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G Protein Coupling and Second Messenger Generation Are Indispensable for Metalloprotease-dependent, Heparin-binding Epidermal Growth Factor Shedding through Angiotensin II Type-1 Receptor
2005; Elsevier BV; Volume: 280; Issue: 28 Linguagem: Inglês
10.1074/jbc.m502906200
ISSN1083-351X
AutoresMizuo Mifune, Haruhiko Ohtsu, Hiroyuki Suzuki, H. Nakashima, Eugen Brailoiu, Nae J. Dun, Gerald D. Frank, Tadashi Inagami, Shigeki Higashiyama, Walter G. Thomas, Andrea D. Eckhart, Peter J. Dempsey, Satoru Eguchi,
Tópico(s)Receptor Mechanisms and Signaling
ResumoA G protein-coupled receptor agonist, angiotensin II (AngII), induces epidermal growth factor (EGF) receptor (EGFR) transactivation possibly through metalloprotease-dependent, heparin-binding EGF (HB-EGF) shedding. Here, we have investigated signal transduction of this process by using COS7 cells expressing an AngII receptor, AT1. In these cells AngII-induced EGFR transactivation was completely inhibited by pretreatment with a selective HB-EGF inhibitor, or with a metalloprotease inhibitor. We also developed a COS7 cell line permanently expressing a HB-EGF construct tagged with alkaline phosphatase, which enabled us to measure HB-EGF shedding quantitatively. In the COS7 cell line AngII stimulated release of HB-EGF. This effect was mimicked by treatment either with a phospholipase C activator, a Ca2+ ionophore, a metalloprotease activator, or H2O2. Conversely, pretreatment with an intracellular Ca2+ antagonist or an antioxidant blocked AngII-induced HB-EGF shedding. Moreover, infection of an adenovirus encoding an inhibitor of Gq markedly reduced EGFR transactivation and HB-EGF shedding through AT1. In this regard, AngII-stimulated HB-EGF shedding was abolished in an AT1 mutant that lacks Gq protein coupling. However, in cells expressing AT1 mutants that retain Gq protein coupling, AngII is still able to induce HB-EGF shedding. Finally, the AngII-induced EGFR transactivation was attenuated in COS7 cells overexpressing a catalytically inactive mutant of ADAM17. From these data we conclude that AngII stimulates a metalloprotease ADAM17-dependent HB-EGF shedding through AT1/Gq/phospholipase C-mediated elevation of intracellular Ca2+ and reactive oxygen species production, representing a key mechanism indispensable for EGFR transactivation. A G protein-coupled receptor agonist, angiotensin II (AngII), induces epidermal growth factor (EGF) receptor (EGFR) transactivation possibly through metalloprotease-dependent, heparin-binding EGF (HB-EGF) shedding. Here, we have investigated signal transduction of this process by using COS7 cells expressing an AngII receptor, AT1. In these cells AngII-induced EGFR transactivation was completely inhibited by pretreatment with a selective HB-EGF inhibitor, or with a metalloprotease inhibitor. We also developed a COS7 cell line permanently expressing a HB-EGF construct tagged with alkaline phosphatase, which enabled us to measure HB-EGF shedding quantitatively. In the COS7 cell line AngII stimulated release of HB-EGF. This effect was mimicked by treatment either with a phospholipase C activator, a Ca2+ ionophore, a metalloprotease activator, or H2O2. Conversely, pretreatment with an intracellular Ca2+ antagonist or an antioxidant blocked AngII-induced HB-EGF shedding. Moreover, infection of an adenovirus encoding an inhibitor of Gq markedly reduced EGFR transactivation and HB-EGF shedding through AT1. In this regard, AngII-stimulated HB-EGF shedding was abolished in an AT1 mutant that lacks Gq protein coupling. However, in cells expressing AT1 mutants that retain Gq protein coupling, AngII is still able to induce