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Activation of MAPKs by Angiotensin II in Vascular Smooth Muscle Cells

2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês

10.1074/jbc.m008570200

ISSN

1083-351X

Autores

Satoru Eguchi, Peter J. Dempsey, Gerald D. Frank, Evangeline D. Motley, Tadashi Inagami,

Tópico(s)

Melanoma and MAPK Pathways

Resumo

In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the Gq-coupled AT1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca2+-dependent "transactivation" of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca2+ ionophore A23187 without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT1 receptor antagonist, RNH6270.A23187 and EGF markedly activate p38MAPK, whereas A23187 but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloprotease-dependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation. In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the Gq-coupled AT1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca2+-dependent "transactivation" of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca2+ ionophore A23187 without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT1 receptor antagonist, RNH6270.A23187 and EGF markedly activate p38MAPK, whereas A23187 but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloprotease-dependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation. angiotensin II extracellular signal-regulated kinase p38 mitogen-activated protein kinase c-Jun N-terminal kinase angiotensin II type-1 receptor G protein-coupled receptor epidermal growth factor EGF receptor heparin-binding EGF-like growth factor platelet-derived growth factor vascular smooth muscle cells protein kinase C phorbol 12-myristate 13-acetate a disintegrin and metalloprotease Angiotensin II (AngII),1the major bioactive peptide of the renin-angiotensin system, plays a fundamental role, not only in controlling cardiovascular and renal homeostasis but also contributing to various cardiovascular diseases such as hypertension, atherosclerosis, and heart failure. In addition to the anti-hypertensive effects, both AngII-converting enzyme inhibitors and AngII type-1 (AT1) receptor antagonists appear to exert tissue-protective effects against these diseases. Therefore, the growth-stimulating activity of the AT1receptor likely contributes to the progression of cardiovascular remodeling (1Goodfriend T.L. Elliott M.E. Catt K.J. N. Engl. J. Med. 1996; 334: 1649-1654Crossref PubMed Google Scholar, 2Dzau V.J. Eur. Heart J. 1998; 19: J2-J6PubMed Google Scholar, 3Dostal D.E. Baker K.M. Circ. Res. 1999; 85: 643-650Crossref PubMed Scopus (302) Google Scholar). In vascular smooth muscle cells (VSMC), AngII is believed to transmit its growth-promoting signal through activation of tyrosine kinases (4Griendling K.K. Ushio F.M. Lassegue B. Alexander R.W. Hypertension. 1997; 29: 366-373Crossref PubMed Google Scholar, 5Berk B.C. J. Am. Soc. Nephrol. 1999; 10: S62-S68Crossref PubMed Google Scholar, 6Inagami T. Eguchi S. Numaguchi K. Motley E.D. Tao H. Matsumoto T. Yamakawa T. J. Am. Soc. Nephrol. 1999; 10: S57-S61PubMed Google Scholar, 7Eguchi S. Inagami T. Regul. Pept. 2000; 91: 13-20Crossref PubMed Scopus (129) Google Scholar) that may involve PYK2 (8Brinson 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, 9Sabri 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, 10Eguchi S. Iwasaki H. Inagami T. Numaguchi K. Yamakawa T. Motley E.D. Owada K.M. Marumo F. Hirata Y. Hypertension. 1999; 33: 201-206Crossref PubMed Google Scholar), c-Src (11Ishida M. Ishida T. Thomas S.M. Berk B.C. Circ. Res. 1998; 82: 7-12Crossref PubMed Scopus (151) Google Scholar), JAK2 (12Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (654) Google Scholar), platelet-derived growth factor (PDGF)-β receptor (13Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), and the epidermal growth factor (EGF) receptor (14Eguchi 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 (507) Google Scholar). We have reported that Ca2+-dependent transactivation of the EGF receptor (EGFR) through the AT1receptor is essential for the activation of downstream Ser/Thr kinases (ERK, Akt/protein kinase B, and p70 S6 kinase), and subsequent c-Fos induction and protein synthesis by AngII in cultured rat VSMC (14Eguchi 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 (507) Google Scholar, 15Eguchi S. Iwasaki H. Motley E.D. Frank G.D. Ueno H. Eguchi K. Marumo F. Hirata Y. Inagami T. J. Biol. Chem. 1999; 274: 36843-36852Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 16Eguchi S. Iwasaki H. Hirata Y. Frank J.D. Motley E.D. Yamakawa T. Numaguchi K. Inagami T. Eur. J. Pharmacol. 