Ethanol disrupts intestinal epithelial tight junction integrity through

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Am J Physiol Gastrointest Liver Physiol 306: G677–G685, 2014. First published February 20, 2014; doi:10.1152/ajpgi.00236.2013.

Ethanol disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated Rho/ROCK activation Elhaseen Elamin,1,2,3 Ad Masclee,1,2,3 Jan Dekker,1,4 and Daisy Jonkers1,2,3 1

Top Institute Food and Nutrition (TIFN), Wageningen; 2Division of Gastroenterology-Hepatology, Department of Internal Medicine and 3School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Center, Maastricht, the Netherlands; and 4Host-Microbe Interactomics Group, Department of Animal Sciences, Wageningen UR, Wageningen, the Netherlands Submitted 22 July 2013; accepted in final form 14 February 2014

Elamin E, Masclee A, Dekker J, Jonkers D. Ethanol disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated Rho/ROCK activation. Am J Physiol Gastrointest Liver Physiol 306: G677–G685, 2014. First published February 20, 2014; doi:10.1152/ajpgi.00236.2013.—Evidence indicates that ethanol-induced intestinal barrier dysfunction and subsequent endotoxemia plays a key role in the pathogenesis of alcoholic liver disease. Recently, it has been demonstrated that ethanol induces RhoA kinase activation in intestinal epithelium, thereby disrupting barrier integrity. In this study, the role of a rise in intracellular calcium concentration ([Ca2⫹]i) in ethanol-induced Rho-associated coiled coil-forming kinase (Rho/ROCK) activation and barrier disruption was investigated in Caco-2 cell monolayers. Treatment of Caco-2 monolayers with 40 mmol/l ethanol induced [Ca2⫹]i release as indicated by increased relative fluorescent units of Fluo-3 from 0.06 ⫾ 0.02 to 2.27 ⫾ 1.96 (P ⬍ 0.0001). Pretreatment with 1,2-bis(2-aminophenoxy) ethaneN,N,N=,N=-tetraacetic acid (BAPTA-AM) completely inhibited the release, whereas the inositol 1,4,5-triphosphate receptor (IP3R)-antagonist, Xestospongin C, partially inhibited the ethanol-induced [Ca2⫹]i release (from 2.27 ⫾ 1.96 to 0.03 ⫾ 0.01; P ⬍ 0.0001 and from 2.27 ⫾ 1.96 to 1.19 ⫾ 1.80; P ⬍ 0.001, respectively). The rise in [Ca2⫹]i was paralleled with increased intestinal permeability, which could be attenuated by either BAPTA-AM or Xestospongin C. Furthermore, ethanol induced Rho/ROCK activation, as indicated by increased phosphorylation of myosin-binding subunit, which could be prevented either by BAPTA, Xestospongin C, or the specific Rho/ ROCK inhibitor Y27632. Finally, inhibition of Rho/ROCK kinase by Y27632 ameliorated the ethanol-induced redistribution of zonula occluden-1, adherens junction proteins including E-cadherin and ␤-catenin, and also disorganization of F-actin. These findings suggest that ethanol-induced [Ca2⫹]i release, mediated by stimulating IP3Rgated Ca2⫹ channel, activates Rho/ROCK in Caco-2 cells, thereby contributing to ethanol-induced intestinal barrier dysfunction. intestinal epithelial barrier; Caco-2; ethanol; inositol 1,4,5-triphosphate receptor; intracellular calcium; Rho kinase; tight junction

integrity is maintained by tight junctions (TJs), a complex of proteins formed by transmembrane and cytoplasmic proteins including occludin and zonula occludens (ZO-1, 2, 3), respectively, linked to actin cytoskeleton (12). In addition, adherens junction (AJ) proteins including E-cadherin and ␤-catenin are required for TJ assembly and maintenance of intestinal epithelial integrity (17). TJs regulate trafficking of ions, molecules, and nutrients and act as a barrier against translocation of harmful allergens and bacterial prod-

INTESTINAL EPITHELIAL BARRIER

Address for reprint requests and other correspondence: E. Elamin, Dept. of Internal Medicine, Division of Gastroenterology and Hepatology, Maastricht Univ. Medical Center, P. Debyelaan 25, 6229 HX Maastricht, the Netherlands (e-mail: [email protected]). http://www.ajpgi.org

