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is required in EGF protection of microtubules and
intestinal barrier integrity against oxidant injury
1 Departments of Internal Medicine (Division of Digestive Diseases), Pharmacology, and Molecular Physiology, Rush University Medical Center, Chicago, Illinois 60612 and 2 Institute of Human Nutrition, Columbia University, New York, New York 10032.
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-
isoform is abundant in wild-type
(WT) Caco-2 cells, we hypothesized that PKC- mediates, at least in
part, EGF protection. Intestinal cells (Caco-2 or HT-29) were
transfected to stably over- or underexpress PKC-. 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-
(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- protection. Most overexpressed PKC- (92%) resided in
membrane and cytoskeletal fractions, indicating constitutive activation of PKC-. Stably inhibiting PKC- 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-
is necessary for EGF-induced protection, 2) PKC- 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.
cytoskeleton; growth factors; epidermal growth factor; Caco-2 cells; gut barrier; protection; transfection; protein kinase C isoforms; inflammatory bowel disease
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