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Laminin-10/11 and Fibronectin Differentially Prevent Apoptosis Induced by Serum Removal via Phosphatidylinositol 3-Kinase/Akt- and MEK1/ERK-dependent Pathways*

Open AccessPublished:March 12, 2002DOI:https://doi.org/10.1074/jbc.M200383200
      Cell adhesion to the extracellular matrix inhibits apoptosis, but the molecular mechanisms underlying the signals transduced by different matrix components are not well understood. Here, we examined integrin-mediated antiapoptotic signals from laminin-10/11 in comparison with those from fibronectin, the best characterized extracellular adhesive ligand. We found that the activation of protein kinase B/Akt in cells adhering to laminin-10/11 can rescue cell apoptosis induced by serum removal. Consistent with this, wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, or ectopic expression of a dominant-negative mutant of Akt selectively accelerated cell death upon serum removal. In contrast to laminin-10/11, fibronectin rescued cells from serum depletion-induced apoptosis mainly through the extracellular signal-regulated kinase pathway. Cell survival on fibronectin but not laminin was significantly reduced by treatment with PD98059, a specific inhibitor of mitogen- or extracellular signal-regulated kinase kinase-1 (MEK1) and by expression of a dominant-negative mutant of MEK1. Laminin-10/11 was more potent than fibronectin in preventing apoptosis induced by serum depletion. These results, taken together, demonstrate laminin-10/11 potency as a survival factor and demonstrate that different extracellular matrix components can transduce distinct survival signals through preferential activation of subsets of multiple integrin-mediated signaling pathways.
      Cell adhesion to the ECM
      The abbreviations used are: ECM
      extracellular matrix
      Cas-ΔSD
      p130cas lacking substrate domain
      DMEM
      Dulbecco's modified Eagle's medium
      DN
      dominant negative
      ERK
      extracellular signal-regulated kinase
      FAK
      focal adhesion kinase
      FN
      fibronectin
      HA
      hemagglutinin
      FRNK
      FAK-related nonkinase
      LN-10/11
      laminin-10/11
      MEK1
      mitogen- or extracellular signal-regulated kinase kinase-1
      PBS
      phosphate-buffered saline
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PLL
      poly-l-lysine
      TUNEL
      terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
      VSV
      vesicular stomatitis virus glycoprotein
      z-VAD
      z-Val-Ala-Asp-fluromethylketone
      BSA
      bovine serum albumin
      1The abbreviations used are: ECM
      extracellular matrix
      Cas-ΔSD
      p130cas lacking substrate domain
      DMEM
      Dulbecco's modified Eagle's medium
      DN
      dominant negative
      ERK
      extracellular signal-regulated kinase
      FAK
      focal adhesion kinase
      FN
      fibronectin
      HA
      hemagglutinin
      FRNK
      FAK-related nonkinase
      LN-10/11
      laminin-10/11
      MEK1
      mitogen- or extracellular signal-regulated kinase kinase-1
      PBS
      phosphate-buffered saline
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PLL
      poly-l-lysine
      TUNEL
      terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
      VSV
      vesicular stomatitis virus glycoprotein
      z-VAD
      z-Val-Ala-Asp-fluromethylketone
      BSA
      bovine serum albumin
      generates intracellular signals that modulate cell proliferation, survival, and differentiation (
      • Giancotti F.G.
      • Ruoslahti E.
      ,
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ). Normal epithelial cells deprived of matrix attachment undergo programmed cell death, a form of apoptosis termed anoikis (
