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Originally published In Press as doi:10.1074/jbc.M709141200 on November 8, 2007

J. Biol. Chem., Vol. 283, Issue 6, 3574-3583, February 8, 2008
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Role of Phospholipase C{gamma}-induced Activation of Protein Kinase C{epsilon} (PKC{epsilon}) and PKCβI in Epidermal Growth Factor-mediated Protection of Tight Junctions from Acetaldehyde in Caco-2 Cell Monolayers*

Takuya Suzuki, Ankur Seth, and Radhakrishna Rao1

From the Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Received for publication, November 7, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidermal growth factor (EGF) protects the intestinal epithelial tight junctions from acetaldehyde-induced insult. The role of phospholipase C{gamma} (PLC{gamma}) and protein kinase C (PKC) isoforms in the mechanism of EGF-mediated protection of tight junction from acetaldehyde was evaluated in Caco-2 cell monolayers. EGF-mediated prevention of acetaldehyde-induced decrease in transepithelial electrical resistance and an increase in inulin permeability, and subcellular redistribution of occludin and ZO-1 was attenuated by reduced expression of PLC{gamma}1 by short hairpin RNA. EGF induced a rapid activation of PLC{gamma}1 and PLC-dependent membrane translocation of PKC{epsilon} and PKCβI. Inhibition of PKC activity or selective interference of membrane translocation of PKC{epsilon} and PKCβI by RACK interference peptides attenuated EGF-mediated prevention of acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. BAPTA-AM and thapsigargin blocked EGF-induced membrane translocation of PKCβI and attenuated EGF-mediated prevention of acetaldehyde-induced disruption of tight junctions. EGF-induced translocation of PKC{epsilon} and PKCβI was associated with organization of F-actin near the perijunctional region. This study shows that PLC{gamma}-mediated activation of PKC{epsilon} and PKCβI and intracellular calcium is involved in EGF-mediated protection of tight junctions from acetaldehyde-induced insult.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tight junction (TJ)2 forms a physical barrier to the diffusion of macromolecules across the epithelial monolayer (1). TJ is assembled by the organization of a variety of proteins, including the transmembrane proteins such as occludin (2), claudins (3), and junctional adhesion molecule (4), and the intracellular plaque proteins such as zonula occludens (ZO-1, ZO-2, and ZO-3) (5-7), cingulin (8), and 7H6 (9). A significant body of evidence indicates that intracellular signaling elements such as protein kinases (10-17), protein phosphatases (18, 19), phosphatidylinositol 3-kinase (17), and GTPase switch proteins (20-22) regulate the integrity of TJ in different epithelia. TJ is disrupted by various injurious factors such as reactive oxygen species (11, 12, 14, 15, 17), toxins (23), cytokines (24, 25), and pathogens (26), and this disruption of TJ appears to play a crucial role in the pathogenesis of a wide spectrum of gastrointestinal (27), pulmonary (28), and renal (29) diseases.

Acetaldehyde, a metabolic product of ethanol oxidation, is highly toxic and carcinogenic (30). Acetaldehyde level has been measured as high as 0.4 mM in the saliva of alcoholics (31), and the plasma acetaldehyde level can be elevated by therapeutic and dietary components (32). The highest level of acetaldehyde was detected in the colonic lumen. The high level of alcohol dehydrogenase and a low level of aldehyde dehydrogenase in enteric bacteria contribute to an accumulation of acetaldehyde in the colonic lumen (33). It is difficult to determine the level of acetaldehyde in biological fluids without the risk of underestimating it because of the volatile property of acetaldehyde. Despite this setback, acetaldehyde concentration in rat colonic lumen after ethanol administration was found to be in millimolar concentrations (33). Previous studies have shown that acetaldehyde (100-500 µM) disrupts TJ and increases paracellular permeability in Caco-2 cell monolayer by a tyrosine kinase-dependent mechanism (13, 34-36). Acetaldehyde induces Tyr phosphorylation of ZO-1, E-cadherin, and β-catenin and dissociates these proteins from the actin-rich detergent-insoluble fractions (34, 37).

Epidermal growth factor (EGF), a gastrointestinal mucosal protective factor, is secreted in saliva and other gastrointestinal secretions (38). EGF protects the gastrointestinal epithelium from a variety of insults (11, 39-42). A recent study demonstrated that EGF prevents acetaldehyde-induced disruption of TJ in the Caco-2 cell monolayer (35). In the present study, we investigated the signaling mechanism involved in this epithelial protective effect of EGF against acetaldehyde. The roles of PLC{gamma}1 and PKC isoforms in the EGF-mediated protection of TJ were determined. The results show that 1) PLC{gamma}1 is required for the EGF-mediated protection of TJ from acetaldehyde in Caco-2 cell monolayers, 2) EGF induces membrane translocation of PKC{epsilon} and PKCβI by a PLC{gamma}-dependent mechanism and the membrane translocation of PKC{epsilon} and PKCβI is required for the EGF-mediated protection of TJ from acetaldehyde, 3) intracellular calcium mediates PLC{gamma}-mediated membrane translocation of PKCβI and EGF-mediated protection of TJ from acetaldehyde, 4) EGF induces organization of F-actin near the intercellular junctions in cells expressing GFP-PKC{epsilon} and GFP-PKCβI, and 5) neither PKC{epsilon} nor PKCβI directly interacts with occludin.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Cell culture reagents and supplies, G418, Lipofectamine®-LTX, and Plus reagent were purchased from Invitrogen. Fluorescein isothiocyanate (FITC)-inulin, GSH (glutathione), leupeptin, aprotinin, bestatin, pepstatin A, phenylmethylsulfonyl fluoride, GSH-agarose, Triton X-100, vanadate, BAPTA-AM, EGF, FITC-inulin, acetaldehyde, AG1478, and protein-A Sepharose were purchased from Sigma. Streptavidin-agarose was obtained from Pierce Biotechnology, Inc. (Rockford, IL). Ro-32-0432 and thapsigargin were purchased from EMD Chemicals (San Diego, CA). All other chemicals were of analytical grade and purchased either from Sigma or Fisher Scientific (Tustin, CA). AlexaFlour-543-conjugated phalloidin was purchased from Molecular Probes (Eugene, OR). pEGFP vectors with or without PKC{epsilon} and PKCβI inserts were purchased from Clontech (Mountain View, CA).

