scholarly journals Sodium butyrate stimulates NHE8 expression via its role on activating NHE8 basal promoter activity

2015 ◽  
Vol 309 (6) ◽  
pp. G500-G505 ◽  
Author(s):  
Hua Xu ◽  
Anthony McCoy ◽  
Jing Li ◽  
Yang Zhao ◽  
Fayez K. Ghishan
Keyword(s):  
Epithelial Cells ◽  
Mrna Expression ◽  
Promoter Region ◽  
Intestinal Epithelial Cells ◽  
Gene Promoter ◽  
Sodium Absorption ◽  
Intestinal Epithelial ◽  
Mobility Shift ◽  
Bacterial Fermentation ◽  
Butyrate Treatment

Butyrate is a major metabolite in colonic lumen. It is produced from bacterial fermentation of dietary fiber. Butyrate has been shown to stimulate electroneutral sodium absorption through its regulation on sodium/hydrogen exchanger 3 (NHE3). Although NHE8, the newest addition of intestinal NHE family, is involved in sodium absorption in the intestinal tract, whether butyrate modulates NHE8 expression in the intestinal epithelial cells is not known. In the current study, we showed that butyrate treatment strongly induced NHE8 protein and NHE8 mRNA expression in human intestinal epithelial cells. Transfection with the human NHE8 promoter reporter constructs showed that butyrate treatment stimulated reporter gene expression at an amount comparable with its stimulation of NHE8 mRNA expression. Interestingly, a similar result was also observed in human NHE8 promoter transfected cells after trichostatin (TSA) treatment. Gel mobility shift assay identified an enhanced Sp3 protein binding on the human NHE8 basal promoter region upon butyrate stimulation. Furthermore, Sp3 acetylation modification is involved in butyrate-mediated NHE8 activation in Caco-2 cells. Our findings suggest that the mechanism of butyrate action on NHE8 expression involves enhanced Sp3 interaction at the basal promoter region of the human NHE8 gene promoter to activate NHE8 gene transcription. Thus butyrate is involved in intestinal regulation of NHE8 resulting enhanced sodium absorption.

scholarly journals Transcriptional inhibition of intestinal NHE8 expression by glucocorticoids involves Pax5

2010 ◽  
Vol 299 (4) ◽  
pp. G921-G927 ◽  
Author(s):  
Hua Xu ◽  
Bo Zhang ◽  
Jing Li ◽  
Huacong Chen ◽  
Chunhui Wang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Promoter Activity ◽  
Intestinal Epithelial Cells ◽  
Gene Promoter ◽  
High Expression ◽  
Male Rats ◽  
Proximal Promoter ◽  
Sodium Absorption ◽  
Intestinal Epithelial ◽  
Intestinal Maturation

Sodium/hydrogen exchangers (NHEs) are a family of proteins that transport sodium ions into the cells by moving protons out of the cells. They play a major role in sodium absorption, cell volume regulation, and intracellular pH regulation. Three out of nine identified NHEs (NHE2, NHE3, and NHE8) are expressed on the apical membrane of intestinal epithelial cells. Glucocorticoids have been found to regulate NHE3 function in the intestine, but it is unknown if they have a similar function on NHE8 expression. Interestingly, high glucocorticoid levels in the intestine coincide chronologically with the change from high expression of NHE8 to high expression of NHE3. Studies were performed to explore the role of glucocorticoids on NHE8 expression during intestinal maturation. Brush-border membrane vesicles were isolated from intestinal epithelia, and Western blotting was performed to determine NHE8 protein expression of suckling male rats treated with methylpredisolone. Real-time PCR was used to quantitate NHE8 mRNA expression in rats and Caco-2 cells. Human NHE8 promoter activity was characterized through transfection of Caco-2 cells. Gel mobility shift assays (GMSAs) were used to identify the promoter sequences and the transcription factors involved in glucocorticoid-mediated regulation. Our results showed that the expression of NHE8 mRNA and protein was decreased in glucocorticoid-treated rats and human intestinal epithelial cells (Caco-2). The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by glucocorticoid treatment. GMSAs suggested that the reduction in promoter activity in the presence of glucocorticoids was due to enhanced transcription factor Pax5 binding on the NHE8 proximal promoter region. In conclusion, this study showed that glucocorticoids inhibit NHE8 gene expression by increasing Pax5 binding on NHE8 gene promoter, suggesting an important role for Pax5 during intestinal maturation.

