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