1999; 376: 203-206Crossref PubMed Scopus (43) Google Scholar). Thus, the EGFR transactivation could play a central role in AngII-mediated vascular remodeling. This new concept of a G protein-coupled receptor (GPCR) signaling, originally reported in Rat1 fibroblasts (17Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1329) Google Scholar), is now supported by a similar transactivation of the EGFR by a variety of GPCRs in many cells (18Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 19Luttrell L.M. Daaka Y. Lefkowitz R.J. Cur. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (612) Google Scholar, 20Carpenter G. J. Cell Biol. 1999; 146: 697-702Crossref PubMed Scopus (247) Google Scholar). Several mechanisms involving an upstream tyrosine kinase, reactive oxygen species, and metalloprotease(s) have been proposed for the transactivation of EGFR by GPCRs (7Eguchi S. Inagami T. Regul. Pept. 2000; 91: 13-20Crossref PubMed Scopus (129) Google Scholar, 18Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 19Luttrell L.M. Daaka Y. Lefkowitz R.J. Cur. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (612) Google Scholar, 20Carpenter G. J. Cell Biol. 1999; 146: 697-702Crossref PubMed Scopus (247) Google Scholar, 21Cunnick 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, 22Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar). Although it has proven difficult to detect GPCR-induced release of endogenous EGFR ligands (14Eguchi 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 (507) Google Scholar, 22Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar, 23Tsai W. Morielli A.D. Peralta E.G. EMBO J. 1997; 15: 4597-4605Crossref Scopus (188) Google Scholar), the metalloprotease-dependent shedding of heparin-binding EGF-like growth factor (HB-EGF) (22Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar) is an attractive mechanism of the EGFR transactivation in VSMC. This is because HB-EGF is a major EGF-like growth factor synthesized in VSMC, is a potent mitogenic and chemotactic factor for VSMC, and is implicated in the pathogenesis of atherosclerosis and restenosis following balloon injury (24Raab G. Klagsbrun M. Biochim. Biophys. Acta. 1997; 1333: F179-F199PubMed Google Scholar). The p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) are members of MAPKs that are preferentially stimulated by environmental stresses and inflammatory cytokines. Like ERK, these stress-activated MAPKs phosphorylate specific subsets of transcriptional factors, thereby regulating cellular processes of inflammation, proliferation, differentiation, apoptosis, and/or survival (25Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2292) Google Scholar, 26Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1386) Google Scholar, 27New L. Han J. Trends. Cardiovasc. Med. 1998; 8: 220-228Crossref PubMed Scopus (152) Google Scholar). They also are likely to play critical roles in cardiovascular disease (27New L. Han J. Trends. Cardiovasc. Med. 1998; 8: 220-228Crossref PubMed Scopus (152) Google Scholar, 28Force T. Pombo C.M. Avruch J.A. Bonventre J.V. Kyriakis J.M. Circ. Res. 1996; 78: 947-953Crossref PubMed Scopus (187) Google Scholar). Indeed, AngII has recently been shown to activate both p38MAPK (29Ushio F.M. Alexander R.W. Akers M. Griendling K.K. J. Biol. Chem. 1998; 273: 15022-15029Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar) and JNK (30Schmitz U. Ishida T. Ishida M. Surapisitchat J. Hasham M.I. Pelech S. Berk B.C. Circ. Res. 1998; 82: 1272-1278Crossref PubMed Scopus (84) Google Scholar) in cultured VSMC. The p38MAPK may positively regulate VSMC growth induced by AngII (29Ushio F.M. Alexander R.W. Akers M. Griendling K.K. J. Biol. Chem. 1998; 273: 15022-15029Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar), whereas JNK was activated in a balloon-injured artery that could be inhibited by an AT1 antagonist (31Kim S. Izumi Y. Yano M. Hamaguchi A. Miura K. Yamanaka S. Miyazaki H. Iwao H. Circulation. 