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• W2010546098 abstract "Shear stress, the tangential component of hemodynamic forces, activates many signal transduction pathways in vascular endothelial cells. The conversion of mechanical stimulation into chemical signals is still unclear. We report here that shear stress (12 dynes/cm2) induced a rapid and transient tyrosine phosphorylation of Flk-1 and its concomitant association with the adaptor protein Shc; these are accompanied by a concurrent clustering of Flk-1, as demonstrated by confocal microscopy. Our results also show that shear stress induced an association of αvβ3 and β1 integrins with Shc, and an attendant association of Shc with Grb2. These associations are sustained, in contrast to the transient Flk-1·Shc association in response to shear stress and the transient association between αvβ3 integrin and Shc caused by cell attachment to substratum. Shc-SH2, an expression plasmid encoding the SH2 domain of Shc, attenuated shear stress activation of extracellular signal-regulated kinases and c-Jun N-terminal kinases, and the gene transcription mediated by the activator protein-1/12-O-tetradecanoylphorbol-13-acetate-responsive element complex. Our results indicate that receptor tyrosine kinases and integrins can serve as mechanosensors to transduce mechanical stimuli into chemical signals via their association with Shc. Shear stress, the tangential component of hemodynamic forces, activates many signal transduction pathways in vascular endothelial cells. The conversion of mechanical stimulation into chemical signals is still unclear. We report here that shear stress (12 dynes/cm2) induced a rapid and transient tyrosine phosphorylation of Flk-1 and its concomitant association with the adaptor protein Shc; these are accompanied by a concurrent clustering of Flk-1, as demonstrated by confocal microscopy. Our results also show that shear stress induced an association of αvβ3 and β1 integrins with Shc, and an attendant association of Shc with Grb2. These associations are sustained, in contrast to the transient Flk-1·Shc association in response to shear stress and the transient association between αvβ3 integrin and Shc caused by cell attachment to substratum. Shc-SH2, an expression plasmid encoding the SH2 domain of Shc, attenuated shear stress activation of extracellular signal-regulated kinases and c-Jun N-terminal kinases, and the gene transcription mediated by the activator protein-1/12-O-tetradecanoylphorbol-13-acetate-responsive element complex. Our results indicate that receptor tyrosine kinases and integrins can serve as mechanosensors to transduce mechanical stimuli into chemical signals via their association with Shc. Cells in the cardiovascular system are exposed to hemodynamic forces as well as chemical factors. Shear stress, the tangential component of hemodynamic forces, acts mainly on vascular endothelial cells (ECs), 1The abbreviations used are: EC, vascular endothelial cell; AP-1, transcription factor activator protein 1; β-gal, β-galactosidase; BAEC, bovine aortic endothelial cell; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FITC, fluorescein isothiocyanate; Grb2, growth factor receptor-binding protein 2; GST, glutathioneS-transferase; HA, hemagglutinin, IB, immunoblotting, IP, immunoprecipitation; JNK, c-Jun N-terminal kinase; mAb, monoclonal antibody; MBP, myelin basic protein; MCP-1, monocyte chemotactic protein-1; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PI, phosphatidylinositol; PLC, phospholipase C; RTK, receptor tyrosine kinase; SH2, Src homology domain-2; Sos, Son of sevenless; TRE, 12-O-tetradecanoylphorbol-13-acetate-responsive element; VEGF, vascular endothelium growth factor.1The abbreviations used are: EC, vascular endothelial cell; AP-1, transcription factor activator protein 1; β-gal, β-galactosidase; BAEC, bovine aortic endothelial cell; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FITC, fluorescein isothiocyanate; Grb2, growth factor receptor-binding protein 2; GST, glutathioneS-transferase; HA, hemagglutinin, IB, immunoblotting, IP, immunoprecipitation; JNK, c-Jun N-terminal kinase; mAb, monoclonal antibody; MBP, myelin basic