Mechanical Stressing of Integrin Receptors Induces Enhanced Tyrosine Phosphorylation of Cytoskeletally Anchored Proteins

Abstract

Physical forces play a fundamental role in the regulation of cell function in many tissues, but little is known about how cells are able to sense mechanical loads and realize signal transduction. Adhesion receptors like integrins are candidates for mechanotransducers. We used a magnetic drag force device to apply forces on integrin receptors in an osteoblastic cell line and studied the effect on tyrosine phosphorylation as a biochemical event in signal transduction. Mechanical stressing of both the β1 and the α2 integrin subunit induced an enhanced tyrosine phosphorylation of proteins compared with integrin clustering. Application of cyclic forces with a frequency of 1 Hz was more effective than a continuous stress. Using Triton X-100 for cell extraction, we found that tyrosine-phosphorylated proteins became physically anchored to the cytoskeleton due to mechanical integrin loading. This cytoskeletal linkage was dependent on intracellular calcium. To see if mechanical integrin stressing induced further downstream signaling, we analyzed the activation of mitogen-activated protein (MAP) kinases and found an increased phosphorylation of MAP kinases due to mechanical stress. We conclude that integrins sense physical forces that control gene expression by activation of the MAP kinase pathway. The cytoskeleton may play a key role in the physical anchorage of activated signaling molecules, which enables the switch of physical forces to biochemical signaling events. Physical forces play a fundamental role in the regulation of cell function in many tissues, but little is known about how cells are able to sense mechanical loads and realize signal transduction. Adhesion receptors like integrins are candidates for mechanotransducers. We used a magnetic drag force device to apply forces on integrin receptors in an osteoblastic cell line and studied the effect on tyrosine phosphorylation as a biochemical event in signal transduction. Mechanical stressing of both the β1 and the α2 integrin subunit induced an enhanced tyrosine phosphorylation of proteins compared with integrin clustering. Application of cyclic forces with a frequency of 1 Hz was more effective than a continuous stress. Using Triton X-100 for cell extraction, we found that tyrosine-phosphorylated proteins became physically anchored to the cytoskeleton due to mechanical integrin loading. This cytoskeletal linkage was dependent on intracellular calcium. To see if mechanical integrin stressing induced further downstream signaling, we analyzed the activation of mitogen-activated protein (MAP) kinases and found an increased phosphorylation of MAP kinases due to mechanical stress. We conclude that integrins sense physical forces that control gene expression by activation of the MAP kinase pathway. The cytoskeleton may play a key role in the physical anchorage of activated signaling molecules, which enables the switch of physical forces to biochemical signaling events. Application of physical forces to cells induces gene expression and proliferation in a variety of cell types (1Wang D.L. Wung B.S. Shyy Y.J. Lin C.F. Chao Y.J. Usami S. Chien S. Circ. Res. 1995; 77: 294-302Google Scholar, 2Diamond S. Eskin S. McIntire L. Science. 1989; 243: 1483-1485Google Scholar, 3Komuro I. Katoh Y. Kaida T. Shibazaki Y. Kurabayashi M. Hoh E. Takaku F. Yazaki Y. J. Biol. Chem. 1991; 266: 1265-1268Google Scholar, 4Hasegawa S. Sato S. Saito S. Suzuki Y. Brunette D.M. Calcif. Tissue Int. 1985; 37: 431-436Google Scholar, 5Wirtz H.R.W. Dobbs L.G. Science. 1990; 250: 1266-1269Google Scholar, 9Hughes-Fulford M. Lewis M.L. Exp. Cell Res. 1996; 224: 103-109Google Scholar). Therefore, mechanical forces are a fundamental physiological factor in regulating structure and function in many tissues. In bone, mechanical loading stimulates the increase of bone mass (6Lanyon L.E. Rubin C.T. J. Biomech. 