      • Frisch S.M.
      • Francis H.
      ). Malignant transformation by oncogenic Ras can prevent this process of apoptosis after denial of ECM attachment (
      • Frisch S.M.
      • Francis H.
      ) or withdrawal of survival factors (
      • Downward J.
      ).
      One approach to study the effects of ECM signals independently of signals from other extracellular sources has been to deprive cells of serum and then to analyze the effects of specific ECM ligands on cellular functions such as adhesion, migration, and survival. Using this approach, many cell biologic functions of ECM signals have been elucidated (
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ). Most studies of integrin-mediated signaling events have been performed on cells adhering to FN through the α5β1 integrin, which seems to be involved in regulating apoptosis triggered by serum deprivation in many cell types (
      • Zhang Z.
      • Vuori K.
      • Wang H.
      • Reed J.C.
      • Ruoslahti E.
      ,
      • O'Brien V.
      • Frisch S.M.
      • Juliano R.L.
      ). This integrin has also been reported to protect neuronal cells against apoptosis triggered by β-amyloid peptide (
      • Matter M.L.
      • Zhang Z.
      • Nordstedt C.
      • Ruoslahti E.
      ). In breast epithelial cells, the α6β1 integrin receptor for laminin-1 has been shown to cooperate with insulin-signaling pathways to protect cells from apoptosis (
      • Farrelly N.
      • Lee Y.J.
      • Oliver J.
      • Dive C.
      • Streuli C.H.
      ). In endothelial cells, functional inhibition of the αvβ3 integrin can lead to programmed cell death (
      • Brooks P.C.
      • Montgomery A.M.
      • Rosenfeld M.
      • Reisfeld R.A., Hu, T.
      • Klier G.
      • Cheresh D.A.
      ). Thus, several distinct integrins have been implicated in protection against apoptosis in different cell types. However, the signaling events transduced by the α3β1integrin, the major receptor for laminin-10/11 (LN-10/11) and LN-5, remain unclear. In addition, it is also not known whether α3β1 integrin-mediated signals differ from those transduced by the α5β1 integrin.
      Several signal transduction components, including focal adhesion kinase (FAK) (
      • Frisch S.M.
      • Vuori K.
      • Ruoslahti E.
      • Chan-Hui P.Y.
      ,
      • Xiong W.
      • Parsons J.T.
      ,
      • Tamura M., Gu, J.
      • Danen E.H.
      • Takino T.
      • Miyamoto S.
      • Yamada K.M.
      ), phosphatidylinositol 3-kinase (PI 3-kinase) (
      • Farrelly N.
      • Lee Y.J.
      • Oliver J.
      • Dive C.
      • Streuli C.H.
      ,
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • Lee J.W.
      • Juliano R.L.
      ), extracellular signal-regulated kinase (ERK) (
      • Le Gall M.
      • Chambard J.C.
      • Breittmayer J.P.
      • Grall D.
      • Pouyssegur J.
      • Van Obberghen-Schilling E.
      ,
      • Jost M.
      • Huggett T.M.
      • Kari C.
      • Rodeck U.
      ,
      • Tran S.E.
      • Holmstrom T.H.
      • Ahonen M.
      • Kahari V.M.
      • Eriksson J.E.
      ), and c-Jun NH2-terminal kinase (
      • Almeida E.A.
      • Ilic D.
      • Han Q.
      • Hauck C.R.
      • Jin F.
      • Kawakatsu H.
      • Schlaepfer D.D.
      • Damsky C.H.
      ), have been implicated in the mechanisms underlying anoikis (
      • Frisch S.M.
      • Screaton R.A.
      ). FAK has been proposed to couple integrins and cytoskeletal proteins to multiple signaling pathways. Several lines of evidence suggest that integrin activation of signaling pathways involving PI 3-kinase, ERK, and c-Jun NH2-terminal kinase require FAK (
      • Tamura M., Gu, J.
      • Danen E.H.
