Periostin mediates vascular smooth muscle cell migration through the integrins ανβ3 and ανβ5 and focal adhesion kinase (FAK) pathway

Smooth muscle cell (SMC) migration involves interactions of integrin receptors with extracellular matrix (ECM) and is an important process of neointimal formation in atherosclerosis and restenosis after vascular interventions. Previous studies have shown that periostin (PN), a novel ECM protein, is upregulated in rat carotid artery after balloon injury, and growth factor-stimulated expression of PN promotes SMC migration in vitro. Here, we address the mechanism by which PN–integrin interaction mediates SMC migration in vitro. Aortic SMCs isolated from PN null mice exhibited a significantly reduced ability to migrate and proliferate in vitro. Endogenous PN protein was absent and very low in the culture medium from the primary cultures of PN?/? and wildtype SMCs, respectively. In both types of SMCs, adenovirus-mediated overexpression of HA-tagged PN to a similar extent, which induced a robust cell migration concomitantly with an increase in β3-integrin expression and phosphorylation of FAK (Tyr397). Furthermore, in cultured human SMCs, specific integrin blocking antibodies showed that interactions of PN-ανβ3 and PN-ανβ5, but not PN-β1 integrins, are required for SMC migration. Inhibition of FAK signaling by overexpression of an endogenous FAK inhibitor termed FRNK (FAK-related nonkinase) significantly attenuated FAK (Tyr397) phosphorylation and the SMC migration induced by PN. These results reveal a mechanism whereby PN mediates vascular SMC migration through an interaction with alphaV-integrins (mainly ανβ3) and subsequent activation of FAK pathway.     

Angiotensin II-augmented migration of VSMCs towards PDGF-BB involves Pyk2 and ERK 1/2 activation

Activation of the local and systemic renin-angiotensin system is directly and indirectly involved in mechanisms of vascular remodeling during chronic hypertension. This study investigated the effect of angiotensin II (AII) on rat vascular smooth muscle cell (VSMC) migration towards platelet-derived growth factor-BB (PDGF-BB) in vitro. Pre-treatment with AII (1 μM) for 48 or 72 h induced a significant increase in PDGF-BB-directed migration by 77 ± 21 % and 58 ± 24 %, respectively (both p < 0.01). This effect was concentration dependent and inhibited by the selective angiotensin receptor type I (AT₁) blocker DUP 753. PDGF-directed migration of VSMCs was significantly inhibited by antibodies against β₃-and β₅-integrins, indicating an important role of these integrins in VSMC migration. However, AII augmented migration was not accompanied by an increased expression of β₃- and β₅-integrin mRNA and protein levels in VSMCs. Inhibition of the mitogen-activated protein kinase ERK 1/2 with PD 98059 (30 μM) completely abolished the effect of AII on PDGF-BB-directed VSMC migration (p < 0.01). The proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK) are cytoskeleton-associated protein kinases participating in integrin-dependent signaling. Therefore, expression and phosphorylation of these kinases was determined 48 h after AII treatment, revealing a significant increase in Pyk2 and FAK protein levels (up to 2-fold, both p < 0.05) and increased phosphorylation of Pyk2 (2-fold, p < 0.05) and ERK 1/2 (4-fold, p < 0.05) as compared to controls. Furthermore, immunofluorescence and Western blot analysis demonstrated a translocation of Pyk2 from the plasma membrane to the cytosol, as well as a perinuclear enrichment of ERK 1/2 protein 48 h after AII treatment. In conclusion, our data suggest that changes in the levels of Pyk2 and ERK 1/2 phosphorylation, responsible for integrin-dependent signaling, as well as their subcellular translocation are important for the enhanced chemotactic response of VSMCs after AII pre-treatment. Received: 16 July 2001/Returned for 1. revision: 2 August 2001/1. Revision received: 12 December 2001/Returned for 2. revision: 21 January 2002/2. Revision received: 14 February 2002/Accepted: 13 March 2002     

Transglutaminase-mediated oligomerization of the fibrin(ogen) alphaC domains promotes integrin-dependent cell adhesion and signaling

