Oxidant-induced intestinal barrier disruption and its prevention by growthfactors in a human colonic cell line: Role of the microtubule cytoskeleton

Reactive oxygen metabolites (ROM) are increased in the inflamed mucosa of i nflammatory bowel disease (IBD) and may contribute to loss of intestinal ba rrier function iir this disorder. Growth factors (GF) are protective. But t he mechanisms of disruption and protection remain elusive. In the present i nvestigation, we hypothesized that the microtubules (a critical cytoskeleta l element) play a key role in the molecular mechanism of intestinal barrier dysfunction induced by ROM and in GF-mediated protection. Utilizing monola yers of a human colonic cell line (Caco-2), we evaluated the effects of ROM (H2O2 or HOCl), in the presence or absence of GF (epidermal growth factor [EGF]; transforming growth factor-alpha [TGF-alpha]), on intestinal barrier function, tubulin (microtubule structural protein), and microtubule stabil ity. Monolayers were also processed for two highly sensitive western immuno blots: fractionated polymerized tubulin (S2; an index of stability); monome ric tubulin (SI; an index of disruption) to detect the oxidation and disass embly/assembly of tubulin. ROM exposure led to a significant increase in th e oxidation of tubulin, decrease in the stable S2 polymerized tubulin, and increase in the unstable S1 monomeric tubulin. In concert, each ROM in a do se dependent manner damaged the microtubule cytoskeleton and disrupted barr ier function. GF pretreatment not only increased the S2 stable tubulin and decreased tubulin oxidation but also, concomitantly, prevented the disrupti on of microtubules and loss of barrier function in monolayers exposed to RO M. Antibody against the GF-receptor and inhibitors of GF-receptor tyrosine kinase abolished GF protection, indicating the involvement of epidermal gro wth factor receptor (EGFR) signaling pathway. As predicted, colchicine, an inhibitor of microtubule assembly, caused barrier dysfunction and prevented GF protection whereas taxol, a microtubule-stabilizing agent, mimicked the protective effects of GF. Thus, organization and stability of the microtub ule cytoskeleton appears to be critical to both oxidant-induced mucosal bar rier dysfunction and protection of intestinal barrier mediated by GF. There fore, microtubules may be useful targets for development of drugs for the t reatment of IBD. (C) 2000 Elsevier Science Inc.