      • Takino T.
      • Miyamoto S.
      • Yamada K.M.
      ,
      • Almeida E.A.
      • Ilic D.
      • Han Q.
      • Hauck C.R.
      • Jin F.
      • Kawakatsu H.
      • Schlaepfer D.D.
      • Damsky C.H.
      ,
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ,
      • Zhao J.H.
      • Reiske H.
      • Guan J.L.
      ,
      • Dolfi F.
      • Garcia-Guzman M.
      • Ojaniemi M.
      • Nakamura H.
      • Matsuda M.
      • Vuori K.
      ,
      • Schlaepfer D.D.
      • Hauck C.R.
      • Sieg D.J.
      ). However, some studies suggest that integrins are able to activate at least some of these pathways independently of FAK (
      • Wary K.K.
      • Mainiero F.
      • Isakoff S.J.
      • Marcantonio E.E.
      • Giancotti F.G.
      ,
      • Lin T.H.
      • Aplin A.E.
      • Shen Y.
      • Chen Q.
      • Schaller M.
      • Romer L.
      • Aukhil I.
      • Juliano R.L.
      ,
      • Wary K.K.
      • Mariotti A.
      • Zurzolo C.
      • Giancotti F.G.
      ). Recently, increasing evidence has emerged showing that PI 3-kinase and its downstream effector Akt play key roles in the regulation of cell survival. For example, signals through the PI 3-kinase/Akt pathway protect Madin-Darby canine kidney cells against apoptosis mediated by denial of cell anchorage or by radiation (
      • Khwaja A.
      • Downward J.
      ). It was found that cell death in this system was inhibited by expression of a constitutively activated form of Akt (
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • Khwaja A.
      • Downward J.
      ). On the other hand, the ERK pathway has also been found to enhance cell survival (
      • Erhardt P.
      • Schremser E.J.
      • Cooper G.M.
      ,
      • Yujiri T.
      • Sather S.
      • Fanger G.R.
      • Johnson G.L.
      ).
      Laminins are the major components of the basement membrane. Cells bind directly to laminins via a subset of integrins (
      • Colognato H.
      • Yurchenco P.D.
      ) and other nonintegrin receptors, such as α-dystroglycans (
      • Montanaro F.
      • Lindenbaum M.
      • Carbonetto S.
      ). All laminins are composed of α, β, and γ chains. The α5-containing laminins, LN-10 (α5β1γ1) and LN-11 (α5β2γ1) are widely expressed in fetal and adult tissues (
      • Miner J.H.
      • Lewis R.M.
      • Sanes J.R.
      ,
      • Sorokin L.M.
      • Pausch F.
      • Frieser M.
      • Kroger S.
      • Ohage E.
      • Deutzmann R.
      ). Recently, we purified LN-10/11 from conditioned medium of A549 human lung carcinoma cells and found that the α3β1 integrin is the preferred receptor for LN-10/11 (
      • Kikkawa Y.
      • Sanzen N.
      • Sekiguchi K.
      ,
      • Kikkawa Y.
      • Sanzen N.
      • Fujiwara H.
      • Sonnenberg A.
      • Sekiguchi K.
      ). LN-10/11 is more active than FN in promoting cell migration, and it preferentially activates Rac, but not Rho, via the p130cas -CrkII-DOCK180 pathway. Cells adhering to FN develop stress fibers and focal contacts, whereas cells adhering to LN-10/11 do not, suggesting that LN-10/11 and FN have distinct effects on integrin-mediated cell spreading and migration (
      • Gu J.
      • Sumida Y.
      • Sanzen N.
      • Sekiguchi K.
      ).
      In this study, the first goal was to determine whether α3β1 integrin-mediated signals from LN-10/11 could rescue A549 cells from apoptosis induced by serum deprivation. We describe here that LN-10/11 has more survival potential than FN. The second goal was to identify pathway(s) that transduce the survival signals from LN-10/11. We report that survival signals from LN-10/11 are mainly through the PI 3-kinase/Akt pathway, whereas survival signals from FN are conveyed by MEK1/ERK through FAK.