ucts, such as endotoxins from intestinal lumen into the circulation and mucosal immune system (5). Therefore, preservation of TJ integrity is crucial for intestinal epithelial homeostasis. Ethanol is known to induce intestinal barrier dysfunction, resulting in endotoxemia, which is suggested to play a key role in the pathogenesis of alcoholic liver disease (ALD) (7, 28, 43). Moreover, detection of high endotoxin levels and its correlation thereof with severity and complications of ALD have been attributed to intestinal barrier dysfunction (15, 20, 46, 52). Our laboratory and others (10, 14, 56) have shown that ethanol at blood concentration as low as low as 0.2% (⬃40 mmol/l), achieved after moderate consumption of two to four drinks (56), can disrupt epithelial TJ integrity in vivo and also increases permeability in Caco-2 three-dimensional (10) and two-dimensional cell culture models (14, 56). Research into potential mechanisms revealed that ethanol disrupts intestinal barrier function via mechanisms involving inducible nitric oxide synthase (iNOS)-induced oxidative stress and activation of myosin light chain kinase (MLCK) signaling pathway (33), thereby mediating disruption of F-actin cytoskeleton and TJ (3, 36). Very recently, activation of RhoA kinase has been found to be implicated in ethanol-induced intestinal barrier disruption (29, 59). Rho kinase family including RhoA is one of the major regulators of actomyosin ring organization and TJ assembly in polarized epithelia (41). The downstream effector of RhoA kinase, Rho-associated coiled-coil forming kinase (Rho/ ROCK), has been shown to activate MLCK, resulting in barrier disruption (42, 61, 64). The mechanisms through which ethanol activates RhoA kinase and subsequent disruption of intestinal barrier integrity have not been clarified. Intracellular calcium ([Ca2⫹]i) homeostasis might be involved, as a rise in [Ca2⫹]i has been shown to activate RhoA kinase, resulting in disruption of intercellular junctions (37, 55). Therefore, this study aimed to examine the role of [Ca2⫹]i homeostasis and RhoA kinase activation in ethanol-induced intestinal hyperpermeability using Caco-2 cell monolayers. We hypothesize that ethanol stimulates inositol 1,4,5-triphosphate receptor (IP3R)-gated [Ca2⫹]i release, thereby causing activation of Rho/ROCK with subsequent barrier disruption. MATERIALS AND METHODS

Chemicals. Cell culture reagents were purchased from Life Technologies (Bleiswijk, the Netherlands) and Lonza Benelux BV (Breda, the Netherlands). Fluorescein isothiocyanate (FITC-D4), dichlorodihydrofluorescein diacetate, and Triton X-100 were purchased from Sigma (Amsterdam, the Netherlands). Cell-based ELISA assay kits were purchased from RayBiotech (Norcross, GA). 1,2-Bis(2-aminophenoxy) ethane-N,N,N=,N=-tetraacetic acid (BAPTA-AM) was pur-

0193-1857/14 Copyright © 2014 the American Physiological Society

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chased from Sigma. Fluo-3 AM and Xestospongin-C were purchased from Life Technologies and Cayman Europe (Tallinn, Estonia), respectively. The Rho/ROCK inhibitor (R)-trans-4-(1-aminoethyl)-N(4-pyridyl) cyclohexanecarboxamide (Y-27632) was purchased from Selleckchem (Munich, Germany). Antibodies. Mouse polyclonal anti-ZO-1 antibodies were purchased from Zymed Laboratories (San Francisco, CA). Rabbit-anti E-cadherin and rabbit-anti ␤-catenin were purchased from Abcam (Cambridge, UK), and rabbit anti-phosphorylated myosin-binding subunit (P-MBS)/myosin phosphatase-targeting subunit 1 (MYPT1) was purchased MBL International (Woburn, MA). Alexa-Fluor 488-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG antibodies were from Jackson Laboratories (Suffolk, UK). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit antibodies were purchased from Dako BV (Heverlee, Belgium). Diamidino-2-phenylindole (DAPI) was purchased from Sigma. Rhodamine-phalloidin was purchased from Life Technologies. Cell line and culture conditions. Caco-2 cells (from passages 29 – 45) purchased from ATCC (Rockville, MD) were grown in either T25 or T75 flasks and were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Lonza Benelux BV) containing 4.5 g/l glucose and L-glutamine, as described previously (11). Microscopic analysis of [Ca2⫹]i. Caco-2 (5 ⫻ 103) cells were plated on glass-bottom dishes in 10% FBS-containing DMEM medium in a humidified atmosphere of 5% CO2-95% air at 37°C for 24 h. Next, cells were loaded with 3 ␮mol/l Fluo-3 AM (Life Technologies Europe) by incubation of the cells at 23°C for 60 min in assay buffer containing 130 mmol/l NaCl, 5 mmol/l KCl, 2 mmol/l CaCl2, 1 mmol/l MgSO4, 8 mmol/l NaOH, and 20 mmol/l HEPES (pH 7.4), followed by a 30-min incubation at 37°C. Fluo-3 AM was chosen as a Ca2⫹ indicator because it exhibits a 40-fold increase in fluorescence intensity with Ca2⫹ and possesses an enhanced resistance to autobleaching (54). Next, Caco-2 monolayers were pretreated (before exposure to ethanol) with either 10 ␮mol/l BAPTA-AM (Sigma-Aldrich) or 1 ␮mol/l Xestospongin C, selective and reversible inhibitor of IP3 receptor (Cayman Europe) for 45 min (4). Thereafter, cells were washed twice with Ca2⫹-free PBS and placed under a Leica TCS SPE confocal laser-scanning microscope (Leica Microsystems, Mannheim, Germany). For imaging of Fluo-3 fluorescence, cells were observed using a Leica ⫻63 oil⫽immersion lens, excitation light was provided by an argon laser at 488 nm and emission at 530 nm, and fluorescence was recorded for 5 min as stable baseline. Next, 40 mmol/l ethanol was added, and changes in fluorescence intensity were monitored over 15 min. Image acquisition frequency was set to one image every 10 s, and images were analyzed for changes in fluorescence intensities within regions of interests (circles drawn over cytosol areas) using the image-analysis software Leica Application Suite Advanced Fluorescence, and values were plotted as changes from baseline as described previously (27). Measurement of transepithelial electrical resistance and paracellular permeability. Caco-2 cells were seeded and grown for 21 days on collagen-coated polycarbonate membrane Transwell inserts with a surface area of 0.33 cm2 and 0.4-␮m pore size (Costar, Cambridge, MA). After monolayers of transepithelial electrical resistance (TEER) ⬎ 600 Ohm·cm2 were developed, monolayers were exposed to 40 mmol/l ethanol either alone for 3 h or after pretreatment with BAPTA-AM (10 ␮mol/l) or Xestospongin C (1 ␮mol/l) for 15 min. In another set of experiments, monolayers were pretreated for 1 h with 100 ␮mol/l of (⫹)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632; Selleckchem, Munich, Germany), the specific inhibitor of both isoforms ROCK2 (also called ROK-␣) and p160ROCK (also known as ROK-␤ or ROCK1) (60). TEER of epithelial monolayers (⍀·cm2) was measured after 3 h by an epithelial voltohmmeter (World Precision Instruments, Berlin, Germany) in each insert and multiplied by the membrane surface area (0.33 cm2), corrected by subtracting background resistance of the blank membrane (no cells;