HB-EGF shedding. Finally, the AngII-induced EGFR transactivation was attenuated in COS7 cells overexpressing a catalytically inactive mutant of ADAM17. From these data we conclude that AngII stimulates a metalloprotease ADAM17-dependent HB-EGF shedding through AT1/Gq/phospholipase C-mediated elevation of intracellular Ca2+ and reactive oxygen species production, representing a key mechanism indispensable for EGFR transactivation. Angiotensin II (AngII) 1The abbreviations used are: AngII, angiotensin II; AP, alkaline phosphatase; ADAM, a disintegrin and metalloprotease; APMA, p-aminophenylmercuric acetate; AT1, AngII type 1 receptor; BiPS, 2R-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide; [Ca2+]i, intracellular Ca2+; dn, dominant-negative; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular signal-regulated kinase; HB-EGF, heparin-binding EGF; GPCR, G protein-coupled receptor; GqI, inhibitor of Gq signaling; MAPK, mitogen-activated protein kinase; m.o.i., multiplicity of infection; m-3M3FBS, 2,4,6-trimethyl-N-(m-3-tri-fluoromethylphenyl)benzenesulfonamide; NAC, N-acetylcysteine; PLC, phospholipase C; ROS, reactive oxygen species; VSMC, vascular smooth muscle cell; WT, wild type. and its G protein-coupled receptor (GPCR), the AngII type-1 receptor (AT1), play critical roles in mediating cardiovascular diseases such as hypertension, atherosclerosis, and restenosis after vascular injury (1Kim S. Iwao H. Pharmacol. Rev. 2000; 52: 11-34PubMed Google Scholar, 2Touyz R.M. Schiffrin E.L. Pharmacol. Rev. 2000; 52: 639-672PubMed Google Scholar). It is widely believed that AngII promotes these diseases by inducing vascular remodeling that involves hypertrophy, hyperplasia, and migration of vascular smooth muscle cells (VSMCs) (3Griendling K.K. Ushio-Fukai M. Lassegue B. Alexander R.W. Hypertension. 1997; 29: 366-373Crossref PubMed Google Scholar, 4Yin G. Yan C. Berk B.C. Int. J. Biochem. Cell Biol. 2003; 35: 780-783Crossref PubMed Scopus (110) Google Scholar). We and others have shown that AngII promotes these cellular effects by “trans”-activation of the epidermal growth factor receptor (EGFR) through the AT1 receptor (5Eguchi S. Frank G.D. Mifune M. Inagami T. Biochem. Soc. Trans. 2003; 31: 1198-1202Crossref PubMed Google Scholar, 6Saito Y. Berk B.C. J. Mol. Cell. Cardiol. 2001; 33: 3-7Abstract Full Text PDF PubMed Scopus (146) Google Scholar). Similar to EGF stimulation, AngII transactivates EGFR, which recruits the adaptor proteins Shc and Grb2, leading to the activation of the extracellular signal-regulated kinase (ERK) cascade (7Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar). Moreover, EGFR transactivation by AngII also leads to critical signaling responses such as activation of Akt/protein kinase B, p70 S6 kinase, and p38 mitogen-activated protein kinase (MAPK) in VSMCs (5Eguchi S. Frank G.D. Mifune M. Inagami T. Biochem. Soc. Trans. 2003; 31: 1198-1202Crossref PubMed Google Scholar, 8Eguchi S. Iwasaki H. Ueno H. Frank G.D. Motley E.D. Eguchi K. Marumo F. Hirata Y. Inagami T. J. Biol. Chem. 1999; 274: 36843-36851Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 9Eguchi S. Dempsey P.J. Frank G.D. Motley E.D. Inagami T. J. Biol. Chem. 2001; 276: 7957-7962Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). These data suggest that EGFR transactivation is one of the main points of convergence by which AngII induces several pathophysiological functions in its target organs (10Suzuki H. Motley E.D. Frank G.D. Utsunomiya H. Eguchi S. Curr. Med. Chem. Cardiovasc. Hematol. Agents. 