The �1 Isoform of Protein Kinase C Mediates the ProtectiveEffects of Epidermal Growth Factor on the Dynamic Assemblyof F-Actin Cytoskeleton and Normalization of CalciumHomeostasis 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 UniversityMedical 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

ABSTRACTUsing intestinal monolayers, we showed that F-actin cytoskel-etal stabilization and Ca2� normalization contribute to epider-mal growth factor (EGF)-mediated protection against oxidantinjury. However, the intracellular mediator responsible for theseprotective effects remains unknown. Since the protein kinaseC-�1 (PKC-�1) isoform is abundant in our naive (N) cells, wehypothesized that PKC-�1 is essential to EGF protection.Monolayers of N Caco-2 cells were exposed to H2O2 � EGF,PKC, or Ca2� modulators. Other cells were transfected toover-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 H2O2. In N monolayersexposed to oxidant, pretreatment with EGF or PKC activatorsactivated PKC-�1, enhanced 45Ca2� efflux, normalized Ca2�,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 wereprotected from injury by even lower doses of EGF or OAG. EGFor OAG rapidly activated the over-expressed PKC-�1. Anti-sense inhibition of PKC-�1 expression (�90%) prevented allmeasures of EGF protection. Inhibitors of Ca2�-ATPase pre-vented EGF protection in N cells as well as protective syner-gism in transfected cells. EGF protects the assembly of theF-actin cytoskeleton in intestinal monolayers against oxidantsin large part through the activation of PKC-�1. EGF normalizesCa2� by enhancing Ca2� efflux through PKC-�1. We haveidentified novel biologic functions, protection of actin and Ca2�

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 etal., 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 etal., 1997) have shown that the integrity of the intestinalbarrier depends on the stability of complex intracellular net-works of the cytoskeletal elements. Among these elements,

the actin cytoskeleton, especially the apical ring of actincortex, plays a key role in the maintenance of an intact GIepithelial barrier (Banan et al., 1996, 2000c, 2001a,e; Unnoet al., 1996; Menconi et al., 1997). Actin is the principalprotein in the cell cortex localized immediately inside theplasma membrane at areas of cell-to-cell contact and repre-sents a critical structural component. As such it plays acrucial role in maintaining the structure of the cytoplasmicmatrix, cell shape, and barrier integrity (permeability).

Intestinal barrier integrity is of clinical and biological im-portance because this barrier normally restricts the passageof harmful pro-inflammatory and toxic molecules (e.g., bac-terial endotoxin, immunoreactive antigens) into the mucosaand systemic circulation (Hollander, 1998; Keshavarzian et

This work was supported in part by a grant from Rush University MedicalCenter, Department of Internal Medicine, and by a grant from the AmericanCollege of Gastroenterology. Portions of this work will be presented at theannual meeting of the American Gastroenterological Association, May 19–25,2002.

ABBREVIATIONS:GI, gastrointestinal; EGF, epidermal growth factor; [Ca�2]i, intracellular Ca2�; 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, fluoresceinsulfonic 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$7.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 301, No. 3Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 0/986120JPET 301:852–866, 2002 Printed in U.S.A.

852

al., 1999). Loss of mucosal barrier integrity, on the otherhand, is characteristic of multiple organ system dysfunction,inflammatory bowel disease, necrotizing enterocolitis, etha-nol- and nonsteroidal anti-inflammatory drug-induced chem-ical injury, and a variety of other GI disorders as well asseveral systemic disorders (e.g., alcoholic liver disease)(Unno et al., 1996; Menconi et al., 1997; Hollander, 1998;Keshavarzian et al., 1999). Although the pathogenesis ofmucosal barrier disruption in these illnesses remains un-clear, several studies, including our own, have shown thatchronic gut inflammation is associated with high levels ofoxidants (e.g., H2O2) and that oxidative damage is a keycontributor to loss of barrier integrity and injury (Keshavar-zian et al., 1992; McKenizie et al., 1996; Kimura et al., 1998;Banan et al., 2000a,b,c, 2001a). Not surprisingly, damage tothe actin cytoskeleton (especially to the cortical ring of F-actin) can lead to a leaky, hyperpermeable gut, and thisdamage has been proposed as a key underlying mechanismfor the initiation and perpetuation of these oxidant-inducedinflammatory disorders. Thus, characterizing the intracellu-lar signaling mechanisms underlying the protection of F-actin is both clinically and biologically important.

In our concerted efforts to enhance our understanding ofendogenous protective mechanisms for the cytoskeleton andto provide new insights that might lead to developing moreeffective treatment regimens for oxidative and inflammatorydisorders associated with loss of intestinal barrier integrity,we have been investigating the underlying protective mech-anisms used by growth factors to stabilize the cytoskeletalnetwork. Utilizing monolayers of human intestinal (Caco-2)cells exposed to oxidants as a model of cytoskeletal andbarrier disruption, we previously showed that growth factors(e.g., EGF or transforming growth factor-�) protect intestinalbarrier integrity in part by stabilizing the assembly of theapical ring of the F-actin cytoskeleton (Banan et al., 2000c,2001a,e). We have also shown that the stability of actin is keyin mucosal healing under in vivo (Banan et al., 1996) and invitro (Banan et al., 2000c, 2001a,e) conditions. Stability isbased on the ability of monomeric G-actin to polymerize andon the stability of F-actin polymers to resist disassembly.Despite the critical importance of the F- and G- actin cy-toskeleton in the maintenance of intestinal barrier integrity,the intracellular signaling mechanism through which EGFstabilizes the actin remains elusive.

In previous studies using naive Caco-2 cells, we alsoshowed that Ca2� is crucial in the maintenance of normalmucosal barrier integrity (Kokoska et al., 1998; Banan et al.,2001b) and that EGF protects the monolayer barrier vianormalization of intracellular Ca2� ([Ca2�]i) levels throughenhancement of protein kinase C (PKC) in general (Banan etal., 2001b). The specific isoform of PKC responsible for thisprotective effect of EGF on Ca2� homeostasis remains un-known. Utilizing the first developed stably transfected intes-tinal (Caco-2) cell lines over-expressing or under-expressingPKC, we recently demonstrated that EGF maintains mono-layer barrier permeability, in large part, by increasing activ-ity and membrane association of the �1 isoform of PKC(Banan et al., 2001c).

In view of the aforementioned considerations, we hypoth-esized that the PKC-�1 isoform not only is essential to EGF-induced protection of the dynamic assembly of the F- andG-actin cellular pools and the stabilization of the cortical

actin ring, but it is key to the normalization of Ca2� ho-meostasis. To this end, we utilized pharmacological and tar-geted molecular interventions employing several novel andstably transfected intestinal cell lines we have recently de-veloped. In several clones the classical isoform PKC-�1 wasreliably over-expressed; in other clones, PKC-�1 expressionwas inhibited. Herein, we report novel biologic functions,protection of actin and Ca2� balance, by the classical �1isoform of PKC.

Materials and MethodsCell Culture. Caco-2 cells, which were obtained from American

Type Culture Collection (Manassas, VA) at passage 15, were chosenbecause they form monolayers that morphologically resemble smallintestinal cells, with defined apical brush borders, junctional com-plexes, and a highly organized actin network (Gilbert et al., 1991;Meunier et al., 1995; Banan et al., 1998b). Cells were maintained at37°C in complete Dulbecco’s minimum essential medium in an at-mosphere of 5% CO2 and 100% relative humidity. Naive or stablytransfected cells (see below) were split at a ratio of 1:6 upon reachingconfluence and set up in either 6- or 24-well plates for experiments orT-75 flasks for propagation. Cells grown for barrier function exper-iments were split at a ratio of 1:2 and seeded at a density of 200,000cells/cm2 into 0.4 �M Biocoat collagen I cell culture inserts (0.3-cm2

growth surface; BD Biosciences-Discovery Labware, Bedford, MA),and experiments were performed at least 7 days post-confluence. Themedia were changed every 2 days. The utility and characterization ofthis cell line has been previously reported (Gilbert et al., 1991;Meunier et al., 1995; Banan et al., 1998b).

Plasmid. The sense and antisense plasmids of PKC-�1 were con-structed as we previously described (Cho et al., 1998; Banan et al.,2001c,d). Expression was controlled by �-actin promoter. The anti-sense PKC-�1 plasmid (p�-actin SP72-As-PKC-�1) was constructedby ligating the 2.3-kb EcoRI fragment of PKC-�1 cDNA from pJ6-PKC-�1 (Cho et al., 1998) into the unique EcoRI sites of the p�-actinSP72 vector. The antisense orientation of the plasmid was confirmedby SamI restriction digestion (Cho et al., 1998).

Stable Transfection. Cultures of Caco-2 cells grown to 50 to 60%confluence were cotransfected with G-418 (selection) resistance plas-mid and expression plasmids encoding either PKC-�1 or antisense-PKC-�1 by Lipofectin (Lipofectin reagent; Invitrogen, Carlsbad, CA)as we previously described (Banan et al., 2001c). Control conditionsincluded vector alone. Briefly, cells were incubated for 16 h at 37°Cwith the plasmid DNA in serum-free media in the presence of Lipo-fectAMINE (25 �l/25-cm2 flask). Subsequently, the DNA-containingsolution was removed and replaced by fresh media containing 10%fetal bovine serum to relieve cells from the shock of exposure toserum-free media. Following transfection, cells were subjected toG-418 selection (0.6 mg/ml) over 4 weeks. Resistant cells were main-tained in Dulbecco’s minimum essential medium/fetal bovine serumand 0.2 mg/ml G-418 (selection medium). PKC protein expression orlack of it was verified by Western blot analysis of cell lysates (seebelow). Multiple clones stably over-expressing PKC-�1 or lackingPKC-�1 were assessed by immunoblotting, plated on cell cultureinserts, allowed to form confluent monolayers, and subsequentlyused for experiments.

