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Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

2008; Elsevier BV; Volume: 283; Issue: 35 Linguagem: Inglês

10.1074/jbc.m709897200

1083-351X

Héctor A. Lucero, Katya Ravid, Jessica L. Grimsby, Celeste B. Rich, Sandra J. DiCamillo, Joni M. Mäki, Johanna Myllyharju, Herbert M. Kagan,

Biochemical Acid Research Studies

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by β-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-β (PDGFR-β), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-β. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-β was diminished by ∼2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-β-dependent signal transduction pathway, including PDGFR-β, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-β. Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by β-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-β (PDGFR-β), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-β. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-β was diminished by ∼2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-β-dependent signal transduction pathway, including PDGFR-β, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-β. Lysyl oxidase (LOX) 3The abbreviations used are:LOXlysyl oxidaseLOXLLOX-likeA7r5embryonic rat aorta SMC lineAKT1v-akt murine thymoma viral oncogene homolog 1BAPNβ-aminoproprionitrileDNPdinitrophenolDNPHdinitrophenylhydrazineERK1/ERK2extracellular signal-regulated kinaseMEFmouse embryonic fibroblastsSMCneonatal rat aorta smooth muscle cellsPDGFR-βplatelet-derived growth factor βSHP2SRC homology 2 domain-containing protein-tyrosine phosphatasePBSphosphate-buffered salineBSAbovine serum albuminTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineMAPKmitogen-activated protein kinase. catalyzes the oxidation of specific lysine residues within extracellular elastin and collagen thus generating residues of α-aminoadipic-δ-semialdehyde within these proteins (1Lucero H.A. Kagan H.M. Cell Mol. Life Sci. 2006; 63: 2304-2316Crossref PubMed Scopus (444) Google Scholar). These peptidyl aldehydes can then undergo condensation with vicinal α-aminoadipic-δ-semialdehyde or unmodified lysine residues to form inter- and intrapeptide covalent cross-linkages that stabilize these fibrous proteins. In addition to the non-ionic aldehyde product, the LOX-catalyzed reaction acting on protonated lysine produces stoichiometric amounts of hydrogen peroxide and ammonium, as shown in Reaction 1.RCH2NH3++H2O+O2→RCHO+H2O2+NH4+REACTION 1(Eq. 1) lysyl oxidase LOX-like embryonic rat aorta SMC line v-akt murine thymoma viral oncogene homolog 1 β-aminoproprionitrile dinitrophenol dinitrophenylhydrazine extracellular signal-regulated kinase mouse embryonic fibroblasts neonatal rat aorta smooth muscle cells platelet-derived growth factor β SRC homology 2 domain-containing protein-tyrosine phosphatase phosphate-buffered saline bovine serum albumin N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine mitogen-activated protein kinase. LOX is synthesized as an N-glycosylated preproprotein, which is secreted as the 50-kDa proprotein, proLOX. proLOX is activated by proteolysis to release the C-terminal moiety of the proprotein as the 32-kDa non-glycosylated functional catalyst (LOX) and the glycosylated propeptide in the extracellular space (2Panchenko M.V. Stetler-Stevenson W.G. Trubetskoy O.V. Gacheru S.N. Kagan H.M. J. Biol. Chem. 1996; 271: 7113-7119Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Deletion of the LOX gene does not alter embryonic development of the knock-out mouse but is perinatal lethal due to major defects in cardiovascular tissue and the respiratory system (3Mäki J.M. Räsänen J. Tikkanen H. Sormunen R. Mäkikallio K. Kivirikko K.I. Soininen R. Circulation. 2002; 106: 2503-2509Crossref PubMed Scopus (406) Google Scholar, 4Mäki J.M. Sormunen R. Lippo S. Kaarteenaho-Wiik R. Soininen R. Myllyharju J. Am. J. Pathol. 