Endothelial Barrier Strengthening by Activation of Focal Adhesion Kinase
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m209922200
ISSN1083-351X
AutoresSadiqa K. Quadri, Mrinal K. Bhattacharjee, Kaushik Parthasarathi, Tatsuo Tanita, Jahar Bhattacharya,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoEndothelial cell barrier (EC) properties regulate blood tissue fluid flux. To determine the role of endothelial-matrix interactions in barrier regulation, we induced cell shrinkage by exposing confluent endothelial monolayers to hyperosmolarity. The dominant effect of a 15-min hyperosmolar exposure was an increase in the trans-endothelial electrical resistance, indicating the induction of barrier strengthening. Hyperosmolar exposure also increased activity of focal adhesion kinase and E-cadherin accumulation at the cell periphery. Concomitantly, the density of actin filaments increased markedly. In EC monolayers stably expressing constitutively active or dominant negative isoforms of Rac1, the actin response to hyperosmolar exposure was enhanced or blocked, respectively, although the response in trans-endothelial resistance was unaffected, indicating that the endothelial barrier enhancement occurred independently of actin. However, in monolayers expressing a kinase-deficient mutant of focal adhesion kinase, the hyperosmolarity-induced increases in activity of focal adhesion and peripheral E-cadherin enhancement were blocked and the induced increase of electrical resistance was markedly blunted. These findings indicate that in EC exposed to hyperosmolar challenge, the involvement of focal adhesion kinase was critical in establishing barrier strengthening. Endothelial cell barrier (EC) properties regulate blood tissue fluid flux. To determine the role of endothelial-matrix interactions in barrier regulation, we induced cell shrinkage by exposing confluent endothelial monolayers to hyperosmolarity. The dominant effect of a 15-min hyperosmolar exposure was an increase in the trans-endothelial electrical resistance, indicating the induction of barrier strengthening. Hyperosmolar exposure also increased activity of focal adhesion kinase and E-cadherin accumulation at the cell periphery. Concomitantly, the density of actin filaments increased markedly. In EC monolayers stably expressing constitutively active or dominant negative isoforms of Rac1, the actin response to hyperosmolar exposure was enhanced or blocked, respectively, although the response in trans-endothelial resistance was unaffected, indicating that the endothelial barrier enhancement occurred independently of actin. However, in monolayers expressing a kinase-deficient mutant of focal adhesion kinase, the hyperosmolarity-induced increases in activity of focal adhesion and peripheral E-cadherin enhancement were blocked and the induced increase of electrical resistance was markedly blunted. These findings indicate that in EC exposed to hyperosmolar challenge, the involvement of focal adhesion kinase was critical in establishing barrier strengthening. endothelial cells focal adhesion kinase monoclonal antibody rat lung microvascular endothelial cells trans-endothelial electrical resistance deleted FAK green fluorescent protein reverse transcriptase phosphate-buffered saline 1,4-piperazinediethanesulfonic acid guanosine 5′-O-(thiotriphosphate) 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′,-tetraacetic acid tetrakis(acetoxymethyl ester) Endothelial cells (EC)1lining blood vessels form the principal barrier to fluid flux from blood to tissue. A decrease in barrier properties increases fluid flux and promotes tissue edema that in vital organs such as lung or brain could potentially be life threatening. Permeability of the EC barrier depends largely on the restriction to fluid transport across the paracellular pathway that contains tight junctions and adherens junctions (1Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar). Tight junctions are the primary determinants of barrier function (2Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2006) Google Scholar). Barrier-deteriorating agents cause EC contraction by activating Ca2+-dependent myosin light chain kinase, thereby widening the junctions and causing hyperpermeability of the EC barrier (3Garcia J.G. Schaphorst K.L. J. Invest. Med. 1995; 43: 117-126PubMed Google Scholar). In a previous study, we reported that exposing lung capillaries to a 1-min hyperosmolar stimulus caused an immediate hyperpermeability response followed by a gradual return of barrier properties to normal (4Ragette R. Fu C. Bhattacharya J. J. Clin. Invest. 