Carbonylation and disassembly of the F-actin cytoskeleton in oxidant induced barrier dysfunction and its prevention by epidermal growth factor and transforming growth factor alpha in a human colonic cell line

Background-Intestinal barrier dysfunction concomitant with high levels of r eactive oxygen metabolites (ROM) in the inflamed mucosa have been observed in inflammatory bowel disease (IBD). The cytoskeletal network has been sugg ested to be involved in the regulation of barrier function. Growth factors (epidermal growth factor (EGF) and transforming growth factor a (TGF-alpha) ) protect gastrointestinal barrier integrity against a variety of noxious a gents. However, the underlying mechanisms of oxidant induced disruption and growth factor mediated protection remain elusive. Aims-To determine: (1) if oxidation and disassembly of actin (a key cytoske letal component) plays a major role in ROM induced epithelial monolayer bar rier dysfunction; and (2) if growth factor mediated protection involves pre vention of theses alterations. Methods-Caco-2 monolayers were preincubated with EGF, TGF-alpha, or vehicle before incubation with (H2O2 or HOCl). Effects on cell integrity, barrier function, and G- and F-actin (oxidation, disassembly, and assembly) were de termined. Results-ROM dose dependently and significantly increased F- and G-actin oxi dation (carbonylation), decreased the stable F-actin fraction (index of sta bility), and increased the monomeric G-actin fraction tinder of disassembly ). Concomitant with these changes were disruption of the actin cytoskeleton and loss of the monolayer barrier function. In contrast, growth factor pre treatment decreased actin oxidation and enhanced the stable F-actin, while in concert prevented actin disruption and restored normal barrier function of monolayers exposed to ROM I. Cytochalasin-D, an inhibitor of actin assem bly, not only caused actin disassembly and barrier dysfunction but also abo lished the protective action of growth factors. Moreover, an actin stabilis ing agent, phalloidin, mimicked the protective actions of the growth factor s. Conclusions-Oxidation, disassembly, and instability of the actin cytoskelet on appears to play a key role in the mechanism of oxidant induced loss of i ntestinal barrier integrity. In contrast, organisation and stabilisation of actin through promotion of its assembly plays a critical role in the mecha nism of growth factor mediated protection.