The involvement of specific PKC isoenzymes in phorbol ester-mediated regulation of steroidogenic acute regulatory protein expression and steroid synthesis in mouse Leydig cells.
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.
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
Pulak R
PR
Manna
Jae-Won
JW
Soh
Douglas M
DM
Stocco
D000818:Animals; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D007527:Isoenzymes; D007985:Leydig Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D010703:Phorbol Esters / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D015398:Signal Transduction; D000098682:Steroidogenic Acute Regulatory Protein
D017362:Cyclic AMP Response Element-Binding Protein; D007527:Isoenzymes; D010703:Phorbol Esters; D010750:Phosphoproteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D000098682:Steroidogenic Acute Regulatory Protein; C089212:protein kinase D; D011493:Protein Kinase C