1984; 17: 897-905Google Scholar, 7Raab-Cullen D.M. Akhter M.P. Kimmel D.B. Recker R.R. J. Bone Miner. Res. 1994; 9: 1143-1152Google Scholar, 8Rubin C.T. Gross T.S. McLeod K.J. Bain S.D. J. Bone Miner. Res. 1995; 10: 488-495Google Scholar) and plays an important role in the therapy of osteoporosis. The cellular mechanisms of mechanically induced signal transduction are largely unknown. Above all, it has remained elusive how cells are able to sense physical forces. Indications exist that integrin receptors may serve as mechanotransducers (10Ingber D. Curr. Opin. Cell Biol. 1991; 3: 841-848Google Scholar, 11Wang N. Butler J.P. Ingber D.E. Science. 1993; 260: 1124-1127Google Scholar, 12Wang N. Ingber D.E. Biophys. J. 1994; 66: 2181-2189Google Scholar, 13Nebe B. Rychly J. Knopp A. Bohn W. Exp. Cell Res. 1995; 218: 479-484Google Scholar, 14Pommerenke H. Schreiber E. Dürr F. Nebe B. Hahnel C. Möller W. Rychly J. Eur. J. Cell Biol. 1996; 70: 157-164Google Scholar). Integrins are heterodimeric transmembrane molecules by which cells adhere to the extracellular matrix (15Hynes R.O. Cell. 1992; 69: 11-25Google Scholar). The β subunit is combined with one of the different α subunits, and both extracellular domains are involved in ligand binding. Engagement and clustering of these receptors induce signal transduction, which involves integrin linkage to the cytoskeleton and the generation of second messengers and biochemical events (13Nebe B. Rychly J. Knopp A. Bohn W. Exp. Cell Res. 1995; 218: 479-484Google Scholar, 16Juliano R.L. Haskill S. J. Cell Biol. 1993; 120: 577-585Google Scholar, 17Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Google Scholar). Activation of protein tyrosine kinases appears to play a central role in integrin-mediated signaling (18Parson J.T. Curr. Opin. Cell Biol. 1996; 8: 146-152Google Scholar, 19Kornberg L. Earp H.S. Parson J.T. Schaller M.D. Juliano R.L. J. Biol. Chem. 1992; 267: 23439-23442Google Scholar, 20Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Google Scholar). Because mechanical strain may act in different frequencies and strength, which appears to have relevance in regulating cell physiology (21Sandy J.R. Meghji S. Scutt A.M. Harvey W. Harris M. Meikle M.C. Bone Miner. 1989; 5: 155-168Google Scholar, 22Lanyon L.E. Calcif. Tissue Int. 1984; 36: S56-S61Google Scholar, 23Neidlinger-Wilje C. Wilke H, J. Claes L. J. Orthop. Res. 1994; 12: 70-78Google Scholar), an important question is whether perception of physical forces by integrin receptors induces a differential intracellular signal transduction. Recently, we developed a method to mechanically stress cell surface receptors (14Pommerenke H. Schreiber E. Dürr F. Nebe B. Hahnel C. Möller W. Rychly J. Eur. J. Cell Biol. 1996; 70: 157-164Google Scholar). Using magnetic beads, drag forces in defined strength and frequency can be applied to receptors of cells in a monolayer. The method enables the application of physical forces to defined integrins, which allows the evaluation of the relevance of specific integrin subunits in mechanical signal transduction. Herein, we report that in the osteosarcoma cell line U-2 OS, mechanical stressing of integrins induces an increased tyrosine phosphorylation of proteins, including the MAP 1The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, acetoxymethyl ester.kinase, compared with integrin clustering and depending on whether a permanent or intermittent stress is applied. We also observed an increased physical anchorage of tyrosine-phosphorylated proteins at the cytoskeleton, which suggests that the cytoskeleton may serve as a structure where mechanical signals can switch into a chemical-signaling pathway. The osteosarcoma U-2 OS cell line was obtained from American Type Culture Collection (Rockville, MD). 