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• W2023302566 abstract "Vascular endothelial cells (ECs), forming a boundary between the circulating blood and the vessel wall, are constantly subjected to fluid shear stress due to blood flow. The aim of this study was to determine the role of the recently identified IκB kinases (IKKs) in shear stress activation of NF-κB and to elucidate the upstream signaling mechanism that mediates IKK activation. Our results demonstrate that IKKs in ECs are activated by shear stress in a rapid and transient manner. This IKK activation is followed by IκB degradation and NF-κB translocation into the nucleus. Transfection of plasmids encoding catalytic inactive mutants of IKKs, i.e. hemagglutinin (HA)-IKKα(K44M) and HA-IKKβ(K44A), inhibits shear stress-induced NF-κB translocation. In addition, constructs encoding antisense IKKs, i.e.HA-IKKα(AS) and HA-IKKβ(AS), attenuate shear stress induction of a promoter driven by the κB enhancer element. Preincubation of the EC monolayer with a monoclonal anti-αvβ3integrin antibody (clone LM609) attenuates shear stress induction of IKK. Inhibition of tyrosine kinases by genistein causes a similar down-regulating effect. These results suggest that the integrin-mediated signaling pathway regulates NF-κB through IKKs in ECs in response to shear stress. Vascular endothelial cells (ECs), forming a boundary between the circulating blood and the vessel wall, are constantly subjected to fluid shear stress due to blood flow. The aim of this study was to determine the role of the recently identified IκB kinases (IKKs) in shear stress activation of NF-κB and to elucidate the upstream signaling mechanism that mediates IKK activation. Our results demonstrate that IKKs in ECs are activated by shear stress in a rapid and transient manner. This IKK activation is followed by IκB degradation and NF-κB translocation into the nucleus. Transfection of plasmids encoding catalytic inactive mutants of IKKs, i.e. hemagglutinin (HA)-IKKα(K44M) and HA-IKKβ(K44A), inhibits shear stress-induced NF-κB translocation. In addition, constructs encoding antisense IKKs, i.e.HA-IKKα(AS) and HA-IKKβ(AS), attenuate shear stress induction of a promoter driven by the κB enhancer element. Preincubation of the EC monolayer with a monoclonal anti-αvβ3integrin antibody (clone LM609) attenuates shear stress induction of IKK. Inhibition of tyrosine kinases by genistein causes a similar down-regulating effect. These results suggest that the integrin-mediated signaling pathway regulates NF-κB through IKKs in ECs in response to shear stress. vascular endothelial cells tumor necrosis factor tumor necrosis factor receptor IκB kinase extracellular matrix bovine aortic endothelial cells hemagglutinin monoclonal antibody glutathione S-transferase reactive oxygen species mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase. Vascular endothelial cells (ECs),1 serving as a barrier between the circulating blood and the vessel wall, are constantly exposed to fluid shear stress. The focal nature of atherosclerotic lesions in the arterial tree demonstrates the critical role of flow conditions in atherogenesis. In vitro experiments using flow channels with cultured ECs have shown that shear stress activates the platelet derived-growth factor gene (1Hsieh H.J. Li N.Q. Frangos J.A. Am. J. Physiol. 1991; 260: H642-H646PubMed Google Scholar), a potent mitogen for vascular smooth muscle cells. The shear stress activation of the platelet derived-growth factor gene is through the action of the transcription factor NF-κB on the shear stress-responsive element GAGACC (2Khachigian L.M. Resnick N. Gimbrone Jr., M.A. Collins T. J. Clin. Invest. 1995; 96: 1169-1175Crossref PubMed Scopus (294) Google Scholar, 3Resnick N. Collins T. Atkinson W. Bonthron D.T. Dewey C.F. Gimbrone Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4591-4595Crossref PubMed Scopus (449) Google Scholar). In addition, electrophoresis mobility shift assay showed that nuclear extracts isolated from ECs exposed to shear stress increase their binding to oligonucleotides containing the κB enhancer element (4Lan Q. Mercurius K.O. Davies P.F. Biochem. Biophys. Res. Commun. 1994; 201: 950-956Crossref PubMed Scopus (230) Google Scholar), and a luciferase reporter driven by the κB enhancer element was shown to be shear-inducible (5Shyy J.Y.-J Li Y.-S. Lin M.C. Chen W. Yuan S. Usami S. Chien S. J. Biomech. 