The steroidogenic acute regulatory (StAR) protein promotes intramitochondrial delivery of cholesterol to the cholesterol side-chain cleavage system, which catalyzes the first enzymatic step in all steroid synthesis. Intriguingly, substrate cholesterol derived from lipoprotein can upregulate StAR gene expression. Moreover, substrate oxysterols have been suggested to also play a role. To investigate whether oxysterols can regulate StAR expression, two steroidogenic cell lines, mouse Y1 adrenocortical and MA-10 Leydig tumor cells, were treated with various oxysterols and steroids, including 25-hydroxycholesterol (25 OHC), 22(R)OHC and 20 OHC. The majority of these compounds rapidly increased StAR protein levels within as little as 1 h. The most potent oxysterols were 20 OHC for Y1 and 25 OHC for MA-10 cells. After 8 h, StAR mRNA abundance also increased whereas there were no detected changes in promoter activity. Thus, in contrast to lipoprotein, oxysterols acutely increase StAR protein levels independently of mRNA abundance, and later increase mRNA levels independently of new gene transcription. Therefore, we propose that oxysterols modulate steroidogenesis at two levels. First, oxysterols may be important in post-transcriptional regulation of StAR activity and production of steroids for paracrine action. Secondly, through direct conversion to steroid, oxysterols may account in part for StAR-independent steroid production in the body.

The production of hormones in steroidogenic tissues is dependent upon the availability of free cholesterol at the inner mitochondrial membrane, the site of the cholesterol side chain cleavage. In adrenocortical cells, adrenocorticotropin (ACTH) 1 stimulates hydrolysis of cholesteryl esters in lipid droplets, and transport of free cholesterol to mitochondria for use in steroid synthesis (1, 2). How free cholesterol in the outer mitochondrial membrane is transferred to the P450-cholesterol side chain cleavage (P450-SCC) complex on the inner mitochondrial membrane is unclear, and the existence of a hormonally regulated, labile protein which facilitates this transfer had been predicted for many years (1, 3). The recently discovered steroidogenic acute regulatory protein (StAR) is a 30-kDa mitochondrial protein which has many of the characteristics of this regulator (3,4). StAR has been demonstrated to promote movement of cholesterol from the outer to the inner mitochondrial membrane where P450-SCC catalyzes the first, and rate-limiting, enzymatic step in steroid synthesis (5, 6). In addition, expression of StAR promotes steroidogenesis, and directly facilitates the transfer of cholesterol and other sterols in reconstituted mitochondria (7-9). Perhaps the most convincing evidence that StAR is critical for this process in vivo is the observation by Miller and others (10, 11) that mutations in the StAR gene cause congenital lipoid adrenal hypoplasia. In this autosomal recessive disorder, gonadal and adrenal steroid production are blocked at the level of cholesterol side chain cleavage, resulting in a massive accumulation of cholesterol in the adrenal glands (10). Likewise, mice in which the StAR gene is disrupted have a severe reduction in corticosterone synthesis and accumulate massive lipid deposits in their adrenal cortex (12).Expression of StAR is acutely regulated in response to hormonal stimulation by a process which does not require de novo protein synthesis (5). Phosphorylation of StAR has also been demonstrated to regulate its steroidogenic activity (13). cAMPdependent protein kinase (PKA), as well as the transcription factors steroidogenic factor-1 (SF-1) and C/EBP␤, are required for basal transcription of StAR (14 -19). Other reports demonstrate that StAR transcription can be regulated by oxysterols (20) and agents known to increase cellular calcium can also regulate StAR transcription (21). Since the net result of ACTH stimulation is an increase in free cholesterol at the outer mitochondrial membrane, the current studies were undertaken to ask if the expression of StAR is regulated directly by lipoproteins, the major source of cholesterol for steroid synthesis in adrenocortical cells. Our studies demonstrate that lipoproteins regulate StAR expression by a mechanism that is dependent on cAMP-dependent protein kinase, and the transcription factor SF-1. The regulation of StAR expression by cholesterol may represent a positive feedback circuit designed to maintain maximal output of steroid hormone. * This work ...

We have previously shown that protein kinase C (PKC) suppresses steroidogenesis in Y-1 adrenocortical cells. To ask directly if the PKCα isoform mediates this suppression, we have developed Y-1 cell lines in which PKCα is expressed from a tetracycline-regulated promoter. Induction of PKCα expression in these cell lines results in decreased P450 cholesterol side-chain cleavage enzyme (P450-SCC) activity as judged by the conversion of hydroxycholesterol to pregnenolone. Transcription of a P450-SCC promoter-luciferase construct is also reduced when PKCα expression is increased. However, expression of PKCα has no effect on 8-bromo-cAMP induction of steroidogenesis, indicating that these pathways function independently to regulate steroidogenesis. To determine the relationship between endogenous PKC activity and steroidogenesis, we examined 12 Y-1 subclones that were isolated by limited dilution cloning. In each of these subclones, steroid production correlates inversely with total PKC activity and with the expression of PKCα but not PKCε or PKCζ. These studies define for the first time the role of a specific PKC isoform (PKCα) in regulating steroidogenesis and P450-SCC activity in adrenocortical cells.