Evidence That Nuclear Factor- B Activation Is Critical in Oxidant-Induced Disruption of the Microtubule Cytoskeleton and Barrier Integrity and That Its Inactivation Is Essential in Epidermal Growth Factor-Mediated Protection of the Monolayers of Intestinal Epithelia

Authors:
  • AFA. Farhadi
    A. Farhadi
  • JFJ. Z. Fields
    J. Z. Fields
  • EME. Mutlu
    E. Mutlu
  • LZL. Zhang
    L. Zhang
  • AKA. Keshavarzian
    A. Keshavarzian
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Abstract
Using monolayers of intestinal (Caco-2) cells, we showed that oxidants disrupt the microtubule cytoskeleton and barrier integrity; epidermal growth factor (EGF) was protective via stabilization of the microtubules. Because proinflammatory conditions activate nuclear factor-κB (NF-κB), we hypothesized that oxidants disrupt barrier integrity through activation of NF-κB and that EGF protects by suppressing NF-κB. Parental cells were pretreated with EGF or NF-κB or inhibitory κBα (I-κBα) modulators. Other cells were stably transfected with varying levels of a dominant negative mutant for the NF-κB inhibitor I-κBα. Both types of cells were grown as monolayers and then exposed to oxidant (H2O2). We then monitored monolayer barrier integrity (permeability), stability of the microtubule cytoskeleton (confocal microscopy, immunoblotting), intracellular levels of the I-κBα (immunoblotting), translocation, and activity of NF-κB (immunoblotting, sensitive enzyme-linked immunosorbent assay). Monolayers were also fractionated and processed to assess alterations in 1) polymerized tubulin (S2; an index of cytoskeletal integrity) and 2) monomeric tubulin (S1; an index of disassembly) (polyacrylamide gel electrophoresis fractionation and immunoblotting). We found the following: 1) Oxidants caused I-κBα degradation, NF-κB translocation, NF-κB (p50 and p65 subunits) activation, tubulin disassembly (↑ S1, ↓S2), microtubule architectural instability, and barrier disruption. I-κBα stabilizers and NF-κB inhibitors [e.g., carbobenzyloxy-leuleu-leucinol (MG-132), lactacystin] suppressed oxidants injurious effects. 2) EGF (10 ng/ml) stabilized I-κBα and prevented both NF-κB translocation and activation while protecting monolayers against oxidants. 3) In stably transfected cells, transfection-induced stabilization of I-κBα by itself led to EGF-like protective effects. In these mutant cells, protection was not potentiated by EGF (10 ng/ml). Conclusions are 1) oxidants induce disruption of the cytoskeleton and intestinal barrier integrity, in part, through I-κBα degradation and subsequent NF-κB activation, 2) I-κBα stabilization is by itself protective, mimicking EGF, and 3) EGF protects cell monolayers through I-κBα stabilization and NF-κB inactivation. To our knowledge, this is the first report that NF-κB can affect the dynamics of cytoskeletal assembly and intestinal barrier integrity.

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