      DISCUSSION

      The α3β1 integrin-mediated signaling events triggered by cell adhesion to LN-10/11 are quite different from those triggered by adhesion to FN. LN-10/11 preferentially activates Rac, but not Rho, through an α3β1integrin-dependent pathway involving a p130cas -CrkII-DOCK180 complex, thereby strongly promoting cell migration through enhanced formation of lamellipodia. FN, however, preferentially activates Rho rather than Rac, leading to enhanced stress fiber and focal contact formation (
      • Gu J.
      • Sumida Y.
      • Sanzen N.
      • Sekiguchi K.
      ). In this study, we analyzed intracellular signaling pathways regulating cell survival of A549 human lung adenocarcinoma cells by focusing on two distinct signaling pathways involving PI 3-kinase/Akt and MEK1/ERK; although separate, these pathways might potentially engage in cross-talk. We found that LN-10/11 is more potent than FN in suppressing apoptosis induced by serum deprivation. The antiapoptotic effects of LN-10/11 could be inhibited by the PI 3-kinase inhibitor wortmannin, whereas the antiapoptotic effects of FN were inhibited by the MEK1 inhibitor PD98059; these contrasting findings indicate that different ECMs selectively modulate different intracellular signaling pathways to sustain cell survival. These findings were further confirmed by expression of dominant negative Akt and MEK1, which compromised the ability of LN-10/11 and FN to transduce survival signals, respectively. Our results provide clear evidence that different signaling pathways leading to cell survival are activated on different ECM ligands (i.e. on FN and LN-10/11) (Fig. 7). Since laminins are the major components of the basement membrane of epithelium, our work supports the notion that a function of the basement membrane is to provide distinctive cell survival signals for establishment and maintenance of epithelial tissue.
      Figure thumbnail gr7
      Figure 7Proposed scheme of distinct survival signaling pathways on LN-10/11 and FN. Cell adhesion to ECM triggers integrin-mediated tyrosine phosphorylation of FAK and p130cas, leading to activation of downstream cascades involving PI 3-kinase/Akt and MEK1/ERK that prevent apoptosis. LN-10/11 preferentially induces phosphorylation of p130cas, whereas FN induces phosphorylation of FAK (Ref.
      • Gu J.
      • Sumida Y.
      • Sanzen N.
      • Sekiguchi K.
      ; also this study). The survival signals from LN-10/11 are mainly through the PI 3-kinase/Akt pathway, whereas FN survival signals are conveyed by FAK through the MEK1/ERK pathway. Cross-talk between PI 3-kinase and Rac has been described (
      • Coniglio S.J.
      • Jou T.S.
      • Symons M.
      ,
      • Zugasti O.
      • Rul W.
      • Roux P.
      • Peyssonnaux C.
      • Eychene A.
      • Franke T.F.
      • Fort P.
      • Hibner U.
      • Coniglio S.J.
      • Jou T.S.
      • Symons M.
      ) (dashed lines). Recently, it has been shown that Rho activation may contribute to sustained ERK activation (
      • Welsh C.F.
      • Assoian R.K.
      ) (dashed line). Cross-talk has also been reported between FAK and PI 3-kinase (
      • Tamura M., Gu, J.
      • Danen E.H.
      • Takino T.
      • Miyamoto S.
      • Yamada K.M.
      ,
      • Zhao J.H.
      • Reiske H.
      • Guan J.L.
      ) (dashed line), although this process was not observed in the present study.

      LN-10/11 Is More Potent Than FN in Protecting Cells against Apoptosis-induced by Serum Depletion

      Interactions of cells with the ECM through integrins are known to suppress apoptosis in many cell types. Mammary epithelial cells cultured on collagen I show extensive apoptosis over periods of several days, whereas the same cells do not when in contact with LN-1 or Matrigel, a basement membrane-like gel containing laminin-1, collagen IV, nidogen, and perlecan (
      • Pullan S.
      • Wilson J.
      • Metcalfe A.
      • Edwards G.M.
      • Goberdhan N.
      • Tilly J.
      • Hickman J.A.
      • Dive C.
      • Streuli C.H.
      ). However, LN-1 may not be a survival ligand for other cells, since endothelial cells undergo apoptosis on an LN-1 substrate while being protected from apoptosis on FN or vitronectin substrates (
      • Wary K.K.
      • Mainiero F.
      • Isakoff S.J.
      • Marcantonio E.E.
      • Giancotti F.G.
      ). Thus, different cell types may have their own favored ECM for protection from apoptosis, depending on the repertoire of integrins expressed on their cell surface, which in turn may define the types of ECM ligands most potent for protection from apoptosis. Our present studies are based on comparisons of the signaling events and abilities of LN-10/11 and FN to rescue A549 cells from serum depletion-induced apoptosis. Since α3β1 and α5β1 integrins serve as the dominant adhesion receptors for LN-10/11 and FN, respectively, the distinct apoptotic responses of cells on LN-10/11 and FN mirror the distinct signaling pathways downstream of the α3β1and α5β1 integrins (Fig. 7). Our data show that LN-10/11 is more potent than FN in preventing apoptosis induced by serum depletion, suggesting that the α3β1integrin transduces potent survival signals when bound to LN-10/11. This is consistent with the closely overlapped distribution of α3β1 integrin and its major ligand LN-10/11. In fact, the α3β1 integrin is predominantly expressed on many kinds of epithelial cells that deposit laminin-10 and laminin-11 as the major components of their basement membrane. It should be noted, however, that signaling pathways on distinct ECM ligands are usually context-dependent and may not be the same in different cell types. Our conclusions based on analyses of A549 lung carcinoma cells remain to be generalized to other cell types.