2005; 167: 927-936Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). In recent years, four lysyl oxidase-like proteins (LOXL), LOXL-1, -2, -3, and -4, have been identified in mammalian cells (5Csiszar K. Prog. Nucleic Acids Res. Mol. Biol. 2001; 70: 1-32Crossref PubMed Google Scholar). LOXL1 activity has recently been implicated in elastin homeostasis (6Liu X. Zhao Y. Gao J. Pawlyk B. Starcher B. Spencer J.A. Yanagisawa H. Zuo J. Li T. Nat. Genet. 2004; 36: 178-182Crossref PubMed Scopus (521) Google Scholar). The spectrum of substrates and biological functions of LOXL-2, -3, and -4 remain to be established. Basic research on LOX began with the demonstration of the oxidation of peptidyl lysine in an elastin substrate by a crude aortic extract (7Pinnell S.R. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1968; 61: 708-716Crossref PubMed Scopus (424) Google Scholar). Subsequent investigations operated on the assumption that the function of this enzyme was restricted to its oxidation of its extracellular elastin and collagen substrates. Thus, it was of considerable interest that purified LOX was found to readily oxidize lysine within a variety of basic globular proteins (pI ≥ 8.0) in vitro, suggesting that its substrate specificity in vivo might be broader than initially thought (8Kagan H.M. Williams M.A. Williamson P.R. Anderson J.M. J. Biol. Chem. 1984; 259: 11203-11207Abstract Full Text PDF PubMed Google Scholar). Nevertheless, the limited view of LOX function pertained until the striking observation was made that LOX acted as a suppressor of ras-induced carcinogenesis (9Kenyon K. Contente S. Trackman P.C. Tang J. Kagan H.M. Friedman R.M. Science. 1991; 253: 802Crossref PubMed Scopus (192) Google Scholar, 10Jeay S. Pianetti S. Kagan H.M. Sonenshein G.E. Mol. Cell Biol. 2003; 7: 2251-2263Crossref Scopus (90) Google Scholar). Subsequently, LOX has been implicated in a number of novel biological functions, including the regulation of the promoter activity of collagen type III (11Giampuzzi M. Botti G. Di Luca M. Arata L. Ghiggeri G. Gusmano R. Ravazzolo R. Di Donato A. J. Biol. Chem. 2000; 275: 36341-36349Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), the control of cell adhesion and growth (12Giampuzzi M. Oleggini R. Di Donato A. Biochim. Biophys. Acta. 2003; 1647: 239-244Crossref PubMed Scopus (20) Google Scholar), the metastatic phenotype of certain tumors in adult animals (13Kirschmann D.A. Seftor E.A. Fong S.F.T Nieva D.R.C Sullivan C.M. Edwards E.M. Sommer P. Csiszar K. Hendrix M.J.C Cancer Res. 2002; 62: 4478-4483PubMed Google Scholar, 14Erler J.T. Bennewithm K.L. Nicolau M. Dornhöfer N. Kong C. Le Q.T. Chi J.T. Jeffrey S.S. Giaccia A.J. Nature. 2006; 440: 1222-1226Crossref PubMed Scopus (1148) Google Scholar), and gene regulation (15Payne S.L. Hendrix M.J. Kirschmann D.A. J. Cell Biochem. 2007; 101: 1338-1354Crossref PubMed Scopus (187) Google Scholar). Of additional interest is the finding that the mature extracellular catalyst can be found within the nuclei of fibrogenic vascular smooth muscle cells and fibroblasts (16Li W. Nellaiappan K. Strassmaier T. Graham L. Thomas K.M. Kagan H.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12817-12822Crossref PubMed Scopus (149) Google Scholar, 17Nellaiappan K. Risitano A. Liu G. Nicklas G. Kagan H.M. J. Cell Biochem. 2000; 79: 576-582Crossref PubMed Scopus (71) Google Scholar). We have previously reported that LOX purified from bovine aorta actively induces the migration of peripheral blood monocytes (18Lazarus H.M. Cruikshank W.W. Narasimhan N. Kagan H.M. Center D.M. Matrix Biol. 1995; 14: 727-731Crossref PubMed Scopus (76) Google Scholar) and vascular smooth muscle cells (SMCs) (19Li W. Liu G. Chou I.N. Kagan H.M. J. Cell Biochem. 2000; 78: 550-557Crossref PubMed Scopus (94) Google Scholar). Moreover, this chemotactic activity of LOX was fully inhibitable by β-aminopropionitrile (BAPN), which inactivates catalysis by LOX, or by the presence of catalase in the chemotactic chamber assays (19Li W. Liu G. Chou I.N. Kagan H.M. J. Cell Biochem. 