Recombinant Proteins—Recombinant GST-occludin-C was produced in the laboratory in Escherichia coli BL21DE cells and purified using GSH-agarose as described before (43). cDNA for the C-terminal tail of human occludin (amino acids 378-522) was amplified using the cDNA clone for human occludin (kind gift from Dr. Van Italie, University of North Carolina, Chapel Hill, NC) and inserted into pGEX2T vector. Recombinant, catalytically active, His-tagged PKC{epsilon} and PKCβI produced in Sf9 cells were purchased from Upstate Biotech Inc. (Charlottesville, VA).

Antibodies—Biotin-conjugated anti-p-Tyr, mouse monoclonal anti-PKC{alpha}, anti-PKC{delta}, anti-PKC{gamma}, and HRP-conjugated anti-GST antibodies were purchased from BD Transduction Laboratories (San Jose, CA). Mouse monoclonal anti-occludin, rabbit polyclonal anti-ZO-1, HRP-conjugated anti-occludin, rabbit polyclonal anti-ERK1/2 and rabbit polyclonal anti-p-Thr antibodies were purchased from Zymed Laboratories Inc. Laboratories (San Francisco, CA). AlexaFluor 488-conjugated anti-mouse IgG and anti-rabbit IgG antibody were obtained from Molecular Probes (Eugene, OR). Cy3-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG, anti-β-actin, rabbit polyclonal anti-PLC{gamma} and HRP-conjugated anti-rabbit IgG antibodies purchased from Sigma. Mouse monoclonal anti-PKCβI antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal anti-PKC{epsilon} and anti-p-PLC{gamma} antibodies were purchased from Upstate Biotech Inc. Mouse monoclonal anti-EGF receptor (EGFR) antibodies were purchased from Takara Bio Inc. (Shiga, Japan). Rabbit polyclonal anti-EGFR (pY1173), anti-GFP, anti-EGFR antibodies, and mouse monoclonal anti-phospho ERK1/2 antibodies were purchased from Invitrogen.

PKC-RACK Interference Peptides—Membrane translocation of PKC is mediated by an isoform-specific interaction with different RACK proteins (44). The specificity of this interaction is determined by the peptide sequence of RACK binding domain of PKC isoforms. Synthetic peptides corresponding to the sequence of RACK binding domains disrupt the interaction between PKC and RACK in an isoform-specific manner. Antennapedia and antennapedia-conjugated PKC-RACK interference peptides specific for PKC{alpha}, PKC{gamma}, PKCβI, and PKC{epsilon} were kind gifts from Dr. Daria Mochly-Rosen (Stanford University). The sequences and their specificity have been described before by Dr. Mochly-Rosen (44).

shRNA for PLC{gamma}—A vector-based short hairpin RNA (shRNA) method was used to silence gene expression of human PLC{gamma}1. Two targeting sequences were chosen against the nucleotide sequence of human PLC{gamma}1 gene (GenBankTM M34667 [GenBank] ) using the Dharmacon web site (Target1: GAATCGTGAGGATCGTATA (nucleotide position, 498-516); Target2: AGAAGTTCCTTCAGTACAA (2993-3011)). The sequences were further verified by BLAST search on the known human genome database, and no matches were found other than PLC{gamma}1, confirming the uniqueness of these sequences. To construct the shRNA vectors, two pairs of oligonucleotides containing the antisense sequence, a hairpin loop region (TTGATATCCG), and the sense sequence with cohesive BamHI and HindIII sites were synthesized (Sigma Genosys, St Louis, MO) as follows: Top strand1, 5'-GATCCCGTATACGATCCTCACGATTCTTGATATCCGGAATCGTGAGGATCGTATATTTTTTCCAAA-3' and bottom strand1, 5'-AGCTTTTGGAAAAAATATACGATCCTCACGATTCCGGATATCAAGAATCGTGAGGATCGTATACGG-3'; top strand2, 5'-GATCCCGTTGTACTGAAGGAACTTCTTTGATATCCGAGAAGTTCCTTCAGTACAATTTTTTCCAAA-3' and bottom strand2, 5'-AGCTTTTGGAAAAAATTGTACTGAAGGAACTTCTCGGATATCAAAGAAGTTCCTTCAGTACAACGG-3'. The top and bottom strands were annealed and cloned into BamHI and HindIII sites of the pRNAtin-H1.2 vector (GenScript Corp., Piscataway, NJ) (pR vector), which induces expression of shRNA by H1.2 promoter and cGFP protein by cytomegalovirus promoter. Successful insertion of the shRNA constructs into the vector was confirmed by releasing the oligonucleotides by digesting with BamHI and HindIII and sequencing. For a control, a mutant form of shRNA1 was designed by replacing nucleotides 39 (adenine) and 42 (guanine) with cytosine and adenine, respectively.

Cell Culture—Caco-2 cells purchased from American Type Cell Collection (Rockville, MD) were grown under standard cell culture conditions as described before (15). Cells were grown on polyester membranes in Transwell inserts (6.5 mm, 12 mm or 24 mm, Costar, Cambridge, MA), and experiments were conducted on days 11-13 (6.5- or 12-mm Transwells) or days 17-19 (24-mm Transwell) post-seeding.

