The molecular mechanisms involved in the regulation of absorption of the human intestinal NaC1 mediated the concerted action of dual ion exchanges of Na+/H+ and Cl-/HCO3 is not well understood. Our prior studies of the human small intestine and colon suggest that the Na+/H+ exchanger, NHE3, may be the predominant isoform involved in Na+ absorption in the small intestine, whereas NHE2 is important for colonic Na+ absorption. It is, therefore, crucial to investigate the regulation of human Na+/H+ exchangers for a better understanding of normal human intestinal physiology and to understand the molecular basis of diarrheal disorders. Our studies of the NHE3 promoter showed that NHE3 expression is regulated by a number of transcription factors including AP-2, Egr-1, and Sp1 family members. The regulation of NHE3 exchanger function and its promoter activity by different stimuli such as serotonin, nitric oxide, IFN-gamma PMA and Enteropathogenic E. coli infection was also demonstrated. We hypothesize that cis-elements in the NHE3 promoter region and their cognate transcription factors mediate the processes involved in NHE3 regulation during basal transcription, tissue-specific expression and intestinal inflammation and propose the following three specific aims to address them.
Specific Aim 1 will elucidate the role of inflammatory mediators in transcriptional regulation of NHE3 by investigating the mechanisms involved in the stimulation of the NHE3 gene expression by EPEC infection and associated inflammatory mediators such as nitric oxide, TNFalpha, IL-8, IL-1beta and IFN-gamma and by characterizing the cis-regulatory elements and their corresponding trans-acting factors that mediate the effects of these agents.
Specific Aim 2 is designed to determine the molecular mechanisms involved in the transcriptional regulation of the NHE3 gene in response to mitogenic stimuli such as PMA and growth factors through characterization of their respective regulatory elements.
Specific Aim 3 will identify the cis and trans-acting factors that regulate cell-specific expression of the NHE3 gene, utilizing C2/bbe, Caco-2, NCM460, HepG-2, MDCK, and NIH3T3 cell lines.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Clinical and Integrative Gastrointestinal Pathobiology Study Section (CIGP)
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May, Michael K
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University of Illinois at Chicago
Internal Medicine/Medicine
Schools of Medicine
United States
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Saksena, Seema; Gill, Ravinder K; Tyagi, Sangeeta et al. (2008) Role of Fyn and PI3K in H2O2-induced inhibition of apical Cl-/OH- exchange activity in human intestinal epithelial cells. Biochem J 416:99-108
Pearse, Ian; Zhu, Ying X; Murray, Eleanor J et al. (2007) Sp1 and Sp3 control constitutive expression of the human NHE2 promoter by interactions with the proximal promoter and the transcription initiation site. Biochem J 407:101-11
Amin, Md Ruhul; Dudeja, Pradeep K; Ramaswamy, Krishnamurthy et al. (2007) Involvement of Sp1 and Sp3 in differential regulation of human NHE3 promoter activity by sodium butyrate and IFN-gamma/TNF-alpha. Am J Physiol Gastrointest Liver Physiol 293:G374-82
Alrefai, Waddah A; Saksena, Seema; Tyagi, Sangeeta et al. (2007) Taurodeoxycholate modulates apical Cl-/OH- exchange activity in Caco2 cells. Dig Dis Sci 52:1270-8
Gill, Ravinder K; Borthakur, Alip; Hodges, Kim et al. (2007) Mechanism underlying inhibition of intestinal apical Cl/OH exchange following infection with enteropathogenic E. coli. J Clin Invest 117:428-37
Malakooti, Jaleh; Sandoval, Ricardo; Amin, Md Ruhul et al. (2006) Transcriptional stimulation of the human NHE3 promoter activity by PMA: PKC independence and involvement of the transcription factor EGR-1. Biochem J 396:327-36
Hodges, Kim; Gill, Ravinder; Ramaswamy, K et al. (2006) Rapid activation of Na+/H+ exchange by EPEC is PKC mediated. Am J Physiol Gastrointest Liver Physiol 291:G959-68
Borthakur, Alip; Gill, Ravinder K; Hodges, Kim et al. (2006) Enteropathogenic Escherichia coli inhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level. Am J Physiol Gastrointest Liver Physiol 290:G30-5
Gill, Ravinder K; Saksena, Seema; Alrefai, Waddah A et al. (2005) Expression and membrane localization of MCT isoforms along the length of the human intestine. Am J Physiol Cell Physiol 289:C846-52
Hadjiagapiou, Christos; Borthakur, Alip; Dahdal, Refka Y et al. (2005) Role of USF1 and USF2 as potential repressor proteins for human intestinal monocarboxylate transporter 1 promoter. Am J Physiol Gastrointest Liver Physiol 288:G1118-26

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