Cytoprotection against Mechanical Forces Delivered through β1 Integrins Requires Induction of Filamin A

Cells in mechanically active environments can activate cytoprotective mechanisms to maintain membrane integrity in the face of potentially lethal applied forces. Cytoprotection may be mediated by expression of membrane-associated cytoskeletal proteins including filamin A, an actin-binding protein that increases the rigidity of the subcortical actin cytoskeleton. In this study, we tested the hypotheses that applied forces induce the expression of filamin A specifically and that this putative protective response inhibits cell death. Magnetically generated forces were applied to protein-coated magnetite beads bound to human gingival fibroblasts, cells with constitutively low basal levels of filamin A mRNA and protein. Forces applied through collagen or fibronectin, but not bovine serum albumin or poly-l-lysine-coated beads, increased mRNA and protein content of filamin A by 3–7-fold. Forces had no effect on the expression of other filamin isotypes or other cytoskeletal proteins. This effect was dependent on the duration of force and was blocked by anti-β1 integrin antibodies. Force also stimulated a 60% increase in expression of luciferase under the control of a filamin A promoter in transiently transfected Rat2 fibroblasts and was dependent on Sp1 transcription factor binding sites located immediately upstream of the transcription start site. Experiments with actinomycin D-treated cells showed that the increased filamin A expression after force application was due in part to prolongation of mRNA half-life. Antisense filamin oligonucleotides blocked force-induced filamin A expression and increased cell death by >2-fold above controls. The force-induced regulation of filamin A was dependent on intact actin filaments. We conclude that cells from mechanically active environments can couple diverse signals from forces applied through β-integrins to up-regulate the production of cytoprotective cytoskeletal proteins, typified by filamin A. Cells in mechanically active environments can activate cytoprotective mechanisms to maintain membrane integrity in the face of potentially lethal applied forces. Cytoprotection may be mediated by expression of membrane-associated cytoskeletal proteins including filamin A, an actin-binding protein that increases the rigidity of the subcortical actin cytoskeleton. In this study, we tested the hypotheses that applied forces induce the expression of filamin A specifically and that this putative protective response inhibits cell death. Magnetically generated forces were applied to protein-coated magnetite beads bound to human gingival fibroblasts, cells with constitutively low basal levels of filamin A mRNA and protein. Forces applied through collagen or fibronectin, but not bovine serum albumin or poly-l-lysine-coated beads, increased mRNA and protein content of filamin A by 3–7-fold. Forces had no effect on the expression of other filamin isotypes or other cytoskeletal proteins. This effect was dependent on the duration of force and was blocked by anti-β1 integrin antibodies. Force also stimulated a 60% increase in expression of luciferase under the control of a filamin A promoter in transiently transfected Rat2 fibroblasts and was dependent on Sp1 transcription factor binding sites located immediately upstream of the transcription start site. Experiments with actinomycin D-treated cells showed that the increased filamin A expression after force application was due in part to prolongation of mRNA half-life. Antisense filamin oligonucleotides blocked force-induced filamin A expression and increased cell death by >2-fold above controls. The force-induced regulation of filamin A was dependent on intact actin filaments. We conclude that cells from mechanically active environments can couple diverse signals from forces applied through β-integrins to up-regulate the production of cytoprotective cytoskeletal proteins, typified by filamin A. monoclonal antibody phosphate-buffered saline reverse transcriptase polymerase chain reaction glyceraldehyde-3-phosphate dehydrogenase base pair(s) kilobase pair(s) bovine serum albumin Cells in biomechanically stressful environments can respond to pericellular changes in mechanical force by transducing physical stimuli into chemical signals that subsequently regulate gene expression (1Watson P.A. FASEB J. 1991; 5: 2013-2019Crossref PubMed Scopus (284) Google Scholar). For example, mechanical force affects the expression of several cytoskeletal genes including the α-skeletal and α-smooth muscle actin isoforms (1Watson P.A. FASEB J. 1991; 5: 2013-2019Crossref PubMed Scopus (284) Google Scholar) as well as the actin-binding proteins talin and vinculin (2Tidball J.G. Spencer M.J. Wehling M. Lavergne E. J. Biol. Chem. 1999; 274: 33155-33160Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Mechanical force induction of cytoskeletal protein expression has been examined most prominently reported for smooth muscle cells (3Birukov K.G. Bardy N. Lehoux S. Merval R. Shirinsky V.P. Tedgui A. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 922-927Crossref PubMed Scopus (72) Google Scholar, 4Brown K.D. Binder L.I. Exp. Cell Res. 1993; 209: 325-332Crossref PubMed Scopus (11) Google Scholar), but the transcriptional regulation of actin-binding proteins in nonmuscle cells has not been evaluated in detail. Notably, cells in mechanically stressed nonmuscle tissues such as bone and periodontium can maintain membrane integrity despite the application of high amplitude applied forces, indicating the existence of adaptive cytoprotective responses that may involve the cytoskeleton. For example, the recruitment of the actin-binding protein filamin A to sites of force transfer in fibroblasts was found to be cytoprotective, since it reduced the potentially lethal influx of calcium ions in response to applied force (5Glogauer M. Arora P. Chou D. Janmey P.A. Downey G.P. McCulloch C.A.G. J. Biol. Chem. 1998; 273: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). As filamin A interacts with the cytoplasmic domains of the β1 and β7integrins (6Pfaff M. Liu S. Erle D.J. Ginsberg M.H. J. Biol. Chem. 1998; 273: 6104-6109Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), it may also be able to transduce matrix-derived extracellular signals including those arising from mechanical forces. Currently, there is no evidence for force-induced regulation of filamin A expression in nonmuscle cells. Filamins are actin-binding proteins originally isolated from chicken gizzard (7Wang K. Biochemistry. 1977; 16: 1857-1865Crossref PubMed Scopus (105) Google Scholar) that organize actin filaments into orthogonal networks and enhance the rigidity of the actin cortex (8Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers H.R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (495) Google Scholar, 9Ohta Y. Suzuki N. Nakamura S. Hartwig J.H. Stossel T.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2122-2128Crossref PubMed Scopus (369) Google Scholar). Three discrete filamins have been described, and alternative splicing may allow for several additional isoforms (10Calderwood D.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 2000; 275: 22607-22610Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Filamin A (actin-binding protein 280) is composed of 23 ∼96-amino acid repeats with flexible hinges and actin binding domains (10Calderwood D.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 2000; 275: 22607-22610Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 11Gorlin J.B. Yamin R. Egan S. Stewart M. Stossel T.P. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1990; 111: 1089-1105Crossref PubMed Scopus (427) Google Scholar, 12Cooper J.A. Schafer D.A. Curr. Opin. Cell Biol. 2000; 12: 97-103Crossref PubMed Scopus (264) Google Scholar, 13Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (492) Google Scholar). The biological importance of filamin A is underscored by evidence showing that the X-linked human disorder periventricular heterotopia is due to mutations at X-q28, theFLN1 locus (14Fox J.W. Lamperti E.D. Eksioglu Y.Z. Hong S.E. Feng Y. Graham D.A. Scheffer I.E. Dobyns W.B. Hirsch B.A. Radtke R.A. Berkovicz S.F. Huttenlocher P.R. Walsch C.A. Neuron. 1998; 21: 1315-1325Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar). This condition is characterized by defects in the migration of cerebral cortical neurons and vascular abnormalities that arise because of a lack of filamin-directed actin assembly and deficient rigidity of cell membranes. While originally described in the context of its ability to organize the actin cytoskeleton, filamin A is now known to interact with many cellular proteins and has multiple including its ability to the for and cell (8Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers H.R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (495) Google Scholar). the of the and other filamin A has been to interact with cytoplasmic factor the factor the the the membrane protein and the β1 integrin (10Calderwood D.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 2000; 275: 22607-22610Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Stossel T.P. Hartwig J.H. 1991; Full Text PDF PubMed Scopus Google Scholar, R.A. C.A. C.C. Hartwig J.H. J. Cell Biol. PubMed Scopus Google Scholar, Y. J. Cell Biol. 1998; PubMed Scopus Google Scholar, A. K. U. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, N. M. A. D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). the of mechanical forces from to and to filamin A and actin filaments may a and for cells in mechanically active In of the of calcium responses by filamin A on cells to high amplitude applied forces (5Glogauer M. Arora P. Chou D. Janmey P.A. Downey G.P. McCulloch C.A.G. J. Biol. Chem. 1998; 273: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), we force regulate filamin A gene expression in human gingival cells low basal levels of filamin A but are to high amplitude forces in may be to force-induced induction of filamin A in cytoprotective that mechanical forces applied through regulate filamin A transcription and that of filamin A expression by oligonucleotides increases force-induced cell death. have for the and of cells in tissues that are to high amplitude physical A monoclonal antibody was from monoclonal antibody muscle actin and were from was from The was from and for was at a of as described N. McCulloch C.A.G. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). and were from gingival fibroblasts were from as described McCulloch C.A.G. J. Cell Sci. 1991; PubMed Google Scholar). Cells from were as in in bovine serum and to cells were and at In fibroblasts were as for gingival fibroblasts as described McCulloch C.A.G. J. PubMed Scopus Google Scholar). Cells were in with bovine serum and to the cells were in at and transfected as described Force through was a described M. Arora J. McCulloch C.A.G. J. Cell Sci. PubMed Google Scholar). magnetite were with collagen or or bovine serum albumin with and with a were by and the cells were with A was on of the to a mechanical force of cell a force that is with that may be by cells in (5Glogauer M. Arora P. Chou D. Janmey P.A. Downey G.P. McCulloch C.A.G. J. Biol. Chem. 1998; 273: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The were for as was a P. N. PubMed Scopus Google Scholar). cells were and by with and in of and was by and were with for The was as described in M. A. J.W. J. FASEB J. 1999; PubMed Scopus Google Scholar). was on of reverse transcriptase and of The was to of in a were to and by to filamin A of was the and The of was with the of the gene glyceraldehyde-3-phosphate dehydrogenase of the oligonucleotides in the were as filamin A filamin A reverse filamin filamin reverse filamin filamin reverse reverse The of the to reaction and the have been described M. A. A. J. J. Biol. 2000; PubMed Scopus Google Scholar). In a control was to the lack of was isolated from or fibroblasts at the in an mRNA mRNA of mRNA were through a a membrane by and as described above for the transfer of and were as described M. A. A. J. J. Biol. 2000; PubMed Scopus Google Scholar) the filamin A and oligonucleotides described The were and for of to gingival fibroblasts were and as described cell cells were and cellular proteins were by and to and as described (5Glogauer M. Arora P. Chou D. Janmey P.A. Downey G.P. McCulloch C.A.G. J. Biol. Chem. 1998; 273: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). in were by the bovine serum albumin as a of protein were on and were as described (5Glogauer M. Arora P. Chou D. Janmey P.A. Downey G.P. McCulloch C.A.G. J. Biol. Chem. 1998; 273: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). was to the The were for of the filamin A luciferase promoter we isolated intact the of S.E. Res. PubMed Scopus Google Scholar). cell were for at with in of intact on a of was with and The was in an of oligonucleotides A and and of The an at by of at at and at with a at for The was to a that sites for into the The the with the of and The was through and into the and the of the was through and at the for for of fibroblasts was with cells were in and of and were transfected to the The luciferase was for of force-induced transcriptional and has been described A. M. McCulloch C.A.G. J. 1999; PubMed Scopus Google Scholar). The were on of cells. We Rat2 cells as for the human gingival fibroblasts, because that Rat2 cells were to human gingival fibroblasts McCulloch C.A.G. J. PubMed Scopus Google Scholar) human fibroblasts, can be transfected A. M. McCulloch C.A.G. J. 1999; PubMed Scopus Google Scholar) for of the promoter in and the filamin A promoter was with the of the upstream of the upstream of the upstream of the upstream of the upstream but of the filamin the promoter mutations at the Sp1 binding oligonucleotides were and to to in was to the of potentially transcription factor binding sites J. Biol. PubMed Scopus (369) Google Scholar). The Sp1 mutations are in The oligonucleotides were into the luciferase and the was through gingival fibroblasts were on with and to force application as described were at and in for by in were with monoclonal filamin A by antibody of filamin with cells were by or by In that examined the of filamin A content to the cell cells were in with and by Antisense oligonucleotides to the filamin A gene and control oligonucleotides were on a The to to and the the base oligonucleotides were generated to described for were on and with of oligonucleotides into the at in to the of cells. for were from and on a cells were with beads as described above and or not to cells were evaluated by and The levels and of filamin A were evaluated by as described above by In cells were For and S.E. of the were were for and was at We examined force-induced filamin A mRNA expression in human gingival fibroblasts by was a increase in filamin A mRNA that was as as force application through collagen of the showed a increase in filamin A mRNA force induction in the of the as the of was isolated from cells at the application of A showed a induction of filamin as the other filamin isoforms were in cells and by force from and cells was to for filamin isoforms and the as for filamin A. was no for filamin and a low of filamin that was not specifically by force application not We force enhance filamin A expression by protein content in and found a increase in filamin A protein of force increases were with for other cytoskeletal proteins including α-smooth muscle and β1 of showed induction by force We filamin A expression at the locus by The in of filamin A in cells but in cells to there was an increased for filamin A at the locus We also examined filamin A levels may be dependent on the cell cells in the of the cell showed filamin A that was cells in the In a to the of force-induced filamin A expression on cells in of cells and cells in of cells were We found by