Urotensin upregulates transforming growth factor-β1 expression of asthma airway through ERK-dependent pathway

Airway smooth muscle cells (ASMCs) play a key role in the process of asthma airway remodeling. Urotensin II (UII) and transforming growth factor (TGF)-β are potent mitogens for ASMCs proliferation. The study was aimed to determine whether UII-upregulated TGF-β-mediated ASMCs proliferation and extracellular signal-regulated kinase (ERK) was required for such an effect. OVA-sensitized rats were challenged to induce asthma. Lung morphology and airway dynamic parameters were monitored. ASMCs from control and asthma rats were purified for the measurement of UII and TGF-β1 expression. In vitro experiments were conducted to determine the direct effect of UII on TGF-β1 expression by ASMCs. Finally, U0126, an ERK inhibitor was used to examine the role of ERK pathway in UII mediated TGF-β1 upregulation. We found that both UII and TGF-β1 were upregulated in asthma lung tissues. In vitro study on ASMCs further revealed that UII may render its effect on ASMCs cells through the upregulation of TGF-β1. Data also supported the conclusion that ERK pathway was required, but not sufficient in UII-induced TGF-β1 upregulation. The current study provides new evidence that UII is involved in the TGF-β mediated mitogenic effect on ASMCs. UII, at least partially, uses ERK pathway to render such effect.     

Role of c-Abl in directing metabolic versus mitogenic effects in insulin receptor signaling

c-Abl is a cytoplasmic tyrosine kinase involved in several signal transduction pathways. Here we report that c-Abl is involved also in insulin receptor signaling. Indeed, c-Abl tyrosine kinase is activated upon insulin stimulation. Inhibition of c-Abl tyrosine kinase by STI571 attenuates the effect of insulin on Akt/GSK-3beta phosphorylation and glycogen synthesis, and at the same time, it enhances the effect of insulin on ERK activation, cell proliferation, and migration. This effect of STI571 is specific to c-Abl inhibition, because it does not occur in Abl-null cells and is restored in c-Abl-reconstituted cells. Numerous evidences suggest that focal adhesion kinase (FAK) is involved in mediating this c-Abl effect. First, anti-phosphotyrosine blots indicate that c-Abl tyrosine kinase activation is concomitant with FAK dephosphorylation in response to insulin, whereas c-Abl inhibition is accompanied by FAK phosphorylation in response to insulin, a response similar to that observed with IGF-I. Second, the c-Abl effects on insulin signaling are not observed in cells devoid of FAK (FAK(-/-) cells). Taken together these results suggest that c-Abl activation by insulin, via a modification of FAK response, may play an important role in directing mitogenic versus metabolic insulin receptor signaling.     

Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal-regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with alpha4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.     

Urotensin-II-Mediated Reactive Oxygen Species Generation via NADPH Oxidase Pathway Contributes to Hepatic Oval Cell Proliferation

Urotensin II (UII), a somatostatin-like cyclic peptide, is involved in tumor progression due to its mitogenic effect. Our previous study demonstrated that UII and its receptor UT were up-regulated in human hepatocellular carcinoma (HCC), and exogenous UII promoted proliferation of human hepatoma cell line BEL-7402. Hepatic progenitor cell (HPCs) are considered to be one of the origins of liver cancer cells, but their relationship with UII remains unclear. In this work, we aimed to investigate the effect of UII on ROS generation in HPCs and the mechanisms of UII-induced ROS in promoting cell proliferation. Human HCC samples were used to examine ROS level and expression of NADPH oxidase. Hepatic oval cell line WB-F344 was utilized to investigate the underlying mechanisms. ROS level was detected by dihydroethidium (DHE) or 2’, 7’-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. For HCC samples, ROS level and expression of NADPH oxidase were significantly up-regulated. In vitro, UII also increased ROS generation and expression of NADPH oxidase in WB-F344 cells. NADPH oxidase inhibitor apocynin pretreatment partially abolished UII-increased phosphorylation of PI3K/Akt and ERK, expression of cyclin E/cyclin-dependent kinase 2. Cell cycle was then analyzed by flow cytometry and UII-elevated S phase proportion was inhibited by apocynin pretreatment. Finally, bromodeoxyuridine (Brdu) incorporation assay showed that apocynin partially abolished UII induced cell proliferation. In conclusion, this study indicates that UII-increased ROS production via the NADPH oxidase pathway is partially associated with activation of the PI3K/Akt and ERK cascades, accelerates G1/S transition, and contributes to cell proliferation. These results showed that UII plays an important role in growth of HPCs, which provides novel evidence for the involvement of HPCs in the formation and pathogenesis of HCC.     

