Negative Feedback Regulation of Cholesterol Biosynthesis by MMAB

Intricate regulatory networks govern the net balance of cholesterol biosynthesis, uptake and efflux; however, the mechanisms surrounding cholesterol homeostasis remain incompletely understood. Here, we develop an integrative genomic strategy to detect regulators of LDLR activity and identify 250 genes whose knockdown affects LDL-cholesterol uptake and whose expression is modulated by intracellular cholesterol levels in human hepatic cells. From these hits, we focus on MMAB, an enzyme which catalyzes the conversion of vitamin B12 to adenosylcobalamin, and whose expression has previously been linked with altered levels of circulating cholesterol in humans. We demonstrate that hepatic levels of MMAB are modulated by dietary and cellular cholesterol levels through SREBP2, the master transcriptional regulator of cholesterol homeostasis. Knockdown of MMAB decreases intracellular cholesterol levels and augments SREBP2-mediated gene expression and LDL-cholesterol uptake in human and mouse hepatic cell lines. Reductions in total sterol content were attributed to increased intracellular levels of propionic and methylmalonic acid and subsequent inhibition of HMGCR activity and cholesterol biosynthesis. Moreover, mice treated with antisense inhibitors of MMAB display a significant reduction in hepatic HMGCR activity, hepatic sterol content and increased expression of SREBP2-mediated genes. Collectively, these findings reveal an unexpected role for the adenosylcobalamin pathway in regulating LDLR expression and identify MMAB as an additional control point by which cholesterol biosynthesis is regulated by its end product. Human hepatic cells (Huh7) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS and 120 μg/ml native LDL (nLDL, to cholesterol enrich), 5 μM statin (to cholesterol deplete) or vehicle control for 24 h. Following this, cells were harvested in Trizol and RNA was purified using the RNeasy Isolation Kit (Qiagen). The purity and integrity of total RNA was verified using the Agilent Bioanalyzer and hybridized to the Affymetrix GeneChip Human Genome U133 Plus 2.0 Array according to the manufacturer’s instructions. Two biological replicates for each condition were used for analysis. Raw data were normalized and analyzed by GeneSpring GX software version 11.5 (Agilent Technologies).