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

• Published in: Endocrinology (Philadelphia) 2011-01, Vol.152 (1), p.313-325
• Main Authors: Manna, Pulak R, Soh, Jae-Won, Stocco, Douglas M
• Format: Article
• Language: English
• Subjects: Acetic acid; Activation analysis; Animals; Biological and medical sciences; c-Fos protein; c-Jun protein; Cell activation; Cell Line, Tumor; Chromatin; CREB-binding protein; Cyclic AMP response element-binding protein; Cyclic AMP Response Element-Binding Protein - genetics; Cyclic AMP Response Element-Binding Protein - metabolism; Ectopic expression; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation - physiology; Immunoprecipitation; In vivo methods and tests; Isoenzymes; Isoforms; Kinases; Leydig cells; Leydig Cells - metabolism; Male; Mice; Phorbol 12-myristate 13-acetate; Phorbol Esters - metabolism; Phosphoproteins - genetics; Phosphoproteins - metabolism; Phosphorylation; Progesterone; Protein biosynthesis; Protein kinase A; Protein kinase C; Protein Kinase C - classification; Protein Kinase C - genetics; Protein Kinase C - metabolism; Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Signal Transduction; Star protein; Steroidogenesis; Steroidogenic acute regulatory protein; Steroids; Synergism; Synthesis; Transcription activation; Transcription factors; Tumor cells; Vertebrates: endocrinology
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2010-0874

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 PKCα, -δ, -ε, and -μ/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKCα, δ, and ε 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 −83/−67-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. The functional relevance of PKCμ/PKD isoenzyme in the regulation of StAR expression and steroidogenesis in mouse Leydig cells is discussed.