Constitutive steroidogenesis in the R2C Leydig tumor cell line is maintained by the adenosine 3',5'-cyclic monophosphate-independent production of a cycloheximide-sensitive factor that enhances mitochondrial pregnenolone biosynthesis.

These studies were designed to characterize constitutive steroidogenesis in Leydig tumor cells. Constitutive steroidogenesis was investigated by comparing constitutively active R2C Leydig tumor cells to trophic hormone-responsive MA-10 Leydig tumor cells. Unlike the MA-10 cells, R2C cells appeared to synthesize steroid hormones independently of the cAMP-protein kinase pathway. Although the adenylate cyclase of R2C cells could be stimulated in the expected manner by cholera toxin, cAMP concentrations in these cells were low, and R2C cell steroidogenesis could be dissociated from other cAMP-dependent processes. Two cAMP-dependent processes in steroidogenic cells, protein kinase activation and lactate formation, showed low basal activities in R2C cells and could be stimulated by (Bu)2cAMP with a dose dependence similar to that detected in MA-10 cells. Steroid hormone biosynthesis parallelled these other cAMP-dependent processes in MA-10 cells, but not in R2C cells. Cycloheximide, however, caused similar dose-dependent inhibition of steroidogenesis in both the R2C and MA-10 cells. Using a cell component bioassay, it was shown that R2C cells constitutively synthesize an extramitochondrial cycloheximide-sensitive factor that is functionally identical to the factor produced in response to hCG in MA-10 cells. This factor enhanced mitochondrial pregnenolone biosynthesis. Thus, constitutive steroidogenesis in R2C cells could be explained by the cAMP-independent but cycloheximide-sensitive constitutive production of an extramitochondrial factor that activated mitochondrial pregnenolone biosynthesis.