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1.
Endocrinology ; 154(4): 1488-500, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23471219

ABSTRACT

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.


Subject(s)
Cholesterol Side-Chain Cleavage Enzyme/metabolism , Chorionic Gonadotropin/metabolism , Leydig Cells/enzymology , Protein Tyrosine Phosphatases/metabolism , RNA, Messenger/metabolism , Receptors, LH/metabolism , 8-Bromo Cyclic Adenosine Monophosphate/metabolism , Animals , Cell Line, Tumor , Male , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation , RNA, Messenger/analysis , Real-Time Polymerase Chain Reaction , Up-Regulation
2.
Endocrinology ; 152(7): 2665-77, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21558315

ABSTRACT

MAP kinases (MAPKs), such as ERK1/2, exert profound effects on a variety of physiological processes. In steroidogenic cells, ERK1/2 are involved in the expression and activation of steroidogenic acute regulatory protein, which plays a central role in the regulation of steroidogenesis. In MA-10 Leydig cells, LH and chorionic gonadotropin (CG) trigger transient ERK1/2 activation via protein kinase A, although the events that lead to ERK1/2 inactivation are not fully described. Here, we describe the hormonal regulation of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates MAPKs, in MA-10 cells. In our experiments, human CG (hCG)/cAMP stimulation rapidly and transiently increased MKP-1 mRNA levels by a transcriptional action. This effect was accompanied by an increase in protein levels in both nuclear and mitochondrial compartments. In cells transiently expressing flag-MKP-1 protein, hCG/cAMP promoted the accumulation of the recombinant protein in a time-dependent manner (10-fold at 1 h). Moreover, hCG/cAMP triggered ERK1/2-dependent MKP-1 phosphorylation. The blockade of cAMP-induced MAPK kinase/ERK activation abated MKP-1 phosphorylation but only partially reduced flag-MKP-1 protein accumulation. Together, these results suggest that hCG regulates MKP-1 at transcriptional and posttranslational level, protein phosphorylation being one of the mechanisms involved in this regulation. Our study also demonstrates that MKP-1 overexpression reduces the effects of cAMP on ERK1/2 phosphorylation, steroidogenic acute regulatory gene promoter activity, mRNA levels, and steroidogenesis, whereas MKP-1 down-regulation by small interfering RNA produces opposite effects. In summary, our data demonstrate that hCG regulates MKP-1 expression at multiple stages as a negative feedback regulatory mechanism to modulate the hormonal action on ERK1/2 activity and steroidogenesis.


Subject(s)
Chorionic Gonadotropin/metabolism , Cyclic AMP/metabolism , Dual Specificity Phosphatase 1/metabolism , Leydig Cells/metabolism , Transcriptional Activation , Animals , Cell Line , Cell Nucleus/metabolism , Dual Specificity Phosphatase 1/antagonists & inhibitors , Dual Specificity Phosphatase 1/genetics , Genes, Reporter , Leydig Cells/cytology , MAP Kinase Signaling System/drug effects , Male , Mice , Mitochondria/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation/drug effects , Promoter Regions, Genetic , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/drug effects , RNA, Messenger/metabolism , RNA, Small Interfering , Recombinant Fusion Proteins/metabolism , Transcriptional Activation/drug effects
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