Regulation of Tight Junction Permeability and Occludin Phosphorylation by RhoA-p160ROCK-dependent and -independent Mechanisms
2001; Elsevier BV; Volume: 276; Issue: 13 Linguagem: Inglês
10.1074/jbc.m007136200
ISSN1083-351X
AutoresTetsuaki Hirase, Seinosuke Kawashima, Elaine Y.M. Wong, Tomomi Ueyama, Yoshiyuki Rikitake, Shöichiro Tsukita, Mitsuhiro Yokoyama, James M. Staddon,
Tópico(s)S100 Proteins and Annexins
ResumoIn epithelial and endothelial cells, tight junctions regulate the paracellular permeability of ions and proteins. Disruption of tight junctions by inflammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophosphatidic acid and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase. Cytoskeletal changes induced by these agents included stimulation of stress fiber formation and myosin light chain phosphorylation. Additionally, occludin, a tight junction protein, was a target for signaling events triggered by lysophosphatidic acid and histamine, events that resulted in its phosphorylation. A dominant-negative mutant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y-27632, prevented stress fiber formation, myosin light chain phosphorylation, occludin phosphorylation, and the increase in tracer flux in response to lysophosphatidic acid. In contrast, although RhoA T19N and Y-27632 blocked the cytoskeletal events induced by histamine, they had no effect on the stimulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability independently of cytoskeletal events. Thus, occludin is a target for receptor-initiated signaling events regulating its phosphorylation, and this phosphorylation may be a key regulator of tight junction permeability. In epithelial and endothelial cells, tight junctions regulate the paracellular permeability of ions and proteins. Disruption of tight junctions by inflammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophosphatidic acid and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase. Cytoskeletal changes induced by these agents included stimulation of stress fiber formation and myosin light chain phosphorylation. Additionally, occludin, a tight junction protein, was a target for signaling events triggered by lysophosphatidic acid and histamine, events that resulted in its phosphorylation. A dominant-negative mutant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y-27632, prevented stress fiber formation, myosin light chain phosphorylation, occludin phosphorylation, and the increase in tracer flux in response to lysophosphatidic acid. In contrast, although RhoA T19N and Y-27632 blocked the cytoskeletal events induced by histamine, they had no effect on the stimulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability independently of cytoskeletal events. Thus, occludin is a target for receptor-initiated signaling events regulating its phosphorylation, and this phosphorylation may be a key regulator of tight junction permeability. tight junction 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid horseradish peroxidase lysophosphatidic acid myosin light chain Thr18- and Ser19-phosphorylated MLC Dulbecco's modified Eagle's medium phosphate-buffered saline polyacrylamide gel electrophoresis immunoprecipitation 4-morpholineethanesulfonic acid The tight junction (TJ)1is localized at cell-cell contact sites in epithelial and endothelial cells. It serves as a paracellular barrier to restrict the movement of ions and proteins across tissue boundaries (1Schneeberger E.E. Lynch R.D. Am. J. Physiol. 1992; 262: L647-L661PubMed Google Scholar, 2Gumbiner B. Am. J. Physiol. 1987; 253: C749-C758Crossref PubMed Google Scholar, 3Gumbiner B.M. J. Cell Biol. 1993; 123: 1631-1633Crossref PubMed Scopus (344) Google Scholar, 4Staddon J.M. Rubin L.L. Curr. Opin. Neurobiol. 