2000; 78: 550-557Crossref PubMed Scopus (94) Google Scholar). The conclusions were drawn, therefore, that the enzymatic activity of LOX and the hydrogen peroxide product of its enzyme activity underlie the chemotactic response. Consistent with the principle that chemokines initiate cell migration by binding to cell membrane receptors, these studies also noted that the chemotactic response of SMCs to LOX did not reflect the interaction of this enzyme with soluble forms of its extracellular substrates secreted into the medium, but more likely required the direct interaction of this enzyme with the cell membrane (19Li W. Liu G. Chou I.N. Kagan H.M. J. Cell Biochem. 2000; 78: 550-557Crossref PubMed Scopus (94) Google Scholar). The chemotactic response of vascular SMCs to LOX may prove to be relevant to certain pathologies. For example, normally quiescent vascular SMCs actively migrate from the arterial media to developing atherosclerotic lesions in the sub-endothelial region of diseased arteries, which, in turn, may relate to the observation that total LOX enzyme activity is significantly increased in the arterial lesions of atherosclerotic rabbits (20Kagan H.M. Raghavan J. Hollander W. Arteriosclerosis. 1981; 1: 287-291Crossref PubMed Google Scholar). A more tangible relationship between LOX-induced migration and pathology has recently been demonstrated. Kirschmann and colleagues (13Kirschmann D.A. Seftor E.A. Fong S.F.T Nieva D.R.C Sullivan C.M. Edwards E.M. Sommer P. Csiszar K. Hendrix M.J.C Cancer Res. 2002; 62: 4478-4483PubMed Google Scholar) have reported that the malignant invasion of breast cancer cells is induced by LOX and noted that this chemotactic response is inhibited by BAPN and by the presence of catalase, supporting the catalysis- and peroxide-dependent mechanism of LOX-induced chemotaxis reported earlier. LOX has also been implicated in the migration of malignant dermal cells in squamous cell carcinoma (21Bouez C. Reynaud C. Noblesse E. Thépot A. Gleyzal C. Kanitakis J. Perrier E. Damour O. Sommer P. Clin. Cancer Res. 2006; 12: 1463-1469Crossref PubMed Scopus (48) Google Scholar) and in the directed movement of neurons (22Laczko R. Szauter K.M. Jansen M.K. Hollosi P. Muranyi M. Molnar J. Fong K.S. Hinek A. Csiszar K. Neuropathol. Appl. Neurobiol. 2007; 33: 631-643Crossref PubMed Scopus (45) Google Scholar). Despite the number of well documented cellular and biological processes dependent on LOX activity, the relevant molecular mechanisms, including the identification of cell protein substrates that might be oxidized by LOX in these instances, has yet to be accomplished. We have approached this issue in part by studying the role of LOX in chemotaxis, a basic cellular response underlying several of the biological functions in which LOX is involved. Specifically, we have focused on the effect of LOX activity on chemotaxis induced by LOX and by PDGF-BB in rat aorta SMCs. The results reveal that oxidation of specific plasma membrane proteins by LOX is required for optimal chemotactic responses of SMCs. This newly documented priming effect of LOX on chemotaxis is clearly distinguishable from the acute chemotactic effect of this enzyme previously described (19Li W. Liu G. Chou I.N. Kagan H.M. J. Cell Biochem. 2000; 78: 550-557Crossref PubMed Scopus (94) Google Scholar). Materials—Rat recombinant, protein carrier-free PDGF-BB (R&D Systems), 125I-Monoiodo Bolton-Hunter reagent (Amersham Biosciences Corporation), rabbit anti-dinitrophenol antibody, fluorescein isothiocyanate-conjugated goat anti-rabbit antibody, and rabbit anti-actin antibody (Invitrogen), oxy-blot protein oxidation detection kit (Chemicon International), rabbit polyclonal anti-PDGFR-β (Santa Cruz Biotechnology Inc.), rabbit polyclonal antibodies against prosphoproteins of the PDGFR-β signal transduction pathway described in the text (Cell Signaling Technology), 30% hydrogen peroxide, glucose oxidase, and catalase (Sigma-Aldrich), and OptiPrep (Axis-Shield) were purchased from the indicated companies. The sources of other reagents are indicated in the text. Cell Culture Preparation—Neonatal rat aorta SMCs were isolated and expanded as described (23Schreiber B.M. Martin B.M. Hollander W. Franzblau C. Atherosclerosis. 