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

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.

[1]  H. Earp,et al.  Paxillin Is Tyrosine-phosphorylated by and Preferentially Associates with the Calcium-dependent Tyrosine Kinase in Rat Liver Epithelial Cells* , 1997, The Journal of Biological Chemistry.

[2]  M. von Lindern,et al.  Enhancement of erythropoietin-stimulated cell proliferation by Anandamide correlates with increased activation of the mitogen-activated protein kinases ERK1 and ERK2. , 2000, The hematology journal : the official journal of the European Haematology Association.

[3]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]/Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .

[4]  D. Freyssenet,et al.  Mitotic activity of rat muscle satellite cells in response to serum stimulation: relation with cellular metabolism. , 2003, Experimental cell research.

[5]  Kenneth M. Yamada,et al.  PTEN Interactions with Focal Adhesion Kinase and Suppression of the Extracellular Matrix-dependent Phosphatidylinositol 3-Kinase/Akt Cell Survival Pathway* , 1999, The Journal of Biological Chemistry.

[6]  P. Davies,et al.  Mechanical stress mechanisms and the cell. An endothelial paradigm. , 1993, Circulation research.

[7]  M. Hughes-Fulford,et al.  A Short Pulse of Mechanical Force Induces Gene Expression and Growth in MC3T3‐E1 Osteoblasts via an ERK 1/2 Pathway , 2003, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[8]  Fenglin Liu,et al.  Distinct Roles of the Adaptor Protein Shc and Focal Adhesion Kinase in Integrin Signaling to ERK* , 2000, The Journal of Biological Chemistry.

[9]  David B. Burr,et al.  In vivo strain measurements to evaluate the strengthening potential of exercises on the tibial bone , 2000 .

[10]  S. Hanks,et al.  Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2 , 1999, Molecular and Cellular Biology.

[11]  C. Turner,et al.  Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly , 1992, The Journal of cell biology.

[12]  K. Mehta,et al.  Differential Roles of Extracellular Signal-regulated Kinase-1/2 and p38MAPK in Interleukin-1β- and Tumor Necrosis Factor-α-induced Low Density Lipoprotein Receptor Expression in HepG2 Cells* , 1998, The Journal of Biological Chemistry.

[13]  J. Guan,et al.  Phosphorylation of Tyrosine 397 in Focal Adhesion Kinase Is Required for Binding Phosphatidylinositol 3-Kinase* , 1996, The Journal of Biological Chemistry.

[14]  J. Guan,et al.  Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. , 1996, Journal of cell science.

[15]  Y. Usson,et al.  Quantitation of cell-matrix adhesion using confocal image analysis of focal contact associated proteins and interference reflection microscopy. , 1997, Cytometry.

[16]  Shu Chien,et al.  Mechanotransduction in Response to Shear Stress , 1999, The Journal of Biological Chemistry.

[17]  E. Peles,et al.  Protein tyrosine kinase PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions , 1995, Nature.

[18]  B. Sumpio,et al.  Regulation of the intestinal epithelial response to cyclic strain by extracellular matrix proteins , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[19]  Su‐Li Cheng,et al.  Erk Is Essential for Growth, Differentiation, Integrin Expression, and Cell Function in Human Osteoblastic Cells* , 2001, The Journal of Biological Chemistry.

[20]  M. Sokabe,et al.  Dynamics of integrin clustering at focal contacts of endothelial cells studied by multimode imaging microscopy. , 2001, Journal of cell science.

[21]  J. Parsons,et al.  Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src , 1994, Molecular and cellular biology.

[22]  H. Yuasa,et al.  Physiological mechanism‐based analysis of dose‐dependent gastrointestinal absorption of L‐carnitine in rats , 1998, Biopharmaceutics & drug disposition.

[23]  L. Lanyon,et al.  Mechanical strain and fluid movement both activate extracellular regulated kinase (ERK) in osteoblast-like cells but via different signaling pathways. , 2002, Bone.

[24]  B. Nebe,et al.  Mechanical Stressing of Integrin Receptors Induces Enhanced Tyrosine Phosphorylation of Cytoskeletally Anchored Proteins* , 1998, The Journal of Biological Chemistry.

[25]  J. Brugge,et al.  Integrins and signal transduction pathways: the road taken. , 1995, Science.

