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 reactive oxygen metabolites (ROM) in the inflamed mucosa have been observed in inflammatory bowel disease (IBD). The cytoskeletal network has been suggested to be involved in the regulation of barrier function. Growth factors (epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha)) protect gastrointestinal barrier integrity against a variety of noxious agents. However, the underlying mechanisms of oxidant induced disruption and growth factor mediated protection remain elusive.


OBJECTIVE
To determine: (1) if oxidation and disassembly of actin (a key cytoskeletal component) plays a major role in ROM induced epithelial monolayer barrier dysfunction; and (2) if growth factor mediated protection involves prevention of theses alterations.


METHODS
Caco-2 monolayers were preincubated with EGF, TGF-alpha, or vehicle before incubation with ROM (H(2)O(2) or HOCl). Effects on cell integrity, barrier function, and G- and F-actin (oxidation, disassembly, and assembly) were determined.


RESULTS
ROM dose dependently and significantly increased F- and G-actin oxidation (carbonylation), decreased the stable F-actin fraction (index of stability), and increased the monomeric G-actin fraction (index of disassembly). Concomitant with these changes were disruption of the actin cytoskeleton and loss of the monolayer barrier function. In contrast, growth factor pretreatment 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. Cytochalasin-D, an inhibitor of actin assembly, not only caused actin disassembly and barrier dysfunction but also abolished the protective action of growth factors. Moreover, an actin stabilising agent, phalloidin, mimicked the protective actions of the growth factors.


CONCLUSIONS
Oxidation, disassembly, and instability of the actin cytoskeleton appears to play a key role in the mechanism of oxidant induced loss of intestinal barrier integrity. In contrast, organisation and stabilisation of actin through promotion of its assembly plays a critical role in the mechanism of growth factor mediated protection.
Banan
A
A
Division of Digestive Disease, Rush University Medical Center, Department of Internal Medicine, Chicago, Illinois, USA. ali_banan@rush.edu
Zhang
Y
Y
Losurdo
J
J
Keshavarzian
A
A
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D016923:Cell Death / Q000502:physiology; D002460:Cell Line; D003108:Colonic Diseases / Q000378:metabolism*; D003599:Cytoskeleton; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005453:Fluorescence; D006801:Humans; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000199:Actins; D014409:Tumor Necrosis Factor-alpha; D004815:Epidermal Growth Factor