Signaling Pathways Transduced through the Elastin Receptor Facilitate Proliferation of Arterial Smooth Muscle Cells
2002; Elsevier BV; Volume: 277; Issue: 47 Linguagem: Inglês
10.1074/jbc.m205630200
ISSN1083-351X
AutoresSatsuki Mochizuki, Bertrand Brassart, Aleksander Hinek,
Tópico(s)Connective tissue disorders research
ResumoIn this report we demonstrate that soluble peptides, elastin degradation products stimulate proliferation of arterial smooth muscle cells. We show that these effects are due to generation of intracellular signals transduced through the cell surface elastin receptor, which consists of peripheral 67-kDa elastin-binding protein (EBP) (spliced variant of β-galactosidase), immobilized to the transmembrane sialidase and the protective protein. We found that elastin receptor-transduced signaling triggers activation of G proteins, opening of l-type calcium channels, and a sequential activation of tyrosine kinases: FAK, c-Src, platelet-derived growth factor-receptor kinase and then Ras-Raf-MEK1/2-ERK1/2 phosphorylation cascade. This, in turn, causes an increase in expression of cyclins and cyclin-dependent kinases, and a consequent increase in cellular proliferation. The EBP-transduced signals also induce tyrosine kinase-dependent phosphorylation of β-tubulin, LC3, microtubule-associated protein 1, and α-actin and troponin-T, which could be linked to reorganization of cytoskeleton. We have also disclosed that induction of these signals can be abolished by anti-EBP antibody or by galactosugars, which cause shedding of EBP from the cell surface. Moreover, elastin-derived peptides did not induce proliferation of EBP-deficient cells derived from patients bearing a nonsense mutation of the β-galactosidase gene or sialidase-deficient cells from patients with congenital sialidosis. In this report we demonstrate that soluble peptides, elastin degradation products stimulate proliferation of arterial smooth muscle cells. We show that these effects are due to generation of intracellular signals transduced through the cell surface elastin receptor, which consists of peripheral 67-kDa elastin-binding protein (EBP) (spliced variant of β-galactosidase), immobilized to the transmembrane sialidase and the protective protein. We found that elastin receptor-transduced signaling triggers activation of G proteins, opening of l-type calcium channels, and a sequential activation of tyrosine kinases: FAK, c-Src, platelet-derived growth factor-receptor kinase and then Ras-Raf-MEK1/2-ERK1/2 phosphorylation cascade. This, in turn, causes an increase in expression of cyclins and cyclin-dependent kinases, and a consequent increase in cellular proliferation. The EBP-transduced signals also induce tyrosine kinase-dependent phosphorylation of β-tubulin, LC3, microtubule-associated protein 1, and α-actin and troponin-T, which could be linked to reorganization of cytoskeleton. We have also disclosed that induction of these signals can be abolished by anti-EBP antibody or by galactosugars, which cause shedding of EBP from the cell surface. Moreover, elastin-derived peptides did not induce proliferation of EBP-deficient cells derived from patients bearing a nonsense mutation of the β-galactosidase gene or sialidase-deficient cells from patients with congenital sialidosis. It has been well established that formation of neointima in vascular diseases is associated with impaired assembly of tropoelastin into insoluble elastin (1Hinek A. Mecham R.P. Keeley F.W. Rabinovitch M. J. Clin. Invest. 1991; 88: 2083-2094Google Scholar, 2Ross R. Nature. 1993; 362: 801-809Google Scholar, 3Raines E. Ross R. Br. Heart J. 1993; 69: 30-37Google Scholar, 4Ju H. Dixon I.M. Can. J. Cardiol. 1996; 12: 1259-1266Google Scholar, 5Macleod D.C. Strauss B.H. De Jong M. J. Am. Coll. Cardiol. 