Interactions of endothelial cells with fibrin(ogen) are implicated in inflammation, angiogenesis, and wound healing. Cross-linking of the fibrinogen alphaC domains with factor XIIIa generates ordered alphaC oligomers mimicking polymeric arrangement of the alphaC domains in fibrin. These oligomers and those prepared with tissue transglutaminase were used to establish a mechanism of the alphaC domain-mediated interaction of fibrin with endothelial cells. Cell adhesion and chemical cross-linking experiments revealed that oligomerization of the alphaC domains by both transglutaminases significantly increases their RGD (arginyl-glycyl-aspartate)-dependent interaction with endothelial alphaVbeta3 and to a lesser extent with alphaVbeta5 and alpha5beta1 integrins. The oligomerization promotes integrin clustering, thereby increasing cell adhesion, spreading, formation of prominent peripheral focal contacts, and integrin-mediated activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways. The enhanced integrin clustering is likely caused by ordered juxtaposition of RGD-containing integrin-binding sites upon oligomerization of the alphaC domains and increased affinity of these domains for integrins. Our findings provide new insights into the mechanism of the alphaC domain-mediated interaction of endothelial cells with fibrin and imply its potential involvement in cell migration. They also suggest a new role for transglutaminases in regulation of integrin-mediated adhesion and signaling via covalent modification of integrin ligands.     

The integrin, alpha6beta1, is necessary for the matrix-dependent activation of FAK and MAP kinase and the migration of human hepatocarcinoma cells.

Expression of the integrin, alpha6beta1, a receptor for laminins, is associated with the progression of hepatocellular carcinoma (HCC). The approach to investigating the alpha6beta1 integrin signaling in HCC cells was to express a deletion mutant of the beta4 integrin cytoplasmic domain (beta4-Deltacyt) in 2 HCC cell lines, HepG2 and Huh7. Expression of this mutant prevents formation of the alpha6beta1 heterodimer. As expected, adhesion of both the HepG2/beta4-Deltacyt and Huh7/beta4-Deltacyt transfectants to laminin, but not to collagen, was reduced compared with the mock transfectants. However, migration of the beta4-Deltacyt transfectants toward both collagen and laminin was inhibited, suggesting a role for alpha6beta1 in the signaling of migration. Migration of HCC cells requires mitogen-activated protein (MAP) kinase. The adhesion of the beta4-Deltacyt transfectants to collagen resulted in a substantial reduction in MAP kinase activation in comparison with the mock transfectants, although their ability to activate MAP kinase in response to epidermal growth factor (EGF) stimulation was not impaired. In addition, matrix adhesion of the beta4-Deltacyt transfectants did not stimulate the tyrosine phosphorylation of focal adhesion kinase (FAK), and this defect correlated with reduced binding of adaptor protein Grb2 to FAK. These results suggest that FAK tyrosine phosphorylation is dependent on alpha6beta1 expression, and that FAK-Grb2 association plays a central role in alpha6beta1-mediated activation of MAP kinase. Moreover, the expression of alpha6beta1 in HCC cells is necessary for FAK/MAP kinase-dependent migration.     

Histological and functional analysis of vascular smooth muscle cells in a novel culture system with honeycomb-like structure

Vascular smooth muscle cells (SMCs) undergo phenotype change with the development of atherosclerosis. The phenotype changes of SMCs have been observed in various culture conditions, such as collagen-coated dishes. Here, we report the morphological and functional features of SMCs in a novel culture system using type I-collagen in a characteristic three-dimensional structure designated as honeycombs. The number of ribosome and mitochondria in SMCs cultured in honeycombs was one half or third of those cultured on collagen-coated plastic plates. DNA and protein synthesis of SMCs cultured in honeycombs were less than 1 and 30-40%, respectively, of those cultured on plastic plates. In addition, PDGF-BB did not increase the amount of DNA synthesis in SMCs in honeycombs. SMCs in honeycombs were shown to express several proteins, which are known to express in SMCs in medial layers of arteries. Particularly, caldesmon heavy chain was expressed in SMCs cultured in honeycombs, whereas not in those on plastic plates. Although focal adhesion kinase (FAK) was clearly detected in SMCs in honeycomb, the phosphotyrosine content of focal adhesion kin ase decreased in the process of culture. Immunoblot analysis showed dear different expression of ERK1 and ERK2 of mitogen-activated protein kinase in SMCs. SMCs in honeycombs expressed ERK2, more abundantly compared to ERK1, whereas SMCs in plates show the same levels of expressions for both proteins. Thus, the histological and functional feature of SMCs in the novel culture system is different from SMCs in plastic plates. The three-dimensional culture system described here may be indicating that cultured SMCs are able to express different proteins responding to the surrounding structures.     

