Hydroxylation site–specific and production-dependent effects of endogenous oxysterols on cholesterol homeostasis: Implications for SREBP-2 and LXR

• Published in: The Journal of biological chemistry 2023-01, Vol.299 (1), p.102733-102733, Article 102733
• Main Authors: Saito, Hodaka, Tachiura, Wakana, Nishimura, Mizuki, Shimizu, Makoto, Sato, Ryuichiro, Yamauchi, Yoshio
• Format: Article
• Language: English
• Subjects: Animals; CH25H; CHO Cells; cholesterol; Cholesterol - metabolism; Cholesterol 24-Hydroxylase; cholesterol regulation; Cricetinae; Cricetulus; CYP27A1; CYP46A1; Homeostasis; Hydroxylation; lipid metabolism; lipid signaling; Liver X Receptors - metabolism; LXR; Mice; oxysterols; Oxysterols - metabolism; Proteins - metabolism; Rats; SREBP-2; Sterol Regulatory Element Binding Protein 2 - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2022.102733

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 in regulating these transcription factors has been well established, whether endogenously synthesized oxysterols similarly control both SREBP-2 and LXR 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 in Chinese hamster ovary cells and rat primary hepatocytes. We then show that 25-hydroxycholesterol (25-HC), 27-hydroxycholesterol, and 24S-hydroxycholesterol endogenously synthesized by CH25H, CYP27A1, and CYP46A1, respectively, suppress SREBP-2 activity at different degrees by stabilizing Insig (insulin-induced gene) proteins, whereas 7α-hydroxycholesterol has little impact on SREBP-2. These results demonstrate the role of site-specific hydroxylation of endogenous oxysterols. In contrast, 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. To induce 25-HC production physiologically, murine macrophages are stimulated with a Toll-like receptor 4 ligand, and its effect on SREBP-2 and LXR is examined. The results also suggest that de novo synthesis of 25-HC preferentially regulates SREBP-2 activity. Finally, we quantitatively determine the specificity of the four cholesterol hydroxylases in living cells. Based on our current findings, we conclude that endogenous side-chain oxysterols primarily regulate the activity of SREBP-2, not LXR.