1994; 23: 59-65Google Scholar, 6Kimura T. Kaburagi S. Tamura T. Circulation. 1997; 96: 475-483Google Scholar) and with extensive degradation of the elastin-rich extracellular matrix by numerous proteinases leaking from the serum and secreted from the infiltrating platelets, leukocytes, and activated vascular cells (7Hornebeck W. Brechemier D. Soleilhac J.M. Bourdillon M.C. Robert L. Hay E. Extracellular Matrix: Structure and Function. Alan R. Liss, Inc., New York, NY1985: 269-282Google Scholar, 8Oho S. Rabinovitch M. Am. J. Pathol. 1994; 145: 202-210Google Scholar, 9Kenagy R.D. Vergel S. Mattsson E. Bendeck M. Reidy M.A. Clowes A.W. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1373-1382Google Scholar, 10Bendeck M.P. Irvin C. Reidy M. Smith L. Mulholland D. Horton M. Giachelli C.M. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1467-1472Google Scholar). It has also been suggested that local accumulation of non-assembled tropoelastin and small elastin-derived peptides may constitute an important factor in the activation of the normally quiescent medial SMC 1The abbreviations used are: SMC, smooth muscle cells; CA, coronary artery; BrdUrd, bromodeoxyuridine; cdk, cyclin-dependent kinase; EBP, elastin-binding protein; EGF, epidermal growth factor; ERK, extracellular signal-regulated protein kinase; FAK, focal adhesion kinase; FCS, fetal calf serum; FBS, fetal bovine serum; LC3, light chain 3 microtubule-associated protein 1; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; PDGF, platelet-derived growth factor; S-GAL, spliced variant of β-galactosidase; PBS, phosphate-buffered saline; JNK, c-Jun N-terminal kinase. into the proliferative and migratory phenotype, which participates in the formation of the occlusive neointima in vascular diseases (2Ross R. Nature. 1993; 362: 801-809Google Scholar, 3Raines E. Ross R. Br. Heart J. 1993; 69: 30-37Google Scholar, 11Boudreau N. Rabinovitch M. Lab. Invest. 1991; 64: 187-199Google Scholar, 12Thyberg J. Int. Rev. Cytol. 1996; 169: 183-265Google Scholar, 13Raines E.W. Koyama H. Carragher N.O. Ann. N. Y. Acad. Sci. 2000; 902: 39-51Google Scholar, 14Hinek A. Wilson S.A. Am. J. Pathol. 2000; 156: 925-938Google Scholar, 15Wachi H. Seyama Y. Yamashita S. Suganami H. Uemura Y. Okamoto K. Yamada H. Tajima S. FEBS Lett. 1995; 368: 215-219Google Scholar). Results ofin vitro studies have also established that elastin-derived peptides can stimulate proliferation of human skin fibroblasts (16Kamoun A. Landeau J.M. Godeau G. Wallach J. Duchesnay A. Pellat B. Hornebeck W. Cell Adhes. Commun. 1995; 4: 273-278Google Scholar), lymphoblasts (17Peterszegi G. Robert A.M. Robert L.C.R. Acad. Sci. III. 1996; 319: 799-803Google Scholar), and several types of cancer cells (18Hinek A. Cell Adhes. Commun. 1994; 2: 185-193Google Scholar, 19Jung S. Rutka J.T. Hinek A. J. Neuropath. Exp. Neurol. 1988; 57: 429-448Google Scholar) as well as cellular chemotaxis and chemokinesis (18Hinek A. Cell Adhes. Commun. 1994; 2: 185-193Google Scholar, 20Hance K.A. Tataria M. Ziporin S.J. Lee J.K. Thompson R.W. J. Vasc. Surg. 2002; 35: 254-261Google Scholar, 21Fulop T. Jacob M.P. Wallach J. Hauck M. Seres I. Varga Z. Robert L.J. Soc. Biol. 2001; 195: 157-164Google Scholar, 22Fujimoto N. Tajima S. Ishibashi A. J. Invest. Dermatol. 2000; 115: 633-639Google Scholar, 23Castiglione Morelli M.A. Bisaccia F. Spisani S. De Biasi M. Traniello S. Tamburro A.M. J. Pept. Res. 1997; 49: 492-499Google Scholar, 24Hinek A. Boyle J. Rabinovitch M. Exp. Cell Res. 1992; 203: 344-353Google Scholar). Elastin does not contain the RGD sequence and does not interact with cell surface integrins. Our previous studies demonstrated that numerous cell types, including vascular myocytes, express the cell surface elastin receptor complex, which consists of three subunits (25Hinek A. Wrenn D.S. Mecham R.P. Barondes S.H. Science. 