Integrin-mediated differentiation of a pancreatic carcinoma cell line is independent of FAK or MAPK activation levels

INTRODUCTION: The extracellular matrix (ECM) plays a salient role for proliferation and differentiation of epithelial cells. It was demonstrated that cell-ECM interactions mediated through integrins control gene expression and the tissue phenotype even in malignant tumors. Alterations of the ECM are a key feature of ductal adenocarcinoma of the pancreas. AIMS: To examine the role of integrins and related signaling events for differentiation. METHODOLOGY AND RESULTS: We established an in vitro model for ECM-induced differentiation of poorly differentiated pancreatic carcinoma cells and found that a specific pattern of ECM proteins resembling basal laminas (matrigel) induces differentiation of the PaTu-II pancreatic carcinoma cell line to a ductal phenotype. Both beta1- and beta4-integrins are required for cellular differentiation. Integrin-associated signaling events include activation of pp125 focal adhesion kinase (FAK) and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinases (JNKs). However, beta1- and beta4-integrin-mediated differentiation of PaTu-II cells was independent from FAK, ERK, and JNK activation levels. Inhibition of MAPK kinases by PD98059 led to a reduction of proliferation but did not interfere with cellular differentiation of PaTu-II cells on matrigel. CONCLUSION: The integrin-mediated differentiation of PaTu-II cells is regulated and maintained through FAK- and MAPK-independent signal transduction pathways.     

Adhesion to the extracellular matrix is positively regulated by retinoic acid in HepG2 cells.

Aims: In this work, we aimed to investigate the possible modulation of cell-matrix interactions by retinoic acid (RA), in view of the well-known role of the extracellular matrix (ECM) and integrins in hepatocyte differentiation and proliferation. For this purpose, we analysed the adhesion ability of HepG2 cells on different substrates in the presence and absence of RA evaluating both the expression and cellular localisation of major proteins involved in focal contacts, using Western blot and confocal microscopy. RESULTS: A positive and substrate-dependent effect of RA on cell-matrix adhesion was observed after long-term culture. The increased adhesiveness in the treated cells was accompanied by an enhanced expression of beta1 and alpha3 integrin subunits, together with a redistribution of beta1 receptors clustered at the basal surface. In contrast, the levels of focal adhesion kinase (FAK), paxillin and alpha-actinin were unchanged, as was the phosphorylation state of FAK. Nonetheless, a stronger association between beta1 integrin and intracytoplasmatic proteins of focal contacts was observed in coimmunoprecipitation experiments after RA treatment, suggesting improved connection with the actin cytoskeleton. These results are consistent with previously described antiproliferative and differentiative effects of RA on transformed hepatocytes, and confirm the hypothesis of a direct influence of RA on specific adhesion molecules.     

Activation of vascular smooth muscle cells by TNF and PDGF: overlapping and complementary signal transduction mechanisms

OBJECTIVE: Because tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of vein graft neointimal hyperplasia, we sought to determine mechanisms by which TNF could induce proliferative and migratory responses in smooth muscle cells (SMCs). METHODS AND RESULTS: In rabbit jugulocarotid interposition vein grafts, SMCs expressed TNF as early as four days postoperatively. In rabbit aortic SMCs, TNF and platelet-derived growth factor (PDGF) elicited comparable migration (1.7-fold/basal), and their effects were partially additive. In contrast, while TNF failed to promote SMC [(3)H]thymidine incorporation alone, it doubled the [(3)H]thymidine incorporation observed with PDGF alone. To gain mechanistic insight into these phenomena, we found that TNF and PDGF each activated p38(mapk) equivalently in SMCs, but that PDGF was two to three times more efficacious than TNF in activating SMC extracellular signal-regulated kinases (ERK) 1 and 2 and phosphoinositide 3-kinase. However, only TNF activated NF kappa B. SMC [(3)H]thymidine incorporation that depended on TNF, but not PDGF, was abolished by overexpression of a dominant-negative I kappa B alpha mutant. Inhibition of ERK activation by U0126 reduced SMC migration stimulated only by PDGF (by 35%, P<0.05), but not by TNF. Inhibition of phosphoinositide 3-kinase by LY294002, however, significantly reduced both TNF- and PDGF-stimulated chemotaxis (by 38-54%, P<0.05). In contrast, both U0126 and LY294002 abolished SMC [(3)H]thymidine incorporation induced by either TNF, PDGF, or both agonists. CONCLUSIONS: In primary rabbit SMCs, TNF promotes migration and mitogenesis through signaling mechanisms that are both distinct from and overlapping with those employed by PDGF. TNF-induced SMC mitogenesis requires complementary co-stimulation with other growth factors.     

