An altered fibronectin matrix induces anoikis of human squamous cell carcinoma cells by suppressing integrin alpha v levels and phosphorylation of FAK and ERK

Fibronectin regulates many cellular processes, including migration, proliferation, differentiation, and survival. Previously, we showed that squamous cell carcinoma (SCC) cell aggregates escape suspension-induced, p53-mediated anoikis by engaging in fibronectin-mediated survival signals through focal adhesion kinase (FAK). Here we report that an altered matrix, consisting of a mutated, nonfunctional high-affinity heparin-binding domain and the V region of fibronectin (V⁺H⁻), induced anoikis in human SCC cells; this response was blocked by inhibitors of caspase-8 and caspase-3. Anoikis was mediated by downregulation of integrin alpha v in a panel of SCC cells and was shown to be proteasome-dependent. Overexpression of integrin alpha v or FAK inhibited the increase in caspase-3 activation and apoptosis, whereas suppression of alpha v or FAK triggered a further significant increase in apoptosis, indicating that the apoptosis was mediated by suppression of integrin alpha v levels and dephosphorylation of FAK. Treatment with V⁺H⁻ decreased the phosphorylation of extracellular signal-regulated kinase (ERK) 1 and 2, and direct activation of ERK by constitutively active MEK1, an ERK kinase, increased ERK1 and ERK2 phosphorylation and inhibited the increase in apoptosis induced by V⁺H⁻. ERK acted downstream from alpha v and FAK signals, since alpha v and FAK overexpression inhibited both the decrease in ERK phosphorylation and the increase in anoikis triggered by V⁺H⁻. These findings provide evidence that mutations in the high-affinity heparin-binding domain in association with the V region of fibronectin, or altered fibronectin matrices, induce anoikis in human SCC cells by modulating integrin alpha v-mediated phosphorylation of FAK and ERK.     

Brain‐derived neurotrophic factor induces migration of endothelial cells through a TrkB–ERK–integrin αVβ3–FAK cascade

Brain‐derived neurotrophic factor (BDNF) promotes the regeneration of periodontal tissue. Since angiogenesis is important for tissue regeneration, investigating effect of BDNF on endothelial cell function may help to reveal its mechanism, whereby, BDNF promotes periodontal tissue regeneration. In this study, we examined the influence of BDNF on migration in human microvascular endothelial cells (HMVECs), focusing on the effects on extracellular signal‐regulated kinase (ERK), integrin α_Vβ₃, and focal adhesion kinase (FAK). The migration of endothelial cells was assessed with a modified Boyden chamber and a wound healing assay. The expression of integrin α_Vβ₃ and the phosphorylation of ERK and FAK were analyzed by immunoblotting and immunofluorescence microscopy. BDNF (25 ng/ml) induced cell migration. PD98059, an ERK inhibitor, K252a, a specific inhibitor for TrkB, a high affinity receptor of BDNF, and an anti‐integrin α_Vβ₃ antibody suppressed the BDNF‐induced migration. BDNF increased the levels of integrin α_Vβ₃ and phosphorylated ERK1/2 and FAK. The ERK inhibitor and TrkB inhibitor also reduced levels of integrin α_Vβ₃ and phosphorylated FAK. We propose that BDNF stimulates endothelial cell migration by a process involving TrkB/ERK/integrin α_Vβ₃/FAK, and this may help to enhance the regeneration of periodontal tissue. J. Cell. Physiol. 227: 2123–2129, 2012. © 2011 Wiley Periodicals, Inc.     

Membrane-type 1 matrix metalloproteinase modulates focal adhesion stability and cell migration