1988; 239: 1539-1541Google Scholar, 26Mecham R.P. Hinek A. Entwistle R. Wrenn D.S. Griffin G. Senior R.M. Biochemistry. 1989; 28: 3716-3722Google Scholar), and that the average cell contains ∼2 × 106 elastin binding sites with the binding affinity (K d) of 8 nm (27Wrenn D.S. Hinek A. Mecham R.P. J. Biol. Chem. 1988; 238: 2280-2284Google Scholar). We found that two of those subunits (55- and 61-kDa) are anchored to the plasma membrane, whereas the third, a peripheral 67-kDa protein, actually binds elastin (25Hinek A. Wrenn D.S. Mecham R.P. Barondes S.H. Science. 1988; 239: 1539-1541Google Scholar). This major functional component of the receptor complex was named the elastin binding protein (EBP). The repeat hexapeptide in tropoelastin, VGVAPG, has been identified as a chief ligand for high affinity binding to this cell surface receptor (25Hinek A. Wrenn D.S. Mecham R.P. Barondes S.H. Science. 1988; 239: 1539-1541Google Scholar, 26Mecham R.P. Hinek A. Entwistle R. Wrenn D.S. Griffin G. Senior R.M. Biochemistry. 1989; 28: 3716-3722Google Scholar, 27Wrenn D.S. Hinek A. Mecham R.P. J. Biol. Chem. 1988; 238: 2280-2284Google Scholar). It has been later established that diverse peptides maintaining GXXPG sequence, including the LGTIPG sequence present on the domain V of B1 chain of laminin, can also bind to the EBP and induce similar cellular effects (28Mecham R.P. Hinek A. Griffin G. Senior R.M. Liotta L.R. J. Biol. Chem. 1989; 264: 16652-16657Google Scholar, 29Mecham R.P. Whitehouse L. Hay M. Hinek A. Sheetz M.P. J. Cell Biol. 1991; 113: 187-194Google Scholar, 30Grosso L.E. Scott M. Matrix. 1993; 13: 157-164Google Scholar, 31Grosso L.E. Scott M. Biochemistry. 1993; 32: 13369-13374Google Scholar, 32Brassart B. Fuchs P. Huet E. Alix A.J. Wallach J. Tamburro A.M. Delacoux F. Haye B. Emonard H. Hornebeck W. Debelle L. J. Biol. Chem. 2001; 276: 5222-5227Google Scholar). Importantly, we found that the EBP has additional galactolectin properties (25Hinek A. Wrenn D.S. Mecham R.P. Barondes S.H. Science. 1988; 239: 1539-1541Google Scholar), hence, it may also bind moieties containing galactosugars through a separate binding domain. We have learned, however, that binding of these carbohydrate-bearing moieties causes conformational changes of the 67-kDa protein, so that it loses its ability to bind elastin and separates from the complex with other receptor subunits (1Hinek A. Mecham R.P. Keeley F.W. Rabinovitch M. J. Clin. Invest. 1991; 88: 2083-2094Google Scholar, 25Hinek A. Wrenn D.S. Mecham R.P. Barondes S.H. Science. 1988; 239: 1539-1541Google Scholar). Sequencing of the EBP isolated from arterial SMC and molecular cloning led us to the discovery that this protein is identical to the 67-kDa, enzymatically inactive, alternatively spliced variant of human β-galactosidase (S-GAL) and that it binds elastin and laminin through the domain located in the unique frameshift-generated sequence (33Hinek A. Rabinovitch M. Keeley F.W. Okamura-Oho J. Callahan J. J. Clin. Invest. 1993; 91: 1198-1205Google Scholar, 34Privitera S. Prody C.A. Callahan J.W. Hinek A. J. Biol. Chem. 1998; 273: 6319-6326Google Scholar). We also characterized the 61-kDa subunit of the elastin receptor as sialidase (neuraminidase) (EC3.2.1.18) and the 55-kDa subunit as the protective protein, also called carboxypeptidase A or cathepsin A (EC 3.4.16.1) (35Hinek A. J. Biol. Chem. 1996; 377: 471-480Google Scholar). Previous studies lead only to a partial disclosure of the signaling pathways that may be linked to the EBP. We, and others, show that interaction between elastin-derived peptides and EBP residing on the surface of several cell types results in a rapid and transient increase in free intracellular Ca2+ (18Hinek A. Cell Adhes. Commun. 