      PI 3-Kinase/Akt Pathway Is Essential for LN-10/11 Survival Signals

      Cell adhesion to ECM triggers integrin-mediated downstream phosphorylation cascades involving the ERK type of mitogen-activated protein kinase and PI 3-kinase, providing possible mechanisms for ECM-dependent cell survival (
      • Giancotti F.G.
      • Ruoslahti E.
      ,
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ). It remains to be determined, however, whether the survival signals from different ECM ligands are transduced by distinct signaling pathways. Activation of the PI 3-kinase/Akt pathway provided a potent antiapoptotic signal in cells adhering to LN-10/11, whereas activation of the MEK1/ERK pathway was necessary for survival of cells adhering to FN. To our knowledge, this is the first report to provide a clear distinction between the signaling pathways that rescue a cell from apoptosis on different ECM ligands.
      Accumulating evidence indicates that the PI 3-kinase/Akt pathway is critical for preventing apoptosis (
      • Downward J.
      ). Anoikis resulting from denial of integrin-mediated adhesion involves reduced signaling through the PI 3-kinase/Akt pathway (
      • Downward J.
      ). Our results show that the α3β1 integrin, when compared with α5β1, selectively activates the PI 3-kinase/Akt pathway, thereby exerting its potent antiapoptotic effects. The evidence for a specific connection between α3β1 integrin and the PI 3-kinase/Akt pathway includes the facts that the antiapoptotic effects of LN-10/11 but not FN are reversed by wortmannin or expression of a dominant-negative Akt mutant. The stronger activation of Rac in cells adhering to LN-10/11 than in those adhering to FN (
      • Gu J.
      • Sumida Y.
      • Sanzen N.
      • Sekiguchi K.
      ) may also contribute to enhance cell survival on LN-10/11; Rac activation has been shown to protect epithelial cells against anoikis through activation of the PI 3-kinase/Akt pathway (
      • Coniglio S.J.
      • Jou T.S.
      • Symons M.
      ,
      • Zugasti O.
      • Rul W.
      • Roux P.
      • Peyssonnaux C.
      • Eychene A.
      • Franke T.F.
      • Fort P.
      • Hibner U.
      • Coniglio S.J.
      • Jou T.S.
      • Symons M.
      ), although the precise mechanisms of the cross-talk between PI 3-kinase and Rac in cell survival remain to be clarified. Protection of Madin-Darby canine kidney cells from anoikis by overexpression of a membrane-anchored, constitutively activated form of FAK has been described (
      • Frisch S.M.
      • Vuori K.
      • Ruoslahti E.
      • Chan-Hui P.Y.
      ); the mechanism might involve cross-talk activation between the hyperactivated FAK and PI 3-kinase pathways (
      • Reiske H.R.
      • Kao S.C.
      • Cary L.A.
      • Guan J.L.
      • Lai J.F.
      • Chen H.C.
      ) or some alternative mechanism.
      Our results do not imply that the α3β1integrin is the only integrin capable of activating the PI 3-kinase/Akt pathway, since cell type-specific differences are known. Mammary epithelial cells utilize the α6β1 integrin to transduce cell survival signals that are dependent on the PI 3-kinase/Akt pathway (
      • Farrelly N.
      • Lee Y.J.
      • Oliver J.
      • Dive C.
      • Streuli C.H.
      ). In contrast to our results, α5β1 integrin regulation of cell survival in rat intestinal epithelial cells has been shown to modulate the PI 3-kinase/Akt pathway (
      • Lee J.W.
      • Juliano R.L.
      ), whereas our data showed that FN protected A549 cells against apoptosis mainly through the MEK1/ERK pathway. The central role of the MEK1/ERK pathway in survival of A549 cells on FN was supported by the proapoptotic effects of PD98059 or the overexpression of dominant negative MEK1 mutant on cells adhering to FN. Consistent with our observations, the Ras/mitogen-activated protein kinase cascade has been shown to function as a survival signaling pathway; thus, sustained activation of this pathway efficiently rescues fibroblasts and epithelial cells from anoikis (
      • Le Gall M.
      • Chambard J.C.
      • Breittmayer J.P.
      • Grall D.
      • Pouyssegur J.
      • Van Obberghen-Schilling E.
      ). Besides PI 3-kinase/Akt and ERK pathways, activation of c-Jun NH2-terminal kinase has also been shown to be involved in apoptosis (
      • Frisch S.M.
      • Vuori K.
      • Kelaita D.
      • Sicks S.
      ,
      • Tournier C.
      • Hess P.
      • Yang D.D., Xu, J.
      • Turner T.K.
      • Nimnual A.
      • Bar-Sagi D.
      • Jones S.N.
      • Flavell R.A.
      • Davis R.J.
      ). Almeida et al. (
      • Almeida E.A.
      • Ilic D.
      • Han Q.
      • Hauck C.R.
      • Jin F.
      • Kawakatsu H.
      • Schlaepfer D.D.
      • Damsky C.H.
      ) reported that activation of the c-Jun NH2-terminal kinase pathway, but not the PI 3-kinase/Akt or ERK signaling pathways, is essential for protecting primary rabbit synovial fibroblasts against apoptosis induced by serum depletion on FN-coated substrates, indicating that distinct signaling pathways play critical roles in integrin-mediated survival signals.