2005; (in press)PubMed Google Scholar). Recently, several interesting observations have been made regarding the possible components involved in EGFR transactivation by GPCRs. First, EGFR transactivation by GPCRs appears to require a second messenger directly and/or signal transduction pathways operated by second messengers, such as elevation of intracellular Ca2+ (11Zwick E. Daub H. Aoki N. Yamaguchi-Aoki Y. Tinhofer I. Maly K. Ullrich A. J. Biol. Chem. 1997; 272: 24767-24770Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), activation of protein kinase C (12Tsai W. Morielli A.D. Peralta E.G. EMBO J. 1997; 16: 4597-4605Crossref PubMed Scopus (188) Google Scholar), and generation of reactive oxygen species (ROS) (13Cunnick J.M. Dorsey J.F. Standley T. Turkson J. Kraker A.J. Fry D.W. Jove R. Wu J. J. Biol. Chem. 1998; 273: 14468-14475Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In this regard, the EGFR transactivation by AngII seems to involve elevation of intracellular Ca2+ concentration and production of ROS in VSMCs (7Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 14Frank G.D. Eguchi S. Inagami T. Motley E.D. Biochem. Biophys. Res. Commun. 2001; 280: 1116-1119Crossref PubMed Scopus (56) Google Scholar, 15Ushio-Fukai M. Griendling K.K. Becker P.L. Hilenski L. Halleran S. Alexander R.W. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 489-495Crossref PubMed Scopus (260) Google Scholar, 16Seshiah P.N. Weber D.S. Rocic P. Valppu L. Taniyama Y. Griendling K.K. Circ. Res. 2002; 91: 406-413Crossref PubMed Scopus (634) Google Scholar). Second, a cytosolic non-receptor tyrosine kinase such as Src or PYK2 may be involved in the EGFR transactivation (17Luttrell L.M. Della Rocca G.J. van Biesen T. Luttrell D.K. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 4637-4644Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 18Andreev J. Galisteo M.L. Kranenburg O. Logan S.K. Chiu E.S. Okigaki M. Cary L.A. Moolenaar W.H. Schlessinger J. J. Biol. Chem. 2001; 276: 20130-20135Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Both kinases have been shown to be activated by AngII in VSMCs (19Ishida M. Ishida T. Thomas S.M. Berk B.C. Circ. Res. 1998; 82: 7-12Crossref PubMed Scopus (150) Google Scholar, 20Sabri A. Govindarajan G. Griffin T.M. Byron K.L. Samarel A.M. Lucchesi P.A. Circ. Res. 1998; 83: 841-851Crossref PubMed Scopus (137) Google Scholar, 21Brinson A.E. Harding T. Diliberto P.A. He Y. Li X. Hunter D. Herman B. Earp H.S. Graves L.M. J. Biol. Chem. 1998; 273: 1711-1718Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Third, an attractive mechanism for the EGFR transactivation by a GPCR was proposed recently that involves metalloprotease-dependent EGFR ligand production from its membrane-bound precursor (9Eguchi S. Dempsey P.J. Frank G.D. Motley E.D. Inagami T. J. Biol. Chem. 2001; 276: 7957-7962Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 22Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1499) Google Scholar). The EGF ligand family consists of EGF, heparin binding-EGF like growth factor (HB-EGF), transforming growth factor-α, epiregulin, amphiregulin, epigen, neuregulins, and betacellulin (23Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell Biol. 2001; 2: 127-137Crossref PubMed Scopus (5633) Google Scholar). Among these, HB-EGF has been most implicated in vascular remodeling because it is a potent mitogen and chemotactic factor for VSMCs and its expression is enhanced in vascular lesions such as atherosclerosis and restenosis following angioplasty (24Raab G. Klagsbrun M. Biochim. Biophys. Acta. 1997; 1333: F179-F199PubMed Google Scholar, 25Berk B.C. Physiol. Rev. 2001; 81: 999-1030Crossref PubMed Scopus (333) Google Scholar). Like other members of the EGF ligand family, HB-EGF is synthesized as a transmembrane precursor “pro-HB-EGF” that is proteolytically cleaved (“shedding”) to release a biologically active soluble growth factor (26Nanba D. Higashiyama S. Cytokine Growth Factor Rev. 2004; 15: 13-19Crossref PubMed Scopus (53) Google Scholar). Recently, many GPCR agonists appear to mediate EGFR transactivation through this metalloprotease-dependent HB-EGF shedding (27Gschwind A. Zwick E. Prenzel N. Leserer M. Ullrich A. Oncogene. 