1994; 2: 185-193Google Scholar, 36Varga Z. Jacob M.P. Robert L. Fulop T. FEBS Lett. 1989; 258: 5-8Google Scholar) and that the EBP-mediated opening of calcium channels involves pertussis toxin-sensitive G proteins and activation of phospholipase C and protein kinase C in fibroblasts and lymphocytes (21Fulop T. Jacob M.P. Wallach J. Hauck M. Seres I. Varga Z. Robert L.J. Soc. Biol. 2001; 195: 157-164Google Scholar, 38Faury G. Ristori M.T. Verdetti J. Jacob M.P. Robert L. J. Vasc. Res. 1995; 32: 112-119Google Scholar). Studies by Kamisato and colleagues (39Kamisato S. Uemura Y. Takami N. Okamoto K. J. Biochem. 1997; 121: 862-867Google Scholar, 40Kamisato S. Irie W. Uemura Y. Takami N. Okamoto K. Peptide Sci. 1999; 12: 61-64Google Scholar) additionally revealed that the EBP-dependent chemotactic response of macrophages to elastin-derived peptides also involves stimulation of cGMP and cGMP-dependent protein kinase. Brassart and colleagues (32Brassart B. Fuchs P. Huet E. Alix A.J. Wallach J. Tamburro A.M. Delacoux F. Haye B. Emonard H. Hornebeck W. Debelle L. J. Biol. Chem. 2001; 276: 5222-5227Google Scholar) reported that pertussis toxin-sensitive G proteins and tyrosine kinase are involved in the EBP-mediated up-regulation of matrix metalloproteinase-2 in HT-1080 cancer cells. Because the abovementioned data clearly suggest that the cell surface EBP functions as a subunit of a true receptor capable of transmitting physiological signals from the extracellular matrix into the cell interior, the present studies have been aimed at a detailed disclosure of the intracellular signaling pathways generated by the association between elastin-derived peptides and the EBP. Culture media, fetal bovine serum, and other tissue culture reagents were obtained from Invitrogen (Burlington, Ontario, Canada). Lactose, VGVAPG peptides, proteinase inhibitors, genistein, pertussis toxin, radicicol, PP2, PD98059, AG1295, AG1498, agarose-linked protein A, and all reagent grade chemicals were purchased from Sigma (St. Louis, MO). Preparation of soluble elastin degradation products, κ-elastin was obtained from Elastin Product Co. (Owensville, MO). AG1478, PP2, and PD 98059 were purchased from Calbiochem Co. (San Diego, CA). Endoproteinase Lys-C was obtained from Roche Molecular Biochemicals (Laval, Quebec, Canada). Monoclonal antibodies against phosphotyrosine (PY20), polyclonal antibodies against FAK, cyclin A, cyclin D1, cdk2, and cdk4 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibody (p44/42) to phosphorylated ERK 1/2 was obtained from Cell Signaling Co. (Beverly, MA). Polyclonal antibody to phospho-Src was obtained from Upstate Biotechnology (Lake Placid, NY). Polyclonal antibody to LC3 microtubule-associated protein 1 was a gift of Dr. J. Hammerback from Winston-Salem, NC. Species and type-specific secondary antibodies conjugated to horseradish peroxidase, enhanced chemiluminescence kit, BrdUrd-based cell proliferation kit RPN 20, and [3H]thymidine were all purchased from Amersham Biosciences Canada Ltd. (Oakville, Ontario, Canada). Fluorescein-conjugated goat-anti rabbit secondary antibody was obtained from ICN Immuno-Biologicals (Lisle, IL). The platelet-derived growth factor BB (PDGF-BB) was from Collaborative Research Inc. (Bedford, MA). SMC were isolated by enzymatic digestion of porcine coronary arteries (CA) and cultured in Medium 199 supplement with 10% FBS as previously described (41Hinek A. Thyberg J. Connect. Tiss. Res. 1981; 8: 181-184Google Scholar). Second passage of CA SMC was then used in all experiments. Normal human skin fibroblasts, EBP-deficient fibroblasts derived from patients with GM1-gangliosidosis bearing a nonsense mutation of the β-galactosidase gene (42Hinek A. Zhang S. Smith A. Callahan J.W. Am. J. Hum. Genet. 