1988; 69: 69-79Abstract Full Text PDF PubMed Scopus (16) Google Scholar). Cells were seeded at a density of 7.5 × 104 cell/ml in 2 ml of medium per well of a 6-well plate. Freshly isolated cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% bovine calf serum, 1 mm sodium pyruvate (Invitrogen), 0.1 mm non-essential amino acids (Invitrogen), and 1% penicillin/streptomycin (Invitrogen). Cell counting was carried out by hemocytometry in triplicate cultures using the trypan blue exclusion method. Wild-type and lox-/- embryos from heterozygous crosses were used to derive mouse embryonic fibroblasts (MEFs). E14.5 embryos were dissociated with sterile scissors, trypsinized, and transferred to 60-mm tissue culture dishes. After 2-3 days, cells were trypsinized and expanded as described (4Mäki J.M. Sormunen R. Lippo S. Kaarteenaho-Wiik R. Soininen R. Myllyharju J. Am. J. Pathol. 2005; 167: 927-936Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Cells of the embryo rat aorta SMC line A7r5 (American Type Culture Collection) were cultured and expanded as described above for SMCs. Lysyl oxidase was purified from bovine aorta (24Kagan H.M. Cai P. Methods Enzymol. 1995; 258: 122-132Crossref PubMed Scopus (28) Google Scholar). Chemotaxis Assays—SMCs, MEFs, and A7r5 cells were grown from seeding to 80-90% confluence in the absence (control) or in the presence of 100 μm BAPN. The cell layers were rinsed three times with Hanks' balanced salt solution. In some experiments, cells were further incubated with pure LOX (5 units/ml) for 30 min in a medium designed for optimal LOX activity and cell viability containing 5.5 mm glucose, 4.2 mm NaHCO3, 0.1 mm CaCl2, 0.8 mm MgSO4 in PBS (pH 7.8). SMCs and A7r5 were starved for 24 h and MEFs for 6 h in Dulbecco's modified Eagle's medium serum-free medium containing 0.2% BSA with or without 100 μm BAPN, and washed three times in Hanks' balanced salt solution. Cell layers were then harvested by incubation at 37 °C for 15 min in a medium containing 2 mm EDTA/Hanks' balanced salt solution, 25 mm HEPES (pH 7.4), and 0.05% TrypZean, a recombinant bovine trypsin (Sigma-Aldrich), and then collected by centrifugation at 1500 rpm for 5 min. This harvesting procedure did not result in the degradation of PDGFR-β as judged by the expected apparent molecular masses of 180-185 kDa determined by Western blots using specific antibody (not shown). Sedimented cells were resuspended in an appropriate volume of quenching medium composed of serum-free Dulbecco's modified Eagle's medium, 5% BSA, 2 mm CaCl2, and 2 mm MgCl2, counted using Trypan blue to assess viability (>90%), and diluted to 1.0 × 106 cells/ml in serum-free Dulbecco's modified Eagle's medium containing 0.2% BSA. Chemotaxis assays, performed in 96-well, 8-μm MultiScreen MIC plates (Millipore), were initiated immediately after cells were harvested, counted, and diluted. Plates and reagents were brought to room temperature (25 °C). Serum-free medium (150 μl) containing or lacking the designated chemoattractants was added to the wells of the feeder tray (lower plate). The cell migration plate (upper plate) was placed on the feeder tray, and 100 μl of serum-free medium containing 5 × 104 cells was added to each well. The mounted plate was covered and incubated for 1-3 h at 37 °C in a 5% CO2 incubator. The cells and media from the migration plate were gently removed and discarded, and the migration plate was rinsed once with Hanks' balanced salt solution and placed onto a new feeder tray containing 150 μl of the cell detachment solution containing 0.25% TrypZean, 1 mm EDTA in Dubecco's PBS without Ca2+ or Mg2+, and