LPP expression during in vitro smooth muscle differentiation and stent-induced vascular injury

Lipoma preferred partner (LPP) has been identified as a protein highly expressed in smooth muscle (SM) tissues. The aim of the present study was to determine mechanisms that regulate LPP expression in an in vitro model of SM cell (SMC) differentiation and in stent-induced pig coronary vessel injury. All trans-retinoic acid treatment of A404 cells induced a strong increase in LPP, as well as SM alpha-actin, SM myosin heavy chain, and smoothelin mRNA levels, in a Rho kinase (ROK)-dependent manner. Adenovirus mediated overexpression of myocardin in A404 cells significantly increased LPP mRNA expression. Interestingly, inactivation of RhoA with C3-exoenzyme or treatment with ROK inhibitors strongly inhibited myocardin mRNA expression in retinoic acid-treated A404 cells or human iliac vein SMCs. LPP silencing with short interfering RNA significantly decreased SMC migration. LPP expression was also markedly decreased in focal adhesion kinase (FAK)-null cells known to have impaired migration but rescued with inducible expression of FAK. LPP expression in FAK-null fibroblasts enhanced cell spreading. In stented pig coronary vessels, LPP was expressed in the neointima of cells lacking smoothelin and showed expression patterns identical to those of SM alpha-actin. In conclusion, LPP appears to be a myocardin-, RhoA/ROK-dependent SMC differentiation marker that plays a role in regulating SMC migration.     

Focal adhesion kinase is involved in angiotensin II-mediated protein synthesis in cultured vascular smooth muscle cells

The rate of vascular smooth muscle cell protein synthesis and cellular hypertrophy in response to angiotensin II (Ang II) is dependent on activation of protein tyrosine kinases (PTKs) and both the extracellular signal-regulated kinase (ERK) 1/2 and p70(S6K) pathways. One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions. We used an actin depolymerizing agent, cytochalasin D (Cyt-D), and a replication-defective adenovirus encoding FAK-related nonkinase (FRNK), an inhibitor of FAK-dependent signaling, as tools to assess whether FAK was upstream of the ERK1/2 and/or the p70(S6K) pathways. Cyt-D reduced basal FAK phosphorylation and blocked Ang II-dependent FAK phosphorylation in a dose-dependent manner. Confocal microscopy indicated that Cyt-D induced actin filament disruption and FAK delocalization from focal adhesions. Cyt-D also reduced Ang II-induced ERK1/2 activation, but p70(S6K) activation was relatively unaffected. Cyt-D reduced basal protein synthetic rate and substantially reduced the Ang II-induced increase in protein synthesis. Similarly, FRNK overexpression blocked Ang II-induced FAK phosphorylation and ERK1/2 activation, but not p70(S6K) phosphorylation, and markedly inhibited protein synthesis. This is the first report to demonstrate that FAK is a critical component of the signal transduction pathways that mediate Ang II-induced ERK1/2 activation, c-fos induction, and enhanced protein synthesis in vascular smooth muscle cells.     

Proline‐rich tyrosine kinase 2 and Src kinase signaling transduce monosodium urate crystal–induced nitric oxide production and matrix metalloproteinase 3 expression in chondrocytes

Objective

Articular deposition of monosodium urate monohydrate (MSU) crystals may promote cartilage and bone erosion. Therefore, the aim of this study was to determine how MSU crystals stimulate chondrocytes.

Methods

Nitric oxide (NO) release, and expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase 3 (MMP‐3) were assessed in cultured chondrocytes treated with MSU. MSU‐induced functional signaling by specific protein kinases (p38, Src, and the focal adhesion kinase [FAK] family members proline‐rich tyrosine kinase 2 [Pyk‐2] and FAK) was also examined using selective pharmacologic inhibitors and transfection of kinase mutants.

Results

MSU induced MMP‐3 and iNOS expression and NO release in chondrocytes in a p38‐dependent manner that did not require interleukin‐1 (IL‐1), as demonstrated by using IL‐1 receptor antagonist. MSU induced rapid tyrosine phosphorylation of Pyk‐2 and FAK, their adaptor protein paxillin, and interacting kinase c‐Src. Pyk‐2 and c‐Src signaling both mediated p38 MAPK activation in response to MSU. Pyk‐2 and c‐Src signaling played a major role in transducing MSU‐induced NO production and MMP‐3 expression. But, despite the observed FAK phosphorylation, a selective pharmacologic FAK inhibitor and a FAK dominant‐negative mutant both failed to block MSU‐induced NO release or MMP‐3 expression in parallel experiments.

