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and TNF-
Regulate the Human NHE3 Gene Expression By Modulating the Sp Family Transcription Factors in Human Intestinal Epithelial Cell Line, C2BBe1
1 Medicine, Section of Digestive Diseases and Nutrition, University of Illinois at Chicago, Chicago, Illinois, United States
2 Medicine, Section of Digestive Diseases and Nutrition, University of Illinois at Chicago, Chicago, Illinois, United States; Jesse Brown VAMC, Chicago, Illinois, United States
* To whom correspondence should be addressed. E-mail: malakoot{at}uic.edu.
Diarrhea associated with inflammatory bowel disease has been attributed to stimulated secretion of proinflammatory cytokines like IFN-
and TNF-
, which have been shown to down-regulate the expression of the sodium hydrogen exchanger3 gene. In this study, we have investigated the mechanism of the NHE3 gene regulation by IFN- and TNF- in C2BBe1 cells. In response to both IFN-(30 ng/ml) and TNF- (20 ng/ml), the construct containing bp -95 to + 5 of the human NHE3 promoter, which harbors a number of cis-elements including four potential Sp1 binding sites, showed a maximum repression of 60%. Knockdown of Sp1 and Sp3 expression using siRNA resulted in a significant inhibition of the NHE3 promoter activity and resistance to cytokines effects. These cytokines showed no effects on the expression of Sp1 and Sp3 mRNA and proteins levels as assessed by RT-PCR and Western blot analyses, respectively. Following treatment with cytokines, the binding of Sp1 and Sp3 proteins to NHE3 promoter decreased significantly, as seen by Gel mobility shift assays and Chromatin Immunoprecipitation assays. The inhibitory effects of both cytokines on the NHE3 promoter were completely blocked by the broad range kinase inhibitor, Staurosporine and selective protein kinase A (PKA) inhibitor, Rp-8-Br-cAMP. The binding affinity of Sp1 and Sp3 proteins for NHE3 Sp1 probe was significantly decreased after in vitro phosphorylation of nuclear proteins by -catalytic subunit of PKA. Our data indicate that IFN- and TNF- may repress the NHE3 promoter activity in C2BBe1 cells by PKA-mediated phosphorylation of Sp1 and Sp3 transcription factors.
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