Experimental Design. In the first series of experiments, post-confluent monolayers of naive Caco-2 cells were preincubated withEGF (1 to 10 ng/ml) or isotonic saline for 10 min and then exposed tooxidant (H2O2, 0.5 mM) or vehicle (saline) for 30 min. As we havepreviously shown (Banan et al., 2000c, 2001a,e), H2O2 at 0.5 mMdisrupts actin and barrier integrity; EGF at 10 ng/ml (but not 1ng/ml) prevents this disruption. These experiments were then re-peated using monolayers composed of cells either stably over-ex-pressing or almost completely lacking PKC-�1. Reagents were ap-plied on the apical side of monolayers unless otherwise indicated.

PKC-�1 Isoform Protects F-Actin Assembly 853

Since our previous studies (Banan et al., 2000a,c) showed that re-gardless of whether apical or basolateral exposure of oxidants wasused, the results were qualitatively similar; all current studies uti-lized apical application. In all experiments, actin cytoskeletal stabil-ity (ring cytoarchitecture, assembly, disassembly), PKC-�1 subcellu-lar distribution, and Ca2� homeostasis ([Ca2�]i and Ca2� efflux)were assessed.

In a second series of experiments, monolayers were preincubated(10 min) with either a PKC activator or a PKC inhibitor and thenincubated with EGF prior to exposure to oxidant. PKC activatorsincluded 1) a synthetic diacylglycerol (OAG; 0.1, 1, 50, and 100 �M)or 2) a phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA;0.1, 1, 30, and 60 nM) or its inactive analog 4�-phorbol 12,13-didecanoate (4�-PDD; 20 nM) (Banan et al., 2001b,c). PKC inhibitorsincluded chelerythrine (1 �M) or bisindolylmaleimide V (GF 109203X; 10 nM) or its inactive analog iGF 109203 X. Controls were treatedwith vehicle (0.02% ethanol). We confirmed (Banan et al., 2001b)that these doses of PKC inhibitors were not injurious to cells .

In a third series of experiments, cell monolayers that were stablyover-expressing PKC-�1 were preincubated (10 min) with low (non-protective) or high (protective) doses of the PKC activator OAG (0.01or 50 �M), EGF (1 or 10 ng/ml), or vehicle prior to exposure (30 min)to damaging concentrations of oxidant (H2O2; 0.5 mM) or vehicle.Vehicle solution for OAG was 0.02% ethanol.

In a fourth series of experiments, monolayers of antisense trans-fected cells lacking PKC-�1 protein were treated with high doses ofEGF or OAG and then oxidant. In all experiments, expression levelsof PKC-�1 were determined by immunoblotting. In corollary exper-iments, we investigated the effects of PKC-�1 under- or over-expres-sion on the state of actin assembly and disassembly and on thestability of the cytoarchitecture of the apical ring of F-actin. Mono-meric and polymerized fractions of actin (the structural proteinsubunit of actin) were isolated and then analyzed by quantitativeimmunoblotting (Banan et al., 2000c, 2001a). Actin integrity wasassessed by 1) immunofluorescent labeling and fluorescence micro-scopy to determine the percentage of cells from monolayers display-ing a normal apical ring of actin; 2) detailed analysis of cortical actinring by high-resolution laser scanning confocal microscopy (LSCM);and 3) quantitative immunoblot analysis of monomeric (G-) andpolymerized (F-) actin fractions.

In a fifth and final series of experiments, we investigated the roleof PKC-�1 in growth factor-mediated normalization of Ca2� ho-meostasis. Outcomes were both [Ca2�]i and Ca2� efflux. Monolayersof either naive or transfected cells (over- or under-expressing PKC-�1) were preloaded with the appropriate Ca2� probe (Fluo-3-AM or45Ca2�), then preincubated for 10 min with EGF or OAG, and finallyexposed to oxidant for 30 min. Where indicated, monolayers werealso preincubated with a membrane-bound Ca2�-ATPase pump in-hibitor (either vanadate or quercetine; 10 �M, 30 min) prior to theEGF or OAG.

Immunofluorescent Staining and High-Resolution LaserScanning Confocal Microscopy of Actin. Cells from monolayerswere fixed in cytoskeletal stabilization buffer and then post-fixed in95% ethanol at �20°C, as we previously described (Banan et al.,2000c, 2001a). Cells were subsequently processed for incubation withfluorescein isothiocyanate-phalloidin (specific for F-actin; Sigma-Al-drich, St. Louis, MO), 1:40 dilution, for 1 h at 37°C. Slides werewashed three times in D-phosphate-buffered saline, once with deion-ized H2O, and subsequently mounted in Aquamount (Fisher Scien-tific, Fair Lawn, NJ). Following staining, cells were observed with anargon laser (� � 488 nm) using a 63� oil immersion plan-apochromatobjective, NA 1.4 (Carl Zeiss GmbH, Jena, Germany). Desired areasof monolayers were processed using the image processing softwareon a Zeiss Ultra high-resolution LSCM (Carl Zeiss). The apical(cortical) rings of actin, which are known to regulate paracellularpermeation through monolayers of Caco-2 cells, were examined in ablinded fashion for their overall morphology and disruption as wepreviously described (Banan et al., 2000c, 2001a,e). At least 1200

cells per group (200 � 6 slides) were examined in four different fieldsby LSCM, and the percentage of cells displaying normal actin ringwas determined. The slides were decoded only after examination wascomplete.

Actin Fractionation and Quantitative Immunoblotting ofF- and G-Actin State of Assembly and/or Disassembly. Poly-merized (F-) and monomeric (G-) fractions of actin were isolated aswe previously described (Banan et al., 2000c, 2001a,e). Cells weregently scraped and pelleted with centrifugation at low speed (700rpm, 7 min, 4°C) and resuspended in actin stabilization-extractionbuffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% dimethyl sulfoxide, 1mM MgSO4, 10 �g/ml anti-protease cocktail, 1 mM EGTA, and 1%Triton X-100) at room temperature for 20 min. Actin fractions wereseparated following a series of centrifugation and extraction steps.Cell lysates were centrifuged at 105,000g for 45 min at 4°C, and thesupernatant containing the soluble monomeric pool of G-actin (or S1)was gently removed. The remaining pellet was then resuspended in0.3 ml of Ca2�-containing depolymerization buffer (0.1 M PIPES, pH6.9, 1 mM MgSO4, 10 �g/ml anti-protease cocktail, and 10 mMCaCl2) and incubated on ice for 60 min. Subsequently, samples werecentrifuged at 48,000g for 15 min at 4°C, and the supernatant (S2- orF-fraction, or cold/Ca2�-soluble fraction) was removed. To ensure thecomplete removal of the F-fraction, the remaining pellet was treatedwith the Ca2�-containing depolymerization buffer twice more byresuspension and centrifugation. The “actin” was recovered by sep-arately incubating (at 37°C for 30 min) the S1 and S2 fractions withstabilizing agents, phalloidin (1 �M) and ATP (0.1 mM), in actinstabilization buffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% dimethylsulfoxide, 10 �g/ml anti-protease cocktail, 1 mM EGTA, 1 mMMgCl2, and 0.1 mM ATP) to promote polymerization of actin. Actinwas then recovered by centrifugation and resuspended in the abovestabilization buffer. Fractionated S1 and S2 samples were thenflash-frozen in liquid N2 and stored at �70°C until immunoblotting.For immunoblotting, samples (5 �g of protein/lane) were placed in astandard SDS sample buffer, boiled for 5 min, and then subjected toPAGE on 7.5% gels. Procedures for Western blotting were performedas previously described (Banan et al., 2000c, 2001a,e). To quantifythe relative levels of actin, the optical density of the bands corre-sponding to immunoradiolabeled actin was measured with a laserdensitometer.

Fractionation and Western Immunoblotting of PKC. Differ-entiated cell monolayers grown in 75-cm2 flasks were processed forthe isolation of the cytosolic, membrane, and cytoskeletal fractions aswe previously described (Banan et al., 2001b,c). Briefly, followingtreatments, post-confluent monolayers were scraped and ultrasoni-cally homogenized in Tris-HCl buffer (20 mM Tris-HCl, pH 7.5, 0.25mM sucrose, 2 mM EDTA, 10 mM EGTA, 2 �g/ml aprotinin, 2 �g/mlpepstatin, 2 �g/ml leupeptin, and 2 �g/ml phenylmethylsulfonylfluoride). The homogenates were then ultracentrifuged (100,000g,for 40 min at 4°C), and the supernatant was removed and used as asource of the cytosolic fraction. Next, pellets were washed with 0.2 mlof Tris-HCl buffer and resuspended in 0.8 ml of buffer containing0.3% Triton X-100 and maintained on ice for 1 h. The samples werethen centrifuged (100,000g, for 1 h at 4°C), and the supernatant wasused as the source of the membrane fraction. To this remainingpellet, 0.3 ml of cold (4°C) lysis buffer (150 mM NaCl, 50 mMTris-HCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 0.1% sodiumdeoxycholate, 0.1% SDS, 2 �g/ml aprotinin, 2 �g/ml pepstatin, 2�g/ml leupeptin, and 2 �g/ml phenylmethylsulfonyl fluoride) wasadded. The samples were then placed on ice for 1 h and ultracentri-fuged as above. The remainder of the lysate or Triton-insolublecytoskeletal fraction was then removed. Protein content of the vari-ous cell fractions was assessed by the Bradford method (Bradford,1976). For total PKC extraction, which provides the fraction used toconfirm total PKC-�1 expression, scraped monolayers were placeddirectly into 1.5 ml of cold lysis buffer and subsequently ultracentri-fuged as described above. The supernatant was used for bulk proteindetermination.