1995; 28: 1451-1457Crossref PubMed Scopus (65) Google Scholar). However, the signal transduction pathway leading to the activation of NF-κB in ECs in response to shear stress is still unclear. The transcription factor NF-κB was first identified as a protein that binds to a specific DNA site in the intronic enhancer of the immunoglobulin κ light chain gene (6Sen R. Baltimore D. Cell. 1986; 47: 921-928Abstract Full Text PDF PubMed Scopus (1462) Google Scholar). It is composed of homo- or heterodimers of members of the Rel family of transcription factors that control the expression of numerous genes involved in the immune and inflammatory responses, cell adhesion, and growth control (see Refs. 7Lenardo M.J. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1257) Google Scholarand 8Baldwin A.J. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5563) Google Scholar for review). NF-κB can be activated by many types of extracellular stimuli, including tumor necrosis factor (TNF), interleukin-1, bacterial endotoxin lipopolysaccharide, viral infection, viral proteins, antigen receptor cross-linking of T and B cells, calcium ionophores, phorbol esters, UV radiation, free radicals, hypoxia, etc. (see Ref. 9Verma I.M. Stevenson J.K. Schwarz E.M. Van A.D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1657) Google Scholar for review). In almost all cell types, NF-κB is sequestered in the cytoplasm through tight association with the inhibitory IκB proteins, including IκB-α and IκB-β. Activation of NF-κB by a variety of stimuli is dependent upon the phosphorylation and subsequent degradation of the IκB proteins; this allows the translocation of NF-κB into the nucleus to activate various target genes. Phosphorylation of IκB proteins occurs at specific residues, Ser-32 and Ser-36 of IκB-α and Ser-19 and Ser-23 of IκB-β (10Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar, 11Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1314) Google Scholar, 12DiDonato J. Mercurio F. Rosette C. Wu L.J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar, 13Traenckner E.B. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (931) Google Scholar). Following phosphorylation, IκB proteins are ubiquitinated and then degraded by a proteasome-dependent pathway (10Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar, 11Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1314) Google Scholar, 12DiDonato J. Mercurio F. Rosette C. Wu L.J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar, 13Traenckner E.B. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (931) Google Scholar, 14Whiteside S.T. Ernst M.K. LeBail O. Laurent W.C. Rice N. Israel A. Mol. Cell. Biol. 1995; 15: 5339-5345Crossref PubMed Google Scholar, 15DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 16Chen Z. Hagler J. Palombella V.J. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1995; 9: 1586-1597Crossref PubMed Scopus (1168) Google Scholar, 17Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Pashut L.I. Ben N.Y. Mol. Cell. Biol. 1995; 15: 1294-1301Crossref PubMed Google Scholar). The IκB kinase (IKK) complex was recently purified and is composed of several subunits (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 19DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar, 20Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1850) Google Scholar, 21Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar). Two of the subunits, 85 and 87 kDa in size, were termed IKKα and IKKβ, respectively. These two proteins (with 52% homology) contain an N-terminal catalytic kinase domain and several putative protein interaction motifs, including a leucine zipper and a helix-loop-helix domain at their C termini (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 19DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar). Integrins are a family of >20 different transmembrane heterodimers composed of α- and β-subunits that are associated noncovalently. All integrins consist of a large extracellular domain, a transmembrane region, and a relatively short cytoplasmic region. The extracellular domain typically binds to an Arg-Gly-Asp (RGD) sequence that is present in various extracellular matrix (ECM) ligands, e.g.fibronectin, vitronectin, and collagen. The cytoplasmic domain, generally consisting of 20–70 amino acids, interacts with cytoskeletal proteins, e.g. actin filaments, and kinases in the focal adhesion sites, e.g. focal adhesion kinase and c-Src (see Refs. 22Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9002) Google Scholar and 23Sastry S.K. Horwitz A.F. Curr. Opin. Cell Biol. 1993; 5: 819-831Crossref PubMed Scopus (407) Google Scholar for review). α4β1 integrin is involved in the NF-κB-mediated gene expression in leukocytes (24McGilvray I.D. Lu Z. Bitar R. Dackiw A.P.B. Davreux C.J. Rotstein O.D. J. Biol. Chem. 1997; 272: 10287-10294Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 25Udagwa T. Woodside D.G. McIntyre B.W. J. Immunol. 