Oxysterol Regulation of Steroidogenic Acute Regulatory Protein Gene Expression

Abstract

Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects of oxysterols on the expression of steroidogenic acute regulatory protein (StAR), which increases the delivery of cholesterol to sterol-metabolizing P450s in the mitochondria. 22(R)-hydroxycholesterol (22(R)-OHC), 25-OHC, and 27-OHC each increased steroidogenic factor-1 (SF-1)-mediated StAR gene transactivation by ϳ2-fold in CV-1 cells. In contrast, cholesterol, progesterone, and the 27-OHC metabolites, 27-OHC-5␤-3-one and 7␣,27-OHC, had no effect. Unlike our findings in CV-1 cells, SF-1-dependent StAR promoter activity was not augmented by 27-OHC in COS-1 cells, Y-1 cells, BeWo choriocarcinoma cells, Chinese hamster ovary (CHO) cells, and human granulosa cells. Studies examining the metabolism of 27-OHC indicated that CV-1 cells formed a single polar metabolite, 3␤-OH-5-cholestenoic acid from radiolabeled 27-OHC. However, this metabolite inhibited StAR promoter activity in CV-1, COS-1 and CHO cells. Because 7␣,27-OHC was unable to increase SF-1-dependent StAR promoter activity, we examined 27-OHC 7␣-hydroxylase in COS-1 and CHO cells. COS-1 cells contained high 7␣-hydroxylase activity, whereas the enzyme was undetectable in CHO cells. The hypothesis that oxysterols act in CV-1 cells to increase StAR promoter activity by reducing nuclear levels of sterol regulatory element binding protein was tested. This notion was refuted when it was discovered that sterol regulatory element binding protein-1a is a potent activator of the StAR promoter in CV-1, COS-1, and human granulosa cells. Human granulosa and theca cells, which express endogenous SF-1, contained more than 5-fold more StAR protein following addition of 27-OHC, whereas StAR mRNA levels remained unchanged. We conclude that 1) there are cell-specific effects of oxysterols on SF-1-dependent transactivation; 2) the ability to increase transactivation is limited to certain oxysterols; 3) there are cell-specific pathways of oxysterol metabolism; and 4) oxysterols elevate StAR protein levels through posttranscriptional actions.

Lipoproteins Regulate Expression of the Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenocortical Cells

“…This is an attractive hypothesis since our data indicates that lipoproteins regulate StAR via the transcription factor SF-1, and oxysterols have been reported to be a ligand for both SF-1 as well as another orphan nuclear receptor, LXR (38,39). Furthermore, a recent report suggests that oxysterols are able to regulate StAR transcription in nonsteroid producing cells (20). While aminoglutethimide and ketoconazole both blocked P450-SCC activity by Ͼ95%, neither inhibitor had any effect on the induction of StAR by lipoprotein.…”

“…As a second approach to verify that PKA is required for the induction of StAR by LDL, we utilized the Y-1 mutant cell line, Y-1 kin-8 which is defective in the activation of PKA due to a mutation in the type I regulatory subunit (23). As seen in Regulation of StAR by Lipoproteins Does Not Require P450-SCC Activity-Oxysterols regulate many key events in cholesterol metabolism (27)(28)(29)(30), and have been demonstrated to regulate StAR transcription in non-steroid producing cells (20). Side chain cleavage of lipoprotein-derived cholesterol is one mechanism by which regulatory oxysterols may be generated in steroidogenic cells.…”

“…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.…”

CCAAT Enhancer-binding Protein β and GATA-4 Binding Regions within the Promoter of the Steroidogenic Acute Regulatory Protein (StAR) Gene Are Required for Transcription in Rat Ovarian Cells

“…At present, the available results are somewhat inconsistent. Using the human, mouse, and rat promoters, an apparent up-regulation of StAR transcription by SF-1 could be demonstrated upon co-transfection of SF-1 cDNA and promoter-reporter plasmids in nonsteroidogenic cells (25)(26)(27)(28)(29). However, other studies analyzing the activity of StAR promoter in SF-1-expressing cells did not support a role for SF-1 in a cAMP-inducible fashion (30).…”

“…Finally, three sera served for negative controls, including antiserum to sterol-responsive element-binding protein (SREBP, Fig. 9, lanes 5 and 6), previously shown to activate StAR transcription in human granulosa cells (29), and ineffective antisera to c-Fos and c-Jun (Fig. 9, lanes 8 and 9).…”