1998; 97: 1731-1737Crossref PubMed Scopus (110) Google Scholar). Thus, there is considerable interest in defining the signal transduction pathways by which AngII activates these stress-activated MAPKs. In the present study, we have examined the hypothesis that a metalloprotease-dependent EGFR transactivation regulates p38MAPK and/or JNK activation by AngII in VSMC. Here we report several lines of evidence indicating that, in addition to ERK, AngII-induced p38MAPK activation requires the metalloprotease-dependent EGFR transactivation, which may involve HB-EGF processing, whereas JNK activation is not mediated by the metalloprotease-dependent system. AngII and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma Chemical Co. AG1478, PD158780, A23187, and anisomycin were obtained from Calbiochem. The metalloprotease inhibitors BB2116 and BB94 were kindly provided by Dr. Helen Mills (British Biotech). RNH-6270 (32Mizuno M. Sada T. Ikeda M. Fukuda N. Miyamoto M. Yanagisawa H. Koike H. Eur. J. Pharmacol. 1995; 285: 181-188Crossref PubMed Scopus (158) Google Scholar), the active form of a prodrug type AT1 antagonist CS-866, was a gift from Sankyo Co., Ltd. EGF, PDGF-BB, and antibodies to phosphotyrosine and JAK2 were obtained from Upstate Biotechnology. The antibodies directed to Tyr204-phosphorylated ERK1/2, ERK2, JNK2 p38MAPK, and EGFR were obtained from Santa Cruz Biotechnology. The antibody directed to Thr180/Tyr182-dually phosphorylated p38MAPK was obtained from New England BioLabs. The antibody directed to Thr183/Tyr185-dually phosphorylated JNK was obtained from Promega Inc. The antibody directed to Tyr1007/Tyr1008-dually phosphorylated JAK2 was obtained from BIOSOURCE International. Anti-PYK2 antibody was obtained from BD Transduction Laboratories. A neutralizing antibody to human HB-EGF was obtained from R & D Systems Inc. The anti-human HB-EGF antibody for immunoblotting was kindly provided by Dr. Judith A. Abraham (Scios Inc). VSMC were prepared from the thoracic aorta of Harlan Sprague-Dawley rats by the explant method and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum as previously described (33Eguchi S. Hirata Y. Imai T. Kanno K. Marumo F. Endocrinology. 1994; 134: 222-228Crossref PubMed Scopus (91) Google Scholar). In all experiments, subcultured VSMC from passages 3–10 were used and showed >99% positive immunostaining of smooth muscle α-actin antibody. The expression of AT1 but not AT2 was confirmed as previously described (34Eguchi 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). Cultured human aortic VSMC were obtained from Clonetics, and subcultured according to the manufacture's manual. Cells at ∼80% confluence in culture wells were made quiescent by serum deprivation for 3 days prior to treatment. After stimulation, cells were lysed with ice-cold immunoprecipitation buffer (50 mm HEPES, pH 7.5, 50 mm NaCl, 1% Triton X-100, 1 mm EDTA, 10 mm sodium pyrophosphate, 1 mmNa3VO4, 30 mm2-(p-nitrophenyl) phosphate, 100 mm NaF, 10% glycerol, 1.5 mm MgCl2, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Lysates were centrifuged at 14,000 × g for 5 min, and the supernatant was immunoprecipitated with the antibody and protein A/G-agarose for 16 h at 4 °C as described previously (14Eguchi 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 (507) Google Scholar). Cell lysates or immunocomplex lysates were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted using an ECL detection kit (Amersham Pharmacia Biotech) as described previously (14Eguchi 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 (507) Google Scholar). Fura-2 was used to monitor changes in intracellular Ca2+ concentration using a previously described procedure (35Yamakawa T. Eguchi S. Yamakawa Y. Motley E.D. Numaguchi K. Utsunomiya H. Inagami T. Hypertension. 