Conclusion

In chondrocytes, MSU crystals activate a signaling kinase cascade typically employed by adhesion receptors that involves upstream Src and FAK family activation and downstream p38 activation. In this cascade, Pyk‐2, Src, and p38 kinases transduce MSU‐induced NO production and MMP‐3 expression. Our results identify Pyk‐2 and c‐Src as novel sites for potential therapeutic intervention in cartilage degradation in chronic gout.

     

Arginine stimulates intestinal cell migration through a focal adhesion kinase dependent mechanism

BACKGROUND: L-Arginine is a nutritional supplement that may be useful for promoting intestinal repair. Arginine is metabolised by the oxidative deiminase pathway to form nitric oxide (NO) and by the arginase pathway to yield ornithine and polyamines. AIMS: To determine if arginine stimulates restitution via activation of NO synthesis and/or polyamine synthesis. METHODS: We determined the effects of arginine on cultured intestinal cell migration, NO production, polyamine levels, and activation of focal adhesion kinase, a key mediator of cell migration. RESULTS: Arginine increased the rate of cell migration in a dose dependent biphasic manner, and was additive with bovine serum concentrate (BSC). Arginine and an NO donor activated focal adhesion kinase (a tyrosine kinase which localises to cell matrix contacts and mediates beta1 integrin signalling) after wounding. Arginine stimulated cell migration was dependent on focal adhesion kinase (FAK) signalling, as demonstrated using adenovirus mediated transfection with a kinase negative mutant of FAK. Arginine stimulated migration was dependent on NO production and was blocked by NO synthase inhibitors. Arginine dependent migration required synthesis of polyamines but elevating extracellular arginine concentration above 0.4 mM did not enhance cellular polyamine levels. CONCLUSIONS: These results showed that L-arginine stimulates cell migration through NO and FAK dependent pathways and that combination therapy with arginine and BSC may enhance intestinal restitution via separate and convergent pathways.     

Tyrosine phosphorylation of paxillin and focal adhesion kinase by activation of muscarinic m3 receptors is dependent on integrin engagement by the extracellular matrix

The G protein-coupled m1 and m3 muscarinic acetylcholine receptors increase tyrosine phosphorylation of several proteins, including the focal adhesion-associated proteins paxillin and focal adhesion kinase (FAK), but the mechanism is not understood. Activation of integrins during adhesion of cells to extracellular matrix, or stimulation of quiescent cell monolayers with G protein-coupled receptor ligands including bradykinin, bombesin, endothelin, vasopressin, and lysophosphatidic acid, also induces tyrosine phosphorylation of paxillin and FAK and formation of focal adhesions. These effects are generally independent of protein kinase C but are inhibited by agents that prevent cytoskeletal assembly or block activation of the small molecular weight G protein Rho. This report demonstrates that tyrosine phosphorylation of paxillin and FAK elicited by stimulation of muscarinic m3 receptors with the acetylcholine analog carbachol is inhibited by soluble peptides containing the arginine–glycine–aspartate motif (the recognition site for integrins found in adhesion proteins such as fibronectin) but is unaffected by peptides containing the inactive sequence arginine–glycine–glutamate. Tyrosine phosphorylation elicited by carbachol, but not by cell adhesion to fibronectin, is reduced by the protein kinase C inhibitor GF 109203X. The response to carbachol is dependent on the presence of fibronectin. Moreover, immunofluorescence studies show that carbachol treatment induces formation of stress fibers and focal adhesions. These results suggest that muscarinic receptor stimulation activates integrins via a protein kinase C-dependent mechanism. The activated integrins transmit a signal into the cell’s interior leading to tyrosine phosphorylation of paxillin and FAK. This represents a novel mechanism for regulation of tyrosine phosphorylation by muscarinic receptors.     

