RESUMO
Cardiac remodeling, a hallmark of heart disease, is associated with intense auto- and paracrine signaling leading to cardiac fibrosis. We hypothesized that the specific mediator of Gq/11-dependent RhoA activation p63RhoGEF, which is expressed in cardiac fibroblasts, plays a role in the underlying processes. We could show that p63RhoGEF is up-regulated in mouse hearts subjected to transverse aortic constriction (TAC). In an engineered heart muscle model (EHM), p63RhoGEF expression in cardiac fibroblasts increased resting and twitch tensions, and the dominant negative p63ΔN decreased both. In an engineered connective tissue model (ECT), p63RhoGEF increased tissue stiffness and its knockdown as well as p63ΔN reduced stiffness. In 2D cultures of neonatal rat cardiac fibroblasts, p63RhoGEF regulated the angiotensin II (Ang II)-dependent RhoA activation, the activation of the serum response factor, and the expression and secretion of the connective tissue growth factor (CTGF). All these processes were inhibited by the knockdown of p63RhoGEF or by p63ΔN likely based on their negative influence on the actin cytoskeleton. Moreover, we show that p63RhoGEF also regulates CTGF in engineered tissues and correlates with it in the TAC model. Finally, confocal studies revealed a closely related localization of p63RhoGEF and CTGF in the trans-Golgi network.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Fibroblastos/metabolismo , Miocárdio/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fator de Resposta Sérica/genética , Proteína rhoA de Ligação ao GTP/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Angiotensina II/genética , Angiotensina II/metabolismo , Animais , Animais Recém-Nascidos , Aorta/cirurgia , Comunicação Autócrina/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Constrição , Feminino , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Cardiovasculares , Miocárdio/patologia , Comunicação Parácrina/genética , Ratos , Ratos Wistar , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Remodelação Ventricular , Proteína rhoA de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestruturaRESUMO
Activation of α(1)-adrenoceptors (α(1)-AR) by high catecholamine levels, e.g. in heart failure, is thought to be a driving force of cardiac hypertrophy. In this context several downstream mediators and cascades have been identified to potentially play a role in cardiomyocyte hypertrophy. One of these proteins is the monomeric G protein Rac1. However, until now it is unclear how this essential G protein is activated by α(1)-AR agonists and what are the downstream targets inducing cellular growth. By using protein-based as well as pharmacological inhibitors and the shRNA technique, we demonstrate that in neonatal rat cardiomyocytes (NRCM) Rac1 is activated via a cascade involving the α(1A)-AR subtype, G(i)ßγ, the phosphoinositide-3'-kinase and the guanine nucleotide exchange factor Tiam1. We further demonstrate that this signaling induces an increase in protein synthesis, cell size and atrial natriuretic peptide expression. We identified the p21-activated kinase 2 (PAK2) as a downstream effector of Rac1 and were able to link this cascade to the activation of the pro-hypertrophic kinases ERK1/2 and p90RSK. Our data thus reveal a prominent role of the α(1A)-AR/G(i)ßγ/Tiam1-mediated activation of Rac1 and its effector PAK2 in the induction of hypertrophy in NRCM.
Assuntos
Cardiomegalia/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Aminoquinolinas/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Eletroforese em Gel Bidimensional , Fatores de Troca do Nucleotídeo Guanina/genética , Immunoblotting , Miócitos Cardíacos/efeitos dos fármacos , Proteínas de Neoplasias/genética , Fenilefrina/farmacologia , Pirimidinas/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células TRESUMO
The purpose of our study was to investigate the role of endogenous p63RhoGEF in G(q/11)-dependent RhoA activation and signaling in rat aortic smooth muscle cells (RASMCs). Therefore, we studied the expression and subcellular localization in freshly isolated RASMCs and performed loss of function experiments to analyze its contribution to RhoGTPase activation and functional responses such as proliferation and contraction. By this, we could show that p63RhoGEF is endogenously expressed in RASMCs and acts there as the dominant mediator of the fast angiotensin II (ANG II)-dependent but not of the sphingosine-1-phosphate (S(1)P)-dependent RhoA activation. p63RhoGEF is not an activator of the concomitant Rac1 activation and functions independently of caveolae. The knockdown of endogenous p63RhoGEF significantly reduced the mitogenic response of ANG II, abolished ANG II-induced stress fiber formation and cell elongation in 2-D culture, and impaired the ANG II-driven contraction in a collagen-based 3-D model. In conclusion, our data provide for the first time evidence that p63RhoGEF is an important mediator of ANG II-dependent RhoA activation in RASMCs and therewith a leading actor in the subsequently triggered cellular processes, such as proliferation and contraction.
