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The Role of Focal Adhesion Kinase-Phosphatidylinositol 3-Kinase-Akt Signaling in Hepatic Stellate Cell Proliferation and Type I Collagen Expression*

Open AccessPublished:December 26, 2002DOI:https://doi.org/10.1074/jbc.M212927200
      Following a fibrogenic stimulus, the hepatic stellate cell (HSC) undergoes a complex activation process associated with increased cell proliferation and excess deposition of type I collagen. The focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is activated by platelet-derived growth factor (PDGF) in several cell types. We investigated the role of the FAK-PI3K-Akt pathway in HSC activation. Inhibition of FAK activity blocked HSC migration, cell attachment, and PDGF-induced PI3K and Akt activation. Both serum- and PDGF-induced Akt phosphorylation was inhibited by LY294002, an inhibitor of PI3K. A constitutively active form of Akt stimulated HSC proliferation in serum-starved HSCs, whereas LY294002 and dominant-negative forms of Akt and FAK inhibited PDGF-induced proliferation. Transforming growth factor-β, an inhibitor of HSC proliferation, did not block PDGF-induced Akt phosphorylation, suggesting that transforming growth factor-β mediates its antiproliferative effect downstream of Akt. Expression of type I collagen protein and α1(I) collagen mRNA was increased by Akt activation and inhibited when PI3K activity was blocked. Therefore, FAK is important for HSC migration, cell attachment, and PDGF-induced cell proliferation. PI3K is positioned downstream of FAK. Signals for HSC proliferation are transduced through FAK, PI3K, and Akt. Finally, expression of type I collagen is regulated by the PI3K-Akt signaling pathway.
      HSC
      hepatic stellate cell
      PDGF
      platelet-derived growth factor
      MEK
      mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase
      ERK
      extracellular signal-regulated kinase
      FAK
      focal adhesion kinase
      PI3K
      phosphatidylinositol 3-kinase
      FBS
      fetal bovine serum
      m.o.i.
      multiplicity of infection
      PBS
      phosphate-buffered saline
      BSA
      bovine serum albumin
      TBS
      Tris-buffered saline
      TGF-β
      transforming growth factor-β
      STAT
      signal transducer and activator of transcription
      JNK
      c-Jun N-terminal kinase
      MAPK
      mitogen-activated protein kinase
      Liver fibrosis represents a wound-healing process in response to a variety of chronic stimuli. Fibrosis is characterized by an excessive deposition of extracellular matrix proteins, of which type I collagen predominates. Activation of the hepatic stellate cell (HSC),1 a perisinusoidal cell that resides in the liver in a quiescent state, is responsible for the increased synthesis and deposition of type I collagen in the liver. Following a fibrogenic stimulus, the HSC undergoes a complex activation process in which the cell changes from a quiescent vitamin A-storing cell to an activated myofibroblast-like cell, which proliferates and becomes fibrogenic (
      • Friedman S.L.
      ,
      • Eng F.J.
      • Friedman S.L.
      ). An increase in DNA synthesis and cell proliferation occurs with HSC activation. Altered collagen synthesis, at both the mRNA and protein levels, is observed with a dramatic increase in type I collagen and smaller but significant increases in type III and IV collagens (
      • Maher J.J.
      • Bissell D.M.
      • Friedman S.L.
      • Roll F.J.
      ,
      • Friedman S.L.
      • Rockey D.C.
      • McGuire R.F.
      • Maher J.J.
      • Boyles J.K.
      • Yamasaki G.
      ,
      • Knittel T.
      • Schuppan D.
      • Meyer zum Buschenfelde K.-H.
      • Ramadori G.
      ).
      Platelet-derived growth factor (PDGF) is the most potent proliferative cytokine for the HSC (
      • Friedman S.L.
      ). Liver fibrosis is associated with an increase in PDGF protein expression and increased PDGF receptor expression (
      • Pinzani M.
      • Marra F.
      • Carloni V.
      ). PDGF receptors contain intrinsic tyrosine kinase activity and, upon binding to its ligand, become autophosphorylated at tyrosine residues (
      • Claesson-Welsh L.
      ). PDGF has been shown to transmit proliferative signals in several cell types, including the HSC (
      • Friedman S.L.
      • Authur M.J.P.
      ,
      • Pinzani M.
      • Gesualdo L.
      • Sabbah G.M.
