RESUMO
In Leydig cells, LH and cAMP promote ERK1/2 activation and MAPK phosphatase-1 (MKP-1) induction. MKP-1 up-regulation, which involves post-translational modifications such as ERK1/2-mediated phosphorylation, reduces ERK1/2 phosphorylation as well as Steroidogenic Acute Regulatory (StAR) protein expression and steroidogenesis. As LH- and cAMP-promoted StAR transcription requires the induction of Nur77, product of Nr4a1 gene, we analyzed the roles of ERK1/2 and MKP-1 in 8Br-cAMP-mediated Nr4a1 expression in MA-10 Leydig cells. Pharmacological blockade of ERK1/2 activation partially reduced the 8Br-cAMP-mediated increase in both Nr4a1 messenger levels and promoter activity. MKP-1 knock-down increased 8Br-cAMP-induced promoter activity, while its over-expression produced the opposite effect. It is concluded that Nr4a1 induction is dependent on ERK1/2 and that MKP-1 negatively regulates this induction. Experiments based on the over-expression of MKP-1 mutated forms revealed that MKP-1 half life is determined by post-translational modifications in ERK-consensus sites, a regulation that modulates the effect of MKP-1 on Nr4a1 expression.
Assuntos
AMP Cíclico/farmacologia , Fosfatase 1 de Especificidade Dupla/metabolismo , Células Intersticiais do Testículo/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Animais , Linhagem Celular , Estabilidade Enzimática/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Modelos Biológicos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Intrarenally-produced dopamine (DA) induces a large increase in urinary sodium excretion mainly due to the inhibition of tubular sodium reabsorption. We aimed to study the participation of reactive oxygen species (ROS) in DA signaling pathway in proximal tubule cells. Our results show that DA increased ROS production in OK cells and indicate the mitochondria as the main source of ROS. DA also increased ERK1/2, superoxide dismutase (SOD) and transcription factor κB (NF-κB) activity. These findings suggest that DA generates mitochondria-derived ROS that activate ERK1/2 and subsequently NF-κB and SOD activity at concentrations that exert a physiological regulation of renal function.
Assuntos
Dopamina/fisiologia , Túbulos Renais Proximais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Catalase/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Túbulos Renais Proximais/citologia , Sistema de Sinalização das MAP Quinases , Mitocôndrias/metabolismo , NF-kappa B/metabolismo , Gambás , Fosforilação , Superóxido Dismutase/metabolismoRESUMO
Luteinizing hormone (LH) activates ERK1/2, MAP kinases (MAPKs) 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 2h) 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.