that cells in increases of filamin A content after force application not that filamin A expression was by mechanical force applied through magnetite We to force of filamin A was mediated through have that and beads not to cells through collagen and specifically through the β1 (5Glogauer M. Arora P. Chou D. Janmey P.A. Downey G.P. McCulloch C.A.G. J. Biol. Chem. 1998; 273: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). were with fibronectin, or and with and force was applied to cells. While there was no in filamin A expression force application through or beads A and beads increased filamin A and protein by a effect as with collagen This was not due to of beads from the of the since and A. M. McCulloch C.A.G. J. 1999; PubMed Scopus Google Scholar) have that application of force through or beads not increased with or beads J. A. McCulloch C.A.G. J. 2000; PubMed Google Scholar). that of filamin A was specifically dependent on β1 cells were with a monoclonal antibody β1 for to the of magnetite This antibody at a has been to of α-smooth muscle actin but not of N. McCulloch C.A.G. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We a of force filamin A production indicating that the β1 integrin is and is for induction of filamin A expression by force Filamin A actin filaments and increases cortical rigidity (8Cunningham C.C. Gorlin J.B. Kwiatkowski D.J. Hartwig J.H. Janmey P.A. Byers H.R. Stossel T.P. Science. 1992; 255: 325-327Crossref PubMed Scopus (495) Google Scholar). Filamin may a in the cell membrane in response to mechanical forces. We have that in filamin cells mechanical force application increases membrane in with the cells but with filamin A expression M. Arora P. Chou D. Janmey P.A. Downey G.P. McCulloch C.A.G. J. Biol. Chem. 1998; 273: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). We to the of filamin A in fibroblasts from force-induced cell death. Antisense filamin A oligonucleotides or oligonucleotides were into fibroblasts, and force was applied for through of filamin A and of cells by filamin A was in the cell cortex and in of and cells with the with the force increased for filamin A. In cells with the filamin A was reduced in We filamin A with an and found that the oligonucleotides reduced filamin A by and blocked the force-induced increase of filamin A We examined of cells after force This is from cells with an intact showed that cells with the oligonucleotides >2-fold of to cells cells with the or those cells that were not to force that force-induced filamin A expression is for of in cells to mechanical Cell is a factor for cytoskeletal gene expression in response to A. M. McCulloch C.A.G. J. 1999; PubMed Scopus Google Scholar) and N. McCulloch C.A.G. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and is dependent on the and assembly of actin filaments. we cell by with or of actin filaments showed that cells on were well and of cortical actin and cells on were and not cortical actin or application of mechanical force through on the cell there was no induction of filamin A or protein cells were on and the were with fibronectin, there was a increase of filamin A mRNA after force at for to after force showed that mechanical force application to fibroblasts on a increase in filamin A after of cells on showed no In of the of force-induced filamin expression after cell and of actin we of actin filaments the mechanical force induction of filamin A. We cells with to the of and examined filamin A inhibits actin assembly in human gingival fibroblasts under but not of beads force application Arora M. Downey McCulloch C.A.G. J. Cell Sci. PubMed Google Scholar). with force-induced production of filamin A mRNA and protein was reduced to levels in cells This with the described that force application through β1 and cortical actin filaments production of filamin A. of filamin A transcriptional by mechanical force not the that force may increase mRNA content by mRNA an effect that has been for α-smooth muscle actin N. McCulloch C.A.G. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). we filamin A mRNA in collagen cells with and force Cells were for filamin A mRNA production after of force actinomycin was to to mRNA the human gingival have that the of actinomycin at is to mRNA N. McCulloch C.A.G. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). For this not to cell death. was isolated at the of actinomycin and filamin A, and were by Cells with actinomycin but force application showed a of filamin A mRNA at at filamin A mRNA was reduced to of levels in cells to force of levels of filamin A mRNA after force filamin mRNA levels were in cells in control cells In to filamin A, the levels of and mRNA this We also the of protein in the induction of filamin A mRNA by Cells were with a that in human gingival fibroblasts of protein McCulloch C.A.G. J. PubMed Scopus Google Scholar). of control cells and cells showed a increase in the of filamin A mRNA after of force a as in there was no force-induced filamin A mRNA expression in the of indicating that protein is for filamin A mRNA induction by We examined the transcriptional of the filamin A gene force fibroblasts were transiently transfected with a base of the filamin A promoter under after cells were with and mechanical force was applied for While the filamin A basal force application a 