2001; 20: 1594-1600Crossref PubMed Scopus (414) Google Scholar). We and others also showed the requirement of HB-EGF for EGFR transactivation through the AT1 receptor (9Eguchi S. Dempsey P.J. Frank G.D. Motley E.D. Inagami T. J. Biol. Chem. 2001; 276: 7957-7962Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 28Fujiyama S. Matsubara H. Nozawa Y. Maruyama K. Mori Y. Tsutsumi Y. Masaki H. Uchiyama Y. Koyama Y. Nose A. Iba O. Tateishi E. Ogata N. Jyo N. Higashiyama S. Iwasaka T. Circ. Res. 2001; 88: 22-29Crossref PubMed Scopus (205) Google Scholar, 29Thomas W.G. Brandenburger Y. Autelitano D.J. Pham T. Qian H. Hannan R.D. Circ. Res. 2002; 90: 135-142Crossref PubMed Scopus (162) Google Scholar). However, the identity of the metalloprotease as well as the detailed signaling mechanisms of HB-EGF shedding by AngII in relation to G protein coupling, second messengers, and upstream kinases are largely unknown. In this study we established a COS7 cell line expressing alkaline phosphatase (AP)-conjugated HB-EGF that enabled us to measure the HB-EGF shedding activity quantitatively. By using this system together with molecular and pharmacological tools including several AT1 receptor mutants, we have elucidated the involvement of heterotrimetric G protein coupling and second messengers (Ca2+ and ROS) in a critical step of a metalloprotease-dependent HB-EGF production. The findings presented here will provide a novel molecular insight by which AngII contributes to cardiovascular diseases. Materials—Phospho-specific antibodies for Tyr1068-phosphorylated EGFR and for Tyr1007-Tyr1008-phosphorylated JAK2 were purchased from BIOSOURCE. Antibody against EGFR was purchased from Santa Cruz Biotechnology. Antibody against hemagglutinin was purchased from Zymed Laboratories Inc.. YM-254890 was a gift from Yamanouchi Pharmaceutical Co. AngII, N-acetylcysteine (NAC), and H2O2 were purchased from Sigma. A23187, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester, GF109203X, TMB-8, phorbol 12-myristate 13-acetate, CRM197, m-3M3FBS, p-aminophenylmercuric acetate (APMA), and 2R-[(4-biphenylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide (BiPS) were purchased from Calbiochem. BiPS was originally described as a matrix metalloprotease-2 and matrix metalloprotease-9 inhibitor (30Tamura Y. Watanabe F. Nakatani T. Yasui K. Fuji M. Komurasaki T. Tsuzuki H. Maekawa R. Yoshioka T. Kawada K. Sugita K. Ohtani M. J. Med. Chem. 1998; 41: 640-649Crossref PubMed Scopus (276) Google Scholar). However, our subsequent findings demonstrated the ability of BiPS to inhibit EGFR transactivation by AngII that was not mediated by matrix metalloproteases (31Saito S. Frank G.D. Motley E.D. Dempsey P.J. Utsunomiya H. Inagami T. Eguchi S. Biochem. Biophys. Res. Commun. 2002; 294: 1023-1029Crossref PubMed Scopus (62) Google Scholar). BiPS shares its structure with CGS27023, which can also inhibit EGFR transactivation by AngII (31Saito S. Frank G.D. Motley E.D. Dempsey P.J. Utsunomiya H. Inagami T. Eguchi S. Biochem. Biophys. Res. Commun. 