2000; 67: 23-36Google Scholar), and sialidase-deficient fibroblasts from patients with sialidosis (43Lukong K.E. Landry K. Elsliger M.A. Chang Y. Lefrancois S. Morales C.R. Pshezhetsky A.V. J. Biol. Chem. 2001; 276: 17286-17290Google Scholar) were also used. The mitogenic effects of the soluble elastin degradation products (κ-elastin) or VGVAPG peptide on cultured CA SMC were determined by immunodetection of BrdUrd with theAmersham Biosciences cell proliferation kit, according to the manufacturer's instructions and by the incorporation of [3H]thymidine (14Hinek A. Wilson S.A. Am. J. Pathol. 2000; 156: 925-938Google Scholar, 19Jung S. Rutka J.T. Hinek A. J. Neuropath. Exp. Neurol. 1988; 57: 429-448Google Scholar). Cells were plated in six-well dishes (1 × 105 cells/dish) and maintained for 24 h in Medium 199 containing 10% FBS. Cells were then transferred to serum-free medium for synchronization of their cell cycle. Seventy-two hours later, cells were transferred again, either to serum-free medium or to medium with 5% FBS and maintained in the presence or absence of 50 μg/ml κ-elastin and 2 μCi/ml [3H]thymidine for the next 48 h. Additionally, estimation of the growth curves in quadruplicate SMC cultures maintained for 1–7 days in the presence or absence of κ-elastin were also performed. At the end of each incubation period, cells from individual cultures (initially plated at 50,000 cells/dish) were trypsinized and counted in a cell counter. Total DNA was assayed at each end point using the DNeasy tissue system. To determine whether the mitogenic signal of κ-elastin was transmitted through the EBP, quadruplicate cultures of CA SMC were preincubated for 3 h with 50 mm lactose, which causes release of the EBP from the cell surface (1Hinek A. Mecham R.P. Keeley F.W. Rabinovitch M. J. Clin. Invest. 1991; 88: 2083-2094Google Scholar, 18Hinek A. Cell Adhes. Commun. 1994; 2: 185-193Google Scholar, 25Hinek A. Wrenn D.S. Mecham R.P. Barondes S.H. Science. 1988; 239: 1539-1541Google Scholar, 26Mecham R.P. Hinek A. Entwistle R. Wrenn D.S. Griffin G. Senior R.M. Biochemistry. 1989; 28: 3716-3722Google Scholar, 27Wrenn D.S. Hinek A. Mecham R.P. J. Biol. Chem. 1988; 238: 2280-2284Google Scholar, 28Mecham R.P. Hinek A. Griffin G. Senior R.M. Liotta L.R. J. Biol. Chem. 1989; 264: 16652-16657Google Scholar, 29Mecham R.P. Whitehouse L. Hay M. Hinek A. Sheetz M.P. J. Cell Biol. 1991; 113: 187-194Google Scholar), or with 5 μg/ml anti-S-GAL antibody, which blocks its elastin binding domain (33Hinek A. Rabinovitch M. Keeley F.W. Okamura-Oho J. Callahan J. J. Clin. Invest. 1993; 91: 1198-1205Google Scholar). They were then incubated with 2 μCi/ml [3H]thymidine for the next 48 h, in the presence or absence of these reagents and 50 μg/ml κ-elastin as described above. To determine whether other factors commonly involved in intracellular mitogenic signaling might also be required for the κ-elastin-induced mitogenic response, parallel quadruplicate cultures of CA SMC maintained for 48 h in the presence and absence of 50 μg/ml κ-elastin were also pretreated for 1 h and further exposed to 100 ng/ml G protein inhibitor, pertussis toxin (44Bokoch G.M. Katada T. Northup J.K. Hewlett E.L. Gilman A.G. J. Biol. Chem. 1983; 258: 2072-2075Google Scholar), 10 μm tyrosine kinase inhibitor, genestin (45Akiyama T. Ishida J. Nakagawa S. Ogawara H. Watanabe S. Itoh N. Shibuya M. Fukami Y. J. Biol. Chem. 1986; 262: 5592-5595Google Scholar), 10 μm L-type calcium channel-blocker, nisoldipine (46Fleischmann B.K. Murray R.K. Kotlikoff M.