Osteopontin stimulates vascular smooth muscle cell migration by inducing FAK phosphorylation and ILK dephosphorylation

Focal adhesion kinase (FAK) and integrin-linked kinase (ILK) are both involved in integrin-mediated cell migration. However, the molecular mechanism, and the relationship between FAK and ILK activity in signaling transduction for the osteopontin (OPN)-induced migration of vascular smooth muscle cells (VSMCs) remain unclear. Here, we show that treating VSMCs with OPN could result in the dissociation of FAK with ILK by inducing phosphorylation of the former and dephosphorylation of the latter. Furthermore, we demonstrate that FAK phosphorylation induced by OPN is coupled with ILK dephosphorylation. We also provide evidence that ILK acts downstream of FAK in the signaling pathways that mediate OPN-induced VSMC migration. These findings suggest that FAK phosphorylation and ILK dephosphorylation play important roles in VSMC migration induced by OPN.     

Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.     

PDGF and FGF induce focal adhesion kinase (FAK) phosphorylation at Ser-910: dissociation from Tyr-397 phosphorylation and requirement for ERK activation

A rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but very little is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with platelet-derived growth factor (PDGF) promoted a striking increase in the phosphorylation of FAK at Ser-910, as revealed by site-specific antibodies that recognized the phosphorylated state of this residue. FAK phosphorylation at Ser-910 could be distinguished from that at Tyr-397 in terms of dose-response relationships and kinetics. Furthermore, the selective phosphoinositide 3-kinase (PI 3-kinase) inhibitors wortmannin and LY 294002 abrogated FAK phosphorylation at Tyr-397 but did not interfere with PDGF-induced FAK phosphorylation at Ser-910. Conversely, treatment with U0126, a potent inhibitor of MEK-mediated ERK activation, prevented FAK phosphorylation at Ser-910 induced by PDGF but did not interfere with PDGF-induced FAK phosphorylation at Tyr-397. These results were extended using growth factors that either stimulate, fibroblast growth factor (FGF), or do not stimulate (insulin) the ERK pathway activation in Swiss 3T3 cells. FGF but not insulin promoted a striking ERK-dependent phosphorylation of FAK at Ser-910. Our results indicate that FAK phosphorylation at Tyr-397 and FAK phosphorylation at Ser-910 are induced in response to PDGF stimulation through different signaling pathways, namely PI 3-kinase and ERK, respectively.     

Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.     

Transactivation of the epidermal growth factor receptor mediates muscarinic stimulation of focal adhesion kinase in intestinal epithelial cells

We have previously shown that the Gq protein coupled receptor (GqPCR) agonist, carbachol (CCh), transactivates and recruits epidermal growth factor receptor (EGFr)-dependent signaling mechanisms in intestinal epithelial cells. Increasing evidence suggests that GqPCR agonists can also recruit focal adhesion-dependent signaling pathways in some cell types. Therefore, the aim of the present study was to investigate if CCh stimulates activation of the focal adhesion-associated protein, focal adhesion kinase (FAK), in intestinal epithelia and, if so, to examine the signaling mechanisms involved. Experiments were carried out on monolayers of T84 cells grown on permeable supports. CCh rapidly induced tyrosine phosphorylation of FAK in T84 cells. This effect was accompanied by phosphorylation of another focal adhesion-associated protein, paxillin, and association of FAK with paxillin. CCh-stimulated FAK phosphorylation was inhibited by a chelator of intracellular Ca2+, BAPTA/AM (20 microM), and was mimicked by thapsigargin (2 microM), which mobilizes intracellular Ca2+ in a receptor-independent fashion. CCh also induced association of FAK with the EGFr and FAK phosphorylation was attenuated by an EGFr inhibitor, tyrphostin AG1478, and an inhibitor of Src family kinases, PP2. The actin cytoskeleton disruptor, cytochalasin D (20 microM), abolished FAK phosphorylation in response to CCh but did not alter CCh-induced EGFr or ERK MAPK activation. In summary, these data demonstrate that agonists of GqPCRs have the ability to induce FAK activation in intestinal epithelial cells. GqPCR-induced FAK activation is mediated by via a pathway involving transactivation of the EGFr and alterations in the actin cytoskeleton.     