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in extracellular matrix-induced cell migration and the activation of extracellular signal-regulated kinase (ERK). We showed here that transfection of the MT1-MMP gene into HeLa cells promoted fibronectin-induced cell migration, which was accompanied by fibronectin degradation and reduction of stable focal adhesions, which function as anchors for actin-stress fibers. MT1-MMP expression attenuated integrin clustering that was induced by adhesion of cells to fibronectin. The attenuation of integrin clustering was abrogated by MT1-MMP inhibition with a synthetic MMP inhibitor, BB94. When cultured on fibronectin, HT1080 cells, which endogenously express MT1-MMP, showed so-called motile morphology with well-organized focal adhesion formation, well-oriented actin-stress fiber formation, and the lysis of fibronectin through trails of cell migration. Inhibition of endogenous MT1-MMP by BB94 treatment or expression of the MT1-MMP carboxyl-terminal domain, which negatively regulates MT1-MMP activity, resulted in the suppression of fibronectin lysis and cell migration. BB94 treatment promoted stable focal adhesion formation concomitant with enhanced phosphorylation of tyrosine 397 of focal adhesion kinase (FAK) and reduced ERK activation. These results suggest that lysis of the extracellular matrix by MT1-MMP promotes focal adhesion turnover and subsequent ERK activation, which in turn stimulates cell migration.     

The Ras-related Protein,Rap1A,Mediates Thrombin-stimulated,Integrin-dependent Glioblastoma Cell Proliferation and Tumor Growth

Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β₁ integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β₁ integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β₁ integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo.     

Involvement of caveolin‐1 in fibronectin‐induced mouse embryonic stem cell proliferation: Role of FAK,RhoA, PI3K/Akt,and ERK 1/2 pathways

Fibronectin (FN) is the foremost proliferation‐associated extracellular matrix component promoting cell adhesion, migration, and survival. We examined the effect of FN on cell proliferation and the related signaling pathways in mouse embryonic stem (ES) cells. FN increased integrin β1, Src, focal adhesion kinase (FAK), and caveolin‐1 phosphorylation levels in a time‐dependent manner. Phosphorylation of Src, FAK, and caveolin‐1 was attenuated by integrin β1 neutralizing antibody. Integrin β1, Src, and FAK coimmunoprecipitated with caveolin‐1 in the presence of FN. In addition, FN increased RhoA and Rho kinase activation, which were completely blocked by PP2, FAK small interfering RNA (siRNA), caveolin‐1 siRNA, or the caveolar disruptor methyl‐β‐cyclodextrin (MβCD). FN also increased phosphorylation of Akt and ERK 1/2, which were significantly blocked by either FAK siRNA, caveolin‐1 siRNA, MβCD, GGTI‐286 (RhoA inhibitor), or Y‐27632 (Rho kinase inhibitor). FN‐induced increase of protooncogenes (c‐fos, c‐myc, and c‐Jun) and cell‐cycle regulatory proteins (cyclin D1/CDK4 and cyclin E/CDK2) expression levels were attenuated by FAK siRNA or caveolin‐1 siRNA. Furthermore, inhibition of each pathway such as integrin β1, Src, FAK, caveolin‐1, RhoA, Akt, and ERK 1/2 blocked FN‐induced [³H]‐thymidine incorporation. We conclude that FN stimulates mouse ES cell proliferation via RhoA‐PI3K/Akt‐ERK 1/2 pathway through caveolin‐1 phosphorylation. J. Cell. Physiol. 226: 267–275, 2010. © 2010 Wiley‐Liss, Inc.     

Rapid expression and activation of MMP-2 and MMP-9 upon exposure of human breast cancer cells (MCF-7) to fibronectin in serum free medium

Interactions between tumour cells and the extracellular matrix (ECM) strongly influence tumour development, affecting cell survival, proliferation and migration. Many of these interactions are mediated through a family of cell surface receptors named integrins. Fibronectin and its integrin receptors play important roles in tumour development. The alpha5beta 1 integrin interacts with the central cell adhesive region of fibronectin and requires both the RGD and synergy sites for maximal binding. Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases. They are capable of digesting the different components of the ECM and basement membrane. The ECM gives structural support to cells and plays a central role in cell adhesion, differentiation, proliferation and migration. Binding of ECM to integrins modulates expression and activity of the different MMPs. Our experimental findings demonstrate that cultivation of human breast cancer cells, MCF-7, in serum free medium in the presence of fibronectin upregulates the activity of MMP-2 and MMP-9. Blocking of alpha5beta 1 integrin with anti-alpha5 monoclonal antibody inhibits the fibronectin-induced MMP activation response appreciably. This strongly indicates alpha5beta 1 mediated signalling events in activation of MMP-2 and MMP-9. Phosphorylation of FAK and PI-3 kinase and the nuclear translocation of ERK and NF-kappaB upon fibronectin binding demonstrate possible participation of the FAK/PI-3K/ERK signalling pathways in the regulation of MMP-2 activity.     

