Abstract
Sterol-regulated HMG-CoA reductase (HMGCR) degradation and SREBP-2 cleavage are two major feedback regulatory mechanisms governing cholesterol biosynthesis. Reportedly, lanosterol selectively stimulates HMGCR degradation, and cholesterol is a specific regulator of SREBP-2 cleavage. However, it is unclear whether other endogenously generated sterols regulate these events. Here, we investigated the sterol intermediates from the mevalonate pathway of cholesterol biosynthesis using a CRISPR/Cas9-mediated genetic engineering approach. With a constructed HeLa cell line expressing the mevalonate transporter, we individually deleted genes encoding major enzymes in the mevalonate pathway, used lipidomics to measure sterol intermediates, and examined HMGCR and SREBP-2 statuses. We found that the C4-dimethylated sterol intermediates, including lanosterol, 24,25-dihydrolanosterol, follicular fluid meiosis activating sterol, testis meiosis activating sterol, and dihydro-testis meiosis activating sterol, were significantly upregulated upon mevalonate loading. These intermediates augmented both degradation of HMGCR and inhibition of SREBP-2 cleavage. The accumulated lanosterol induced rapid degradation of HMGCR, but did not inhibit SREBP-2 cleavage. The newly synthesized cholesterol from the mevalonate pathway is dispensable for inhibiting SREBP-2 cleavage. Together, these results suggest that lanosterol is a bona fide endogenous regulator that specifically promotes HMGCR degradation, and that other C4-dimethylated sterol intermediates may regulate both HMGCR degradation and SREBP-2 cleavage.
Highlights
Sterol-regulated HMG-CoA reductase (HMGCR) degradation and SREBP-2 cleavage are two major feedback regulatory mechanisms governing cholesterol biosynthesis
We first sought to establish a system to evaluate HMGCR degradation and SREBP-2 cleavage in response to endogenous regulators generated from mevalonate
To increase the metabolic flux and augment the regulatory effects of mevalonate on HMGCR and SREBP-2, we generated a cell line stably expressing the monocarboxylate transporter 1 (MCT1) with a phenylalanine-to-cysteine substitution at the amino acid site 360 (MCT1F360C), which allows for efficient uptake of mevalonate into cells [34, 38]
Summary
Sterol-regulated HMG-CoA reductase (HMGCR) degradation and SREBP-2 cleavage are two major feedback regulatory mechanisms governing cholesterol biosynthesis. Lanosterol selectively stimulates HMGCR degradation, and cholesterol is a specific regulator of SREBP-2 cleavage It is unclear whether other endogenously generated sterols regulate these events. We found that the C4-dimethylated sterol intermediates, including lanosterol, 24,25-dihydrolanosterol, follicular fluid meiosis activating sterol, testis meiosis activating sterol, and dihydro-testis meiosis activating sterol, were significantly upregulated upon mevalonate loading These intermediates augmented both degradation of HMGCR and inhibition of SREBP-2 cleavage. The newly synthesized cholesterol from the mevalonate pathway is dispensable for inhibiting SREBP-2 cleavage Together, these results suggest that lanosterol is a bona fide endogenous regulator that promotes HMGCR degradation, and that other C4-dimethylated sterol intermediates may regulate both HMGCR degradation and SREBP-2 cleavage.—Chen, L., M-Y. When the cellular sterol level is Abbreviations: CYP51A1, cytochrome P450 family 51 subfamily A member 1; DHCR24, 24-dehydrocholesterol reductase; 24,25-DHL, 24,25-dihydrolanosterol; DKO, double KO; FDFT1, farnesyl diphosphate farnesyltransferase 1; FF-MAS, follicular fluid meiosis activating sterol; 25-HC, 25-hydroxycholesterol; HeLa/MT, HeLa cells stably expressing the mevalonate transporter; HMGCR, HMG-CoA reductase; MCT1, monocarboxylate transporter 1; MCT1F360C, monocarboxylate transporter 1 with a phenylalanine-to-cysteine substitution at the amino acid site 360; MSMO1, methylsterol monooxygenase 1; MT, mevalonate transporter; PC, phosphatidylcholine; SCAP, SREBP cleavage activating protein; T-MAS, testis meiosis activating sterol
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