Cellular characterization of a novel focal adhesion kinase inhibitor

Focal adhesion kinase (FAK) is a member of a family of non-receptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC(50) of 4 nM. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr(397) with an IC(50) of 30-100 nM. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr(397) phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials.     

Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation

The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner.     

RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes

Focal adhesion kinase (FAK) has been shown to be activated in cardiac myocytes exposed to mechanical stress. However, details of how mechanical stimuli induce FAK activation are unknown. We investigated whether signaling events mediated by the RhoA/Rho-associated coiled coil-containing kinase (ROCK) pathway are involved in regulation of stretch-induced FAK phosphorylation at Tyr(397) in neonatal rat ventricular myocytes (NRVMs). Immunostaining showed that RhoA localized to regions of myofilaments alternated with phalloidin (actin) staining. The results of coimmunoprecipitation assays indicated that FAK and RhoA are associated in nonstretched NRVMs, but cyclic stretch significantly reduced the amount of RhoA recovered from anti-FAK immunoprecipitates. Cyclic stretch induced rapid and sustained (up to 2 h) increases in phosphorylation of FAK at Tyr(397) and ERK1/2 at Thr(202)/Tyr(204). Blockade of RhoA/ROCK signaling by pharmacological inhibitors of RhoA (Clostridium botulinum C3 exoenzyme) or ROCK (Y-27632, 10 micromol/l, 1 h) markedly attenuated stretch-induced FAK and ERK1/2 phosphorylation. Similar effects were observed in cells treated with the inhibitor of actin polymerization cytochalasin D. Transfection of NRVMs with RhoA antisense oligonucleotide attenuated stretch-induced FAK and ERK1/2 phosphorylation and expression of beta-myosin heavy chain mRNA. Similar results were seen in cells transfected with FAK antisense oligonucleotide. These findings demonstrate that RhoA/ROCK signaling plays a crucial role in stretch-induced FAK phosphorylation, presumably by coordinating upstream events operationally linked to the actin cytoskeleton.     

p38 MAP kinase mediates platelet-derived growth factor-stimulated migration of hepatic myofibroblasts

Although the migration of hepatic myofibroblasts (HMFs) contributes to the development of fibrosis, the signals regulating migration of these cells are poorly understood. In this study, we tested the hypothesis that HMF migration is stimulated by platelet-derived growth factor-BB (PDGF-BB) through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) signaling pathways. This hypothesis was addressed by directly visualizing the migration of cultured human HMFs into a wound. PDGF-BB stimulated membrane ruffling, migration, and proliferation. PDGF-BB also induced activation of p38 MAP kinase, its downstream effector, heat shock protein (HSP) 27, ERK 1 and ERK 2, and p125 focal adhesion kinase (FAK). Selective antagonism of p38 MAP kinase blocked PDGF-BB-stimulated HSP 27 phosphorylation, membrane ruffling, and migration, but did not alter PDGF-BB-induced proliferation. Selective antagonism of ERK kinase inhibited PDGF-BB-induced ERK phosphorylation and proliferation, but did not affect PDGF-BB-stimulated migration. Concentrations of PDGF-BB that stimulated migration and proliferation did not influence myosin-dependent contractility. Neither selective inhibition of p38 MAP kinase nor ERKs altered PDGF-BB-induced activation of FAK. In conclusion, these results provide novel evidence indicating that (1) HMF migration is stimulated by PDGF-BB through the regulation of membrane ruffling by a p38 MAP kinase signaling pathway, (2) whereas p38 MAP kinase mediates PDGF-BB-stimulated migration, but not proliferation, ERKs mediate PDGF-induced proliferation, but not migration, and (3) increases in myosin-dependent contractility are not required for PDGF-BB-stimulated migration.     