⬃30 ⍀·cm2). Data were collected from duplicate inserts per treatment in three experiments and expressed as percentage of basal TEER obtained before treatment. By the end of TEER measurements, FITClabeled 4-kDa dextran (1 mg/ml FITC-D4; Sigma-Aldrich) was added to the apical side of cells and incubated for 1 h at 37°C. Monolayer permeability was assessed by measuring the fluorescence in the basal medium compartment of FITC-D4 spectrophotometrically using SpectraMax M2 spectrofluorometer (Molecular Devices, Sunnyvale, CA) at excitation and emission spectra of 485 nm and 540 nm, respectively, and data were reported as relative fluorescent units. Immunofluorescent microscopic analysis of cellular proteins. Cell monolayers on membranes were fixed in the inserts with 10% (wt/vol) trichloroacetic acid (TCA), permeabilized with PBS and glycine (30 mmol/l) and 1% (vol/vol) Triton X-100, and blocked with a blocking buffer containing PBS and glycine with 3% (vol/vol) FCS. Primary antibodies including mouse anti-ZO-1 (Zymed Laboratories), rabbit anti-P-MBS at Thr853, known also as MYPT1, an indicator of Rho/ROCK activation (31) (P-MBS/MYPT1, MBL International), mouse anti-E-cadherin (Abcam), or rabbit anti-␤-catenin (Abcam) were added 1:100 overnight in blocking buffer. Following the washing, fluorochrome-conjugated secondary antibodies including AlexaFluor 488-conjugated anti-mouse (Invitrogen) and Cy3-conjugated anti-rabbit IgG (Invitrogen) antibodies were used as secondary antibodies (1:200). F-actin was stained with rhodamine-phalloidin (500 ng/ml; Invitrogen) at room temperature. Thereafter, monolayers were stained for 5 min with DAPI (1:10,000 dilution in PBS; Sigma Chemical). Finally, the monolayers on the semipermeable membrane were transferred to glass slides and mounted with VectaShield mounting medium (Vector Laboratories, Burlingame, CA). The slides were examined under a Leica TCS SPE confocal laser-scanning microscope equipped with a 50-mW Argon laser and a 1-mW HeNe laser (Leica Microsystems). Confocal images obtained were processed and analyzed by using Image J software (1). Dot blotting of phosphorylated MBS/MYPT1. Caco-2 cell lysates were prepared with radioimmunoprecipitation assay buffer [150 mM NaCl, 1.0% (vol/vol) Triton X-100, 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) sodium dodecyl sulfate, and 50 mM Tris, plus protease and phosphates inhibitor cocktails], and protein levels were determined using bicinchoninic acid protein assay protein assay (BioRad Laboratories, Hercules, CA). With the use of narrow-mouth pipette tips, 2 ␮l of samples were spotted onto a nitrocellulose membrane and allowed to dry to the air. Nonspecific sites were blocked by soaking the membrane in 5% (wt/vol) BSA in Trisbuffered saline and Tween 20 buffer (TBST) for 1 h at room temperature. The membrane was then incubated with either rabbit anti-total MBS/MYPT1 or rabbit anti-phosphorylated MBS/MYPT1 at Thr853 (10 ␮g/ml dissolved in BSA/TBS, MBL International) for 30 min at room temperature. Thereafter, the membrane was washed thrice in TBS and incubated with swine anti-rabbit HRP-conjugated secondary antibody (1:1,000 dilution; Dako, Glostrup, Denmark) for 30 min at RT, followed by washing in TBST and TBS, respectively. Finally, the membrane was incubated with chemiluminescence kit (GE Healthcare Europe GmbH, Diegem, Belgium) for 1 min, covered with Saran wrap, and proteins were visualized by chemidoc XRS (Bio-Rad, Hercules, CA). Quantification of the dots was done using Image J software (1). Determination of MBS/MYPT1 phosphorylation. Phosphorylation of MBS/MYPT1 as indicative of Rho/ROCK activation was determined by assessment using cell-based ELISA kits (Ray Biotech), according to manufacturer’s instructions. Briefly, Caco-2 cells (20 ⫻ 103) were seeded in 96 well-plates (Corning BV Life Sciences, Amsterdam, the Netherlands) and incubated overnight at 37°C, 5% CO2. Monolayers were treated as described earlier. Next, monolayers were fixed and blocked and were incubated with rabbit anti: phosphorylated MBS/MYPT1 (1:100 dilution in the blocking solution; Ray Biotech), followed by HRP-conjugated mouse anti-rabbit IgG

