RESUMEN
OBJECTIVE: The investment of newly formed endothelial cell tubes with differentiated smooth muscle cells (SMC) is critical for appropriate vessel formation, but the underlying mechanisms remain unknown. We previously showed that depletion of focal adhesion kinase (FAK) in the nkx2.5 expression domain led to aberrant outflow tract (OFT) morphogenesis and strove herein to determine the cell types and mechanisms involved. METHODS AND RESULTS: We crossed fak(loxp) targeted mice with available Cre drivers to deplete FAK in OFT SMC (FAK(wnt) and FAK(nk)) or coronary SMC (FAK(cSMC)). In each case, depletion of FAK led to defective vasculogenesis that was incompatible with postnatal life. Immunohistochemical analysis of the mutant vascular structures revealed that FAK was not required for progenitor cell proliferation, survival, or differentiation into SMC but was necessary for subsequent SMC recruitment to developing vasculature. Using a novel FAK-null SMC culture model, we found that depletion of FAK did not influence SMC growth or survival, but blocked directional SMC motility and invasion toward the potent endothelial-derived chemokine, platelet-derived growth factor PDGFBB. FAK depletion resulted in unstable lamellipodial protrusions due to defective spatial-temporal activation of the small GTPase, Rac-1, and lack of Rac1-dependent recruitment of cortactin (an actin stabilizing protein) to the leading edge. Moreover, FAK null SMC exhibited a significant reduction in stimulated extracellular matrix degradation. CONCLUSIONS: FAK drives PDGFBB-stimulated SMC chemotaxis/invasion and is essential for SMC to appropriately populate the aorticopulmonary septum and the coronary vascular plexus.
Asunto(s)
Quimiotaxis , Quinasa 1 de Adhesión Focal/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Neovascularización Fisiológica , Animales , Aorta/embriología , Aorta/enzimología , Apoptosis , Becaplermina , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Quimiotaxis/genética , Vasos Coronarios/embriología , Vasos Coronarios/enzimología , Cortactina/metabolismo , Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Quinasa 1 de Adhesión Focal/deficiencia , Quinasa 1 de Adhesión Focal/genética , Regulación del Desarrollo de la Expresión Génica , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Músculo Liso Vascular/embriología , Neovascularización Fisiológica/genética , Neuropéptidos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-sis , Seudópodos/enzimología , Arteria Pulmonar/embriología , Arteria Pulmonar/enzimología , Codorniz/embriología , Interferencia de ARN , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1RESUMEN
OBJECTIVE: Smooth muscle cell (SMC) differentiation is a dynamic process that must be tightly regulated for proper vascular development and to control the onset of vascular disease. Our laboratory previously reported that a specific focal adhesion kinase (FAK) inhibitor termed FRNK (FAK Related Non-Kinase) is selectively expressed in large arterioles when SMCs are transitioning from a synthetic to contractile phenotype and that FRNK inhibits FAK-dependent SMC proliferation and migration. Herein, we sought to determine whether FRNK expression modulates SMC phenotypes in vivo. METHODS AND RESULTS: We present evidence that FRNK(-/-) mice exhibit attenuated SM marker gene expression during postnatal vessel growth and after vascular injury. We also show that FRNK expression is regulated by transforming growth factor (TGF)-beta and that forced expression of FRNK in cultured cells induces serum- and TGF-beta-stimulated SM marker gene expression, whereas FRNK deletion or expression of a constitutively activated FAK variant attenuated SM gene transcription. CONCLUSIONS: These data highlight the possibility that extrinsic signals regulate the SMC gene profile, at least in part, by modulating the expression of FRNK and that tight regulation of FAK activity by FRNK is important for proper SMC differentiation during development and after vascular injury.