Treatment with Acetaldehyde and Inhibitors—Acetaldehyde was administered by exposing cell monolayers in PBS (Dulbecco's saline containing 1.2 mM CaCl2, 1 mM MgCl2, and 0.6% bovine serum albumin) to vapor phase acetaldehyde as described previously (13) to achieve acetaldehyde concentration of 500 µM in the buffer bathing the cell monolayer. EGF and inhibitors were administered to the apical and basal compartments. Cell monolayers were incubated with 10 or 20 µM BAPTA-AM, 1 or 2 µM thapsigargin, 3 µM AG1478, 1 µM Ro-32-0432, and 1 µM chelerythrine 50 min prior to EGF administration. RACK interference peptides (0.3-3.0 µg/ml) were administered to apical wells 20 min prior to EGF administration. EGF (1-60 nM) was administered to both apical and basal wells 10 min prior to acetaldehyde treatment.


Figure 1
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  		FIGURE 1.
EGF induces rapid translocation and activation of PLC{gamma}1. A, Caco-2 cell monolayers were incubated with 30 nM EGF for varying times. Whole cell extract was immunoblotted for p-PLC{gamma}1 and PLC{gamma}1. B, Caco-2 cell monolayers were incubated with varying concentrations of EGF for 5 min. Whole cell extract was immunoblotted for p-PLC{gamma}1 and PLC{gamma}1. C, Caco-2 cell monolayers were incubated with 30 nM EGF for varying times. Membrane and soluble fractions were prepared and immunoblotted for p-PLC{gamma}1 and PLC{gamma}1. D, Caco-2 cells were incubated with 30 nM EGF for varying times with or without AG1478 (3 µM) pretreatment for 1 h. Whole cell extracts were immunoblotted for p-PLC{gamma}1 and PLC{gamma}1. E, Caco-2 cell monolayers were incubated with or without 3 µM AG1478 for 1 h followed by incubation with or without 30 nM EGF for varying times. EGFR was immunoprecipitated from native protein extracts and immunoblotted for p-PLC{gamma}1 and EGFR. F, Caco-2 cell monolayers were incubated with 30 nM EGF for 10 min in cells pretreated with or without acetaldehyde for 30 min, or EGF was co-administered with acetaldehyde. Whole cell extract was immunoblotted for p-PLC{gamma}1 and PLC{gamma}1.

 
Transfection of Expression Constructs—Caco-2 cells (125,000 cells/well) were seeded on 6-well plates a day before transfection. The cells were transfected using 1 ml of antibiotics-free Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1 µg of DNA plasmid (Empty vector, shRNA1, or shRNA2), 1 µl of Plus reagent, and 3 µl of Lipofectamine-LTX for each well. After 20 h, the cell monolayers were trypsinized and seeded on 100-mm plates. The cells were subjected to G-418 selection (0.7 mg/ml) for 2 weeks. Resistant cells were sorted to obtain only GFP-expressing cells by fluorescence-activated cell sorting. Cells were maintained in the medium supplemented with 0.4 mg/ml G418. Reduction in PLC{gamma}1 protein expression was verified by immunoblot analysis. Similarly, pEGFP-PKCβI and pEGFP-PKC{epsilon} were transfected to Caco-2 cells. Transiently transfected cells were fixed and double labeled for GFP by immunofluorescence staining and for F-actin using AlexaFluor-543-conjugated phalloidin.

Measurement of Transepithelial Electrical Resistance—TER was measured as described previously (15) using a Millicell-ERS Electrical Resistance System (Millipore, Bedford, MA). TER was calculated as ohms ({Omega})·cm2 by multiplying it with the surface area of the monolayer. The resistance of the polyester membrane in Transwells (~30 {Omega}·cm2) was subtracted from all readings.

Unidirectional Flux of Inulin—Transwells with the cell monolayers were incubated under different experimental conditions in the presence of FITC-inulin (0.5 mg/ml) in the basal well. At varying times during the experiment, 100 µl of apical and basal media was withdrawn and the fluorescence was measured using a fluorescence plate reader (BioTEK Instruments, Winooski, VT). The flux into the apical well was calculated as the percentage of total fluorescence administered into the basal well per hour per cm2 surface area.

Immunofluorescence Microscopy—Cell monolayers (12 mm) were washed with PBS and fixed in acetone:methanol (1:1) at 0 °C for 5 min. Cell monolayers were blocked in 3% nonfat milk in TBST (20 mM Tris, pH 8.0, containing 150 mM NaCl and 0.5% Tween 20) and incubated for 1 h with primary antibodies; rabbit polyclonal anti-ZO-1 and mouse monoclonal anti-occludin, followed by incubation for 1 h with secondary antibodies, goat AlexaFluor 488-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG antibodies. The fluorescence was visualized using a Zeiss LSM 5 Laser Scanning Confocal Microscope, and images from Z-series sections (1 µm) were collected by using Zeiss LSM 5 Pascal Confocal Microscopy Software (Release 3.2). Images were stacked using the software, Image J (NIH) and processed by Adobe Photoshop (Adobe Systems Inc., San Jose, CA).

Preparation of Membrane Fraction—Caco-2 cell monolayers (24-mm Transwells) were washed twice with ice-cold PBS and once with lysis buffer-F (PBS containing 10 mM β-glycerophosphate, 2 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml bestatin, 10 µg/ml pepstatin-A, and 1 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride). Cells were scraped and homogenized as described before (15). The membrane pellet was suspended in 50 µl of lysis buffer D (0.3% SDS in 10 mM Tris, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride). Protein was measured by BCA method (Pierce). The membrane fraction was mixed with an equal volume of Laemmli sample buffer (2x concentrated) and heated at 100 °C for 5 min.