1997; 100: 685-692Crossref PubMed Scopus (20) Google Scholar). These findings suggest that even while challenged by barrier-deteriorating stimuli, EC institute repair mechanisms that reestablish adequacy of the barrier. However, these repair processes remain inadequately understood. In this regard, the role of focal adhesions requires consideration. EC exposed to barrier deteriorating stimuli develop focal adhesion complexes at points of cell-matrix contact (5Bhattacharya S. Fu C. Bhattacharya J. Greenberg S. J. Biol. Chem. 1995; 270: 16781-16787Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 6Abedi H. Zachary I. J. Biol. Chem. 1997; 272: 15442-15451Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). Although the barrier regulatory role of this response remains unclear, evidence from other cell types indicates that focal adhesion formation causes activation of focal adhesion kinase (FAK) (1Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar, 6Abedi H. Zachary I. J. Biol. Chem. 1997; 272: 15442-15451Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). It is proposed that in EC exposed to the barrier-deteriorating agent, thrombin, actin-induced translocation of FAK to focal adhesions reduces barrier deterioration (7Mehta D. Tiruppathi C. Sandoval R. Minshall R.D. Holinstat M. Malik A.B. J. Physiol. (Lond.). 2002; 539: 779-789Crossref Scopus (80) Google Scholar). The actin cytoskeleton may be particularly relevant in this regard, because receptor-mediated enhancement of actin (8Garcia J.G. Liu F. Verin A.D. Birukova A. Dechert M.A. Gerthoffer W.T. Bamberg J.R. English D. J. Clin. Invest. 2001; 108: 689-701Crossref PubMed Scopus (739) Google Scholar) or actin stabilization by phallicidin (9Phillips P.G. Semin. Thromb. Hemostasis. 1994; 20: 417-425Crossref PubMed Scopus (8) Google Scholar) strengthens, whereas actin depolymerization by cytochalasins deteriorates the barrier (10Shasby D.M. Shasby S.S. Sullivan J.M. Peach M.J. Circ. Res. 1982; 51: 657-661Crossref PubMed Scopus (241) Google Scholar). However, the specific mechanisms induced by FAK leading to barrier strengthening in EC remain unclear. Here we considered the possibility that FAK may be responsible for cross-talk between focal adhesions and cadherins. Dynamic regulation of the EC barrier is attributable to E-cadherin (1Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar, 11Rubin L.L. Hall D.E. Porter S. Barbu K. Cannon C. Horner H.C. Janatpour M. Liaw C.W. Manning K. Morales J. Tanner L.I. Tomaselli K.J. Bard F. J. Cell Biol. 1991; 115: 1725-1735Crossref PubMed Scopus (642) Google Scholar, 12Moy A.B. Winter M. Kamath A. Blackwell K. Reyes G. Giaever I. Keese C. Shasby D.M. Am. J. Physiol. 2000; 278: L888-L898PubMed Google Scholar) or VE-cadherin (13Lampugnani M.G. Resnati M. Raiteri M. Pigott R. Pisacane A. Houen G. Ruco L.P. Dejana E. J. Cell Biol. 1992; 118: 1511-1522Crossref PubMed Scopus (548) Google Scholar). At intercellular junctions, the cadherins form homophilic interactions between their extracellular domains on adjacent cell membranes, whereas their cytoplasmic domains bind β-catenin or plakoglobin (γ-catenin) that in turn associates with the actin-binding protein, α-catenin, thereby establishing a linkage between the cadherin-catenin complex and the actin cytoskeleton (1Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2917) Google Scholar,14Conacci-Sorrell M. Zhurinsky J. Ben Ze'ev A. J. Clin. Invest. 2002; 109: 987-991Crossref PubMed Scopus (517) Google Scholar). Evidence that this linkage is important for barrier regulation comes from findings that barrier-deteriorating stimuli deplete both the cadherin-catenin complex (15Rabiet M.J. Plantier J.L. Rival Y. Genoux Y. Lampugnani M.G. Dejana E. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 488-496Crossref PubMed Scopus (256) Google Scholar) as well as actin (16Ehringer W.D. Yamany S. Steier K. Farag A. Roisen F.J. Dozier A. Miller F.N. Microcirculation. 1999; 6: 291-303Crossref PubMed Scopus (29) Google Scholar) from the cell periphery. The FAK substrate α-actinin also binds β-catenin (17Tsukatani Y. Suzuki K. Takahashi K. J. Cell. Physiol. 1997; 173: 54-63Crossref PubMed Scopus (36) Google Scholar), thus providing a link between focal adhesions and the cadherin-catenin complex and thereby raising the possibility that focal adhesion assembly stabilizes the cadherin complex. We considered these issues in the context of hyperosmolar exposure that causes a cell shrinkage-induced activation of focal adhesion proteins as indicated by increased tyrosine phosphorylation of FAK (18Malek A.M. Goss G.G. Jiang L. Izumo S. Alper S.L. Stroke. 1998; 29: 2631-2640Crossref PubMed Scopus (91) Google Scholar). This response provided an opportunity to test whether in EC FAK is involved in the stabilization of the cadherin complex and possibly of barrier properties. Accordingly, we generated EC expressing a truncated form of deleted FAK (del-FAK) in which cadherin expression in the plasma membrane was markedly diminished. Our findings indicate that although hyperosmolarity increased barrier properties and peripheral cadherin recruitment in wild type EC, both effects were markedly blunted in EC-expressing del-FAK, indicating that EC employ FAK-dependent signaling mechanisms as a means to barrier strengthening. Chemicals were obtained from Sigma unless otherwise stated. Cell culture media and growth supplements, M199 medium, Lipofectin, G418, and Opti-MEM were obtained from Invitrogen. All reagents for immunofluorescence studies were obtained from Molecular Probes Inc. (Eugene, OR). Anti-phosphotyrosine mAb PY99 (mouse and monoclonal) and protein A/G-agarose beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-paxillin mAb was purchased from Zymed Laboratories Inc. (South San Francisco, CA). Anti-FAK and E-cadherin mAbs were obtained from Transduction Laboratories, Inc. (Lexington, KY). Anti-FAK polyclonal antibody (BC3) was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-actin rabbit polyclonal antibody and α-tubulin mAb were obtained from Sigma. Rat lung microvascular endothelial cells (RLMEC) were cultured as described previously (4Ragette R. Fu C. Bhattacharya J. J. Clin. Invest. 1997; 100: 685-692Crossref PubMed Scopus (20) Google Scholar, 5Bhattacharya S. Fu C. Bhattacharya J. Greenberg S. J. Biol. Chem. 1995; 270: 16781-16787Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) under 5% CO2 in M199 medium supplemented with 5% fetal bovine serum and 5% bovine calf serum. Cells were plated at a density of 1 × 105/cm2. EC phenotype was confirmed by cell uptake of fluorescent labeled-acetylated low density lipoprotein in imaged monolayers. For EC barrier quantification, we determined TER in RLMEC monolayers grown on sterile polycarbonate inserts held at 37 °C (Endohm, World Precision Instruments, Sarasota, FL). After a 30-min base-line period, experimental solutions were added and TER was determined every 15 s for the first 10 min and then at 1-min intervals for the subsequent 20 min. The data were corrected for the resistance of the insert alone. The plasmid pBS-FAK (a gift of Dr. James Parsons, Department of Microbiology, School of Medicine, University of Virginia, VA) was prepared by cloning the full-length FAK cDNA into the pBluescript, K5 vector (Stratagene, La Jolla, CA) (19Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1290) Google Scholar). This plasmid was used for the generation of del-FAK DNA by deleting sequences among the EaeI sites at 1176 and 2793 bp (amino acids 392–931) in the FAK gene (Fig.1A). This deletion includes a segment containing tyrosine residues that are critical for FAK activation. These residues include Tyr-397 at which FAK autophosphorylates (20Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar), Tyr-576 and Tyr-577 at which phosphorylation determines the kinase activity of FAK (21Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar), and Tyr-925 at which phosphorylation leads to activation of Src (22Schlaepfer D.D. Hanks