1996; 157: 1965-1972PubMed Google Scholar, 26Rosales C. Juliano R. Cancer Res. 1996; 56: 2302-2305PubMed Google Scholar). The RGD motif of the ECM appears to be involved in the NF-κB activation during cell-cell interaction (27Faure E. Lecine P. Imbert J. Champion S. Cell. Mol. Biol. 1996; 42: 811-823PubMed Google Scholar). There is ample evidence demonstrating that integrin-mediated signaling regulates the mitogen-activated protein kinase pathways including extracellular signal-regulated kinase and c-Jun N-terminal kinase (see Ref. 28Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1467) Google Scholar for review). We have proposed that integrins in ECs can serve as mechanosensors (29Shyy J.Y.-J. Chien S. Curr. Opin. Cell Biol. 1997; 9: 707-713Crossref PubMed Scopus (294) Google Scholar). In this study, we present evidence that integrins such as αvβ3 transduce mechanical stimuli into biochemical signals to activate NF-κB through IKKs in ECs in response to shear stress. Bovine aortic endothelial cells (BAECs) prior to passage 10 were used in all experiments. The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified 5% CO2 and 95% air incubator at 37 °C. BAECs were cultured on glass slides (38 × 76 mm) to confluence. 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). The BAECs in the 12.5-cm2 area was exposed to the applied shear stress, which was generated by circulating the tissue culture medium through a hydrostatic pump connected to the upper and lower reservoirs (30Frangos J.A. Eskin S.G. McIntire L.V. Ives C.L. Science. 1985; 227: 1477-1479Crossref PubMed Scopus (1001) Google Scholar). The pH of the system was kept constant by gassing with humidified 95% air and 5% CO2, and the temperature was maintained at 37 °C by keeping the flow system in a temperature-controlled hood. The shear stress, determined by the flow rate perfusing the channel and the channel dimensions, was 12 dynes/cm2, which is comparable to the physiological range in the human major arteries and has been found to induce the expression of many immediately early genes in vitro (31Hsieh H.J. Li N.Q. Frangos J.A. J. Cell. Physiol. 1993; 154: 143-151Crossref PubMed Scopus (196) Google Scholar, 32Shyy J.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). Static control experiments were performed on BAECs kept on slides for the same duration without being exposed to shear stress. The expression plasmids hemagglutinin (HA)-IKKα and HA-IKKβ, which encode HA epitope-tagged IKKα, and IKKβ, respectively, and their catalytic inactive mutants, HA-IKKα(K44M) and HA-IKKβ(K44A), were described previously (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar). HA-IKKα(AS) and HA-IKKβ(AS) are the antisense forms of HA-IKKα and HA-IKKβ, respectively (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 19DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar). HIV(LTR)-Luc is a luciferase reporter driven by the human immunodeficiency virus long terminal repeat that contains two binding sites for NF-κB (33Nabel G. Baltimore D. Nature. 1987; 326: 711-713Crossref PubMed Scopus (1451) 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 Dulbecco's modified Eagle's medium and incubated in fresh Dulbecco's modified Eagle's medium to reach confluence. Within 48 h after transfection, the BAEC monolayer was either subjected to shear stress or kept as a static control. BAECs were lysed in a lysis buffer containing 25 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 1% Triton X-100. The lysate was centrifuged, and the protein concentration of the supernatant was determined using the Bio-Rad protein assay reagent. The protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. 