Immunoprecipitation—Caco-2 cell monolayers (24-mm Transwells) were washed with ice-cold PBS, and proteins were extracted in immunoprecipitation buffer (50 mM Tris buffer, pH 7.4, containing 1% Nonidet P-40, 2 mM EDTA, 2 mM EGTA, 10 mM sodium fluoride, 1 mM vanadate, and protease inhibitor mixture as described above). Protein extracts (1.0 mg of protein/ml) were incubated with 2 µg of anti-occludin or anti-EGFR antibodies at 4 °C for 16 h. Immunocomplexes were isolated by precipitation using protein-A Sepharose (for 1 h at 4 °C).


Figure 2
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  		FIGURE 2.
Reduced expression of PLC{gamma}1 attenuates EGF-mediated prevention of acetaldehyde-induced disruption of barrier function. A, Caco-2 cells were transfected with two different shRNAs to PLC{gamma}1 or a control shRNA as described under "Experimental Procedures." Protein extracts from cells transfected with vector, shRNA1, or shRNA2 were immunoblotted for PLC{gamma}1 or actin. B, 12 days after transfection (control shRNA or shRNA1), cells were incubated with EGF for 5 or 10 min. Protein extracts from cells were immunoblotted for pEGFR, pERK1/2, total EGFR, and total ERK1/2. C, 12 days after transfection, cells were incubated with EGF for 10 min followed by acetaldehyde treatment for 5 h. TER and inulin flux were measured. Values are mean ± S.E. (n = 6). Asterisks indicate the values that are significantly (p < 0.05) different from values for corresponding acetaldehyde-treated cells. D, following acetaldehyde treatment in the presence or absence of EGF, cell monolayers were fixed and stained for ZO-1 by immunofluorescence methods, and images were collected by confocal microscopy.

 
Immunoblot Analysis—Proteins were separated by 7% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blotted for occludin, ZO-1, p-Thr, PKC{alpha}, PKCβI, PKC{epsilon}, PKC{gamma}, PLC{gamma}1, p-PLC{gamma}1, EGFR, EGFR(pY1173), ERK1/2, phospho-ERK1/2, and β-actin by using specific antibodies in combination with HRP-conjugated anti-mouse IgG or HRP-conjugated anti-rabbit IgG antibodies. HRP-conjugated anti-GST antibody was used for immunoblot analysis of GST or GST-occludin-C. The blot was developed using the ECL chemiluminescence method (Amersham Biosciences). Quantitation was performed by densitometric analysis of specific bands on immunoblots by using the software, Image J.

Pairwise Binding Assay—To determine the direct interaction between occludin and PKCβI or PKC{epsilon}, GST-occludin-C (10 µg) was incubated with recombinant, His-tagged PKCβI (0.1-1.0 µg) or PKC{epsilon} (0.1-1.0 µg) in PBS containing 0.2% Triton X-100, 1 mM vanadate, and 10 mM sodium fluoride for 16 h at 4 °C on an inverter. GST-occludin-C was pulled down by binding to 20 µl of 50% nickel-agarose slurry at 4 °C for 1 h. The amounts of PKCβI and PKC{epsilon} bound to nickel-agarose pulled down were determined by immunoblot analysis. Nonspecific binding was determined by carrying out the binding with GST, instead of GST-occludin-C.

Occludin Phosphorylation in Vitro—GST-occludin-C (10 µg) was incubated with 1.0 µg of active PKC{epsilon} or PKCβI in 20 mM MOPS, pH 7.2, containing 25 mM β-glycerophosphate, 2.25 mM MgCl2, 0.2 mM ATP, and 1 mM dithiothreitol. Following 3-h incubation at 30 °C, the reaction mixture was immunoblotted for p-Thr.

Statistics—Comparison between two groups was made by Student's t-tests for grouped data. Significance in all tests was set at 95% or greater confidence level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
EGF Rapidly Activates PLC{gamma}1 by an EGF Receptor Tyrosine Kinase-dependent Mechanism—To determine the effect of EGF on activation of PLC{gamma}1, protein extracts prepared from cells incubated with EGF for varying times were immunoblotted for p-PLC{gamma}1 and total PLC{gamma}1. EGF rapidly increased p-PLC{gamma}1 in whole cell protein extracts (Fig. 1A), whereas the level of total PLC{gamma}1 remained unaffected. This effect of EGF on p-PLC{gamma}1 level was dose-dependent (Fig. 1B). p-PLC{gamma}1 was predominantly distributed in the membrane fraction, whereas only a trace amount was present in the cytosolic fractions (Fig. 1C). The non-phosphorylated PLC{gamma}1 was predominantly localized in the cytosolic fraction. AG1478, an EGF receptor (EGFR) tyrosine kinase-selective inhibitor, abrogated the EGF-induced increase in p-PLC{gamma}1 (Fig. 1D). Immunoprecipitation of EGFR followed by immunoblot analysis showed that p-PLC{gamma}1 in EGF-treated cells can be co-immunoprecipitated with EGFR, which was completely prevented by AG1478 (Fig. 1E). Acetaldehyde treatment did not induce PLC{gamma}1 activation nor did it influence the EF-mediated activation of PLC{gamma} (Fig. 1F).


Figure 3
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  		FIGURE 3.
EGF induces membrane translocation of PKCβI and PKC{epsilon} by PLC{gamma}1-dependent mechanism. Caco-2 cell monolayers were transfected with shRNA to PLC{gamma}1 or a control shRNA as described under "Experimental Procedures." On day 4 after transfection, cell monolayers were incubated with 30 nM EGF for varying times. Membrane fractions were prepared and immunoblotted for PKCβI (A), PKC{epsilon} (B), PKC{gamma} (C), and PKC{alpha} (D). All immunoblots were reblotted for β-actin. PKC bands were semi-quantitated by densitometric analysis. Density values were normalized to value for corresponding control cell that was incubated without EGF. Values are mean ± S.E. (n = 3). Asterisks indicate the values that are significantly (p < 0.05) different from values for corresponding control cells, and # indicates values different from values for control RNA-treated cells.

