Our veteran patient population is very vulnerable to diarrheal illnesses such as associated with inflammatory bowel diseases (IBD) due to their age and numerous co-morbidities. In spite of significant medical advances, the treatment of IBD-associated diarrhea still remains challenging. Therefore, it is important to understand the mechanisms involved in the pathophysiology of diarrhea, the most debilitating symptom associated with IBD. Diarrhea results from increased intestinal secretion and/or decreased absorption of water and electrolytes. A major route of electrolyte absorption in the human intestine involves coupled operation of Na+/H+ (NHE) and Cl- /HCO3- exchangers. Studies have shown NHE3 to play a critical role in mediating intestinal sodium absorption as NHE3 knockout mice exhibit diarrheal phenotype. Also, it has been shown that NHE3 KO mice are more susceptible to colitis. To date, however, very little is known about the molecular mechanisms involved in the downregulation of NHE3 in diarrhea-associated with inflammation. In order to elucidate the mechanisms underlying the down-regulation of NHE3 expression in IBD-associated diarrhea, our current studies focused on the role of transcription factor, HNF4? (hepatic nuclear factor) and microRNAs (miRNAs) known to play a crucial role in the pathogenesis of IBD. Our extensive preliminary data provide strong evidence for the regulation of HNF4?-mediated NHE3 expression by HNF4? co-activator, p300 [possess intrinsic histone acetylase (HAT) activity] and inhibition of histone deacetylases (HDACs). Our preliminary studies also showed the modulation of intestinal NHE3 by miRNAs. Based on these data, we hypothesize that HNF4?, its associated co-regulators and miRNAs via both transcriptional and post-transcriptional mechanisms play important roles in modulating NHE3 gene expression that underlie the pathophysiology of diarrhea. The current application is, therefore, designed to investigate the regulation of NHE3 gene expression by HNF4?, its associated co-regulators and miRNAs utilizing both in vitro cell culture, ex-vivo human/mouse enteroid-derived monolayers (Aims 1 & 2) and in vivo mouse models (Aim 3).
The Specific Aims are: 1. Elucidate the role of co-activator (s), co-repressor (s) and HNF4? acetylation in the modulation of HNF4?- mediated NHE3 gene expression and determine the mechanisms by which HNF4? attenuates IFN?/TNF?- induced repression of NHE3; 2: Investigate the role of NHE3 3?UTR (3? untranslated region) in response to miRNA mimics on NHE3 and establish the role of miR binding to 3?UTR of NHE3 in regulating NHE3 expression in response to mutations in the miR-binding sites or miR silencing by respective antigomirs and 3: Examine the therapeutic role of increased HNF4? levels (by feeding conjugated linoleic acid rich diet) and specific miRNA mimics or antigomirs on NHE3 expression and function under normal and inflammatory conditions utilizing TNBS-induced colitis mouse model. The outcome of these studies will not only enhance our understanding of the importance of NHE3 as a therapeutic target for IBD-associated diarrhea, but will also establish for the first time, the role of HNF4?, its associated co-regulators and miRNAs in the modulation of intestinal NHE3 under normal and inflammatory conditions.
Inflammatory bowel diseases (IBD)-associated diarrhea remains a major health challenge to our veteran patient population. Diarrhea occurs due to increased intestinal secretion and/or decreased absorption of water and electrolytes. Dysregulation of NHE3, the predominant protein involved in Na+ absorption has been implicated in the pathophysiology of inflammatory bowel diseases (IBD)-associated diarrhea. Therefore, a detailed understanding of the mechanisms of regulation of NHE3 is necessary to ascertain the role of this important transporter in health and disease. The studies are proposed to provide crucial insights into the mechanisms of intestinal NHE3 regulation by the transcription factor, HNF4? and microRNAs under normal and inflammatory states. The findings from these studies could be exploited for developing novel and efficient therapeutics to attenuate the decrease in NHE3 expression and treat diarrhea associated with inflammation.
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