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Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation

2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês

10.1074/jbc.m313244200

ISSN

1083-351X

Autores

Nadia Boutahar, Alain Guignandon, Laurence Vico, Marie‐Hélène Lafage‐Proust,

Tópico(s)

Protease and Inhibitor Mechanisms

Resumo

The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner. The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner. It is now well evidenced that mechanical stimulation can increase bone mass in vivo (1Oxlund H. Andersen N.B. Ortoft G. Orskov H. Andreassen T.T. Endocrinology. 1998; 139: 1899-1904Crossref PubMed Scopus (43) Google Scholar) through an increase in the number of osteoblasts, the bone-forming cells. In vitro experiments have revealed that physical forces act directly at the cellular level and showed that bone cells are able to respond physiologically to mechanical stress (2Banes A.J. Tsuzaki M. Yamamoto J. Fischer T. Brigman B. Brown T. Miller L. Cell Biol. 1995; 73: 349-365Google Scholar, 3Wozniak M. Fausto A. Carron C.P. Meyer D.M. Hruska K.A. J. Bone Miner. Res. 2000; 15: 1731-1745Crossref PubMed Scopus (119) Google Scholar). It was shown that cyclic mechanical loading is more effective in inducing osteoblast proliferation and gene expression than is static loading (4Damien E. Price J.S. Lanyon L.E. J. Bone Miner. Res. 2000; 15: 2169-2177Crossref PubMed Scopus (146) Google Scholar). 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Chem. 2000; 275: 3335-3342Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and provides specific osteoblastic responses (18Smalt R. Mitchell F.T. Howard R.L. Chambers T.J. Am. J. Physiol. 1997; 273: E751-E758PubMed Google Scholar). The molecular mechanisms that control mechanically induced ERK activation in osteoblasts remain obscure. Integrins are strong candidates as mechanotransducers in mechanical strain-induced ERK activation (19Schmidt C. Pommerenke H. Durr F. Nebe B. Rychly J. J. Biol. Chem. 1998; 273: 5081-5085Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 20Zhang J. Li W. Sanders M.A. Sumpio B.E. Panja A. Basson M.D. FASEB J. 2003; 17: 926-928PubMed Google Scholar). Indeed, integrin-mediated cell attachment activates many intracellular signaling pathways such as tyrosine phosphorylation cascades, calcium influx, inositol lipid turnover, and mitogen-activated protein kinase (MAPK) activation, (21Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2822) Google Scholar, 22Alahari S.K. Reddig P.J. Juliano R.L. Int. Rev. Cytol. 2002; 220: 145-184Crossref PubMed Scopus (64) Google Scholar). Thus, the first aim was to determine whether ERK1/2 activation mediated stretch-induced mitogenicity in a cyclic strain model using cell stretch. In particular, we focused on tyrosine-phosphorylated forms of proteins activated by mechanical strain. Focal adhesion kinase (FAK) is a cytoplasmic protein tyrosine kinase that has been reported to play an important role in integrin-mediated signal transduction pathways (22Alahari S.K. Reddig P.J. Juliano R.L. Int. Rev. Cytol. 2002; 220: 145-184Crossref PubMed Scopus (64) Google Scholar). Indeed, some studies showed that the adhesion of suspended cells onto components of the extracellular matrix induces the activation of proteins such as FAK at multiples sites, including Tyr-397 (23Owen J.D. Ruest P.J. Fry D.W. Hanks S.K. Mol. Cell. Biol. 1999; 19: 4806-4818Crossref PubMed Scopus (343) Google Scholar, 24Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar), the only apparent autophosphorylation site (25Cobb B.S. Schaller M.D. Leu T.H. Parsons J.T. Mol. Cell. Biol. 1994; 14: 147-155Crossref PubMed Scopus (486) Google Scholar, 26Xing Z. Chen H.C. Nowlen J.K. Taylor S.J. Shalloway D. Guan J.L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (285) Google Scholar, 27Sieg D.J. Hauck C.R. Ilic D. Klingbeil C.K. Schaefer