96-well Fluoro Nunc modules from Nunc A/S (Roskilde/Denmark) were used for plating the cells. Paramagnetic microbeads (size 2.8 μm, coated with streptavidin) were purchased from Dynal (Hamburg, Germany). For coating of the microbeads, biotinylated anti-β1 (clone 2A4) and anti-α2 (clone AK7) integrin antibodies were from Southern Biotechnology Associates, Inc. (Birmingham, AL); biotinylated anti-CD71 (transferrin receptor) antibody was from Oncogene Science, Inc. (Uniondale, NY). For immunoprecipitation of MAP kinases, anti-ERK-1 (p44) antibody (clone C-16), which also reacts with ERK-2 (p42), was used from Santa Cruz Biotechnology. Protein A-agarose was also purchased from Santa Cruz Biotechnology. Recombinant anti-phosphotyrosine antibody (clone RC-20) conjugated with alkaline phosphatase was from Transduction Laboratories. CDP-star for chemiluminescence was obtained from Boehringer Mannheim. U-2 OS cells were cultured in Dulbecco's modified Eagle's medium and supplemented with 10% fetal calf serum at 37 °C and in 5% CO2 atmosphere. For the experiments, 100 μl of cells in complete medium containing 105 cells were seeded into wells of a 96-well culture module and grown to near confluence. 2 h before mechanical strain was applied, the cells were depleted of serum. The procedure to strain integrin receptors was described in detail elsewhere (14Pommerenke H. Schreiber E. Dürr F. Nebe B. Hahnel C. Möller W. Rychly J. Eur. J. Cell Biol. 1996; 70: 157-164Google Scholar). In brief, the cell monolayer was incubated with paramagnetic microbeads coated with anti-β1 or anti-α2 antibodies. This is termed here as clustering. In average, five beads bound at the surface of one cell. To apply mechanical drag forces, each of the wells was then placed between the poles of the magnetic device, which induces an inhomogeneous magnetic field (14Pommerenke H. Schreiber E. Dürr F. Nebe B. Hahnel C. Möller W. Rychly J. Eur. J. Cell Biol. 1996; 70: 157-164Google Scholar). The forces subjected to one bead were adjusted to 10 dyne/cm2. Analyses revealed that this strength was exerted to most of the beads, whereas a smaller part of the beads in the vicinity of the flat pole was subjected to lower forces. The forces were applied either with a frequency of 1 Hz if not otherwise mentioned or permanently during the indicated time. In general, the following experimental designs were compared: 1) control cells without any treatment (−); 2) clustered samples (c), i.e. the cell monolayer was incubated with anti-integrin antibody-coated beads for 50 min; 3) mechanically stressed samples, i.e. the cell monolayer was incubated (in a control experiment with anti-CD71) with anti-integrin antibody-coated beads for 20 min followed by application of forces for 30 min. After integrin stimulation as described above, adherent cells in each well were washed in phosphate-buffered saline and lysed in 10 μl of SDS sample buffer. Lysates of several wells were pooled and then boiled. The samples were subjected to a 7.5% SDS-polyacrylamide gel electrophoresis. For immunoblotting, the proteins were transferred to polyvinylidene difluoride membranes. To block nonspecific binding, membranes were incubated with 5% milk powder in buffer containing 0.01% Tween 20, 100 mm Tris/HCl, pH 9.0, 155 mm NaCl for 30 min at 37 °C. Immunoblotting was performed with alkaline phosphatase-labeled anti-phosphotyrosine antibody at a dilution of 1:20,000 and then visualized with chemiluminescence (CDP star). To analyze activation of MAP kinases, cells were lysed in precipitation buffer containing protease inhibitor, SDS, and Triton X-100 (0.1%). After centrifugation, the supernatant was incubated with 100 μl of anti-ERK antibody for 1 h at room temperature followed by adding 50 μl of protein A-agarose. After centrifugation, the pellet was washed in precipitation buffer and analyzed for phosphotyrosine as described above. The cell monolayer was incubated with cell extraction buffer containing 1% Triton X-100, 20 mm imidazole, 2 mm MgCl2, 80 mm KCl, 2 mm EGTA for 5 min at 4 °C. The Triton nonsoluble fractions were then collected in SDS sample buffer and subjected to gel electrophoresis as described above. To disrupt the actin filaments of the cytoskeleton, the cell monolayer was treated with 25 nm cytochalasin D for 20 min at 37 °C. The cells were then washed and incubated with the microbeads to perform the procedure for mechanical loading. For chelating intracellular calcium, the cells were preincubated with 5 μm of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, acetoxymethyl ester (BAPTA-AM) for 15 min. Mechanical strain was then applied