The rate-limiting enzymatic reaction in steroid biosynthesis (i.e., the conversion of free cholesterol to pregnenolone) is catalyzed by the cytochrome P450 side chain cleavage enzyme (CYP11A1; herein called P450scc). However, the physiologically relevant rate-limiting step is the transfer of free cholesterol from the outer mitochondrial membrane to the inner membrane where the P450scc resides. This transfer is mediated in part by the steroidogenic acute regulatory (StAR) protein. Steroidogenesis in tissues such as the adrenal, ovary, and testis cannot occur without ongoing StAR synthesis. Inhibition of protein synthesis by cycloheximide blocks both hormone-induced StAR synthesis and steroid production in steroidogenic cells.
R2C rat Leydig tumor cells constitutively synthesize steroids. This constitutive steroidogenesis has been studied in several laboratories and compared with that in MA-10 cells, a mouse Leydig tumor cell line. MA-10 cells produce steroids and StAR protein when stimulated by trophic hormones, while R2C cells do not require external stimulation and exhibit inherently high basal levels of steroids and StAR protein. Earlier, we investigated the manner by which R2C cells are equipped to constitutively produce steroids at such high levels. We found that the scavenger receptor-type B class I (SCARBI or SRBI), hormone-sensitive lipase (LIPE or HSL), and StAR, all of which are used in the trafficking of cholesterol for steroidogenesis, are constitutively expressed in R2C cells at levels much higher than those observed in MA-10 cells under basal conditions. We further demonstrated that the levels of the peripheral benzodiazepine receptor (BZRP; herein called PBR), a protein also involved in the transfer of free cholesterol into the inner mitochondrial membrane, were, surprisingly, much higher in MA-10 cells than that in R2C cells. Because it has been shown that PBR is an essential component of cholesterol transfer in steroidogenic cells, perhaps it is the proposed relationship between StAR protein and PBR that is altered in R2C cells and thus responsible for constitutive steroidogenesis.
Given these observations, we sought to determine the mechanism for the constitutively high basal levels of proteins supporting steroid production in R2C cells and, as a target, we chose to focus on the StAR protein in the present study. Because a recent investigation from our laboratory demonstrated that the absence of the dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (NR0B1 or DAX1) transcription factor in R2C cells is only partially responsible for the inherently high steroidogenic output of the cells, we assumed that other factors are also implicated.
Leydig cells are specialized interstitial cells in the testis that produce the testosterone required for spermatogenesis. The signal that initiates steroidogenesis in Leydig cells is the binding of LH to the LH receptor (LHCGR) and the activation of numerous downstream pathways that in turn regulate steroid biosynthesis. There are many lines of evidence indicating that LH can activate both the protein kinase A (PKA) and protein kinase C (PKC) signaling pathways. Using preovulatory follicles from immature rats, Morris and Richards showed that the PKA inhibitor H8Q, and the PKC inhibitor calphostin-C, were able to inhibit LH-induced progesterone synthesis in granulosa cells. Furthermore, they found that following activation of the LH receptor, phospholipase C (PLC) is able to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) liberating diacylglycerol (DAG), which can fully activate PKC, and inositol 1,4,5-triphosphate (IP3), which can trigger the release of calcium from the endoplasmic reticulum. The authors suggested that granulosa cells respond to an LH surge through both the PKA and PKC pathways. An investigation using porcine endometrial cells also demonstrated that stimulation with LH increased cAMP and IP3 concentrations. Oxytocin was even more effective at increasing the concentrations of cAMP. In addition, turnover of IP3 was accelerated by both LH and oxytocin. Unlike PKA, whose role in steroidogenesis and StAR expression has been studied extensively, the involvement of PKC in the pathways controlling StAR and steroid biosynthesis has only recently been implicated. The precise mechanism through which the PKC pathway is involved in steroid production and Star gene regulation has not been demonstrated in the Leydig cell.
When R2C cells, which have a 2-fold higher PKA activity under basal conditions than MA-10 cells, were treated with H8Q to inactivate PKA, constitutive synthesis of Star mRNA and steroids was significantly inhibited, indicating that a basal level of PKA activity is crucial for the cells’ constitutive steroidogenic phenotype. Therefore, we hypothesize that in the basal state, R2C cells have activated signaling pathways that render the R2C cells constitutively steroidogenic. To test this hypothesis, we attempted to determine the role of the PKC and PKA pathways in constitutive StAR and steroid production. Using the R2C, MA-10, and mLTC-1 Leydig tumor cell lines as well as primary cultures of immature rat Leydig cells, we activated PKC and PKA with phorbol-12-myristate-13-acetate (PMA; a synthetic analog of DAG) and (Bu)2cAMP, respectively, to determine the role of each pathway in the regulation of StAR and steroid biosynthesis.
Category: Leydig Cells
Tags: signal transduction, StAR, steroid biosynthesis, testosterone