1,25-dihydroxyvitamin D regulation of glutamine synthetase and glutamine metabolism in human mammary epithelial cells

Genomic profiling has identified a subset of metabolic genes that are altered by 1,25-dihydroxy-vitamin D (1,25D) in breast cells, including GLUL, the gene that encodes glutamine synthetase (GS). In this study, we explored the relevance of vitamin D modulation of GLUL and other metabolic genes in the context of glutamine utilization and dependence. We show that exposure of breast epithelial cells to glutamine deprivation or a GS inhibitor reduced growth and these effects were exacerbated by cotreatment with 1,25D. 1,25D downregulation of GLUL was sufficient to reduce abundance and activity of GS. Flow cytometry demonstrated that glutamine deprivation induced S phase arrest, likely due to reduced availability of glutamine for DNA synthesis. In contrast, 1,25D induced G₀/G₁ arrest, indicating that its effects are not solely due to reduced glutamine synthesis. Indeed, 1,25D also reduced expression of GLS1 and GLS2 genes, which code for glutaminases that shunt glutamine into the tricarboxylic acid (TCA) cycle. Consistent with reduced entry of glutamine into the TCA cycle, 1,25D inhibited glutamine oxidation and the metabolic response to exogenous glutamine as analyzed by Seahorse Bioscience extracellular flux assays. Effects of 1,25D on GLUL/GS expression and glutamine oxidation were retained in human mammary epithelial (HME) cells that express SV-40 (HME-LT cells) but not in those that express SV-40 and oncogenic H-Ras (HME-PR cells). Furthermore, HME-PR cells exhibited glutamine independence and expressed constitutively high levels of GLUL/ GS, which were unaffected by 1,25D. Collectively, these data suggest that 1,25D alters glutamine availability, dependence, and metabolism in nontransformed and preneoplastic mammary epithelial cells in association with cell cycle arrest.