The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression

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-beta, an inhibitor of HSC proliferation, did not block PDGF-induced Akt phosphorylation, suggesting that transforming growth factor-beta mediates its antiproliferative effect downstream of Akt. Expression of type I collagen protein and alpha1(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.     

v-Crk activates the phosphoinositide 3-kinase/AKT pathway by utilizing focal adhesion kinase and H-Ras

v-Crk, an oncogene product of avian sarcoma virus CT10, efficiently transforms chicken embryo fibroblasts (CEF). We have recently reported that constitutive activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway plays a critical role in the v-Crk-induced transformation of CEF. In the present study we investigated the molecular mechanism by which v-Crk activates the PI3K/AKT pathway. First, we found that v-Crk promotes the association of the p85 regulatory subunit of PI3K with focal adhesion kinase (FAK) by inducing the phosphorylation of the Y397 residue in FAK. This FAK phosphorylation needs activation of the Src family tyrosine kinase(s) for which the v-Crk SH2 domain is responsible. v-Crk was unable to activate the PI3K/AKT pathway in FAK-null cells, indicating the functional importance of FAK. In addition, we found that H-Ras is also required for the activation of the PI3K/AKT pathway. The v-Crk-induced activation of AKT was greatly enhanced by the overexpression of H-Ras or its guanine nucleotide exchange factor mSOS, which binds to the v-Crk SH3 domain, whereas a dominant-negative mutant of H-Ras almost completely suppressed this activation. Furthermore, we showed that v-Crk stimulates the interaction of H-Ras with the Ras binding domain in the PI3K p110 catalytic subunit. Our data indicated that the v-Crk-induced activation of PI3K/AKT pathway was cooperatively achieved by two distinct interactions. One is the interaction of p85 with tyrosine-phosphorylated FAK promoted by the v-Crk SH2 domain, and another is the interaction of p110 with H-Ras dictated by the v-Crk SH3 domain.     

Survivin expression induced by endothelin-1 promotes myofibroblast resistance to apoptosis

Fibrosis of the lungs and other organs is characterized by the accumulation of myofibroblasts, effectors of wound-repair that are responsible for the deposition and organization of new extracellular matrix (ECM) in response to tissue injury. During the resolution phase of normal wound repair, myofibroblast apoptosis limits the continued deposition of ECM. Mounting evidence suggests that myofibroblasts from fibrotic wounds acquire resistance to apoptosis, but the mechanisms regulating this resistance have not been fully elucidated. Endothelin-1 (ET-1), a soluble peptide strongly associated with fibrogenesis, decreases myofibroblast susceptibility to apoptosis through activation of phosphatidylinositol 3′-OH kinase (PI3K)/AKT. Focal adhesion kinase (FAK) also promotes myofibroblast resistance to apoptosis through PI3K/AKT-dependent and -independent mechanisms, although the role of FAK in ET-1 mediated resistance to apoptosis has not been explored. The goal of this study was to investigate whether FAK contributes to ET-1 mediated myofibroblast resistance to apoptosis and to examine potential mechanisms downstream of FAK and PI3K/AKT by which ET-1 regulates myofibroblast survival. Here, we show that ET-1 regulates myofibroblast survival by Rho/ROCK-dependent activation of FAK. The anti-apoptotic actions of FAK are, in turn, dependent on activation of PI3K/AKT and the subsequent increased expression of Survivin, a member of the inhibitor of apoptosis protein (IAP) family. Collectively, these studies define a novel mechanism by which ET-1 promotes myofibroblast resistance to apoptosis through upregulation of Survivin.     