Specific adhesion molecules bind anchoring filaments and endothelial cells in human skin initial lymphatics

Anchoring filaments are a characteristic feature of initial lymphatic vessels. They connect the abluminal membrane of endothelial cells to the surrounding elastic fibers. The main molecular component of anchoring filaments is fibrillin. Initial lymphatic vessels of human skin were stained with monoclonal antibodies to fibrillin, integrins alpha 2 beta 1, alpha 3 beta 1 and alpha v beta 3, vinculin, talin, beta-actin and focal adhesion kinase (FAK). A double-labeling immunofluorescence method was used to simultaneously stain fibrillin and alpha 3 beta 1 integrin or FAK. Close contiguities between integrins and anchoring filaments were observed. These results suggest that the anchoring filaments connect the extracellular matrix and the endothelial cell cytoskeleton through the transmembrane integrin and FAK molecule. The results also demonstrate the presence of focal adhesions in the wall of initial lymphatic vessels. These connections possibly enable transmission of chemical and/or mechanical stimuli from the extracellular matrix to the endothelial cells. Here, they are transformed in cytoskeleton rearrangements and intracellular signaling events, some of which may contribute to the initial formation of lymph.     

Phosphatidylinositol-3-kinase activation mediates proline-rich tyrosine kinase 2 phosphorylation and recruitment to beta1-integrins in human CD34+ cells

OBJECTIVE: beta1-integrins mediate hematopoietic stem and progenitor cell homing and retention in the bone marrow (BM) and inhibit hematopoietic proliferation and differentiation. Having no intrinsic kinase activity, integrins recruit intracellular kinases, such as the focal adhesion kinase (FAK) or the related proline-rich tyrosine kinase 2 (PYK2), to initiate signal transduction. Phosphatidylinositol-3-kinase (PI3K), which is involved in beta1-integrin signaling in many cell types, is physically and functionally associated with FAK in anchorage-dependent cells. Because PYK2 is the principal focal adhesion kinase expressed in primary human CD34+ cells, we assessed its functional relationship with PI3K in CD34+ cells in response to integrin engagement. METHODS: beta1-integrins on primary mobilized peripheral blood CD34+ cells and CD34+ KG1A cells were engaged by adhesion to fibronectin (FN) or by cross-linking with an anti-beta1 integrin antibody, respectively. PI3K activity and PYK2 phosphorylation were then assessed in the presence or absence of the PI3K inhibitor, wortmannin. Association between PI3K, PYK2, and the beta1-integrin subunit were also evaluated in co-immunoprecipitation experiments. RESULTS: beta1-integrin engagement induced PI3K activation, which was required for, and temporally preceded, PYK2 phosphorylation, indicating that PI3K lies upstream of PYK2 in CD34+ cells. Furthermore, although PYK2 and PI3K were constitutively associated, interaction of the PYK2/PI3K complex with beta1-integrins required prior integrin engagement and PI3K activation. CONCLUSION: Activation of PI3K following beta1-integrin engagement on human CD34+ cells results in subsequent phosphorylation of PYK2, and is required for the recruitment of the PI3K/PYK2 complex to beta1-integrins at the cell surface.     

Selective involvement of p130Cas/Crk/Pyk2/c-Src in endothelin-1-induced JNK activation

Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds on which the G protein-coupled receptor (GPCR)-induced signaling complex might assemble. We have recently reported that Ca2+-sensitive tyrosine kinase, Pyk2, and epidermal growth factor receptor (EGFR) act as independently regulated scaffolds in cardiomyocytes. In this report, we investigated the activation and regulation of p130Cas, Crk, Pyk2, and c-Src by a well-known hypertrophic agonist, endothelin-1 (ET), and determined their contributions to the activation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in cardiomyocytes. Like Pyk2, ET-induced tyrosine phosphorylation of p130Cas was significantly inhibited by either chelating intracellular Ca2+ ([Ca2+]i) or a protein kinase C inhibitor, calphostin C. This activation of p130Cas was also abrogated by the tetrapeptide RGDS, which disrupts integrin heterodimerization; cytochalasin D, which depolymerizes the actin cytoskeleton; or a selective Src family kinase inhibitor, PP2, but not by an EGFR inhibitor, AG1478. We also observed ET-induced temporal associations of Pyk2 with active c-Src, followed by p130Cas with Pyk2, c-Src, and Crk. Overexpression of a dominant-negative mutant of p130Cas (CasDeltaSD), Crk (CrkSH2m), Pyk2 (PKM), or C-terminal Src kinase (Csk), but not of a deletion mutant of EGFR (533delEGFR), attenuated ET-induced JNK activation. Similarly, an ET-induced increase in c-jun promoter luciferase activity was inhibited by overexpression of CasDeltaSD, CrkSH2m, PKM, or Csk. In contrast, ET-induced ERK activation and c-fos gene expression were predominantly regulated by EGFR. Collectively, the focal adhesion-dependent p130Cas/Crk/Pyk2/c-Src-mediated pathway is selectively involved in ET-induced JNK activation in cardiomyocytes.