S.K. Hunter T. van der G.P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1442) Google Scholar). Del-FAK DNA (1.6 kb) was subcloned into the plasmid vector pBK-CMV (7.76 kb) atBamH1-XhoI sites and then introduced into the bacterial strain MV10. We selected the clone (kanamycin, 50 μg/ml) containing the 1.6-kb segment (pSLRCU33) corresponding to del-FAK variant (Qiagen, Valencia, CA) using restriction enzymes (BamH1-XhoI). Each 2 μg of plasmid pSLRCU33 or the empty vector was stably transfected in RLMEC using nominal procedures (Lipofectin, Invitrogen). To confirm transfection of the del-FAK construct in RLMEC, we prepared primers based on the full-length FAK cDNA (GenBankTMaccession number M86656) (19Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5192-5196Crossref PubMed Scopus (1290) Google Scholar): sense primer (873–895 nucleotides), 5′-CCC AGA GGA AGG AAT CAG CTA C-3′, and antisense primer (3085–3065 nucleotides), 5′-GCT GGT CAT GAC GTA CTG CTG-3′ (Fig. 1A). For PCR, the parameters were: denaturing at 95 °C for 15 min followed by 35 cycles of denaturing, annealing, and extension at 95, 59, and 72 °C for 1, 1, and 2 min, respectively, in 3 mmMgCl2. In the presence of these primers, amplification by RT-PCR is expected to yield a 600-bp product only in cells containing the del-FAK construct (Fig. 1B), confirming successful transfection of the del-FAK construct. In wild type and empty vector-transfected cells, the same primers yielded only the expected RT-PCR product of 2.2 kb (Fig. 1B). V12Rac1GFP and N17Rac1GFP constructs were generously provided by Dr. P. Jurdic (Laboratoire de Biologie Moléculaire et Cellulaire, Ecole Normale Supérieure de Lyon, Lyon, France) (23Ory S. Munari-Silem Y. Fort P. Jurdic P. J. Cell Sci. 2000; 113: 1177-1188Crossref PubMed Google Scholar). Chimeras between enhanced GFP and GTPases were derived by insertion of the mutated GTPase open reading frames betweenEcoR1/SalI into the pEGFP-Cl expression vector downstream to the enhanced green fluorescent protein (GFP) coding sequence (Clontech, Palo Alto, CA). The resulting cDNA encoded chimeric GTPase-GFP expression vector. Plasmid DNA, V12Rac1GFP, N17Rac1GFP, and vector pEGFP-Cl were amplified by the standard protocol and adjusted at a final concentration of 1 mg/ml in water. All of the plasmids were expressed by stable transfection using Lipofectin reagent and following the manufacturer's instructions. These procedures are routine in our laboratory (4Ragette R. Fu C. Bhattacharya J. J. Clin. Invest. 1997; 100: 685-692Crossref PubMed Scopus (20) Google Scholar, 5Bhattacharya S. Fu C. Bhattacharya J. Greenberg S. J. Biol. Chem. 1995; 270: 16781-16787Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Cells were exposed to isosmolar medium, or medium was made hyperosmolar by the addition of sucrose (except where stated) at 37 °C under 5% CO2 in M199 for indicated periods. Subsequently, cells were washed twice with ice-cold PBS and lysed in radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 2 mm EDTA, 50 mmNaF, 0.5% SDS, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride) containing 1 mm sodium orthovanadate, 10 mm sodium pyrophosphate, 25 mm β-glycerophosphate, 0.1% SDS, and 1% Triton X-100 at 4 °C. Total cell lysate was clarified by centrifugation at 10,000 × g for 10 min. Protein concentrations were determined using a protein analysis kit (BCA, Pierce). Cells were rinsed 2× PBS and solubilized in Triton X-100 buffer (50 mm NaCl, 10 mm Pipes, pH 6.8, 3 mm MgCl2, 0.5% Triton X-100, 300 mm sucrose, 1.2 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin) for 20 min at 4 °C on a rocking platform. The cells were scraped from the plate and centrifuged (10 min). The supernatant was collected. The cell pellet was suspended in 100 μl of SDS immunoprecipitation buffer (15 mm Tris, pH 7.5, 5 mm EDTA, 2.5 mmEGTA, 1% SDS) and then boiled for 10 min and diluted to 300 μl with Triton X-100 buffer. Equal amounts of extracted proteins were immunoprecipitated and loaded for SDS-PAGE gel electrophoresis. Immunoprecipitation and immunoblotting were performed as described previously (4Ragette R. Fu C. Bhattacharya J. J. Clin. Invest. 