in the presence of 5 μm of BAPTA. The osteosarcoma cell line expressed the β1 as well as the α2 integrin subunits on the cell surface (14Pommerenke H. Schreiber E. Dürr F. Nebe B. Hahnel C. Möller W. Rychly J. Eur. J. Cell Biol. 1996; 70: 157-164Google Scholar). Therefore, we examined the effect of mechanical stress applied to both integrin subunits on tyrosine phosphorylation of proteins as a mechanism in integrin-mediated signal transduction. First, we were interested in the time course of tyrosine phosphorylation due to clustering of the β1 integrin subunit by incubation of the cells with anti-β1-coated microbeads. We observed an increase of phosphorylation during the time of incubation, which reached the maximum after 60 min (Fig. 1). Based on this finding, mechanical stress was applied to integrins for 30 min after an incubation time of 20 min to bind the beads to the receptors. Application of forces to the β1 as well as to the α2 subunit induced an increased tyrosine phosphorylation of proteins compared with integrin clustering alone (Fig. 2). Stressing the β1 chain, the effect was more pronounced than with α2. To prove whether the mechanically induced cellular reactions are specific for integrins, we stressed the transferrin receptor (CD71) for comparison. Although a slightly increased tyrosine phosphorylation was observed compared with untreated cells, the effect was distinctly lower than after stressing an integrin receptor (Fig. 3). Next we compared the effect of permanent mechanical loading with an intermittent stress of 1 Hz on tyrosine phosphorylation. Application of a stress with a frequency of 1 Hz induced a more profound phosphorylation than permanent drag forces (Fig. 2). To exclude the possibility that the influence of different modes of magnetic field application alone and not the mechanical receptor stress provoked the differences in tyrosine phosphorylation, we compared controls in which pure cells were subjected to a permanent and a cyclic magnetic field. This experiment clearly demonstrated that the magnetic field alone did not influence tyrosine phosphorylation, independent of the mode of the magnetic field (Fig. 3).Figure 2Tyrosine phosphorylation induced by mechanical stressing of integrins. Cells in a monolayer were incubated with microbeads to bind at the β1 or α2 integrin subunit followed by application of drag forces (s). These samples are compared with clustering of the integrins by incubation with the microbeads without subsequent mechanical loading (c), and untreated control cells (−). Total cell lysates were electrophoresed and blotted for anti-phosphotyrosine. Most pronounced differences are observed in the 40-kDa region. In all cases, physical forces induced a significantly enhanced tyrosine phosphorylation compared with clustering and controls. Mechanical stressing of β1 induced a higher response than loading the α2 subunit (compare β1/1 Hz/sversus α2/1 Hz/s; or β1/P/s versus α2/P/s). A cyclic stress with a frequency of 1 Hz was more effective than a permanent stress in both integrin subunits (compare β1/1 Hz/sversus β1/P/s; and α2/1 Hz/s versusα2/P/s). The results are representative of four independent experiments.View Large of tyrosine phosphorylation due to mechanical stress to integrins, mechanical to the transferrin and application of the magnetic field alone to the cells. Cells in a monolayer were mechanically stressed at the β1 integrin with 1 Hz as described 2) or stressed in the at the transferrin receptor (CD71) 3). Cells in a monolayer without magnetic beads were subjected to a permanent magnetic field or a cyclic magnetic field with 1 Hz for 30 min. cells were also examined 1). Total cell lysates were electrophoresed and blotted for anti-phosphotyrosine. with application of stress to the β1 mechanical stress to the transferrin receptor induced a but significantly lower of tyrosine phosphorylation. modes of the magnetic field applied to untreated cells effect on tyrosine Large To the role of the cytoskeleton in mechanically induced tyrosine phosphorylation, we examined whether tyrosine-phosphorylated proteins are anchored to the