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 using horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and the ECL detection system (Amersham Pharmacia Biotech). The translocation of NF-κB was investigated by immunostaining. Confluent monolayers of BAECs were fixed in methanol at −20 °C for 5 min and incubated with 100% goat serum at 4 °C overnight. The specimens were washed three times with phosphate-buffered saline, followed by incubation in phosphate-buffered saline containing 1% bovine serum albumin, 0.2% Triton X-100, and polyclonal anti-NF-κB p65 antibody (1:100, v/v; Santa Cruz Biotechnology) for 2 h at 37 °C. After being washed three times with phosphate-buffered saline containing 0.2% Triton X-100, the specimens were incubated with fluorescein-conjugated anti-rabbit IgG (1:200, v/v; Sigma) for 2 h at room temperature. In the inhibition experiments, BAECs were transfected with HA-IKKα(K44M) or HA-IKKβ(K44A). An anti-HA mAb (Boehringer Mannheim) and anti-NF-κB p65 antibody were used in double immunostaining (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar). These antibodies were detected using rhodamine-conjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG, respectively. The immunostaining was observed under an epifluorescence microscope. Fluorescein was excited at a wavelength of 488 nm and detected between 506 and 538 nm, whereas rhodamine was excited at 568 nm and detected between 589 and 621 nm. BAECs transfected with HA-IKKα and HA-IKKβ were lysed in a lysis buffer containing 20 mm Tris-HCl, pH 7.6, 0.3 m NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2 mm dithiothreitol, 20 mm β-glycerophosphate, 1 mm Na3VO4, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin. HA-IKKα and HA-IKKβ were immunoprecipitated from the cell lysate using protein A-Sepharose beads and anti-HA mAb. After centrifugation, the pelleted immunocomplex was washed with the lysis buffer, followed by washing first with a lysis buffer containing 1m urea and then with an IKK assay buffer containing 20 mm Hepes, pH 7.4, 20 mm MgCl2, 20 mm dithiothreitol, 20 mm β-glycerophosphate, 0.1 mm Na3VO4, and 10 μg/ml aprotinin. The immunocomplex was then resuspended in 20 μl of the kinase buffer containing 5 μCi of [γ-32P]ATP, 20 μm ATP, and 1 μg of glutathioneS-transferase (GST)-IκB-α-(1–54). After incubation at 30 °C for 30 min, the kinase reaction mixture was resolved on a 10% SDS-polyacrylamide gel, and phosphorylated GST-IκB-α was detected by autoradiography (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar). HIV(LTR)-Luc, HA-IKKα(AS), and HA-IKKβ(AS) were cotransfected into BAECs for the luciferase induction assay. The pSV-β-galactosidase plasmid, which contains a β-galactosidase gene driven by the SV40 promoter and enhancer, was also cotransfected to monitor the transfection efficiency. To release the reporter luciferase and β-galactosidase, the cells were lysed with a lysis buffer containing 1% Triton X-100. ATP and luciferin were then added to the lysate in a luminometer for measuring the total light output. The level of β-galactosidase was assayed by adding the substrate o-nitrophenyl-β-galactopyranoside to the cell lysate and incubating at 37 °C for 1 h. The reaction was then quenched by the addition of Na2CO3, and the absorbance at 410 nm was recorded. The luciferase activity was normalized with that of β-galactosidase. Khachigian et al. (2Khachigian L.M. Resnick N. Gimbrone Jr., M.A. Collins T. J. Clin. Invest. 1995; 96: 1169-1175Crossref PubMed Scopus (294) Google Scholar) and Lan et al. (4Lan Q. Mercurius K.O. Davies P.F. Biochem. Biophys. Res. Commun. 1994; 201: 950-956Crossref PubMed Scopus (230) Google Scholar) have previously shown that shear stress increases the binding activity of NF-κB, and we have demonstrated that shear stress increases the transcriptional activity of promoters containing the κB element (5Shyy J.Y.-J Li Y.-S. Lin M.C. Chen W. Yuan S. Usami S. Chien S. J. Biomech. 1995; 28: 1451-1457Crossref PubMed Scopus (65) Google Scholar). To test whether the shear stress induction of NF-κB transcriptional activity results from a degradation of IκB proteins and the ensuing NF-κB translocation into the nucleus, BAECs were subjected to a shear stress of 12 dynes/cm2 for various time periods. Immunoblotting with polyclonal anti-IκB-α antibody revealed that shear stress caused IκB-α degradation in ECs in a transient manner (Fig. 1 A). Compared with static controls, the amount of IκB-α decreased in cells after 10 min of shearing, reached a minimal level at 30 min, and began to increase at 60 min. At 2 h after shearing, cellular IκB was at the same level as static controls. Immunostaining of the p65 subunit of NF-κB (Fig. 1 B) demonstrated that the temporal response of NF-κB translocation was comparable to that of IκB-α degradation. In static ECs, NF-κB was mainly distributed in the cytoplasm, but 15 min of shearing caused some of the nuclei to become