Gene Expression in Inflammatory Bowel Disease

The idiopathic inflammatory bowel diseases (IBD)—Crohn's disease (CD) and ulcerative colitis (UC)—are chronic, frequently disabling diseases of the gastrointestinal tract. CD and UC have an estimated combined prevalence of 0.2% to 0.3% in the United States, and are two to eight times more prevalent in Jewish Americans of Central or Eastern European descent (Ashkenazi Jews) as compared to non-Jewish Americans. CD may involve any part of the gastrointestinal tract, but most often the colon and terminal ileum. Bowel inflammation is discontinuous, transmural, and may contain granulomas. CD is also associated with bowel stenoses and intestinal and perianal fistulas. UC, by contrast, involves continuous, non-granulomatous inflammation of the colon and rectum, limited to the mucosal layers. Fistulas are not observed. Colonic disease that cannot be clearly identified as CD or UC is designated "indeterminate colitis".

IBD has a prevalence of ~ 0.2% of the Western population. In the United States alone, there are more than a million diagnosed IBD patients. IBD results in enormous suffering and health-care costs. In addition, repeated GI inflammation puts individuals at an increased risk of developing colon cancer. Although there are currently a variety of clinical treatments to alleviate the symptoms of IBD patients, and increasing therapeutic options are being developed as a result of ongoing research, there is no cure for the diseases. In addition, long-term usage of some of the current therapies, such as glucocorticoids and immunosuppressive agents, can be associated with serious side-effects. Progress has been made in recent years in understanding the pathological mechanisms of IBD, particularly in the search of IBD susceptibility genes. However, due to the extreme complexity of the diseases, there is still a long way ahead in elucidating detailed molecular mechanisms of IBD pathogenesis and identifying more effective therapeutic targets. Therefore, it is the goal of this research study to discover biomarkers and protein factors involved in the pathogenesis of IBD which may pave the way for the identification of more effective therapeutic targets.

Hypothesis: In addition to exploration of differential gene expression using microarray technology, the investigators wish to identify protein factors that are involved in the pathogenesis of IBD. Initial proteomic studies of IBD patients with ulcerative colitis (UC) and Crohn's disease (CD) are proposed. The hypothesis to be tested is that, by identifying proteins that are differentially expressed or modified in IBD vs normal intestine, the investigators will increase the current understanding of the pathogenesis of IBD and of the associated diarrhea. Using 1-D and 2-D gel electrophoresis, mass spectrometry, and Western blot screening, the preliminary studies have demonstrated that 1) a large number of proteins are differentially expressed in IBD intestinal mucosa vs normal mucosa. Some of these protein have been identified, and 2) three membrane transport proteins, Na+/H+ exchanger 3 (NHE3), ClC Cl- channel 5 (ClC5) and Na+/K+-ATPase, are significantly down-regulated in IBD mucosa, suggesting a potential explanation of IBD-associated diarrhea. The investigators propose to extend the preliminary studies with two specific aims. AIM 1. Identify proteins that are changed in expression and post-translational modification in the intestinal mucosa of patients with active UC or CD compared to i) uninvolved intestinal mucosa from the same patients, ii) normal intestinal mucosa in control subjects, and iii) infectious/Inflammatory colitis (C. difficile colitis). To increase the chances of identifying changes in relatively low abundant proteins, the investigators will first separate total cellular proteins into several subfractions according to their detergent-solubility and the sizes of protein complexes. Protein expression and modification will then be analyzed by either 1-D/2-D gel/DIGE/MS technology (DIGE, Differential Image Gel Electrophoresis; MS, Mass Spectrometry), or electrospray LC-MS/MS (liquid chromatography MS/MS), from either total mucosa, or villus and crypt cells or lamina proprial cells isolated by laser capture microdissection (LCM). AIM 2. Identify changes in the expression of intestinal membrane transporters for Na absorption and Cl secretion, including NHE3, in the intestinal mucosa of patients with active UC or CD compared to i) uninvolved intestinal mucosa from the same patients, ii) normal intestinal mucosa in control subjects, and and iii) infectious/Inflammatory colitis (C. difficile colitis). Transporters of interest will be identified using large-scale targeted screening by Western blot analysis. The targeted screening will also include several intestinal epithelial brush border-associated PDZ-containing proteins that have been recently shown to regulate trafficking and activity of membrane transporters. The ultimate goal is to identify therapeutic targets to cure IBD or alleviate the symptoms of IBD.