 


Figure 4
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  		FIGURE 4.
PKC inhibitors attenuate EGF-mediated protection of TJ from acetaldehyde. Caco-2 cell monolayers were preincubated with or without 1 µM Ro-32-0432 or 1 µM chelerythrine for 1 h followed by incubation with or without 30 nM EGF. Ten minutes after EGF administration cell monolayers were incubated with acetaldehyde as described under "Experimental Procedures." A and B, TJ integrity was evaluated by measuring TER (A) and unidirectional flux of FITC-inulin (B). Values are mean ± S.E. (n = 8). Asterisks indicate the values that are significantly (p < 0.05) different from values for acetaldehyde-treated cells, whereas # indicates that the values are significantly different from values for cells treated with EGF and acetaldehyde. C, cell monolayers incubated with or without acetaldehyde, EGF, and Ro-32-0432 were fixed and stained for ZO-1 by the immunofluorescence method, and images were collected by confocal microscopy.

 
Reduced Expression of PLC{gamma}1 by shRNA Prevents EGF-mediated Protection of TJ from Acetaldehyde—To confirm the role of endogenous PLC{gamma}1 in EGF-mediated protection of TJ from acetaldehyde we transfected Caco-2 cells with shRNA specific for the nucleotide sequence of human PLC{gamma}1 gene inserted in pR vector. The effect of EGF on acetaldehyde-induced disruption of TJ was evaluated in cells transfected with shRNA or the control shRNA. In shRNA-transfected cells, PLC{gamma}1 levels were significantly lower than that in cells transfected with a control shRNA, indicating that the shRNA was effective in reducing the expression of PLC{gamma}1 (Fig. 2A). Transfection with shRNA did not affect the EGF-mediated tyrosine phosphorylation of EGFR or ERK1/2 (Fig. 2B). Acetaldehyde significantly reduced TER and increased inulin permeability (Fig. 2C) in both shRNA and control shRNA-transfected cell monolayers. EGF treatment significantly prevented acetaldehyde-induced changes in TER and inulin flux in control shRNA-transfected cells, but EGF failed to prevent acetaldehyde-induced effect in shRNA-transfected cells. EGF also prevented acetaldehyde-induced redistribution of occludin and ZO-1 (Fig. 2D) from the intercellular junctions in vector-transfected cell monolayer, but not in shRNA-transfected cells.

EGF Induces Membrane Translocation of PKC{epsilon} and PKCβI by a PLC{gamma}-dependent Mechanism—The intracellular signaling molecules generated by the activation of PLC{gamma}1 are IP3 and diacylglycerol, which are known to induce translocation of PKC isoforms to the membrane fraction (44, 45). To determine the EGF-mediated activation of PKC isoforms and the role of PLC{gamma}1 activity in PKC translocation, we evaluated the effect of EGF on the level of PKC isoforms in the membrane fractions. EGF rapidly increased the levels of PKCβI (Fig. 3A), PKC{epsilon} (Fig. 3B), and PKC{gamma} (Fig. 3C), but not PKC{alpha} (Fig. 3D), in the membrane fraction prepared from cells transfected with mutant shRNA. Reduced expression of PLC{gamma}1 by shRNA prevented EGF-induced translocations of PKCβI (Fig. 3A) and PKC{epsilon} (Fig. 3B), however, it did not affect the translocation of PKC{gamma} (Fig. 3C). ET-18-OCH3 by itself produced no effect on the basal level of PKC isoforms.


Figure 5
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  		FIGURE 5.
PKC-RACK interference peptides (PRIP) specific for PKCβI and PKC{epsilon} prevent EGF-mediated protection of TJ from acetaldehyde. A-C, Caco-2 cell monolayers were preincubated with or without 3 µg/ml antennapedia-conjugated, isoform-selective PRIP for 20 min followed by incubation with or without 30 nM EGF for 5 min. Membranes prepared from these cells were immunoblotted for PKC{epsilon} (A), PKCβI (B), or PKC{gamma} (C). D and E, Caco-2 cell monolayers were preincubated with or without 3 µg/ml antennapedia-conjugated, isoform-selective PRIP for 20 min. Cell monolayers were incubated with acetaldehyde as described under "Experimental Procedures" 10 min after EGF administration. TJ integrity was evaluated by measuring TER (D) and unidirectional flux of FITC-inulin (E). Values are mean ± S.E. (n = 4). Asterisks indicate the values that are significantly (p < 0.05) different from values for acetaldehyde-treated cells, while # indicates that the values are significantly different from values for cells treated with EGF and acetaldehyde. F and G, Caco-2 cell monolayers were preincubated with or without varying concentrations of antennapedia-conjugated, isoform-selective PRIP for 20 min. Cell monolayers were incubated with acetaldehyde as described under "Experimental Procedures" 10 min after EGF administration. TJ integrity was evaluated by measuring TER (F) and unidirectional flux of FITC-inulin (G). Values are mean ± S.E. (n = 4). Asterisks indicate the values that are significantly (p < 0.05) different from values for acetaldehyde-treated cells, whereas # indicates that the values are significantly different from values for cells treated with EGF and acetaldehyde.

 
PKC Activity Is Required for EGF-mediated Protection of TJ from Acetaldehyde—To determine the role of PKC activity in EGF-mediated prevention of acetaldehyde-induced disruption of TJ, we evaluated the effect of Ro-32-0432 and chelerythrine (the PKC-selective inhibitors) on EGF effect. Both Ro-32-0432 and chelerythrine significantly attenuated EGF-mediated prevention of acetaldehyde-induced decrease in TER (Fig. 4A) and increase in inulin flux (Fig. 4B). PKC inhibitors also blocked EGF-mediated prevention of acetaldehyde-induced redistribution of ZO-1 from the intercellular junctions (Fig. 4C).