protein; MCP-1, monocyte chemotactic protein-1; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PI, phosphatidylinositol; PLC, phospholipase C; RTK, receptor tyrosine kinase; SH2, Src homology domain-2; Sos, Son of sevenless; TRE, 12-O-tetradecanoylphorbol-13-acetate-responsive element; VEGF, vascular endothelium growth factor.whereas circumferential stress is borne primarily by vascular smooth muscle cells. The mechanotransduction processes by which these vascular cells convert mechanical stimuli into biochemical signals have gained increasing attention. Several laboratories, including ours, have performed in vitro experiments using flow channels to study the responses of ECs to applied shear stress (see Refs. 1Davies P.F. Physiol. Rev. 1995; 75: 519-560Crossref PubMed Scopus (2332) Google Scholar, 2Takahashi M. Ishida T. Traub O. Corson M.A. Berk B.C. J. Vasc. Res. 1997; 34: 212-219Crossref PubMed Scopus (157) Google Scholar, 3Gimbrone Jr., M.A. Nagel T. Topper J.N. J. Clin. Invest. 1997; 100: S61-S65PubMed Google Scholar, 4Shyy J.Y. Chien S. Curr. Opin. Cell Biol. 1997; 9: 707-913Crossref PubMed Scopus (294) Google Scholar for review). Mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), also known as stress-activated protein kinase, are rapidly activated by shear stress (5Tseng H. Peterson T.E. Berk B.C. Circ. Res. 1995; 77: 869-878Crossref PubMed Scopus (269) Google Scholar, 6Li Y.S. Shyy J.Y. Li S. Lee J. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (206) Google Scholar, 7Jo H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Crossref PubMed Scopus (237) Google Scholar). This results in the transcriptional activation of immediate early genes such as those encoding monocyte chemotactic protein-1 (MCP-1) and c-Fos (8Hsieh H.J. Li N.Q. Frangos J.A. J. Cell. Physiol. 1993; 154: 143-151Crossref PubMed Scopus (196) Google Scholar, 9Shyy Y.J. Hsieh H.J. Usami S. Chien S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4678-4682Crossref PubMed Scopus (322) Google Scholar, 10Jalali S. Li Y.S. Sotoudeh M. Yuan S. Li S. Chien S. Shyy J.Y. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 227-234Crossref PubMed Scopus (218) Google Scholar). On the upstream side, the shear stress activation of ERK and JNK is modulated by Ras, which in turn is regulated by Son of sevenless (Sos), a guanine nucleotide exchange factor, as evidenced by the findings that negative mutants of Ras and Sos can block the shear stress induction of ERK and JNK (6Li Y.S. Shyy J.Y. Li S. Lee J. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (206) Google Scholar, 7Jo H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Crossref PubMed Scopus (237) Google Scholar). Conceptually, shear stress acts on the EC membrane to activate putative shear stress sensors or receptors which then lead to the activation of the Sos-Ras pathway. To date, several mechanism of mechanotransduction involving the EC membrane have been suggested. Shear stress activates the seven-span-receptor-coupled G-protein (11Gudi S.R. Clark C.B. Frangos J.A. Circ. Res. 1996; 79: 834-839Crossref PubMed Scopus (226) Google Scholar), ion channels such as K+ channel (12Olesen S.P. Clapham D.E. Davies P.F. Nature. 1988; 331: 168-170Crossref PubMed Scopus (742) Google Scholar), and the transforming growth factor-β receptor-related Smad6 and Smad7 (13Topper J.N. Cai J. Qiu Y. Anderson K.R. Xu Y.Y. Deeds J.D. Feeley R. Gimeno C.J. Woolf E.A. Tayber O. Mays G.G. Sampson B.A. Schoen F.J. Gimbrone Jr., M.A. Falb D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9314-9319Crossref PubMed Scopus (290) Google Scholar). Several recent studies showed that tyrosine kinases, i.e. focal adhesion kinase (FAK) and c-Src in the focal adhesion site constitute a part of the mechanotransduction in ECs in response to shear stress (10Jalali S. Li Y.S. Sotoudeh M. Yuan S. Li S. Chien S. Shyy J.Y. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 227-234Crossref PubMed Scopus (218) Google Scholar, 14Takahashi M. Berk B.C. J. Clin. Invest. 1996; 98: 2623-2631Crossref PubMed Scopus (189) Google Scholar, 15Li S. Kim M. Hu Y.-L. Jalali S. Schlaepfer D.D. Hunter T. Chien S. Shyy Y.