854 Banan et al.

For immunoblotting, samples (75 �g of protein/lane) were added toSDS buffer (250 mM Tris-HCl, pH 6.8, 2% glycerol, and 5% mercap-toethanol), boiled for 5 min, and then separated on 7.5% SDS-PAGE(Banan et al., 2001c). Subsequently, proteins were transferred tonitrocellulose membranes (0.2-�m pore size), and then blocked in 3%bovine serum albumin for 1 h, followed by several washes withTris-buffered saline. The immunoblotted proteins were incubated for2 h in Tween 20, Tris-buffered saline, 1% bovine serum albumin, andthe primary mouse monoclonal anti-PKC-�1 (Santa Cruz Biotech-nology, Inc., Santa Cruz, CA) at 1:1000 dilution for 1 h at roomtemperature. A horseradish peroxidase-conjugated goat anti-mouseantibody (Molecular Probes, Eugene, OR) was used as a secondaryantibody at 1:3000 dilution. Proteins on membranes were visualizedby enhanced chemiluminescence (Amersham Biosciences, ArlingtonHeights, IL) and autoradiography, and subsequently analyzed bydensitometry. The identity of the PKC-�1 band was confirmed as wepreviously described (Banan et al., 2001c) by using the PKC-�1blocking peptide (Santa Cruz Biotechnology, Inc.) in combinationwith the anti-PKC-�1 antibody that prevents the appearance of thecorresponding “major” band in Western blots. Additionally, in theabsence of the primary antibody to PKC-�1, no corresponding bandfor PKC-�1 was observed. The PKC-�1 band ran at the expectedmolecular mass of 78 kDa, as confirmed by a known positive controlfor PKC-�1 (from rat brain lysates). Prestained molecular weightmarkers (Mr 67,000 and 93,000) were run in adjacent lanes. Usingtotal PKC extracts, we previously showed (Banan et al., 2001c) thatover-expression of PKC-�1 or antisense inhibition of PKC-�1 expres-sion did not affect the relative expression levels of other PKC iso-forms or injure the Caco-2 cells.

Measurement of [Ca2� ]i. Alterations in [Ca2�]i were deter-mined using the sensitive fluorescence Ca2�-indicator Fluo-3-AM(Molecular Probes) as we described previously (Kokoska et al., 1998;Banan et al., 2001b). Briefly, monolayers were washed two timeswith Hanks’ balanced salt solution prior to loading with Fluo-3 for 60min (final concentration of 4 �M). Monolayers were then washedthree times to remove excess Fluo-3 followed by treatment regimens.At the desired time points [Ca2�]i was quantitated using the equa-tion: [Ca2�]i (nM) � Kd [(F � Fmin)/(Fmax � F)], where Fmin (theminimum Fluo-3 signal) is equal to FMnCl2 minus 0.25 Fmax, and Kd

(the dissociation constant) equals 400 nM (Vandenberghe and Ceup-pens, 1990; Kokoshka et al., 1998). The maximum Fluo-3 signal(Fmax) was obtained by permeabilizing Caco-2 cells with 50 �Mdigitonin (Sigma-Aldrich). The Fluo-3 signal was quenched to ob-tain FMnCl2 using 2 mM MnCl2 and 50 �M digitonin. The heavymetal scavenger, N,N,N�,N�-tetrakis(2-pyridylmethyl)-ethylenediamine(50 �M), was used in all solutions. Fluorescent signals from sampleswere quantitated by a fluorescence multiplate reader (FL 600; Bio-Tek Instruments, Winooski, VT) at 37°C, employing excitation andemission wavelengths of 485 and 530 nm, respectively.

Measurement of 45Ca2� Efflux. Caco-2 cells were preloadedwith 45Ca2� (10 �Ci/ml) for 1 h at 37°C and then incubated with thetest agents. After centrifugation, radioactivity in the supernatantand within the lysed cells was determined by scintillation counting(Kokoska et al., 1998; Banan et al., 2001b). The 45Ca2� efflux wasexpressed as: Ca2� efflux (%) � [CPMsupernatant /(CPMsupernatant �CPMcells)], where CPM is counts per minute.

Statistical Analysis. Data are presented as mean � S.E.M. Allexperiments were carried out with a sample size of at least sixobservations per treatment group that were run in triplicate (or insome cases in duplicate) on 2 to 3 different days. Statistical analysiscomparing treatment groups was performed using analysis of vari-ance followed by Dunnett’s multiple range test (Harter, 1960). Cor-relational analyses were done using the Pearson test for parametricanalysis or, when applicable, the Spearman test for nonparametricanalysis; p values �0.05 were deemed statistically significant.

ResultsProtection of the F-Actin by EGF and PKC Activa-

tors against Oxidant-Induced Damage. Figure 1 demon-strates that preincubation of Caco-2 monolayers with EGF orPKC activators (OAG or TPA) prior to subsequent exposureto oxidant (H2O2) dose dependently and significantly pro-tected the F-actin against oxidant-induced disruption asmeasured by increases in the percentage of cells with normalactin. Since EGF (10 ng/ml), OAG (50 �M), and TPA (30 nM),which we previously reported to prevent oxidant-induceddisruption of the monolayer barrier and increased paracellu-lar permeability (Banan et al., 2001b), provided maximalprotection of actin, we utilized these doses in subsequentpharmacological studies. OAG or TPA protection of actin wasnot significantly different from EGF-induced protection.

Figure 2 shows that pretreatment with a biologically inac-tive phorbol ester 4�-PDD, as expected, did not protect theactin. Preincubation with known pharmacological inhibitorsof PKC (chelerythrine or GF 109203 X) prevented the protec-tive effects of EGF or PKC activators. As expected, the inac-tive analog of the latter PKC inhibitor, iGF 109203 X, wasnot protective. PKC inhibitors by themselves did not affect

Fig. 1. Protective effects of EGF or PKC activators (OAG and TPA) on thepercentage of naive Caco-2 cells displaying a normal actin cytoskeleton.Monolayers of naive cells were exposed to the shown doses of EGF or theOAG and TPA for 10 min prior to exposure to oxidant H2O2 (0.5 mM) for30 min. Cell monolayers were processed for actin staining by cellularfixation followed by incubation with fluorescein-conjugated phalloidin(specific for F-actin), and the percentage of cells displaying a normal actinwas then assessed as described under Materials and Methods. Note thatpretreatment with the protective agents (EGF, OAG, or TPA) dose de-pendently maintains a high percentage of cells with normal actin againstexposure to oxidant injury. �, p � 0.05 versus vehicle (control). �, p �0.05 versus H2O2. &, p � 0.05 versus EGF (10 ng/ml) � H2O2 or OAG (50�M) � H2O2 or TPA (30 nM) � H2O2. n � 6 per treatment group in allexperiments including those shown in other figures.

PKC-�1 Isoform Protects F-Actin Assembly 855

the actin. Moreover, a combination of each PKC inhibitor(i.e., chelerythrine or GF 109203 X) with H2O2 did not causeany significant increase in H2O2-induced damage to the actin(percentage of normal actin was 49 � 4 for H2O2 alonecompared with 48 � 5 for chelerythrine � H2O2 or 50 � 3 forGF 109203 X � H2O2).

Fluorescent images obtained by high-resolution laser scan-ning confocal microscopy from the apical cortical ring of F-actin, which is known to regulate monolayer paracellularpermeability (Banan et al., 2000c, 2001a,e), corroborated theaforementioned findings on the actin (Fig. 3, a to f). Controlcells from untreated monolayers showed a normal andsmooth distribution of the actin ring at the areas of cell-to-cell contact (Fig. 3a). Exposure of cell monolayers to H2O2

produced extensive fragmentation, disorganization, andbeading of the actin ring (Fig. 3b). On the other hand, pre-incubation with EGF prevented this disruption as shown bya smooth and continuous pattern of the actin ring (Fig. 3c).Not surprisingly, growth factor-pretreated monolayers wereindistinguishable from controls (Fig. 3a). Furthermore, PKCactivators (e.g., OAG) had protective effects on the actin ringsimilar to that of EGF (Fig. 3e). Moreover, preincubation

with PKC inhibitors (e.g., GF 109203 X) prevented the pro-tection of actin by EGF (Fig. 3d) or by PKC activator (Fig. 3f).

To investigate the underlying cause of actin stabilityand/or instability, we performed quantitative Western immu-noblotting of the polymerized F-actin pool (S2 fraction, indexof actin assembly) and monomeric G-actin pool (S1 fraction,index of actin disassembly) in response to various treatmentregimens (Fig. 4). EGF and PKC activators (OAG, TPA)increased the stable F-actin fraction and decreased the un-stable G-actin in monolayers exposed to oxidant, indicatingstabilization of actin assembly. Oxidant alone reduced the

Fig. 2. Prevention of the protective effects of EGF or PKC activators(OAG and TPA) on the percentage of naive Caco-2 cells with normal actin.EGF (10 ng/ml) or PKC activators (OAG, 50 �M or TPA, 30 nM) wereadded to the monolayers 10 min before subsequent exposure to oxidant(H2O2, 0.5 mM) for 30 min. In other experiments, monolayers werepreincubated with either PKC inhibitors (chelerythrine, 1 �M, or GF109203 X, 10 nM, or the inactive analog iGF 109203 X) or a biologicallyinactive phorbol ester (4�-PDD, 20 nM). �, p � 0.05 versus vehicle(control). �, p � 0.05 versus H2O2. &, p � 0.05 versus EGF (or OAG orTPA) � H2O2. †, p � 0.05 versus corresponding GF 109203 X � EGF (orOAG or TPA) � H2O2.