anti-NF-κB antibody immunostaining-positive, indicating the translocation of NF-κB from the cytoplasm into the nucleus. At 30 min after shearing, NF-κB was mainly localized in the nucleus, and at 45 min, NF-κB began to reappear in the cytoplasm. Antibody specificity was verified by the absence of NF-κB immunostaining in control experiments in which nonimmune serum was used instead of the primary antibody (i.e. polyclonal anti-NF-κB p65 antibody). Recent findings indicate that IκB proteins are specifically phosphorylated by IKKs, leading to their ubiquitination and degradation by proteasome (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 19DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar, 20Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1850) Google Scholar, 21Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 34Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1067) Google Scholar). To investigate whether shear stress activates IKKs to up-regulate the NF-κB signaling pathway in ECs, BAECs were transfected with plasmids encoding HA-IKKα and HA-IKKβ and subjected to shear stress experiments. Using GST-IκB-α-(1–54) as the substrate, immunocomplex kinase activity assay showed that IKK activity associated with HA-IKKα and HA-IKKβ was increased by shear stress, similar to the IKK activity induced by the treatment of TNF-α (Fig. 2). The shear stress activation of IKKs occurred as early as 5 min, peaked at 30 min, and returned to the basal level 2 h after shearing. Densitometric analysis showed that the peak activity was three times the static controls. The temporal change of IKK activity is similar to that of IκB degradation (Fig. 1 A), suggesting that shear stress activates IKKs, which phosphorylate IκB proteins to cause their degradation. To test whether inhibition of IKKs abolishes shear stress-induced NF-κB translocation, BAECs were transfected with plasmids encoding HA-IKKα(K44M) and HA-IKKβ(K44A), the respective mutants of catalytic inactive HA-IKKα and HA-IKKβ (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 19DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar). In parallel control experiments, cells were transfected with wild-type HA-IKKα and HA-IKKβ. The transfected BAECs were subjected to a shear stress of 12 dynes/cm2 for 30 min or kept as static controls. Double immunostaining using anti-HA and anti-NF-κB p65 antibodies was performed to detect NF-κB translocation in the plasmid-transfected cells. Whereas anti-HA antibody identified the transfected cells, anti-NF-κB p65 antibody revealed the distribution of NF-κB in these transfected cells. As shown in Fig. 3, shear stress induced the translocation of NF-κB from the cytoplasm into the nucleus in the non-transfected cells. In contrast, the transfection of HA-IKKα(K44M) and HA-IKKβ(K44A) blocked the NF-κB translocation induced by shear stress. In parallel control experiments, the transfection of wild-type HA-IKKα and HA-IKKβ did not affect the shear-induced translocation of NF-κB (data not shown). HA-IKKα(AS) and HA-IKKβ(AS) encode the antisense forms of HA-IKKα and HA-IKKβ, respectively (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 19DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar). HIV(LTR)-Luc is a shear-inducible construct, with its induction mediated by the κB element (5Shyy J.Y.-J Li Y.-S. Lin M.C. Chen W. Yuan S. Usami S. Chien S. J. Biomech. 1995; 28: 1451-1457Crossref PubMed Scopus (65) Google Scholar). To further confirm that the shear stress-increased NF-κB transcriptional activity is regulated by the IKKs, HIV(LTR)-Luc was cotransfected with HA-IKKα(AS) or HA-IKKβ(AS). As shown in Fig. 4, in BAECs cotransfected with HIV(LTR)-Luc and the pSRα3 parental vector, shear stress caused an increase in luciferase activity to 3.2-fold of the static controls. However, cotransfection with HA-IKKα(AS) or HA-IKKβ(AS) or a combination of both abolished this shear stress induction of luciferase activity. The data presented in Figs. Figure 1, Figure 2, Figure 3, Figure 4 show that application of shear stress to ECs activates NF-κB through the induction of IKKs. An important question is what are the upstream molecules that mediate the mechanotransduction to activate IKKs. The activation of mitogen-activated protein kinases by shear stress is similar to that induced by attachment of cells to the ECM or incubation of cells with beads coated with integrin ligands or anti-integrin antibodies (see Ref. 29Shyy J.Y.