2002; 294: 1023-1029Crossref PubMed Scopus (62) Google Scholar). CGS27023 was demonstrated to block the catalytic activity of ADAM9 and ADAM17 (32Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjument-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar), suggesting that BiPS could act as an ADAM inhibitor as well. Cell Lines—COS7 cells were obtained from the American Type Culture Collection and subcultured as described previously (33Frank G.D. Mifune M. Inagami T. Ohba M. Sasaki T. Higashiyama S. Dempsey P.J. Eguchi S. Mol. Cell. Biol. 2003; 23: 1581-1589Crossref PubMed Scopus (100) Google Scholar). COS7 cells permanently expressing HB-EGF were established by antibiotic selection after transfection with the AP-tagged HB-EGF (HBEGF-AP) plasmid (34Tokumaru S. Higashiyama S. Endo T. Nakagawa T. Miyagawa J.I. Yamamori K. Hanakawa Y. Ohmoto H. Yoshino K. Shirakata Y. Matsuzawa Y. Hashimoto K. Taniguchi N. J. Cell Biol. 2000; 151: 209-220Crossref PubMed Scopus (264) Google Scholar) as described previously (33Frank G.D. Mifune M. Inagami T. Ohba M. Sasaki T. Higashiyama S. Dempsey P.J. Eguchi S. Mol. Cell. Biol. 2003; 23: 1581-1589Crossref PubMed Scopus (100) Google Scholar). A Chinese hamster ovary cell line stably expressing wild type rat AT1 (AT1WT) and its deletion mutants, AT1-(1-309) and AT1-(1-317), were established as described previously (35Sano T. Ohyama K. Yamano Y. Nakagomi Y. Nakazawa S. Kikyo M. Shirai H. Blank J.S. Exton J.H. Inagami T. J. Biol. Chem. 1997; 272: 23631-23636Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The AT1 receptors expressed in these cells have a comparable Kd and Bmax (35Sano T. Ohyama K. Yamano Y. Nakagomi Y. Nakazawa S. Kikyo M. Shirai H. Blank J.S. Exton J.H. Inagami T. J. Biol. Chem. 1997; 272: 23631-23636Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Adenoviral Infection—Adenovirus constructs encoding wild type rat AT1 receptor and a carboxyl-terminal mutant, AT1Y319F, in which carboxyl-terminal Tyr319 was replaced with Phe319 were generated as described previously (29Thomas W.G. Brandenburger Y. Autelitano D.J. Pham T. Qian H. Hannan R.D. Circ. Res. 2002; 90: 135-142Crossref PubMed Scopus (162) Google Scholar, 36Seta K. Nanamori M. Modrall J.G. Neubig R.R. Sadoshima J. J. Biol. Chem. 2002; 277: 9268-9277Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 37Seta K. Sadoshima J. J. Biol. Chem. 2003; 278: 9019-9026Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The adenoviral vector containing the inhibitor of Gq signaling (GqI), comprised of the amino acids 305-359 of murine Gαq, was constructed as described previously (38Akhter S.A. Skaer C.A. Kypson A.P. MacDonald P.H. Peppel K.C. Glower D.D. Lefkowitz R.J. Koch W.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12100-12105Crossref PubMed Scopus (169) Google Scholar). Each adenovirus titer (m.o.i.) was determined by Adeno-X™ rapid titer kit (BD Biosciences). Confluent COS7 cells were infected with adenovirus at 50-100 m.o.i. for 2 days as described previously (33Frank G.D. Mifune M. Inagami T. Ohba M. Sasaki T. Higashiyama S. Dempsey P.J. Eguchi S. Mol. Cell. Biol. 2003; 23: 1581-1589Crossref PubMed Scopus (100) Google Scholar). Transfection efficiency was estimated to be >95% as defined by infection with adenovirus (50 m.o.i.) encoding green fluorescent protein. Retroviral Infection—C-terminal hemagglutinin-tagged catalytically inactive/dominant negative ADAM17 (dnADAM17), in which Glu406 was replaced with Ala (39Garton K.J. Gough P.J. Blobel C.P. Murphy G. Greaves D.R. Dempsey P.J. Raines E.W. J. Biol. Chem. 2001; 276: 37993-38001Abstract Full Text Full Text PDF PubMed Google Scholar), was cloned into the pBM-IRES-PURO retroviral vector (40Sanderson M.P. Erickson S.N. Gough P.J. Garton K.J. Wille P.T. Raines E.W. Dunbar A.J. Dempsey P.J. J. Biol. Chem. 2005; 280: 1826-1837Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). For retroviral infection, 4 × 105 cells were