Fig. 3. Fluorescent staining of the apical cortical ring of F-actin byfluorescein-conjugated (fluorescein isothiocyanate) phalloidin revealingits intracellular organization in confluent intestinal cell monolayers.Naive monolayers were treated with (panel a) isotonic saline/control or(panel b) 0.5 mM H2O2. In panels c and e, monolayers were pretreated,prior to exposure to H2O2, with protective agents EGF (10 ng/ml; panel c)or PKC activator OAG (50 �M; panel e). In panels d and f, monolayerswere pretreated with PKC inhibitor GF 109203 X and then either EGF(panel d) or OAG and subsequently H2O2 (panel f). Ultra high-resolutionLSCM reveals that control cells (panel a) show a normal, continuous, andsmooth distribution of actin ring (apical cortex) at areas of cell-to-cellcontact. In cells exposed to 0.5 mM H2O2 alone (panel b), the actin ringappears disrupted, condensed, beaded, and fragmented. In contrast, incells pretreated with EGF (panel c) normal actin cytoarchitecture ap-pears intact and preserved. Similarly, actin ring architecture in cellspretreated with OAG (panel e) is highly preserved and resembles themorphology observed in the control group. Preincubation of monolayerswith PKC inhibitor GF 109203 X abolished protection of actin in EGF �H2O2 (panel d) or OAG � H2O2 (panel f) groups as shown by a disruptedappearance of the actin cytoskeleton.

856 Banan et al.

F-actin pool while increasing the G-actin pool. Moreover, asexpected, pretreatment with an inactive phorbol ester, 4�-PDD, did not prevent actin disassembly in H2O2-exposedmonolayers (37 � 1.2% for 4�-PDD-pretreated versus 38 �0.4% for H2O2-exposed). In additional experiments, PKC in-hibitors abolished the stabilizing effects of protective agents(EGF, OAG, TPA) on the dynamics of actin assembly. Forexample, in monolayers incubated with H2O2, preincubationwith the PKC inhibitor, GF 109203 X, inhibited most of theincrease in actin assembly by EGF (41 � 0.35%), OAG (42 �0.80%), or TPA (43 � 0.65%). As expected, the inactive analogiGF 109203 X was ineffective when preadministered beforeEGF (51 � 0.20%), OAG (52 � 0.70%), or TPA (54 � 0.40%)and H2O2 insult. As with their effects on the percentage ofnormal actin, combinations of PKC inhibitors with H2O2 didnot potentiate H2O2-induced actin disassembly (37 � 0.6%for chelerythrine � H2O2 and 39 � 0.9% for GF 109203 X �H2O2 compared with 38 � 0.4% for H2O2 alone).

A representative Western blot of actin fractions fromCaco-2 monolayers (Fig. 5) also showed that EGF and PKCactivators (e.g., OAG) increased the F-actin lane density,indicating enhanced actin polymerization (and actin stabili-ty). Once again, these protective effects on actin assemblywere prevented by the PKC inhibitors (e.g., GF 109203 X) butnot the inactive analog iGF 109203 X. These findings on the

dynamic alterations in actin polymerization and depolymer-ization parallel the above-noted protective effects of EGF andPKC activators on the protection of actin ring cytoarchitec-ture.

We then investigated the question as to which PKC isoformis key to EGF-induced protection of the actin. Because ourprevious studies (Banan et al., 2001c) indicate that the �1isoform of PKC is not only a major isoform of PKC in Caco-2cells but also key to the maintenance of an intact monolayerbarrier function, we explored the possible role of this isoformin the underlying mechanism of EGF protection of the integ-rity of the actin, including dynamic assembly of both G- andF-actin pools.

Key Role of PKC-�1 Isoform in Protection of theF-Actin Cytoskeleton. To this end, we used novel and sta-bly transfected intestinal cell lines we developed (Banan etal., 2001c) that either over- or under-express PKC-�1 com-pared with naive Caco-2 cells. Figure 6 and Table 1 show thatover-expression (3.1-fold) of PKC-�1-potentiated protectionby EGF or PKC activators (e.g., OAG) of the F-actin againstoxidant-induced injury. In cells stably over-expressing �1isoform, actin integrity (as assessed by the percentage of cellsdisplaying normal F-actin ring) was protected against oxi-dant damage by a low dose of EGF (1 ng/ml). This same doseof EGF did not protect the actin in naive cells. A similarsynergy was seen for protection of actin by a low dose of aPKC activator (OAG, 0.01 �M) (Fig. 6). TPA caused identicaleffects (not shown). In all instances, the extent of protectionof PKC-�1-over-expressing cells was not significantly differ-ent from protection of naive cells by higher doses of thesesame agents (10 ng/ml EGF; 50 �M OAG). This did notappear to be due to changes in the ability of oxidants to causedamage to actin as PKC-�1-over-expressing cells (withoutEGF or OAG) and naive cells responded comparably to H2O2,both with similar and significant damage to actin (Fig. 6).EGF or OAG alone did not affect actin compared with vehicle(percentage of normal F-actin was 99 � 1 for vehicle versus98 � 2 for EGF or 96 � 4 for OAG). Furthermore, PKC-�1over-expression by itself did not protect or injure actin. Asexpected, transfection of only the vector (SP-72) did not pro-tect actin against oxidant injury (e.g., percentage of normalF-actin was 98 � 2 for vector-transfected cells exposed tovehicle; 48 � 4 for vector-transfected cells exposed to H2O2

alone; and 49 � 5 for vector-transfected cells incubated with1 ng/ml EGF � H2O2 versus 90 � 5 for PKC-�1 sense-transfected cells incubated with 1 ng/ml EGF � H2O2).

Multiple clones of Caco-2 cells transfected with varyingamounts (1, 2, 4, and 5 �g) of PKC-�1 sense cDNA showed

Fig. 4. Quantitative immunoblotting analysis of the polymerized F-actinpool (S2, index of actin stability) and monomeric G-actin pool (S1, indexof actin disassembly) in Caco-2 monolayers. Conditions were described inFigs. 2 and 3. Percentage of polymerized actin � [(S2)/(S2 � S1)], whereS2 � S1 is the total cellular actin pool. �, p � 0.05 versus vehicle (control).�, p � 0.05 versus H2O2. &, p � 0.05 versus EGF (or OAG or TPA) �H2O2. †, p � 0.05 versus corresponding GF 109203 X � EGF (or OAG orTPA) � H2O2.

Fig. 5. Representative Western immunoblot photomicrograph of the po-lymerized actin (S2, Triton-insoluble) extracts from naive cell monolayersfollowing treatments. F-actin fractions were analyzed by SDS-PAGE andWestern immunoblots using a monoclonal anti-actin primary antibodyfollowed by a horseradish peroxidase-conjugated secondary antibody andthen autoradiographed. The lanes from left to right correspond to: a,vehicle; b, 0.5 mM H2O2 challenge; c, EGF (10 ng/ml) � 0.5 mM H2O2; d,PKC inhibitor (GF 109203 X) � EGF (10 ng/ml) � 0.5 mM H2O2; e,inactive analog of PKC inhibitor (iGF 109203 X) � EGF (10 ng/ml) � 0.5mM H2O2; f, OAG (50 �M) � 0.5 mM H2O2; g, PKC inhibitor (GF 109203X) � OAG (50 �M) � 0.5 mM H2O2; h, inactive PKC inhibitor (iGF 109203X) � OAG (50 �M) � 0.5 mM H2O2; i, actin standard (43 kDa).

PKC-�1 Isoform Protects F-Actin Assembly 857

(Table 1) dose-dependent synergistic protection of the actin.Because the clone transfected with 4 �g of PKC-�1 senseDNA provided almost complete (90%) protection of actin (Fig.6 and Table 1), we used this clone in all subsequent experi-ments. In terms of the responses of these various transfectedclones to H2O2 exposure (alone), we did not observe anysignificant differences from one clone to the next clone (1 to 5�g) or even when compared with the naive cells that wereexposed to the same oxidant (4 �g clone shown in Fig. 6).Thus, an average (mean � S.E.M.) of these groups is pre-sented as H2O2 alone in Table 1.

High-resolution fluorescent images obtained by laser scan-ning confocal microscopy from the apical cortical ring of F-actin corroborated the above findings. Figure 7 shows thatover-expression of PKC-�1 potentiates protection by lowdoses of EGF (Fig. 7e) or OAG (Fig. 7f). This synergy is shown

by the appearance of normal, intact, and smooth architectureof the actin ring at the areas of cell-to-cell contact (Fig. 7, eand f). The appearance of the actin ring in these transfectedcells was indistinguishable from the untreated normal cells,which also showed an intact pattern of the actin ring (Fig.7a). Without the synergy afforded by PKC-�1 over-expres-sion, naive cells pretreated with the same low doses of EGFand OAG and exposed to H2O2 showed extensive disorgani-zation, condensation, and beading of the actin ring at areasassociated with the plasma membrane (Fig. 7, c and d, re-spectively) as did naive cells exposed to H2O2 alone (Fig. 7b).

Immunoblotting analysis of the F-actin fraction (S2, anindex of actin integrity) and the G-actin fraction (S1, an indexof actin disruption) (Fig. 8A) further demonstrates that onlythe transfected cells over-expressing PKC-�1 exhibit a syn-ergy between low doses of EGF (1 ng/ml) or OAG (0.01 �M)and PKC-�1 as indicated by normal dynamics of actin poly-merization. Transfected cells exposed to low doses of EGF orOAG and then H2O2 had enhanced levels of the polymerizedF-actin and reduced levels of monomeric G-actin; this wascomparable with the normal controls. In contrast, H2O2 alonereduced polymerized F-actin and increased monomeric G-actin in both naive cells and PKC-�1-over-expressing cells(without added EGF or OAG), indicating disruption of actin.Pretreatment of naive cells with only the higher doses of EGF(10 ng/ml) or OAG (50 �M) resulted in normal steady-statelevels of actin polymerization. Similar to the above-notedeffects on actin architecture, transfection of vector alone wasnot effective in protecting or maintaining normal pools ofactin (not shown).

