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β1 Integrin Regulates Fibroblast Viability during Collagen Matrix Contraction through a Phosphatidylinositol 3-Kinase/Akt/Protein Kinase B Signaling Pathway*

  • Bin Tian
    Affiliations
    From the Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
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  • Khashayar Lessan
    Affiliations
    From the Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
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  • Judy Kahm
    Affiliations
    From the Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
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  • Jill Kleidon
    Affiliations
    From the Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
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  • Craig Henke
    Correspondence
    To whom correspondence should be addressed: Pulmonary and Critical Care Div., Dept. of Medicine, FUMC Box 276, University of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455. Tel.: 612-624-0999; Fax: 612-625-2174l;
    Affiliations
    From the Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
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  • Author Footnotes
    * This work was supported by Grant P50 HL50152 (Specialized Center of Research in Acute Lung Injury) from the National Institutes of Health and by a career investigator award from the American Lung Association (to C. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:May 01, 2002DOI:https://doi.org/10.1074/jbc.M203565200
      Integrins regulate cell viability through their interaction with the extracellular matrix. Integrins can sense mechanical forces arising from the matrix and convert these stimuli to chemical signals capable of modulating intracellular signal transduction. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is a major regulator of cell survival. It is not known, however, whether integrins, acting as mechanoreceptors, regulate cell survival via the PI3K/Akt pathway. Here, we show that in response to a matrix-derived mechanical stimulus, β1integrin regulated cell viability by regulating Akt activity in a PI3K-dependent fashion. To accomplish this, we employed fibroblasts cultured in collagen gels. During contraction of collagen matrices, fibroblasts underwent apoptosis. We demonstrate that ligation of β1 integrin with anti-β1 integrin antibodies protected fibroblasts from apoptosis. The nature of the survival signal activated by β1 integrin engagement with antibody was mediated by PI3K acting through Akt/protein kinase B. We show that Akt phosphorylation decreased during collagen contraction and that this decrease correlated precisely with the onset of fibroblast apoptosis. Fibroblasts transfected with constitutively active PI3K displayed increased Akt phosphorylation and were protected from anoikis and collagen gel contraction-induced apoptosis. Our data identify a novel role for β1 integrin in regulating fibroblast viability through a PI3K/Akt/protein kinase B signaling pathway in response to a matrix-derived mechanical stimulus.
      ECM
      extracellular matrix
      PI3K
      phosphatidylinositol 3-kinase
      PKB
      protein kinase B
      mAb
      monoclonal antibody
      TUNEL
      terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling
      CA-p110
      constitutively active p110 subunit of PI3K
      IRES
      internal ribosome entry site
      GFP
      green fluorescent protein
      FACS
      fluorescence-activated cell sorter
      Integrins are a family of αβ-heterodimeric cell-surface receptors that mediate attachment to the extracellular matrix (ECM)1 (
      • Ruoslahti E.
      ). Integrins associate with signaling molecules in the focal adhesion complex, which serves as a signaling device and provides a direct link to the cytoskeleton (
      • Ruoslahti E.
      ,
      • Giancotti F.G.
      • Ruoslahti E.
      ). The continuous link between the ECM, integrins, signaling molecules, and the cytoskeleton has led to the discovery that integrins are capable of serving as mechanoreceptors (
      • Davies P.F.
      ,
      • Wilson E.
      • Sudhir K.
      • Ives H.E.
      ,
      • Ishida T.
      • Peterson T.E.
      • Kovach N.L.
      • Berk B.C.
      ,
      • MacKenna D.A.
      • Dolfi F.
      • Vuori K.
      • Ruoslahti E.
      ). As mechanoreceptors, integrins detect matrix-derived mechanical stimuli and convert these to chemical signals capable of modulating signal transduction. Integrin-ECM interaction activates signal transduction pathways that regulate a variety of cellular functions, including cell viability (
      • Meredith J.E., Jr.
      • Fazeli B.
      • Schwartz M.A.
      ,
      • Frisch S.M.
      • Francis H.
