The 1 Isoform of Protein Kinase C Mediates the Protective Effects of Epidermal Growth Factor on the Dynamic Assembly of F-Actin Cytoskeleton and Normalization of Calcium Homeostasis in Human Colonic Cells A. BANAN, J. Z. FIELDS, A. FARHADI, D. A. TALMAGE, L. ZHANG, and A. KESHAVARZIAN Departments of Internal Medicine (Section of Gastroenterology and Nutrition), Pharmacology, and Molecular Physiology, Rush University Medical Center, Chicago, Illinois (A.B., J.Z.F., A.F., L.Z., A.K.); and Institute of Human Nutrition, Columbia University, New York, New York (D.A.T.) Received January 15, 2002; accepted February 19, 2002 This article is available online at http://jpet.aspetjournals.org ABSTRACT Using intestinal monolayers, we showed that F-actin cytoskel- etal stabilization and Ca 2+ normalization contribute to epider- mal growth factor (EGF)-mediated protection against oxidant injury. However, the intracellular mediator responsible for these protective effects remains unknown. Since the protein kinase C-1 (PKC-1) isoform is abundant in our naive (N) cells, we hypothesized that PKC-1 is essential to EGF protection. Monolayers of N Caco-2 cells were exposed to H 2 O 2  EGF, PKC, or Ca 2+ modulators. Other cells were transfected to over-express PKC-1 or to inhibit its expression and then pre- treated with low or high doses of EGF or a PKC activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), before H 2 O 2 . In N monolayers exposed to oxidant, pretreatment with EGF or PKC activators activated PKC-1, enhanced 45 Ca 2+ efflux, normalized Ca 2+ , decreased monomeric G-actin, increased stable F-actin, and protected the cytoarchitecture of the actin. PKC inhibitors pre- vented these protective effects. Transfected cells stably over- expressing PKC-1(+3.1-fold) but not N cell monolayers were protected from injury by even lower doses of EGF or OAG. EGF or OAG rapidly activated the over-expressed PKC-1. Anti- sense inhibition of PKC-1 expression (-90%) prevented all measures of EGF protection. Inhibitors of Ca 2+ -ATPase pre- vented EGF protection in N cells as well as protective syner- gism in transfected cells. EGF protects the assembly of the F-actin cytoskeleton in intestinal monolayers against oxidants in large part through the activation of PKC-1. EGF normalizes Ca 2+ by enhancing Ca 2+ efflux through PKC-1. We have identified novel biologic functions, protection of actin and Ca 2+ homeostasis, among the classical isoforms of PKC. A fundamental property of the epithelium of the gastroin- testinal (GI) mucosa is the ability to maintain a highly selec- tive permeability barrier (Unno et al., 1996; Menconi et al., 1997; Hollander, 1998; Banan et al., 1999; Keshavarzian et al., 1999). We (Banan et al., 1996, 1998a,b, 1999, 2000a,b,c, 2001a,b,c,d,e, 2002) and others (Unno et al., 1996; Menconi et al., 1997) have shown that the integrity of the intestinal barrier depends on the stability of complex intracellular net- works of the cytoskeletal elements. Among these elements, the actin cytoskeleton, especially the apical ring of actin cortex, plays a key role in the maintenance of an intact GI epithelial barrier (Banan et al., 1996, 2000c, 2001a,e; Unno et al., 1996; Menconi et al., 1997). Actin is the principal protein in the cell cortex localized immediately inside the plasma membrane at areas of cell-to-cell contact and repre- sents a critical structural component. As such it plays a crucial role in maintaining the structure of the cytoplasmic matrix, cell shape, and barrier integrity (permeability). Intestinal barrier integrity is of clinical and biological im- portance because this barrier normally restricts the passage of harmful pro-inflammatory and toxic molecules (e.g., bac- terial endotoxin, immunoreactive antigens) into the mucosa and systemic circulation (Hollander, 1998; Keshavarzian et This work was supported in part by a grant from Rush University Medical Center, Department of Internal Medicine, and by a grant from the American College of Gastroenterology. Portions of this work will be presented at the annual meeting of the American Gastroenterological Association, May 19 –25, 2002. ABBREVIATIONS:GI, gastrointestinal; EGF, epidermal growth factor; [Ca +2 ] i , intracellular Ca 2+ ; PKC, protein kinase C; OAG, 1-oleoyl-2-acetyl- sn-glycerol; TPA, 12-O-tetradecanoylphorbol-13-acetate; LSCM, laser scanning confocal microscopy (microscope); PIPES, 1,4-piperazinedi- ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; Fluo-3, 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)phenoxy]-2-[2- amino-5-methylphenoxy]ethane-N,N,N',N'-tetraacetic acid; Fluo-3-AM, 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)phenoxy]-2-[2- amino-5-methylphenoxy]ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester; 4-PDD, 4-phorbol-12,13-didecanoate; FSA, fluorescein sulfonic acid; GF 109203 X, bisindolylmaleimide V; iGF 109203 X, inactive GF 109203 X; MARCKS, myristoylated, alanine-rich PKC substrate. 0022-3565/02/3013-852–866$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 301, No. 3 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 0/986120 JPET 301:852–866, 2002 Printed in U.S.A. 852