      • Abboud H.E.
      ). The activated PDGF receptor acts as a high affinity binding site for several signaling molecules leading to activation of Ras, followed by activation of Raf, MEK, and ERK (
      • Marshall C.J.
      ).
      Focal adhesion kinase (FAK) has a key role in the synergistic interaction between growth factor and integrin signaling pathways. FAK, a 125-kDa cytoplasmic protein-tyrosine kinase, is a member of the focal adhesion family that mediates integrin-mediated signal transduction leading to a variety of cellular functions, including cell proliferation, migration, and adhesion (
      • Gilmore A.P.
      • Romer L.H.
      ). PDGF induces tyrosine phosphorylation of FAK (
      • Carloni V.
      • Pinzani M.
      • Giusti S.
      • Romanelli R.G.
      • Parola M.
      • Bellomo G.
      • Failli P.
      • Hamilton A.D.
      • Sebti S.M.
      • Laffi G.
      • Gentilini P.
      ). Association of FAK and phosphatidylinositol 3-kinase (PI3K) is stimulated by PDGF and is required for PDGF stimulation of HSC proliferation (
      • Marra F.
      • Gentilini A.
      • Pinzani M.
      • Choudhury G.G.
      • Parola M.
      • Herbst H.
      • Dianzani M.U.
      • Laffi G.
      • Abboud H.E.
      • Gentilini P.
      ,
      • Chen H.-C.
      • Guan J.-L.
      ). Focal adhesion non-kinase (FAK-CD) represents a kinase-deficient splice variant of FAK that contains only the C-terminal portion of FAK and that can inhibit phosphorylation of endogenous FAK, therefore acting as a dominant-negative form of FAK (
      • Gilmore A.P.
      • Romer L.H.
      ,
      • Richardson A.
      • Malik R.K.
      • Hildebrand J.D.
      • Parsons J.T.
      ).
      PI3K represents another key signaling molecule that is recruited to the activated PDGF receptor (
      • Parker P.J.
      • Waterfield M.D.
      ). Activated PI3K generates several phosphoinositols, leading to Akt activation by phosphorylation at Thr308 and Ser473 by phosphoinositide-dependent kinase-1 (
      • Chan T.O.
      • Rittenhouse S.E.
      • Tsichlis P.N.
      ). Activated Akt is considered a key downstream survival factor by stimulating cell proliferation and inhibiting apoptosis (
      • Chan T.O.
      • Rittenhouse S.E.
      • Tsichlis P.N.
      ). Akt is activated not only by growth factors that stimulate tyrosine kinase activity, but also by other signals that can activate PI3K, such as integrins and stimulators of G-protein-coupled receptors (
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • Shaw M.
      • Cohen P.
      • Alessi D.R.
      ,
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ,
      • Muraga C.
      • Fukuhara S.
      • Gutkind J.S.
      ).
      We report here that FAK, PI3K, and Akt are involved in transmitting proliferative signals induced by serum and PDGF in the HSC. We show that both serum and PDGF induce phosphorylation of FAK, PI3K, and Akt and that PI3K and Akt signal downstream of FAK in the HSC. Blocking either FAK or PI3K inhibits HSC adhesion and migration. We demonstrate that PI3K activation is important for both type I collagen mRNA and protein expression in the HSC. Inhibition of PI3K reduces secreted type I collagen protein expression, whereas the intracellular unprocessed collagen intermediates remain unchanged. Therefore, this signaling pathway is critical for HSC proliferation and type I collagen gene expression in the activated HSC.

      DISCUSSION

      Two major events occur following HSC activation that promote the fibrogenic response of these cells. First, HSCs change their pattern of gene expression, increasing the synthesis and deposition of extracellular matrix proteins and especially that of type I collagen; hence, these cells become directly fibrogenic. Second, the proliferation rate of HSCs increases, thereby effectively amplifying the number of fibrogenic cells present in the liver. Therefore, it is believed that effective treatment regimes aimed at reducing or inhibiting either the fibrogenic or proliferative responses of HSCs would reduce the deleterious effects of HSCs in the progression of fibrosis.
      PDGF is the most potent mitogenic factor for HSC proliferation (
      • Friedman S.L.
      ). Several signaling pathways have been described that regulate HSC proliferation involving cross-talk between the different signaling pathways (Fig. 12). PDGF treatment of HSCs activates Ras, followed by the sequential activation of Raf, MEK, and ERK in HSCs (
      • Pinzani M.
