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Am J Physiol Gastrointest Liver Physiol (January 9, 2002). doi:10.1152/ajpgi.00284.2001
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Articles in PresS, published online ahead of print January 9, 2002
Am J Physiol Gastrointest Liver Physiol, 10.1152/ajpgi.00284.2001
Submitted on June 28, 2001
Accepted on December 19, 2001

Requirement for PKC-{zeta} in EGF's Protection of The Microtubule Cytoskeleton and Intestinal Barrier Integrity

Ali Banan1*, Jeremy Z Fields1, David A Talmage2, Lei Zhang1, and Ali Keshavarzian1

1 Medicine (Digestive Diseases)/Pharmacology/Molecular Physiology, Rush University Medical Center, Chicago, IL, USA
2 Institute of Human Nutrition, Columbia University, New York, NY, USA

* To whom correspondence should be addressed. E-mail: ali_banan{at}rush.edu.

Using monolayers of human intestinal (Caco-2) cells, we previously showed that EGF protects intestinal barrier integrity against oxidant injury by protecting the microtubule cytoskeleton and that activation of PKC is required. Because PKC activity has been associated with models of cell injury, we surmised that specific isoforms of PKC might mediate the protective actions of PKC. Since isoform PKC-zeta (PKC-{zeta}), an atypical diacylglycerol (DAG) independent isoform, is abundant in wild type (WT) Caco-2 cells, we hypothesized that PKC-{zeta} mediates, at least in part, EGF's protective effects. Cells were transfected to either stably over-express the PKC-{zeta} isoform or to inhibit its expression. These cells were then preincubated with EGF (1 or 10 ng/ml) or a PKC activator/DAG analog (OAG), or vehicle, prior to exposure to oxidant (H2O2, 0.5 mM). We monitored monolayer barrier function (FSA clearance), stability of the microtubule cytoskeleton (laser confocal microscopy; immunoblotting), and subcellular distribution of the PKC-{zeta} isoform (immunoblotting). Monolayers were also fractionated and processed for quantitative western immunoblotting to assess dynamic alterations in the structural protein of microtubules, polymerized tubulin (S2; an index of stability) and monomeric tubulin (S1; an index of disruption). Relative to WT cells exposed to oxidant, monolayers of transfected, PKC-{zeta} over-expressing cells (2.9 fold increase) were protected as indicated by increases in barrier integrity (62% less leakiness), enhancement of polymerized tubulin / decreases in monomeric tubulin, and microtubule cytoskeletal stability. Over-expression-induced protection was OAG-independent and even EGF-independent, but EGF potentiated PKC-{zeta} protection. Most of the over-expressed PKC-{zeta} resided in membrane and cytoskeletal fractions, indicating constitutive PKC-{zeta} activation. Inhibiting PKC-{zeta} expression (-95%) with anti-sense transfection attenuated EGF protection as demonstrated by reduced tubulin assembly and increased microtubule disassembly; instability of microtubules; loss of monolayer barrier integrity. Conclusions: 1) activation of PKC-{zeta} is necessary but not sufficient for maximal EGF protection; 2) PKC-{zeta} appears to be an endogenous stabilizer of microtubule and intestinal barrier function; 3) we have identified a novel biologic function - protection - among the PKC family of isoforms; 4) increasing the activity of protective PKC isoforms may be useful in the development of novel therapies for the treatment of a wide variety of oxidant-induced inflammatory disorders of the GI tract such as inflammatory bowel disease.




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