Abstract-Previously we reported that the rapid activation of the Fak/Src multicomponent signaling complex mediates load-induced activation of growth and survival signaling pathways in adult rat heart. In this study, we report that 5% to 20% (10-minute) cyclic stretch (1 Hz) of neonatal rat ventricular myocytes (NRVMs) was paralleled by increases of Fak phosphorylation at Tyr-397 (from 1.5-to 2.8-fold), as detected by anti-Fak-pY 397 phosphospecific antibody. Moreover, 15% cyclic stretch lasting from 10 to 120 minutes increased Fak phosphorylation at Tyr-397 by 2.5-to 3.5-fold. This activation was accompanied by a dramatic change in Fak localization in NRVMs from densely concentrated in the perinuclear regions in nonstretched cells to aggregates regularly distributed along the myofilaments in stretched cells. Furthermore, a 4-hour cyclic stretch enhanced the activity of an atrial natriuretic factor (ANF) promoter-luciferase reporter gene by 2.7-fold. Disrupting endogenous Fak/Src signaling either by expression of a dominant-negative Fak mutant with phenylalanine substituted for Tyr-397 or by treatment with a c-Src pharmacological inhibitor (PP-2) markedly attenuated stretch-induced Fak activation and clustering at myofilaments and inhibited stretch-induced ANF gene activation. On the other hand, overexpression of wild-type Fak potentiated the stretchinduced Fak phosphorylation but did not enhance either baseline or stretch-induced ANF promoter-luciferase reporter gene activity compared with the responses of nontransfected NRVMs. These findings identify Fak as an important element in the early responses induced by stretch in cardiac myocytes, indicating that it may coordinate the cellular signaling machinery that controls gene expression program associated with load-induced cardiac myocyte hypertrophy. Key Words: focal adhesion kinase Ⅲ mechanotransduction Ⅲ cell signaling Ⅲ hypertrophy M echanical overload is both cause and consequence of most heart diseases. 1 Cardiac myocytes respond to increased mechanical load by hypertrophic growth, but mechanical stress is also an important stimulus for triggering the initial steps toward cardiac myocyte degeneration and death, which play a critical role in the maladaptive myocardial remodeling and heart failure. 1,2 A major goal in this field is to decipher the mechanisms that link biomechanical forces to the activation of signaling pathways that mediate the hypertrophic as well as maladaptive responses of cardiac myocytes to mechanical stress.The mechanistic pathways that link mechanical stimuli to biochemical signals in cardiac myocytes are presently unclear, but a growing body of evidence indicates that costameres (complex structures constituted by integrins and cytoskeletal proteins at the junction of sarcolemma and Z-discs) have a critical role in sensing and transducing mechanical stress into biochemical signals that coordinate growth responses to hypertrophic stimuli in both cardiac and skeletal muscle. [3][4][5][6][7] The prominent location of integrins at the junc...

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Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. We recently reported that reexpression of CAS in CAS-deficient mouse embryo fibroblasts transformed by oncogenic Src promoted an invasive phenotype associated with enhanced cell migration through Matrigel, organization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin. We have now extended these studies to examine the mechanism by which CAS achieves these changes and to evaluate the potential role for CAS in promoting in vivo tumor growth and metastasis. Whereas the presence or absence of CAS did not alter the primary growth of subcutaneous-injected Src-transformed mouse embryo fibroblasts, CAS expression was required to promote lung metastasis following removal of the primary tumor. The substrate domain YxxP tyrosines, the major sites of CAS phosphorylation by Src that mediate interactions with Crk, were found to be critical for promoting both invasive and metastatic properties of the cells. The ability of CAS to promote Matrigel invasion, formation of large podosome structures, and tyrosine phosphorylation of Src substrates, including FAK, paxillin, and cortactin, was also strictly dependent on the YxxP tyrosines. In contrast, matrix metalloproteinase-2 activation was most dependent on the CAS SH3 domain, whereas the substrate domain YxxP sites also contributed to this property. Thus multiple CAS-mediated signaling events are implicated in promoting invasive and metastatic properties of Src-transformed cells. (Mol Cancer Res 2005;3(6):307 -15)

Abstract-The increased expression of immediate-early genes is a key feature of the myocardial response to hypertrophic stimuli. In this study, we investigated whether pressure overload or phenylephrine treatment stimulated myocyte enhancer factor 2 (MEF2)-dependent transcriptional activation of c-jun in cardiac myocytes. Western blotting and immunohistochemical analysis of rat myocardium demonstrated that p70 MEF2 is highly expressed in the rat heart and is predominantly located at the nuclei of cardiac myocytes. Electrophoretic mobility shift assays of myocardial nuclear extracts revealed a consistent DNA binding activation of MEF2 after 1 and 2 hours of pressure overload. We further showed that pressure overload induced a progressive nuclear translocation and activation of extracellular signalregulated kinase 5 (ERK5). Coimmunoprecipitation and in vitro kinase assays indicated that the activation of ERK5 was paralleled by increased association of ERK5/p70 MEF2 and by enhanced ability of ERK5 to phosphorylate p70 MEF2 . Experiments with in vivo transfection of the left ventricle with the c-jun promoter reporter gene showed that pressure overload induced a consistent increase of c-jun transcriptional activity in the rat myocardium. Rendering the MEF2 site of the c-jun plasmid inactive by mutation abolished the load-induced activation of the c-jun promoter reporter gene. Mutation of the MEF2 site also abolished the phenylephrine-induced c-jun promoter activation in neonatal rat ventricular myocytes. In addition, we demonstrated that neonatal rat ventricular myocyte transfection with ERK5-antisense oligodeoxynucleotide inhibited the phenylephrine-induced c-jun promoter activation. These findings identify MEF2 as a potential regulator of c-jun transactivation and suggest that ERK5 might be an important mediator of MEF2 and c-jun promoter activation in response to hypertrophic stimuli in cardiac myocytes. L ong-term hemodynamic overload induces myocardial hypertrophy and remodeling of cardiac chambers that assume a key role in the compensation of the increased hemodynamic burden. 1 The initial response of cardiac myocytes to mechanical stress includes a coordinated rapid and transient activation of immediate-early genes, which is followed by activation of the fetal gene program and a timedependent increase in protein synthesis. 2 The immediateearly genes (ie, c-jun, c-fos, and c-myc) encode for transcription factors that are normally expressed at low levels in cardiac myocytes. Nevertheless, the signaling mechanisms involved in the regulation of these genes by hypertrophic stimuli remain to be determined.One such gene, c-jun, has been shown to be rapidly and transiently activated by mechanical stress in isolated myocytes 3 and myocardium. 4 The c-jun promoter contains binding sites for transcription factors such as Sp1, CTF, activator protein-1 (AP-1), and myocyte enhancer factor 2 (MEF2). 5 Binding of the AP-1 complex (c-Fos/ATF or c-Jun/c-Jun dimer) to the tissue plasminogen activator-responsive element in...