1998; 31: 248-253Crossref PubMed Scopus (64) Google Scholar). After incubation in serum-free medium for 48 h, cells were trypsinized, incubated with 4 μm fura-2 acetoxymethylester for 30 min at 37 °C in Krebs-Ringer HEPES solution (20 mm HEPES, pH 7.4, 130 mm NaCl, 5 mm KCl, 1 mmMgCl2, 1.5 mm CaCl2, 10 mm glucose, 0.1% bovine serum albumin), and resuspended to 2 × 106 cells/ml. Measurements of fluorescence were made at 37 °C using a SPEX dual wavelength fluorometer (excitation at 340 and 380 nm; emission at 510 nm). After cell-surface proteins of human VSMC were biotinylated by 1 mg/ml Sulfo-NHS-LC-biotin (Pierce) for 30 min at 4 °C in Hanks' balanced salt solution, VSMC were rinsed and incubated in the cultured medium for 30 min at 37 °C and stimulated with or without AngII. Culture medium was collected and precipitated with immobilized streptavidin-agarose (Pierce), and immunoblotted with anti-human-HB-EGF antibody. Unless stated otherwise, results are representative of at least three experiments giving similar results. A recent study indicates that the transactivation of EGFR by several GPCR agonists requires pro-HB-EGF processing that can be blocked by a synthetic metalloprotease inhibitor, BB94 (22Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar). We have shown that AngII mainly activates ERK through the EGFR transactivation in VSMC (14Eguchi 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 (507) Google Scholar). To determine whether this cascade involves metalloprotease activation, the effect of two hydroxamic acid-based metalloprotease inhibitors BB2116 and BB94 on AngII-induced ERK activation was examined in cultured rat VSMC. As shown in Fig. 1 (A andB), BB2116 markedly and concentration-dependently inhibited AngII-induced ERK1/2 activation as assessed by their phosphorylation. By contrast, BB2116 had no effect on ERK activation induced by EGF or PDGF-BB (Fig.1 B). BB94 also inhibited AngII-induced ERK activation in VSMC to a similar extent as BB2116 (Fig. 1 C). The EGFR transactivation and subsequent ERK activation by AngII require intracellular Ca2+ elevation through the Gq-coupled AT1 receptor but not protein kinase C (PKC) (14Eguchi 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 (507) Google Scholar, 34Eguchi 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). In agreement with our previous observations (14Eguchi 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 (507) Google Scholar, 34Eguchi 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), ERK activation by the Ca2+ ionophore A23187 was markedly inhibited by BB2116, whereas PMA-induced ERK activation was minimally affected (Fig. 2 A). BB2116 did not alter AngII-induced intracellular Ca2+ elevation (Fig.2 B), indicating that the metalloprotease inhibitor had no effect on AngII binding to the AT1 receptor or subsequent Gq activation. In addition, BB2116 had no effect on AngII-induced nonreceptor tyrosine kinase activation as assessed by tyrosine phosphorylation of PYK2 or JAK2 (Fig. 2 C). As shown in Fig.3 A, AngII-induced EGFR transactivation was markedly inhibited by BB2116 in rat VSMC. To determine whether HB-EGF shedding is involved in the transactivation, the effect of a HB-EGF neutralizing antibody (human specific) was examined. Pretreatment with the antibody almost completely inhibited AngII-induced EGFR transactivation in cultured human VSMC (Fig.3 B). We next examined the AngII-induced release of endogenous HB-EGF from VSMC using cell surface biotinylation assay. However, it proved difficult to detect the release of soluble HB-EGF from VSMC (data not shown). The inability to biochemically detect soluble HB-EGF may be due to the low endogenous expression levels of the ligand, and this may be further exacerbated in the presence of functional EGFRs, which bind and internalize the ligand (36Dempsey P. Coffey R. J. Biol. Chem. 1994; 269: 16878-16889Abstract Full Text PDF PubMed Google Scholar). Alternatively, it could be due to cleaved HB-EGF remaining associated with the heparan sulfate proteoglycan matrix, preventing the immediate release of the mature growth factor into the conditioned medium. Taken together, these data suggest that processing of HB-EGF as a cell-associated ligand by a BB2116-sensitive metalloprotease may be an essential component for the EGFR transactivation and subsequent ERK activation induced by AngII. Cultured rat VSMC is an interesting model where a GPCR agonist, AngII, can activate three major MAPKs (ERK, JNK, and p38MAPK) (29Ushio F.M. Alexander R.W. Akers M. Griendling K.K. J. Biol. Chem. 1998; 