1996; 6: 622-627Crossref PubMed Scopus (96) Google Scholar). This barrier function is essential for the maintenance of tissue environments. Dysfunction of the TJ occurs in response to a variety of inflammatory stimuli and also during ischemia, leading to tissue edema and damage. Therefore, analysis of TJ regulation could lead to an understanding of normal physiology as well as pathology and to the identification of novel therapeutic targets. The molecular components of the TJ are being discovered and so far include ZO-1 (5Stevenson B.R. Siliciano J.D. Mooseker M.S. 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Cell Biol. 1996; 134: 1003-1018Crossref PubMed Scopus (267) Google Scholar). In addition to these peripheral membrane proteins, occludin was discovered as an integral membrane protein of the TJ having four transmembrane domains (12Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2160) Google Scholar). The carboxy tail of occludin is linked to the actin cytoskeleton via ZO-1, ZO-2 and ZO-3 (13Furuse M. Itoh M. Hirase T. Nagafuchi A. Yonemura S. Tsukita S. J. Cell Biol. 1994; 127: 1617-1626Crossref PubMed Scopus (816) Google Scholar, 14Fanning A.S. Jameson B.J. Jesaitis L.A. Anderson J.M. J. Biol. Chem. 1998; 273: 29745-29753Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar, 15Itoh M. Morita K. Tsukita S. J. Biol. Chem. 1999; 274: 5981-5986Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Recent work has also identified members of the claudin family as TJ components that have four transmembrane domains but no sequence similarity to occludin (16Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1746) Google Scholar, 17Morita K. Furuse M. Fujimoto K. Tsukita S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 511-516Crossref PubMed Scopus (985) Google Scholar, 18Tsukita S. Furuse M. Trends Cell Biol. 1999; 9: 268-273Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). It has not yet been defined how these novel proteins interact with occludin or other TJ components. However, as the protein architecture of the TJ is revealed, analysis of function of the TJ on a molecular basis becomes possible. The formation and maintenance of the TJ has been considered to be regulated not only by the specific proteins of cell-cell junctions but also by the perijunctional actin cytoskeleton (19Madara J.L. Annu. Rev. Physiol. 1998; 60: 143-159Crossref PubMed Scopus (465) Google Scholar). Botulinum C3 toxin, which ADP-ribosylates and inactivates Rho, has been shown to disrupt perijunctional actin, resulting in TJ dysfunction in epithelial cells (20Nusrat A. Giry M. Turner J.R. Colgan S.P. Parkos C.A. Carnes D. Lemichez E. Boquet P. Madara J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10629-10633Crossref PubMed Scopus (359) Google Scholar). Also, mutants of RhoA and Rac1 disrupted TJ functions (21Jou T.S. Schneeberger E.E. Nelson W.J. J. Cell Biol. 1998; 142: 101-115Crossref PubMed Scopus (309) Google Scholar). Thus, signaling pathways transduced by the Ras-related small GTPase Rho family members like Rho and Rac1, which control the actin cytoskeleton, have been implicated in the regulation of the TJ (see Refs. 19Madara J.L. Annu. Rev. Physiol. 1998; 60: 143-159Crossref PubMed Scopus (465) Google Scholar and 22Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5242) Google Scholar), whereas involvement of downstream signaling events of Rho in TJ function is still lacking. Recently, several downstream targets of Rho have been identified (see Refs. 22Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5242) Google Scholar and 23Narumiya S. Ishizaki T. Watanabe N. FEBS Lett. 1997; 410: 68-72Crossref PubMed Scopus (331) Google Scholar). Among these, p160ROCK/Rho kinase (24Matsui T. Amano M. Yamamoto T. Chihara K. Nakafuku M. Ito M. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. EMBO J. 1996; 15: 2208-2216Crossref PubMed Scopus (946) Google Scholar, 25Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2562) Google Scholar), one of the key effectors of Rho, has been shown to be a serine-threonine kinase that is involved in the regulation of actin organization, cellular morphology, and cellular transformation (26Ishizaki T. Naito M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. FEBS Lett. 1997; 404: 118-124Crossref PubMed Scopus (458) Google Scholar,27Sahai E. Ishizaki T. Narumiya S. Treisman R. Curr. Biol. 1999; 9: 136-145Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). In this study, the basis of effects of inflammatory stimuli on TJ permeability was investigated. The role of the cytoskeleton and the possibility of direct effects on occludin were explored. Using ECV304 cells as a model system, lysophosphatidic acid (LPA) and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase (HRP). LPA is a glycerophospholipid that is secreted from activated platelets, mediating tissue regeneration and wound healing (28Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar), and can induce an increase in TJ permeability in brain endothelial cells (29Schulze C. Smales C. Rubin L.L. Staddon J.M. J. Neurochem. 1997; 68: 991-1000Crossref PubMed Scopus (158) Google Scholar). Also, histamine causes vascular leakinessin vivo, but mechanisms are not completely clear yet (see Ref. 30McDonald D.M. Thurston G. Baluk P. Microcirculation. 1999; 6: 7-22Crossref PubMed Google Scholar). Because LPA activates Rho and its targets (22Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5242) Google Scholar, 28Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar), the involvement of these was studied using a dominant-negative mutant of RhoA (RhoA T19N) considered to be the inactive GDP-bound form of RhoA (31Gebbink M.F.B.G. Kranenburg O. Poland M. van Horck F.P.G. Houssa B. Moolenaar W.H. J. Cell Biol. 1997; 137: 1603-1613Crossref PubMed Scopus (140) Google Scholar, 32Kranenburg O. Poland M. Gebbink M. Oomen L. Moolenaar W.H. J. Cell Sci. 1997; 110: 2417-2427Crossref PubMed Google Scholar) and a specific p160ROCK inhibitor Y-27632 (25Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2562) Google Scholar). Evidence is provided that TJ permeability is regulated by RhoA-p160ROCK-dependent and -independent mechanisms and that occludin is a target for receptor-initiated signaling events regulating its phosphorylation. All tissue culture materials were from Life Technologies, Inc. Gel electrophoresis reagents were from Bio-Rad. HRP was from Sigma. Cytochalasin D and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) were from Calbiochem. Other reagents used were of the highest grade commercially available. Y-27632 (25Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2562) Google Scholar), a specific inhibitor of p160ROCK, was a generous gift from WelFide (Osaka, Japan). Pervanadate was prepared as described previously (33Staddon J.M. Herrenknecht K. Smales C. Rubin L.L. J. Cell Sci. 1995; 108: 609-619Crossref PubMed Google Scholar). The rat monoclonal antibody (MOC37) and rabbit polyclonal antibody were both raised against a fragment of occludin fused to glutathione S-transferase (34Saitou M. Ando-Akatsuka Y. Itoh M. Furuse M. Inazawa J. Fujimoto K. Tsukita S. Eur. J. Cell Biol. 1997; 73: 222-231PubMed Google Scholar). The anti-ZO-1 monoclonal antibody and the anti-phosphotyrosine antibody PY20 were from Transduction Laboratories (Lexington, KY). The anti-phosphotyrosine antibody 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). The monoclonal antibody against RhoA was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A purified polyclonal antibody recognizing Thr18- and Ser19-phosphorylated myosin light chain (MLC) was raised against the synthetic peptide RPQRApTpSNVFAMK (where p indicates phosphorylation), as described previously (35Ratcliffe M.J. Smales C. Staddon J.M. Biochem. J. 1999; 338: 471-478Crossref PubMed Scopus (61) Google Scholar). ECV304 cells were obtained from the European Collection of Animal Cell Cultures (Salisbury, UK) and cultured at 37 °C under an atmosphere of 5% CO2 in DME containing 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. RhoA T19N, from Professor Yoshimi Takai (Osaka University, Osaka, Japan), or LacZ was placed into pAdex1CAwt under a CA promoter comprising a