seeded into 25-cm2 tissue culture flasks and cultured for 24 h prior to infection. Cells were incubated with 5 ml of virus stock for 12 h in the presence of 4 μg/ml Polybrene and then replenished with fresh media. Cells were then grown for 48 h prior to passaging into media containing 6 μg/ml puromycin. Resistant cells were used in subsequent experiments (40Sanderson M.P. Erickson S.N. Gough P.J. Garton K.J. Wille P.T. Raines E.W. Dunbar A.J. Dempsey P.J. J. Biol. Chem. 2005; 280: 1826-1837Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Plasmid Transfection—Plasmids encoding rat AT1WT and AT1Y319F were generated as described previously (35Sano T. Ohyama K. Yamano Y. Nakagomi Y. Nakazawa S. Kikyo M. Shirai H. Blank J.S. Exton J.H. Inagami T. J. Biol. Chem. 1997; 272: 23631-23636Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 41Ohyama K. Yamano Y. Sano T. Nakagomi Y. Wada M. Inagami T. Biochem. Biophys. Res. Commun. 2002; 292: 362-367Crossref PubMed Scopus (32) Google Scholar). COS7 cells were transiently transfected with the plasmids by using FuGENE6 (Roche Applied Science) for 24 h with 10% serum, and then the cells were serum-starved for 24 h before stimulation. HB-EGF Shedding Assay—48 h after AT1 receptor transfection, COS7-HBEGF-AP cells were pre-incubated in fresh phenol red-free Dulbecco's modified Eagle's medium for 30 min in the presence or absence of inhibitors and then stimulated by agonists up to 60 min. The HB-EGF-AP secreted into the medium was assessed by measuring alkaline phosphatase activity as described previously (33Frank G.D. Mifune M. Inagami T. Ohba M. Sasaki T. Higashiyama S. Dempsey P.J. Eguchi S. Mol. Cell. Biol. 2003; 23: 1581-1589Crossref PubMed Scopus (100) Google Scholar). Western Blotting—Cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose membrane as described previously (42Eguchi S. Matsumoto T. Motley E.D. Utsunomiya H. Inagami T. J. Biol. Chem. 1996; 271: 14169-14175Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). The membranes were then exposed to primary antibodies overnight at 4 °C. After incubation with the peroxidase-linked secondary antibody for 1 h at room temperature, the immunoreactive proteins were visualized by a chemiluminescence reaction kit (Chemicon). Intracellular Ca2+ ([Ca2+]i) Measurements—[Ca2+]i was measured as described previously (43Filipeanu C.M. Brailoiu E. Le Dun S. Dun N.J. J. Neurochem. 2002; 83: 879-884Crossref PubMed Scopus (42) Google Scholar). VSMCs subcultured on coverslips were loaded in Hanks' balanced salt solution with 3 μm fura 2/AM at room temperature for 45 min in the dark and then washed three times with fura 2-free Hanks' balanced salt solution to allow for complete de-esterification of the dye for 15-60 min. Under these conditions, compartmentalization of the dye was minimal. The coverslips were mounted in a custom-designed bath in the stage of a S300 Axiovert Nikon inverted microscope equipped with a C & L Instruments fluorometer system. The fura 2 fluorescence was acquired at a frequency of 1 Hz, and [Ca2+]I values were then obtained as described (43Filipeanu C.M. Brailoiu E. Le Dun S. Dun N.J. J. Neurochem. 