The dioxin receptor controls β1 integrin activation in fibroblasts through a Cbp–Csk–Src pathway

Recent studies have suggested a regulatory role for the dioxin receptor (AhR) in cell adhesion and migration. Following our previous work, we report here that the C-terminal Src kinase-binding protein (Cbp) signaling pathway controls β1 integrin activation and that this mechanism is AhR dependent. T-FGM AhR ?/? fibroblasts displayed higher integrin β1 activation, revealed by the increased binding of the activation reporter 9EG7 anti-β1 mAb and of a soluble fibronectin fragment, as well as by enhanced talin-β1 association. AhR ?/? fibroblasts also showed increased fibronectin secretion and impaired directional migration. Notably, interfering Cbp expression in AhR ?/? fibroblasts reduced β1 integrin activation, improved cell migration and rescued wild-type cell morphology. Cbp over-expression in T-FGM AhR ?/? cells enhanced the formation of inhibitory Csk–Cbp complexes which in turn reduced c-Src p-Tyr⁴¹⁶ activation and focal adhesion kinase (FAK) phosphorylation at the c-Src-responsive residues p-Tyr⁵⁷⁶ and p-Tyr⁵⁷⁷. The c-Src target and migration-related protein Cav1 was also hypophosphorylated at p-Tyr¹⁴ in AhR ?/? cells, and such effect was rescued by down-modulating Cbp levels. Thus, AhR regulates fibroblast migration by modulating β1 integrin activation via Cbp-dependent, Src-mediated signaling.     

Adhesion-dependent activation of CaMKII and regulation of ERK activation in vascular smooth muscle

Cell adhesion-dependent activation of ERK1/2 has been linked functionally to focal adhesion dynamics. We previously reported that in adherent vascular smooth muscle (VSM) cells, CaMKII mediates ERK1/2 activation in response to Ca(2+)-mobilizing stimuli. In the present study, we tested whether CaMKII regulates ERK1/2 signaling in response to VSM cell adhesion. Using an antibody that specifically recognizes CaMKII autophosphorylated on Thr(287), we determined that CaMKII is rapidly activated (within 1 min) after the adherence of cells on multiple ECM substrates. Activation of CaMKII on fibronectin was unaffected in cells overexpressing focal adhesion kinase (FAK)-related nonkinase (FRNK), an endogenous inhibitor of FAK. Furthermore, CaMKII was rapidly and robustly activated in VSM cells plated on poly-l-lysine. These results suggest that adhesion-dependent CaMKII activation is integrin independent. Adhesion-dependent FAK activation on fibronectin was not affected in cells treated with the selective CaMKII inhibitor KN-93 (30 muM) or in cells in which the expression of CaMKII with small interfering RNA (siRNA) was suppressed, although tyrosine phosphorylation of paxillin was inhibited in CaMKII-delta(2)-suppressed cells. Sustained ERK1/2 activation that was dependent on FAK activation (inhibited by FRNK) was also attenuated by CaMKII inhibition or siRNA-mediated gene silencing. Rapid ERK1/2 activation that preceded FAK and paxillin activation was detected upon VSM cell adhesion to poly-l-lysine, and this response was inhibited by CaMKII gene silencing. These results indicate that integrin-independent CaMKII activation is an early signal during VSM cell adhesion that positively modulates ERK1/2 signaling through FAK-dependent and FAK-independent mechanisms.     

MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21^WAF1 and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin α_vβ₃ were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.     

Osteopontin Promotes Mesenchymal Stem Cell Migration and Lessens Cell Stiffness via Integrin β1, FAK, and ERK Pathways