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(Dako). Finally, 3,3=,5,5=-Tetramethylbenzidine was added, followed by stop solution, and optical density was read at 450 nm by SpectraMax M2 spectrophotometer (Molecular Devices). Assessment of cellular F-actin contents. Cellular F-actin levels were determined by a fluorescent phalloidin-binding assay as described previously (19). Briefly, Caco-2 cells grown on 96-well plates (Corning BV), were rinsed with PBS, and then incubated with medium only, with 40 mmol/l ethanol alone for 3 h, or after prior incubation with 100 ␮mol/L of the ROCK inhibitor, Y-27632, for 1 h (followed by 3-h ethanol incubation). Next, the cells were fixed with acetone/methanol (1:1), and then actin was stained with rhodaminephalloidin (500 ng/ml) for 20 min. Stained cells were extracted with 200 ␮l methanol and measured spectrophotometrically at excitation and emission wavelengths of 545 and 578 nm, respectively. Statistical analysis. All data analyses were conducted with GraphPad Prism software package (GraphPad Software, San Jose, CA). Data are expressed as means ⫾ SD of triplicate experiments. One-way ANOVA and Tukey’s post hoc tests were performed to compare between different experimental conditions, considering difference of P ⬍ 0.05 as statistically significant. RESULTS

Effects of ethanol on intracellular calcium [Ca2⫹]i and barrier disruption. In these experiments, Caco-2 monolayers were loaded with the Ca2⫹-sensing indicator Fluo-3 AM, and free intracellular Ca2⫹ [Ca2⫹]i was evaluated before and during 40 mmol/l ethanol exposure. We observed an increase in

free [Ca2⫹]i after application of 40 mmol/l ethanol, reaching a maximum by 5 min (Fig. 1A). However, pretreatment with 10 ␮mol/l BAPTA-AM (a cell-permeable Ca2⫹ chelator) completely prevented the ethanol-elicited rise in [Ca2⫹]i (Fig. 1, A–C). To verify whether the IP3R functional activity is involved, we analyzed the IP3R-gated [Ca2⫹]i release in response to stimulation with ethanol. Pretreatment of Caco-2 cells with 1 ␮mol/l Xestospongin C (selective and reversible IP3R antagonist) for 5 min delayed and partially inhibited the ethanolinduced elevation in [Ca2⫹]i (Fig. 1, A–C). Ethanol significantly reduced TEER and increased FITC-D4 permeation (Fig. 2, A and B; both P ⬍ 0.0001 vs. control), which was significantly attenuated after pretreatment of cell monolayers with BAPTA-AM (Fig. 2, A and B; both P ⬍ 0.0001 vs. ethanol). BAPTA-AM pretreatment also attenuated ethanol-induced redistribution of ZO-1 and occludin from the intercellular junctions into the intracellular compartment (Fig. 2E). BAPTA-AM treatment by itself did not have a significant effect on TEER, FITC-D4 permeation, or distribution of ZO-1 or occludin (data not shown). IP3R-gated intracellular Ca2⫹ release is involved in ethanol-induced TJ disruption. Next, the effects of IP3R inhibition on barrier function were explored. Pretreatment of cell monolayers with Xestospongin C significantly attenuated the ethanol-induced decrease in TEER and increase in FITC-D4 per-