cytomegalovirus enhancer and a chicken β-actin promoter (36Niwa H. Yamamura K. Miyazaki J. Gene. 1991; 108: 193-199Crossref PubMed Scopus (4626) Google Scholar) to give pAdex RhoA T19N and pAdex LacZ, respectively. A recombinant adenovirus was constructed by in vitro homologous recombination in 293 cells using pAdex RhoA T19N or pAdex LacZ and the adenovirus DNA-terminal protein complex by a method previously described (37Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (789) Google Scholar). After ECV304 cells had attained confluence, they were infected with recombinant adenovirus expressing either LacZ or RhoA T19N. The viruses were diluted in serum-depleted medium at a multiplicity of infection of 30 particles/cell and incubated for 60 min. The viral suspension was removed by washing twice with serum-depleted DME, and the cells were cultured with serum-depleted DME for 48 h. ECV304 cells were seeded onto 0.4-μm polycarbonate Transwell filters (Costar Corp., Cambridge, MA). After attaining confluence, the cells were incubated with serum-depleted DME with or without recombinant adenoviral gene transfer for 48 h. For pretreatment with compound, vehicle or Y-27632 at a final concentration of 10 μm was added, and the incubation was continued for 60 min. Medium was then replaced with fresh serum-free DME in the presence or absence of 10 μm Y-27632 and agonists at the indicated concentrations. To the upper chambers, HRP dissolved in serum-free DME was added to give a final concentration of 0.5 μm. The upper chambers contained 200 μl of medium, and the lower chambers contained 800 μl of medium. One hour after the start of the experiment, 50 μl of medium was collected from the lower chambers. The HRP content of the samples was evaluated spectrophotometrically by assaying peroxidase activity in buffer containing 0.5 mm guaiacol, 50 mmNa2HPO4, and 0.6 mmH2O2 and measuring absorbance at 470 nm. Data from five independent experiments, each in triplicate, are shown as the means ± S.E. Statistical significance, calculated using Student's t test, was taken as p < 0.001. All procedures were performed at room temperature. Cells were fixed in 3% paraformaldehyde in PBS for 15 min. After fixation, the cells were rinsed and permeabilized by incubation with 0.2% Triton X-100 in PBS for 15 min. After rinsing, the cells were blocked in 1% BSA in PBS for 15 min and incubated with 100 ng/ml fluorescein isothiocyanate-conjugated phalloidin (Molecular Probes, Inc., Eugene, OR) in blocking solution for 60 min. After the final rinse, the cells were mounted with fluorescent mounting medium (DAKO, Carpinteria, CA) and examined using a AxioskopTMfluorescence microscope (Carl Zeiss, Inc., Thornwood, NY) fitted with 100× objectives. Photographs were taken using 400 ASA T-MAX film (Eastman Kodak Co.). The protein content of samples was determined using the Bio-Rad protein assay. Samples were resolved by one-dimensional SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride membranes (0.2 μm pore size; ATTO, Tokyo, Japan). The membrane was subjected to immunoblotting as described previously (38Hirase T. Staddon J.M. Saitou M. Ando-Akatsuka Y. Itoh M. Furuse M. Fujimoto K. Tsukita S. Rubin L.L. J. Cell Sci. 1997; 110: 1603-1613Crossref PubMed Google Scholar). Cells were rinsed twice with ice-cold PBS containing 0.9 mm CaCl2 and 0.5 mm MgCl2 and then lysed with boiling-hot SDS-IP buffer (25 mm Hepes/NaOH, pH 7.4, 4 mm EDTA, 25 mm NaF, 1% SDS, 1 mmNa3VO4). After the lysates had been heated at 100 °C for 3 min and cooled, a 9-fold volume of ice-cold Nonidet P-40-IP buffer (25 mm Hepes/NaOH, pH 7.4, 150 mm NaCl, 4 mm EDTA, 25 mm NaF, 1% Nonidet P-40, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin) was added. Lysates were passed 10 times through a 27-gauge needle and then gently mixed for 30 min at 4 °C. After centrifugation (10,000 × g for 30 min), the supernatant was collected. For immunoprecipitation, 4 μl of anti-occludin polyclonal antibody and a 15-μl bed volume of GammaBind