273: 15022-15029Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 30Schmitz U. Ishida T. Ishida M. Surapisitchat J. Hasham M.I. Pelech S. Berk B.C. Circ. Res. 1998; 82: 1272-1278Crossref PubMed Scopus (84) Google Scholar, 34Eguchi 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). We measured the activation of these MAPKs by immunoblotting with their phospho-specific antibodies. This method allows us to assess activation of these different MAPKs on the same membrane. AngII stimulated p38MAPK phosphorylation starting at 5 min, and it peaked at 10 min, which resembled its time course of ERK activation in VSMC. AngII also activated JNK. It peaked at 30 min as assessed by p54 JNK phosphorylation (Fig.4 A). For longer exposure of the membrane, we observed a similar phosphorylation pattern of p46 JNK (data not shown). These data are comparable with previous publications in which MAPKs activation by AngII in VSMC was measured by kinase activities (37Kusuhara M. Takahashi E. Peterson T.E. Abe J. Ishida M. Han J. Ulevitch R. Berk B.C. Circ. Res. 1998; 83: 824-831Crossref PubMed Scopus (131) Google Scholar). VSMC normally express Gq-coupled AT1 receptors but not AT2 receptors. As expected, AngII-induced activation of these MAPKs is completely inhibited by the selective AT1 receptor antagonist RNH6270 (32Mizuno M. Sada T. Ikeda M. Fukuda N. Miyamoto M. Yanagisawa H. Koike H. Eur. J. Pharmacol. 1995; 285: 181-188Crossref PubMed Scopus (158) Google Scholar) (Fig. 4, Band C), indicating that AngII activates these MAPKs solely through the AT1 receptor in VSMC. To define the signaling relay leading to p38MAPK and JNK activation by AngII, we compared the respective time courses of their activation by AngII with those by PMA, A23187, PDGF, or EGF. PMA preferentially activated ERK, whereas A23187 markedly activated all three MAPKs (Fig. 5 A). PDGF and EGF markedly activated p38MAPK and ERK, whereas JNK was markedly activated by PDGF but not by EGF (Fig. 5 B). These differences may be due to different effects of PDGF and EGF on intracellular Ca2+ concentration, because PDGF, but not EGF, elevated Ca2+ concentration in VSMC (Fig.5 C). These data suggest that the Ca2+-dependent EGFR transactivation by AngII could also signal to p38MAPK as well as ERK. In contrast, JNK activation by AngII may require a distinct Ca2+ effector other than the EGFR. To define whether the metalloprotease-dependent EGFR transactivation participates in AngII-induced p38MAPK activation, effects of BB2116, BB94, and the selective EGFR kinase inhibitor AG1478 on AngII-induced activation of p38MAPK and JNK were studied. As shown in Fig.6 (A and B), BB2116 markedly inhibited AngII-induced p38MAPK phosphorylation, whereas it had no effect on the JNK phosphorylation. AngII-induced p38MAPK activation was similarly inhibited by pretreatment with BB94 (Fig.6 C). The HB-EGF neutralizing antibody also markedly inhibited AngII-induced p38MAPK activation in cultured human VSMC (Fig.6 D). Moreover, two different EGFR kinase inhibitors, AG1478 as well as PD158780, markedly inhibited both AngII- and EGF-induced p38MAPK phosphorylation, whereas they had no significant effect on the activation by the well-known p38MAPK agonist, anisomycin (Fig.7 A). Consistent with our previous publications (14Eguchi 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 (507) Google Scholar), AngII-induced ERK activation was also markedly inhibited by AG1478. By contrast, AngII-induced JNK activation was not affected by the EGFR inhibitor (Fig. 7 B). These data strongly indicate that, in addition to ERK activation, the metalloprotease-dependent EGFR transactivation involving HB-EGF shedding mediates the p38MAPK activation by AngII in VSMC.Figure 7Involvement of EGFR in AngII-induced p38MAPK activation in VSMC. A, VSMC were pretreated with or without AG1478 (250 nm) or PD158780 (100 nm) for 30 min, and stimulated with AngII (100 nm), EGF (100 ng/ml), or anisomycin (20 μg/ml) for 10 min. B, VSMC were pretreated with AG1478 (250 nm) or its solvent dimethyl sulfoxide (DMSO, 0.1%) for 30 min and stimulated with AngII (100 nm) for indicated durations. Cell lysates were analyzed by immunoblotting with antibodies as indicated by repeated reprobing.