Abstract
Ensuring continuous intracellular supply of thiamine is essential to maintain metabolism. Cellular homeostasis requires the function of the membrane bound thiamine transporters THTR1 and THTR2. In the absence of increased dietary intake of thiamine, varying intracellular levels to meet metabolic demands during pathophysiological stressors, such as hypoxia, requires adaptive regulatory mechanisms to increase thiamine transport capacity. Previous work has established the up-regulation of SLC19A3 (THTR2) gene expression and activity during hypoxic stress through the activity of the hypoxia inducible transcription factor 1 alpha (HIF-1α). However, it is unknown whether HIF-1α acts directly or indirectly to trans-activate expression of SLC19A3. This work utilized the breast cancer cell line BT-474 treated with 1% O(2) or a hypoxia chemical mimetic deferoxamine to determine the minimal promoter region of SLC19A3 responsible for hypoxia responsiveness. In silico sequence analysis determined two contiguous hypoxia responsive elements in close proximity to the transcriptional start site of the SLC19A3 gene. Using a HIF-1α transcriptional factor ELISA assay, HIF-1α was capable of binding to a dsDNA construct of the SLC19A3 minimal promoter. Chromatin immunoprecipitation assay established that SP1 was bound to the SLC19A3 minimal promoter region under normoxic conditions. However, HIF-1α binding to the minimal promoter region occurred during hypoxic treatments, while no SP1 binding was observed under these conditions. This work demonstrates the direct binding and activation of SLC19A3 expression by HIF-1α during hypoxic stress, suggesting an important adaptive regulatory role for HIF-1α in maintaining thiamine homeostasis.