Membrane Translocation of PKCβI and PKC{epsilon} Is Involved in EGF-mediated Protection of TJ from Acetaldehyde—Translocation from cytosol to membrane is an important event in the activation of PKC isoforms during cellular stimulation (4). Peptides that specifically interfere with PKC and isoform-specific RACK protein have been successfully used to prevent stimulated membrane translocation of PKC in an isoform-specific manner (44). In the present study, we used such peptides to prevent EGF-mediated translocation of PKCβI, PKC{epsilon}, PKC{gamma}, and PKC{alpha}. Pretreatment of cell monolayers with antennapedia-conjugated RACK interference peptides selectively blocked translocation of the corresponding PKC isoform (Fig. 5, A-C). RACK interference peptides specific for PKC{epsilon} and PKCβI abrogated EGF-mediated prevention of acetaldehyde-induced decrease in TER (Fig. 5D) and increase in inulin permeability (Fig. 5E). The effects of PKC{epsilon}- and PKCβI-specific RACK interference peptides on TER (Fig. 5F) and inulin flux (Fig. 5G) were dose-dependent. RACK interference peptide specific for PKC{gamma} and PKC{alpha} or antennapedia did not alter EGF effects on TER or inulin flux. PKC{epsilon}- and PKCβI-specific RACK interference peptides also attenuated EGF-mediated prevention of acetaldehyde-induced redistribution of ZO-1 from the intercellular junctions (Fig. 6A). PKC inhibitors, Ro-32-0432 and chelerythrine, did not affect EGF-induced phosphorylation of EGFR and PLC{gamma}1 (Fig. 6B).

Intracellular Calcium Is Required for EGF-mediated Protection of TJ from Acetaldehyde—Both IP3 and diacylglycerol are involved in the activation of PKC isoforms (44, 45). IP3 induces release of intracellular calcium from endoplasmic reticular calcium store into the cytosol, and the cytosolic calcium is involved in membrane translocation of several PKC isoforms (44). We evaluated the effect of BAPTA-AM (depletes cytosolic calcium by chelation) and thapsigargin (depletes stored calcium) on EGF-mediated PKC translocation and the protection of TJ and barrier function from acetaldehyde. Results show that both BAPTA-AM and thapsigargin prevented EGF-mediated translocation of PKCβI, without affecting the translocation of PKC{epsilon} (Fig. 7A), and attenuated the EGF-mediated prevention of acetaldehyde-induced increase in inulin permeability (Fig. 7B). BAPTA-AM and thapsigargin also attenuated the EGF-mediated prevention of acetaldehyde-induced redistribution of occludin and ZO-1 from the intercellular junctions (Fig. 7C).


Figure 6
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  		FIGURE 6.
PRIPs specific for PKCβI and PKC{epsilon} prevent EGF-mediated protection of TJ from acetaldehyde. A, cell monolayers incubated with or without acetaldehyde, EGF, and PRIP were fixed and stained for ZO-1 by immunofluorescence method, and images were collected by confocal microscopy. B, Caco-2 cell monolayers were preincubated with or without Ro-32-0432 or U0126 for 1 h, followed by incubation with or without EGF for 5 min. Cell extracts were immunoblotted for pEGFR, p-PLC{gamma}, and β-actin.

 
EGF Induces Redistribution of PKCβI, PKC{epsilon}, and F-actin—EGF-induced translocation of PKCβI and/or PKC{epsilon} were determined by transient transfection of Caco-2 cells with p-EGFP-PKCβI or pEGFP-PKC{epsilon}. Four days after transfection cell monolayers were stimulated with EGF for 5 or 15 min. Fixed cell monolayers were then fluorescently labeled for GFP and F-actin. In unstimulated cells, GFP-PKC{epsilon} was localized predominantly in the intracellular compartment (Fig. 8A), whereas stimulation with EGF resulted in redistribution to perijunctional regions, and it appeared to co-localize with F-actin. In GFP-positive unstimulated cells, the F-actin organization appeared disrupted compared with that in GFP-negative, unstimulated cells. However, in EGF-stimulated cells, the F-actin was organized predominantly at the perijunctional regions compared with that in unstimulated cells. Similarly, EGF treatment also induced redistribution of GFP-PKCβI to the perijunctional regions (Fig. 8B).

To determine whether PKCβI and/or PKC{epsilon} directly interact with occludin to phosphorylate it on Thr residues, we evaluated the interaction of PKCβI and PKC{epsilon} with occludin. Immunoprecipitation of occludin followed by immunoblot analysis showed that occludin pulls down only trace amounts of PKCβI or PKC{epsilon} in unstimulated and EGF-stimulated cells (Fig. 9A). The pairwise binding assay using GST-occludin-C (the C-terminal tail) and recombinant PKCβI (Fig. 9B) or PKC{epsilon} (Fig. 9C) also failed to demonstrate specific binding of these PKC isoforms to the C-terminal tail of occludin. Incubation of GST-occludin-C with PKCβI or PKC{epsilon} in the presence of ATP failed to show direct phosphorylation of occludin by these PKC isoforms (Fig. 9D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
EGF is known to protect the gastrointestinal epithelium from various insults (38). EGF protects the barrier function and TJ of Caco-2 cell monolayers from oxidative stress and acetaldehyde (35, 42). The present study demonstrates that EGF-induced membrane translocations of PKC{epsilon} and PKCβI are mediated by PLC{gamma}1 activation and that intracellular calcium is involved in the translocation of PKCβI. EGFR-mediated activation of signaling cascade that involves PLC{gamma}1, intracellular calcium, PKC{epsilon}, and PKCβI prevents acetaldehyde-induced Thr-dephosphorylation of occludin, loss of association of occludin and ZO-1 with the actin-rich detergent-insoluble fraction and cellular redistribution of occludin and ZO-1, and thus protects the integrity of TJ from acetaldehyde-induced insult.