Plus Sepharose (Amersham Pharmacia Biotech) were added to each sample and mixed for 3 h at 4 °C. Beads were washed five times with 1 ml of Nonidet P-40-IP buffer, from which immunoprecipitates were eluted by boiling in Laemmli sample buffer (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar) for 5 min. Samples were then separated by gel electrophoresis followed by immunoblotting. After immunoprecipitation, the beads were washed three times with 1 ml of Nonidet P-40-IP buffer and three times with 1 ml of either AP buffer (50 mmTris-HCl, pH 9.0, 1 mm MgCl2, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride) for alkaline phosphatase treatment or LP buffer (50 mmTris-HCl, pH 7.5, 1 mm MnCl2, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride) for λ protein phosphatase treatment. They were then resuspended in 100 μl of AP buffer or LP buffer and incubated with or without calf intestine alkaline phosphatase (Takara Shuzo Co., Ltd., Ohtsu, Japan) or with or without λ protein phosphatase (New England Biolabs, Inc., Beverly, MA), respectively. To block phosphatase activity, a phosphatase inhibitor mixture (100 mm β-glycerophosphate, 25 mm NaF, 4 mm EDTA, 1 mmNa3VO4) was used. After a 1-h incubation at 30 °C with occasional mixing, beads were washed three times with 1 ml of Nonidet P-40-IP buffer and boiled with Laemmli sample buffer to elute the immunoprecipitates. Confluent cultures of ECV304 cells on 9-cm diameter dishes were rinsed twice with phosphate-free M199 containing 0.5% fetal calf serum and 2 mm glutamine. The cells were then incubated in 10 ml of this medium containing 1 mCi [32P]orthophosphate (ICN Biomedicals, Costa Mesa, CA) for 4 h under an atmosphere of 5% CO2. Vehicle or factors were then added as required, and the incubation was continued for an additional 10 min. At 4 °C, the cultures were then rapidly rinsed twice in PBS (magnesium- and calcium-free) and lysed in 1 ml of extraction buffer containing 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 25 mm Hepes, 2 mm EDTA, 0.1 m NaCl, 25 mm NaF, 1 mm Na3VO4, pH 7.6 (adjusted with NaOH), 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml soybean trypsin inhibitor, 0.1 units/ml α2-macroglobulin, and 10 μg/ml leupeptin. The extracts were collected by scraping the dish and then centrifuged at 10,000 × g for 10 min. After preclearing with protein A-Sepharose (Amersham Pharmacia Biotech), occludin was immunoprecipitated using 2.5 μg of anti-occludin antibody (Zymed Laboratories Inc., South San Francisco, CA) in the presence of protein A-Sepharose. The beads were washed five times with extraction buffer, and protein was eluted into Laemmli sample buffer, resolved by SDS-PAGE (8% acrylamide), and transferred to Immobilon P membrane (Millipore, Bedford, MA). [32P]Phosphate-labeled protein was detected by autoradiography, and occludin protein was then detected by probing the filters with the anti-occludin antibody. In this manner, after quantitation of band intensity by densitometry, the relative amount of radiolabeled phosphate per occludin protein could be estimated. Phosphoamino acid analysis was performed on the labeled bands according to the procedures described previously (40Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). For two-dimensional gel electrophoresis, immunoprecipitated occludin from [32P]orthophosphate-labeled cells was solubilized in 50 μl of buffer containing 0.5% SDS, 0.1 mdithiothreitol, 1 mm EDTA, 25 mm Tris/HCl, pH 8.0, and heated at 100 °C for 5 min. The eluted occludin was then freeze-dried and redissolved in 50 μl of a solution containing 9.5m urea, 4% Triton X-100, 0.1 m dithiothreitol, 2% Pharmalyte® 3.5–10 (Amersham Pharmacia Biotech), 0.05% bromphenol blue. Isoelectric focusing (400 V for 16 h) was performed in tube gels containing 9.5 m urea, 4% Triton X-100, 1% Pharmalyte 4–6.5, 1% Pharmalyte 5–8 in polymerized 3% acrylamide, 0.15% bisacrylamide. The proteins in the tube gels were then equilibrated in Laemmli sample buffer, transferred to slab gels, and resolved by SDS-PAGE (8% polyacrylamide). After transfer to nitrocellulose, [32P]phosphate incorporated into protein was detected by autoradiography, and signal corresponding to occludin protein was then revealed by subsequent immunoblotting of the filter with anti-occludin antibody. Phosphatase treatment of radiolabeled occludin was performed essentially as described by Meisenhelder and Hunter (41Meisenhelder J. Hunter T. Methods Enzymol. 