      Prolonged Activation of Akt or ERK Is Substrate-dependent

      Although this study showed that the initial activation of Akt or ERK (e.g. 10 min after replating; see Fig. 3) was observed in cells adhering to either LN-10/11 or FN, prolonged basal activation of Akt or ERK was observed only in cells adhering to LN-10/11 or to FN, respectively. The basal levels of activated Akt and/or ERK seem to be important for protecting cells against apoptosis induced by serum removal, as demonstrated by the experiments using wortmannin and PD98059 and expression of their dominant negative mutants. The precise mechanisms of specific activation of Akt and ERK by different ECM ligands remain to be elucidated. Several lines of evidence indicate that integrins with different α subunits activate mitogen-activated protein kinases via different signaling pathways (
      • Giancotti F.G.
      • Ruoslahti E.
      ). For example, a subset of integrins including the α5β1 integrin can recruit the transmembrane protein caveolin-1 and the adaptor protein Shc, thereby activating the ERK pathway. Recently, it has been reported that Rho has an essential role in integrin- and growth factor receptor-mediated signaling pathways that lead to sustained ERK activation and subsequent cyclin D1 regulation (
      • Welsh C.F.
      • Assoian R.K.
      ). Thus, the prolonged basal activation of ERK on FN could be explained by the observations that in certain cells, FN preferentially activates Rho but not Rac, whereas LN-10/11 preferentially activates Rac but not Rho (
      • Gu J.
      • Sumida Y.
      • Sanzen N.
      • Sekiguchi K.
      ). The α subunits of α3β1 and α6β1associate with a group of TM4SF proteins (
      • Hemler M.E.
      ). The TM4SF proteins may associate with protein kinase C and phosphatidylinositol 4-kinase, linking these integrins to phosphoinositide signaling pathways (
      • Yauch R.L.
      • Berditchevski F.
      • Harler M.B.
      • Reichner J.
      • Hemler M.E.
      ). It remains to be examined, however, whether TM4SF proteins are involved in the prolonged basal activation of Akt on LN-10/11 via association with α3β1.

      FN Survival Signals Are FAK-dependent, whereas LN-10/11 Survival Signals Are FAK-independent

