NF-κB activation as a key mechanism in ethanol-induced disruption of the F-actin cytoskeleton and monolayer barrier integrity in intestinal epithelium

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-kappa B), we hypothesized that EtOH induces disruption of cytoskeletal assembly and barrier integrity by activating NF-kappa B. Parental cells were pretreated with pharmacological modulators of NF-kappa B. Other cells were stably transfected with a dominant negative mutant for the NF-kappa B inhibitor, I-kappa B alpha. 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 irnmunoblotting); intracellular levels of the I-kappa B alpha (immunoblotting); subcellular distribution and activity of NF-kappa B (immunoblotting and sensitive ELISA); and intracellular alterations in the 43 kDa protein of the actin cytoskeleton, polymerized F-actin, and monomeric G-actin (SDS-PAGE fractionation). EtOH caused destabilizing alterations, including I-kappa B alpha. degradation, NF-kappa B nuclear translocation, NF-kappa B subunit (p50 and p65) activation, actin disassembly (up arrow G-, down arrow F-), actin cytoskeleton instability, and barrier disruption. Inhibitors of NF-kappa B and stabilizersof I-kappa B alpha(e.g.,MG-132, lactacystin,etc) prevented NF-kappa B activation while protecting against EtOH-induced injury. In transfected I-kappa B alpha mutant clones, stabilization Of I-kappa B alpha to inactivate NF-kappa B protected against all measures of EtOH-induced injury. Our data support several novel mechanisms where NF-kappa B 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-kappa B alpha degradation and NF-kappa B activation; (2) The mechanism underlying this pathophysiological effect of the NF-kappa B appears to involve instability of the assembly of the subunit components of actin network. (c) 2007 Elsevier Inc. All rights reserved.