Sp1 and Sp3 mediate NHE2 gene transcription in the intestinal epithelial cells

2007 ◽  
Vol 293 (1) ◽  
pp. G146-G153 ◽  
Author(s):  
Ping Hua ◽  
Hua Xu ◽  
Jennifer K. Uno ◽  
Maciej A. Lipko ◽  
Jiali Dong ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Epithelial Cells ◽  
Gene Transcription ◽  
Promoter Region ◽  
Promoter Activity ◽  
Intestinal Epithelial Cells ◽  
Gene Promoter ◽  
Minimal Promoter ◽  
Intestinal Epithelial ◽  
Minimal Promoter Region

Our previous studies have identified a minimal Sp1-driven promoter region (nt −36/+116) directing NHE2 expression in mouse renal epithelial cells. However, this minimal promoter region was not sufficient to support active transcription of NHE2 gene in the intestinal epithelial cells, suggesting the need for additional upstream regulatory elements. In the present study, we used nontransformed rat intestinal epithelial (RIE) cells as a model to identify the minimal promoter region and transcription factors necessary for the basal transcription of rat NHE2 gene in the intestinal epithelial cells. We identified a region within the rat NHE2 gene promoter located within nt −67/−43 upstream of transcription initiation site as indispensable for the promoter function in intestinal epithelial cells. Mutations at nt −56/−51 not only abolished the DNA-protein interaction in this region, but also completely abolished NHE2 gene promoter activity in RIE cells. Supershift assays revealed that Sp1 and Sp3 interact with this promoter region, but, contrary to the minimal promoter indispensable for renal expression of NHE2, both transcription factors expressed individually in Drosophila SL2 cells activated rat NHE2 gene promoter. Moreover, Sp1 was a weaker transactivator and when coexpressed in SL2 cells it reduced Sp3-mediated NHE2 basal promoter activity. Furthermore, DNase I footprinting confirmed that nt −58/−51 is protected by nuclear protein from RIE cells. We conclude that the mechanism of basal control of rat NHE2 gene promoter activity is different in the renal and intestinal epithelium, with Sp3 being the major transcriptional activator of NHE2 gene transcription in the intestinal epithelial cells.

scholarly journals HNF4α and CDX2 Regulate Intestinal YAP1 Promoter Activity

2019 ◽  
Vol 20 (12) ◽  
pp. 2981 ◽  
Author(s):  
Larsen ◽  
Davidsen ◽  
Dahlgaard ◽  
Pedersen ◽  
Troelsen
Keyword(s):  
Epithelial Cells ◽  
Promoter Activity ◽  
Intestinal Epithelial Cells ◽  
Hippo Pathway ◽  
Bioinformatic Analysis ◽  
Intestinal Epithelial ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
The Hippo Pathway

The Hippo pathway is important for tissue homeostasis, regulation of organ size andgrowth in most tissues. The co‐transcription factor yes‐associated protein 1 (YAP1) serves as a maindownstream effector of the Hippo pathway and its dysregulation increases cancer development andblocks colonic tissue repair. Nevertheless, little is known about the transcriptional regulation ofYAP1 in intestinal cells. The aim of this study to identify gene control regions in the YAP1 gene andtranscription factors important for intestinal expression. Bioinformatic analysis of caudal typehomeobox 2 (CDX2) and hepatocyte nuclear factor 4 alpha (HNF4α) chromatin immunoprecipitatedDNA from differentiated Caco‐2 cells revealed potential intragenic enhancers in the YAP1 gene.Transfection of luciferase‐expressing YAP1 promoter‐reporter constructs containing the potentialenhancer regions validated one potent enhancer of the YAP1 promoter activity in Caco‐2 and T84cells. Two potential CDX2 and one HNF4α binding sites were identified in the enhancer by in silicotranscription factor binding site analysis and protein‐DNA binding was confirmed in vitro usingelectrophoretic mobility shift assay. It was found by chromatin immunoprecipitation experimentsthat CDX2 and HNF4α bind to the YAP1 enhancer in Caco‐2 cells. These results reveal a previouslyunknown enhancer of the YAP1 promoter activity in the YAP1 gene, with importance for highexpression levels in intestinal epithelial cells. Additionally, CDX2 and HNF4α binding areimportant for the YAP1 enhancer activity in intestinal epithelial cells.

scholarly journals Regulation of NF-κB responses by epigenetic suppression of IκBα expression in HCT116 intestinal epithelial cells

2010 ◽  
Vol 299 (1) ◽  
pp. G96-G105 ◽  
Author(s):  
Angela O'Gorman ◽  
Amy Colleran ◽  
Aideen Ryan ◽  
Jelena Mann ◽  
Laurence John Egan
Keyword(s):  
Epithelial Cells ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Α ◽  
Intestinal Epithelial Cells ◽  
Gene Promoter ◽  
Intestinal Epithelial ◽  
Epigenetic Factors ◽  
Factor Α ◽  
Necrosis Factor ◽  
Hct116 Cells