The use of mesenchymal stem cells (MSCs) for therapeutic applications has attracted great attention because MSCs home to and engraft to injured tissues after in vivo administration. The expression of osteopontin (OPN) is elevated in response to injury and inflammation, and its role on rat bone marrow-derived mesenchymal stem cells (rMSCs)-directed migration has been elucidated. However, the signaling pathways through the activation of which OPN promotes rMSCs migration and the involvement of cell mechanics during OPN-mediating rMSCs migration have not been well studied. In this study, we found that OPN activated focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways by the ligation of integrin β1 in rMSCs. Inhibitors of FAK and ERK pathways inhibited OPN-induced rMSCs migration, indicating the possible involvement of FAK and ERK activation in OPN-induced migration in rMSCs. In addition, atomic force microscopy analysis showed that OPN reduced cell stiffness in rMSCs via integrin β1, FAK, and ERK pathways, suggesting that the promotion of rMSCs migration might partially be contributing to the decrease in cell stiffness stimulated by OPN. To further examine the role of OPN on cell motility and stiffness, actin cytoskeleton of rMSCs was observed. The reduced well-defined F-actin filaments and the promoted formation of pseudopodia in rMSCs induced by OPN explained the reduction in cell stiffness and the increase in cell migration. The current study data have shown for the first time that OPN binding to integrin β1 promotes rMSCs migration through the activation of FAK and ERK pathways, which may be attributed to the change in cell stiffness caused by the reduction in the amount of organized actin cytoskeleton.     

CB1 cannabinoid receptors promote maximal FAK catalytic activity by stimulating cooperative signaling between receptor tyrosine kinases and integrins in neuronal cells

Tyrosine phosphorylation (Tyr-P) of focal adhesion kinase (FAK) regulates FAK activation. Phosphorylated FAK Tyr 397 binds Src family kinases (Src), which in turn directly phosphorylate FAK Tyr 576/577 to produce maximal FAK enzymatic activity. CB₁ cannabinoid receptors (CB₁) are abundantly expressed in the nervous system and influence FAK activation by presently unknown mechanisms. The current investigation determined that CB₁-stimulated maximal FAK catalytic activity is mediated by G_i/o proteins in N18TG2 neuronal cells, and that G_12/13 regulation of Rac1 and RhoA occurs concomitantly. Immunoblotting analyses using antibodies against FAK phospho-Tyr 397 and phospho-Tyr 576/577 demonstrated that the time-course of CB₁-stimulated FAK 576/577 Tyr-P occurred in three phases: Phase I (0–2 min) maximal Tyr-P, Phase II (5–20 min) rapid decline in Tyr-P, and Phase III (> 20 min) plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B and Shp1/Shp2)-mediated Src activation, Protein Kinase A (PKA) inhibition, and integrin activation. Phase I maximal FAK 576/577 Tyr-P also required cooperative signaling between receptor tyrosine kinases (RTKs) and integrins. The integrin antagonist RGDS peptide, Flk-1 vascular endothelial growth factor receptor (VEGFR) antagonist SU5416, and epidermal growth factor receptor (EGFR) antagonist AG 1478 blocked Phase I FAK 576/577 Tyr-P. CB₁ agonists failed to stimulate FAK Tyr-P in the absence of integrin activation upon suspension in serum-free culture media. In contrast, cells grown on the integrin ligands fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB₁ agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416. Taken together, these studies have identified a complex integrative pathway utilized by CB₁ to stimulate maximal FAK 576/577 Tyr-P in neuronal cells.     

Extracellular matrix protein fibronectin induces matrix metalloproteinases in human prostate adenocarcinoma cells PC-3

Abstract

Studies on interaction of tumor cells with ECM components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human prostate adenocarcinoma PC-3 cells–fibronectin (FN) interaction on MMPs and the underlying signaling pathways. Culturing of PC-3 cells on FN-coated surface upregulated MMP-9 and MMP-1. This response is abrogated by the blockade of α₅ integrin. siRNA and inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI-3K), focal adhesion kinase (FAK) and nuclear factor-kappaB (NF-κB) in FN-induced upregulation of MMPs. FN treatment also enhanced phosphorylation of FAK, PI3K, protein kinase B (PKB or Akt), nuclear translocation of NF-κB, surface expression of CD-44, and cell migration. Our findings indicate that, binding of PC-3 cells to FN, possibly via α_5β1 integrin, induces signaling involving FAK, PI-3K, Akt, NF-κB followed by upregulation of MMP-9 and MMP-1. CD-44 may have role in modulating MMP-9 activity.     