Fig. 6. Protective actions of PKC-�1 over-expression on the percentage ofCaco-2 cells from transfected monolayers displaying a normal F-actin inthe presence of a low concentration of EGF or PKC activator (OAG). Anovel sense-transfected cell line previously developed in our laboratory(see Materials and Methods) that over-expresses PKC-�1 by 3.1-fold wasutilized. Transfected “(T)” cells stably over-expressing PKC-�1 were in-cubated in low doses of EGF (1 ng/ml) or the PKC activator OAG (0.01�M) prior to the subsequent exposure to oxidant H2O2 (0.5 mM). Theselow doses do not protect F-actin against oxidant injury in naive “(N)” cells,but they do protect F-actin in transfected cells over-expressing PKC-�1.Only high doses of EGF (10 ng/ml) or OAG (50 �M) protected actin innaive monolayers (not over-expressing �1). Also note synergy-inducedprotection of the actin in PKC-�1-over-expressing (T) cells that wereexposed to low doses of EGF or OAG. In the absence of these low doses ofEGF or OAG, both PKC-�1-over-expressing cells and naive cells re-sponded comparably to oxidant insult to actin, both displaying significantreduction in the percentage of cells with normal actin. �, p � 0.05 versusvehicle. �, p � 0.05 versus H2O2. &, p � 0.05 versus corresponding lowdoses of EGF or OAG � H2O2 in naive cells.

TABLE 1Effects of transfection of varying amounts of PKC-�1 sense orantisense DNA on F-actin cytoskeleton in Caco-2 monolayersValues are means � S.E.M. following treatments. Cells stably transfected withvarying amounts of PKC-�1 sense DNA (1, 2, 4, or 5 �g) were preincubated (10 min)with a low dose of EGF (1 ng/ml) or OAG (a synthetic diacylglycerol and a PKCactivator, 0.01 �M) before subsequent exposure to oxidant (H2O2, 0.5 mM) for 30min. In separate studies, cells transfected with varying amounts of PKC-�1 anti-sense (1, 4, or 5 �g) were treated with a high dose of EGF (10 ng/ml) or OAG (50 �M)prior to oxidant. Select treatments from naive (untransfected) cell monolayers arealso shown. One hundred fifty to 200 cells per slide (well) were examined byhigh-resolution laser confocal and the percentage of cells displaying normal actincytoskeleton was determined. N � 6 per group.

Treatment % Normal Actin

Vehicle 98 � 2H2O2 alone 47 � 4*EGF (1 ng/ml) � H2O2 49 � 3*EGF (1 ng/ml) � H2O2 (1 �g of sense DNA) 56 � 5*,‡

EGF (1 ng/ml) � H2O2 (2 �g of sense DNA) 72 � 2*,†,‡

EGF (1 ng/ml) � H2O2 (4 �g of sense DNA) 90 � 5†

EGF (1 ng/ml) � H2O2 (5 �g of sense DNA) 92 � 3†

OAG (0.01 �M) � H2O2 48 � 5*OAG (0.01 �M) � H2O2 (1 �g of sense DNA) 55 � 6*,‡

OAG (0.01 �M) � H2O2 (2 �g of sense DNA) 73 � 2*,†,‡

OAG (0.01 �M) � H2O2 (4 �g of sense DNA) 87 � 4†

OAG (0.01 �M) � H2O2 (5 �g of sense DNA) 86 � 3†

EGF (10 ng/ml) � H2O2 91 � 4†

EGF (10 ng/ml) � H2O2 (1 �g of antisense DNA) 88 � 5†,‡

EGF (10 ng/ml) � H2O2 (4 �g of antisense DNA) 52 � 2*EGF (10 ng/ml) � H2O2 (5 �g of antisense DNA) 50 � 7*OAG (50 �M) � H2O2 86 � 4†

OAG (50 �M) � H2O2 (1 �g of antisense DNA) 84 � 3*,†,‡

OAG (50 �M) � H2O2 (4 �g of antisense DNA) 53 � 5*OAG (50 �M) � H2O2 (5 �g of antisense DNA) 54 � 6*

* p � 0.05 compared to the vehicle.† p � 0.05 compared to H2O2.‡ p � 0.05 compared to corresponding EGF/OAG (low dose) prior to H2O2 in cells

transfected with 4 �g of sense DNA or EGF/OAG (high dose) and H2O2 in cellstransfected with 4 �g of anti-sense DNA.

858 Banan et al.

A representative immunoblot of the polymerized actin frac-tion from Caco-2 monolayers is shown in Fig. 8B. It demon-strates, once again, that PKC-�1 over-expression synergizeswith low doses of EGF or OAG to increase the F-actin lane(band) density, indicating enhancement of actin assemblyand stability. These various findings on dynamic alterationsin actin polymerization and depolymerization parallel thesynergistic effects of PKC-�1 over-expression on the protec-tion of actin ring architecture.

Activation of Over-Expressed PKC-�1 Correlateswith Three Different Indices of Actin Integrity. Weinitially confirmed our recent findings (Banan et al., 2001b,c)

in both naive and transfected, PKC-�1-over-expressing intes-tinal cells that EGF or PKC activators translocate the �1(�78 kDa) isoform of PKC from the cytosol to both mem-brane- and cytoskeletal-bound fractions. In particular, fol-lowing pretreatment with low doses of EGF or OAG, there isa rapid redistribution of over-expressed PKC-�1 isoform froma mostly cytosolic distribution into particulate fractions (i.e.,particulate � membrane � cytoskeletal fractions), indicatingthe induced activation of the �1 isoform (Fig. 9A). This figurealso shows a temporal relationship between PKC-�1 (optical

Fig. 7. The intracellular architecture of the apical cortical ring of F-actincytoskeleton from confluent monolayers as captured by ultra high-reso-lution LSCM. Monolayers of naive cells were incubated with vehicle(isotonic saline; panel a), 0.5 mM H2O2 (panel b), EGF (1 ng/ml) plus 0.5mM H2O2 (panel c), or OAG (0.01 �M) plus 0.5 mM H2O2 (panel d).PKC-�1-over-expressing cell monolayers (panels e and f) were also ex-posed to the same low doses of EGF (panel e) or OAG (panel f) and thenincubated with the same H2O2 concentration. Normal cells (panel a)reveal an intact and smooth architecture of apical actin cortex (i.e., ring)on the inner side of the plasma membrane (areas of cell-to-cell contact),whereas cells exposed to H2O2 (panel b) show fragmentation, beading,and disruption of the actin ring. Cells over-expressing PKC-�1, whichwere exposed to low doses of EGF (panel e) or OAG (panel f) beforeoxidant exposure, exhibit a highly preserved appearance of the apicalactin ring, which is indistinguishable from the normal cells (panel a). Incontrast, this protection did not occur in naive cells (not over-expressingPKC-�1; panels c and d) that were pre-exposed to these same low doses ofEGF (panel c) or OAG (panel d), as shown by the abnormal cytoarchitec-ture of the actin ring.

Fig. 8. A, immunoblotting analysis of the stable polymerized F-actinfraction (S2, index of actin integrity) and the monomeric G-actin fraction(S1, index of actin instability) in Caco-2 monolayers. Conditions as in Fig.6. Percentage of polymerized actin � [(S2)/(S2 � S1)], where S2 � S1 isthe total intracellular actin pool. �, p � 0.05 versus vehicle. �, p � 0.05versus H2O2. &, p � 0.05 versus low doses of EGF or OAG � H2O2 innaive cells. (N), naive. (T), transfected. B, representative blot of thepolymerized actin fractions from Caco-2 cell monolayers following treat-ments. F-actin fractions were isolated and immunoblotted and then pro-cessed for X-ray film exposure by enhanced chemiluminescence reagents.The lanes from left to right correspond to: a, vehicle; b, 0.5 mM H2O2challenge in naive cells; c, 0.5 mM H2O2 challenge in PKC-�1-over-expressing cells; d, EGF (1 ng/ml) � 0.5 mM H2O2 in PKC-�1-over-expressing cells; e, EGF (1 ng/ml) � 0.5 mM H2O2 in naive cells; f, OAG(0.01 �M) � 0.5 mM H2O2 in PKC-�1-over-expressing cells; g, OAG (0.01�M) � 0.5 mM H2O2 in naive cells; h, EGF (10 ng/ml) � 0.5 mM H2O2 innaive cells; i, OAG (50 �M) � 0.5 mM H2O2 in naive cells; and j, actinstandard (43 kDa). In transfected cells the over-expressed PKC-�1 in thepresence of a low dose of EGF (1 ng/ml) or OAG (0.01 �M) increases thepolymerized F-actin band density to almost normal levels. In naive cells,on the other hand, preincubation with these same low doses of EGF orOAG does not increase actin polymerization, which is similar to that ofthe oxidant-exposed groups. Also shown are high concentrations of EGF(10 ng/ml) or OAG (50 �M), which are the only doses that increase actinassembly in naive cells.

PKC-�1 Isoform Protects F-Actin Assembly 859

density from the particulate fraction) and actin ring integ-rity. When these two variables were plotted against eachother, we found a robust correlation (r � 0.93, p � 0.05).When two other markers of actin stability, F-actin polymer-ization and G-actin disassembly, were plotted againstPKC-�1 (Fig. 9, B and C), additional robust correlations wereobserved (r � 0.92 and 0.91, respectively, p � 0.05 for each),further suggesting that increased activation of �1 isoform iskey in protection of actin integrity.

Stable Antisense Inhibition of PKC-�1 and Its Inhi-bition of EGF-Induced Protection of Actin.

To further investigate a possible role for PKC-�1 in EGF-mediated protection of actin, we utilized Caco-2 cells thatwere transfected with PKC-�1 antisense plasmid and cDNAencoding G-418 resistance. We confirmed our earlier reports

(Banan et al., 2001c) that this manipulation substantially(�90%) and stably reduces the steady-state levels of PKC-�1protein.

