HIF‐1a‐Mediated Induction of Insig‐2 Regulates Cholesterol Metabolism by Accelerating Degradation of HMGCR

HMG CoA reductase (HMGCR) catalyzes a rate‐limiting reaction in cholesterol synthesis. In cultured cells, sterols exert tight feedback control on HMGCR by inhibiting its gene transcription and by accelerating its proteasomal degradation. Both of these processes require endoplasmic reticulum membrane proteins called Insig‐1 and Insig‐2. Although the transcriptional component of the HMGCR regulatory system has been scrutinized in livers of mice through various transgenic and knockout animals, regulated degradation of HMGCR in animal livers has not been studied in detail. HMGCR protein accumulates disproportionately to its mRNA in livers of mice genetically deficient in both Insigs, suggesting that Insig‐mediated degradation significantly contributes to feedback regulation of the enzyme. As cholesterol synthesis is an oxygen‐intensive process, the current study is aimed at elucidating whether hypoxia modulates Insig‐dependent degradation of HMGCR in liver of mice. To determine if hypoxia affects Insig‐dependent degradation of HMGCR in vivo , a liver‐specific transgenic mouse model was developed which expresses a T7‐tagged membrane domain of HMGCR, which is necessary and sufficient for degradation of HMGCR. As the transcription of the transgene is driven by a constitutive apoE promoter, the feedback regulation of the transgene product is exclusively dependent on proteasomal degradation. In this mouse model, we demonstrated that mice exposed to hypoxia stabilized hypoxia‐inducible factor (HIF)‐1a, increased Insig‐2 mRNA and protein, and accelerated degradation of HMGCR. Similar results were observed in mice administered with dimethyloxalylglycine (DMOG), a pharmacological inhibitor of prolyl hydroxylase that mimics hypoxia. DMOG failed to reduce the levels of HMGCR protein in mice that express the mutant HMGCR which is resistant to Insig‐induced ubiquitination, indicating that degradation of the enzyme significantly contributes to DMOG‐induced inhibition of HMGCR. In cultured human cell lines, DMOG treatment stabilized HIF‐1a, up‐regulated Insig‐2, and accelerated degradation of HMGCR in a dose‐dependent manner. RNAi‐mediated knockdown of HIF‐1a, but not of HIF‐2a, impaired DMOG‐induced expression of Insig‐2 and degradation of HMGCR. Moreover, DMOG‐induced degradation of HMGCR was blocked in cells lacking Insig‐2, whereas DMOG continued to augment HMGCR degradation in Insig‐1 deficient cells. Using chromatin immunoprecipitation and luciferase promoter assays, we show that Insig‐2 is a target gene of HIF‐1 in human and mouse cells. This is consistent with the presence of a putative HRE in the promoters of the human and mouse Insig‐2 gene. Overall, these findings suggest that Insig‐2 is a direct transcriptional target of HIF‐1 and that hypoxia‐induced degradation of HMGCR is mediated through HIF‐1a stabilization and subsequent up‐regulation of Insig‐2. Support or Funding Information NIH Grants HL20948, GM112409