Endothelin-1 promotes MMP-13 production and migration in human chondrosarcoma cells through FAK/PI3K/Akt/mTOR pathways

Tumor malignancy is associated with several cellular properties including proliferation and ability to metastasize. Endothelin-1 (ET-1) the most potent vasoconstrictor plays a crucial role in migration and metastasis of human cancer cells. We found that treatment of human chondrosarcoma (JJ012 cells) with ET-1 increased migration and expression of matrix metalloproteinase (MMP)-13. ET-1-mediated cell migration and MMP-13 expression were reduced by pretreatment with inhibitors of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), as well as the NF-κB inhibitor and the IκB protease inhibitor. In addition, ET-1 treatment induced phosphorylation of FAK, PI3K, AKT, and mTOR, and resulted in increased NF-κB-luciferase activity that was inhibited by a specific inhibitor of PI3K, Akt, mTOR, and NF-κB cascades. Taken together, these results suggest that ET-1 activated FAK/PI3K/AKT/mTOR, which in turn activated IKKα/β and NF-κB, resulting in increased MMP-13 expression and migration in human chondrosarcoma cells.     

Focal adhesion kinase is upstream of phosphatidylinositol 3-kinase/Akt in regulating fibroblast survival in response to contraction of type I collagen matrices via a beta 1 integrin viability signaling pathway

The beta(1) integrin, functioning as a mechanoreceptor, senses a mechanical stimulus generated during collagen matrix contraction and down-regulates the phosphatidylinositol 3-kinase (PI3K)/Akt survival signal triggering apoptosis. The identities of integrin-associated signal molecules in the focal adhesion complex that are responsible for propagating beta(1) integrin viability signals in response to collagen matrix contraction are not known. Here we show that in response to collagen contraction focal adhesion kinase (FAK) is dephosphorylated. In contrast, enforced activation of beta(1) integrin by anti-beta(1) integrin antibody, which protects fibroblasts from apoptosis, preserves FAK phosphorylation. We demonstrate that ligation of beta(1) integrin by type I collagen or by enforced activation of beta(1) integrin by antibody promotes phosphorylation of FAK, p85 subunit of PI3K, and serine 473 of Akt. Wortmannin inhibited Akt but not FAK phosphorylation in response to enforced activation of beta(1) integrin by antibody. Blocking FAK by pharmacologic inhibition or by dominant negative FAK attenuated phosphorylation of p85 subunit of PI3K and Akt. Dominant negative FAK augmented fibroblast apoptosis during collagen contraction, and this was associated with diminished Akt activity. Constitutively active FAK augmented levels of p85 subunit of PI3K and Akt phosphorylation, and fibroblasts were protected from apoptosis. Our data identify a novel role for FAK, functioning upstream of PI3K/Akt, in transducing a beta(1) integrin viability signal in collagen matrices.     

Density- and serum-dependent regulation of the Reck tumor suppressor in mouse embryo fibroblasts

Reck is a membrane-anchored glycoprotein identified as a transformation suppressor. Accumulating evidence indicates that Reck negatively regulates a wide spectrum of matrix metalloproteinases and is commonly down-regulated in a variety of malignant solid tumors. Physiological cues that regulate Reck expression, however, remained unknown. In this study, we found that Reck expression was up-regulated at high cell density, low serum, or after treatment with some kinase inhibitors, such as PP2 (Src inhibitor), LY294002 (PI3-kinase inhibitor), and PF573228 (FAK inhibitor), in mouse embryo fibroblasts. Curve fitting indicated that the levels of Reck protein and Reck mRNA are quadratic in the cell density. Other factors, including serum, extracellular matrix components (type I collagen and fibronectin), the kinase inhibitors, and some of their oncogenic targets (v-Src and PIK3CA mutants), modify the shape of the quadratic curve. Comparison of these modifications implicated Src in Reck down-regulation under sparse conditions, PI3-kinase in serum-induced Reck down-regulation, and FAK in Reck down-regulation at high cell density. Fibronectin and type I collagen down-regulated Reck, supporting the role of integrin-FAK signaling in Reck down-regulation at high cell density. Our study has revealed multiple signaling pathways impinging on Reck in cultured mouse embryo fibroblasts and sets a foundation for future studies to find effective Reck inducers of potential value in cancer therapy.     

Metastatic function of BMP-2 in gastric cancer cells: the role of PI3K/AKT, MAPK, the NF-κB pathway, and MMP-9 expression

Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-κB, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of IκBα and the nuclear translocation/activation of NF-κB. Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-κB. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-κB inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-κB and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.     