To determine the role of a specific PLC isoform, PLC{gamma}1, in the protection of TJ from acetaldehyde we silenced the PLC{gamma}1 gene expression by designing specific shRNA. Reduction in the expression of PLC{gamma}1 did not alter the basal TJ integrity, confirming that PLC{gamma}1 is not involved in the maintenance of basal TJ integrity. However, knockdown of PLC{gamma}1 resulted in the prevention of EGF-mediated protection of TJ from acetaldehyde. These data demonstrate that PLC{gamma}1 is involved in EGF-mediated protection of TJ. Previous studies, using selective inhibitors, showed that PLC{gamma} and PLCβ activities are involved in the regulation of TJ integrity in Madin-Darby canine kidney cell monolayers (46, 47), whereas PLC{gamma} is involved in the protection of TJ from hydrogen peroxide in Caco-2 cells (48). Although the previous study showed that U73122 [GenBank] prevented the protection of TJ from hydrogen peroxide (48), our present study (data not shown) showed that U73122 [GenBank] disrupted the TJ by itself. It was also previously demonstrated that U73122 [GenBank] is a selective inhibitor of PLCβ and it increases the TJ permeability in Madin-Darby canine kidney cell monolayers (47). This is consistent with our present observation indicating that PLCβ activity is required for the maintenance of basal integrity of TJ. ET-18-OCH3 (PLC{gamma}-selective inhibitor), on the other hand, did not alter the basal TJ integrity, suggesting that PLC{gamma} activity is not involved in the maintenance of basal TJ integrity, but it is required for EGF-mediated protection of the TJ from acetaldehyde.


Figure 7
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  		FIGURE 7.
Intracellular calcium mediates EGF-induced translocation of PKCβI and protection of TJ from acetaldehyde. A, Caco-2 cells were pretreated for 1 h with varying doses of BAPTA-AM or thapsigargin. Membrane fractions isolated from these cells were immunoblotted for PKCβI, PKC{epsilon}, and β-actin. B, following treatment with or without BAPTA-AM or thapsigargin for 1 h, cell monolayers were incubated with 30 nM EGF. After EGF treatment for 10 min, cell monolayers were exposed to acetaldehyde. TER and inulin permeability were measured at 4 h after acetaldehyde treatment. Values are mean ± S.E. (n = 6). Asterisks indicate the values that are significantly different from values for corresponding cells treated with acetaldehyde, and # represents values different from those for cells treated with EGF and acetaldehyde. C, following treatment with or without acetaldehyde, in the presence or absence of EGF, BAPTA-AM and thapsigargin cell monolayers were fixed and stained for occludin and ZO-1 by immunofluorescence methods. Images were collected using a confocal microscope.

 
The appearance of p-PLC{gamma} as early as 2 min after EGF administration indicates that EGF rapidly activates PLC{gamma}1 in Caco-2 cell monolayers. The activated PLC{gamma}1 was distributed in the membrane fraction and co-immunoprecipitated with EGFR. Membrane translocation and the activation of PLC{gamma}1 could be attenuated by AG1478, an EGFR tyrosine kinase inhibitor. Therefore, PLC{gamma} is activated by its interaction with activated EGFR. Acetaldehyde by itself did not induce activation of PLC{gamma} nor did it influence the EGF-mediated activation of PLC{gamma}. Additionally, knockdown of PLC{gamma}1 did not affect the activation of EGFR by phosphorylation on Tyr-1173, demonstrating that PLC{gamma}1 activation is a downstream event to EGFR autophosphorylation. Furthermore, PLC{gamma}1 knockdown did not affect EGF-mediated activation of ERK1/2, indicating that EGF-induced ERK activation is independent of PLC{gamma}1 activation.

The PLC{gamma}-mediated protection of TJ from acetaldehyde may involve activation of downstream signaling molecules. Translocation of PKC isoforms to the membrane is an established downstream signaling event. Calcium and diacylglycerol, the second messengers released by PLC{gamma}-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate, regulate the membrane translocation and activation of PKC isoforms. The attenuation of EGF-mediated prevention of acetaldehyde-induced disruption of barrier function and redistribution of ZO-1 by PKC inhibitors, Ro-32-0432 and chelerythrine, indicates that PKC activity is involved in EGF-mediated protection of TJ from acetaldehyde. EGF rapidly induced membrane translocation of PKC{epsilon}, PKC{gamma}, and PKCβI, but not PKC{alpha}. This EGF-induced translocation of PKC{epsilon} and PKCβI was completely attenuated by the PLC{gamma} inhibitor, demonstrating that PLC{gamma} does activate PKC{epsilon} and PKCβIin Caco-2 cell monolayers. However, PLC{gamma} inhibitor failed to prevent the EGF-mediated membrane translocation of PKC{gamma}. PKC inhibitors also failed to prevent EGF-induced phosphorylation of EGFR and PLC{gamma}1.

To determine the role of membrane translocation of PKC isoforms in EGF-mediated protection of the TJ from acetaldehyde we used isoform-specific RACK interference peptides. EGF-mediated membrane translocations of PKC{epsilon}, PKCβI, and PKC{gamma} were selectively blocked by corresponding RACK interference peptide. The RACK interference peptides for PKC{epsilon} and PKCβI attenuated EGF-mediated prevention of acetaldehyde-induced increase in permeability and redistribution of ZO-1. However, RACK interference peptide for PKC{gamma} failed to prevent EGF effect. These studies demonstrate that EGF-mediated membrane translocations of PKC{epsilon} and PKCβI are involved in the mechanism of EGF-mediated protection of TJ from acetaldehyde.

