The Involvement of Specific PKC Isoenzymes in Phorbol Ester-Mediated Regulation of Steroidogenic Acute Regulatory Protein Expression and Steroid Synthesis in Mouse Leydig Cells

Abstract

Protein kinase C (PKC) is a multigene family of serine/threonine kinases. PKC is involved in regulating adrenal and gonadal steroidogenesis; however, the functional relevance of the different PKC isoenzymes remains obscure. In this study, we demonstrate that MA-10 mouse Leydig tumor cells express several PKC isoforms to varying levels and that the activation of PKC signaling, by phorbol 12-myristate 13-acetate (PMA) elevated the expression and phosphorylation of PKC␣, -␦, -, and -/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKC␣, ␦, and and PKD, using small interfering RNAs, resulted in deceases in basal and PMA-mediated StAR and steroid levels and demonstrated the importance of PKD in steroidogenesis. PKD was capable of controlling PMA and cAMP/PKA-mediated synergism involved in the steroidogenic response. Further studies pointed out that the regulatory events effected by PKD are associated with cAMP response element-binding protein (CREB) and c-Jun/c-Fos-mediated transcription of the StAR gene. Chromatin immunoprecipitation studies revealed that the activation of phosphorylated CREB, c-Jun, and c-Fos by PMA was correlated with in vivo protein-DNA interactions and the recruitment of CREB-binding protein, whereas knockdown of PKD suppressed the association of these factors with the StAR promoter. Ectopic expression of CREB-binding protein enhanced the trans-activation potential of CREB and c-Jun/c-Fos in StAR gene expression. Using EMSA, a Ϫ83/Ϫ67-bp region of the StAR promoter was shown to bind PKD-transfected MA-10 nuclear extract in a PMA-responsive manner, targeting CREB and c-Jun/c-Fos proteins. These findings provide evidence for the presence of multiple PKC isoforms and demonstrate the molecular events by which selective isozymes, especially PKD, influence PMA/PKC signaling involved in the regulation of the steroidogenic machinery in mouse Leydig cells. (Endocrinology 152: 313-325, 2011)

Primary Aldosteronism: Changing Definitions and New Concepts of Physiology and Pathophysiology Both Inside and Outside the Kidney

“…An IP 3 -induced efflux of calcium from the ER also contributes to the rise in intracellular calcium, which leads to activation of calcium/calmodulin-dependent protein kinases (CaMK) (173,398) and ultimately activation by CaMK of cAMP-response element binding protein (CREB) which promotes StAR activity (482). DAG-induced activation of phospholipase D (PLD) (51) and protein kinases C (PKC) and D (PKD) (52) may represent an additional means by which CREB is activated and StAR activity enhanced (222,307).…”

Mechanisms of Action of Hormone-sensitive Lipase in Mouse Leydig Cells

“…MA-10 cells were cultured in either 35-or 60-mm plates to 60 -70% confluence and cells were transfected using Lipofectamine 2000 reagent (Invitrogen), under optimized conditions (28,32). The amount of DNA used in different transfections was equalized with pcDNA3.1 empty expression vector (Invitrogen).…”

“…Silencing-Knockdown of endogenous HSL and LXR␣/␤ was performed using small interfering RNAs (siRNAs) with either X-tremeGENE siRNA transfection reagent (Roche Diagnostics Corp.) or Lipofectamine 2000 reagent (Invitrogen), under optimized conditions (32,33). Silencer negative control and mouse HSL (#1, 5Ј-GCUCAUCUCCUAUGACCUA-3Ј, and #2, 5Ј-GCAAGAGUAUGUCACGCUA-3Ј), mouse LXR␣ (5Ј-GGA-GUGUCGACUUCGCAAA-3Ј), and LXR␤ (5Ј-AGACAGAA-UGCAUCACGUU-3Ј) siRNAs were obtained as annealed oligos (Ambion Inc., Austin, TX).…”

“…Immunoblotting-Immunoblotting studies were carried out using total cellular protein (32,33). Briefly, cells were homogenized in lysis buffer, and equal amounts of protein (20 -30 g) were solubilized in sample buffer and loaded onto 6 -12% SDS-PAGE (Mini Protean II System, Bio-Rad).…”

Calcium-dependent Nr4a1 expression in mouse Leydig cells requires distinct AP1/CRE and MEF2 elements

“…Activated cJUN and CREB have been correlated with maximal steroidogenesis and activation of Nr4a1 promoter activity , Manna et al 2011. As CREB and cJUN dimerize and bind to target promoters (Hai & Curran 1991), we assessed the impact of CREB knockdown on NR4A1 expression and steroidogenesis.…”