Assuntos
Angiotensina II/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Benzimidazóis/farmacologia , Benzoatos/farmacologia , Cálcio/metabolismo , Células Cultivadas , Endotelina-1/farmacologia , Imunofluorescência , Fatores de Troca do Nucleotídeo Guanina/genética , Imidazóis/farmacologia , Immunoblotting , Imuno-Histoquímica , Lisofosfolipídeos/farmacologia , Piridinas/farmacologia , Ratos , Ratos Wistar , Serotonina/farmacologia , Esfingosina/análogos & derivados , Esfingosina/farmacologia , TelmisartanRESUMO
INTRODUCTION: RhoA has been shown to be beneficial in cardiac disease models when overexpressed in cardiomyocytes, whereas its role in cardiac fibroblasts (CF) is still poorly understood. During cardiac remodeling CF undergo a transition towards a myofibroblast phenotype thereby showing an increased proliferation and migration rate. Both processes involve the remodeling of the cytoskeleton. Since RhoA is known to be a major regulator of the cytoskeleton, we analyzed its role in CF and its effect on myofibroblast characteristics in 2 D and 3D models. RESULTS: Downregulation of RhoA was shown to strongly affect the actin cytoskeleton. It decreased the myofibroblast marker α-sm-actin, but increased certain fibrosis-associated factors like TGF-ß and collagens. Also, the detailed analysis of CTGF expression demonstrated that the outcome of RhoA signaling strongly depends on the involved stimulus. Furthermore, we show that proliferation of myofibroblasts rely on RhoA and tubulin acetylation. In assays accessing three different types of migration, we demonstrate that RhoA/ROCK/Dia1 are important for 2D migration and the repression of RhoA and Dia1 signaling accelerates 3D migration. Finally, we show that a downregulation of RhoA in CF impacts the viscoelastic and contractile properties of engineered tissues. CONCLUSION: RhoA positively and negatively influences myofibroblast characteristics by differential signaling cascades and depending on environmental conditions. These include gene expression, migration and proliferation. Reduction of RhoA leads to an increased viscoelasticity and a decrease in contractile force in engineered cardiac tissue.
Assuntos
Miofibroblastos/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas/fisiologia , Reação em Cadeia da Polimerase , Ratos , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Cells can adapt to hypoxia by various mechanisms. Yet, hypoxia-induced effects on the cytoskeleton-based cell architecture and functions are largely unknown. Here we present a comprehensive analysis of the architecture and function of L929 fibroblasts under hypoxic conditions (1% O2). Cells cultivated in hypoxia showed striking morphological differences as compared to cells cultivated under normoxic conditions (20% O2). These changes include an enlargement of cell area and volume, increased numbers of focal contacts and loss of cell polarization. Furthermore the ß- and γ-actin distribution is greatly altered. These hypoxic adjustments are associated with enhanced cell spreading and a decline of cell motility in wound closure and single cell motility assays. As the hypoxia-inducible factor-1α (HIF-1α) is stabilised in hypoxia and plays a pivotal role in the transcriptional response to changes in oxygen availability we used an shRNA-approach to examine the role of HIF-1α in cytoskeleton-related architecture and functions. We show that the observed increase in cell area, actin filament rearrangement, decrease of single cell migration in hypoxia and the maintenance of p-cofilin levels is dependent on HIF-1α stabilisation.