Adenovirus Endocytosis via αv Integrins Requires Phosphoinositide-3-OH Kinase

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by α_v integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas α_v integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with α_v integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.Adenovirus entry into host cells depends on α_v integrin binding to the penton base viral coat protein (2, 20, 48). A highly mobile protrusion on the adenovirus penton base contains the arginine-glycine-aspartic acid (RGD) sequence which mediates α_v integrin binding (42). Integrins are more noted for their ability to mediate cell surface recognition of the extracellular matrix, thereby facilitating adhesion, migration (24), and cell growth and differentiation (28). These interactions have been associated with cell differentiation and tissue development, angiogenesis, wound repair, cancer, and inflammation (22).A number of cell signaling molecules that are associated with integrin-mediated cellular processes, including adhesion, survival, and motility, have recently been identified (18, 32, 34). For example, the signaling molecule pp125^FAK focal adhesion kinase (FAK) (35) is localized to clustered integrins following ligation by extracellular matrix proteins. Engagement (clustering) of integrins by its ligands increases tyrosine phosphorylation and activation of FAK (29). Potential downstream substrates of FAK are the ERK1/ERK2 mitogen-activated protein (MAP) kinases (8, 40) and phosphoinositide-3-OH kinase (PI3K) (7, 17).Recent studies have demonstrated that ligation of α_v and β1 integrins by the extracellular matrix leads to engagement of the ERK1/ERK2 MAP kinase pathway (24). Integrin-mediated regulation of the ERK1/ERK2 MAP kinase pathway results in the activation of myosin light chain kinase and subsequently to phosphorylation of myosin light chains. These molecular events culminate in enhanced cell motility. Cell motility, but not cell adhesion or spreading, can be blocked by ERK antisense oligonucleotides or by the compound PD98059, a specific inhibitor of MEK MAP kinase (24), indicating that the ERK1/ERK2 MAP kinase pathway plays a specific role in cell movement.PI3K (44) is another downstream effector of FAK. PI3K is a member of a family of lipid kinases comprised of a p85 regulatory subunit and a p110 catalytic subunit. The p85 subunit of PI3K binds directly to phosphorylated FAK (6). The products of PI3K activation, phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIP₃), are increased in the plasma membrane of activated but not quiescent cells and have been proposed to act as second messengers for a number of cell functions (5), including cell cycle progression (9) and cytoskeletal changes underlying the cell plasma membrane (47). PI3K activation also modulates intracellular protein trafficking (41), although a direct role of PI3K in receptor-mediated endocytosis has not been established.While integrins play an important role in adenovirus entry and in cell migration, the precise mechanisms by which these receptors promote these distinct biological functions are not known. In the studies reported here, we demonstrate that a specific signaling event is involved in the cell entry of a human viral pathogen. Evidence is provided that PI3K is activated upon adenovirus interaction with α_v integrins and that this event is required for adenovirus internalization. Surprisingly, activation of ERK1/ERK2 following integrin ligation was necessary for cell migration but not for internalization of adenovirus.     

SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover

Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway.     

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.     

Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration.

The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.     

CCN2 promotes keratinocyte adhesion and migration via integrin α5β1

Background

CCN2, (a.k.a. connective tissue growth factor and CTGF) has emerged as a regulator of cell migration. While the importance of CCN2 for the fibrotic process in wound healing has been well studied, the effect of CCN2 on keratinocyte function is not well understood. In this study, we investigated the mechanism behind CCN2-driven keratinocyte adhesion and migration.Materials and methods: Adhesion assays were performed by coating wells with 10 μg/ml fibronectin (FN) or phosphate-buffered saline (PBS). Keratinocytes were seeded in the presence or absence of 200 ng/ml CCN2, 5 mmol/l ethylenediaminetetraacetic acid, 10 mmol/l cations, 500 μl arginine–glycine–aspartic acid (RGD), 500 μM arginine–glycine–glutamate–serine (RGES), and 10 μg/ml anti-integrin blocking antibodies. Migration studies were performed using a modified Boyden chamber assay. Quantitative PCR was used to study the effect of CCN2 on integrin subunit mRNA expression. To block intracellular pathways, keratinocytes were pretreated with 20 μM PD98059 (MEK-1 inhibitor) or 20 μM PF573228 (FAK inhibitor) for 60 min prior the addition of CCN2. Western blot was used to measure CCN2, p-ERK1/2, and ERK1/2.Results: CCN2 enhanced keratinocyte adhesion to fibronectin via integrin α5β1. The addition of anti-integrin α5β1 antibodies reduced CCN2-mediated keratinocyte migration. In addition, CCN2 regulated mRNA and protein expression of integrin subunits α5 and β1. CCN2 activated the FAK-MAPK signaling pathway, and pretreatment with MEK1-specific inhibitor PD98059 markedly reduced CCN2-induced keratinocyte migration.Conclusions: Our results demonstrate that CCN2 enhances keratinocyte adhesion and migration through integrin α5β1 and activation of the FAK-MAPK signaling cascade.     