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The major finding of the present study is that AngII-stimulated ERK and p38MAPK activation requires metalloprotease-dependent EGFR transactivation in cultured VSMC, whereas JNK activation does not. In addition, studies using a neutralizing HB-EGF antibody suggest that processing of HB-EGF by the AngII-induced metalloprotease activation is involved in the EGFR transactivation. These data will provide new insight into the signaling mechanism by which AngII orchestrates several transcriptional events that contribute to vascular remodeling as illustrated in Fig.8. The transactivation of the EGFR by GPCRs is an important mechanism that may explain cell growth promotion by GPCRs (7Eguchi S. Inagami T. Regul. Pept. 2000; 91: 13-20Crossref PubMed Scopus (129) Google Scholar, 18Zwick E. Hackel P.O. Prenzel N. Ullrich A. Trends Pharmacol. Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 19Luttrell L.M. Daaka Y. Lefkowitz R.J. Cur. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (612) Google Scholar, 20Carpenter G. J. Cell Biol. 1999; 146: 697-702Crossref PubMed Scopus (247) Google Scholar). Recently, the metalloprotease-dependent generation of HB-EGF has been reported to mediate the EGFR transactivation (22Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar) and ERK activation (38Kalmes A. Vesti B.R. Daum G. Abraham J.A. Clowes A.W. Circ. Res. 2000; 87: 92-98Crossref PubMed Scopus (167) Google Scholar) by certain GPCRs. In COS-7 cells, insulin-like growth factor 1 also induces HB-EGF-dependent EGFR transactivation through a metalloprotease activation (39Roudabush F.L. Pierce K.L. Maudsley S. Khan K.D. Luttrell L.M. J. Biol. Chem. 2000; 275: 22583-22589Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). In the present study, we have shown that a synthetic metalloprotease inhibitor BB2116 as well as the HB-EGF neutralizing antibody attenuated EGFR transactivation by AngII. The metalloprotease inhibitor BB2116 as well as BB94 also inhibited ERK activation by AngII. The specificity of BB2116 as a synthetic hydroxamate metalloprotease inhibitor that can inhibit processing of membrane-anchored proteins (40Gearing A.J. Beckett P. Christodoulou M. Churchill M. Clements J. Davidson A.H. Drummond A.H. Galloway W.A. Gilbert R. Gordon J.L. et al.Nature. 1994; 370: 555-557Crossref PubMed Scopus (1111) Google Scholar, 41Hansen H.P. Kisseleva T. Kobarg J. Horn-Lohrens O. Havsteen B. Lemke H. Int. J. Cancer. 1995; 63: 750-756Crossref PubMed Scopus (61) Google Scholar, 42Parvathy S. Hussain I. Karran E.H. Turner A.J. Hooper N.M. Biochemistry. 1998; 37: 1680-1685Crossref PubMed Scopus (115) Google Scholar), including EGFR ligand precursors (43Dong J. Opresko L.K. Dempsey P.J. Lauffenburger D.A. Coffey R.J. Wiley H.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6235-6240Crossref PubMed Scopus (216) Google Scholar, 44Dong J. Wiley H.S. J. Biol. Chem. 2000; 275: 557-564Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), has been previously published by several researchers, including ourselves. In addition, we have shown that BB2116 had no nonspecific effect on the growth factor receptor signaling (Fig.1 B), the AngII receptor-Gq coupling (Fig.2 B), or AngII-induced nonreceptor tyrosine kinase activation such as PYK2 or JAK2 (Fig. 2 C) further supporting the specificity of BB2116. Taken together, we submit that the metalloprotease-dependent processing of pro-HB-EGF is a point of convergence by which several growth-promoting factors activate ERK by the transactivated EGFR from their respective receptors that cannot directly couple to the ERK cascade. We and others have previously failed to detect the release of endogenous EGFR ligand in response to GPCR agonists (14Eguchi 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 (507) Google Scholar, 22Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar, 23Tsai W. Morielli A.D. Peralta E.G. EMBO J. 1997; 15: 4597-4605Crossref Scopus (188) Google Scholar). Consistent with these findings, we could not detect the release of soluble HB-EGF by AngII in the present study. In general, detection of cell surface processing of endogenous EGFR ligands has proven difficult due to their low levels of expression and the lack of sensitive assays to