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11914-11918Google Scholar), 10 μm inhibitor of C-Src PP2 (47Salazar E.P. Rozengurt E. J. Biol. Chem. 1999; 274: 28371-28378Google Scholar), 5 μminhibitor of Ras, radicicol (48Soga S. Kozawat T. Narumi H. Akinaga S. Iriel K. Matsumoto K. Sharmall S.V. Nakanot H. Mizukamit T. Harat M. J. Biol. Chem. 1998; 273: 822-828Google Scholar), 50 μm MEK1/2 inhibitor, and PD98059 (49Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Google Scholar, 50Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Google Scholar) or with 10 μm PDGF receptor kinase inhibitor, AG1295 (51Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Google Scholar), and 10 μm EGF receptor kinase inhibitor, AG1498 (51Levitzki A. Gazit A. Science. 1995; 267: 1782-1788Google Scholar). At the end of each experiment, the amount of thymidine incorporation was determined by liquid scintillation counting. All the above listed inhibitors were used in concentrations that were proven to be non-cytotoxic, when tested in a series of pilot experiments, in which SMC were treated with the increased concentrations of each inhibitor for different times ranging from 30 min to 12 h. In those experiments cell viability was assessed by the trypan blue exclusion test (marking dead cells) and by the neutral read incorporation into the living cells. The appearance of 20% more dead cells in the inhibitor-treated cultures than in untreated controls was considered as cytotoxic. The selected concentrations of inhibitors also allowed for reversibility of their anti-mitogenic effect 24 h after cessation of treatment and transfer to the media containing κ-elastin alone (tested by [3H]thymidine incorporation). To definitively demonstrate involvement of EBP and sialidase in the transduction of mitogenic signals, EBP-deficient cells derived from patients with GM1-gangliosidosis bearing a nonsense mutation of the β-galactosidase gene (42Hinek A. Zhang S. Smith A. Callahan J.W. Am. J. Hum. Genet. 2000; 67: 23-36Google Scholar) and sialidase-deficient cells from sialidosis patients (43Lukong K.E. Landry K. Elsliger M.A. Chang Y. Lefrancois S. Morales C.R. Pshezhetsky A.V. J. Biol. Chem. 2001; 276: 17286-17290Google Scholar) were also exposed to κ-elastin and [3H]thymidine as described above. To demonstrate the lack of EBP expression in GM1-gangliosidosis fibroblasts bearing a nonsense mutation of the β-galactosidase gene, subconfluent cultures of these cells and normal fibroblasts were immunostained with anti-S-GAL antibody and counterstained with propidium iodide to visualize nuclei, as previously described (42Hinek A. Zhang S. Smith A. Callahan J.W. Am. J. Hum. Genet. 2000; 67: 23-36Google Scholar). Cultures of CA SMCs (initially plated 1 × 105 cells/dish) incubated for 72 h in serum-free medium were exposed for different periods of time ranging from 15 min to 24 h to 50 μg/ml κ-elastin in the presence or absence of the various reagents inhibiting steps of intracellular signaling, as specified in the figure legends. At the end of each experiment cells were lysed by boiling in 62.5 mmTris-HCl buffer, pH 6.8, containing 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromphenol blue. Proteins were resolved by 12% SDS-PAGE, transferred to nitrocellulose membranes, and then immunoblotted with anti-p-Tyr:PY20 antibody, with anti-FAK antibody, with anti-LC3 antibody, or with antibodies recognizing phosphorylated forms of c-Src and ERK 1/2 (anti-p-Src and anti-p-ERK 1/2) (all at 0.5 μg/ml). Cyclins D1, A, and E and cyclin-dependent kinases (cdk)4 and cdk2 were detected with specific anti-human polyclonal antibodies (all at 0.2 μg/ml). Expression of the EBP was also detected with anti-SGAL antibody in lysates of normal and GM1 fibroblasts. All blots were then treated with the appropriate secondary antibodies and examined using the enhanced chemiluminescence detection system. The degree of expression or phosphorylation of immunodetected signaling molecules was measured by densitometry. Lysates of control and κ-elastin-treated cells were also immunoprecipitated with 3 μg/ml polyclonal anti-FAK antibody and subsequently treated with 50 μl of 4% Protein A-beaded agarose as previously described (33Hinek A. Rabinovitch M. Keeley F.W. Okamura-Oho J. Callahan J. J. Clin. Invest. 