E. Damsky C.H. Schlaepfer D.D. Nat. Cell Biol. 2000; 2: 249-256Crossref PubMed Scopus (1068) Google Scholar). This autophosphorylation is critical, because it creates a high affinity binding site for the Src homology 2 domain of Src family kinases, leading to a signaling complex between FAK and Src family kinases (24Calalb M.B. Polte T.R. Hanks S.K. Mol. Cell. Biol. 1995; 15: 954-963Crossref PubMed Google Scholar, 28Schlaepfer D.D. Hauck C.R. Sieg D.J. Prog. Biophys. Mol. Biol. 1999; 71: 435-478Crossref PubMed Scopus (1036) Google Scholar, 29Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1121) Google Scholar). This association of FAK-Src, together with the Grb2 adaptor protein, represents one mechanism of activation of ERK by FAK (30Schlaepfer D.D. Hanks S.K. Hunter T. van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1448) Google Scholar). This cascade of phosphorylation events and protein-protein interaction was reported upon integrin binding. However, it is not known whether similar events are induced by the mechanical strain applied to adherent cells. Among strain-induced tyrosine phosphorylated proteins we found expression of the related adhesion focal tyrosine kinase, also known as proline-rich tyrosine kinase 2 (PYK2) (31Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar), cell adhesion kinase β (32Sasaki H. Nagura K. Ishino M. Tobioka H. Kotani K. Sasaki T. J. Biol. Chem. 1995; 270: 21206-21219Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), or calcium-dependent protein-tyrosine kinase (33Yu H. Li X. Marchetto G.S. Dy R. Hunter D. Calvo B. Dawson T.L. Wilm M. Anderegg R.J. Graves L.M. Earp H.S. J. Biol. Chem. 1996; 271: 29993-29998Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar) (for convenience, it is referred to here as PYK2). PYK2 (116 kDa) shares a 45% overall sequence similarity with FAK (125 kDa), and four of the six FAK tyrosine phosphorylation sites (Tyr-397, -576, -577, and -925) are conserved at analogous positions in PYK2 (Tyr-402, -579, -580, and -881). Interestingly, PYK2 was found to be activated in osteoblasts by fluoroaluminate, a strong bone anabolic agent (34Jeschke M. Standke G.J. Scaronuscarona M. J. Biol. Chem. 1998; 273: 11354-11361Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), suggesting that this tyrosine kinase might play a critical role in the stimulation of osteoblast proliferation (35Kassem M. Mosekilde L. Eriksen E.F. J. Bone Miner. Res. 1993; 8: 1453-1458Crossref PubMed Scopus (78) Google Scholar). It has been shown that the tyrosine phosphorylation of PYK2 was markedly enhanced when the cytoplasmic free Ca2+ concentration is increased (31Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1254) Google Scholar). In our study, we therefore began to analyze the potential involvement of Ca2+ regulatory role in PYK2 activation by mechanical strain. Materials—Y402F-c-Myc-tagged PYK2 and Y397F-HA-tagged FAK were kindly provided by Dr. Shelton Earp (University of North Carolina Chapel Hill, NC). Horseradish peroxidase-conjugated goat anti-rabbit P0448 and horseradish peroxidase-conjugated horse anti-mouse P0447 were obtained from DAKO, and the phosphospecific antibodies anti-ERK-Tyr(P)-187, anti-FAK-Tyr(P)-397, anti-FAK-Tyr(P)-925, anti-Src-Tyr(P)-418, and anti-PYK2-Tyr(P)-402 were purchased from Biosource International (Camarillo, CA). Enhanced chemiluminescence detection reagents were obtained from Pierce. The stripping solution Re-Blot Plus was obtained from Chemicon International (Temecula, CA). ERK2 (D-2), c-Src (B-12), Tyr(P) (PY99), and FAK (H-1) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Pyrazolopyrimidine 2 (PP-2) and PD-98059 inhibitor were obtained from Calbiochem. Anti-HA and anti-c-Myc mouse monoclonal antibodies were obtained from Santa Cruz Biotechnology. Cells—We used ROS 17/2.8 osteoblast-like cells, a well defined cell line (36Majeska R.J. Rodan S.B. Rodan G.A. Endocrinology. 