Abstract
G-protein coupled receptor 43 (GPR43) recognizes short chain fatty acids and is implicated in obesity, colitis, asthma and arthritis. Here, we present the first full characterization of the GPR43 promoter and 5′-UTR. 5′-RACE of the GPR43 transcript identified the transcription start site (TSS) and a 124 bp 5′-UTR followed by a 1335 bp intron upstream of the ATG start codon. The sequence spanning -4560 to +68 bp relative to the GPR43 TSS was found to contain strong promoter activity, increasing luciferase reporter expression by >100-fold in U937 monocytes. Stepwise deletions further narrowed the putative GPR43 promoter (−451 to +68). Site-directed mutagenesis identified XBP1 as a core cis element, the mutation of which abrogated transcriptional activity. Mutations of predicted CREB, CHOP, NFAT and STAT5 binding sites, partially reduced promoter activity. ChIP assays confirmed the binding of XBP1 to the endogenous GPR43 promoter. Consistently, GPR43 expression is reduced in monocytes upon siRNA-knockdown of XBP1, while A549 cells overexpressing XBP1 displayed elevated GPR43 levels. Based on its ability to activate XBP1, we predicted and confirmed that TNFα induces GPR43 expression in human monocytes. Altogether, our findings form the basis for strategic modulation of GPR43 expression, with a view to regulate GPR43-associated diseases.
Highlights
G-protein coupled receptor 43 (GPR43) recognizes short chain fatty acids and is implicated in obesity, colitis, asthma and arthritis
XBP1 is part of the core promoter while CREB, CHOP, NFAT and STAT5 act as enhancers
In this study, we identified and characterized the GPR43 promoter, revealing that XBP1 acts as a core cis promoter element while CREB, CHOP, NFAT and STAT5 act as enhancers (Fig. 2, 4a, 4b)
Summary
G-protein coupled receptor 43 (GPR43) recognizes short chain fatty acids and is implicated in obesity, colitis, asthma and arthritis. While the liver metabolism of ethanol can generate micromolar concentrations of acetate in the blood, by far the most abundant source of SCFAs in the human body is the colonic lumen, where hundreds of millimolars are continuously being produced during the anaerobic fermentation of dietary fibre by saccarolytic gut bacteria[9,10] These gut SCFAs have been found to beneficially modulate blood glucose and lipid levels, the colonic environment, and immune functions[11,12,13]. GPR43 expression appears to be modulated during inflammation since immune challenge by lipopolysaccharide (LPS) or treatment with granulocyte-macrophage colony stimulating factor (GM-CSF) raises GPR43 transcript levels in human monocytes[17]. This tissue-specificity suggests that GPR43 expression is tightly regulated and may be important for its function. Gpr[43] knockout mice phenotype: Exacerbated colitis, arthritis and asthma Reduced neutrophil recruitment Reduced colitis Increased neutrophil recruitment Reduced colitis Reduced ERK and p38 activation in epithelial cells Exacerbated colitis Reduced Treg cell count Increased lipolysis and plasma free fatty acids Improved glucose control and reduced body fat mass on a high fat diet Impaired glucagon-like peptide-1 secretion and glucose tolerance Increased fat accumulation and obesity on a normal diet
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