      ,
      • Brooks P.C.
      • Montgomery A.M.P.
      • Rosenfeld M.
      • Reisfeld R.A., Hu, T.
      • Klier G.
      • Cheresh D.A.
      ,
      • Zhang Z.
      • Vuori K.
      • Reed J.
      • Ruoslahti E.
      ,
      • Frisch S.M.
      • Vuori K.
      • Kelaita D.
      • Sicks S.
      ,
      • Stromblad S.
      • Becker J.C.
      • Yebra M.
      • Brooks P.C.
      • Cheresh D.A.
      ). Not all integrins appear to be capable of regulating cell viability. However, β1 integrin, which mediates attachment to type I collagen, can regulate cell survival (
      • Meredith J.E., Jr.
      • Fazeli B.
      • Schwartz M.A.
      ).
      The biological effects of integrin-ECM binding are mediated through the interaction of the cytoplasmic domain of integrins with associated lipid and protein kinases. Specific lipid and protein kinase molecules known thus far to promote cell survival include pp125FAK(
      • Crouch D.H.
      • Fincham V.J.
      • Frame M.C.
      ,
      • Frisch S.M.
      • Vuori K.
      • Ruoslahti E.
      • Chan-Hui P.Y.
      ,
      • Hungerford J.E.
      • Compton M.T.
      • Matter M.L.
      • Hoffstrom B.G.
      • Otey C.A.
      ,
      • Levkau B.
      • Herren B.
      • Koyama H.
      • Ross R.
      • Raines E.
      ,
      • Chen H.C.
      • Guan J.L.
      ,
      • Ilic D.
      • Almeida E.A.C.
      • Schlaepfer D.D.
      • Dazin P.
      • Aizawa S.
      • Damsky C.H.
      ), integrin-linked kinase (
      • Hannigan G.E.
      • Leung-Hagesteijn C.
      • Fitz-Gibbon L.
      • Coppolini M.
      • Radeava G.
      • Filmus J.
      • Bell J.C.
      • Dedhar S.
      ,
      • Wu C.
      ,
      • Delcommenne M.
      • Tan C.
      • Gray V.
      • Rue L.
      • Woodgett J.
      • Dedhar S.
      ), and phosphatidylinositol 3-kinase (PI3K) and Akt/PKB (
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • Lee J.W.
      • Juliano R.J.
      ,
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ). Evidence has emerged demonstrating the important role of an integrin/PI3K/Akt/PKB signaling pathway in regulating epithelial cell survival (
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • Lee J.W.
      • Juliano R.J.
      ). Attachment of these cells to the underlying basement membrane via integrins activates the PI3K/Akt/PKB survival signal. However, upon detachment, epithelial cells undergo anoikis. Unlike epithelial cells, which are polarized and attach to the underlying basement membrane at their basal surface, mesenchymal cells are frequently surrounded by the ECM. Because mesenchymal cells are surrounded by the matrix, it is unlikely that unnecessary mesenchymal cells are eliminated by detachment and anoikis. However, recent studies indicate that matrix-derived mechanical signals can regulate cell survival (
      • Dimmeler S.
      • Assmus B.
      • Hermann C.
      • Haendeler J.
      • Zeiher A.M.
      ,
      • Chen K.D., Li, Y.S.
      • Kim M., Li, S.
      • Yuan S.
      • Chien S.
      • Shyy J.Y.
      ). Currently, it is not known whether integrins, acting as mechanoreceptors, can regulate cell survival via the PI3K pathway in response to a matrix-derived mechanical stimulus.
      To begin to examine this issue, we employed three-dimensional type I collagen gels. We used three-dimensional collagen gels for our studies because it has been found that three-dimensional matrix adhesions differ in structure, localization, and function from two-dimensional adhesions; and therefore, three-dimensional cell-matrix interactions may be more relevant biologically (
      • Cukierman E.
      • Pankov R.
      • Stevens D.R.
      • Yamada K.M.