1980; 107: 1494-1503Crossref PubMed Scopus (418) Google Scholar) with the characteristics of mature osteoblasts (alkaline phosphatase activity, osteocalcin and type I collagen expression, and the ability to mineralize), and the mouse osteoblastic cell line MC3T3-E1, which consists of primary osteoblastic cells from old male Wistar rats. Briefly, after removing soft tissues, metaphyses reduced to ∼2-mm-wide fragments were submitted to fractional digestions every 15 min with 1 mg/ml clostridium histolyticum neutral collagenase (Sigma) in medium at 37 °C three times with stirring. Collagenase was neutralized with medium supplemented with 15% fetal calf serum (FCS). Cell suspension was then centrifuged at 1300 rpm for 5 min and resuspended in α-minimum Eagle's medium supplemented with 10% FCS (Sigma), 50 μg/ml ascorbic acid, and 2 mg/ml β-glycerophosphate. The medium was changed after the first 24 h to remove non-adherent cells. Cells were grown in humidified atmosphere of 5% CO2 at 37 °C. ROS 17/2.8 cells were cultured in Dulbecco's modified Eagle's medium, whereas MC3T3-E1 and primary rodent osteoblastic cells were cultured in α-minimum Eagle's medium supplemented with 10% FCS, 2 mm l-glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. After reaching a subconfluent state, cells were trypsinized with 1× trypsin-EDTA and plated onto flexible type I collagen-coated, silicon-bottomed, six-well culture plates (Bioflex; Flexcell Corp., McKeesport, PA). Cyclic Deformation—The Flexcell Strain Unit Fx-3000 (Flexcell Corp.) was used for the application of mechanical stretch to osteoblasts (37Banes A.J. Gilbert J. Taylor D. Monbureau O. J. Cell Sci. 1985; 75: 35-42Crossref PubMed Google Scholar). Cells were plated in six-well tissue culture dishes with type I collagen-coated, flexible silicone bottoms (Bioflex; Flexcell Corp.) at 5·104 cells per well density for proliferation studies and 1·105 cells per well for signal transduction studies. Seventy-two hours after seeding, cells were serum-starved overnight and then subjected to mechanical deformation. Mechanical deformation was induced with a Flexercell strain unit, which consists of a vacuum manifold regulated by solenoid valves that are controlled by a computer timer program. The Bioflex baseplate contain the Bioflex loading station that consists of six buttons per plate that insert into each plate, allowing a uniform magnitude of the strain across 85% of the surface of the flexible well. For these experiments a negative pressure of 80 kilopascals was applied through an air pump to the culture plate, bottoms were deformed to a known percentage of elongation, and then the membranes were released to their original conformation. The experimental regimens used in this study delivered 1% elongation at a rate of 15 cycles/min (a 2-s deformation period followed by a 2-s neutral position). Cells remained adherent, and the deformation of the membrane is directly transmitted to cells. Unstretched cells grown on Bioflex plates were used as controls. Proliferation Studies—ROS 17/2.8 cell proliferation was analyzed with PKH26 (38Yamamura Y. Rodriguez N. Schwartz A. Eylar E. Bagwell B. Yano N. Cell. Mol. Biol. (Noisy-Le-Grand). 1995; 41: S121-S132PubMed Google Scholar, 39Barani A.E. Sabido O. Freyssenet D. Exp. Cell Res. 2003; 283: 196-205Crossref PubMed Scopus (19) Google Scholar) according to the manufacturer's instructions (Sigma). PKH26 is a fluorescent reporter molecule that incorporates into the cell membrane and is equally distributed to daughter cells following division. It was shown that PKH26 labeling is stable and reproducible and has no effect on cell proliferation (40Boutonnat J. Muirhead K.A. Barbier M. Mousseau M. Ronot X. Seigneurin D. Anticancer Res. 1998; 18: 4243-4251PubMed Google Scholar). Cell suspension was washed in PBS/EDTA (5 μm) and incubated in 4·10-6m PKH26 staining solution for 4 min