PKC-ζ is required in EGF protection of microtubules and intestinal barrier integrity against oxidant injury

Abstract

Using monolayers of human intestinal (Caco-2) cells, we showed that epidermal growth factor (EGF) protects intestinal barrier integrity against oxidant injury by protecting the microtubules and that protein kinase C (PKC) is required. Because atypical PKC-ζ isoform is abundant in wild-type (WT) Caco-2 cells, we hypothesized that PKC-ζ mediates, at least in part, EGF protection. Intestinal cells (Caco-2 or HT-29) were transfected to stably over- or underexpress PKC-ζ. These clones were preincubated with low or high doses of EGF or a PKC activator [1-oleoyl-2-acetyl- sn-glycerol (OAG)] before oxidant (0.5 mM H2O2). Relative to WT cells exposed to oxidant, only monolayers of transfected cells overexpressing PKC-ζ (2.9-fold) were protected against oxidant injury as indicated by increases in polymerized tubulin and decreases in monomeric tubulin, enhancement of architectural stability of the microtubule cytoskeleton, and increases in monolayer barrier integrity toward control levels (62% less leakiness). Overexpression-induced protection was OAG independent and even EGF independent, but EGF significantly potentiated PKC-ζ protection. Most overexpressed PKC-ζ (92%) resided in membrane and cytoskeletal fractions, indicating constitutive activation of PKC-ζ. Stably inhibiting PKC-ζ expression (95%) with antisense transfection substantially attenuated EGF protection as demonstrated by reduced tubulin assembly and increased microtubule disassembly, disruption of the microtubule cytoskeleton, and loss of monolayer barrier integrity. We conclude that 1) activation of PKC-ζ is necessary for EGF-induced protection, 2) PKC-ζ appears to be an endogenous stabilizer of the microtubule cytoskeleton and of intestinal barrier function against oxidative injury, and 3) we have identified a novel biological function (protection) among the atypical isoforms of PKC.