Analysis of the percentage of cells with a normal actinindicates that antisense inhibition of PKC-�1 expression sub-stantially inhibited the protection of actin by high doses ofEGF (10 ng/ml) or PKC activator (OAG, 50 �M) (Fig. 10 andTable 1), doses that almost completely protected actin innaive cells against exposure to oxidant. Under-expression ofthe PKC-�1 isoform by itself did not damage actin. Table 1also shows the effects of varying amounts of �1 antisensecDNA (1, 4, and 5 �g) on the attenuation of EGF or OAG-mediated protection of actin. Similar to our over-expression(sense) studies, the antisense data also indicate a dose-de-pendent phenomenon. The clone transfected with 4 �g of �1

Fig. 9. A, actin integrity and PKC-�1 particulate-associated fractionversus time. PKC-�1-over-expressing cells were pretreated with EGF (1ng/ml) or OAG (0.01 �M) prior to incubation with H2O2 (0.5 mM). Vari-ables depicted are optical density (O.D.) of particulate-associated PKC-�1band and percentage of cells with normal actin. B and C, polymerizedF-actin assembly (B) or monomeric G-actin disassembly (C) versus theO.D. of particulate-associated PKC-�1 band, �1 isoform activation.PKC-�1 levels (O.D.) in the particulate bands from PKC-�1-over-express-ing cells were correlated with two other markers of the condition of actinintegrity, namely the percentage of the pool of polymerized F-actin, anindex of actin assembly, and the percentage of the pool of monomericG-actin, an index of actin disassembly.

860 Banan et al.

antisense cDNA led to maximum inhibition of EGF- or OAG-induced protection of actin. Accordingly, this antisense clonewas used in all subsequent inhibition experiments. Quanti-tative immunoblotting analysis of the actin fractions fromantisense transfected cells further demonstrates (Fig. 11)that stable under-expression of PKC-�1 isoform preventedEGF- or OAG-induced enhancement of the stable F-actin pooland the reduction of the monomeric G-actin pool.

Normalization of [Ca2�]i Concentration: Preventionof Oxidant-Induced Rise in [Ca2�]i in PKC-�1 Trans-fected Cells. Because our previous studies showed that EGFnormalizes [Ca2�]i in the face of oxidant challenge, we sur-mised that normalization of [Ca2�]i might be a key mecha-nism for PKC-�1-induced, EGF-mediated protection. Indeed,measurement of [Ca2�]i in monolayers prelabeled with theCa2�-sensitive dye Fluo-3 (Fig. 12A) showed that PKC-�1over-expression synergized with low doses of EGF (1 ng/ml)or OAG (0.01 �M) to maintain [Ca2�]i at almost controllevels. These same low doses of EGF or OAG did not normal-ize [Ca2�]i in naive cells. Additionally, the extent of normal-ization of [Ca2�]i in transfected cells exposed to these lowdoses was not significantly different from the extent of nor-malization of [Ca2�]i in naive cells by higher doses of EGF orOAG (Fig. 12A). This did not appear to be due to changes inthe ability of oxidants to markedly increase [Ca2�]i sincePKC-�1-over-expressing cells (without EGF or OAG) and

naive cells responded similarly to H2O2, both with compara-ble and significant increases in [Ca2�]i. For example, naivemonolayers exposed to H2O2 exhibited [Ca2�]i levels at 327 �10 nM compared with 122 � 7 nM for vehicle. Furthermore,transfection of vector alone was ineffective ([Ca2�]i � 125 �10 nM for vector-transfected cells exposed to vehicle, 321 � 9nM for vector-transfected cells exposed to H2O2, and 324 �15 nM for vector-transfected cells incubated with 1 ng/mlEGF � H2O2 versus 138 � 15 nM for PKC-�1 sense-trans-fected cells incubated with 1 ng/ml EGF � H2O2).

Figure 12B shows that preincubation with two differentinhibitors of the membrane Ca2�-ATPase pump, quercetineor vanadate, abrogated the synergy-induced normalization of[Ca2�]i in PKC-�1-over-expressing cells exposed to low dosesof EGF or OAG, as [Ca2�]i levels remained elevated. TheseCa2�-ATPase inhibitors by themselves had no significanteffects on baseline Ca2� levels.

To further investigate the possibility that PKC-�1 plays akey role in the normalization of [Ca2�]i by EGF, stable anti-sense inhibition of this PKC isoform was utilized. Figure 13shows that under-expression of PKC-�1 inhibited the nor-malization of [Ca2�]i by high doses of EGF or PKC activatorOAG, doses that almost completely normalized [Ca2�]i innaive cells against oxidant challenge. Antisense inhibition ofPKC-�1 isoform by itself did not affect [Ca2�]i.

Induction of Ca2� Efflux under Protective Condi-tions in Stably Transfected Intestinal Cells. Since two

Fig. 10. Stable antisense under-expression of PKC-�1 isoform inhibitsthe protective effects of high doses of EGF (10 ng/ml) or PKC activator(OAG, 50 �M) on the actin cytoskeleton as determined by the percentageof cells displaying normal F-actin. A novel antisense-transfected cell linepreviously developed in our laboratory (see Materials and Methods),which almost completely lacks PKC-�1 protein, was grown as monolayersand then exposed to a high dose of EGF (10 ng/ml) or OAG (50 �M) andthen to H2O2. �, p � 0.05 versus vehicle. �, p � 0.05 versus H2O2. &, p �0.05 versus high doses of EGF or OAG � H2O2 in naive cells. (N), naive.(AS), antisense inhibition of PKC-�1.

Fig. 11. Stable antisense (AS) inhibition of PKC-�1 prevents the protec-tive effects of EGF or OAG on the enhancement of actin polymerization.Immunoblotting analysis of the polymerized F-actin (S2) and monomericG-actin (S1) from cellular extracts was performed following treatmentregimens similar to those shown in Fig. 10. Percentage of polymerizedactin � [(S2)/(S2 � S1)]. �, p � 0.05 versus vehicle. �, p � 0.05 versusH2O2. &, p � 0.05 versus high dose of EGF (10 ng/ml) � H2O2 or high doseof OAG (50 �M) � H2O2 in naive (N) cells.

PKC-�1 Isoform Protects F-Actin Assembly 861

different Ca2�-ATPase inhibitors abolished synergy-inducednormalization of [Ca2�]i, thereby maintaining [Ca2�]i at highlevels, we hypothesized that Ca2� efflux is an essential mech-anism for PKC-�1-induced (EGF) normalization of [Ca2�]i.Indeed, assessment of Ca2� efflux from monolayers prela-beled with 45Ca2� (Fig. 14A) showed that PKC-�1 over-expression synergized with the low doses of EGF (1 ng/ml)or OAG (0.01 �M) to markedly and significantly enhanceCa2� efflux. These same doses were ineffective in naivecells; only high doses were effective. Furthermore, twodifferent inhibitors of the membrane Ca2�-ATPase pump(Fig. 14B), quercetine and vanadate, abolished this en-hancement in Ca2� efflux. We did not observe any signif-icant changes in Ca2� efflux in vector (SP-72)-alone trans-fected cell monolayers (Ca2� efflux � 51 � 4% for vector-transfected cells exposed to vehicle, 43 � 2% for vector-transfected cells exposed to H2O2, and 49 � 5% for vector-transfected cells incubated with 1 ng/ml EGF � H2O2

versus 84 � 3% for PKC-�1 sense-transfected incubatedwith 1 ng/ml EGF � H2O2). These data on efflux parallelour data on [Ca2�]i.

Finally, we observed (Fig. 15) that the antisense inhibitionof PKC-�1 expression prevents the increases in Ca2� effluxinduced by high (protective) doses of EGF or PKC activator.

Antisense inhibition by itself did not affect baseline Ca2�

efflux.

DiscussionIn the current study, we demonstrate that activation of the

�1 isoform of PKC is required for EGF-mediated protection ofthe cortical F-actin cytoskeletal assembly and cytoarchitec-ture in intestinal cells. It is also required for EGF-inducednormalization of Ca2� homeostasis against oxidant-inducedinjury in enterocytes. The PKC-�1 isoform also appears to bea critical stabilizer of the dynamic alterations in polymerized(F-) actin and monomeric (G-) actin cytoskeletons in theintestinal epithelium. To our knowledge, this is the firstdemonstration of this novel mechanism for the protection ofCa2� balance and actin integrity in GI cells. The followingindependent lines of evidence support and elaborate on theseconclusions.

First, preincubation of naive intestinal monolayers withhigh doses of EGF activates PKC-�1 and evokes a cascade ofalterations that are consistent with the proposed mechanism.EGF activates a specific PKC isoform, �1, increases Ca2�

efflux, normalizes cytosolic Ca2�, increases the size of thepolymerized F-actin pool while reducing the size of the mo-

Fig. 12. Intracellular Ca2� alterations assessed by the Ca2�-sensitive probe Fluo-3-AM in PKC-�1-over-expressing cells following treatments withEGF or OAG (A). Cells preloaded with Fluo-3-AM were treated under similar conditions to those in Fig. 6. In other experiments (B), cells werepretreated with known membrane “pump” Ca2�-ATPase inhibitors (vanadate or quercetine) and subsequently incubated with H2O2 in the presenceor absence of pretreatment with EGF or OAG. A, in transfected (T) cells that were exposed to oxidants, PKC-�1 over-expression synergizes with thelow dose of EGF (1 ng/ml) or OAG (0.01 �M) to markedly and significantly normalize intracellular Ca2�; this did not occur in naive (N) cells (notover-expressing �1). Only high doses of EGF (10 ng/ml) or OAG (50 �M) normalize intracellular Ca2� in naive monolayers. B, inhibitors of membraneCa2�-ATPase prevent the normalization of intracellular calcium. �, p � 0.05 versus vehicle. �, p � 0.05 versus H2O2. &, p � 0.05 versus the low doseof EGF or OAG � H2O2 in naive cells. #, p � 0.05 versus the corresponding EGF or OAG � H2O2 in naive or transfected cells.

862 Banan et al.

nomeric G-actin pool, and increases the number of cells dis-playing a normal actin ring at the apical plane of monolayers(an area known to regulate intestinal paracellular perme-ation; Banan et al., 2000c, 2001a,e).