at room temperature. The reaction was stopped by the addition of 2 ml of FCS and 4 ml of proliferation medium. Cells were centrifuged at 400 × g for 10 min and washed with complete medium to remove cells from the staining solution. Then, cells were seeded at 5·104 cells onto type I collagen-precoated flexible membranes. An aliquot was fixed at day 0 in 2% paraformaldehyde and kept at 4 °C until analysis as a time 0 reference. For experimental studies, cells were allowed to recover for 72 h after seeding. Cells were then placed in serum-free media overnight and then submitted to static or strained conditions as described in the previous paragraph (“Cyclic Deformation”) in the absence or presence of 20 μm PD-98059 added 30 min before the mechanical stimulus (41Kumar A. Middleton A. Chambers T.C. Mehta K.D. J. Biol. Chem. 1998; 273: 15742-15748Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). When indicated, cells were fixed in 2% paraformaldehyde and kept at 4 °C until analysis. Analysis was performed on a FACStarplus cell sorter with a 585/42-nm bandpass filter using the Cell Quest program. Each analysis was carried out on a minimum of 5 × 104 cells. Deconvolution of the cell fluorescence into peaks was performed using the Cell Proliferation Model program ran on Modftit 2.0 software. Parameters calculated included the percentage of cells in each subsequent generation, the proliferation index (PI), and the goodness of fit measured by a reduced χ2. The PI, a measure of the proliferation activity of total cell population, was calculated by ModFit 2.0 using Equation 1,PI=∑K=0K=nAk/∑K=0K=nAk2k(Eq. 1) in which Ak is the summation of the Gaussian areas in each generation and k equals the generation number. PI represents the total expansion of the parent generation as described by Nk = 2k·N0 or Nk = PI·N0, where N0 is the number of cells in the parent generation. Therefore, PI = 2k with k = t/Tg, where t is the time point considered and Tg is the generation time. Data are representative of a minimum of two separate experiments performed in triplicate with statistically significant and similar results. Immunoprecipitation—Cells were lysed in lysis buffer (25 mm Tris, pH 7.5, 150 mm NaCl, 1% Igepal CA-630 (Sigma), 1% sodium deoxycholate, 0.1% SDS, 50 mm sodium fluoride, 10 mm sodium pyrophosphate, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1 unit/ml aprotinin, 10 μg/ml leupeptin, and 1 mm sodium orthovanadate). Lysates were clarified by centrifugation at 14,000 rpm for 30 min. Supernatants were transferred to fresh tubes. Protein concentration of lysates was determined by using the bicinchoninic acid assay (Pierce). Immunoprecipitations were typically performed by using 1–2 mg of cell lysate and 2 μg of purified monoclonal or polyclonal antibodies for Tyr(P), FAK, PYK2, Src, and ERK2. After incubation at 4 °C for 2 h on a rotating device, immune complexes were precipitated at 4 °C overnight on a rotating device by using protein A-Sepharose (Sigma). Immunoprecipitates were washed three times with lysis buffer, extracted in 2× SDS-PAGE sample buffer (125 mm Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, and 0.025% bromphenol blue) by boiling for 5 min, electrophoresed by SDS-PAGE, and then analyzed by Western blotting. Western Blot Analysis—After SDS-PAGE, proteins were transferred to nitrocellulose membrane (Bio-Rad). After transfer, membranes were blocked using 5% nonfat dried milk in PBS, pH 7.4, and incubated overnight at 4 °C with the anti-Tyr(P) (PY99), anti-ERK-Tyr(P)-187, anti-FAK-Tyr(P)-397, anti-FAK-Tyr(P)-925, anti-Src-Tyr(P)-418, and anti-PYK2-tyr(P)-402 antibodies. All of these antibodies were used at a concentration of 0.1 μg/ml. Membranes were washed three times with Tris-buffered saline (with 0.1% Tween 20) and then incubated with secondary antibodies (peroxidase-conjugated goat anti-rabbit (1:2000) or peroxidase-conjugated horse