Crosstalk between Raf/MEK/ERK and PI3K/AKT in suppression of Bax conformational change by Grp75 under glucose deprivation conditions

During glucose deprivation (GD)-induced cellular stress, the molecular chaperone glucose-regulated protein 75 (Grp75)/Mortalin/PBP74/mtHSP70 (hereafter termed “Grp75”) plays an important role in the suppression of apoptosis by inhibiting the Bax conformational change that delays the release of cytochrome c. The molecular pathways by which it carries out these functions are still unclear. We hypothesize that the anti-apoptotic effect by the overexpression of Grp75 was through the signal of AKT activated by classic phosphoinositide 3-kinase (PI3K) and also involved PI3K-independent pathways. Using the PC12 cell GD model, we demonstrated a novel mechanism of Grp75 activating AKT, which may be PI3K independent and associated with Raf/MEK (mitogen-activated protein kinase/ERK kinase)/ERK signaling. The PI3K inhibitor LY294002 did not influence the activation of AKT by the Grp75 overexpression under GD; however, the MEK inhibitor U0126 dramatically inhibited AKT phosphorylation in the same assay. In addition to the PI3K/AKT signal pathway, Grp75 overexpression also inhibited the Bax conformational change through the Raf/MEK/ERK signal pathway. In conclusion, Grp75 overexpression in activating AKT can be PI3K independent and associated with Raf/MEK/ERK signaling under GD. At the same time, PI3K may also crosstalk with Raf-1, in which the prosurvival signal of PI3K maintains the expression of Raf-1. The activated AKT and extracellular signal-regulated protein kinases 1 and 2 by Grp75 inhibited the Bax conformational change and subsequent apoptosis.     

PTEN improve renal fibrosis in vitro and in vivo through inhibiting FAK/AKT signaling pathway

Renal fibrosis, the ultimate common pathway of progressive nephropathy, is characterized by excess accumulation and deposition of extracellular matrix (ECM) within the renal interstitium and glomeruli, finally resulting in end-stage kidney failure. TGFβ1 is not only abnormally increased during fibrosis but also involved in ECM induction and accumulation. Based on the bioinformative analyses, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and focal adhesion kinase (FAK) signaling pathway might be involved in TGFβ1 functions on renal fibrosis development. In the present study, fibrosis was induced in HK-2 cells using TGFβ1 and PTEN expression was significantly suppressed by 24 or 48 hours TGFβ1 treatment. PTEN overexpression in HK-2 cells improved TGFβ1-induced fibrosis within α-SMA and E-cadherin. According to the KEGG signaling pathway annotation analyses on microarray profiles (GSE23338 and GSE20247) and immunoblotting validation, FAK signaling might be involved in PTEN functions in TGFβ1-induced fibrosis. PTEN overexpression significantly inhibited TGFβ1- or unilateral ureteral obstruction (UUO)-induced FAK signaling pathway activation both in vitro and in vivo; more importantly, PTEN silence enhanced TGFβ1- or UUO-induced fibrosis, while FAK inhibitor PF567721 significantly reversed the effects of PTEN silence, indicating that PTEN exerted its effects on TGFβ1- and UUO-induced fibrotic development in vitro and in vivo via inhibiting FAK signaling pathway. In summary, these findings indicate that PTEN could improve cellular fibrotic changes and renal fibrosis via inhibiting FAK/AKT signaling pathway. Restoring PTEN expression to target FAK/AKT signaling pathway might be a potent strategy for renal fibrosis treatment.     

Periostin Induces Intracellular Cross-talk between Kinases and Hyaluronan in Atrioventricular Valvulogenesis