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Fig. 1. Ethanol increases free intracellular Ca2⫹ in Caco-2 cell monolayers. A: real-time change in free intracellular calcium levels was monitored for up to 20 min following Fluo-3 AM loading, before and after addition of 40 mmol/l ethanol (EtOH) in the presence or absence of 10 ␮mol/l 1,2-Bis(2-Aminophenoxy) ethane-N,N,N=,N=-tetraacetic acid (BAPTA-AM) or 1 ␮mol/l Xestospongin C (Xe-C). B: relative fluorescence intensity levels after 5 min are quantified and shown. Data represent means ⫾ SD of 3 independent experiments. *P ⬍ 0.0001 vs. baseline values, #P ⬍ 0.0001 and **P ⬍ 0.001 vs. ethanol. C: representative images of intracellular Ca2⫹ release. Caco-2 cells were loaded with Fluo-3, exposed to ethanol for 5 min, and examined by confocal microscopy. Scale bars ⫽ 10 ␮m. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00236.2013 • www.ajpgi.org

INTRACELLULAR CALCIUM IN ETHANOL-INDUCED BARRIER DYSFUNCTION

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Fig. 2. Free intracellular Ca2⫹ and inositol 1,4,5-triphosphate (IP3) receptor-mediated intracellular calcium release are required for ethanol-induced tight junction disruption. Caco-2 cell monolayers were pretreated with either 10 ␮mol/L BAPTA-AM or 1 ␮mol/L Xestospongin C, followed by exposure to 40 mmol/l ethanol or medium only as controls for 3 h. Thereafter, transepithelial electrical resistance (TEER) (A and C) and FITC-D4 permeation (B and D) were measured, and values represent means ⫾ SD of 3 independent experiments. *P ⬍ 0.0001 vs. control and #P ⬍ 0.0001 vs. ethanol. E: monolayers exposed to ethanol with or without BAPTA and Xe-C were fixed and stained for zonula occludens (ZO)-1 (green) and occludin (red) by immunofluorescence staining. Scale bars ⫽ 10 ␮m.

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meability (Fig. 2, C and D; both P ⬍ 0.001 vs. ethanol). Xestospongin C also prevented ethanol-induced redistribution of ZO-1 and occludin from intercellular junctions to intracellular compartments (Fig. 2E). Xestospongin C, by itself, did not alter TEER or FITC-D4 in the absence of ethanol (Fig. 2, C and 2D).

Ethanol-induced Rho/ROCK activation is mediated by IP3Rgated endoplasmic reticulum Ca2⫹ release. To test whether ethanol can activate ROCK and whether that Rho/ROCK activation is mediated by IP3R-gated intracellular Ca2⫹ release, phosphorylation of MBS/MYPT1 protein was evaluated by dot-blot analysis. The effects of ethanol on total and

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phosphorylated MBS/MYPT1 are shown in a representative dot blot (Fig. 3A). Densitometric analysis revealed no significant differences in the total MBS/MYPT1 between treatment conditions. However, MBS/MYPT1 phosphorylation was significantly increased in Caco-2 cells after incubation with 40 mmol/l ethanol compared with control (Fig. 3B). The ethanol-induced MBS/MYPT1 phosphorylation was significantly attenuated by pretreatment with Xestospongin C and BAPTA-AM compared with ethanol alone (Fig. 3, A and B). In accordance with dot-plot data, ethanol enhanced immunofluorescent staining of MBS/MYPT1 phosphorylation mainly at cortical regions, which was reduced on pretreatments with either Xestospongin C or BAPTA-AM (Fig. 3C). Protein levels of phosphorylated MBS/MYPT1, determined by cell-based ELISA, were significantly increased after exposure to ethanol compared with the total Snail levels in untreated controls (P ⬍ 0.0001; Fig. 3D). Pretreatment with either Xestospongin C or BAPTA-AM significantly attenuated the ethanol-induced increase in phosphorylated MBS/MYPT1 protein levels (both P ⬍ 0.0001; Fig. 3D). Rho/ROCK activation is involved in ethanol-induced barrier disruption. We evaluated the effect of Y27632, a potent and selective Rho/ROCK inhibitor, on ethanol-induced barrier dysfunction. The ethanol-induced decrease in TEER (Fig. 4A) and increases in FITC-D4 permeation (Fig. 4B) were almost completely reversed by pretreatment with Y27632. Immunofluorescent staining showed that the ethanol-induced permeability was associated with ZO-1 redistribution, correlating with increased MBS/MYPT1 phosphorylation (Fig. 4). CY27632 attenuated ZO-1 redistribution and prevented ethanol-induced MBS/MYPT1 phosphorylation (Fig. 4C). Y27632 by itself had no significant effect on TEER, permeability, or distribution of