      ). This is especially true in the context of examination of mesenchymal cells such as fibroblasts, which typically exist encompassed by the matrix. Recently, it has been discovered that during collagen gel contraction, fibroblasts undergo apoptosis in response to changes in mechanical tension in the collagen matrix (
      • Grinnell F.
      ,
      • Grinnell F.
      ,
      • Fluck J.
      • Querfeld C.
      • Cremer A.
      • Niland S.
      • Krieg T.
      • Sollberg S.
      ,
      • Grinnell F.
      • Zhu M.
      • Carlson M.A.
      • Abrams J.M.
      ). Furthermore, it is known that α2β1 integrin mediates collagen gel contraction and that β1 integrin can function as a mechanoreceptor and can also regulate cell viability. Therefore, we examined whether β1 integrin in response to a mechanical stimulus generated by collagen contraction can regulate fibroblast apoptosis. Using anti-β1 integrin antibodies, we show that ligation of β1 integrin protected fibroblasts from undergoing apoptosis in response to collagen gel contraction. The nature of the survival signal activated by β1 integrin engagement with anti-β1 integrin antibody was mediated by PI3K acting through Akt/PKB. We demonstrate that Akt activity precipitously declined during collagen matrix contraction and that this decrease correlated precisely with the onset of collagen matrix contraction-induced fibroblast apoptosis. Up-regulation of PI3K activity using fibroblasts transfected with the catalytically active p110 subunit of PI3K protected fibroblasts from undergoing anoikis in suspension cultures and apoptosis in response to collagen gel contraction. These studies identify a novel role for β1integrin in regulating fibroblast viability via modulation of a PI3K/Akt signaling pathway in response to contraction of type I collagen matrices.

      DISCUSSION

      Attachment of epithelial cells to the underlying basement membrane via integrins activates the survival signaling protein Akt/PKB in a PI3K-dependent manner. Detachment of epithelial cells is associated with a loss of the Akt/PKB survival signal and anoikis. Unlike polarized epithelial cells, which attach only to the matrix at their basal surface, mesenchymal cells such as fibroblasts are surrounded by the ECM. When unnecessary mesenchymal cells are eliminated, it is unlikely that they detach from the matrix and undergo anoikis. However, the molecular mechanism regulating mesenchymal cell viability remains to be elucidated. Here, we show that in response to a matrix-derived mechanical stimulus, β1 integrin regulates mesenchymal cell viability by regulating Akt activity in a PI3K-dependent fashion. Our data suggest that β1 integrin is capable of acting as a mechanoreceptor and that in response to a mechanical stimulus generated by contraction of the type I collagen matrix, it regulates fibroblast viability through a PI3K/Akt/PKB signaling pathway.
      During the evolution of tissue development and repair after injury, unnecessary mesenchymal cells need to be eliminated for normal tissue patterning to occur (
      • Darby I.
      • Skalli O.
      • Gabbiani G.
      ,
      • Polunovsky V.A.
      • Chen B.
      • Henke C.
      • Snover D.
      • Wendt C.
      • Ingbar D.
      • Bitterman P.B.
      ,
      • Desmouliere A.
      • Redard M.
      • Darby I.
      • Gabbiani G.
      ,
      • Nakagawa S.
      • Pawelek P.
      • Grinnell F.
      ,
      • Xu J.
      • Zutter M.M.
      • Santora S.A.
      • Clark R.A.F.
      ). However, the physiological signal that initiates apoptosis of cells surrounded by the matrix remains obscure. In 1999, Grinnell et al. (
      • Grinnell F.
      • Zhu M.
      • Carlson M.A.
      • Abrams J.M.
      ) determined that a decrease in mechanical tension in collagen matrices during gel contraction triggers fibroblast apoptosis. Consistent with this report, we show that the degree of collagen contraction is dependent upon the concentration of collagen used to make the gels. Furthermore, we demonstrate that the magnitude of fibroblast apoptosis is directly proportional to the degree of gel contraction. The degree of gel contraction appears to be the determining factor for apoptosis, and not the concentration of collagen. Fibroblasts embedded in non-contractile collagen gels composed of concentrations of collagen equivalent to those used in contractile gels do not undergo apoptosis. Together, these data suggest that changes in mechanical tension in the ECM serve as the trigger for initiation of fibroblast apoptosis and that the degree of collagen contraction is an important factor in determining the magnitude of the apoptotic response.
