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 approximately 2-fold in CV-1 cells. In contrast, cholesterol, progesterone, and the 27-OHC metabolites, 27-OHC-5beta-3-one and 7alpha,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, 3beta-OH-5-cholestenoic acid from radiolabeled 27-OHC. However, this metabolite inhibited StAR promoter activity in CV-1, COS-1 and CHO cells. Because 7alpha,27-OHC was unable to increase SF-1-dependent StAR promoter activity, we examined 27-OHC 7alpha-hydroxylase in COS-1 and CHO cells. COS-1 cells contained high 7alpha-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.
Highlights
Oxysterols exert a major influence over cellular cholesterol homeostasis
Because steroidogenic factor-1 (SF-1) is essential for cAMP responsiveness of the human steroidogenic acute regulatory protein (StAR) gene promoter as well as the promoters of many of the steroid hydroxylase genes [7, 11], we examined the effect of 27-OHC on 8-bromo-cAMP-stimulated StAR promoter activity
CV-1 cells co-transfected with the SF-1 expression vector and the StAR promoter construct were treated with a maximal stimulatory dose of 27-OHC (10 M) and increasing concentrations of 8-bromo-cAMP (Fig. 2). 27-OHC alone caused a 2.3-fold increase in StAR promoter activity
Summary
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. 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. Certain oxysterols reduce nuclear levels of the key transcription factors controlling genes regulating cholesterol homeostasis by suppressing the proteolytic processing of sterol regulatory element binding proteins (SREBPs)1 [1, 2]. They activate cholesterol ester synthesis by allosterically modulating acyl-CoA cholesterol acyl transferase activity [3]. Because of the pivotal role that StAR plays in controlling one arm of the cholesterol metabolic pathway, we wished to determine whether oxysterols influence StAR expression
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