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ZO-1 (Fig. 4, A–C). To complement these data, dot blotting was performed, showing that expression of phosphorylated MBS/MYPT1 increased in Caco-2 cells incubated with ethanol and decreased after pretreatment with Y27632 (Fig. 4D). Densitometric analysis of the dots showed that ethanol significantly enhanced phosphorylated MBS/MYPT1 protein expression compared with control cells (P ⬍ 0.0001), which could be attenuated by Y27632 (P ⬍ 0.01 vs. ethanol alone; Fig. 4E). Rho/ROCK is involved in ethanol-induced changes in AJs and F-actin. Because TJ formation is dependent on AJs (16) and actin remodeling plays a crucial role in junction assembly (24), we examined whether Rho/ROCK plays a role in ethanolinduced disruption of AJs and F-actin cytoskeleton. Immunofluorescence staining for the AJ proteins indicated that ethanol exposure induced redistribution of E-cadherin and ␤-catenin from the intercellular junctions into intracellular compartments (indicated with white arrowheads), and this effect of ethanol was attenuated by Y27632 treatment (Fig. 5A). In addition, exposure to ethanol resulted in a significant increase in intracellular F-actin content compared with control (P ⬍ 0.0001; Fig. 5B), which was significantly reduced by pretreatment with Rho/ROCK inhibitor, Y27632 (P ⬍ 0.05; Fig. 5B), and was found to be comparable with the control condition. Figure 5C shows that, under control conditions, F-actin microfilaments were assembled into a prominent perijunctional F-actin belt that encircled cell borders. In contrast, after ethanol exposure, the F-actin microfilaments were markedly disorganized, in which the perijunctional actin belt was transformed into an array of disordered filaments and stress fiber-like bundles (indicated with white arrowheads), which could be attenuated by pretreatment with Y27632 (Fig. 5C).

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Fig. 3. Ethanol induces myosin-binding subunit/myosin phosphatase-targeting subunit 1 (MBS/MYPT1) phosphorylation (Rho/ROCK activation) by an intracellular Ca2⫹-dependent mechanism. Caco-2 cells grown on glass-bottom dishes were treated with 40 mmol/l ethanol in the presence or absence of 10 ␮mol/l BAPTA-AM or Xestospongin C for 3 h. A: representative dot blot analysis of total and phosphorylated MBS/MYPT1 expression. B: densitometric analysis of 3 different experiments. Values are means ⫾ SD of, n ⫽ 3. *P ⬍ 0.0001 vs. control, &P ⬍ 0.001 and #P ⬍ 0.0001 vs. ethanol only-treated cell monolayers. C: representative images of phosphorylated MBS/MYPT1 from 3 independent experiments. Nuclei were stained with DAPI, and the scale bar ⫽ 10 ␮m. D: Cell-based ELISA analysis of *P ⬍ 0.0001 vs. control, &P ⬍ 0.0001 and #P ⬍ 0.0001 vs. ethanol-treated cells in the absence of BAPTA-AM. AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00236.2013 • www.ajpgi.org

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Fig. 4. Rho/ROCK activation mediates ethanol-induced disruption of the tight junction in Caco-2 monolayers. Caco-2 cell monolayers were pretreated with Rho/ROCK inhibitor Y27632 (100 ␮mol/l) for 1 h, followed by exposure to ethanol (40 mmol/l) for 3 h. TEER (A) and FITC-D4 permeation (B) were measured, and values represent means ⫾ SD of 3 independent experiments. *P ⬍ 0.05 vs. control and #P ⬍ 0.05 vs. ethanol-treated cells in the absence of Y27632. C: cell monolayers incubated with ethanol for 3 h in the presence or absence of Y27632 were fixed and stained for ZO-1 (green), phosphorylated MBS/MYPT1 (red), and nuclei (blue) by immunofluorescence. Images are representative of at least 3 independent experiments. Scale bars ⫽ 10 ␮m. D: representative dot blot analysis of phosphorylated MBS/MYPT1 protein expression in Caco-2 cells incubated with EtOH in the absence or presence of pretreatment with Y27632. E: densitometric analysis of 3 different experiments. Values are means ⫾ SD of, n ⫽ 3. *P ⬍ 0.0001 vs. control and #P ⬍ 0.01 vs. ethanol-treated cell monolayers in the absence of Y27632.

DISCUSSION

The present study indicates that ethanol triggers a rise in free intracellular [Ca2⫹]i, mediated at least in part by IP3R-gated release of Ca2⫹ from endoplasmic reticulum (ER). Furthermore, our study also shows that free [Ca2⫹]i mediates ethanolinduced activation of Rho/ROCK with subsequent loss of TJ and AJ integrity and disruption of F-actin organization and, consequently, barrier dysfunction. Research on the role of [Ca2⫹]i in ethanol-induced cell injury has mainly focused on gastric epithelium, providing evidence that accumulation of [Ca2⫹]i mediates ethanol-induced gap junction dysfunction, resulting in cell shrinkage (40) and gastric epithelial cell injury (38). So far, studies investigating the role of [Ca2⫹]i in ethanol-induced intestinal epithelial barrier function are lacking. Herein, abolishment of ethanol-induced rapid increase in [Ca2⫹]i and attenuation of increased permeability by BAPTA-AM indicate that [Ca2⫹]i mediates ethanol-induced TJ disruption. Our observations are in line with previous studies demonstrating that elevation in intracellular calcium [Ca2⫹]i disrupts TJ integrity in Caco-2 cells and lowers transepithelial resistance in T84 monolayers via mechanisms involving protein kinase C activation (34, 49). In addition, it has recently been shown that rise in [Ca2⫹]i is required for osmotic stress-induced TJ disruption in Caco-2 monolayers (51). The finding that rise in free [Ca2⫹]i mediates ethanolinduced TJ disruption raised the question of the source of the observed elevation. One option could be the ER, which can contribute to elevation of free [Ca2⫹]i through IP3R-gated