      • Marra F.
      • Carloni V.
      ,
      • Marshall C.J.
      ,
      • Gentilini A.
      • Marra F.
      • Gentilini P.
      • Pinzani M.
      ). Activation of ERK is observed following PDGF stimulation in culture-activated HSCs and in HSCs isolated from animals treated with a single dose of CCl4 (
      • Marra F.
      • Arrighi M.C.
      • Fazi M.
      • Caligiuri A.
      • Pinzani M.
      • Romanelli R.G.
      • Efsen E.
      • Laffi G.
      • Gentilini P.
      ). PDGF-induced ERK activation in human HSCs is followed by transient up-regulation of c-fos expression and AP-1 and STAT1 binding activities (
      • Marra F.
      • Pinzani M.
      • DeFranco R.
      • Laffi G.
      • Gentilini P.
      ,
      • Marra F.
      • Choudhury G.G.
      • Abboud H.E.
      • Reeves H.L.
      • Thompson M.G.
      • Dack C.L.
      • Burt A.D.
      • Day C.P.
      ). Blocking ERK activity with the pharmacological inhibitor PD98059 inhibits HSC proliferation along with AP-1 and STAT1 DNA-binding activities, thus supporting a role for ERK activation in HSC proliferation (
      • Marra F.
      • Arrighi M.C.
      • Fazi M.
      • Caligiuri A.
      • Pinzani M.
      • Romanelli R.G.
      • Efsen E.
      • Laffi G.
      • Gentilini P.
      ).
      Figure thumbnail gr12
      Figure 12Proliferative signaling pathways in HSCs. PDGF-R, PDGF receptor; ECM, extracellular matrix; PDK-1, phosphoinositide-dependent kinase-1; D3PPIs, D3-phosphorylated phosphoinositols; PtdIns, phosphatidylinositol; LY, LY294002; TAK, transforming growth factor-β-activated kinase-1.
      c-Jun and JNK are positive regulators of cell proliferation in several cell types, including HSCs (
      • Mitsui H.
      • Takuwa N.
      • Kurokawa K.
      • Exton J.H.
      • Takuwa Y.
      ,
      • Bost F.
      • McKay R.
      • Dean N.
      • Mercola D.
      ,
      • Schnabl B.
      • Bradham C.A.
      • Bennett B.L.
      • Manning A.M.
      • Stefanovic B.
      • Brenner D.A.
      ). Blocking JNK activity in quiescent HSCs or in culture-activated HSCs with a dominant-negative form of JNK prevents cell proliferation (
      • Schnabl B.
      • Bradham C.A.
      • Bennett B.L.
      • Manning A.M.
      • Stefanovic B.
      • Brenner D.A.
      ). Interestingly, inhibition of p38 MAPK in either quiescent or activated HSCs with the pharmacological inhibitor SB203580 actually increases cell proliferation, implying that activation of p38 inhibits HSC proliferation (
      • Schnabl B.
      • Bradham C.A.
      • Bennett B.L.
      • Manning A.M.
      • Stefanovic B.
      • Brenner D.A.
      ). An inhibitory role for p38 in cell proliferation has been shown in other cell types, perhaps by inhibiting cyclin D1 (
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Muller R.
      • Pouyssegur J.
      ). Indeed, culture-induced proliferation of HSCs is associated with increased mRNA and protein levels of cyclins D1, D2, and E (
      • Kawada N.
      • Ikeda K.
      • Seki S.
      • Kuroki T.
      ).
      PDGF also activates FAK, a member of the focal adhesion complex family (
      • Carloni V.
      • Pinzani M.
      • Giusti S.
      • Romanelli R.G.
      • Parola M.
      • Bellomo G.
      • Failli P.
      • Hamilton A.D.
      • Sebti S.M.
      • Laffi G.
      • Gentilini P.
      ). This complex interacts with extracellular matrix proteins through integrin interactions, providing a direct sensor to the integrity and composition of the extracellular environment. Following integrin activation, FAK is activated by autophosphorylation (
      • Kornberg L.
      • Earp H.S.
      • Parsons J.T.
      • Schaller M.
      • Juliano R.L.
      ). PDGF treatment of HSCs leads to FAK phosphorylation that is blocked by a dominant-negative form of Ras (
      • Carloni V.
      • Pinzani M.