anti-mouse (1:6000) for 1 h at room temperature. After washing three times with TBS with 0.1% Tween 20, the immunoreactive bands were visualized using enhanced chemiluminescence detection reagents. Immunoblot were stripped by using mild antibody stripping solution and reprobed with another antibody. Band intensities were determined by densitometry using Scion Image program (Scion Corporation, Frederick, Maryland). Immunofluorescence Image Analysis—Cells were exposed to mechanical strain, and protein tyrosine phosphorylation was studied by indirect immunofluorescence staining. Cells (1·105 cells per well) were grown for 72 h on flexible type I collagen-coated, silicone-bottomed, six-well culture plates. Cells were then placed in serum-free media overnight and then submitted to static or mechanical strain conditions as described above in the paragraph entitled “Cyclic Deformation.” Immunostaining was performed on stretched and control cells at various time points after the end of the stimulation. Cells were fixed in 4% paraformaldehyde for 10 min, washed with cold PBS, and permeabilized with 0.1% Triton X-100 in PBS for 2 min. Mouse monoclonal anti-phosphotyrosine (PY99; 1:100 in 1% PBS/bovine serum albumin) and rabbit polyclonal anti-FAK-Tyr(P)-397 (1:100 in 1% PBS/bovine serum albumin) antibodies were applied for 1 h at 37 °C, and the cells were rewashed in PBS on a rocking platform. Cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse or anti-rabbit IgG (1:100 in PBS/bovine serum albumin 1%) for 1h at 37 °C Phosphorylation of tyrosine residues were observed with fluorescence microscope (Leica DMRB, Lyon, France) and acquired with a coded CCD camera (CoolSNAP.fx, Roper Scientific, Evry, France) and Metaview system software (Universal Imaging Corp., Downingtown, PA). Image Analysis—Cells were analyzed with a semi-automatic image analyzer (Samba-Alcatel, France) on ∼120 cells per condition. Quantification was performed on confocal scanning laser images of phosphotyrosine staining (details are reported in Ref. 42Usson Y Guignandon A. Laroche N. Lafage-Proust M.H. Vico L. Cytometry. 1997; 28: 298-304Crossref PubMed Scopus (19) Google Scholar). Several morphological features were calculated by the image analysis program. They included the mean area or number of phosphotyrosine-positive spots (42Usson Y Guignandon A. Laroche N. Lafage-Proust M.H. Vico L. Cytometry. 1997; 28: 298-304Crossref PubMed Scopus (19) Google Scholar). Plasmid DNA and Purification—pcDNA3.1/lacZ control vector (Invitrogen), mutated PYK2 cDNAs, pcDNA3-PYK2Y402F, and the mutated FAK cDNA pcDNA3-FAKY397F vector were amplified in ultraMAX™DH5a-FT™ competent cells (Invitrogen), purified on Qiagen (Courtaboeuf, France) columns, and then resuspended in sterile endotoxin-free saline solution (Qiagen; EndoFree plasmid mega kit). Plasmid DNA concentration was spectrophotometrically measured at 260/280 nm. Plasmids were digested by the specific restriction endonucleases EcoRI and PvuI (Invitrogen), and the quality of non-digested and digested DNA was assessed by 1% agarose gel electrophoresis. At least 90% of non-digested plasmid DNA was supercoiled. Plasmid was stocked at -20 °C before use. Transient Transfection in Rat ROS17/2.8 Cells—pcDNA3.1/lacZ control vector (pcDNA3 vector), pcDNA3-PYK2Y402F, and the pcDNA3-FAKY397F vector were transfected into ROS 17/2.8 cells with LipofectAMINE™ 2000 according to the manufacturer's recommendation (Invitrogen). After 24 h, cells were fed fresh medium and allowed to grow for 1 day. Cells were then placed in serum-free medium overnight and then submitted to static or strained conditions as described above in the paragraph entitled “Cyclic Deformation.” Cells were then lysed in lysis buffer. Lysates were analyzed by immunoprecipitation followed by immunoblotting with specific antibodies. On average, 30% of the cells were highly stained as determined by 