1997; 100: 685-692Crossref PubMed Scopus (20) Google Scholar). Cell lysates containing equal amounts of protein were precleared for 30 min with 20 μl of protein A/G-agarose beads followed by incubation with primary antibodies (4 μg for 2 h). Antibody-antigen complexes were precipitated with 30 μl of protein A/G-agarose beads overnight at 4 °C. Nonspecific bound proteins were removed by washing the agarose beads three times with radioimmune precipitation assay buffer and one time with PBS. Bound proteins were eluted in 40 μl of 4× Laemmli's loading buffer. The proteins were resolved by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membrane, and analyzed by immunoblotting. For the analysis of FAK autophosphorylation, we used the reported immune complex kinase assay (21Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar). We immunoprecipitated p125FAK from wild type or del-FAK-transfected RLMEC. The immunoprecipitated complexes were washed three times with radioimmune precipitation assay buffer and once with kinase buffer (pH 7.4, 20 mm Hepes, 50 mm NaCl, 5 mmMgCl2, 5 mm MnCl2). The pellet was resuspended in 30 μl of the kinase buffer containing 10 μCi of [γ-32P]ATP (6000 Ci/mmol), and the sample was incubated for 30 min at 37 °C with frequent agitation. The reaction was terminated by the addition of 10 μl of 4× SDS-PAGE gel sample buffer followed by boiling for 5 min, and all of the products were resolved by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad). Direct exposures were used to visualize FAK autophosphorylation. The amount of FAK present in the reactions was visualized by immunoblotting of the same membrane with anti-FAK monoclonal antibody. Using glutathioneS-transferase-tagged p21- activated kinase-p21 binding domain (PAK-PBD) protein beads that specifically bind active Rac1, we obtained immunoprecipitates from EC lysates as per the methods reported previously (24Lewis A. Di Ciano C. Rotstein O.D. Kapus A. Am. J. Physiol. 2002; 282: C271-C279Crossref PubMed Scopus (53) Google Scholar) using the assay kit from Cytoskeleton Inc. (Denver, CO). For positive and negative controls for the active and inactive small GTPases, the non-hydrolyzable GTP analog, GTPγS, and GDP were used, respectively. Cell lysates obtained from untreated cells were incubated with 100 μm GTPγS or 1 mm GDP in the presence of 10 mm EDTA for 15 min at 30 °C to ensure efficient loading with the added nucleotide. To terminate the reaction, the lysates were placed on ice and supplemented with 60 mm MgCl2. These control samples were then incubated with the glutathione S-transferase-tagged PAK-PBD protein beads and washed in the same manner as the other samples. Captured proteins were removed from the beads by boiling the samples in Laemmli buffer, and the samples were subjected to SDS-PAGE and Western blotting. RLMEC monolayers grown on glass coverslips were fixed (4% formaldehyde in PBS, pH 7.4, 20 min, 22 °C), rinsed (3× PBS), permeabilized (0.1% Triton X-100), and stained using rhodamine-phalloidin. For immunofluorescence, cells were incubated with diluted primary antibodies (1:50) in blocking solution, 4% goat serum in PBS (1 h at 22 °C), and washed with 3× PBS. Fluorescence-conjugated antibodies then were added (1:500, 1 h at 22 °C) and washed with 3× PBS. The glass coverslips were mounted upside down on object slides using fluorescent-mounting medium (Dako Corporation Carpinteria, CA). Confocal images were obtained by means of a laser-scanning microscope (Pascal LSM, Carl Zeiss) and subjected to image analysis as described below (MCID 5, Imaging Research, St. Catherine, Canada). TER of RLMEC monolayers was stable at 37 ± 3 ohm/cm2 for up to 40 min (mean ± S.E.,n = 6). Exposure of monolayers to isosmolar medium (300 mosm) did not change TER from base line (Fig.2A). However, in monolayers exposed to medium-made hyperosmolar by the addition of sucrose (350 mosm), TER decreased in the first minute and then increased in the subsequent ∼10 min (Fig. 2A). The increase was sustained at a steady level for 5–7 min after which TER gradually returned to base line in 20–30 min. The increase of TER was abrogated by the addition of isosmolar medium (Fig. 2B), and it could be repeated subsequently in the same monolayer (data not shown). These findings indicated that subsequent to an initial decrease of barrier properties, the dominant effect of hyperosmolar exposure was to enhance the EC barrier. Because hyperosmolar exposure increases protein tyrosine phosphorylation in EC (18Malek A.M. Goss G.G. Jiang L. Izumo S. Alper S.L. Stroke. 