      A rapid increase in the tyrosine phosphorylation of FAK at multiple sites has been identified as a prominent early event in integrin-mediated cell adhesion that regulates cell proliferation, migration, and apoptosis (
      • Schlaepfer D.D.
      • Hauck C.R.
      • Sieg D.J.
      ,
      • Hanks S.K.
      • Polte T.R.
      ). Autophosphorylation of FAK at Tyr-397 has emerged as a crucial event in FAK-mediated signal transduction, since the phosphorylation of FAK at Tyr-397 triggers the formation of molecular complexes with other signaling proteins including Src family kinases (
      • Cobb B.S.
      • Schaller M.D.
      • Leu T.H.
      • Parsons J.T.
      ,
      • Xing Z.
      • Chen H.C.
      • Nowlen J.K.
      • Taylor S.J.
      • Shalloway D.
      • Guan J.L.
      ), the p85 regulatory subunit of PI 3-kinase (
      • Chen H.C.
      • Appeddu P.A.
      • Isoda H.
      • Guan J.L.
      ), Shc (
      • Schlaepfer D.D.
      • Jones K.C.
      • Hunter T.
      ), and tumor suppressor PTEN (
      • Gu J.
      • Tamura M.
      • Pankov R.
      • Danen E.H.
      • Takino T.
      • Matsumoto K.
      • Yamada K.M.
      ,
      • Tamura M., Gu, J.
      • Matsumoto K.
      • Aota S.
      • Parsons R.
      • Yamada K.M.
      ). Our data showed that FAK phosphorylation at Tyr-397 was more prominently induced in cells adhering to FN than to LN-10/11, supporting the previous observation that the level of overall tyrosine phosphorylation of FAK was lower in cells adhering to LN-10/11 than in those adhering to FN (
      • Gu J.
      • Sumida Y.
      • Sanzen N.
      • Sekiguchi K.
      ). Together with the observation that expression of FRNK substantially impaired survival of cells on FN with minimal effects when cells were on LN-10/11, our results suggest that FAK is an essential component in survival signaling on FN but not on LN-10/11.
      The role of FAK in integrin-mediated ERK activation is complex. Schlaepfer et al. (
      • Schlaepfer D.D.
      • Jones K.C.
      • Hunter T.
      ,
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      ) found that FAK was involved in integrin-triggered ERK signaling, but differing findings have been reported in other systems (
      • Wary K.K.
      • Mainiero F.
      • Isakoff S.J.
      • Marcantonio E.E.
      • Giancotti F.G.
      ,
      • Lin T.H.
      • Aplin A.E.
      • Shen Y.
      • Chen Q.
      • Schaller M.
      • Romer L.
      • Aukhil I.
      • Juliano R.L.
      ,
      • Wary K.K.
      • Mariotti A.
      • Zurzolo C.
      • Giancotti F.G.
      ). Since the time course of phosphorylation of FAK did not correlate with the time course of ERK activation (Figs. 3 and 6), FAK may act collaboratively by other mechanisms with other signaling molecules to ensure prolonged basal activation of ERK on FN. In fact, B-Raf has been shown to be required for the sustained activation of ERK in a FAK-dependent manner (
      • Barberis L.
      • Wary K.K.
      • Fiucci G.
      • Liu F.
      • Hirsch E.
      • Brancaccio M.
      • Altruda F.
      • Tarone G.
      • Giancotti F.G.
      ). On the other hand, p130cas appeared to be involved in both LN-10/11 and FN survival signaling pathways, since the expression of p130cas lacking the substrate domain significantly reduced cell viability on both LN-10/11 and FN. Consistent with this observation, the role of p130cas in integrin α3β1-dependent Rac activation on LN-10/11 has been demonstrated (
      • Gu J.
      • Sumida Y.
      • Sanzen N.
      • Sekiguchi K.
      ), while the involvement of p130cas in integrin α5β1-dependent ERK activation has also been demonstrated in different cell types (
      • Barberis L.
      • Wary K.K.
      • Fiucci G.
      • Liu F.
      • Hirsch E.
      • Brancaccio M.
      • Altruda F.
      • Tarone G.
      • Giancotti F.G.
      ).
      In conclusion, our results strongly suggest that different ligands differentially activate integrin-mediated signaling pathways to protect against apoptosis in A549 cells, although it remains to be examined whether the distinct signaling pathways transduced by those different ECMs exist in other cell types. LN-10/11 was more potent than FN for protection against apoptosis induced by serum depletion by selectively activating the PI 3-kinase/Akt pathway rather than the MEK1/ERK pathway. The importance of anchorage to the basement membrane is well established for the maintenance of epithelial architecture and survival of epithelial cells; this study provides insight into the molecular basis of basement membrane-triggered signaling events regulating epithelial cell function.

      ACKNOWLEDGEMENT

      We are grateful to Kazue Matsumoto for generating the VSV-FAK and VSV-FRNK plasmids.

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