channels (51). IP3R upon activation stimulates Ca2⫹ release from the ER lumen to the cytoplasm (6). We observed that pretreatment of Caco-2 monolayers with Xestospongin C, a selective antagonist of IP3R, can attenuate the ethanol-induced increase in [Ca2⫹]i and TJ disruption, indicating involvement of IP3R-operated ER Ca2⫹ release. This interpretation is compatible with findings from a prior study demonstrating in brain microvascular endothelial cell monolayers that IP3R-mediated increase in [Ca2⫹]i is required for ethanol-induced impairment of blood-brain-barrier function (52). In our study, effects of antagonizing IP3R on the ethanol-induced rise in [Ca2⫹]i and barrier dysfunction were partial, indicating involvement of other Ca2⫹ sources and mechanism(s) in ethanol-induced barrier disruption. Samak et al. (51) have demonstrated that influx of Ca2⫹ through extracellular calcium channels located on the apical membrane of intestinal epithelial cells such as L-type voltage-gated channels, i.e., Cav 1.3, can result in a rise in intracellular calcium [Ca2⫹]i and, consequently, disruption of the TJs via mechanisms involving JNK2. Because we used a Ca-free buffer in this study, influx of Ca2⫹ through extracellular calcium channels is excluded. However, other intracellular sources such as mitochondria should be considered (22). Whether mitochondrial calcium release can contribute to the observed increase in [Ca2⫹]i cannot be answered at this time, and it merits further investigations. In our study, how ethanol modulates IP3R was not examined. However, ethanol has been shown to upregulate and increase IP3R gene and protein levels, respectively. These effects could be abolished by treatment with either the inhibitor

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INTRACELLULAR CALCIUM IN ETHANOL-INDUCED BARRIER DYSFUNCTION E-cadherin

β-catenin

DAPI

B

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Relative F-actin Content

A Control

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Fig. 5. Rho/ROCK activation mediates ethanol-induced disruption of the adherens junctions in Caco-2 monolayers. Caco-2 cell monolayers incubated with ethanol (40 ␮/l) for 3 h with or without Y27632 (100 ␮mol/l) pretreatment were fixed and triple stained for E-cadherin (green), ␤-catenin (red), and nuclei (blue) by immunofluorescence (A). Images are representative of at least 3 independent experiments. Scale bars ⫽ 10 ␮m. F-actin contents were assessed by a cell-based fluorescent phalloidin-binding assay (B). Data bars represent means ⫾ SD of 3 independent experiments. *P ⬍ 0.05 vs. control and #P ⬍ 0.05 vs. ethanol-treated cells in the absence of Y27632. Representative images of immunostaining of F-actin microfilaments (C). Caco-2 cell monolayers incubated with ethanol for with or without Y27632 pretreatment were fixed and stained with rhodamine-phalloidin dye (red) in 3 independent experiments. Scale bar ⫽ 10 ␮m.

of the ethanol-metabolizing enzyme alcohol dehydrogenase (ADH) 4-methylpyrazole (4-MP) or the antioxidant uric acid (52), indicating involvement of acetaldehyde and oxidative stress. Because Caco-2 cells used in or study do not express ADH as capable of metabolizing ethanol into acetaldehyde (30), it is justifiable that our observations are due to ethanol itself rather than acetaldehyde. Because acetaldehyde also disrupts intestinal barrier function (47), future studies examining modular effects of acetaldehyde on IP3R-operated ER Ca2⫹ release are required. Elevation of free [Ca2⫹]i achieved by either intracellular or extracellular sources has been shown to induce Rho/ROCK kinase activation with subsequent TJ disruption (44). A significant body of evidence indicates that Rho kinase regulates cytoskeleton proteins including microtubules (57, 58) and, indirectly, assembly of the TJ in T84 (63) and Caco-2 cells (53). Among many effectors of Rho, Rho/ROCK has been shown to mediate RhoA-induced disassembly of intercellular junctions and formation of stress fibers (2, 18). Such effects have been attributed to Rho/ROCK-induced P-MBS/MYPT1 (23), the regulatory units of MLC, thereby inhibiting MLC phosphatase and, subsequently, MLC phosphorylation (42). In the present study, we observed that ethanol phosphorylates MBS/MYPT1 at Thr853, indicating Rho/ROCK activation. This activation could be attenuated by pretreatment with Xestospongin C and BAPTA-AM. Therefore, we reasoned that [Ca2⫹]i mediates ethanol-induced ROCK activation. The observed changes can also be considered a rapid defensive response to overcome the noxious effects of ethanol. This would be in line with observations of Rao et al. (45) and Ray et al. (48), showing that a raise in free [Ca2⫹]i is necessary for activating RhoA and that activation of RhoA plays a pivotal