After clustering and mechanically loading of the integrins, cells were with Triton X-100, and the analyzed for tyrosine-phosphorylated proteins (Fig. clustering and stress induced a linkage of tyrosine-phosphorylated mechanical was more effective than clustering. This the β1 whereas the linkage of α2 to the cytoskeleton was the effect of clustering and mechanical anchored proteins were in the higher The anchorage of proteins to the cytoskeleton was further studied by of the actin filaments by cytochalasin D (Fig. with cytochalasin D the cytoskeletal linkage of tyrosine-phosphorylated proteins the of actin of the cytoskeletal anchorage of tyrosine-phosphorylated proteins by cytochalasin D. Cells were treated by mechanical stressing of the β1 integrin subunit clustering of β1 (c), or without treatment in the presence of cytochalasin D to disrupt the actin filaments of the For the cells were treated in the in the of cytochalasin D (−). The cells were then with Triton X-100 to the cytoskeletal This was then for anti-phosphotyrosine D the cytoskeletal linkage of tyrosine-phosphorylated In the of cytochalasin mechanical stressing of integrins induced a distinctly enhanced anchorage of This the results of four independent a in the samples, the in these were to the Large Because intracellular is induced by integrin we whether intracellular plays a role in the of tyrosine-phosphorylated proteins to the mechanical loading of integrins, cells were treated with the intracellular The following of anchored proteins, which are revealed that significantly the physical anchorage of these proteins to the cytoskeleton (Fig. This that the linkage of activated proteins to the cytoskeleton during integrin-mediated mechanically induced signal transduction. we were interested in whether the increased tyrosine phosphorylation of proteins due to mechanical integrin stressing may to downstream signaling, which be for gene Therefore, we examined whether the tyrosine-phosphorylated proteins the MAP kinases are also of MAP kinases and of tyrosine phosphorylation demonstrated that mechanical loading of both the β1 and the α2 integrin subunits induced a distinctly higher of activation of the MAP kinases compared with integrin clustering (Fig. This suggests that regulation of gene expression by physical forces is by differential activation of MAP kinases and by integrins. phosphorylation of several cellular proteins appears to play an role in integrin-mediated signal transduction gene expression C. K. Haskill S. Juliano R.L. J. Biol. Chem. 1995; Scholar). The mechanisms by which extracellular of integrins tyrosine phosphorylation We that application of physical forces to integrin receptors enhanced the tyrosine phosphorylation of proteins compared with integrin clustering These results the that integrin receptors function as N. Butler J.P. Ingber D.E. Science. 1993; 260: 1124-1127Google Scholar, 12Wang N. Ingber D.E. Biophys. J. 1994; 66: 2181-2189Google Scholar, D.E. Curr. Opin. Cell Biol. 1991; 3: 841-848Google Scholar). that physical forces applied to integrin receptors the of tyrosine phosphorylation that may have in gene tyrosine phosphorylation was also by the mode of receptor forces are more effective than a permanent stress. have that application of an intermittent mechanical on is more effective to than a permanent strain L.E. Rubin C.T. J. Biomech. 1984; 17: 897-905Google Scholar, J. M. J. Scholar). Therefore, increased tyrosine phosphorylation due to cyclic integrin stressing a mechanism for the differential effect of mechanical loads on of the of a mechanical stress to integrins is the of cytoskeletal This has demonstrated in cells by integrin or mechanical of the integrins N. Butler J.P. Ingber D.E. Science. 1993; 260: 1124-1127Google Scholar, B. Bohn W. H. Rychly J. Exp. Cell Res. 1996; Scholar, C.E. M.P. J. Cell Biol. 1993; Scholar). is an that the strength of this linkage is and on forces. Using an to integrin bound beads in a of the integrin cytoskeletal linkage D. Cell. Scholar). In the line of these we that in mechanical forces induced an increased strength of the integrin cytoskeletal have revealed that the of the ligand and control of the integrin-mediated signal transduction in Scholar, H. Cell. 1996; Scholar, S. Science. 