incubated for 30 min at 37 °C. During this time, the migration plate was gently lifted from and then reassembled onto the feeder tray to allow complete dislodging of the cells from the underside of the membrane. Finally, 100 μl taken from each well of the feeder tray was transferred to the corresponding wells of a new 96-well plate. Cell content in the wells was determined in a Tecan Infinite M200 microplate reader using the CyQuant NF cell proliferation kit assay (Invitrogen). Oxy-immunofluorescence Microscopy—Cells were grown in the wells of the Nunc Lab-Tek II CC2 Chamber Slide System to ∼80% confluence under the conditions as described herein, including the starvation in serum-free medium and washing steps. Cells were fixed in PBS containing 3% formaldehyde for 20 min at room temperature, washed three times with PBS, incubated for 10 min at room temperature in PBS containing 10 mm ammonium chloride to quench the excess of formaldehyde, and then washed twice with PBS. Carbonyl residues generated by the oxidative deamination of lysyl residues in cell surface proteins of cell monolayers were covalently derivatized with 1 ml/well of 1.5 mm dinitrophenylhydrazine (DNPH) in 0.5 m trifluoroacetic acid for 15 min at room temperature with gentle shaking. Cells not derivatized with DNPH were incubated in 0.5 m trifluoroacetic acid for 15 min at room temperature in the absence of DNPH. The DNPH solution was aspirated and 0.5 m Tris base (1 ml/well) was added to neutralize the residual trifluoroacetic acid. The fact that neither anti-core histone antibody nor rhodamine-phalloidin were able to bind to their target proteins when fixed cells were derivatized but not permeabilized (not shown) indicated that the acidic treatment of the fixed cell monolayer, under the conditions described above, did not result in cell permeabilization. Wells were rinsed two times with PBS, incubated for 2 h at room temperature in blocking buffer (PBS/3% BSA). The resulting dinitrophenyl adducts were reacted with anti-dinitrophenol antibodies (anti-DNP) in PBS/3% BSA buffer for 2 h at 4 °C. Wells were rinsed three times for 5 min each with PBS and then incubated with a 1/500 dilution of fluorescein isothiocyanate-labeled secondary antibody in PBS/3% BSA buffer for 1 h at 4 °C. Fluorescent images were captured in a Nikon Eclipse E400 equipped with ocular lens CFIUW 10×/25 and objective lenses 40×/0.65 Ph2 DL and 20×/0.40 Ph1 DL, using a Spot RT KE digital camera (Diagnostic Instruments, Inc.) driven by the Spot imaging software V 4.6. Plasma Membrane Purification—Cultured cells were grown in 150 mm plates, rinsed and then harvested by gently scraping each plate in 5 ml of lysis buffer (10 mm NaCl, 20 mm Tris-HCl, pH 7.5), containing 1 μl/ml medium of protease inhibitor mixture for use with mammalian cell extracts (Sigma) and 0.5 mm phenylmethylsulfonyl fluoride (PMSF). The resulting cell suspension was left on ice for 15 min to allow swelling and then disrupted by 20 strokes in a 12 ml Dounce homogenizer. The homogenate was subjected to zonal rate centrifugation twice for 5 min at 700 × g to sediment the nuclear fraction and cell debris. The post-nuclear supernatant (S700 × g) was either concentrated by ultracentrifugation at 150,000 × g for 1 h or loaded directly onto a 6-40% OptiPrep (Iodixanol) gradient. When S700 × g was concentrated by ultracentrifugation, the supernatant (S150K × g) consisted of the cytoplasmic fraction while the pellet (P150K × g) was the total membrane fraction of the cells depleted of nuclear membranes. This P150K × g was then gently resuspended in 500 μl of lysis buffer and loaded onto a 6-40% OptiPrep gradient composed of 150 mm NaCl, Tricine-NaOH (pH 7.4), and 1 mm EDTA. A linear gradient was formed by overlaying 18 fractions (211 μl) of 40.0%, 38.0%, 36.0%, 34.0%, 32.0%, 30.0%, 28.0%, 26.0%, 24.0%, 22.0%, 20.0%, 18.0%, 16.0%, 14.0%, 12.0%, 10.0%, 8.0%, and 6.0% in a 4.3-ml tube of the SW 60Ti rotor. Equilibrium sedimentation