Asunto(s)
Vasos Sanguíneos/citología , Vasos Sanguíneos/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Animales , Vasos Sanguíneos/crecimiento & desarrollo , Vasos Sanguíneos/lesiones , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas Tirosina Quinasas/deficiencia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Leupaxin is a LIM domain-containing adapter protein belonging to the paxillin family that has been previously reported to be preferentially expressed in hematopoietic cells. Herein, we identified leupaxin in a screen for focal adhesion kinase binding partners in aortic smooth muscle, and we show that leupaxin is enriched in human and mouse vascular smooth muscle and that leupaxin expression is dynamically regulated during development. In addition, our studies reveal that leupaxin can undergo cytoplasmic/nuclear shuttling and functions as an serum response factor cofactor in the nucleus. We found that leupaxin forms a complex with serum response factor and associates with CArG-containing regions of smooth muscle promoters and that ectopic expression of leupaxin induces smooth muscle marker gene expression in both 10T1/2 cells and rat aortic smooth muscle cells. Subsequent studies indicated that enhanced focal adhesion kinase activity (induced by fibronectin or expression of constitutively active focal adhesion kinase) attenuates the nuclear accumulation of leupaxin and limits the ability of leupaxin to enhance serum response factor-dependent gene transcription. Thus, these studies indicate that modulation of the subcellular localization of serum response factor cofactors is 1 mechanism by which extracellular matrix-dependent signals may regulate phenotypic switching of smooth muscle cells.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Miocitos del Músculo Liso/metabolismo , Fosfoproteínas/fisiología , Elemento de Respuesta al Suero/fisiología , Animales , Aorta/citología , Aorta/embriología , Aorta/crecimiento & desarrollo , Transporte Biológico , Moléculas de Adhesión Celular/farmacología , Diferenciación Celular , Células Cultivadas/efectos de los fármacos , Vasos Coronarios/citología , Femenino , Quinasa 1 de Adhesión Focal/fisiología , Adhesiones Focales/fisiología , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Persona de Mediana Edad , Miocitos del Músculo Liso/efectos de los fármacos , Miometrio/citología , Especificidad de Órganos , Fosfoproteínas/farmacología , Mapeo de Interacción de Proteínas , Ratas , Proteínas Recombinantes de Fusión/fisiología , Elemento de Respuesta al Suero/efectos de los fármacos , Factor de Respuesta Sérica/fisiología , Transducción de Señal/fisiología , Factores de Transcripción , Transcripción GenéticaRESUMEN
The Rac1/Cdc42 effector p21-activated kinase (PAK) is activated by various signaling cascades including receptor-tyrosine kinases and integrins and regulates a number of processes such as cell proliferation and motility. PAK activity has been shown to be required for maximal activation of the canonical Ras/Raf/MEK/ERK Map kinase signaling cascade, likely because of PAK co-activation of Raf and MEK. Herein, we found that adhesion signaling also stimulates an association between PAK1 and ERK1/2. PAK1 and ERK1/2 co-immunoprecipitated from rat aortic smooth muscle cells (SMC) plated on fibronectin, and the two proteins co-localized in membrane ruffles and adhesion complexes following PDGF-BB or sphingosine 1-phosphate treatment, respectively. Far Western analysis demonstrated a direct association between the two proteins, and peptide mapping identified an ERK2 binding site within the autoinhibitory domain of PAK1. Interestingly, deletion of a major ERK binding site in PAK attenuates activation of an ERK-dependent serum-responsive element (SRE)-luciferase reporter gene, indicating that association between PAK and ERK is required to facilitate ERK signaling. We also show that ERK2 phosphorylates PAK1 on Thr(212) in vitro and that Thr(212) is phosphorylated in smooth muscle cells following PDGF-BB treatment in an adhesion- and MEK/ERK-dependent fashion. Expression of a phosphomimic variant, PAK-T212E, does not alter ERK association, but markedly attenuates downstream ERK signaling. Taken together, these data suggest that PAK1 may facilitate ERK signaling by serving as a scaffold to recruit Raf, MEK, and ERK to adhesion complexes, and that subsequent growth factor-stimulated phosphorylation of PAK-Thr(212) by ERK may serve to provide a negative feedback signal to control coordinate activation of ERK by growth factor- and matrix-induced signals.