MAP kinase phosphatase-3 (MKP-3) is transcriptionally and post-translationally up-regulated by hCG and modulates cAMP-induced p21 expression in MA-10 Leydig cells

Abstract Luteinizing hormone (LH) activates ERK1/2, MAP kinases (MAPK) necessary for its action on steroidogenesis and cell proliferation, and also induces MAPK phosphatase-1 (MKP-1), which rapidly dephosphorylates nuclear ERK1/2. MKP-3 is a cytoplasmic ERK-phosphatase up-regulated by proliferative stimuli. MKP-3 also dephosphorylates transcription factor FOXO1, promoting its transport to the nucleus. Here we analyzed MKP-3 expression in MA-10 Leydig cells and demonstrated that LH receptor (LHR) activation with human gonadotropin hormone (hCG) and an analog of its second messenger, 8Br-cAMP, up-regulates MKP-3 by transcriptional and post-translational mechanisms. It is known that FOXO1 drives the Expression of the cell cycle inhibitor p21. Since the activation of this transcription factor by MKP-3 has been reported, we assessed the effect of shRNA against MKP-3 on p21mRNA levels. 8Br-cAMP increased these levels (2-fold at 2 h) and MKP-3 down-regulation reduced this effect. Our work demonstrates that LH/hCG tightly up-regulates MKP-3 which in turn, dephosphorylates ERK1/2 and drives p21 expression. These events could contribute to counteract hormonal action on cell proliferation.Fil: Mori Sequeiros Garcia, Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Gómez, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Gorostizaga, Alejandra Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Acquier, Andrea Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología; ArgentinaFil: González Calvar, Silvia I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Mendez, Carlos Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología; ArgentinaFil: Paz, Cristina del Valle. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentin

MAPK phosphatase-2 (MKP-2) is induced by hCG and plays a role in the regulation of CYP11A1 expression in MA-10 Leydig cells

MAPKs such as ERK1/2 are dephosphorylated, and consequently inactivated, by dual specificity phosphatases (MKPs). In Leydig cells, LH triggers ERK1/2 phosphorylation through the action of protein kinase A. We demonstrate that, in MA-10 Leydig cells, LH receptor activation by human chorionic gonadotropin (hCG) up-regulates MKP-2, a phosphatase that dephosphorylates ERK1/2, among other MAPKs. After 2 hours, hCG and 8-bromo-cAMP (8Br-cAMP) significantly increased MKP-2 mRNA levels (3-fold), which declined to basal levels after 6 hours. MKP-2 protein accumulation exhibited a similar kinetic profile. In cells transiently expressing flag-MKP-2 protein, hCG/8Br-cAMP stimulation promoted the accumulation of the chimera (2.5-fold after 3 h of stimulation). Pharmacologic and biochemical approaches showed that the accumulation of flag-MKP-2 involves a posttranslational modification that increases MKP-2 half-life. MKP-2 down-regulation by a short hairpin RNA (MKP-2 shRNA) raised the levels of phosphorylated ERK1/2 reached by 8Br-cAMP stimulation. This effect was evident after 180 min of stimulation, which suggests that MKP-2 down-regulates the late phase of cAMP-induced ERK1/2 activity. Also, MKP-2 down-regulation by MKP-2 shRNA increased the stimulatory effect of 8Br-cAMP on both promoter activity and messenger levels of CYP11A1, which encodes for the steroidogenic enzyme P450scc and is induced by LH/hCG through protein kinase A and ERK1/2 activities. Our findings demonstrate, for the first time, that LH/hCG tightly regulates MKP-2 expression, which modulates the induction of CYP11A1 by 8Br-cAMP. MKP-2 up-regulation might control ERK1/2 activity in a specific temporal frame to modulate the expression of a finite repertory of ERK-dependent genes.Fil: Gómez, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Gorostizaga, Alejandra Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Mori Sequeiros, María de Las Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Brion, Laura. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Acquier, Andrea Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología; ArgentinaFil: González Calvar, Silvia I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Mendez, Carlos Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires. Facultad de Odontología; ArgentinaFil: Podesta, Ernesto Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Paz, Cristina del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin