Our earlier studies have shown that the electroneutral NaCl absorption in the human ileum and colon involves the operation of dual ion exchanges of Na+/H+ and Cl-/HCO3-. Our studies utilizing purified plasma membrane vesicles isolated from human organ donor small intestinal and colonic mucosa demonstrated that the Na+ and Cl- transport processes in the human intestine exhibit many distinct characteristics compared to those of the rat and rabbit intestine. A total of seven molecular isoforms of Na+/H+ exchangers, have been reported, and detailed studies of the rat and rabbit NHE-1, 2 and 3 isoforms have been carried out. Of the human NHE's, only the NHE-1 has been extensively investigated, while very little information is available for the other human isoforms. Our recent studies have focused on the human specific NHE-2 and NHE-3, to gain an understanding of their function and regulation. Our recent studies emphasize that the NHE-2 isoform might be the predominant colonic sodium-absorbing transporter, supporting our earlier hypothesis that the NHE-2 isoform is involved in sodium absorption in the human colon. Therefore, it is critical to understand the mechanisms underlying its basal transcriptional regulation, factors influencing tissue-specific expression and changes in response to colonocyte differentiation. We have recently cloned the full-length cDNA of the human NHE-2 and its 5' regulatory region. The objective of the current proposal is to analyze the 5'- regulatory region of the human NHE2 gene and to identify cis-elements and the cognate regulatory factors to investigate its transcriptional regulation.
The Specific Aims of this proposal are: 1a. To elucidate the molecular mechanisms involved in the basal transcriptional activity of the NHE2 gene promoter, 1b. To analyze the mechanisms involved in the transcriptional regulation of the NHE2 gene expression in response to PMA and serum, 1c. To identify the transcription factor(s) involved in the functioning of the silencer element of the NHE2 promoter; 2a. To identify the cis-elements and the regulatory factors responsible for tissue and cell-specific expression of the NHE2 gene. 2b. To investigate the mechanisms of transcriptional regulation of the NHE2 gene in response to enterocyte differentiation. Our proposed studies of the regulation of NHE-2 isoform may increase our understanding of the mechanisms of sodium absorption in the human colon and the potential role of this NHE isoform in pathophysiology of diarrheal disorders. ? ?
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