of the sub-cellular membrane fraction P150K × g was achieved by centrifugation at 200,000 × g for 130 min at 4 °C. Twenty fractions (215 μl) from the top of the tube were collected in new tubes. The refractive index of each fraction (50 μl) was determined using a digital display refractometer, and the corresponding density, ρ, was calculated from ρ = (η × A) - B, where η is the refractive index and the constant values used, A = 3.344 and B = 3.458, are for an OptiPrep gradient in NaCl/Tricine solution. To identify the actual sedimentation density of the PM fraction in the gradient, SMC surface proteins were biotinylated as follows: rinsed cell monolayers in 150-mm plates were incubated in 5 ml of biotinylation buffer (1 mg/ml biotin (Pierce), 10 mm triethanolamine (pH 8.3), and 150 mm NaCl), and incubated for 25-30 min at 4 °C to prevent endocytosis. Plates were rinsed three times with PBS, and the excess biotin was quenched by incubation in 5 ml of quenching buffer (100 mm glycine (pH 8.3) in PBS) for an additional 15 min at 4 °C. Surface biotinylated cells were then subjected to lysis, zonal rate centrifugation, and equilibrium sedimentation in an OptiPrep gradient as described above. Western blot analysis using horseradish peroxidase-conjugated streptavidin (Invitrogen) to probe gradient fractions revealed that plasma membranes sedimented at a density of 1.065 ± 0.021 g/ml corresponding to fractions 6 (8% Opti-Prep) to 10 (16% OptiPrep) withdrawn from the top of the tube. Alternatively, plasma membranes were recovered after equilibrium sedimentation in the 6-18% interface of a stepwise gradient consisting of 6%, 18%, and 30% OptiPrep layers. Ten 150-mm plates were processed to obtain 100-150 μg of purified plasma membrane, free of antigenic signals toward anti-Sec61β (ER marker), anti-syntaxin 6 (Golgi marker), and anti-histone H1 (nucleus marker) (not shown). Detection of oxidized proteins in solution was done using an oxy-blot protein oxidation detection kit (Chemicon International) following the manufacturer's instructions. Enzyme Assays and Immunoprecipitation—Lysyl oxidase and glucose oxidase activities were determined fluorometrically by monitoring the production of H2O2 through the oxidation of Amplex Red (Invitrogen) as described for lysyl oxidase activity (25Palamakumbura A.H. Trackman P.C. Anal. Biochem. 2002; 300: 245-251Crossref PubMed Scopus (186) Google Scholar) with modification. The substrate used for lysyl oxidase was a synthetic lysine:tyrosine (4:1) heteropolymer present at 5 μg/ml in the assay mixture. Glucose oxidase activity was measured in PBS containing 5 mm glucose. One unit of activity of either enzyme is the amount of enzyme required to produce 50 pmol of H2O2/min. The kinetics of fluorescence change was monitored using a TECAN Infinite M200 microplate reader. PDGFR-β was immunoprecipitated with a rabbit polyclonal antibody against rat PDGFR-β (Santa Cruz Biotechnology) using a protein A/G-agarose immunoprecipitation kit (KPL) following the manufacturer's instructions. Iodination of PDGF-BB and Binding of 125I-PDGF-BB to Cell Monolayers—PDGF-BB was iodinated using the Bolton-Hunter reagent (26Bolton A.E. Hunter W.M. Biochem. J. 1973; 133: 529-539Crossref PubMed Scopus (2398) Google Scholar). Briefly, 50 μg of freeze-dried, recombinant rat, carrier-free PDGF-BB (R&D Systems) was resuspended in 50 μl of 100 mm sodium borate, pH 8.5, and added to 125I-Monoiodo Bolton-Hunter reagent (1000 μCi) that had been dried under nitrogen and incubated for 15 min at 4 °C. 200 μl of quenching solution (0.1 m sodium borate, pH 8.5, and 0.2 m glycine) was added and the mixture was incubated for an additional 10 min at 4 °C followed by the addition of 50 μl of 100 mm sodium borate containing 5 mg/ml BSA. Two 5-μl aliquots of the mixture were used to assess the efficiency of iodination, and the remaining 295 μl was loaded onto a D-Salt polyacrylamide desalting column (6-kDa cut-off, Pierce) that had been