role in polyamine-induced epithelial cell migration after wounding of an epithelial cell layer. Evidence is accumulating for a role of Rho/ROCK signaling in mediating disruption of AJs and TJs in vitro (50). We confirmed that ethanol decreased TEER and increased FITC-D4 permeation in association with redistribution of ZO-1, E-cadherin, and ␤-catenin, which was effectively attenuated by the ROCK inhibitor Y27632. While the present work was in progress, Tong et al. (29, 59) have reported in Caco-2 monolayers that upregulation of RhoA mRNA expression and its activation via iNOS mediate ethanol MLC phosphorylation and, consequently, TJ disruption. However, in these studies, the role of the downstream effector of RhoA, ROCK, was not examined. Our study reported for the first time that ethanol-induced increase in [Ca2⫹]i activates Rho/ROCK, resulting in the loss of TJ and AJ integrity and, consequently, barrier dysfunction. Our data also demonstrate that ethanol increases cellular F-actin contents and induces disorganization of the F-actin ring, which could also be abrogated by Y27632, indicating involvement of Rho/ROCK activity in ethanol-induced remodeling of cytoskeleton. These observations are in line with previous findings showing that ethanol increases paracellular permeability by disrupting the F-actin ring in Caco-2 cells (33) and are in agreement with prior data showing that ROCK activity, at least in endothelial cells, is required for F-actin disruption and barrier dysfunction (25, 62). Previously, changes in actin filament organization has been demonstrated to be regulated through globular G-/F-actin equilibria (8), alterations in the amount and type of actin-binding proteins (35), and assembly of myosin filaments (9). Because activation of ROCK modulates F-actin organization and causes stress fiber formation but not actin disruption (21), disassembly

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observed in our study can be attributed to other cell-signaling messengers and pathways, including increased free [Ca2⫹]i (26), protein kinase C and MLCK (39), and phosphatases (13). Mechanistic studies have indicated key roles for iNOS-mediated stress (3) and cytochrome-P450 2E1 (CYP2E1)-mediated oxidative stress (36), remodeling of actin and microtubules (3, 10), and activation of MLCK in ethanol-induced intestinal barrier disruption (32, 33). In the present study, a possible crosstalk between MLCK and Rho/ROCK pathways cannot be excluded, as each pathway can be either selectively induced or coinduced. Previously, it has been suggested that MLCK activation may initiate impairment of epithelial barrier, which is then followed by Rho/ROCK-dependent junctional disassembly and, consequently, barrier disruption (25). The mechanism(s) associated with Ca2⫹-induced Rho/ROCK activation and how MLCK interacts with Rho/ROCK in ethanol-induced intestinal hyperpermeability merit further investigation. In summary, the results of the current study demonstrate that ethanol induces a rapid increase in [Ca2⫹]i, mediated in part by IP3R-gated ER Ca2⫹ release. This increase in [Ca2⫹]i activates ROCK with subsequent disruption of apical junctional complex and actin cytoskeleton and, consequently, intestinal epithelial hyperpermeability. Because the crosstalk between intracellular Ca2⫹ homeostasis and Rho kinase contributes to many intracellular signaling networks, further delineation of their roles in ethanol-induced gut leakiness may shed light on their potential as therapeutic or preventive targets for ethanol-related gut-liver axis diseases including ALD. GRANTS This work was funded by the Top Institute Food and Nutrition. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: E.E.E., A.A.M., J.D., and D.M.J. conception and design of research; E.E.E. performed experiments; E.E.E. analyzed data; E.E.E., A.A.M., J.D., and D.M.J. interpreted results of experiments; E.E.E. prepared figures; E.E.E. and D.M.J. drafted manuscript; E.E.E., A.A.M., J.D., and D.M.J. edited and revised manuscript; E.E.E. and A.A.M. approved final version of manuscript. REFERENCES 1. Abramoff MD, Magelhaes PJ, Ram SJ. Image Processing with ImageJ. Biophotonics Int 11: 36 –42, 2004. 2. Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y, Kaibuchi K. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 275: 1308 –1311, 1997. 3. Banan A, Choudhary S, Zhang Y, Fields JZ, Keshavarzian A. Ethanolinduced barrier dysfunction and its prevention by growth factors in human intestinal monolayers: evidence for oxidative and cytoskeletal mechanisms. J Pharmacol Exp Ther 291: 1075–1085, 1999. 4. Barhoumi R, Awooda I, Mouneimne Y, Safe S, Burghardt RC. Effects of benzo-a-pyrene on oxytocin-induced Ca2⫹ oscillations in myometrial cells. Toxicol Lett 165: 133–141, 2006. 5. Baumgart DC, Dignass AU. Intestinal barrier function. Curr Opin Clin Nutr Metab Care 5: 685–694, 2002. 6. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4: 517–529, 2003. 7. Bode C, Bode JC. Activation of the innate immune system and alcoholic liver disease: effects of ethanol per se or enhanced intestinal translocation of bacterial toxins induced by ethanol? Alcohol Clin Exp Res 29: 166S– 171S, 2005.

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