-J. J. Biol. Chem. 1997; 272: 30455-30462Crossref PubMed Scopus (356) Google Scholar). FAK, by forming a complex with growth factor receptor-binding protein 2 (Grb2), regulates the shear stress induction of ERK and JNK (15Li S. Kim M. Hu Y.-L. Jalali S. Schlaepfer D.D. Hunter T. Chien S. Shyy Y.-J. J. Biol. Chem. 1997; 272: 30455-30462Crossref PubMed Scopus (356) Google Scholar). The involvement of these signaling molecules in the focal adhesion sites may be correlated with the dynamic reorientation of focal adhesions in ECs under shear stress (16Davies P.F. Robotewskyj A. Griem M.L. J. Clin. Invest. 1994; 93: 2031-2038Crossref PubMed Google Scholar). Considering the multiplicity of the signaling molecules engaged in the EC responses to shear stress, there is a missing link to integrate the various pathways into an unified theme. Shc is an adaptor protein containing a C-terminal Src homology domain-2 (SH2) domain and a central glycine/proline-rich sequence (17Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Grignani F. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1138) Google Scholar). In response to many growth factors such as platelet-derived growth factor and epidermal growth factor (EGF), Shc is tyrosine-phosphorylated and associates with phosphotyrosines of the cognate receptor tyrosine kinases (RTK) through SH2 binding (17Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Grignani F. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1138) Google Scholar, 18Cutler R.L. Liu L. Damen J.E. Krystal G. J. Biol. Chem. 1993; 268: 21463-21465Abstract Full Text PDF PubMed Google Scholar, 19Ruff-Jamison S. McGlade J. Pawson T. Chen K. Cohen S. J. Biol. Chem. 1993; 268: 7610-7612Abstract Full Text PDF PubMed Google Scholar, 20Batzer A.G. Rotin D.J. Urena M. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar). Tyrosine-phosphorylated Shc also associates with Grb2 through SH2 interaction (21Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (826) Google Scholar, 22van der Geer P. Hunter T. EMBO J. 1993; 12: 5161-5172Crossref PubMed Scopus (124) Google Scholar). The assembly of Shc·Grb2·Sos provides an alternative mechanism in addition to the Grb2·Sos pathway for the activation of Ras. Recently, it has been shown that Shc is involved in the integrin-mediated signal transduction. In A431 cells, Shc is recruited to α1β1, α5β1, and αvβ3 when these integrins have been conjugated to their corresponding antibodies (23Wary K.K. Mainiero F. Isakoff S.J. Marcantonio E.E. Giancotti F.G. Cell. 1996; 87: 733-743Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). In the same study, it was also shown that Shc is necessary and sufficient for the activation of ERK in response to integrin ligation. These results suggest that both growth factors and integrins can regulate the ERK pathway via Shc. In the current study, we show for the first time that fetal liver kinase 1 (Flk-1), an RTK specific for vascular endothelium growth factor (VEGF), and integrins (αvβ3,β1, and β5 integrins) can both function as mechanosensors in ECs, and that shear stress causes both to be associated with Shc. The interaction of Shc with Flk-1 is rapid and transient, whereas its association with the various integrin is sustained. These findings provide new insights into the roles of RTKs and integrins in the transduction of shear stress into chemical signals. Bovine aortic endothelial cells (BAECs) were isolated from bovine aorta and cultured in a humidified 95% air, 5% CO2 incubator at 37 °C. The culture medium was Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 2 mm l-glutamine, and 1 mm each of penicillin-streptomycin and sodium pyruvate. All experiments were conducted with cultures prior to passage 10. A flow system was used to impose shear stress on cultured ECs as described previously (24Frangos J.A. Eskin S.G. McIntire L.V. Ives C.L. Science. 