Delta-opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases

Integrin-mediated cell adherence to extracellular matrix proteins results in stimulation of ERK1/2 activity, a mechanism involving focal adhesion tyrosine kinases (pp125FAK, Pyk-2) and epidermal growth factor receptors (EGFRs). G protein-coupled receptors (GPCRs) may also mediate ERK1/2 activation in an integrin-dependent manner, the underlying signaling mechanism of which still remains unclear. Here we demonstrate that the δ-opioid receptor (DOR), a typical GPCR, stimulates ERK1/2 activity in HEK293 cells via integrin-mediated transactivation of EGFR function. Inhibition of integrin signaling by RGDT peptides, cytochalasin, and by keeping the cells in suspension culture both blocked [D-Ala², D-Leu⁵]enkephalin (DADLE)- and etorphine-stimulated ERK1/2 activity. Integrin-dependent ERK1/2 activation does not involve FAK/Pyk-2, because over-expression of the FAK/Pyk-2 inhibitor SOCS-3 failed to attenuate DOR signaling. Exposure of the cells to the EGFR inhibitors AG1478 and BPIQ-I blocked DOR-mediated ERK1/2 activation. Because RGDT peptides also prevented DOR-mediated EGFR activation, the present findings indicate that in HEK293 cells DOR-stimulated ERK1/2 activity is mediated by integrin-stimulated EGFRs. Further studies with the phospholipase C (PLC) inhibitors U73122 and ET-18-OCH₃ revealed that opioid-stimulated integrin activation is sensitive to PLC. In contrast, integrin-mediated transactivation of EGFR function appears to be dependent on PKC-δ, as indicated by studies with rottlerin and siRNA knock-down. A similar ERK1/2 signaling pathway was observed for NG108-15 cells, a neuronal cell line endogenously expressing the DOR. In these cells, the nerve growth factor TrkA receptor replaces the EGFR in connecting DOR-activated integrins to the Ras/Raf/ERK1/2 pathway. Together, these data describe an alternative ERK1/2 signaling pathway in which the DOR transactivates the growth factor receptor associated mitogen-activated protein kinase cascade in an integrin-dependent manner.     

Requirement of PEA3 for Transcriptional Activation of FAK Gene in Tumor Metastasis

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase critically involved in cancer metastasis. We found an elevation of FAK expression in highly metastatic melanoma B16F10 cells compared with its less metastatic partner B16F1 cells. Down-regulation of the FAK expression by either small interfering RNA or dominant negative FAK (FAK Related Non-Kinase, FRNK) inhibited the B16F10 cell migration in vitro and invasiveness in vivo. The mechanism by which FAK activity is up-regulated in highly metastatic cells remains unclear. In this study, we reported for the first time that one of the Est family proteins, PEA3, is able to transactivate FAK expression through binding to the promoter region of FAK. We identified a PEA3-binding site between nucleotides −170 and +43 in the FAK promoter that was critical for the responsiveness to PEA3. A stronger affinity of PEA3 to this region contributed to the elevation of FAK expression in B16F10 cells. Both in vitro and in vivo knockdown of PEA3 gene successfully mimicked the cell migration and invasiveness as that induced by FAK down-regulation. The activation of the well-known upstream of PEA3, such as epidermal growth factor, JNK, and ERK can also induce FAK expression. Furthermore, in the metastatic human clinic tumor specimens from the patients with human primary oral squamous cell carcinoma, we observed a strong positive correlation among PEA3, FAK, and carcinoma metastasis. Taking together, we hypothesized that PEA3 might play an essential role in the activation of the FAK gene during tumor metastasis.