title: NF-kappaB activation as a key mechanism in ethanol-induced disruption of the F-actin cytoskeleton and monolayer barrier integrity in intestinal epithelium.
abstract: Intestinal barrier disruption has been implicated in several intestinal and systemic disorders including alcoholic liver disease (ALD). Using monolayers of intestinal (Caco-2) cells, we showed that ethanol (EtOH) disrupts the barrier integrity via destabilization of the cytoskeleton. Because proinflammatory conditions are associated with activation of NF-kappa B (NF-kappaB), we hypothesized that EtOH induces disruption of cytoskeletal assembly and barrier integrity by activating NF-kappaB. Parental cells were pretreated with pharmacological modulators of NF-kappaB. Other cells were stably transfected with a dominant negative mutant for the NF-kappaB inhibitor, I-kappaBalpha. Monolayers of each cell type were exposed to EtOH and we then monitored monolayer barrier integrity (permeability); cytoskeletal stability and molecular dynamics (confocal microscopy and immunoblotting); intracellular levels of the I-kappaBalpha (immunoblotting); subcellular distribution and activity of NF-kappaB (immunoblotting and sensitive ELISA); and intracellular alterations in the 43kDa protein of the actin cytoskeleton, polymerized F-actin, and monomeric G-actin (SDS-PAGE fractionation). EtOH caused destabilizing alterations, including I-kappaBalpha degradation, NF-kappaB nuclear translocation, NF-kappaB subunit (p50 and p65) activation, actin disassembly (upward arrow G-, downward arrow F-), actin cytoskeleton instability, and barrier disruption. Inhibitors of NF-kappaB and stabilizers of I-kappaBalpha (e.g., MG-132, lactacystin, etc) prevented NF-kappaB activation while protecting against EtOH-induced injury. In transfected I-kappaBalpha mutant clones, stabilization of I-kappaBalpha to inactivate NF-kappaB protected against all measures of EtOH-induced injury. Our data support several novel mechanisms where NF-kappaB can affect the molecular dynamics of the F-actin cytoskeleton and intestinal barrier integrity under conditions of EtOH injury. (1) EtOH induces disruption of the F-actin cytoskeleton and of intestinal barrier integrity, in part, through I-kappaBalpha degradation and NF-kappaB activation; (2) The mechanism underlying this pathophysiological effect of the NF-kappaB appears to involve instability of the assembly of the subunit components of actin network.
authors.affiliation: Department of Internal Medicine, Section of Gastroenterology & Nutrition, Rush University Medical Center, Chicago, IL 60612, USA. ali_banan@rush.edu
authors.forename: A
authors.initials: A
authors.lastname: Banan
authors.forename: A
authors.initials: A
authors.lastname: Keshavarzian
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: Shaikh
authors.forename: C B
authors.initials: CB
authors.lastname: Forsyth
authors.forename: Y
authors.initials: Y
authors.lastname: Tang
authors.forename: J Z
authors.initials: JZ
authors.lastname: Fields
mesh_terms: D000199:Actins / Q000737:chemistry*; D018938:Caco-2 Cells; D003599:Cytoskeleton / Q000187:drug effects*; D000431:Ethanol / Q000633:toxicity*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism; D008108:Liver Diseases, Alcoholic / Q000209:etiology / Q000628:therapy; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000199:Actins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha; D000431:Ethanol