1985; 227: 1477-1479Crossref PubMed Scopus (1001) Google Scholar). In brief, a 75 × 38-mm glass slide was seeded with BAECs, which were cultured until reaching a confluent monolayer. A silicone gasket was sandwiched between the glass slide and an acrylic plate to create a rectangular flow channel (0.025 cm in height, 2.5 cm in width, and 5.0 cm in length) with inlet and outlet for exposing the cultured BAECs to shear stress. A high reservoir, the flow channel, a low reservoir, and a peristaltic pump were connected to form a circulation loop. Steady, laminar flow across the channel was generated as a result of the height difference between the two reservoirs. During the flow experiments, the system was kept at 37 °C in a constant temperature cabinet and equilibrated with 95% humidified air plus 5% CO2. The antibodies used in immunoprecipitation and immunoblotting were PY20 anti-phosphotyrosine monoclonal antibody (mAb) (Transduction Laboratories, Lexington, KY), polyclonal anti-Shc (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal anti-Grb2/Sem5 (Santa Cruz Biotechnology), anti-c-Myc mAb (Santa Cruz Biotechnology), polyclonal anti-Flk-1 (Santa Cruz Biotechnology), anti-αvβ3 LM609 mAb, polyclonal anti-β5 (Chemicon, Temecula, CA), anti-β1CD29 mAb (PharMingen, San Diego, CA), and anti-hemagglutinin (HA) mAb (Roche Molecular Biochemicals). For immunoprecipitation, cells were scraped into a lysis buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 1% Triton X-100); the lysate was centrifuged, and the supernatant was immunoprecipitated with the appropriate antibodies and protein A-Sepharose beads (Amersham Pharmacia Biotech) at 4 °C overnight. The immunoprecipitated complexes were washed and used for either kinase activity assays or immunoblotting. After SDS-PAGE, proteins in the gel were transferred to a nitrocellulose membrane for immunoblotting. The membrane was blocked with 5% bovine serum albumin followed by incubation with the primary antibody in 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.05% Tween 20, containing 0.1% bovine serum albumin. The bound primary antibodies were detected by using a goat anti-mouse or a goat anti-rabbit IgG-horseradish peroxidase conjugate (Santa Cruz Biotechnology) and the ECL detection system (Amersham Pharmacia Biotech). Confluent BAEC monolayers were fixed in a phosphate-buffered saline (PBS) containing 3% paraformaldehyde at room temperature for 10 min. The cells were then incubated in PBS containing the polyclonal anti-Flk-1 at a concentration of 1:200 (v/v) for 1 h at room temperature. The specimens were washed in PBS and incubated with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR). The immunostaining of Flk-1 was observed with a confocal microscopy system (MRC-1000, Bio-Rad) equipped with an argon/krypton laser line, a scan head, and a Nikon Diaplot 300 inverted microscope. FITC was excited at a wavelength of 488 nm and detected within a band between 506 and 538 nm. The pixel intensity of the confocal images was measured and analyzed by Lasersharp Processing software (Bio-Rad). To construct plasmid Shc-SH2 encoding the SH2 domain of Shc, the full-length Shc cDNA was first obtained from mRNA isolated from HeLa cells by reverse transcription-polymerase chain reaction using 5′-ATGAACAAGCTGAGTGGAGGC-3′ and 5′-GAGCGCTAGGGCAGATCA-3′ as the forward and reverse primers. The obtained Shc cDNA was then used as the template for the polymerase chain reaction synthesis of the SH2 domain (from Trp-378 to Ser-475) using 5′-TGGTTCCATGGGAAGCTG-3′ as the forward primer and 5′-GAGCGCTAGGGCAGATCA-3′ as the reverse primer. The 0.3-kilobase pair amplicons obtained after purification was ligated into plasmid pCRII (Invitrogen, San Diego, CA), and the fragments flanked by HindIII and EcoRI sites were then subcloned into the pcDNA3 vector (Invitrogen). Plasmids HA-JNK1, Myc-ERK2, 4XTRE-Pl-Luc, and MCP1-Luc-540 have been described previously (25Dérijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2954) Google Scholar, 26Shyy J.Y. Lin M.C. Han J. Lu Y. Petrime M. Chien S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8069-8073Crossref PubMed Scopus (219) Google Scholar). The various DNA plasmids were transfected into BAECs at 80% confluence using the LipofectAMINE method (Life Technologies, Inc.). After incubation for 6 h, the transfected cells were washed with DMEM and incubated in fresh complete DMEM to reach confluence. Within 48 h after transfection, the BAEC monolayer was subjected to shear stress or kept as static controls. The epitope-tagged Myc-ERK2 was co-transfected