title: Critical role of the atypical {lambda} isoform of protein kinase C (PKC-{lambda}) in oxidant-induced disruption of the microtubule cytoskeleton and barrier function of intestinal epithelium.
abstract: Oxidant injury to epithelial cells and gut barrier disruption are key factors in the pathogenesis of inflammatory bowel disease. Studying monolayers of intestinal (Caco-2) cells, we reported that oxidants disrupt the cytoskeleton and cause barrier dysfunction (hyperpermeability). Because the lambda isoform of protein kinase C (PKC-lambda), an atypical diacylglycerol-independent isozyme, is abundant in parental (wild type) Caco-2 cells and is translocated to the particulate fractions upon oxidant exposure, we hypothesized that PKC-lambda is critical to oxidative injury to the assembly and architecture of cytoskeleton and the intestinal barrier function. To this end, Caco-2 cells were transfected with an inducible plasmid, a tetracycline-responsive system, to create novel clones stably overexpressing native PKC-lambda. Other cells were transfected with a dominant-negative plasmid to stably inhibit the activity of native PKC-lambda. Cells were exposed to oxidant (H(2)O(2)) +/- modulators. Parental Caco-2 cells were treated similarly. We then monitored barrier function (fluorescein sulfonic acid clearance), microtubule cytoskeletal stability (confocal microscopy, immunoblotting), subcellular distribution of PKC-lambda (immunofluorescence, immunoblotting, immunoprecipitation), and PKC-lambda isoform activity (in vitro kinase assay). Monolayers were also processed to assess alterations in tubulin assembly, polymerized tubulin (S2, an index of cytoskeletal integrity), and monomeric tubulin (S1, an index of cytoskeletal disassembly) (polyacrylamide gel electrophoresis fractionation and immunoblotting. In parental cells, oxidant caused: 1) translocation of PKC-lambda from the cytosol to the particulate (membrane + cytoskeletal) fractions, 2) activation of native PKC-lambda, 3) tubulin pool instability (increased monomeric S1 and decreased polymerized S2), 4) disruption of cytoskeletal architecture, and 5) barrier dysfunction (hyperpermeability). In transfected clones, overexpression of the atypical (74 kDa) PKC-lambda isoform by itself ( approximately 3.2-fold increase) led to oxidant-like disruptive effects, including cytoskeletal and barrier hyperpermeability. Overexpressed PKC-lambda was mostly found in particulate cell fractions (with a smaller cytosolic distribution) indicating its activation. Disruption by PKC-lambda overexpression was also potentiated by oxidant challenge. Stable inactivation of endogenous PKC-lambda ( approximately 99.6%) by a dominant-negative protected against all measures of oxidant-induced disruption. We conclude that: 1) oxidant induces disruption of epithelial barrier integrity by disassembling the cytoskeleton, in large part, through the activation of PKC-lambda isoform; and 2) activation of PKC-lambda by itself appears to be sufficient for disruption of cellular cytoskeleton and monolayer barrier permeability. The unique ability to mediate an oxidant-like injury and cytoskeletal depolymerization and instability is a novel mechanism not previously attributed to the atypical subfamily of PKC isoforms.
authors.affiliation: Section of Gastroenterology and Nutrition, Rush University of Chicago, College of Medicine, Division of Digestive Diseases, 1725 W. Harrison, Suite 206, Chicago, IL 60612, USA. ali_banan@rush.edu
authors.forename: A
authors.initials: A
authors.lastname: Banan
authors.forename: L J
authors.initials: LJ
authors.lastname: Zhang
authors.forename: A
authors.initials: A
authors.lastname: Farhadi
authors.forename: J Z
authors.initials: JZ
authors.lastname: Fields
authors.forename: M
authors.initials: M
authors.lastname: Shaikh
authors.forename: C B
authors.initials: CB
authors.lastname: Forsyth
authors.forename: S
authors.initials: S
authors.lastname: Choudhary
authors.forename: A
authors.initials: A
authors.lastname: Keshavarzian
mesh_terms: D015153:Blotting, Western; D018938:Caco-2 Cells; D003599:Cytoskeleton / Q000187:drug effects* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047468:Immunoprecipitation; D007408:Intestinal Absorption / Q000187:drug effects; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000201:enzymology; D007527:Isoenzymes; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D016877:Oxidants / Q000633:toxicity*; D010957:Plasmids / Q000235:genetics; D011493:Protein Kinase C / Q000502:physiology*; D013347:Subcellular Fractions / Q000187:drug effects / Q000648:ultrastructure; D014162:Transfection; D014404:Tubulin / Q000096:biosynthesis / Q000378:metabolism; D000099293:Protein Kinase C-lambda
chemical_list: D006861:Hydrogen Peroxide; D007527:Isoenzymes; D016877:Oxidants; D011493:Protein Kinase C; D014404:Tubulin; D000099293:Protein Kinase C-lambda