2002; 83: 879-884Crossref PubMed Scopus (42) Google Scholar). Statistic—Data were analyzed by using the Student's t test. The mean ± S.E. was determined with a significance level of p < 0.05. Results are representative of at least three separate experiments. Metalloprotease and HB-EGF-dependent EGFR Transactivation by AngII in COS7 Cells—Using COS7 cells, we have established a system to examine the mechanism of EGFR transactivation through a GPCR, AT1. For this purpose, COS7 cells were infected with an adenovirus encoding AT1. Compared with the COS7 cells infected with the control adenovirus encoding LacZ, AngII stimulation resulted in marked phosphorylation of the EGFR at Tyr1068, a Grb2-binding site, in COS7 cells expressing AT1 in a time-dependent manner (Fig. 1A). To determine the involvement of metalloprotease activation in AngII-induced EGFR transactivation in COS7 cells, the effect of a metalloprotease inhibitor, BiPS, on EGFR phosphorylation at Tyr1068 was examined. As we observed previously in VSMCs (31Saito S. Frank G.D. Motley E.D. Dempsey P.J. Utsunomiya H. Inagami T. Eguchi S. Biochem. Biophys. Res. Commun. 2002; 294: 1023-1029Crossref PubMed Scopus (62) Google Scholar), BiPS completely inhibited AngII-induced EGFR transactivation in COS7 cells (Fig. 1B). To demonstrate the critical involvement of HB-EGF in AngII-induced EGFR transactivation, the effect of a diphtheria toxin analogue, CRM197, which acts as a specific inhibitor against primate HB-EGF was examined in regard to EGFR phosphorylation. Pretreatment of CRM197 markedly inhibited AngII-induced EGFR transactivation in COS7 cells expressing AT1 (Fig. 1B). In contrast, both BiPS and CRM197 did not affect JAK2 phosphorylation at Tyr1007-Tyr1008 stimulated by AngII. These results clearly demonstrated that COS7 cells expressing AT1 provide an interesting model for studying the mechanism of AngII-induced EGFR transactivation involving metalloprotease-dependent HB-EGF production. To examine the detailed mechanism of metalloprotease-dependent HB-EGF shedding, we took advantage of a reporter assay system using transfection of the HB-EGF-AP plasmid, an established assay for HB-EGF shedding (34Tokumaru S. Higashiyama S. Endo T. Nakagawa T. Miyagawa J.I. Yamamori K. Hanakawa Y. Ohmoto H. Yoshino K. Shirakata Y. Matsuzawa Y. Hashimoto K. Taniguchi N. J. Cell Biol. 2000; 151: 209-220Crossref PubMed Scopus (264) Google Scholar). In COS7 cells permanently expressing this plasmid, we evaluated the shedding activity of HB-EGF by measuring AP activity secreted into the medium. In these cells, there is a gradual but statistically significant accumulation of AP activity in a non-stimulated condition during pre-incubation, suggesting a presence of basal shedding activity of HB-EGF. However, basal shedding was not further enhanced up to 60 min, suggesting a saturated nature of the basal shedding activity. In contrast, a marked and time-dependent enhancement of AP activity was observed when cells were treated with AngII, thus demonstrating the ability of AngII to stimulate HB-EGF shedding. In addition, AngII-induced HB-EGF shedding was completely inhibited by BiPS (data not shown). These findings further indicated that the HB-EGF-AP assay system in a cell line expressing AT1 would be an ideal tool to study the signal transduction mechanism of metalloprotease-dependent HB-EGF shedding and subsequent EGFR transactivation by AngII. AngII Stimulates HB-EGF Shedding through Intracellular Ca2+ Elevation and ROS Production—Previous studies have shown the involvement of Ca2+ and ROS as critical signal intermediates in EGFR transactivation through AT1 in VSMCs (7Eguchi S. Numaguchi K. Iwasaki H. Matsumoto T. Yamakawa T. Utsunomiya H. Motley E.D. Kawakatsu H. Owada K.M. Hirata Y. Marumo F. Inagami T. J. Biol. Chem. 1998; 273: 8890-8896Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 14Frank G.D. Eguchi S. Inagami T. Motley E.D. Biochem. Biophys. Res. Commun. 2001; 280: 1116-1119Crossref PubMed Scopus (56) Google Scholar, 15Ushio-Fukai M. Griendling K.K. Becker P.L. Hilenski L. Halleran S. Alexander R.W. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 489-495Crossref PubMed Scopus (260) Google Scholar). Also, protein kinase C may exist upstream of the ROS production (16Seshiah P.N. Weber D.S. Rocic P. Valppu L. Taniyama Y. Griendling K.K. Circ. Res. 2002; 91: 406-413Crossref PubMed Scopus (634) Google Scholar). In COS7 cells, AngII-induced HB-EGF shedding was completely blocked by pretreatment with TMB-8, an intracellular Ca2+ antagonist, as well as by NAC, a potent antioxidant (Fig. 2A). In contrast, a protein kinase C inhibitor, GF109203X, had no significant effect on AngII-induced HB-EGF shedding (Fig. 2B), whereas it markedly inhibited 100 nm phorbol 12-myristate 13-acetate-induced HB-EGF shedding in the COS7 cells (data not shown). Stimulation with a metalloprotease activator, APMA, a Ca2+ ionophore, A23187, and H2O2 resulted in enhanced HB-EGF shedding that was inhibited by pretreatment with BiPS. The basal shedding activity was also partially inhibited by BiPS (Fig. 2C). In addition, we confirmed that APMA and A23187 as well as H2O2 induced EGFR transactivation in COS7 cells (data not shown). Moreover, HB-EGF shedding stimulated by A23187 was completely blocked by NAC (Fig. 2D), whereas H2O2-induced HB-EGF shedding was minimally affected by TMB-8 (data not shown). These results suggest that, in COS7 cells, intracellular Ca2+ elevation and subsequent ROS production are required for a metalloprotease activation that is responsible for EGFR transactivation through AT1. Requirement of Gq Coupling for HB-EGF Shedding through AT1—In addition to Gq,AT1 has been shown to couple Gi,G12, and/or G13, depending on cell type (44Ushio-Fukai M. Griendling K.K. Akers M. Lyons P.R. Alexander R.W. J. Biol. Chem. 1998; 273: 19772-19777Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 45Gohla A. Schultz G. Offermanns S. Circ. Res. 2000; 87: 221-227Crossref PubMed Scopus (199) Google Scholar). Although intracellular Ca2+ elevation through AT1 is primarily believed to require Gαq/PLC-β, additional mechanisms involving Gα12/13, Gβγ, PLC-γ, and/or l-type Ca2+ channel have been proposed (44Ushio-Fukai M. Griendling K.K. Akers M. Lyons P.R. Alexander R.W. J. Biol. Chem. 1998; 273: 19772-19777Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Activation of Gq has been proposed to participate in a GPCR-induced EGFR transactivation (46Seo B. Choy E.W. Maudsley S. Miller W.E. Wilson B.A. Luttrell L.M. J. Biol. Chem. 2000; 275: 2239-2245Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), whereas possible participation of the Gβγ subunit was reported in a metalloprotease/EGFR-dependent ERK activation by α2A-adrenergic receptor in human embryonic kidney cells (47Pierce K.L. Tohgo A. Ahn S. Field M.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 23155-23160Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Therefore, we examined the contribution of Gq signaling in HB-EGF shedding induced by AngII by using an adenovirus encoding the C-terminal fragment of Gαq termed GqI, a selective Gq inhibitor (48Akhter S.A. Luttrell D.K. Rockman H.A. Iaccarino G. Lefkowitz R.J. Koch W.J. Science. 1998; 280: 574-577Crossref PubMed Scopus (393) Google Scholar), as well as a novel Gq selective pharmacological inhibitor, YM-254890 (49Takasaki J. Saito T. Taniguchi M. Kawasaki T. Moritani Y. Hayashi K. Kobori M. J. Biol. Chem. 2004; 279: 47438-47445Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). GqI markedly inhibited intracellular Ca2+ elevation (Fig. 3A), HB-EGF shedding (Fig. 3B), and EGFR transactivation (Fig. 3C) induced by AngII through AT1. YM-254890 also inhibited HB-EGF shedding induced by AngII but not by A23187 (Fig. 3D). In addition, a selective PLC activator, m-3M3FBS (50Bae Y.S. Lee T.G. Park J.C. Hur J.H. Kim Y. Heo
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