      β1 integrin mediates attachment to collagen and contraction of collagen matrices and can regulate cell survival. However, whether β1 integrin is capable of regulating fibroblast viability during collagen gel contraction is not known. In this report, we show that β1 integrin regulates fibroblast viability during collagen gel contraction. We demonstrate that direct engagement of β1 integrin with anti-β1 integrin antibodies protects fibroblasts from contraction-induced apoptosis. We used two different anti-β1 integrin monoclonal antibodies that recognize the same ligand-binding domain of β1 integrin (
      • Seltzer J.L.
      • Lee A.Y.
      • Akers K.T.
      • Sudbeck B.
      • Southon E.A.
      • Wayner E.A.
      • Eisen A.Z.
      ,
      • Takada Y.
      • Puzon W.
      ). These antibodies have been used to study β1 integrin-mediated signal transduction in collagen matrices. Consistent with a previous report (
      • Seltzer J.L.
      • Lee A.Y.
      • Akers K.T.
      • Sudbeck B.
      • Southon E.A.
      • Wayner E.A.
      • Eisen A.Z.
      ), we showed that anti-β1 integrin antibody M-13 inhibited collagen gel contraction, whereas the P5D2 antibody had little effect on collagen gel contraction. Despite this, we found that both antibodies were capable of protecting fibroblasts from collagen gel contraction-induced apoptosis. We expected that collagen gels treated with the M-13 antibody would have decreased apoptosis. This is because the M-13 antibody completely inhibits gel contraction, thereby eliminating the stimulus for induction of apoptosis. The surprising finding was that the P5D2 antibody also protected against contraction-induced apoptosis. Because the P5D2 antibody had little effect on gel contraction, this suggested that the antibody may function by either inhibiting a pro-apoptotic pathway or alternatively by stimulating a survival signal. Therefore, the results with the P5D2 antibody suggest that the effects we observed were due to β1 integrin signaling and not from the contraction of the gel itself.
      Attachment of epithelial cells to the ECM via integrins provides a survival signal. Detachment from the matrix precipitates anoikis (
      • Meredith J.E., Jr.
      • Fazeli B.
      • Schwartz M.A.
      ,
      • Frisch S.M.
      • Francis H.
      ). Fibroblasts are more resistant than epithelial cells to the process of anoikis (
      • Hadden H.L.
      • Henke C.A.
      ). However, if deprived of matrix contact for prolonged time periods, fibroblasts also undergo anoikis (
      • Hadden H.L.
      • Henke C.A.
      ,
      • Henke C.A.
      • Bitterman P.
      • Roongta U.
      • Polunovsky V.
      ). Because, in our system, fibroblasts were embedded in three-dimensional collagen gels, loss of attachment to the ECM is unlikely to be the mechanism by which fibroblasts undergo apoptosis. In this report, we show that prior to gel contraction, fibroblasts are well spread. During collagen gel contraction, the cells become progressively round in appearance. Because cell spreading and shape have been closely linked to survival (
      • Chen C.S.
      • Mrksich M.
      • Huang S.
      • Whitesides G.M.
      • Ingber D.E.
      ), it is possible that changes in cell shape may be responsible for apoptosis of fibroblasts during collagen gel contraction. Indeed, recent work suggests that chemical signaling events induced by integrin interaction with the ECM can be modulated by changes in the actin cytoskeleton in response to alterations in the ECM (
      • Plopper G.E.
      • McNamee H.P.
      • Dike L.E.
      • Bojanowski K.
      • Ingber D.E.
      ,
      • Chicurel M.E.
      • Chen C.S.
      • Ingber D.E.