FAK和ILK介导骨桥蛋白诱导的血管平滑肌细胞黏附和迁移

黏着斑激酶（FAK）和整合素偶联激酶（ILK）是整合素信号途径中的重要信号转导分子,为阐明两者在血管平滑肌细胞（VSMC）黏附和迁移中的作用,以骨桥蛋白（OPN）作为VSMC黏附和迁移的诱导剂,检测其对FAK和ILK磷酸化以及对两者之间结合的影响.在此基础上,用FAK磷酸化特异性抑制剂黏着斑相关非激酶（FRNK）或ILK反义RNA分别阻断FAK磷酸化或ILK表达,进一步探讨两者在VSMC黏附和迁移中所起的作用.结果显示,OPN诱导可促进FAK磷酸化,诱导10 min后FAK磷酸化水平升高到对照组的2.4倍;与此同时,ILK的磷酸化受到抑制,30 min降至对照细胞的44.6％.OPN诱导FAK磷酸化的同时使FAK与ILK的结合减少.外源性FRNK在VSMC中的过表达显著降低FAK的磷酸化水平,促进ILK磷酸化和FAK与ILK之间的结合,抑制VSMC的黏附和迁移.用ILK反义RNA抑制ILK表达使VSMC在OPN上的黏附增加1.8倍,迁移细胞数降低45.5％.结果提示,FAK和ILK介导OPN诱导的VSMC黏附和迁移过程,两者通过对同一刺激信号产生不同的磷酸化变化而对VSMC的黏附和迁移产生不同的影响.     

Signaling through focal adhesion kinase

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130^Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK⁻) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK⁻ cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.     

Src and focal adhesion kinase mediate mechanical strain-induced proliferation and ERK1/2 phosphorylation in human H441 pulmonary epithelial cells

Pulmonary epithelial cells are exposed to repetitive deformation during physiological breathing and mechanical ventilation. Such deformation may influence pulmonary growth, development, and barotrauma. Although deformation stimulates proliferation and activates extracellular signal-regulated kinases (ERK1/2) in human pulmonary epithelial H441 cells, the upstream mechanosensors that induce ERK activation are poorly understood. We investigated whether c-Src or focal adhesion kinase (FAK) mediates cyclic mechanical strain-induced ERK1/2 activation and proliferation in human pulmonary epithelial (NCI-H441) cells. The H441 and A549 cells were grown on collagen I-precoated membranes and were subjected to an average 10% cyclic mechanical strain at 20 cycles/min. Cyclic strain activated Src within 2 min by increasing phosphorylation at Tyr⁴¹⁸, followed by rapid phosphorylation of FAK at Tyr³⁹⁷ and Tyr⁵⁷⁶ and ERK1/2 at Thr²⁰²/Tyr²⁰⁴ (n = 5, P < 0.05). Twenty-four (A549 cells) and 24–72 h (H441 cells) of cyclic mechanical strain increased cell numbers compared with static culture. Twenty-four hours of cyclic strain also increased H441 FAK, Src, and ERK phosphorylation without affecting total FAK, Src, or ERK protein. The mitogenic effect was blocked by Src (10 µmol/l PP2 or short interfering RNA targeted to Src) or MEK (50 µmol/l PD-98059) inhibition. PP2 also blocked strain-induced phosphorylation of FAK-Tyr⁵⁷⁶ and ERK-Thr²⁰²/Tyr²⁰⁴ but not FAK-Tyr³⁹⁷. Reducing FAK by FAK-targeted short interfering RNA blocked mechanical strain-induced mitogenicity and significantly attenuated strain-induced ERK activation but not strain-induced Src phosphorylation. Together, these results suggest that repetitive mechanical deformation induced by ventilation supports pulmonary epithelial proliferation by a pathway involving Src, FAK, and then ERK signaling. extracellular signal-regulated kinase; mitogenic; signaling     

Divergent signaling pathways link focal adhesion kinase to mitogen-activated protein kinase cascades. Evidence for a role of paxillin in c-Jun NH(2)-terminal kinase activation.