      • Giusti S.
      • Romanelli R.G.
      • Parola M.
      • Bellomo G.
      • Failli P.
      • Hamilton A.D.
      • Sebti S.M.
      • Laffi G.
      • Gentilini P.
      ). Using a novel approach with an adenovirus expressing a dominant-negative form of FAK, FAK-CD, we have shown that PDGF treatment activates and requires FAK and Akt, a downstream target of FAK, for PDGF-induced HSC proliferation as assessed by [3H]thymidine incorporation. We also showed that FAK is positioned upstream of PI3K and Akt in the proliferative response based on PI3K activity and the phosphorylation status of Akt. FAK-CD overexpression also results in decreased DNA synthesis in endothelial cells (
      • Gilmore A.P.
      • Romer L.H.
      ). Overexpression of wild-type FAK increases cyclin D1 expression, decreases p21 expression, and accelerates G1-to-S phase transition in NIH3T3 cells (
      • Zhao J.H.
      • Reiske H.
      • Guan J.-L.
      ). Overexpression of dominant-negative FAK blocks cyclin D1 up-regulation, induces p21 expression, and inhibits DNA synthesis in human foreskin fibroblast cells (
      • Zhao J.H.
      • Reiske H.
      • Guan J.-L.
      ). Our data demonstrate that FAK plays a role in DNA synthesis and cell proliferation in activated HSCs.
      Integrin signaling through FAK has a role in regulating cell spreading, migration, and proliferation. Cultured fibroblasts isolated from FAK null mouse embryos, which demonstrate a lethal phenotype, show reduced cell motility (
      • Ilic D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Takeda N.
      • Sobue K.
      ). Overexpressing a dominant-negative form of FAK decreases cell spreading and migration in endothelial umbilical vein cells (
      • Gilmore A.P.
      • Romer L.H.
      ). Similarly, we demonstrated that inhibiting FAK blocks HSC migration. A role of FAK in cell adhesion has not been well established. Cultured FAK−/− cells do not show altered adhesion to fibronectin (
      • Ilic D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Takeda N.
      • Sobue K.
      ). Overexpressing FAK in Chinese hamster ovary cells has no effect on cell adhesion (
      • Cary L.A.
      • Chang J.F.
      • Guan J.-L.
      ). However, it was recently demonstrated that FAK is required for attachment of a human melanoma cell line (
      • Maung K.
      • Easty D.J.
      • Hill S.P.
      • Bennett D.C.
      ). We showed that FAK is required for attachment of activated HSCs. Therefore, activated HSCs show a similar requirement of FAK for cell attachment as shown in other nontransformed cells.
      The PI3K-Akt pathway is also activated following PDGF stimulation of HSCs (
      • Marra F.
      • Gentilini A.
      • Pinzani M.
      • Choudhury G.G.
      • Parola M.
      • Herbst H.
      • Dianzani M.U.
      • Laffi G.
      • Abboud H.E.
      • Gentilini P.
      ). Akt can be activated not only by growth factors that trigger tyrosine kinase activity or activation of cytokine receptors, but also by other signals that activate PI3K, including integrins, thus linking FAK activation to this signaling pathway (
      • Khwaja A.
      • Rodriguez-Viciana P.
      • Wennstrom S.
      • Warne P.H.
      • Downward J.
      ,
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ). Activated Akt is a key downstream survival factor that stimulates cell proliferation and inhibits apoptosis (
      • Madge L.A.
      • Pober J.S.
      ,
      • Kim A.L.
      • Khursigara G.
      • Sun X.
      • Franke T.F.
      • Chao M.V.
      ,
      • Bulik G.
      • Klippel A.
      • Weber M.J.
      ). Our studies confirm that activation of PI3K is important for HSC proliferation and chemotaxis (
      • Marra F.
      • Gentilini A.
      • Pinzani M.
      • Choudhury G.G.
      • Parola M.
      • Herbst H.
      • Dianzani M.U.
      • Laffi G.
      • Abboud H.E.
      • Gentilini P.
      ). A role for PI3K in HSC proliferation is supported by in vivo studies showing that, in rats, CCl4 treatment leads to autophosphorylation of the PDGF receptor and increased PI3K activity. Furthermore, inhibition of PI3K by wortmannin blocks mitogenesis in response to PDGF, supporting the involvement of this pathway in HSC proliferation (
      • Marra F.