PKCβI is a calcium-dependent PKC isoform, whereas PKC{epsilon} is calcium-independent (44). IP3, one of the second messengers generated by PLC{gamma} activation is known to increase intracellular calcium (45). The attenuation of EGF-mediated prevention of acetaldehyde-induced disruption of barrier function and redistribution of occludin and ZO-1 by BAPTA-AM and thapsigargin indicates that intracellular calcium release does play a role in the EGF-mediated protection of TJ from acetaldehyde. Both BAPTA-AM and thapsigargin prevented EGF-induced membrane translocation of PKCβI. Therefore, EGF-mediated activation of PLC{gamma} induced membrane translocation of PKCβI by releasing IP3 and intracellular calcium. BAPTA-AM and thapsigargin, however, did not alter EGF-mediated membrane translocation of PKC{epsilon}. The dependence of EGF-mediated translocation of PKC{epsilon} on PLC{gamma} activity suggests that diacylglycerol or other PLC{gamma}-mediated mechanism may play a role in inducing membrane translocation of PKC{epsilon}.


Figure 8
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  		FIGURE 8.
EGF induces redistribution of PKC{epsilon} and PKCβI. Caco-2 cells transiently transfected with pEGFP-PKC{epsilon} (A) or pEGFP-PKCβI (B) were incubated with or without 30 nM EGF for 5 or 15 min. Fixed cell monolayers were double labeled for GFP (green) and F-actin (red).

 


Figure 9
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  		FIGURE 9.
PKC{epsilon} and PKCβI do not directly interact with occludin. A, occludin from native protein extracts from Caco-2 cells were immunoprecipitated, and immunocomplexes were immunoblotted for PKC{epsilon} and PKCβI. B, 10 µg of GST-occludin-C (GST-Ocl) was incubated with varying amounts of active His-tagged PKC{epsilon}, and nickel-agarose pulldown was immunoblotted for GST. C, 10 µg of GST-occludin-C (GST-Ocl) was incubated with varying amounts of active His-tagged PKCβI, and nickel-agarose pulldown was immunoblotted for GST. D, 10 µg of GST-occludin-C (GST-Ocl) was incubated with varying amounts of active PKC{epsilon} or PKCβI in the presence of ATP, and the reaction mixture was immunoblotted for p-Thr.

 
EGF-induced redistribution of PKC{epsilon} and PKCβI could be confirmed by transient transfection of GFP-PKC{epsilon} and GFP-PKCβI in Caco-2 cells. EGF induced translocation of both PKC{epsilon} and PKCβI to the perijunctional regions. The confocal analysis of z-sections of immunofluorescence images indicated that neither PKC{epsilon} nor PKCβI localized at the TJ, rather they were localized at the perijunctional region. Interestingly, co-staining of these cell monolayers for F-actin showed that F-actin is disorganized in GFP-positive cells compared with that in GFP-negative cells in the absence of EGF. This indicates that excess of intracellular PKC{epsilon} and PKCβI has disruptive influence on the actin cytoskeleton. However, EGF stimulation leads to reorganization of F-actin near the perijunctional region. These observations suggest that EGF-mediated translocation of PKC{epsilon} and PKCβI may play a role in reorganization of F-actin at the perijunctional region, likely the actomyosin ring. The EGF-mediated stabilization of the actinomycin ring may serve as one of the mechanisms involved in the protection of TJ integrity from acetaldehyde-induced insult.

Occludin is known to be hyperphosphorylated on Ser/Thr residues, and this phosphorylation appears to be required for the assembly of TJ (49, 50). Occludin undergoes dephosphorylation during the disruption of TJ by calcium depletion (49), phorbol ester (51), and pathogenic infection (52). These observations suggest that PKC{epsilon} and PKCβI may interact directly with occludin and maintain Thr-phosphorylation of occludin. To test this hypothesis, we investigated the possibility of a direct interaction between occludin and PKC{epsilon} or PKCβI by co-immunoprecipitation and pairwise binding assay using recombinant proteins (GST-Occludin-C, PKC{epsilon} and PKCβI). However, results of both assays demonstrated that PKCβI and PKC{epsilon} do not directly interact with the C-terminal tail of occludin. PKC{epsilon} and PKCβI also failed to induce Thr-phosphorylation of GST-occludin-C in vitro. Therefore, this study suggests that PKC{epsilon} and PKCβI may have other downstream targets in the plasma membrane, which may indirectly affect the Thr phosphorylation of occludin and the protection of the integrity of TJ.

In summary, this study demonstrates that PLC{gamma} activation, intracellular calcium, and membrane translocation of PKCβI and PKC{epsilon} are involved in the mechanism of EGF-mediated protection of TJ of Caco-2 cell monolayers from acetaldehyde-induced insult.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants R01-DK55532 and R01-AA12307. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Physiology, University of Tennessee, 894 Union Ave., Memphis, TN 38163. Tel.: 901-448-3235; Fax: 901-448-7126; E-mail: rkrao{at}physio1.utmem.edu.

2 The abbreviations used are: TJ, tight junction; PBS, phosphate-buffered saline; HRP, horseradish peroxidase, ZO-1, ZO-2, and ZO-3, zonula occludens 1, 2, and 3; TER, transepithelial electrical resistance; EGF, epidermal growth factor; ET-18-OCH3, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine; BAPTA-AM, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; EGFR, EGF receptor; AG1478, 4-(3-achloroanillino)-6,7-dimethoxyquinazoline; Ro-32-0432, 2-(8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl)-3-(1-methylinfol-3-yl)maleimide; PLC, phospholipase C; PKC, protein kinase C; IP3, inositol 1,4,5-trisphosphate; GST, glutathione S-transferase; GST-Occludin-C, GST-conjugated C-terminal tail of occludin; p-Thr, phospho-threonine; p-Ser, phospho-serine; GST-Occludin-C, C-terminal tail region of occludin fused with GST; FITC, fluorescein isothiocyanate; GFP, green fluorescence protein; pR vector, pRNATinH1.2 vector; PRIP, PKC-RACK interference peptide; ERK, extracellular signal-regulated kinase; shRNA, short hairpin RNA; MOPS, 4-morpholinepropanesulfonic acid. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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