Adaptive regulation of the vitamin B1 transporter, SLC19A3

Abstract

Thiamine (vitamin B1) is an essential nutrient requiring continuous dietary intake to meet cellular demands in support of metabolism. The activated form of thiamine, thiamine pyrophosphate, serves as a cofactor for enzymes involved in the TCA cycle and the non-oxidative portion of the pentose phosphate pathway. Due to the hydrophilicity of thiamine, it does not easily diffuse across biological membranes and requires the activity of Solute Carrier (SLC) transporters to facilitate uptake into cells. Cells have altered nutrient and growth factor requirements in order to maintain cellular metabolism and conserve anabolic energy under conditions of physiological stress such as hypoxia and nutrient deprivation. In the absence of dietary changes, cells require adaptive regulatory processes to conserve intracellular thiamine levels. Therefore, the purpose of this research is to characterize the effects of hypoxia and thiamine deficiency on thiamine transporter expression and investigate the mechanism of adaptive transcriptional regulation. Using a RT-PCR array strategy to evaluate changes in transporter expression within a chronic hypoxia breast cancer cell line model, we have identified various SLC transporters that are differentially regulated. In particular, the thiamine transporter SLC19A3 was found to be significantly upregulated during chronic and acute hypoxia. SLC19A3 was also upregulated after exposure to thiamine deficient conditions. Interestingly, SLC19A2 was not adaptively upregulated during either hypoxia or thiamine deficient conditions. A decrease in the upregulation of SLC19A3 was found after HIF-1α shRNA treatment indicating the potential involvement of HIF-1α in SLC19A3 adaptive regulation to hypoxia. We report stabilization of HIF-1α protein levels and an increase in the expression of genes involved in glycolytic metabolism during thiamine deficiency. To further investigate the mechanism of SLC19A3 adaptive regulation, we performed in silico sequence analysis and identified four putative hypoxic response elements within the promoter region of SLC19A3. Luciferase assays were used to analyze the promoter activity of deletion and mutation promoter constructs of SLC19A3. We identified two putative hypoxic response elements in the promoter region of SLC19A3 that are involved in the adaptive regulation to hypoxic conditions. Overall, these results suggest a transcriptional congruency between hypoxia and thiamine deficiency centering on the stabilization of HIF-1α.