Second, these protective effects of EGF on the actin aremimicked by known PKC activators but not by a biologicallyinactive phorbol ester. Third, the protective effects of EGF orPKC activators on the actin are prevented by specific PKCinhibitors. Fourth, transfected cells that over-expressPKC-�1 are severalfold more sensitive to protection of theF-actin ring structure by EGF or the PKC activator OAG.Indeed, in these stably transfected cells, PKC-�1 over-ex-pression synergized with the exogenously added EGF or OAGto increase the stability of F-actin, to decrease the unstableG-actin, to protect a high percentage of cells with normal-appearing cortical actin ring, and to maintain cytosolic Ca2�

at nearly normal levels via increases in Ca2� efflux. Theenhanced sensitivity to EGF in transfected Caco-2 cells ap-pears to require not only over-expression, which is not pro-tective by itself, but also enhanced activation of PKC-�1.Fifth, antisense to PKC-�1, which causes under-expression ofPKC-�1 at only 10% of normal levels, almost completelyabrogated all the various protective effects of EGF and PKCactivator.

Finally, quantitative considerations such as correlationsbetween several outcome measures further support our con-clusions. Our previous work in naive intestinal cells showed

significant correlations between protection of the integrity ofmonolayer barrier permeability and actin ring stability (Ba-nan et al., 2000c, 2001a,e) and between the integrity of theintestinal barrier and enhanced general PKC activation (Ba-nan et al., 2001b). In the current study, we show correlationsbetween protection against actin ring disruption and in-creased PKC-�1 isoform activation (i.e., membrane associa-tion) (r � 0.93, p � 0.05) as well as between protectionagainst actin disassembly (increase in G-actin) and enhancedPKC-�1 activation (r � 0.91, p � 0.05), and between protec-tion of actin assembly (increase in F-actin) and PKC-�1 ac-tivation (r � 0.92, p � 0.05).

In our previous investigations using the same transfectedand naive monolayer models (e.g., Banan et al., 2000a,c,2001a,c, 2002), we utilized several membrane-impermeablefluorescent probes such as fluorescein sulfonic acid (FSA;0.478 kDa) for assessing intestinal barrier permeability(leakiness). Our findings showed that first there is a para-cellular permeation of permeability probes in Caco-2 mono-layers following exposure to oxidants. Second, there is aninverse relationship between the probe size and leakiness.Third, PKC-�1 activation prevents the passage of these smallpermeability probes (e.g., FSA) through the paracellularroute, indicating protection/maintenance of monolayer bar-rier integrity. We also showed significant correlations be-tween protection of the integrity of the monolayer barrier(FSA clearance) and actin ring stability (Banan et al., 2000c,2001a,e) and between the integrity of the intestinal barrierand general PKC activity (Banan et al., 2001b,c), and now inthe present study between protection of the actin ring andincreased �1 isoform activation.

Our present and previous findings are consistent with amodel in which enhanced translocation and activation ofPKC-�1 results in increased polymerization of F-actin andconcomitant reduction in G-actin pools and subsequentlyleads to increased stability of the actin architecture and themonolayer barrier. Furthermore, protection against oxidant-induced rise in [Ca2�]i and increased PKC-�1 activation (r �0.91, p � 0.05) and increase in Ca2� efflux and enhancedPKC-�1 activation (r � 0.89, p � 0.05) provide other robustcorrelations in the current report. The high strength of thesecorrelations indicates that increased PKC-�1 activation isapparently essential to the protective effects of EGF (and thePKC activator OAG) on calcium balance and actin. In thisview, enhanced activation of PKC-�1 leads to the normaliza-tion of [Ca2�]i and the protection of F-actin. Other PKCisoforms also appear to be protective, as we have recentlydiscovered (Banan et al., 2002), and these other isoforms maymodulate additional signaling mechanisms.

Our findings using targeted molecular interventions areconsistent with previous pharmacological reports (Boner etal., 1992; Saxon et al., 1994; McKenna et al., 1995; Birkenfeldet al., 1996; Wang et al., 1996), including our own (Banan etal., 2001b), in which general PKC activation (translocation)was shown to be necessary for the observed effects of PKC.An immunofluorescent study in non-GI cells such as fibro-blasts showed that OAG (a synthetic version of diacylglyc-erol) causes the activation of a constitutively expressedPKC-�1 isoform (Goodnight et al., 1995). Our current find-ings on the �1 isoform of PKC utilizing more specific andtargeted molecular approaches further expand on these pre-vious pharmacological reports and, we believe, now establish

Fig. 13. Antisense (AS) inhibition of PKC-�1 isoform substantially atten-uates the normalization of intracellular Ca2� by high doses of EGF (10ng/ml) or by PKC activator (OAG, 50 �M). Treatment conditions were asin Fig. 10. �, p � 0.05 versus vehicle. �, p � 0.05 versus H2O2. &, p � 0.05versus high doses of EGF or OAG � H2O2 in naive (N) cells.

PKC-�1 Isoform Protects F-Actin Assembly 863

a novel biologic function—protection of Ca2� balance andactin—among the classical isoforms of PKC. Furthermore,we have more recently identified activation of EGF-receptortyrosine kinase and then phospholipase C-�1 as the up-stream signal for EGF-induced, PKC-mediated, protection ofintestinal barrier and cytoskeletal integrity (Banan et al.,2001d).

Despite the fundamental importance of PKC signal trans-duction in protection as our previous and current studiesindicate, the role of the PKC-�1 isoform in cell function,especially in epithelial cells, has remained poorly under-stood. Although PKC in general has been implicated in reor-ganization of the cytoskeleton (Hartwig et al., 1992; Good-night et al., 1995; Babich et al., 1997; Chun et al., 1997; He etal., 1997), it is not known which PKC isoforms are importantin this process. Our study indicates that PKC-�1 is one suchisoform. The mechanism through which PKC-�1 isoform pro-tects the actin is unclear. Studies in chromaffin cells suggestthat one possible target of PKC might be MARCKS (myris-toylated, alanine-rich PKC substrate), which has been pro-posed to rearrange the actin cytoskeleton (Hartwig et al.,1992). Moreover, PKC has been suggested to be a MARCKSkinase. Alternatively, PKC-�1 may directly phosphorylatethe actin molecule itself, leading to dynamic redistribution ofactin cortex. It should be noted that other proteins can also be

involved in maintaining the integrity of an epithelial barrierin the GI tract, including microtubules (Banan et al., 1999,2000a), E-cadherin, adherin, connexin 43, �-catenin, EGF-receptor, and integrins, and some of these proteins (e.g.,microtubules) are also phosphorylated by PKC, includingPKC-�1 (e.g., Banan et al., 2001c). It remains to be seenwhether there are additional molecular mechanisms under-lying the interactions between PKC-�1 and F-actin in GIepithelial cells.

Utilizing a pharmacological approach, we previouslyshowed in naive intestinal cells that known PKC activators(e.g., OAG) attenuate oxidant-induced increases in cytoso-lic Ca2� through stimulation of Ca2� efflux, leading to thenormalization of intracellular Ca2� (Banan et al., 2001b).The nature of the PKC isoform responsible for this protec-tive phenomenon remained elusive until the current re-port. The effects of the �1 isoform of PKC on cellular Ca2�

trafficking as reported herein are potentially importantbecause we and others have shown that the cytoskeleton isexquisitely sensitive to changes in intracellular Ca2� lev-els and that it can be extensively injured by oxidant-induced increases in intracellular Ca2�, leading to mono-layer barrier hyperpermeability (Kakiuchi and Sobue,1981; Babich et al., 1997; Banan et al., 2001b). Our studiesusing Ca2�-ATPase pump inhibitors show the inhibition of

Fig. 14. A, 45Ca2� efflux from Caco-2 monolayers over-expressing PKC-�1 in the presence of a low concentration of EGF or PKC activator (OAG).PKC-�1 over-expression synergizes with a low dose of EGF or OAG to result in an enhancement of Ca2� efflux from transfected monolayers. In naive(N) cells, in contrast, these same low doses of EGF or OAG did not cause Ca2� efflux. In these naive cells higher doses of EGF or OAG were requiredto increase Ca2� efflux. B, prevention of calcium efflux by known inhibitors of Ca2�-ATPase pump (vanadate or quercetine). �, p � 0.05 versus vehicle.�, p � 0.05 versus H2O2. &, p � 0.05 versus the low dose of EGF or OAG � H2O2 in naive cells. #, p � 0.05 versus the corresponding EGF or OAG� H2O2 in naive or transfected (T) cells.

864 Banan et al.

EGF-induced, PKC-�1-mediated, enhancement of Ca2� ef-flux and subsequent attenuation of [Ca2�]i. It is possible,therefore, that alterations of Ca2�-ATPase by increasedPKC activation, either directly or indirectly, activate Ca2�

efflux, which is then responsible for the return of [Ca2�]i tonormal levels. For example, direct phosphorylation of theCa2�-ATPase via the pharmacological manipulation ofPKC was suggested by two recent studies in non-GI (non-epithelial) cells such as HL-60 leukemia cells and neutro-phils (Enyedi et al., 1997; Verma et al., 1999). Consistentwith our current report in epithelial cells, in neutrophilsand in HL-60 cells, ligand-initiated (e.g., fMLP) signalingelicits a PKC-induced down-regulation of the rise in[Ca2�]i (Enyedi et al., 1997; Verma et al., 1999). Studiesare under way in our laboratory to determine to whatextent PKC isoform-activated protection of calcium bal-ance is mediated by effects on membrane Ca2�-ATPases.

In summary, our findings demonstrate for the first timethat the ability of growth factors to protect the actin cytoskel-eton and maintain normal intracellular calcium homeostasisin intestinal cell monolayers is substantially dependent onincreased activation of the PKC-�1 isoform. This new knowl-edge may prove useful because increasing the activity ofprotective PKC isoforms through activation of endogenousPKC or using PKC mimetics may lead to unique therapeuticstrategies for the treatment of a wide variety of oxidant-

induced inflammatory disorders of the GI tract, includinginflammatory bowel disease.

ReferencesBabich M, Foti LR, and Mathias KL (1997) Protein kinase C modulator effects on

parathyroid hormone-induced intracellular calcium and morphologic changes inUMR 106-H5 osteoblastic cells. J Cell Biochem 65:276–285.