[26]  A. E. El Haj,et al.  Selected contribution: regulatory pathways involved in mechanical induction of c-fos gene expression in bone cells. , 2000, Journal of applied physiology.

[27]  H J Donahue,et al.  Osteopontin Gene Regulation by Oscillatory Fluid Flow via Intracellular Calcium Mobilization and Activation of Mitogen-activated Protein Kinase in MC3T3–E1 Osteoblasts* , 2001, The Journal of Biological Chemistry.

[28]  A. Banes,et al.  A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro. , 1985, Journal of cell science.

[29]  P. Sperryn,et al.  Blood. , 1989, British journal of sports medicine.

[30]  K. Lau,et al.  Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways. , 2003, Bone.

[31]  B. K. English,et al.  Specific inhibitors of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-gamma. , 1999, The Journal of infectious diseases.

[32]  J. Guan,et al.  Association of Focal Adhesion Kinase with Grb7 and Its Role in Cell Migration* , 1999, The Journal of Biological Chemistry.

[33]  T. Yoshimoto,et al.  Activation of cell adhesion kinase beta by mechanical stretch in vascular smooth muscle cells. , 2003, Endocrinology.

[34]  K. Muirhead,et al.  PKH26 probe in the study of the proliferation of chemoresistant leukemic sublines. , 1998, Anticancer research.

[35]  S. Hanks,et al.  Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases , 1995, Molecular and cellular biology.

[36]  E. Rozengurt,et al.  Src Family Kinases Are Required for Integrin-mediated but Not for G Protein-coupled Receptor Stimulation of Focal Adhesion Kinase Autophosphorylation at Tyr-397* , 2001, The Journal of Biological Chemistry.

[37]  T. Ogata Fluid flow‐induced tyrosine phosphorylation and participation of growth factor signaling pathway in osteoblast‐like cells , 2000, Journal of cellular biochemistry.

[38]  C. Carron,et al.  Mechanically Strained Cells of the Osteoblast Lineage Organize Their Extracellular Matrix Through Unique Sites of αVβ3‐Integrin Expression , 2000 .

[39]  J. Hanke,et al.  Discovery of a Novel, Potent, and Src Family-selective Tyrosine Kinase Inhibitor , 1996, The Journal of Biological Chemistry.

[40]  C. Damsky,et al.  FAK integrates growth-factor and integrin signals to promote cell migration , 2000, Nature Cell Biology.

[41]  K. Thai,et al.  NO Inhibits Stretch-induced MAPK Activity by Cytoskeletal Disruption* , 2000, The Journal of Biological Chemistry.

[42]  T. Hunter,et al.  Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase , 1994, Nature.

[43]  Terukatsu Sasaki,et al.  Cloning and Characterization of Cell Adhesion Kinase β, a Novel Protein-tyrosine Kinase of the Focal Adhesion Kinase Subfamily (*) , 1995, The Journal of Biological Chemistry.

[44]  D. Seigneurin [Cytometry]. , 2020, Annales de Pathologie.

[45]  R. Juliano,et al.  Biological aspects of signal transduction by cell adhesion receptors. , 2002, International review of cytology.

[46]  J. Parsons,et al.  pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[47]  H. Weinans,et al.  ERK activation and αvβ3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts , 2002 .

[48]  L. Lanyon,et al.  Mechanical Strain Stimulates Osteoblast Proliferation Through the Estrogen Receptor in Males as Well as Females , 2000, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[49]  S. J. Taylor,et al.  Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain. , 1994, Molecular biology of the cell.

[50]  T. Peterson,et al.  MAP kinase activation by flow in endothelial cells. Role of beta 1 integrins and tyrosine kinases. , 1996, Circulation research.

[51]  R. Anderegg,et al.  Activation of a Novel Calcium-dependent Protein-tyrosine Kinase , 1996, The Journal of Biological Chemistry.

[52]  B. Berk,et al.  Mechanotransduction in endothelial cells: temporal signaling events in response to shear stress. , 1997, Journal of vascular research.

[53]  T. Yamakawa,et al.  Mechanotransduction of rat aortic vascular smooth muscle cells requires RhoA and intact actin filaments. , 1999, Circulation research.

[54]  J. Heersche,et al.  Characteristics of in vitro osteoblastic cell loading models. , 2002, Bone.