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal) staining. We also obtained similar results when we transfected ROS 17/2.8 with HA-FAKY397F and then immunostained with the HA antibody (not shown). Statistics—All data were obtained from at least two independent experiments performed in triplicate. Results were expressed as mean ± S.E. Statistical analyses were performed with Mann-Whitney's nonparametric test for unpaired samples. A p value of <0.05 was considered significant. Strain-induced Proliferation—In this experiment, ROS 17/2.8 cell proliferation was analyzed 24 and 48 h after strain stimulation. As shown in Fig. 1A, the fraction of cells in each generation can be assessed by the PI, a measure of the proliferation activity of total cell population. ROS 17/2.8 cells submitted to cyclic strain were more numerous than unstrained cells at 24 h as well as at 48 h after a 10-min strain period (Fig 1A). Indeed, in a typical 48-h experiment as shown by both the FL2 histogram (i.e. PKH-26 profile; Fig 1B, upper panel) and the percentages of cells in each generation (Fig 1B, table), the strained cell numbers were 24.9 ± 1.4% and 52.4 ± 2.4% in the second and the third generation, respectively, whereas the unstrained cell numbers were 66.14 ± 1.9% and 9.3 ± 3.2% in the second and the third generation, respectively. MEK Inhibition Blocked Strain-induced Proliferation—We hypothesized that the mitogenic cyclic strain was mediated by MEK pathway activation. Treatment of the culture medium with the MEK inhibitor PD-98059 (20 μm) during the 24- or 48-h incubation period did not affect the cell morphology, at least not at the light microscopic level, but completely blocked strain-induced proliferation. PD-98059-treated static monolayers in the same experiment proliferated equivalently to static monolayers in control or Me2SO-treated media, but no increase in proliferation was observed in response to strain (Fig 1A). No effect on either basal or strain-stimulated proliferation was observed with the Me2SO vehicle control. Cyclic Strain Phosphorylated ERK—Because cyclic strain stimulated ROS 17/2.8 proliferation, we measured ERK phosphorylation in time course experiments using phosphospecific antibodies. Cyclic strain induced ERK2 phosphorylation in a time-dependent manner in contrast to the case with ERK1, which was weakly phosphorylated. Strain-induced ERK2 at Tyr-187 phosphorylation increased progressively from 0 min to 4 h after strain up to 429 ± 84% of control (p < 0.05; n = 3) as measured by densitometric scanning (Fig. 2). Identification of Phosphotyrosine-containing Proteins Induced by Cyclic Strain—To investigate the involvement of upstream protein tyrosine phosphorylation involved in stretch-induced ERK activation, tyrosine phosphorylation of cellular proteins was examined by immunoblotting using an anti-phosphotyrosine monoclonal antibody. In response to cyclic strain, a time-dependent tyrosine phosphorylation of several proteins was observed in ROS 17/2.8 cells (Fig. 3) as well as in the mouse osteoblastic cell line MC3T3-E1 (data not shown). These proteins included broad bands of 125, 116, 68, and 60 kDa. The increase in the tyrosine phosphorylation level was detected as early as the end of strain and was extended up to 4 h. Under basal conditions (i.e. after an overnight serum starvation), a significant level of protein tyrosine phosphorylation was observed, probably because of integrin-mediated protein tyrosine phosphorylation linked to the extracellular matrix adhesion (43Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1185) Google Scholar). Because major tyrosine-phosphorylated proteins were detected at 60–70 and 110–125 kDa, we examined the involvement of the tyrosine phosphorylations of pp125FAK (125 kDa) (44Schaller M.D. Borgman C.A. Cobb B.S. Vines R.R. Reynolds A.B. Parsons J.T. Proc. Natl. Acad.

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