1991; 197: 288-305Crossref PubMed Scopus (9) Google Scholar). Thus, an immune complex containing occludin from [32P]orthophosphate-labeled cells was prepared as described above. To remove interfering salts and detergents, the beads (50-μl packed volume) were washed twice with 1 ml of wash buffer containing 1% Triton X-100, 0.1 m NaCl, 25 mmHepes-NaOH, pH 7.4. They were then switched to phosphatase buffer by washing twice with a solution containing 20 mm Mes-NaOH, 1 mm MgCl2, 0.8 mm dithiothreitol, 4 μg/ml leupeptin, 4 μg/ml soybean trypsin inhibitor, pH 5.5. The beads were then incubated with 50 μl of the phosphatase buffer in the absence or presence of 0.2 units of potato acid phosphatase (Calbiochem). Phosphatase activity was blocked as described (41Meisenhelder J. Hunter T. Methods Enzymol. 1991; 197: 288-305Crossref PubMed Scopus (9) Google Scholar). After 1 h at 37 °C, the reaction was quenched by the addition of 1 ml of ice-cold wash buffer. The suspension was briefly (<10 s) centrifuged to pellet the beads. Protein was eluted into Laemmli sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose as described above. [32P]Phosphate incorporated into protein was detected by autoradiography, and occludin protein was revealed by immunoblotting. The effects of LPA on the TJ permeability of ECV304 cell monolayers were investigated by measuring paracellular flux of HRP. Using cultures on Transwell filters, HRP was added to the apical chambers in the presence or absence of LPA. HRP that passed via the paracellular pathway to enter the basolateral chamber was quantified by assaying peroxidase activity spectrophotometrically. The HRP activity detected was compared with that of control cells. As shown in Fig.1 a, LPA induced a greater flux of HRP in a dose-dependent manner. The involvement of RhoA was examined by using adenovirus-mediated overexpression of RhoA T19N, a dominant-negative mutant of RhoA. LacZ overexpression, confirmed by X-gal (5-bromo-4-chloro-3-indolyl β-d-galactopyranoside) staining (data not shown), was used as a control. In these experiments, the increase in HRP flux in response to LPA was similar in noninfected cells and LacZ-overexpressing cells. In contrast, LPA failed to stimulate an increase in HRP flux in RhoA T19N-overexpressing cells (Fig. 1 b). Next, the role of p160ROCK was investigated using the specific p160ROCK inhibitor Y-27632 (25Uehata M. Ishizaki T. Satoh H. Ono T. Kawahara T. Morishita T. Tamakawa H. Yamagami K. Inui J. Maekawa M. Narumiya S. Nature. 1997; 389: 990-994Crossref PubMed Scopus (2562) Google Scholar). Pretreatment with 10 μm Y-27632 blocked the increase in HRP flux induced by LPA (Fig. 1 b). These data indicate that RhoA and its target p160ROCK transduce the action of LPA to increase TJ permeability in ECV304 cells. The effects of histamine on HRP flux in ECV304 cells were then examined. Like LPA, histamine increased HRP flux in a dose-dependent manner in ECV304 cells (Fig.2 a). The roles of RhoA and p160ROCK were again studied. In noninfected cells and LacZ-overexpressing cells, histamine increased HRP flux to similar levels (Fig. 2 b). However, overexpression of RhoA T19N had no significant effect on the increase in HRP flux induced by histamine (Fig. 2 b). Also, inhibition of p160ROCK using Y-27632 failed to inhibit the increase in HRP flux in response to histamine (Fig.2 b). Thus, in contrast to the response to LPA, histamine appears to stimulate an increase in TJ permeability in ECV304 cells independently of RhoA and p160ROCK. To