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To whom correspondence should be addressed: Division of Digestive Diseases and Nutrition, Dept. of Medicine, University of North Carolina, CB 7038, Glaxo Bldg., Rm. 134, Chapel Hill, NC 27599. Tel.: 919-966-7469; Fax: 919-966-7468;
* This work was supported in part by National Institutes of Health Grants AA10459 (to R. A. R.) and DK34987 (to R. A. R. and D. A. B.) and National Institute on Alcohol Abuse and Alcoholism Alcohol Center Grant AA11605 (to R. A. R. and D. A. B.).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.
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.
hepatic stellate cell
platelet-derived growth factor
mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase
extracellular signal-regulated kinase
focal adhesion kinase
fetal bovine serum
multiplicity of infection
bovine serum albumin
transforming growth factor-β
signal transducer and activator of transcription
c-Jun N-terminal kinase
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 (
). 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 (
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 (
). 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 (
). 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 (
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.
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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
), 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 (
). 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 (
). 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.