Understanding the underlying molecular mechanisms of liver fibrosis is important to develop effective therapy. Herein, we show that focal-adhesion-kinse (FAK) plays a key role in promoting hepatic stellate cells (HSCs) activation in vitro and liver fibrosis progression in vivo. FAK activation is associated with increased expression of α-smooth muscle actin (α-SMA) and collagen in fibrotic live tissues. Transforming growth factor beta-1 (TGF-β1) induces FAK activation in a time and dose dependent manner. FAK activation precedes the α-SMA expression in HSCs. Inhibition of FAK activation blocks the α-SMA and collagen expression, and inhibits the formation of stress fibers in TGF-β1 treated HSCs. Furthermore, inhibition of FAK activation significantly reduces HSC migration and small GTPase activation, and induces apoptotic signaling in TGF-β1 treated HSCs. Importantly, FAK inhibitor attenuates liver fibrosis in vivo and significantly reduces collagen and α-SMA expression in an animal model of liver fibrosis. These data demonstrate that FAK plays an essential role in HSC activation and liver fibrosis progression, and FAK signaling pathway could be a potential target for liver fibrosis.

Myofibroblasts are effector cells in fibrotic disorders that synthesize and remodel the extracellular matrix (ECM). This study investigated the role of the Src kinase pathway in myofibroblast activation in vitro and fibrogenesis in vivo. The profibrotic cytokine, transforming growth factor β1 (TGF-β1), induced rapid activation of Src kinase, which led to myofibroblast differentiation of human lung fibroblasts. The Src kinase inhibitor AZD0530 (saracatinib) blocked TGF-β1–induced Src kinase activation in a dose-dependent manner. Inhibition of Src kinase significantly reduced α-smooth muscle actin (α-SMA) expression, a marker of myofibroblast differentiation, in TGF-β1–treated lung fibroblasts. In addition, the induced expression of collagen and fibronectin and three-dimensional collagen gel contraction were also significantly inhibited in AZD0530-treated fibroblasts. The therapeutic efficiency of Src kinase inhibition in vivo was tested in the bleomycin murine lung fibrosis model. Src kinase activation and collagen accumulation were significantly reduced in the lungs of AZD0530-treated mice when compared with controls. Furthermore, the total fibrotic area and expression of α-SMA and ECM proteins were significantly decreased in lungs of AZD0530-treated mice. These results indicate that Src kinase promotes myofibroblast differentiation and activation of lung fibroblasts. Additionally, these studies provide proof-of-concept for targeting the noncanonical TGF-β signaling pathway involving Src kinase as an effective therapeutic strategy for lung fibrosis.

Transforming growth factor (TGF)-β1 induces fibroblast transdifferentiation to myofibroblasts, a process that requires the involvement of integrin-mediated signaling and focal adhesion kinase (FAK). FAK-related non-kinase (FRNK) is known for its role in inhibiting integrin-mediated cell migration; however, its role in myofibroblast differentiation has not been defined. Here, we report that FRNK abrogates TGF-β1-induced myofibroblast differentiation in vitro and in vivo. TGF-β1 can induce α-smooth muscle actin (α-SMA) expression in the presence or absence of FAK; however, TGF-β1-induced α-SMA expression is reduced (∼73%) in FAK-deficient fibroblasts. Although both ERK and p38 MAPK activation is required for maximal TGF-β1-induced α-SMA expression, ERK is the major signaling intermediate in cells that express FAK. In contrast, p38 MAPK is the dominant mediator of TGF-β1-induced α-SMA expression in FAK-deficient cells. FRNK overexpression blocks TGF-β1-induced ERK or p38 MAPK activation in the presence, and surprisingly, in the absence of FAK. The loss of FRNK was tested in vivo during experimentally induced pulmonary fibrosis in mice. FRNK knock-out mice have a greater increase in α-SMA-expressing cells in response to a pulmonary fibrotic stimulus in vivo, as compared with congenic wild type mice. This is the first time that FRNK loss has been shown to modify the pathobiology in any animal disease model. Together, the data demonstrate that FRNK negatively regulates myofibroblast differentiation in vitro and in vivo. These data further suggest that modulation FRNK expression may be a novel avenue for therapeutic intervention in tissue fibrosis.

S100A4 expression is associated with poor clinical outcomes of patients with pancreatic cancer. The effects of loss or gain of S100A4 were examined in pancreatic cancer cell lines. S100A4 downregulation remarkably reduces cell migration and invasion, inhibits proliferation and induces apoptosis in pancreatic tumor cells. S100A4 downregulation results in significant cell growth inhibition and apoptosis in response to TGF-β1, supporting a non-canonical role of S100A4 in pancreatic cancer. The role of S100A4 in tumor progression was studied by using an orthotopic human pancreatic cancer xenograft mouse model. Tumor mass is remarkably decreased in animals injected with S100A4-deficient pancreatic tumor cells. P27^Kip1 expression and cleaved caspase-3 are increased, while cyclin E expression is decreased, in S100A4-deficient pancreatic tumors in vivo. S100A4-deficient tumors have lower expression of vascular endothelial growth factor, suggesting reduced angiogenesis. Biochemical assays revealed that S100A4 activates Src and focal adhesion kinase (FAK) signaling events and inhibition of both kinases is required to maximally block the tumorigenic potential of pancreatic cancer cells. These findings support that S100A4 plays an important role in pancreatic cancer progression in vivo and S100A4 promotes tumorigenic phenotypes of pancreatic cancer cells through the Src-FAK mediated dual signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease whose underlying molecular mechanisms are largely unknown. Herein, we show that focal adhesion kinase-related nonkinase (FRNK) plays a key role in limiting the development of lung fibrosis. Loss of FRNK function in vivo leads to increased lung fibrosis in an experimental mouse model. The increase in lung fibrosis is confirmed at the histological, biochemical, and physiological levels. Concordantly, loss of FRNK function results in increased fibroblast migration and myofibroblast differentiation and activation of signaling proteins that drive these phenotypes. FRNK-deficient murine lung fibroblasts also have an increased capacity to produce and contract matrix proteins. Restoration of FRNK expression in vivo and in vitro reverses these profibrotic phenotypes. These data demonstrate the multiple antifibrotic actions of FRNK. More important, FRNK expression is down-regulated in human IPF, and down-regulation of FRNK in normal human lung fibroblasts recapitulates the profibrotic phenotype seen in FRNK-deficient cells. The effect of loss and gain of FRNK in the experimental model, when taken together with its down-regulation in human IPF, suggests that FRNK acts as an endogenous negative regulator of lung fibrosis by repressing multiple profibrotic responses.