measure the release of soluble ligands. In the case of GPCR-induced soluble HB-EGF generation, the lack of detection may also be due to cleaved HB-EGF remaining associated with the heparan sulfate proteoglycan matrix, which prevents the immediate release of the growth factor into the conditioned medium. Additionally, AngII-induced EGFR transactivation requires the presence of functional EGFRs, which may immediately bind and internalize the soluble ligand upon its release and therefore greatly decrease the concentration of soluble ligand (36Dempsey P. Coffey R. J. Biol. Chem. 1994; 269: 16878-16889Abstract Full Text PDF PubMed Google Scholar). Despite the inability to detect HB-EGF, our data together with recent findings of GPCR-induced EGFR transactivation reported by other groups (22Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar, 38Kalmes A. Vesti B.R. Daum G. Abraham J.A. Clowes A.W. Circ. Res. 2000; 87: 92-98Crossref PubMed Scopus (167) Google Scholar) suggest that pro-HB-EGF processing occurs in response to GPCR activation at the cell surface. The activation mechanism and identity of the metalloprotease mediating the cleavage of pro-HB-EGF leading to the transactivation of EGFR by AngII remains uncertain. The ADAM (adisintegrin and metalloprotease) family of metalloproteases is believed to mediate proteolysis of the EGFR ligands precursors (45Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 46Blobel C.P. Curr. Opin. Cell Biol. 2000; 12: 606-612Crossref PubMed Scopus (225) Google Scholar). Among the family, ADAM17/TACE and ADAM9/MDC9 can be inhibited by hydroxamic acid-based metalloprotease inhibitors (47Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2728) Google Scholar, 48Moss M.L. Jin S.L. Milla M.E. Burkhart W. Carter H.L. Chen W.J. Clay W.C. Didsbury J.R. Hassler D. Hoffman C.R. Kost T.A. Lambert M.H. Leesnitzer M.A. McCauley P. McGeehan G. Mitchell J. Moyer M. Pahel G. Rocque W. Overton L.K. Schoenen F. Seaton T. Su J.L. Becherer J.D. et al.Nature. 1997; 385: 733-736Crossref PubMed Scopus (1490) Google Scholar, 49Roghani 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). ADAM17/TACE has been shown to cleave pro-transforming growth factor α (50Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1371) Google Scholar). Interestingly, ADAM17/TACE knockout mice have a phenotype very similar to that observed in EGFR knockout mice, indicating its possible role in the processing of other EGFR ligands (50Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1371) Google Scholar). Recently, ADAM9/MDC9 was shown to mediate PKC-dependent cleavage of pro-HB-EGF (51Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (475) Google Scholar). However, the PKC-dependent metalloprotease does not contribute to the EGFR transactivation by GPCRs (22Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1501) Google Scholar), and our present data showed that BB2116 preferentially inhibited Ca2+-induced but not PMA-stimulated ERK activation. Additionally, Ca2+ influx was shown to stimulate HB-EGF release independent from PKC that was specifically inhibited by the metalloprotease inhibitors (52Dethlefsen S.M. Raab G. Moses M.A. Adam R.M. Klagsbrun M. Freeman M.R. J. Cell. Biochem. 1998; 69: 143-153Crossref PubMed Scopus (108) Google Scholar). These observations are consistent with our previous observation and by others that AngII-induced EGFR transactivation is independent of PKC (14Eguchi 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 (507) Google Scholar, 53Li X. Lee J.W. Graves L.M. Earp H.S. EMBO J. 1998; 17: 2574-2583Crossref PubMed Scopus (144) Google Scholar) and requires intracellular Ca2+ elevation (14Eguchi 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 (507) Google Scholar). The identification and characterization of the putative Ca2+-sensitive metalloprotease that is indispensable for the EGFR transactivation by AngII must await further investigation. Recently, much progress has been accomplished in defining the signal transduction pathway(s) by which GPCRs activate ERK. However, little is known regarding the mechanism of activation of other MAPKs by GPCRs (19Luttrell L.M. Daaka Y. Lefkowitz R.J. Cur. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (612) Google Scholar, 54Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 55English J. Pearson G. Wilsbacher J. Swantek J. Karandikar M. Xu S. Cobb M.H. Exp. Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (377) Google Scholar). The present results strongly indicate that the metalloprotease-dependent EGFR activation through HB-EGF generation also contributes to p38MAPK activation in the Gq-coupled AT1 receptor cascade in VSMC. A recent publication implicated an Src family tyrosine kinase in p38MAPK activation by Gq (56Nagao M. Yamauchi J. Kaziro Y. Itoh H. J. Biol. Chem. 1998; 273: 22892-22898Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Additionally, p38MAPK activation by AngII in VSMC was shown to involve generation of reactive oxygen species (29Ushio F.M. Alexander R.W. Akers M. Griendling K.K. J. Biol. Chem. 1998; 273: 15022-15029Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar). Because we have previously shown the inducible association of EGFR and c-Src by AngII (14Eguchi 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 (507) Google Scholar) and that c-Src is activated by reactive oxygen species (57Abe J. Berk B.C. Trends. Cardiovasc. Med. 1998; 8: 59-64Crossref PubMed Scopus (247) Google Scholar), c-Src could be a possible link that connects the transactivated EGFR to p38MAPK in response to AngII in VSMC. Although we showed that PDGF activates JNK, and AngII has been shown to transactivate PDGF receptor (13Linseman D.A. Benjamin C.W. Jones D.A. J. Biol. Chem. 1995; 270: 12563-12568Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), the involvement of PDGF receptor in AngII-induced JNK activation is unlikely, because AngII failed to induce tyrosine phosphorylation of PDGF receptor in our VSMC (14Eguchi 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 (507) Google Scholar). How does AngII activate JNK in VSMC? A recent study indicated that a Ca2+-sensitive tyrosine kinase mediates the JNK activation by AngII in cultured VSMC (30Schmitz U. Ishida T. Ishida M. Surapisitchat J. Hasham M.I. Pelech S. Berk B.C. Circ. Res. 1998; 82: 1272-1278Crossref PubMed Scopus (84) Google Scholar), and the present study showed that intracellular Ca2+ elevation by AngII is sufficient to activate JNK. Thus, a Ca2+-sensitive tyrosine kinase other than the EGFR (8Brinson 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, 10Eguchi S. Iwasaki H. Inagami T. Numaguchi K. Yamakawa T. Motley E.D. Owada K.M. Marumo F. Hirata Y. Hypertension. 1999; 33: 201-206Crossref PubMed Google Scholar) is a candidate involved in JNK activation by AngII. HB-EGF has long been implicated in vascular diseases (24Raab G. Klagsbrun M. Biochim. Biophys. Acta. 1997; 1333: F179-F199PubMed Google Scholar). Interestingly, the metalloprotease inhibitor BB94 inhibited VSMC migration and DNA synthesis after arterial injury (58Zempo N. Koyama N. Kenagy R.D. Lea H.J. Clowes A.W. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 28-33Crossref PubMed Scopus (282) Google Scholar). We and others recently reported that not only AngII (14Eguchi 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 (507) Google Scholar) but also endothelin (59Iwasaki H. Eguchi S. Ueno H. Marumo F. Hirata Y. Endocrinology. 1999; 140: 4659-4668Crossref PubMed Google Scholar), thrombin (38Kalmes A. Vesti B.R. Daum G. Abraham J.A. Clowes A.W. Circ. Res. 2000; 87: 92-98Crossref PubMed Scopus (167) Google Scholar), oxidized low density lipoproteins (60Suc I. Meilhac O. Lajoie-Mazenc I. Vandaele J. Jurgens G. Salvayre R. Negre-Salvayre A. FASEB. J. 1998; 12: 665-671Crossref PubMed Scopus (133) Google Scholar), and mechanical stretch (61Iwasaki H. Eguchi S. Ueno H. Marumo F. Hirata Y. Am. J. Physiol. 2000; 278: H521-H529Crossref PubMed Google Scholar) induce the transactivation of EGFR in cultured VSMC, suggesting the pathogenic role of the transactivation in vascular remodeling. Our data presented here connect the extracellular signaling through metalloprotease-dependent processing of EGFR ligands such as HB-EGF and the transactivation of EGFR by which various vascular pathogens including AngII exert their function. We thank Kunie Eguchi and Trinita Fizgerald for their excellent technical assistance.

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