Periostin (PN), a novel fasciclin-related matricellular protein, has been implicated in cardiac development and postnatal remodeling, but the mechanism remains unknown. We examined the role of PN in mediating intracellular kinase activation for atrioventricular valve morphogenesis using well defined explant cultures, gene transfection systems, and Western blotting. The results show that valve progenitor (cushion) cells secrete PN into the extracellular matrix, where it can bind to INTEGRINs and activate INTEGRIN/focal adhesion kinase signaling pathways and downstream kinases, PI3K/AKT and ERK. Functional assays with prevalvular progenitor cells showed that activating these signaling pathways promoted adhesion, migration, and anti-apoptosis. Through activation of PI3K/ERK, PN directly enhanced collagen expression. Comparing PN-null to WT mice also revealed that expression of hyaluronan (HA) and activation of hyaluronan synthase-2 (Has2) are also enhanced upon PN/INTEGRIN/focal adhesion kinase-mediated activation of PI3K and/or ERK, an effect confirmed by the reduction of HA synthase-2 in PN-null mice. We also identified in valve progenitor cells a potential autocrine signaling feedback loop between PN and HA through PI3K and/or ERK. Finally, in a three-dimensional assay to simulate normal valve maturation in vitro, PN promoted collagen compaction in a kinase-dependent fashion. In summary, this study provides the first direct evidence that PN can act to stimulate a valvulogenic signaling pathway.     

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

Focal adhesion kinase negatively regulates neuronal insulin resistance

Focal adhesion kinase (FAK), a non-receptor protein kinase, is known to be a phosphatidyl inositol 3-kinase (PI3K) pathway activator and thus widely implicated in regulation of cell survival and cancer. In recent years FAK has also been strongly implicated as a crucial regulator of insulin resistance in peripheral tissues like skeletal muscle and liver, where decrease in its expression/activity has been shown to lead to insulin resistance. However, in the present study we report an altogether different role of FAK in regulation of insulin/PI3K signaling in neurons, the post-mitotic cells. An aberrant increase in FAK tyrosine phosphorylation was observed in insulin resistant Neuro-2a (N2A) cells. Downregulation of FAK expression utilizing RNAi mediated gene silencing in insulin resistant N2A cells completely ameliorated the impaired insulin/PI3K signaling and glucose uptake. FAK silencing in primary cortical neurons also showed marked enhancement in glucose uptake. The results thus suggest that in neurons FAK acts as a negative regulator of insulin/PI3K signaling. Interestingly, the available literature also demonstrates cell-type specific functions of FAK in neurons. FAK that is well known for its cell survival effects has been shown to be involved in neurodegeneration. Along with these previous reports, present findings highlight a novel and critical role of FAK in neurons. Moreover, as this implicates differential regulation of insulin/PI3K pathway by FAK in peripheral tissues and neuronal cells, it strongly suggests precaution while considering FAK modulators as possible therapeutics.     

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

Involvement of PI3K and MAPK signaling in bcl-2-induced vascular endothelial growth factor expression in melanoma cells

We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1alpha expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways.     

p38 and EGF receptor kinase-mediated activation of the phosphatidylinositol 3-kinase/Akt pathway is required for Zn2+-induced cyclooxygenase-2 expression

Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus and cell type specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction of COX-2 expression. This study aims to elucidate the role of intracellular signaling pathways in Zn2+-induced COX-2 expression in human bronchial epithelial cells. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) potently block Zn2+-induced COX-2 mRNA and protein expression. Overexpression of adenoviral constructs encoding dominant-negative Akt kinase downstream of PI3K or wild-type phosphatase and tensin homolog deleted on chromosome 10, an important PI3K phosphatase, suppresses COX-2 mRNA expression induced by Zn2+. Zn2+ exposure induces phosphorylation of the tyrosine kinases, including Src and EGF receptor (EGFR), and the p38 mitogen-activated protein kinase. Blockage of these kinases results in inhibition of Zn2+-induced Akt phosphorylation as well as COX-2 protein expression. Overexpression of dominant negative p38 constructs suppresses Zn2+-induced increase in COX-2 promoter activity. In contrast, the c-Jun NH2-terminal kinase and the extracellular signal-regulated kinases have minimal effect on Akt phosphorylation and COX-2 expression. Inhibition of p38, Src, and EGFR kinases with pharmacological inhibitors markedly reduces Akt phosphorylation induced by Zn2+. However, the PI3K inhibitors do not show inhibitory effects on p38, Src, and EGFR. These data suggest that p38 and EGFR kinase-mediated Akt activation is required for Zn2+-induced COX-2 expression and that the PI3K/Akt signaling pathway plays a central role in this event.