1998; 29: 2631-2640Crossref PubMed Scopus (91) Google Scholar) and because actin polymerization is proposed to cause barrier strengthening (8Garcia J.G. Liu F. Verin A.D. Birukova A. Dechert M.A. Gerthoffer W.T. Bamberg J.R. English D. J. Clin. Invest. 2001; 108: 689-701Crossref PubMed Scopus (739) Google Scholar, 9Phillips P.G. Semin. Thromb. Hemostasis. 1994; 20: 417-425Crossref PubMed Scopus (8) Google Scholar), we exposed monolayers to inhibitors of tyrosine kinase and actin polymerization. The TER increase was inhibited by the tyrosine kinase inhibitor, genistein, and by the inhibitors of actin polymerization, latrinculin B (Fig.2C) and cytochalasin D (data not shown). Although hyperosmolar exposure is reported to increase the cytosolic Ca2+ (25Paemeleire K. de Hemptinne A. Leybaert L. Exp. Brain Res. 1999; 126: 473-481Crossref PubMed Scopus (35) Google Scholar), the intracellular calcium chelator, BAPTA-AM had no effect on the present TER response to hyperosmolarity (Fig. 2C). Not shown are results from experiments in which we incubated monolayers separately with the p38 mitogen-activated protein kinase blocker SB203580 (25 μm), the protein kinase C blocker calphostin C (500 nm), or the phosphatidylinositol 3-kinase blocker wortmannin (50 nm) (n = 3 for each inhibitor). A single monolayer was pretreated with the nitric-oxide synthase inhibitor l-NAME (30 μm). None of these treatments affected the TER response to hyperosmolarity. The maximum increase in TER correlated non-linearly with an osmolar concentration (Fig. 2D) with 80% of the response being established at 350 mosm. Hence, for the studies described below, we exposed EC to this hyperosmolar concentration for 15 min. Because genistein blocked the TER response to hyperosmolarity, we considered that hyperosmolar cell shrinkage might increase cell-matrix interactions, leading to activation of focal adhesion proteins. By confocal microscopy of wild type cells under base-line conditions, immunofluorescence of the focal adhesion marker protein paxillin was largely localized to the nuclear and perinuclear regions (Fig.3A, left image). Following hyperosmolar exposure, the fluorescence became pronounced as aggregates localized to the cell periphery in a pattern characteristic of focal adhesion formation (Fig. 3A, right image) (6Abedi H. Zachary I. J. Biol. Chem. 1997; 272: 15442-15451Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). This focal adhesion response was absent in del-FAK-expressing monolayers (Fig. 3B). Exposure of plated EC to hyperosmolar sucrose also increased tyrosine phosphorylation of FAK and paxillin (Fig. 3C). FAK activity, which was less in del-FAK-transfected monolayers than in wild type monolayers under base-line conditions (Fig. 3, D and E), increased 3-fold in wild type monolayers exposed to hyperosmolar medium (Fig. 3,D and E). By contrast, hyperosmolar exposure caused no enhancement of FAK activity in del-FAK-transfected cells (Fig. 3, D and E). Under non-stimulated conditions, TER was 30 ± 5% less in del-FAK-expressing monolayers than in wild type monolayers or in monolayers expressing vector alone (p < 0.05; n = 4). Following hyperosmolar exposure, the increase of TER was blunted in del-FAK-expressing monolayers to 60 ± 5% of that of wild type monolayers (Fig. 3F). Taking these findings together, we interpret that hyperosmolar exposure increased focal adhesion formation and that inhibition of this response inhibited the hyperosmolarity-induced barrier enhancement. To further consider the role of the cytosolic Ca2+, we carried out immunoprecipitations from monolayers treated with the intracellular Ca2+ chelator BAPTA-AM. Predictably, BAPTA-AM blocked the tyrosine phosphorylation of the Ca2+-dependent focal adhesion protein Pyk2 (Fig.4A) (26Tokiwa G. Dikic I. Lev S. Schlessinger J. Science. 