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-κB), we hypothesized that EtOH induces disruption of cytoskeletal assembly and barrier integrity by activating NF-κB. Parental cells were pretreated with pharmacological modulators of NF-κB. Other cells were stably transfected with a dominant negative mutant for the NF-κB inhibitor, I-κBα. 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-κBα (immunoblotting); subcellular distribution and activity of NF-κB (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-κBα degradation, NF-κB nuclear translocation, NF-κB subunit (p50 and p65) activation, actin disassembly (↑G-, ↓F-), actin cytoskeleton instability, and barrier disruption. Inhibitors of NF-κB and stabilizers of I-κBα (e.g., MG-132, lactacystin, etc) prevented NF-κB activation while protecting against EtOH-induced injury. In transfected I-κBα mutant clones, stabilization of I-κBα to inactivate NF-κB protected against all measures of EtOH-induced injury. Our data support several novel mechanisms where NF-κB 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-κBα degradation and NF-κB activation; (2) The mechanism underlying this pathophysiological effect of the NF-κB appears to involve instability of the assembly of the subunit components of actin network.