Regulation of basal promoter activity of the human thiamine pyrophosphate transporter SLC44A4 in human intestinal epithelial cells

Abstract

Microbiota of the large intestine synthesize considerable amount of vitamin B1 in the form of thiamine pyrophosphate (TPP). There is a specific high-affinity regulated carrier-mediated uptake system for TPP in human colonocytes (product of the SLC44A4 gene). The mechanisms of regulation of SLC44A4 gene expression are currently unknown. In this study, we characterized the SLC44A4 minimal promoter region and identified transcription factors important for basal promoter activity in colonic epithelial cells. The 5'-regulatory region of the SLC44A4 gene (1,022 bp) was cloned and showed promoter activity upon transient transfection into human colonic epithelial NCM460 cells. With the use of a series of 5'- and 3'-deletion luciferase reporter constructs, the minimal genomic region that required basal transcription of the SLC44A4 gene expression was mapped between nucleotides -178 and +88 (using the distal transcriptional start site as +1). Mutational analysis performed on putative cis-regulatory elements established the involvement of ETS/ELF3 [E26 transformation-specific sequence (ETS) proteins], cAMP-responsive element (CRE), and SP1/GC-box sequence motifs in basal SLC44A4 promoter activity. By means of EMSA, binding of ELF3 and CRE-binding protein-1 (CREB-1) transcription factors to the SLC44A4 minimal promoter was shown. Contribution of CREB into SLC44A4 promoter activity was confirmed using NCM460 cells overexpressing CREB. We also found high expression of ELF3 and CREB-1 in colonic (NCM460) compared with noncolonic (ARPE19) cells, suggesting their possible contribution to colon-specific pattern of SLC44A4 expression. This study represents the first characterization of the SLC44A4 promoter and reports the importance of both ELF3 and CREB-1 transcription factors in the maintenance of basal promoter activity in colonic epithelial cells.

Mechanism(S) Involved in the Colon-Specific Expression of the Thiamine Pyrophosphate (Tpp) Transporter

“…An in silico CpG island prediction/primer design Methprimer algorithm [ 25 ] was used to predict the CpG islands in the promoter region of the SLC44A4 gene. Experimentally verified 1,022 bp SLC44A4 promoter [ 15 ] was subjected to prediction algorithm analysis using the default parameters. The analysis identified a single CpG island that was defined in accordance with the classical sequence parameters of the CpG islands [ 27 , 28 ].…”

“…Recent studies from our laboratory have shown that human colonocytes possess an efficient and specific carrier-mediated process for uptake of the microbiota generated TPP [ 13 ]. We have also established the molecular identity of the colonic TPP transporter (TPPT) as the product of the SLC44A4 gene [ 14 ], and have further cloned and characterized its 5’-regulatory region and showed an important role for the ETS/ELF3 (E74-Like Factor 3 (Ets Domain Transcription Factor, Epithelial-Specific)) and CREB-1 (cAMP-responsive element-binding protein-1) transcriptional factors in regulating the basal activity of the SLC44A4 promoter [ 15 ]. While examining the pattern of expression of the TPPT along the human and mouse GI tract, we found that expression of this system is mainly restricted to the colon with negligible expression in other parts of the GI tract [ 14 ].…”

Genetic defects of thiamine transport and metabolism: A review of clinical phenotypes, genetics, and functional studies

“…This carrier consists of 710 amino acids and is highly expressed in epithelial cells of the colon, prostate, trachea, and lungs. TPPT has an important role in TPP absorption from microbiota and contributes to cellular nutrition of the local colonocytes …”

“…TPPT has an important role in TPP absorption from microbiota and contributes to cellular nutrition of the local colonocytes. 28 The whole body content of thiamine is estimated in 25 to 30 mg, mostly located in skeletal muscles, heart, brain, liver, and kidneys. In the blood, 80% of thiamine is located in erythrocytes as TPP.…”

Metabolic functions of the human gut microbiota: the role of metalloenzymes

“…195 Indeed, human colonocytes express a thiamine pyrophosphate-specic transport that would allow for uptake of this vitamin by the host. 184,[197][198][199] Interestingly, variants in this gene have been identied as susceptibility markers for ulcerative colitis in Northern Indian populations, which are known to exhibit thiamine deciency. 200,201 This connection between thiamine uptake and ulcerative colitis remains to be explored further.…”