Banan A, Choudhary S, Zhang Y, Fields JZ, and Keshavarzian A (1999) Ethanol-induced barrier dysfunction and its prevention by growth factors in human intes-tinal monolayers: evidence for oxidative and cytoskeletal mechanisms. J Pharma-col Exp Ther 291:1075–1085.

Banan A, Choudhary S, Zhang Y, Fields JZ, and Keshavarzian A (2000a) Role of themicrotubule cytoskeleton in protection by epidermal growth factor and transform-ing growth factor-� against oxidant-induced barrier disruption in a human coloniccell line. Free Radic Biol Med 28:727–738.

Banan A, Fields JZ, Talmage DA, Zhang L, and Keshavarzian A (2002) The atypicalPKC-Zeta (�) isoform protects the human intestinal monolayer barrier integrity.Am J Physiol, in press.

Banan A, Fields JZ, Zhang Y, and Keshavarzian A (2000b) Nitric oxide and itsmetabolites mediate ethanol-induced microtubule disruption and intestinal bar-rier dysfunction. J Pharmacol Exp Ther 294: 997–1008.

Banan A, Fields JZ, Zhang Y, and Keshavarzian A (2001a) iNOS upregulationmediates oxidant-induced disruption of F-actin and the permeability barrier ofintestinal monolayers. Am J Physiol 280:G1234–G1246.

Banan A, Fields JZ, Zhang Y, and Keshavarzian A (2001b) Key role of generalprotein kinase C activation and calcium in EGF-induced protection of the micro-tubule cytoskeleton and intestinal epithelial barrier against oxidant injury. Am JPhysiol 280:G828–G843.

Banan A, Fields JZ, Zhang Y, and Keshavarzian A (2001c) The �1 isoform of proteinkinase C mediates epidermal growth factor protection of the barrier integrity ofintestinal monolayers against oxidants. Am J Physiol 281:G833–G847.

Banan A, Fields JZ, Zhang Y, and Keshavarzian A (2001d) Targeted molecularinhibition of phospholipase C-� prevents EGF-mediated protection of the microtu-bule cytoskeleton and intestinal epithelial barrier function against oxidant injury.Am J Physiol 281:G412–G423.

Banan A, Fitzpatrick L, Zhang Y, and Keshavarzian A (2001e) OPC-compoundsprevent oxidant-induced carbonylation and depolymerization of the F-actin cy-toskeleton and intestinal barrier hyperpermeability. Free Radic Biol Med 30:287–298.

Banan A, McCormack SA, and Johnson LR (1998a) Polyamines are required formicrotubule formation during mucosal ulcer healing. Am J Physiol 274:G879–G885.

Banan A, Smith GS, Rickenberg C, Kokoska ER, and Miller TA (1998b) Protectionagainst ethanol injury by prostaglandins in a human intestinal cell line: role ofmicrotubules. Am J Physiol 274:G111–G121.

Banan A, Wang J-Y, McCormack SA, and Johnson LR (1996) Relationship betweenpolyamines, actin distribution, and gastric mucosal ulcer healing in rats. Am JPhysiol 34:G893–G903.

Banan A, Zhang Y, Losurdo J, and Keshavarzian A (2000c) Carbonylation anddisassembly of the F-actin in oxidant-induced barrier dysfunction and its preven-tion by epidermal growth factor and transforming growth factor-� in a humanintestinal cell line. Gut 46:830–837.

Birkenfeld PA, McIntyre BS, Biriski KP, and Sylvester PW (1996) Role of proteinkinase C in modulating epidermal growth factor- and phorbol ester-induced mam-mary epithelial cell growth in vitro. Exp Cell Res 233:183–191.

Boner C, Guadagno SN, Fabbro D, and Weinstein IB (1992) Expression of fourprotein kinase C isoforms in rat fibroblasts. J Biol Chem 267:12892–12899.

Bradford MA (1976) A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of dye binding. Anal Biochem 72:224–254.

Cho Y, Klein MG, and Talmage DA (1998) Distinct functions of protein kinaseC-alpha and protein kinase C-beta during retinoic acid-induced differentiation ofF9 cells. Cell Growth Differ 9:147–154.

Chun J, Auer KA, and Jacobson BS (1997) Arachidonate initiated protein kinase Cactivation regulates HeLa cell spreading on a gelatin substrate by inducing F-actinformation and exocytotic upregulation of beta 1 integrin. J Cell Physiol 173:361–370.

Enyedi A, Elweiss NL, Filoteo AG, Verma AK, Paszty K, and Penniston JT (1997)Protein kinase C phosphorylates the “a” forms of plasma membrane Ca2� pumpisoforms 2 and 3 and prevents binding of calmodulin. J Biol Chem 272:27525–27528.

Gilbert T, Le Bivic A, Quaroni A, and Rodreguez-Boulan E (1991) Microtubuleorganization and its involvement in the biogenic pathways of plasma membraneproteins in Caco-2 intestinal epithelial cells. J Cell Biol 133:275–288.

Goodnight J, Mischak H, Kolch W, and Mushinski JF (1995) Immunocytochemicallocalization of eight PKC isoenzymes over-expressed in NIH 3T3 fibroblasts. J BiolChem 270:9991–10001.

Harter JL (1960) Critical values for Dunnett’s new multiple range test. Biometrics16:671–685.

Hartwig JH, Thelen M, Rosen A, Janmey PA, Nairn AC, and Aderem A (1992)MARCKS is an actin filament crosslinking protein regulated by protein kinase Cand calcium-calmodulin. Nature (Lond) 356:618–622.

He Q, Dent EW, and Meiri KF (1997) Modulation of actin filament behavior byGAP-43 (neuromodulin) is dependent on the phosphorylation status of serine 41,the protein kinase C site. J Neurosci 17:3515–3524.

Hollander D (1998) Crohn’s disease—a permeability disorder of the tight junction?Gut 26:1621–1624.

Kakiuchi S and Sobue K (1981) Ca2� and calmodulin-dependent flip-flop mechanismin cytoskeletal assembly-disassembly. FEBS Lett 132:141–143.

Fig. 15. Under-expressing PKC-�1 prevents 45Ca2� efflux from monolay-ers incubated with high doses of EGF or PKC activator (OAG). Conditionswere similar to those in Fig. 13. �, p � 0.05 versus vehicle. �, p � 0.05versus H2O2. &, p � 0.05 versus high doses of EGF or OAG � H2O2 innaive (N) cells. (AS), antisense inhibition of PKC-�1.

PKC-�1 Isoform Protects F-Actin Assembly 865

Keshavarzian A, Holmes EW, Patel M, Iber F, and Pethkar S (1999) Leaky gut inalcoholic cirrhosis: a possible mechanism for alcohol induced liver damage. Am JGastroenterol 94:200–207.

Keshavarzian A, Sedghi S, Kanofsky J, List T, Robinson C, Ibrahim C, and WinshipD (1992) Excessive production of reactive oxygen metabolites by inflamed colon:analysis by chemiluminescence probe. Gastroenterology 103:177–185.

Kimura H, Hokari R, Miura S, Shigematsu T, Hirokawa M, Akiba Y, Kurose I,Higuchi H, Fujimori H, Tsuzuki Y, et al. (1998) Increased expression of an induc-ible isoform of nitric oxide synthase and the formation of peroxynitrite in colonicmucosa of patients with active ulcerative colitis. Gut 42:180–187.

Kokoska ER, Smith GS, Banan A, and Miller TA (1998) The role of calcium inadaptive cytoprotection and cell injury induced by deoxycholate in a human gastriccell line. Am J Physiol 275:G322–G330.

McKenizie SJ, Baker MS, Buffington GD, and Doe WF (1996) Evidence for oxidant-induced injury to epithelial cells during inflammatory bowel disease. J Clin Invest98:136–141.

McKenna JP, Williams JM, and Hanson PJ (1995) Protein kinase C inhibits hista-mine-stimulated adenylate cyclase activity in a particulate fraction of the humangastric cancer cell line HGT-1. Inflamm Res 44: 66–69.

Menconi MJ, Salzman AL, Unno N, Ezzell RB, Casey DM, Brown DA, Tsuji Y, andFink MP (1997) Acidosis induces hyperpermeability in Caco-2BBe cultured intes-tinal epithelial monolayers. Am J Physiol 272:G1007–G1021.

Meunier VM, Bourrie Y, Berger Y, and Fabre G (1995) The human intestinal

epithelial cell line Caco-2: pharmacological and pharmacokinetics applications.Cell Biol Toxicol 11:187–194.

Saxon ML, Zho X, and Black JD (1994) Activation of protein Kinase C is associatedwith post-mitotic events in intestinal epithelial cells in situ. J Cell Biol 126:747–763.

Unno N, Menconi MJ, Salzman AL, and Fink MP (1996) Hyperpermeability and ATPdepletion induced by chronic hypoxia or glycolytic inhibition in Caco-2BBe mono-layers. Am J Physiol 270:G1010–G1021.

Vandenberghe PA and Ceuppens JL (1990) Flow cytometric measurement of cyto-plasmic free calcium in human peripheral blood T lymphocyte with Fluo-3, a newfluorescent calcium indicator. J Immunol Methods 127:197–205.

Verma AK, Paszty K, Filoteo AG, Penniston JT, and Enyedi A (1999) Protein kinaseC phosphorylates plasma membrane Ca2� pump isoform 4a. J Biol Chem 274:527–532.

Wang L, Wilson EJ, Osburn J, and DelValle J (1996) Epidermal growth factorinhibits carbachol stimulated canine parietal cell function via protein kinase C.Gastroenterology 110:469–477.

Address correspondence to: Dr. Ali Banan, Rush University Medical Cen-ter, Division of Digestive Diseases, 1725 W. Harrison, Suite 206, Chicago, IL60612. E-mail: ali_banan@rush.edu

866 Banan et al.