determine whether LPA and histamine caused changes in the actin cytoskeleton in ECV304 cells, F-actin was visualized by staining with fluorescein isothiocyanate-conjugated phalloidin (Fig.3). In noninfected cells and LacZ-overexpressing cells as controls, pericellular actin bundles were seen, and stress fibers were hardly detectable in the cell bodies (Fig.3, a and b), whereas subtle reorganization of pericellular actin bundles was observed in RhoA T19N-overexpressing cells and cells pretreated with 10 μm Y-27632 (Fig. 3,c and d). Noninfected cells and LacZ-overexpressing cells that were treated with 1 μm LPA showed F-actin bundles in stress fibers and some gaps between cells (Fig. 3, e and f). In contrast, in the cells overexpressing RhoA T19N, LPA failed to induce F-actin bundles (Fig.3 g). Pretreatment with 10 μm Y-27632 also prevented stress fiber formation in response to LPA (Fig.3 h). Similar to the effect of LPA, 1 μm histamine induced F-actin bundles in stress fibers in noninfected cells and LacZ-overexpressing cells (Fig. 3, i and j). Both overexpression of RhoA T19N and pretreatment with Y-27632 also blocked formation of stress fibers in response to histamine (Fig. 3,k and l). These data show that LPA and histamine induce reorganization of pericellular actin bundles and stimulation of stress fiber formation in ECV304 cells and that both events are mediated by RhoA and p160ROCK. Although LPA and histamine clearly had effects on the actin-based cytoskeleton, the possibility of direct effects on TJ proteins was also explored. The main reason for this was because the cytoskeletal effects of histamine were blocked by inhibition of RhoA-p160ROCK signaling, whereas effects on TJ permeability were unaffected. By immunocytochemistry, it was shown that the localization of occludin or ZO-1 was not altered in response to either LPA or histamine (data not shown). The possibility of effects of LPA and histamine on biochemical changes in the TJ protein occludin was then examined. Occludin immunoprecipitates from cells stimulated with either LPA or histamine were resolved by SDS-PAGE and then analyzed by immunoblotting with an anti-occludin monoclonal antibody. This revealed that the electrophoretic mobility of occludin in gels was altered within minutes in response to both LPA and histamine in a dose-dependent manner. This alteration was visualized as a retarded mobility during SDS-PAGE (Fig.4, a and b). The dose dependences were similar to those that caused an increase in HRP flux (see Figs. 1 and 2). As a control, expression levels and electrophoretic mobility of ZO-1 were not changed in response to LPA or histamine (Fig. 4, a and b). The biochemical basis of the change in electrophoretic mobility of occludin was investigated. One possibility was that this was due to changes in phosphorylation of the protein. In initial experiments, cells were metabolically labeled with [32P]orthophosphate, and phosphorylation of occludin was examined after immunoprecipitation and resolution by SDS-PAGE. In control cells, phosphate incorporation into occludin was detected. However, after stimulation with histamine or LPA, even though a band shift in occludin was observed, an increase in phosphate labeling of the protein was not detected (Fig.5 a). The intensity of labeled bands was quantitated by densitometry, and similar results were obtained in other experiments. Amino acid residues phosphorylated in occludin were then characterized and investigated to see if these changed in response to cell stimulation. Both immunological and labeling procedures were used. Occludin immunoprecipitates from cells treated with either 1 μm LPA or 1 μmhistamine were resolved by SDS-PAGE and immunoblotted with anti-occludin antibody, revealing the mobility shift, and then with the anti-phosphotyrosine antibodies PY20 or 4G10 (Fig. 5 b). As a control, cells were treated with pervanadate, a membrane-permeable peroxide derivative of vanada
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