[55]  G. Rodan,et al.  PYK2 Autophosphorylation, but Not Kinase Activity, Is Necessary for Adhesion-induced Association with c-Src, Osteoclast Spreading, and Bone Resorption* , 2003, The Journal of Biological Chemistry.

[56]  A. Freedman,et al.  The Related Adhesion Focal Tyrosine Kinase Is Tyrosine-phosphorylated after β1-Integrin Stimulation in B Cells and Binds to p130cas* , 1997, The Journal of Biological Chemistry.

[57]  K. Thai,et al.  Mesangial cell signaling cascades in response to mechanical strain and glucose. , 1999, Kidney international.

[58]  A. Grodzinsky,et al.  Mechanical Regulation of Mitogen-activated Protein Kinase Signaling in Articular Cartilage* , 2003, Journal of Biological Chemistry.

[59]  S. Hanks,et al.  Focal adhesion kinase promotes phospholipase C-γ1 activity , 1999 .

[60]  Y. Yazaki,et al.  Pulsatile stretch activates mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase (p125(FAK)) in cultured rat cardiac myocytes. , 1999, Biochemical and biophysical research communications.

[61]  Tony Hunter,et al.  Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events , 1998, Molecular and Cellular Biology.

[62]  M. Šuša,et al.  Fluoroaluminate Induces Activation and Association of Src and Pyk2 Tyrosine Kinases in Osteoblastic MC3T3-E1 Cells* , 1998, The Journal of Biological Chemistry.

[63]  Micah Dembo,et al.  Focal adhesion kinase is involved in mechanosensing during fibroblast migration , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[64]  A. Samarel,et al.  Vascular Endothelial Growth Factor Regulates Focal Adhesion Assembly in Human Brain Microvascular Endothelial Cells through Activation of the Focal Adhesion Kinase and Related Adhesion Focal Tyrosine Kinase* , 2003, Journal of Biological Chemistry.

[65]  Sábata S Constancio,et al.  Focal Adhesion Kinase Is Activated and Mediates the Early Hypertrophic Response to Stretch in Cardiac Myocytes , 2003, Circulation research.

[66]  S. Rodan,et al.  Parathyroid hormone-responsive clonal cell lines from rat osteosarcoma. , 1980, Endocrinology.

[67]  B. Geiger,et al.  Assembly and mechanosensory function of focal contacts. , 2001, Current opinion in cell biology.

[68]  D. Schlaepfer,et al.  Signaling through focal adhesion kinase. , 1999, Progress in biophysics and molecular biology.

[69]  S. Palle,et al.  Physical exercise during remobilization restores a normal bone trabecular network after tail suspension‐induced osteopenia in young rats , 1995, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[70]  M. Dieudonné,et al.  Leptin mediates a proliferative response in human MCF7 breast cancer cells. , 2002, Biochemical and biophysical research communications.

[71]  B. Nebe,et al.  The Mode of Mechanical Integrin Stressing Controls Intracellular Signaling in Osteoblasts , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[72]  J. Parsons,et al.  Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK , 1994, Molecular and cellular biology.

[73]  H. Avraham,et al.  Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes. , 1996, Blood.

[74]  W. Cance,et al.  Interactions between Two Cytoskeleton-associated Tyrosine Kinases: Calcium-dependent Tyrosine Kinase and Focal Adhesion Tyrosine Kinase* , 1999, The Journal of Biological Chemistry.

[75]  N. Matsuda,et al.  Proliferation and Differentiation of Human Osteoblastic Cells Associated with Differential Activation of MAP Kinases in Response to Epidermal Growth Factor, Hypoxia, and Mechanical Stressin Vitro , 1998 .

[76]  M. Šuša,et al.  Fluoroaluminate stimulates phosphorylation of p130 Cas and Fak and increases attachment and spreading of preosteoblastic MC3T3-E1 cells. , 2002, Bone.

[77]  K. Chihara,et al.  Parathyroid Hormone-activated Volume-sensitive Calcium Influx Pathways in Mechanically Loaded Osteocytes* , 2000, The Journal of Biological Chemistry.

[78]  L. Mosekilde,et al.  1,25‐dihydroxyvitamin D3 potentiates fluoride‐stimulated collagen type I production in cultures of human bone marrow stromal osteoblast‐like cells , 1993, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.