-J. Chien S. Curr. Opin. Cell Biol. 1997; 9: 707-713Crossref PubMed Scopus (294) Google Scholar for review). Thus, we investigated whether integrins regulate the shear stress activation of IKKs in ECs. Confluent monolayers of BAECs transfected with HA-IKKα and HA-IKKβ were preincubated for 3 h with LM609, a mAb against the abundant endothelial αvβ3 integrin. With such an incubation, the applied antibody has been shown to gain access to the abluminal side of the cells (35Li S. Kim M. Hu Y.L. Jalali S. Schlaepfer D.D. Hunter T. Chien S. Shyy J.Y.-J. J. Biol. Chem. 1997; 272: 30455-30462Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). A shear stress of 12 dynes/cm2 was applied to these LM609-incubated cells for 30 min, followed by immunocomplex kinase assay for HA-IKKα and HA-IKKβ. As shown in Fig. 5, preincubation of BAECs with LM609 attenuated shear stress activation of these HA-IKKs, as indicated by the decreased phosphorylation of GST-IκB-α-(1–54) compared with cells that had been exposed to mouse IgG. These results suggest that αvβ3 integrin is involved in the mechanotransduction that mediates the shear stress activation of IKK pathways. Protein-tyrosine kinases in the focal adhesions are commonly involved in integrin-mediated signal transduction (see Ref. 36Parsons J.T. Schaller M.D. Hildebrand J. Leu T.H. Richardson A. Otey C. J. Cell Sci. 1994; 18 (suppl.): 109-113Crossref Google Scholar for review). To examine the roles of protein-tyrosine kinases in shear stress activation of IKKs, confluent monolayers of BAECs transfected with HA-IKKα and HA-IKKβ were pretreated with a protein-tyrosine kinase inhibitor (genistein), followed by shear stress experiments and immunocomplex kinase assay. As shown in Fig. 5, pretreatment of BAECs with genistein attenuated shear stress activation of HA-IKKs. Integrin-mediated signal transduction is usually investigated in cells adhered to the ECM. Many of these signaling events are similar to those involved in cellular responses to shear stress (see Ref. 29Shyy J.Y.-J. Chien S. Curr. Opin. Cell Biol. 1997; 9: 707-713Crossref PubMed Scopus (294) Google Scholar for review). To further confirm that IKK can be activated by integrin-mediated signal transduction, HA-IKKα- and HA-IKKβ-transfected BAECs in suspension were allowed to adhere to a fibronectin-coated surface and then subjected to immunocomplex kinase assay. As shown in Fig. 6, the temporal response of these HA-IKKs during EC adhesion was similar to that in ECs exposed to shear stress. The peak activity occurred at 15 min; by 1 h after cell attachment, the activity was at a basal level similar to that in the suspension. In addition to fibronectin, the activation of IKKs was also observed in cells adhered to vitronectin and collagen, but not to poly-l-lysine (data not shown). Reperfusion injury results in many responses, including the release of reactive oxygen species (ROS) and the expression of genes that are mediated by the transcription factor NF-κB (see Ref. 37Blake D.R. Winyard P.G. Marok R. Ann. N. Y. Acad. Sci. 1994; 723: 308-317Crossref PubMed Scopus (43) Google Scholar for review). The sudden application of shear stress to ECs cultured in a flow channel mimics the reperfusion process in the vessels. Using such an in vitro model, we (5Shyy J.Y.-J Li Y.-S. Lin M.C. Chen W. Yuan S. Usami S. Chien S. J. Biomech. 1995; 28: 1451-1457Crossref PubMed Scopus (65) Google Scholar) and others (2Khachigian L.M. Resnick N. Gimbrone Jr., M.A. Collins T. J. Clin. Invest. 1995; 96: 1169-1175Crossref PubMed Scopus (294) Google Scholar, 3Resnick N. Collins T. Atkinson W. Bonthron D.T. Dewey C.F. Gimbrone Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4591-4595Crossref PubMed Scopus (449) Google Scholar, 4Lan Q. Mercurius K.O. Davies P.F. Biochem. Biophys. Res. Commun. 1994; 201: 950-956Crossref PubMed Scopus (230) Google Scholar) have previously shown that shear stress increases the transcriptional activity of NF-κB. In this investigation on the upstream signal transduction pathway leading to the shear stress induction of NF-κB in ECs, we found that shear stress activates an IKK/NF-κB pathway and that this is at least in part mediated by integrins such as αvβ3 integrin. The recently identified IKKα and IKKβ constitute two components of the IKK complex that phosphorylates the serine residues in IκB (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 19DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar, 20Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1850) Google Scholar, 21Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 34Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1067) Google Scholar). Specific serine phosphorylation, such as at Ser-32 and Ser-36 of IκB-α, leads to the ubiquitination and degradation of IκB (18Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1582) Google Scholar, 19DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar, 21Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar). The temporal responses of ECs to shear stress in terms of IKK activation, IκB degradation, NF-κB translocation, and NF-κB-mediated transcriptional activation reveal the following events. Under static condition, NF-κB in ECs is sequestered in the cytoplasm by the binding of IκB. Shear stress activates IKKs, which phosphorylate IκB to lead to its degradation. As a consequence, the NF-κB released from the NF-κB·IκB complex translocates into the nucleus to activate its target genes. In addition to the augmented expression of the NF-κB-mediated genes, the functional consequences of shear stress activation of the IKK/NF-κB pathway in ECs may also include the modulation of cell survival, apoptosis, and motility. During the preparation of this manuscript, Scatena et al. (38Scatena M. Almerida M. Chasson M.L. Nelson R.F. Giachelli C.M. J. Cell Biol. 1998; 141: 1083-1093Crossref PubMed Scopus (446) Google Scholar) reported that adhesion of ECs to osteopontin activates NF-κB and thus rescues cells from apoptosis induced by serum deprivation. It was further suggested that the NF-κB activation through αvβ3integrin mediates this EC survival since anti-β3 integrin mAb F11 blocks NF-κB activity and induces EC apoptosis. The activation of NF-κB by TNF, ionizing radiation, or the rasproto-oncogene was found to protect cells from apoptosis (39Mayo M.W. Wang C.Y. Cogswell P.C. Rogers G.K. Lowe S.W. Der C.J. Baldwin A.J. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (506) Google Scholar, 40Wang C.Y. Mayo M.W. Baldwin A.J. Science. 1996; 274: 784-787Crossref PubMed Scopus (2509) Google Scholar, 41Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2447) Google Scholar). Inhibition of NF-κB nuclear translocation enhances apoptotic killing by these reagents, but not by apoptotic stimuli that do not activate NF-κB. Recent studies showed that shear stress protects ECs from apoptosis: TNF- or H2O2-induced EC apoptosis is inhibited by preconditioning the EC monolayer with a shear stress of 15 dynes/cm2 (42Hermann C. Zeiher A.M. Dimmeler S. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3588-3592Crossref PubMed Scopus (183) Google Scholar, 43Dimmeler S. Haendeler J. Nehls M. Zeiher A.M. J. Exp. Med. 1997; 185: 601-607Crossref PubMed Scopus (786) Google Scholar). Although the anti-apoptotic effects of shear stress have been linked to the production of nitric oxide (42Hermann C. Zeiher A.M. Dimmeler S. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3588-3592Crossref PubMed Scopus (183) Google Scholar), it would be interesting to investigate whether the augmented IKK/NF-κB pathway is also involved. αvβ3integrin, in conjunction with activated protein kinase C, promotes the migration of FG carcinoma cells on vitronectin (44Klemke R.L. Yebra M. Bayna E.M. Cheresh D.A. J. Cell Biol. 1994; 127: 859-866Crossref PubMed Scopus (248) Google Scholar). An oligonucleotide-containing κB element, when introduced into FG carcinoma cells, inhibited the NF-κB-mediated cell motility (45Yebra M. Filardo E.J. Bayna E.M. Kawahara E. Becker J.C. Cheresh D.A. Mol. Biol. Cell. 1995; 6: 841-850Crossref PubMed Scopus (77) Google Scholar). In a disturbed flow field, there is a net EC migration directed away from the region of the high shear stress gradient (46Tardy Y. Resnick N. Nagel T. Gimbrone M.J. Dewey C.J. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3102-3106Crossref PubMed Scopus (223) Google Scholar). This organized migration pattern under disturbed flow conditions is accompanied by a >2-fold increase in cell motility. Thus, shear stress activation of the IKK/NF-κB pathway and of protein kinase C (47Kuchan M.J. Frangos J.A. Am. J. Physiol. 1993; 264: H150-H156PubMed Google Scholar) may regulate EC motility, which would be important for the morphological remodeling of ECs. Many extracellular stimuli activate NF-κB, presumably acting through IKK due to its specificity in phosphorylating IκB (19DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1910) Google Scholar, 20Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1850) Google Scholar). However, the upstream signaling events activated by the various stimuli that converge at IKKs have not yet been clearly established. There is increasing evidence to indicate that integrins are important in mechanotransduction in cells in response to mechanical stimuli (see Ref. 29Shyy J.Y.