Diarrhea is a predominant symptom of inflammatory bowel diseases (IBD) or intestinal infections caused by food-borne pathogens. Our veteran patient population is particularly more vulnerable to diarrheal illnesses due to their age and numerous co-morbidities. In spite of significant medical advances, the treatment of diarrheal disorders still remains challenging. Therefore, it is important to understand the mechanisms involved in the pathophysiology of diarrhea associated with these diseases. 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 inflammation as NHE3 deficiency in these mice resulted in diarrhea associated with colitis. To date, however, very little is known about the molecular mechanisms involved in decreasing NHE3 expression in diarrheal disorders. In order to elucidate the mechanisms underlying the down-regulation of NHE3 expression in IBD-related diarrhea, our current studies are focused on the role of epigenetic mechanisms such as DNA methylation and histone modifications shown to be implicated in the pathogenesis of IBD. Our extensive preliminary data provides strong evidence for the epigenetic regulation of NHE3 expression by changes in DNA methylation and histone modifications. Based on these data, we hypothesize that changes in DNA methylation, histone modifications and chromatin remodeling 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 DNA methylation and histone modifications utilizing both in vitro (Aims 1 & 2) and in vivo models (Aim 3) as follows:
Aim 1. Elucidate in detail the effect of DNA methylation on NHE3 promoter activity, identify the specific CpG dinucleotide involved in the modulation of NHE3 promoter and determine the roles of DNA methyltransferase (DNMT) isoforms;
Aim 2 : Investigate the role of histone acetyl transferase, p300 & histone deacetylase (HDAC) isoforms in the modulation of NHE3 expression, changes in histone acetylation marks on NHE3 gene and chromatin remodeling in the stimulation of NHE3 expression;
and Aim 3 : Examine the regulation of NHE3 expression by epigenetic mechanisms under normal and inflammatory conditions utilizing wild type, DSS-induced colitis and NHE3 knockout mice (diarrheal phenotype). The outcome of these studies should provide novel insights into the epigenetic regulation of NHE3 and define important mechanistic link between alterations in DNA methylation/histone modifications of NHE3 gene and diarrhea associated with IBD. Also, these studies will establish for the first time the identity of a particular DNMT or HDAC isoforms involved in the dysregulation of NHE3 in diarrheal disorders.
Diarrhea associated with inflammatory bowel diseases (IBD) remains a leading cause of morbidity and mortality in our veteran patient population. Diarrhea occurs due to either increased secretion or decreased absorption of water and electrolytes or both. Dysregulation of NHE3, the predominant isoform involved in Na+ absorption has been implicated in the pathophysiology of diarrhea associated with inflammation. The proposed studies are focused at understanding the mechanisms involved in the epigenetic regulation of NHE3 gene expression in the intestine in models of intestinal inflammation & diarrhea. The findings from these studies could be exploited for developing potential targets to block the decrease in NHE3 expression and treat diarrhea associated with inflammation.