equilibrated in 100 mm acetic acid containing 1 mg/ml BSA. Fractions of 500 μl were collected and 125I-PDGF-BB eluted in the first peak of radioactivity (fractions 4th to 6th) with a specific radioactivity of 25,000 cpm/ng. Binding of 125I-PDGF-BB was measured in monolayers of cells grown in 24-well cluster culture plates. Cells were seeded on the plates at a density of 5 × 104 cells/well, grown to near confluence, starved in serum-free medium for 48 h, washed three times with cold binding buffer (25 mm Hepes, pH 7.4, 125 mm NaCl, 5 mm MgSO4, 5 mm KCl, and 1 mm CaCl2, containing 2 mg/ml BSA) and incubated in 1 ml of binding buffer for 30 min at 4 °C. The binding buffer was aspirated, and 1 ml of fresh binding buffer containing the indicated concentrations of 125I-PDGF-BB at a specific radioactivity of 88,000 cpm/ng was added and further incubated at 4 °C for 4 h. After washing four times with 1 ml of binding buffer at 4 °C, cells were solubilized with 1 ml of 1% Triton X-100, 10% glycerol, 25 mm Hepes, pH 7.5, 1 mg/ml BSA. Radioactivity was measured in a gamma counter. Nonspecific binding was determined in the presence of unlabeled PDGF-BB at 500 ng/ml. Specific binding was obtained by subtracting the nonspecific binding from the total binding. The dissociation constant (Kd) and the maximum binding capacity (Bmax) for 125I-PDGF-BB were determined by least-squares, non-linear regression fitting of the hyperbolic function for ligand binding to one site saturation. Cell numbers were determined in triplicate parallel cultures by trypsinization and counting the detached cells in a hemocytometer. PDGF-BB Signal Transduction Pathway—Activation of SHP2, Akt, and ERK1/ERK2 (p44/42 MAPK), assessed as downstream components of PDGFR-β in response to PDGF-BB binding, was assayed in cell monolayers by monitoring their phosphorylated state using specific anti-phosphoprotein antibodies. Cells grown on 6-well plates were incubated for 24 h (SMC and A7r5) or for 6 h (MEF) in serum-free medium and then stimulated with PDGF-BB (25 ng/ml) for the indicated times at 37 °C in a CO2 incubator. The reaction was stopped by aspirating the media, and cells were immediately lysed with 200 μl/well extraction buffer containing 20 mm Tris, pH 7.5, 5 mm EGTA, 150 mm NaCl, 1% Nonidet P-40, 0.1 mm Na3VO4, 1 mm NaF, 10 mm sodium β-glycerophosphate, 0.5 mm phenylmethylsulfonyl fluoride, 1 μl/ml protease inhibitor mixture (Sigma). Cell extracts were clarified by centrifugation at 10,000 × g for 10 min at 4 °C, and the supernatants were saved. Protein concentrations were determined by the bicinchoninic acid method (Pierce) using BSA as the standard. To assess phosphorylation of SHP2, Akt, and ERK1/ERK2 (p44/42 MAPK) and PDGFR-β, total protein extracts (30 μg/lane) were separated by 4.0-20.0% gradient SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes in 25 mm Tris, 192 mm glycine, 20% methanol using a Mini Trans-Blot blotter (Bio-Rad) at 30 V overnight and under continuous cooling at 4 °C. Blots were blocked in PBS buffer containing 0.1% Tween 20 and 5% w/v nonfat dry milk for 1 h and then incubated overnight with gentle agitation at 4 °C with a mixture of the anti-phosphoprotein antibodies, anti-P-SHP2, anti-P-Akt, anti-P-ERK1/ERK2(p44/42 MAPK), and anti-P-PDGFR-β, each diluted 1/1000 in PBS buffer containing 0.1% Tween 20 and 5% w/v BSA. Membranes were washed three times for 5 min each with PBS/T buffer and incubated for 1 h with horseradish peroxidase-conjugated, goat anti-rabbit-IgG antibody (1:2000) in PBS buffer containing 0.1% Tween 20 and 5% w/v BSA. After washing four times for 10 min each with PBS/T, membranes were developed with a chemiluminescence substrate for detection of horseradish peroxidase (Pierce), and images were captured in a Chemi Doc XRS imager (Bio-Rad) using the Quantity One v 4.6 imaging software. Real-time PCR—Total RNA was extracted from the cells in 4 m guanidinium thiocyanate as described previously (27Wolfe L.B. Rich C.B. Go