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

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 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.

SUBMITTER: Saito H

PROVIDER: S-EPMC9792893 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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

Saito Hodaka H Tachiura Wakana W Nishimura Mizuki M Shimizu Makoto M Sato Ryuichiro R Yamauchi Yoshio Y

The Journal of biological chemistry 20221122 1

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 explo ...[more]

PMID: 36423680

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Project description:Cholesterol accumulation and removal are regulated by two different transcription factors. SREBP-2 (sterol-regulatory-element-binding protein-2) is best known to up-regulate genes involved in cholesterol biosynthesis and uptake, whereas LXR (liver X receptor) is best known for up-regulating cholesterol efflux genes. An important cholesterol efflux gene that is regulated by LXR is the ATP-binding cassette transporter, ABCA1 (ATP-binding cassette transporter-A1). We have previously shown that statin treatment down-regulated ABCA1 expression in human macrophages, probably by inhibiting synthesis of the LXR ligand 24(S),25-epoxycholesterol. However, it was subsequently reported that ABCA1 expression is down-regulated by SREBP-2 through binding of SREBP-2 to an E-box element in ABCA1's proximal promoter. As statin treatment induces SREBP-2 activation, this may provide an alternative explanation for the statin-mediated down-regulation of ABCA1. In the present study, we employed a set of CHO (Chinese-hamster ovary) mutant cell lines to investigate the role of SREBP-2 in the regulation of ABCA1. We observed increased ABCA1 mRNA levels in SREBP-2-overexpressing cells and decreased levels in cells lacking a functional SREBP-2 pathway, which were restored when the SREBP-2 pathway was reinstated. Moreover, ABCA1 gene expression was positively associated with synthesis of 24(S),25-epoxycholesterol in these cell lines. In studies using a human ABCA1 promoter reporter assay, mutation of the E-box motif had a similar response as the wild-type construct to either statin treatment or addition of 24(S),25-epoxycholesterol. By contrast, these responses were completely ablated when the DR4 element to which LXR binds was mutated. These results support the idea that 24(S),25-epoxycholesterol and statin treatment influence ABCA1 transcription via supply of an LXR ligand and not through an SREBP-2/E-box-related mechanism. In addition, our results indicate a critical role of SREBP-2 as a positive regulator of ABCA1 gene expression by enabling the generation of oxysterol ligands for LXR.

Dataset Information

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

Publications

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

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