Probing the characteristics of constitutive steroid biosynthesis in R2C Leydig tumor cells

The Steroidogenic Acute Regulatory (StAR) protein plays an important role in the acute regulation of steroid hormone biosynthesis through its function in mediating cholesterol transfer to the inner mitochondrial membrane where the cholesterol side chain cleavage enzyme system resides. The StAR protein was identified and cloned in 1994, a process simplified through studies in two different Leydig tumor cell lines: MA-10 and R2C cells. MA-10 cells synthesize steroids and StAR protein only following trophic hormone stimulation, but R2C cells produce abundant steroids and StAR protein in a constitutive manner. In this work, we attempted to identify the differential regulation of steroidogenesis in both cell lines and to explore the mechanism(s) behind the highly active steroidogenic phenotype in R2C cells.

In studying the alterations in cholesterol homeostasis found in the R2C cell, we first examined the levels of cholesterol esters and free cholesterol. The results indicated that R2C mitochondria were four-fold enriched in free cholesterol content when compared to MA-10 mitochondria. In support of this observation, in addition to the high levels of StAR protein found in R2C cells, R2C cells express much higher levels of SR-BI (the physiological receptor for the binding and uptake of HDL cholesterol in steroidogenic tissues of rodents), and thus have a higher capacity to take up extracellular cholesteryl esters when compared to MA-10 cells. R2C cells also show enhanced expression of Hormone Sensitive Lipase, the enzyme involved in hydrolysis of cholesteryl esters to release free cholesterol. Therefore, the high level of steroid biosynthesis in R2C cells is a result of the constitutive expression of the components involved in the uptake of cholesteryl esters (SR-BI), their conversion to free cholesterol (HSL) and its mobilization to the inner mitochondrial membrane (StAR).

Next, we performed experiments to determine the levels of both positive and negative transcription factors known to bind the StAR promoter in order to determine if one of them might be responsible for the constitutive steroid production observed. Several transcription factors in R2C cells were measured and the results demonstrated that DAX-1 protein, a negative regulator of several steroidogenic genes, was undetectable in R2C cells and therefore might account for the observed increase in StAR synthesis and steroidogenesis. We utilized both a Tet-on inducible gene expression system and a retroviral infection system to determine the effects of DAX-1 expression in R2C cells and observed that overexpression of DAX-1 protein in R2C cells repressed both steroid and StAR synthesis by --60% and -40% respectively. These observations suggest that the high levels of constitutive steroid biosynthesis and StAR expression in R2C cells may be, in part, a result of the absence of the DAX-1 transcription factor. Finally, we focused on the PKA and PKC signal transduction pathways in order to determine whether these pathways were actively driving steroidogenesis and StAR expression. R2C cells had marginally elevated PKA activity and an active PKC pathway under basal conditions since inhibitors of PKA and PKC resulted in decreases in steroid production and StAR levels. To study the roles of both the PKA and PKC pathways in steroid and StAR synthesis we tested other Leydig tumor cell lines, MA-10 and mLTC-1 cells, using PMA (10 nM, a PKC activator) and a cAMP analog ((Bu)2cAMP 0.05 mM, a PKA activator). While neither treatment alone produced significant increases in steroid synthesis, PMA could induce high amounts of StAR protein. Treatment with both PMA and 0.05 mM (Bu)2cAMP resulted in significant increases in steroids. These studies determined that addition of 0.05 mM (Bu)2cAMP was required for the phosphorylation of StAR protein and was necessary to activate the PMA-induced StAR.