1993; 91: 1198-1205Google Scholar). Immunoprecipitated proteins were then resolved at 12% SDS-PAGE, transferred to nitrocellulose membranes, and probed with 2.5 μg/ml monoclonal PY20 antibody recognizing phosphotyrosine. The reverse experiment was also performed. The lysates of κ-elastin-treated cells were resolved at 12% SDS-PAGE and transferred to nitrocellulose membranes. Parallel membranes were then immunoblotted with anti-p-Tyr antibody or stained with Coomassie Blue. The blue bands matching those recognized with anti-p-Tyr antibody were dissected from the membranes, and proteins were extracted with a 1:1 mixture of 10% formic acid and 100% acetonitrile as described before (52Szewczyk B. Summers D.F. Anal. Biochem. 1988; 168: 48-55Google Scholar). Extracted individual proteins were then dissolved in 50 μl of 25 mm Tris, pH 8.5, containing 1 mm EDTA and additionally subjected to further cleavage with 1 μg of endoproteinase Lys-C. The obtained cleavage products were separated by C-18 reversed-phase high-performance liquid chromatography, and the major isolated peptide peaks were sequenced by gas phase protein sequencing (Porton Instruments, Tarzana, CA). The sequences obtained were then subjected to homology search by the Advanced BLAST at NCBI protein data base. Subconfluent cultures of SMC plated on cover slips were preincubated for 30 min in PBS containing 5 μm of Ca2+ ion-binding fluorochrome, Fura-2AM according to the manufacturer's instructions. Cultures were then washed extensively, covered with Medium 199 (containing 1% FBS, 1.8 mmCa2+), transferred to the incubation chamber, and monitored under an inverted fluorescent microscope (516-nm emission filter) connected to a charge-coupled device camera and a computerized video analysis system (Image-Pro Plus software for Macintosh, Media Cybernetics, Silver Spring, MD) allowing the real-time analysis of the captured images. Images of 20 single cells were then captured in real-time after addition of 50 μg/ml κ-elastin to the incubation chamber. Intracellular influxes of free calcium ions were also analyzed by the perforated-patch method in voltage-clamp experiments.l-type Ca2+ currents were recorded at 37 °C from single SMCs and analyzed using microelectrode amplifiers (Axopatch 200B, Axon Instruments, Union City, CA) under software control (pCLAMP 7.0, Axon Instruments, Union City, CA), before and after addition of 50 μg/ml κ-elastin to the external solution as previously described (53Suto F. Habuchi Y. Yamamoto T. Tanaka H. Hamaoka J. Eur. J. Pharmacol. 2000; 409: 213-221Google Scholar). Cultured SMC were scraped from the plastic dishes and suspended in PBS (pH 7.4) containing 5% bovine serum albumin and preincubated for 30 min with the fluorescent, Ca2+ -binding dye Fluo-4-AM diluted to a final concentration 3 μm. Cells were then washed in PBS, and separate aliquots containing 4 × 105 cells/ml were re-suspended in Medium 199 (containing 1.8 mmCa2+) and incubated for 10 min in the presence or absence of 0.1 m lactose, 100 ng/ml pertussis toxin, or the calcium channel blocker nisoldipine (1 μm). Influxes of Ca2+, marked by fluorescence, were monitored by a spectrometer at an emission wavelength of 488 nm and excitation wavelength of 516 nm. All parameters, immunodetection of incorporated BrdUrd, assessment of growth curves, and measurements of [3H]thymidine