with Shc-SH2 into BAECs. After shear stress experiments, the cells were lysed in a kinase lysis buffer (25 mm HEPES, pH 7.4, 0.5 m NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 5 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 50 mmNaF, 10 mm Na3VO4, and 2 mm β-glycerophosphate). Myc-ERK2 was immunoprecipitated with the anti-c-Myc mAb and protein A-Sepharose beads. To perform immunocomplex kinase assays, the immunoprecipitates were washed twice in the lysis buffer and twice in a kinase assay buffer (25 mm HEPES, pH 7.4, 20 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 20 mm β-glycerophosphate, 1 mmNa3VO4, and 2 mm dithiothreitol). Two micrograms of myelin basic protein (MBP) and 10 μCi of [γ-32P]ATP (ICN, Irvine, CA) in 30 μl of kinase assay buffer containing 25 μm ATP were added to each immunocomplex pellet for kinase reaction at 30 °C for 20 min. The phosphoproteins were separated by SDS-PAGE, and the gels were dried for autoradiography. The kinase activities of the epitope-tagged HA-JNK1 were assessed by using essentially the same method as those for ERK, except that HA-JNK1 was immunoprecipitated by anti-HA mAb and that glutathione S-transferase (GST)-c-Jun-(1–79) fusion protein was used as the substrate in the immunocomplex kinase assays. pcDNA3 or Shc-SH2 was co-transfected with either 4xTRE-Pl-Luc or MCP1-Luc-540 into BAECs at 70% confluence by using the transient transfection protocols. The pSVβ-gal plasmid, which contains a β-galactosidase (β-gal) gene driven by SV40 promoter and enhancer, was included in the co-transfection to monitor the transfection efficiency. The cells were then subjected to shear stress experiments or kept as static controls. The luciferase reporter activities normalized for transfection efficiency were used to assess the effects of Shc-SH2 on shear stress-induced transcription activation mediated by AP-1/TRE. The binding of growth factors to their cognate RTKs induces the tyrosine phosphorylation of the cytoplasmic domains of RTKs, leading to the recruitment of the SH2-containing adaptor molecules such as Shc to the phosphorylated tyrosine. To test whether shear stress can activate endothelial RTKs as in the case of growth factor binding, confluent BAEC monolayers were subjected to a shear stress of 12 dynes/cm2 for various lengths of time. In parallel positive control experiments, however, BAECs were stimulated with 10 nm VEGF in the absence of shear. The cell lysates from the various experiments were immunoprecipitated with a polyclonal antibody against Flk-1, a VEGF receptor (27de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1892) Google Scholar). The immunoprecipitated protein complexes were then immunoblotted with PY20 mAb to detect the change in tyrosine phosphorylation of Flk-1 which has a molecular mass of 210 kDa. As shown in Fig. 1 A, shear stress induced the tyrosine phosphorylation of Flk-1 as early as 1 min, reached a peak level at 5 min, decreased afterward, and returned to the basal level at 30 min. The temporal response of Flk-1 tyrosine phosphorylation induced by shear stress was similar to that found in cells stimulated by VEGF (Fig. 1 B). Shear stress induction of Flk-1 tyrosine phosphorylation occurred both in the absence or presence of serum supplements and was not inhibited by pretreating BAEC monolayer with a polyclonal anti-VEGF antibody, indicating that the effect of shear stress was not due to background growth factors stimulation or to a paracrine or autocrine induction of VEGF. To investigate whether shear stress induction of Flk-1 tyrosine phosphorylation was accompanied by an increased association of Flk-1 with Shc, the cell lysates were immunoprecipitated with a polyclonal antibody against Shc followed by immunoblotting with polyclonal anti-Flk-1. As shown in Fig.2 A, shear stress increased the association of Flk-1 and Shc in a rapid and transient manner with a time course parallel to that of tyrosine phosphorylation of Flk-1 shown in Fig. 1 A. As a control, VEGF treatment also induced the association of Flk-1 and Shc in BAECs (Fig. 2 B). In contrast, using anti-rabbit IgG as a negative control in the immunoblotting, the association of FlK-1 with Shc was