Stimulation of a number of cell surface receptors, including integrins and G protein-coupled receptors, results in the activation of a non-receptor tyrosine kinase known as focal adhesion kinase (FAK). In turn, this kinase is believed to play a critical role in signaling to intracellular kinase cascades controlling gene expression such as extracellular signal-regulated kinases (ERKs), by a yet poorly defined mechanism. Furthermore, whether this tyrosine kinase also mediates the activation of other mitogen-activated protein kinase family members, such as c-Jun NH(2)-terminal kinases (JNKs), is still unclear. We show here that the activation of FAK by anchoring to the cell membrane is itself sufficient to stimulate potently both ERK and JNK. These effects were found to be phosphatidylinositol 3-kinase-independent, as FAK effectively stimulated Akt, and wortmannin suppressed Akt but not ERK or JNK activation. As previously reported by others, activation of ERK correlated with the ability of FAK to induce tyrosine phosphorylation of Shc. Surprisingly, however, stimulation of JNK was not dependent on the kinase activity of FAK or on the ability to induce tyrosine phosphorylation of FAK substrates. Instead, we provide evidence that FAK may stimulate JNK through a novel pathway involving the recruitment of paxillin to the plasma membrane and the subsequent activation of a biochemical route dependent on small GTP-binding proteins of the Rho family.     

Focal adhesion kinase controls aggressive phenotype of androgen-independent prostate cancer

Overexpression of focal adhesion kinase (FAK) has been well correlated with tumor development and/or the maintenance of tumor phenotype. In addition, inappropriate activation of the extracellular regulated kinase (ERK) signaling pathway is common to many human cancers. In the present study, we investigated the interplay between FAK and ERK in androgen-independent prostate cancer cells (PC3 and DU145 cells). We observed that suppression of FAK expression using small interfering RNA-mediated knockdown decreased the clonogenic activity, whereas overexpression of FAK increased it. We also observed that detachment of PC3 and DU145 cells from their substrate induced tyrosine phosphorylation of FAK. ERK knockdown diminished FAK protein levels and tyrosine phosphorylation of FAK as well as FAK promoter-reporter activity. We also tested the effect of MEK inhibitors and small interfering RNA-mediated knockdown of ERK1 and/or ERK2 on cell proliferation, invasiveness, and growth in soft agar of PC3 and DU145 cells. Inhibition of ERK signaling grossly impaired clonogenicity as well as invasion through Matrigel. However, inhibition of ERK signaling resulted in only a modest inhibition of 3H-thymidine incorporation and no effect on overall viability of the cells or increased sensitivity to anoikis. Taken together, these data show, for the first time, a requirement for FAK in aggressive phenotype of prostate cancer cells; reveal interdependence of FAK and ERK1/2 for clonogenic and invasive activity of androgen-independent prostate cancer cells; suggest a role for ERK regulation of FAK in substrate-dependent survival; and show for the first time, in any cell type, the regulation of FAK expression by ERK signaling pathway.     

Src介导骨桥蛋白诱导的血管平滑肌细胞黏附和迁移过程

为阐明酪氨酸激酶Src在整合素被骨桥蛋白(OPN)激活所触发的细胞黏附和迁移信号途径中所起的作用,应用Src特异性抑制剂PP2阻断Src,观察OPN诱导的血管平滑肌细胞(VSMC)黏附和迁移活性的改变,并利用免疫沉淀检查PP2对整合素下游信号分子黏着斑激酶(FAK)和整合素偶联激酶(ILK)磷酸化及其相互作用的影响。结果显示,PP2可明显抑制OPN诱导的VSMC黏附和伤口愈合(黏附和迁移活性分别为对照组的76.6%和33.8%);OPN可显著诱导FAK磷酸化(磷酸化水平达对照组的1.9倍),促进ILK去磷酸化,并使FAK与ILK的结合减少(降至对照组的46.4%)。10μmol/LPP2可明显抑制OPN诱导的FAK磷酸化、拮抗OPN诱导对ILK的去磷酸化作用、促进FAK与ILK之间的结合。研究结果表明,Src作为OPN-整合素-FAK信号途径中的信号分子,通过影响FAK和ILK的磷酸化以及两者之间的相互作用来调节VSMC的黏附和迁移活性。