      ). Thus, in three-dimensional culture conditions, fibroblast apoptosis does not appear to be triggered by cell detachment from the ECM. Rather, fibroblast apoptosis in response to collagen matrix contraction may occur due to down-regulation of a survival signal as a result of integrin-ECM interaction-induced alterations in the actin cytoskeleton and associated signal transduction pathways.
      A function of β1 integrin as a mechanotransducer is to convert mechanical stimuli into chemical signals. For β1integrin to regulate fibroblast viability during collagen gel contraction, it must be capable of modulating either pro-apoptotic or survival signal transduction pathways. It has been established that integrins can promote epithelial cell survival through the PI3K/Akt pathway (
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • Lee J.W.
      • Juliano R.J.
      ). Our data demonstrate that ligation of β1 integrin with anti-β1 integrin antibody P5D2 in both adherent and non-adherent human fibroblasts results in activation of Akt/PKB in a PI3K-dependent manner. Using a pharmacological inhibitor of PI3K (wortmannin), we have demonstrated that phosphorylation of Akt/PKB in response to ligation of β1 integrin by antibody is abrogated. These data indicate that direct engagement of β1 integrin with the P5D2 antibody is capable of up-regulating the activity of the Akt/PKB survival signal. It is important to note that the P5D2 antibody, when preincubated with cells prior to their attachment, can block adhesion and therefore has blocking properties under these conditions. However, the fact that the P5D2 antibody can increase Akt activity upon binding cells in suspension or already attached indicates that under these culture conditions, the antibody functions in an agonist fashion. It also suggests that the mechanism by which the P5D2 antibody protects fibroblasts from apoptosis is by β1 integrin-mediated activation of a PI3K/Akt survival signal transduction pathway. The M-13 antibody also protects against contraction-induced apoptosis. However, the mechanism by which it does so was not by augmenting the Akt survival signal because Akt activity was not increased after ligation of β1 integrin with antibody. Instead, the M-13 antibody likely inhibited apoptosis by eliminating the stimulus for apoptosis, namely gel contraction.
      To determine whether the PI3K/Akt survival signal regulates fibroblast viability during contraction of type I collagen matrices, we examined Akt activity during matrix contraction. Akt phosphorylation precipitously declined during collagen gel contraction, and the decrease in Akt activity temporally mirrored precisely the timing of fibroblast apoptosis in response to gel contraction. These results led us to examine whether modulation of PI3K activity could regulate fibroblast viability in collagen gels. To approach this issue, we up-regulated PI3K activity by transfecting our fibroblasts with the constitutively active p110 subunit of PI3K. We found that constitutively active PI3K was capable of markedly up-regulating the activity of Akt/PKB and that it protected fibroblasts from both anoikis as well as collagen gel contraction-induced apoptosis. Furthermore, we have shown that unlike normal fibroblasts, whose level of Akt activity markedly declined during collagen matrix contraction, the level of Akt activity in CA-p110 fibroblasts stayed relatively steady during matrix contraction. Collectively, our data suggest that a stimulus generated by collagen gel contraction is detected by β1 integrin, which in turn down-regulates the PI3K/Akt survival signal, triggering fibroblast apoptosis. Up-regulation of PI3K/Akt activity by either stimulation of β1 integrin by antibody binding or by expression of constitutively active PI3K protects fibroblasts against contraction-induced apoptosis.
      In summary, the data presented here support the idea that β1 integrin can function as a mechanoreceptor that is capable of sensing a mechanical stimulus generated by collagen matrix contraction. Our data indicate that induction of apoptosis during collagen gel contraction is controlled by β1 integrin, which is capable of modulating the activity of the PI3K/Akt survival signal. Understanding the control of fibroblast viability in collagen matrices provides insight into the regulation of mesenchymal cell regression during tissue development and repair.

      Acknowledgments

      We thank Dr. Julian Downward for the constitutively active PI3K (p110) plasmid, Dr. Leo Furcht for anti-β1 integrin antibody P5D2, and Dr. Kenneth Yamada for anti-β1 integrin antibody M-13. We also thank Dr. Peter Bitterman for helpful discussions and critical review of the manuscript.

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