      • Gentilini A.
      • Pinzani M.
      • Choudhury G.G.
      • Parola M.
      • Herbst H.
      • Dianzani M.U.
      • Laffi G.
      • Abboud H.E.
      • Gentilini P.
      ). We and others have shown similar results in HSC proliferation using a more specific PI3K inhibitor, LY294002 (
      • Gentilini A.
      • Marra F.
      • Gentilini P.
      • Pinzani M.
      ). However, we provide additional support showing that the phosphorylation of Akt at Ser473 is induced by serum or PDGF and is inhibited by LY294002 and that this inhibits cell proliferation. Inhibition of PI3K with wortmannin also reduces ERK activity and c-Fos mRNA levels, suggesting that cross-talk occurs between the PI3K and MAPK pathways following PDGF stimulation in HSCs (
      • Marra F.
      • Pinzani M.
      • DeFranco R.
      • Laffi G.
      • Gentilini P.
      ). The phosphorylation status of Akt correlated with an increase in HSC proliferation, α1(I) collagen mRNA levels, and type I collagen protein levels, demonstrating a regulatory role of Akt in these processes. Inhibition of both PI3K with LY294002 and Akt by adenovirus-mediated transduction of a dominant-negative form of Akt markedly reduced HSC proliferation. Transduction of HSCs with an adenovirus expressing a constitutively active form of Akt induced HSC proliferation in low serum conditions, thereby demonstrating that Akt is positioned downstream of PI3K and reaffirming its role in HSC proliferation.
      TGF-β, the most potent fibrogenic cytokine in the HSC, did not induce Akt phosphorylation in the HSC. This might be related to the antiproliferative property of TGF-β in HSCs (
      • Saile B.
      • Mathes N.
      • Knittel T.
      • Ramadori G.
      ). TGF-β failed to block Akt activation by PDGF when administered prior to, at the same time as, or following PDGF treatment. These results are similar to those of Chen et al. (
      • Chen R.H.
      • Su Y.H.
      • Chuang R.L.
      • Chang T.Y.
      ), who found that suppression of the antiproliferative effect of TGF-β in human hepatoma cells (Hep3B) is dependent on the PI3K-Akt pathway. This suggests that PDGF, as found in our study, may impair the antiproliferative effect of TGF-β.
      HSC activation produces a dramatic induction of type I collagen gene expression. We show here that PI3K is important in regulating α1(I) collagen gene expression. Inhibition of PI3K with LY294002 or Akt by adenovirus-mediated transduction of a dominant-negative form of Akt markedly reduced α1(I) collagen mRNA and type I collagen protein levels. Blocking PI3K with LY294002 inhibited collagen expression at both the mRNA and protein levels, similar to previously reported observations in lung fibroblasts (
      • Ricupero D.A.
      • Poliks C.F.
      • Rishikof D.C.
      • Cuttle K.A.
      • Kuang P.-P.
      • Goldstein R.H.
      ). However, mRNA levels in HSCs decreased at a much slower rate than in lung fibroblast cells. This may reflect different half-lives of the α1(I) collagen mRNA in the two cell types. Increased stability of α1(I) collagen mRNA occurs following HSC activation mediated through protein interactions within the 3′-untranslated region of the mRNA molecule and is an important mechanism for controlling α1(I) collagen gene expression in the HSC (
      • Stefanovic B.
      • Hellerbrand C.
      • Brenner D.A.
      ,
      • Lindquist J.N.
      • Marzluff W.F.
      • Stefanovic B.
      ). Interestingly, extracellular collagen protein levels were significantly reduced within 24 h after inhibiting PI3K activity, whereas the intracellular unprocessed collagen levels remained essentially unchanged even after 72 h of treatment. This may reflect an inhibition of collagen secretion following LY294002 treatment with an accumulation of unprocessed intracellular collagen. Because collagenase activity is primarily found extracellularly, there may be no mechanism to degrade the unprocessed intracellular collagen.
      In summary, we have shown that the FAK-PI3K-Akt signaling pathway is a critical pathway for PDGF-induce HSC proliferation. Fig. 12 represents current knowledge for intracellular signaling leading to HSC proliferation. In addition, we have demonstrated that PI3K is an important modulator of α1(I) collagen gene expression. Together, this information indicates that this signaling pathway may provide a potential therapeutic target to modulate the fibrogenic response in the liver.

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