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Involvement of Protein Kinase C and Cyclic Adenosine 3',5'-Monophosphate-Dependent Kinase in Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis in Leydig Cells¹

Department of Cell Biology and Biochemistry,³ Texas Tech University Health Sciences Center, Lubbock, Texas 79430 Department of Urology,⁴ Baylor College of Medicine, Houston, Texas 77030 Center for Cancer Research and Therapeutic Development,⁵ Clark Atlanta University, Atlanta, Georgia 30314

This study investigated the roles of the protein kinase C (PKC) and protein kinase A (PKA) pathways in regulating constitutive steroidogenesis and steroidogenic acute regulatory (STAR; herein designated by its common name, StAR) protein in R2C Leydig tumor cells. Inhibition of PKC and phospholipase C resulted in significant decreases in steroid production, phosphorylation of cAMP-responsive element binding (CREB) protein, and Star gene transcription under basal conditions in R2C cells. These observations were corroborated in MA-10 and mLTC-1 Leydig tumor cell lines, in which activation of PKC by phorbol-12-myristate-13-acetate (PMA, 10 nM) increased CREB phosphorylation and total StAR (tot-StAR) protein expression. However, induction of StAR protein by PMA did not result in the expected concomitant increase in steroids because PKC failed to phosphorylate StAR, the biologically active form of the protein. However, in conjunction with PMA, minor increases in PKA activity using submaximal doses of (Bu)₂cAMP (0.05–0.1 mM; a concentration range insufficient for induction of StAR), were able to stimulate dramatic increases in both phospho-StAR (P-StAR) and steroid production. Human chorionic gonadotropin stimulation also resulted in a further enhancement in P-StAR and progesterone production when added to PMA-treated MA-10 cells. Similar results for tot-StAR and P-StAR expression were observed in primary cultures of immature rat Leydig cells treated with PMA and submaximal doses of (Bu)₂cAMP. In summary, the present study demonstrates that basal activities of both PKC and PKA play important roles in the constitutive steroidogenic characteristics of R2C cells. This study also demonstrates for the first time a role for PMA-induced PKC in StAR protein regulation and the requirement for submaximal doses of cAMP to produce steroids in Leydig cells.

cAMP, CREB, kinases, Leydig cells, PKA, PKC, PMA, progesterone, signal transduction, StAR, steroid biosynthesis, testosterone

² Correspondence: Douglas M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430. FAX: 806 743 2990; doug.stocco{at}ttuhsc.edu

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