not observed (data not shown). Neither tyrosine phosphorylation of Flk-1 nor its association with Shc was due to metabolites released from the shear stress-stimulated ECs, since these responses were not found in ECs incubated with the shearing media (data not shown). Binding of the cognate ligands induces the dimerization and thus the activation of various RTKs. To test the hypothesis that shear stress activates Flk-1 by causing its clustering, confluent monolayers of BAECs were kept static or subjected to a shear stress of 12 dynes/cm2 followed by anti-Flk-1 immunostaining. Confocal microscopy revealed that Flk-1 was mainly distributed on the luminal side of BAECs (Fig. 3). Quantification of images from static and sheared samples showed that the application of shear stress for 1 min enhanced the clustering of Flk-1. This focal pattern of clustering peaked at 5 min and reduced to the level comparable to that in the static controls at 30 min after shearing. This time course is similar to those of Flk-1 tyrosine phosphorylation and Flk-1·Shc association. When ECs are exposed to shear stress, focal adhesion plaques move dynamically on the abluminal membrane (16Davies P.F. Robotewskyj A. Griem M.L. J. Clin. Invest. 1994; 93: 2031-2038Crossref PubMed Google Scholar). We have previously demonstrated that the αvβ3 integrin in focal adhesion sites is involved in the shear stress activation of ERK and JNK (15Li S. Kim M. Hu Y.-L. Jalali S. Schlaepfer D.D. Hunter T. Chien S. Shyy Y.-J. J. Biol. Chem. 1997; 272: 30455-30462Crossref PubMed Scopus (356) Google Scholar). To investigate the role of Shc in the integrin-mediated signal transduction in response to shear stress, αvβ3 was immunoprecipitated from BAEC lysates by LM609 mAb, and this was followed by immunoblotting with polyclonal anti-Shc. As shown in Fig.4 A, Shc association with αvβ3 was not detectable in static BAECs. Application of a shear stress of 12 dynes/cm2 rapidly augmented the formation of a complex of αvβ3·Shc, as demonstrated by their co-immunoprecipitation. This association was already detectable 10 min after the cells were exposed to shear stress, reached a peak level at 30 min, and sustained for the duration of this experiment (6 h). In two other separate experiments, the αvβ3·Shc association lasted for at least 18 h (data not shown). In contrast, neither αvβ3 integrin nor ERK was found to be associated with Shc in control experiments using anti-rat IgG or anti-ERK for the immunoprecipitation (data not shown). The αvβ3·Shc association during the endothelial attachment to fibrinogen is transient (Fig. 4 B), becoming undetectable by 2 h. Thus, the integrin-mediated signaling in response to shear stress differs from that caused by cell adhesion in that its association with Shc is sustained. It is possible that mechanotransduction causes the recruitment of Shc to various types of integrins in ECs. Thus, we also investigated whether shear stress increases the association of Shc with integrins containing the β1 or β5 subunit by immunoprecipitating the cell lysates with polyclonal anti-Shc followed by immunoblotting with anti-β1 mAb CD29 or polyclonal anti-β5. As shown in Fig.5, β1- or β5-containing integrins were not associated with Shc in the static cells. Exposure of BAEC monolayer to shear stress increased the association of Shc with β1 or β5 with a time course similar to that for αvβ3. Together, the data presented in Figs. 2, 4, and 5 demonstrate that Shc associates with both RTKs and integrins in ECs in response to shear stress. In contrast to the transient Shc·Flk-1 association, the Shc·integrin association is much more sustained. When cells are stimulated by growth factors, tyrosine phosphorylation of Shc coincides with its recruitment to RTKs. To investigate whether Shc is tyrosine phosphorylated in response to shear stress, the anti-Shc immunoprecipitates were immunoblotted with PY20 mAb. As shown in Fig.6 A, shear stress caused a sustained increase in tyrosine phosphorylation of Shc, which lasted for at least 6 h after the exposure to shear stress. FAK regulates Grb2·Sos-Ras pathway in the EC response to shear stress, which was demonstrated by the association of FAK with Grb2 (15Li S. Kim M. Hu Y.