-J. Chien S. Curr. Opin. Cell Biol. 1997; 9: 707-713Crossref PubMed Scopus (294) Google Scholar for review). Indeed, the results in Fig. 5 suggest that integrins are directly involved in the shear stress activation of IKKs in ECs. The integrin-mediated signaling during cell adhesion to the ECM often results in an increase in the activity of tyrosine kinases in the focal adhesion sites, including focal adhesion kinase and Src family proteins (see Ref. 28Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1467) Google Scholar for review). Previous studies have shown that shear stress activates focal adhesion kinase and Src family kinases (35Li S. Kim M. Hu Y.L. Jalali S. Schlaepfer D.D. Hunter T. Chien S. Shyy J.Y.-J. J. Biol. Chem. 1997; 272: 30455-30462Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 48Takahashi M. Berk B.C. J. Clin. Invest. 1996; 98: 2623-2631Crossref PubMed Scopus (189) Google Scholar, 49Jalali S. Li Y.-S. Sotoudeh M. Yuan S. Li S. Chien S. Shyy J.Y.-J. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 227-234Crossref PubMed Scopus (218) Google Scholar). The inhibition of IKKs by genistein (Fig. 5) and the activation of IKKs when ECs attached to the ECM (Fig. 6) further confirmed that integrins are upstream molecules modulating IKKs. The observations that the isolated IKK complex from unstimulated cells can be activated in vitro by MEKK and that overexpression of MEKK in cells leads to the phosphorylation of IκB-α (50Lee F.S. Hagler J. Chen Z.J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar) indicate that the IKK/NF-κB pathway can be activated by MEKK. We have previously shown that Ras and MEKK modulate shear stress activation of c-Jun N-terminal kinase since dominant-negative mutants of Ras and MEKK block c-Jun N-terminal kinase activation in ECs (51Li Y.-S. Shyy J.Y.-J. Li S. Lee J. Su B. Karin M. Chien S. Mol. Cell. Biol. 1996; 16: 5947-5954Crossref PubMed Scopus (206) Google Scholar). Taken together, the previous studies suggest that integrins are activated by shear stress. As a consequence, the Ras/MEKK pathway is activated in a focal adhesion kinase- and c-Src-dependent manner, which in turn causes the activation of IKKs. In addition to integrins, other upstream signaling molecules such as the TNF receptor (TNFR), CD95 (Fas/Apo-1), and ROS may also be involved in the shear stress activation of IKKs. In the TNF induction of NF-κB, the signal transduces through the TNFR, TNFR-associated factor 2, and NF-κB-inducing kinase (52Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1162) Google Scholar, 53Hsu H. Solovyev I. Colombero A. Elliott R. Kelley M. Boyle W.J. J. Biol. Chem. 1997; 272: 13471-13474Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). NF-κB-inducing kinase is an IKK kinase that has been shown to activate IKKs, possibly through a direct interaction (21Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar, 34Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1067) Google Scholar). In addition to the TNFR, NF-κB-inducing kinase is also involved in CD95-mediated NF-κB activation (52Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1162) Google Scholar). We have found that shear stress causes the clustering of membrane-associated proteins such as Flk-1, which is a receptor for the vascular endothelium growth factor. 2K.-D. Chen and J. Y.-J. Shyy, unpublished results. It is likely that shear stress activates the TNFR and CD95 through a similar clustering-dependent mechanism and that this in turn leads to the activation of the NF-κB-inducing kinase/IKK pathway. The NF-κB/IκB pathway is potentiated by ROS, but attenuated by antioxidants (see Ref. 37Blake D.R. Winyard P.G. Marok R. Ann. N. Y. Acad. Sci. 1994; 723: 308-317Crossref PubMed Scopus (43) Google Scholar for review). Application of shear stress to ECs induces the production of ROS (54Chiu J.J. Wung B.S. Shyy J.Y.-J. Hsieh H.J. Wang D.L. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3570-3577Crossref PubMed Scopus (166) Google Scholar). Although the nature of the shear stress induction of ROS is still unknown, it is likely that the shear-generated ROS also modulates the activation of IKKs." @default.
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