1996; 273: 792-794Crossref PubMed Scopus (285) Google Scholar), but it did not modify phosphorylation responses for FAK or paxillin (data not shown). These results together with the above lack of an inhibitory effect on TER by BAPTA-AM indicate that the hyperosmolarity-induced TER increase was Ca2+-independent. In EC lysates, bands were evident at 125 and 68 kDa (Fig. 4, B and C), corresponding to FAK and paxillin, respectively (Fig. 3A). However, as opposed to the responses in plated EC, no increase of tyrosine phosphorylation was evident in lysates prepared either from suspended EC exposed to hyperosmolar sucrose (Fig. 4B,lanes 3 and 4) or from plated EC exposed to hyperosmolar urea (Fig. 4C, lane 3). The implications of these findings are discussed below. In considering the barrier regulatory function of FAK, we addressed the role of cadherins. Immunoprecipitation studies using mAbs against either VE- or E-cadherin indicated that although E-cadherin was well expressed in RLMEC (Fig.5A), the expression of VE-cadherin was weak (Fig. 5B), indicating that E-cadherin was the dominant cadherin type expressed in these EC. We confirmed that the mAb against VE-cadherin was capable of recognizing rat antigens (Fig. 5B) and that as reported by others (27Hinck L. Nathke I.S. Papkoff J. Nelson W.J. J. Cell Biol. 1994; 125: 1327-1340Crossref PubMed Scopus (556) Google Scholar) it recognized a band in human umbilical vein endothelial cells (Fig.5B). Detergent-based fractionation of EC lysates into cytosolic and membrane fractions followed by quantitative immunoprecipitation and immunoblotting revealed that in wild type EC, E-cadherin content was greater in the membrane than the cytosolic fraction (Fig.5A, lanes 1 and 2). Hyperosmolar exposure increased the membrane content (Fig. 5A,lanes 2 and 4) while decreasing the cytosolic content (Fig. 5A, lanes 1 and 3). These compartmental changes were approximately equal as quantified by band densitometry (Fig. 5C). Accordingly, hyperosmolarity increased the membrane-cytosol ratio for E-cadherin (Fig.5C). In del-FAK-expressing cells, E-cadherin content was less under control conditions than in wild type cells (p < 0.01) (Fig. 5, A and C). Moreover, the membrane content was less than the cytosolic (Fig.5A, lanes 5 and 6, and C). Furthermore, in contrast to the wild type response, in del-FAK-expressing cells, E-cadherin failed to increase following hyperosmolar exposure (Fig. 5A, lanes 6 and 8), resulting in similar membrane-cytosol ratios under control and hyperosmolar conditions (Fig. 5C). Responses in monolayers expressing the empty vector were similar to those of wild type monolayers (Fig. 5A, lanes 9–12, and C). In wild type cells under base-line conditions, confocal microscopy revealed the distribution of E-cadherin as a discontinuous line of fluorescence that marked the cell periphery (Fig. 5D,left image). Following hyperosmolar exposure, this peripheral fluorescence became pronounced and was now evident as a continuous line (Fig. 5D, right image), indicating increased E-cadherin expression. In del-FAK-expressing monolayers, the peripheral fluorescence was poorly developed under both base-line and hyperosmolar conditions (Fig. 5E), indicating abrogation of E-cadherin expression and pointing to FAK as critical factor in the hyperosmolarity-induced enhancement of E-cadherin. Because latrinculin B inhibited the hyperosmolarity-induced TER increase (Fig. 2C), we considered the involvement of the actin cytoskeleton in the present barrier response. Confocal microscopy of untreated wild type cells revealed actin distribution as reflected in rhodamine-phalloidin fluorescence as a thin band at the cell periphery and a perinuclear condensation (Fig. 6A,left image). A 15-min hyperosmolar exposure markedly enhanced the density of filamentous actin in wild type cells but not in del-FAK-expressing cells (Fig. 6, A–C). Immunoblotting with an actin-recognizing mAb indicated that, as expected, actin content was higher in the membrane than in the cytosolic fraction under control conditions (Fig. 7, A and B). In wild type cells, hyperosmolar exposure increased membrane actin content almost 2-fold (Fig. 7B, comparesecond and fourth bars) whi
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