Butyrate Transcriptionally Enhances Peptide Transporter PepT1 Expression and Activity

Background: PepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte. Results: We found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1) promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by,2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles

Transcription factors Cdx2 and CREB are crucial for butyrate-induced hPepT1 promoter activity in Caco2-BBE cells.

<p>Caco2-BBE cells were transfected with different constructs of hPepT1 promoter that are point mutated at CREB, Sp1, Cdx2, or AP1 sites. Cells were then treated with 5 mM butyrate for 24 h and luciferase activity relative to hPepT1 promoter activity was measured. Data were normalized by Renilla activity and expressed as fold increase in response to butyrate. Values represent means±S.E. of three determinations. *<i>P</i><0.05; ***<i>P</i><0.001.</p

Butyrate increases hPepT1 protein expression in Caco2-BBE monolayers.

<p>A) Caco2-BBE cells grown on filters were treated with 5 mM butyrate for 24 h and membrane and cytosol hPepT1 protein expression was analyzed by Western blot. Expressions of Na⁺/K⁺ ATPase and GAPDH were used as loading controls. B) Bar graphs represent the densitometric quantification of hPepT1 blots shown in (A). Values represent means±S.E. of four blots from independent experiments. **<i>P</i><0.005.</p

Butyrate transcriptionally up-regulates hPepT1 expression in Caco2-BBE cells.

<p>Caco2-BBE cells were treated with 5 mM butyrate for 24 h and hPepT1 mRNA levels were assessed by A) semi-quantitative RT-PCR and B) real-time RT-PCR. Values represent means±S.E. of three determinations. **<i>P</i><0.005 <i>vs</i> control. To examine butyrate effect on the stability of hPepT1 mRNA, cells were pre-incubated with 5 µg/ml Actinomycin D (AcD) for 30 min and then treated with butyrate for 24 h. C) Total RNA was analyzed by Northern blot using a probe specific to the hPepT1 transcript. RNA loading controls were shown as bottom panel. D) hPepT1 mRNA levels were quantified using real-time RT-PCR. Values expressed as normalized cycling threshold values relative to untreated (0 h) cells represent means±S.E. of three determinations.</p