60% increase of luciferase production promoter were generated described under transfected into cells and to force that of the filamin A promoter induction with cells In an of the filamin A promoter with J. Biol. PubMed Scopus (369) Google Scholar) in of transcription factor binding the most of sites was the most transcription binding sites the a of the was found to be by force application despite the of of promoter we generated a filamin promoter at the most Sp1 transfected this into and to force The that of Sp1 sites at and specifically the force-induced production of luciferase that Sp1 sites the force-induced expression of filamin transcription factor binding are from J. Biol. PubMed Scopus (369) Google in a are from J. Biol. PubMed Scopus (369) Google cells by and of forces, the cells in mechanically active The mechanisms by nonmuscle cells in mechanically active environments maintain and are that filamin A mRNA and protein are in human gingival fibroblasts application of mechanical forces through β1 and actin filaments. Notably, we showed that induction of filamin A cells death. filamin A may be in to maintain cell in the face of applied forces. and a of cells in mechanically active environments are to extracellular by cell the of the extracellular proteins and binding to an in biological responses to a of extracellular signals and cell bound to proteins can applied physical forces through the to the Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the not as a but may also signals for transcriptional regulation by mechanical forces. that in well cells with cortical mechanical force application through specifically filamin A This to be since force application through poly-l-lysine-coated beads not filamin A and the force-induced increases of filamin A mRNA and protein were blocked by with anti-β1 integrin antibodies. As filamin A to the cytoplasmic of the β1 integrin (10Calderwood D.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 2000; 275: 22607-22610Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), and the showing the increased of filamin A at the locus evidence for a physical forces applied through the extracellular to filamin A and the transcriptional of a protective to a in a cell can cytoskeletal and respond by the transcription and of filamin A, an actin protein that cells applied forces. This may involve the of force from through actin filaments to the as Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of the importance of cell and the ability of cells to forces the cell membrane and into the we in of beads were on the of cells on or of cells on cell cell and inhibits force-induced and of factor E. K. J. 96: PubMed Scopus Google Scholar). In cells on force application through beads filamin A to levels that were with those in cells on cell In the lack of on the cell in cells the of the forces to the cell that is for filamin A since this of cortical actin filaments. the importance of intact actin filaments for of force to the cell and are with to actin the induction of filamin A and protein in response to Notably, of cell and the of force to sites that may be in The increased of filamin A mRNA after force application is by promoter that force the basal of a of the filamin A a filamin A we that Sp1 binding sites located immediately upstream of the start were for the force-induced of filamin A. Notably, Sp1 a of genes R. 2000; PubMed Scopus Google and and filamin A can now be to this As an of a gene that is in the cell for the is known to be by binding sites located at M. D. J.A. PubMed Scopus Google Scholar). In the gene is also known to multiple Sp1 binding sites that this as by S. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). to a promoter was by the of to the gene located of the start M. R. A. M. D. M. 5: PubMed Scopus Google Scholar). the filamin and genes are by a promoter of is that the that we most of the that is for filamin A We of the filamin A promoter on of the gene that α-smooth muscle cytoskeletal protein that has been examined in detail. have of the upstream of α-smooth muscle actin D. J. 1998; 275: PubMed Google Scholar). from this have on located from the transcription start Res. 1999; PubMed Scopus Google Scholar), are to on in the filamin A gene reported The ability of force to induce mRNA and increased protein levels of filamin A is not to increased promoter of filamin A that mRNA also to this there were in mRNA since cells of mRNA content cells. In this the of mRNA for α-smooth muscle actin actin found in is by applied mechanical forces in N. McCulloch C.A.G. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In we have that the force-induced expression of filamin A in human gingival fibroblasts is mediated through cells low basal levels of filamin A and are to high amplitude forces in have to enhance the cytoprotective effect of filamin A the ability of force to increase filamin A protein content of fibroblasts is in on prolongation of the of filamin A of a cytoprotective gene that is found in cells that may be to high amplitude mechanical forces. the of force-induced transcription of proteins in a of cell that In the context of applied we that tissues that may be to high amplitude physical may be for by the of cells with expression of filamin A.

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