Abstract

The cholesterol metabolites oxysterols play central roles in cholesterol feedback control. They modulate the activity of two master transcription factors that control cholesterol homeostatic responses, sterol regulatory element-binding protein-2 (SREBP-2) and liver X receptor (LXR). Although the role of exogenous oxysterols has been well-established, whether endogenously synthesized oxysterols similarly control both SREBP-2 and LXR to those added exogenously remains poorly explored. Here, we carefully validate the role of oxysterols enzymatically synthesized within cells in cholesterol homeostatic responses. We first show that SREBP-2 responds more sensitively to exogenous oxysterols than LXR. We then show that endogenous 25-hydroxycholesterol (25-HC), 27-HC, and 24S-HC synthesized by CH25H, CYP27A1, and CYP46A1, respectively, suppress SREBP-2 activity at different degrees by stabilizing Insig proteins whereas 7a-HC has little impact on SREBP-2. The results explain the hydroxylation site-specific role of endogenous oxysterols. On the other hand, the expression of CH25H, CYP46A1, CYP27A1, or CYP7A1 fails to induce LXR target gene expression. We also show the 25-HC production-dependent suppression of SREBP-2 using a tetracycline-inducible CH25H expression system. Moreover, we quantitatively determine the specificity of the four cholesterol hydroxylases in living cells. Based on these results, we propose that endogenous side-chain oxysterols primarily regulate the activity of SREBP-2, not LXR.