Molecular mechanisms mediating the adaptive regulation of human colonic thiamine pyrophosphate (TPP) uptake process

Abstract

Thiamin(vitamin B1) is indispensable for normal cellular metabolism due to itsinvolvement in critical metabolic pathways (e.g., energy metabolism, maintenanceof normal cellular redox state, normal mitochondrial structure and function). Human(mammals) cannot synthesize thiamin, and thus, must obtain the vitamin fromexogenous sources, i. e., diet and gut microbiota. A substantial amount of themicrobiota‐generated thiamin exists in the form of TPP. We have previously shown the existence of a specific and high‐affinity carrier‐mediated uptake process for TPP in human colonocytes (Am J Physiol Gastrointest Liver Physiol. 2012; 303: G389‐95), and have recently determined the molecular identity of the system involved (product of the SLC44A4 gene) (J Biol Chem. 2014; 289: 4405‐16). We have also shown that the colonic TPP uptake process is adaptively regulated by the extracellular substrate level, but nothing is known about the molecular mechanism(s) that mediates this regulation. We addressed this issue in the current investigation using the human colonepithelial NCM460 cells as a model. Maintaining these cells in the presence of high levels of TPP (1 mM) for 2 and 9 days was found to lead to a significant(p ≤ 0.01) reduction in the rate of TPP uptake compared to uptake by cells maintained in the absence of added TPP. While no change in the level of expression of the SLC44A4 mRNA was found in cells maintained for 2 days in the presence of high TPP level [suggesting possible involvement of post translation mechanism(s)], a significant (p ≤ 0.01) decrease in level of SLC44A4 mRNA was found in cells maintained for 9 days. We also observed a decrease in activity of the SLC44A4 promoter in cells maintained for 9 days in high TPP level. The latter was associated with clear histone modifications at the SLC44A4 promoter that involve significant decrease in histone H3K4‐trimethylation and H3K9‐acetylation (euchromatin/activating markers)and a significant increase in histone H3K9‐trimethylation and H3K27‐trimethylation (heterochromatin/repressive markers). While maintaining the cells for 9 days in the presence of high TPP level did not affect the level of expression of the two transcription factors, ELF3 and CREB‐1, that drive the activity of the SLC44A4promoter, their binding to the SLC44A4promoter was decreased as shown by Chip‐qPCR analysis. These studies demonstrate, for the first time, that multiple mechanisms are involved in the adaptive regulation of colonic TPP uptake by extracellular substrate level. At early stages, this regulation involves post‐translational mechanism(s), while long‐term adaptive regulation involves transcriptional and epigenetic mechanisms.Support or Funding InformationSupported by grants from the DVA and the NIH (DK56061 and DK58057).