incorporation (Fig.1), demonstrated that CA SMC significantly increased their proliferation rate after exposure to κ-elastin or to the synthetic VGVAPG peptide, mimicking a major ligand of the elastin receptor. The magnitude of elastin peptide-induced increase in cellular proliferation was more visible in cultures maintained in FBS-containing medium than in cultures maintained in serum-free medium. Treatment with κ-elastin also enhanced the magnitude of mitogenic response of CA SMC maintained in serum-free medium to PDGF-BB (Fig. 1 C). Conversely, the proliferative response to κ-elastin was significantly decreased when cells were both treated with lactose, which causes release of EBP molecules from the cell surface, or with anti-S-GAL antibody, which blocks the elastin binding domain of the EBP (Fig. 1, B andD). Further experiments demonstrated that κ-elastin-stimulated [3H]thymidine incorporation was blocked by preincubation of cultured SMC with inhibitors of G protein (pertussis toxin),l-type calcium channels (nisoldipine), tyrosine kinases (genistein), c-Src (PP2), Ras (radicicol), or MEK1/2 kinase (PD98059), and all abolished the κ-elastin-induced up-regulation (Fig.1 D). This suggested that EBP-induced mitogenic signaling requires G protein, l-type calcium channels, certain tyrosine kinase(s), and ERK1/2 MAPKs. We have also established that inhibitor of PDGF receptor kinase (AG1295), but not with EGF receptor kinase inhibitor (AG1498), caused a significant decrease in the magnitude of the κ-elastin-induced up-regulation of [3H]thymidine incorporation. Involvement of the EBP in the transduction of proliferative signals was additionally confirmed by the fact that EBP-deficient fibroblasts derived from patients with the congenital GM1-gangliosidosis (bearing a nonsense mutation of the β-galactosidase gene) did not increase their [3H]thymidine incorporation in response to κ-elastin or VGVAPG peptides (Fig. 2). Real-time fluorescence microscopy and patch clamping confirmed that SMC exposed to κ-elastin demonstrated a rapid transient influx of Ca2+ into the cytoplasm (Fig.3, A and B). Fluorometry additionally indicated that such κ-elastin-induced Ca2+ influxes were inhibited by nisoldipine and were not observed in cells deprived of cell surface EBP (pretreatment with lactose) or after blocking of G proteins with pertussis toxin (Fig.3 C). Western blot analysis with anti-phosphotyrosine antibody indicated that even 5-min exposure of SMC to κ-elastin led to tyrosine phosphorylation of multiple proteins and that such κ-elastin-induced phosphorylation did not occur in cells preincubated with anti-EBP antibody, lactose, pertussis toxin, nisoldipine, or genistein (Fig.4 A). Immunoprecipitation with the anti-phosphotyrosine antibody followed by immunoblotting with several antibodies recognizing proteins involved in the intracellular signaling further demonstrated that treatment with κ-elastin up-regulated levels of tyrosine phosphorylation of 125-kDa FAK, 60-kDa c-Src, as well as 42- to 44-kDa ERK1/2. Treatment with κ-elastin did not cause tyrosine phosphorylation of other MAPKs (JNK or P38) commonly involved in intracellular signaling (Fig. 4 B). Results depicted in Fig. 5 A show that κ-elastin-induced phosphorylation of 60-kDa c-Src did not occur in cells pretreated with lactose or in cells exposed to pertussis toxin and genistein but was not diminished in cells treated with an inhibitor of Ras (radicicol), nor an inhibitor of MEK1/2 (PD98059). These data indicated that elastin peptide-induced phosphorylation of c-Src depends on the presence of EBP and occurs downstream of G protein, but upstream of the Ras-Raf-MEK1/2-ERK1/2 phosphorylation cascade.Figure 5A, We
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