MAPK Phosphatase-2 (MKP-2) Is Induced by hCG and Plays a Role in the Regulation of CYP11A1 Expression in MA-10 Leydig Cells

• Published in: Endocrinology (Philadelphia) 2013-04, Vol.154 (4), p.1488-1500
• Main Authors: Gómez, Natalia V, Gorostizaga, Alejandra B, Mori Sequeiros García, María M, Brion, Laura, Acquier, Andrea, González-Calvar, Silvia I, Méndez, Carlos F, Podestá, Ernesto J, Paz, Cristina
• Format: Article
• Language: English
• Subjects: 8-Bromo Cyclic Adenosine Monophosphate - metabolism; Accumulation; Animals; Biological and medical sciences; Cell activation; Cell Line, Tumor; Chimeras; Cholesterol Side-Chain Cleavage Enzyme - metabolism; Chorionic gonadotropin; Chorionic Gonadotropin - metabolism; Cyclic AMP; Down-regulation; Extracellular signal-regulated kinase; Flags; Fundamental and applied biological sciences. Psychology; Gene regulation; Gonadotropins; Kinases; Leydig cells; Leydig Cells - enzymology; Male; MAP kinase; Mice; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; MKP-2 protein; mRNA; Phosphatase; Phosphorylation; Pituitary (anterior); Protein folding; Protein kinase A; Protein Tyrosine Phosphatases - metabolism; Proteins; Real-Time Polymerase Chain Reaction; Receptor mechanisms; Receptors, LH - metabolism; RNA, Messenger - analysis; RNA, Messenger - metabolism; Stimulation; Up-Regulation; Vertebrates: endocrinology
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2012-2032

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.