-L. Jalali S. Schlaepfer D.D. Hunter T. Chien S. Shyy Y.-J. J. Biol. Chem. 1997; 272: 30455-30462Crossref PubMed Scopus (356) Google Scholar). To investigate the possible engagement of Shc in the shear stress activation of the Grb2·Sos-Ras pathway, we examined whether Shc associates with Grb2 in the sheared BAECs. As shown in Fig.6 B, there was an increase in the amount of Grb2 co-immunoprecipitated with Shc in ECs subjected to shear stress for 1 min. This increased association of Grb2 with Shc was sustained. In a separate experiment, cell lysates immunoprecipitated with a polyclonal anti-Shc and immunoblotted with the polyclonal anti-Sos revealed that Shc was also associated with Sos in sheared cells (data not shown). The results in Fig. 6 demonstrate that shear stress induces a sustained interaction of integrins with Shc, which not only results in the tyrosine phosphorylation of Shc, but also the association of Shc with the Grb2·Sos complex. Shear stress activates mitogen activated protein kinases, including ERK and JNK (5Tseng H. Peterson T.E. Berk B.C. Circ. Res. 1995; 77: 869-878Crossref PubMed Scopus (269) Google Scholar, 6Li Y.S. Shyy J.Y. Li S. Lee J. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (206) Google Scholar, 7Jo H. Sipos K. Go Y.-M. Law R. Rong J. McDonald J.M. J. Biol. Chem. 1997; 272: 1395-1401Crossref PubMed Scopus (237) Google Scholar), which in turn cause the transcriptional activation of AP-1 acting on the TRE in the 5′ promoter of some of the shear-inducible genes, e.g. the MCP-1 gene (6Li Y.S. Shyy J.Y. Li S. Lee J. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (206) Google Scholar, 26Shyy J.Y. Lin M.C. Han J. Lu Y. Petrime M. Chien S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8069-8073Crossref PubMed Scopus (219) Google Scholar). Through its association with Grb2·Sos, Shc can be upstream of these events. We constructed Shc-SH2 that functions as a negative mutant of Shc (28Gotoh N. Muroya K. Hattori S. Nakamura S. Chida K. Shibuya M. Oncogene. 1995; 11: 2525-2533PubMed Google Scholar) to investigate its inhibitory effects on the shear stress activation of ERK, JNK, and on the TRE-driven luciferase reporter. Shc-SH2 was co-transfected with either Myc-ERK2 or HA-JNK1 into BAECs, and pcDNA3 parental plasmid was used as parallel controls. The transfected cells were either kept under static condition or subjected to a shear stress of 12 dynes/cm2 for 10 min (for Myc-ERK2 assay) or 30 min (for HA-JNK1 assay) followed by immunocomplex kinase assays using MBP or GST-c-Jun-(1–79) fusion protein as the respective substrate. As shown in Fig. 7, shear stress activated Myc-ERK2 and HA-JNK1 in BAECs transfected with pcDNA3 by 2- and 3-fold, respectively. Co-transfection of Shc-SH2 drastically attenuated the shear stress activation of Myc-ERK2 and HA-JNK1. These results indicate that Shc is involved in the upstream signaling for the shear stress induction of ERK and JNK. BAECs were co-transfected with Shc-SH2 and the chimeric construct 4XTRE-Pl-Luc consisting of luciferase reporter driven by four copies of TRE linked to the rat prolactin minimum promoter. In parallel experiments, cells were co-transfected with pcDNA3 together with 4XTRE-Pl-Luc. A shear stress of 12 dynes/cm2 caused 33-fold induction of luciferase activities (relative to those in the static controls kept for 8 h); this shear stress induction of luciferase activity was drastically reduced in cells co-transfected with Shc-SH2 (Fig. 8 A). We also tested whether Shc-SH2 can attenuate shear stress induction of MCP1-Luc-540 by using a chimeric construct which contains luciferase under the control of the 540-base pair 5′ promoter of the MCP-1 g" @default.
• W2010546098 created "2016-06-24" @default.
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• W2010546098 creator A5047330789 @default.
• W2010546098 creator A5048522454 @default.
• W2010546098 creator A5064441182 @default.
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• W2010546098 date "1999-06-01" @default.
• W2010546098 modified "2023-10-16" @default.
• W2010546098 title "Mechanotransduction in Response to Shear Stress" @default.
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