Intestinal epithelial cells play critical roles in regulating mucosal immunity. Since epigenetic factors such as DNA methylation and histone modifications are implicated in aging, carcinogenesis, and immunity, we set out to assess any role for epigenetic factors in the regulation of intestinal epithelial cell immune responses. Experiments were conducted using the HCT116 cell line, and a subclone was genetically engineered to lack DNA methyltransferases (DNMT). The induction of the chemokine interleukin-8 and the antiapoptotic protein cFLIP by tumor necrosis factor-α were markedly less in HCT116 cells lacking DNMT than in parental cells. These effects were accompanied by lower monocyte chemotaxis and higher caspase signaling in HCT116 cells lacking DNMT than parental cells. Tumor necrosis factor-α-induced NF-κB activation was blocked and IκBα expression was higher in HCT116 cells lacking DNMT than in parental cells. A CpG island in the IκBα gene promoter region was found to contain variable levels of methylation in parental HCT116 cells. Chromatin immunoprecipitation analysis of histone proteins bound to the IκBα gene promoter revealed that higher levels of IκBα expression in HCT116 cells lacking DNMT compared with parental cells were accompanied by more chromatin marks permissive to gene transcription. These findings show that epigenetic factors influence the NF-κB system in intestinal epithelial cells, resulting in a previously unrecognized mechanism of innate immune regulation.

scholarly journals Surface Expression of Toll-Like Receptor 9 Is Upregulated on Intestinal Epithelial Cells in Response to Pathogenic Bacterial DNA

2007 ◽  
Vol 75 (5) ◽  
pp. 2572-2579 ◽  
Author(s):  
Julia B. Ewaschuk ◽  
Jody L. Backer ◽  
Thomas A. Churchill ◽  
Florian Obermeier ◽  
Denis O. Krause ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mrna Expression ◽  
Intestinal Epithelial Cells ◽  
Surface Expression ◽  
Intestinal Epithelial ◽  
Toll Like Receptor ◽  
Colonic Epithelial Cells ◽  
Bacterial Dna ◽  
Tlr9 Expression ◽  
Ht 29

ABSTRACT Colonic epithelial cells are constantly exposed to high levels of bacterial DNA in the intestinal lumen and must recognize and respond appropriately to pathogens, while they maintain a tolerance to nonpathogenic commensal bacterial strains. Bacterial DNA is recognized by Toll-like receptor 9 (TLR9). The aim of this study was to investigate TLR9 expression and localization in colonic epithelial cells under basal conditions and in response to bacterial DNA. HT-29 cells were exposed to DNA from various strains of commensal and pathogenic microbes. TLR9 mRNA expression was determined by real-time reverse transcription-PCR, and interleukin-8 (IL-8) secretion was measured by an enzyme-linked immunosorbent assay. Localization of TLR9 was determined by flow cytometry in HT-29 cells and by immunofluorescence in HT-29 cells and mouse colonic tissue. Immunofluorescence and flow cytometric analyses demonstrated that there was intracellular and surface expression of TLR9 in HT-29 cells under basal conditions. Exposure of cells to DNA from pathogenic strains of Salmonella and Escherichia coli resulted in a significant increase in TLR9 mRNA expression. Salmonella enterica serovar Dublin DNA increased surface TLR9 protein and IL-8 secretion. There was no change in mRNA levels or localization of TLR9 in response to Bifidobacterium breve. Chloroquine did not block IL-8 secretion in response to S. enterica serovar Dublin DNA. TLR9 was expressed on the colonic apical surface in wild-type mice but not in germfree mice. These results demonstrate that intestinal epithelial cells recognize pathogenic bacterial DNA and respond by increasing surface localization and expression of TLR9, suggesting that the epithelial inflammatory response to pathogenic DNA is mediated at least in part by increased TLR9 expression.

scholarly journals Regulation of nuclear factor-κB in intestinal epithelial cells in a cell model of inflammation

2003 ◽  
Vol 12 (5) ◽  
pp. 277-283 ◽  
Author(s):  
Fadia R. Homaidan ◽  
Iman Chakroun ◽  
Marwan E. El-sabban
Keyword(s):  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Cell Model ◽  
Interleukin 1 ◽  
Nuclear Factor Κb ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Intestinal Epithelial ◽  
Mobility Shift ◽  
Apoptotic Proteins

Background:Interleukin-1 (IL-1), an inflammatory cytokine whose levels are elevated in inflamed mucosa, causes part of its effect on intestinal epithelial cells (IEC) through inducing ceramide production.Aim:To study the role of nuclear factor-κB (NF-κB), a pro-inflammatory and anti-apoptotic factor, in IL-1- treated IEC.Methods:NF-κB activity and levels of apoptotic proteins were assessed by electrophoretic mobility shift assay and RNA-protection assay, respectively.Results:IL-1 and ceramide, which have been shown to partially mediate IL-l effects on IEC, activated NF-κB levels significantly. This activation was due to a decrease in IκB-α and IκB-β protein levels. Moreover, the ratio of mRNA levels of anti-apoptotic to pro-apoptotic proteins was significantly increased in IL-1-treated IEC.Conclusion:NF-κB may play a key role in the regulation of the expression of pro-inflammatory and/or apoptotic genes in inflammatory bowel disease, making this protein an attractive target for therapeutic intervention.

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