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 to 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 and 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. Overall design: mRNA profiles from liver tissue isolated from WT mice treated with antisense inhibitors of MMAB (MMAB ASO) or antisense inhibitor control (Control ASO). Mice received one i.p. injection per week of 50 mg/kg Control ASO or MMAB ASO for 6 weeks. 48 hours after the last injection animals were sacrificed and liver sample were collected. Total RNA from liver samples of mice treated with 50 mg/kg Control ASO or MMAB ASO was extracted and purified using the RNeasy isolation Kit (Qiagen) according to the manufacturer's instructions and treated with DNAse to remove genomic contamination (RNA MinElute Cleanup, Qiagen). rRNA was depleted from RNA samples using Ribo-Zero rRNA Removal Kit (Illumina). RNA libraries were performed using TrueSeq Small RNA Library preparation (Illumina). Libraries were sequenced for 45 cycles on Illumina HiSeq 2000 platform (1 x 100bp read length).