phosphatase-1 (MKP-1) gene transcription by a PKA mediated mechanism. Given that MKP-1 controls nuclear MAP kinase activity, the action of this phosphatase has important consequences for gene transcription. In the present work we analyzed the post-translational regulation of MKP-1 and its functional role on the expression of steroidogenesis-related genes. Western blot analysis showed that ACTH stimulation (0-4 h) up-regulates MKP-1 protein in Y1 adrenocortical cells transiently overexpressing the protein. The magnitude of this effect was higher than that observed in mock transfected cells, suggesting a post-translation effect of ACTH on the MKP-1 protein. The effect of ACTH on MKP-1 protein level is dependent on ERK1/2 mediated phosphorylation, since it was reduced by the MEK inhibitor PD98059. In Leydig cells we also detected an ERK1/2-mediated post translational up-regulation of MKP-1. The exposure of non-stimulated Y1 and MA-10 Leydig cells overexpressing MKP-1 to a proteasome inhibitor also produces MKP-1 accumulation. However, the protein level reached by this treatment was not higher than the levels reached in stimulated cells exposed to proteasome inhibitor. Thus, our results support the notion that in adrenocortical and Leydig cells ERK1/2–mediated phosphorylation of MKP-1 impairs its proteasomal degradation. We also tested the role of MKP-1 on cAMP-stimulated StAR promoter activity using a reporter (luciferase) system. 8Br-cAMP stimulation (6 h) enhanced promoter activity (in relative luciferase units, Control = 0.36ｱ0.03 vs. 8Br = n1.0ｱ0.1, P<0.01) and MKP-1 overexpression reduced the effect (8Br/MKP1 = 0.46ｱ0.09, P<0.01 vs 8Br). We conclude that cAMP exerts a fine control of MKP-1 levels that sets a temporal limit to the regulation of MAP kinase-induced gene transcription.