1995; 267: Scholar). For integrin clustering induced a of signaling molecules into the cytoskeletal compared with of the receptor which to a of a in signal transduction S. H. J.S. J. Cell Biol. 1995; Scholar). the physical of the matrix was integrin activation and signal transduction, which proliferation H. Cell. 1996; Scholar). This clearly suggests that how integrins with a ligand is of for the induced signal transduction. Although the β and α subunits of integrins a we have found a in mechanically induced tyrosine phosphorylation between the integrin Mechanical on the β subunit provoked a more profound tyrosine phosphorylation than stressing the α We exclude that this was due to a higher expression of β1 compared with α2 that we have found in these cells (14Pommerenke H. Schreiber E. Dürr F. Nebe B. Hahnel C. Möller W. Rychly J. Eur. J. Cell Biol. 1996; 70: 157-164Google Scholar), but it a different mechanism of the intracellular of the domains of both integrin The β can with the cytoskeletal proteins α and K. J. Cell Biol. 1990; Scholar, A. K. C. M.C. K. 1985; Scholar). In the kinase can with the β1 and further downstream signaling C. L. M. J. J. S. 1996; Scholar). Mechanical stress to the β subunit provoked a anchorage of tyrosine-phosphorylated proteins to the cytoskeleton, which was increased compared with integrin clustering. phosphorylation of anchored proteins be a to the cytoskeletal M. Y. J. M. J. Biol. Chem. 1996; Scholar), and a higher of phosphorylation may be a for the higher between receptors and the that the of activated signaling molecules to the cytoskeleton, we have found that intracellular is an important of the of proteins to the This not proteins but also the cytoskeletal anchorage of integrins to the cytoskeleton B. Bohn W. H. Rychly J. Exp. Cell Res. 1996; Scholar). The role of for a mechanically induced signal transduction is also stressed by that have that intracellular with force applied to integrins M. J. J. 1995; Scholar). that the differential cytoskeletal anchorage of tyrosine-phosphorylated proteins and integrin subunits due to stimulation of β1 compared with the α subunit is not by differences in the of the of cells with anti-integrin antibodies to mechanical stimulation of the cells (13Nebe B. Rychly J. Knopp A. Bohn W. Exp. Cell Res. 1995; 218: 479-484Google Scholar), as well as results the of the due to mechanical stress applied with magnetic beads to β1 and revealed differences in downstream signaling, we that the cytoskeleton a structure where physical forces are into a biochemical signal pathway. The differential anchorage of tyrosine-phosphorylated proteins due to physically integrins may downstream events. of these events is the activation of MAP kinases as a key mechanism to control the activation of which gene The of this in integrin signaling has E.A. R.O. J. Biol. Chem. 1996; Scholar, N. T. K. K. K. K. K. T. Yazaki Y. Y. J. Biol. Chem. 1995; Scholar). We found that activation of the MAP kinases was significantly increased due to physical forces compared with integrin clustering. to the key role of the MAP kinases, that physical forces by integrins cell proliferation and the expression of the MAP kinase The that activation of MAP kinase by integrins on an cytoskeleton N. T. K. K. K. K. K. T. Yazaki Y. Y. J. Biol. Chem. 1995; Scholar, Lin K. Juliano J. Biol. Chem. 1994; Scholar) and the of signaling molecules like kinase T. J. Biol. Chem. Scholar) the of a cytoskeletal anchorage of tyrosine-phosphorylated proteins for in cell Because the integrin-mediated MAP kinase with C. Rubin K. J. Cell Biol. 1996; Scholar), suggests a effect of mechanical forces and in the regulation of cell In integrins physical forces and may physiological in the cell by a well of the of tyrosine phosphorylation of A is the cytoskeletal anchorage of activated signaling proteins, which on the of intracellular calcium. The relevance of these mechanisms is by the of an enhanced activation of MAP kinases due to mechanical integrin