The long-term goal of this project is to determine the mechanisms whereby Epithelial Neutrophil-Activating peptide-78 (ENA-78), produced by activated enterocytes, regulates neutrophil recruitment in inflammatory bowel disease. The experimental goals for this proposal are to define the molecular mechanisms that regulate ENA-78 gene expression in Caco-2 intestinal epithelial cells. ENA-78 is a C-X-C chemokine that binds to the CXCR2 receptor and stimulates neutrophil chemotaxis. ENA-78 also facilitates cellular regeneration and is a potent angiogenic factor. The investigator has shown previously that activation of intestinal epithelial cells by IL-1beta or TNFalpha induces prolonged ENA-78 production. The investigator has also shown that enterocytes are the main site of ENA-78 production in normal human colon and that colonic mucosal ENA-78 production is substantially increased in ulcerative colitis. Based on these findings, the investigator hypothesizes that the ENA-78 gene is specifically adapted for the prolonged production of ENA-78 protein by activated enterocytes. The sustained production of ENA-78 by inflamed intestinal epithelial cells is likely to regulate neutrophil recruitment into the colonic epithelial layer in IBD. The preliminary studies demonstrate that an NF-kappaB binding site in the ENA-78 5' promoter region plays a major role in regulating IL-lbeta-induced gene transcription in Caco-2 cells. The investigator has also identified a second 5 regulatory element (-118 to -146 bp) designated """"""""Site A."""""""" Site A regulates basal ENA-78 gene transcription in Caco-2 cells and binds the zinc finger transcription factor Sp-l in addition to another, as yet unidentified, nuclear factor(s). The first specific aim is to define the functional Sp-l-binding element in the ENA-78 promoter by scanning and site-directed mutagenesis of Site A using EMSA and luciferase reporter gene assays. Our second specific aim is to characterize the other transactivator(s) that bind to Site A.
This aim will also examine our hypothesis that nuclear factor binding to Site A can regulate cell-type-specific (enterocyte) ENA-70 gene expression. The preliminary data indicate that a post-transcriptional mechanism, ENA-78 mRNA stability, accounts for the prolonged kinetics of ENA-78 protein production. The third specific aim will test the hypothesis that the organization of AU-rich binding elements within the 3' untranslated region of ENA-78 mRNA confers message stability and determines the sustained production of ENA-78 by activated enterocytes. An in-depth, mechanistic understanding of the regulation of ENA-78 gene expression in human enterocytes may lead to novel therapeutic approaches to IBD.
