RESUMEN
Heart outflow tract septation in mouse embryos carrying mutations in retinoic acid receptor genes fails with complete penetrance. In this mutant background, ectopic TGFß signaling in the distal outflow tract is responsible for septation failure, but it was uncertain what tissue was responsive to ectopic TGFß and why this response interfered with septation. By combining RAR gene mutation with tissue-specific Cre drivers and a conditional type II TGFß receptor (Tgfbr2) allele, we determined that ectopic activation of TGFß signaling in the endocardium is responsible for septation defects. Ectopic TGFß signaling results in ectopic mesenchymal transformation of the endocardium and thereby in improperly constituted distal OFT cushions. Our analysis highlights the interactions between myocardium, endocardium, and neural crest cells in outflow tract morphogenesis, and demonstrates the requirement for proper TGFß signaling in outflow tract cushion organization and septation.
Asunto(s)
Endocardio/patología , Insuficiencia Cardíaca/patología , Defectos de los Tabiques Cardíacos/patología , Mesodermo/patología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Endocardio/embriología , Endocardio/metabolismo , Insuficiencia Cardíaca/embriología , Insuficiencia Cardíaca/metabolismo , Defectos de los Tabiques Cardíacos/embriología , Defectos de los Tabiques Cardíacos/metabolismo , Mesodermo/embriología , Ratones , Mutación/genética , Especificidad de Órganos , Fenotipo , Receptores de Ácido Retinoico/metabolismoRESUMEN
Fibroblast growth factor receptor 2 (FGFR2) is a crucial regulator of bone formation during embryonic development. Both gain and loss-of-function studies in mice have shown that FGFR2 maintains a critical balance between the proliferation and differentiation of osteoprogenitor cells. We have identified de novo FGFR2 mutations in a sporadically occurring perinatal lethal skeletal dysplasia characterized by poor mineralization of the calvarium, craniosynostosis, dysmorphic facial features, prenatal teeth, hypoplastic pubis and clavicles, osteopenia, and bent long bones. Histological analysis of the long bones revealed that the growth plate contained smaller hypertrophic chondrocytes and a thickened hypercellular periosteum. Four unrelated affected individuals were found to be heterozygous for missense mutations that introduce a polar amino acid into the hydrophobic transmembrane domain of FGFR2. Using diseased chondrocytes and a cell-based assay, we determined that these mutations selectively reduced plasma-membrane levels of FGFR2 and markedly diminished the receptor's responsiveness to extracellular FGF. All together, these clinical and molecular findings are separate from previously characterized FGFR2 disorders and represent a distinct skeletal dysplasia.
Asunto(s)
Enfermedades del Desarrollo Óseo/genética , Anomalías Craneofaciales/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Secuencia de Aminoácidos , Enfermedades del Desarrollo Óseo/metabolismo , Huesos/anomalías , Huesos/embriología , Huesos/metabolismo , Condrocitos/metabolismo , Anomalías Craneofaciales/metabolismo , Feto/anomalías , Feto/metabolismo , Factores de Crecimiento de Fibroblastos/deficiencia , Heterocigoto , Humanos , Datos de Secuencia Molecular , Mutación , Mutación Missense , Osteoblastos/metabolismo , Osteogénesis/genética , Transducción de Señal , EsqueletoRESUMEN
Members of the bone morphogenetic protein (BMP) superfamily, including transforming growth factor-betas (TGFß), regulate multiple aspects of chondrogenesis. Smad7 is an intracellular inhibitor of BMP and TGFß signaling. Studies in which Smad7 was overexpressed in chondrocytes demonstrated that Smad7 can impact chondrogenesis by inhibiting BMP signaling. However, whether Smad7 is actually required for endochondral ossification in vivo is unclear. Moreover, whether Smad7 regulates TGFß in addition to BMP signaling in developing cartilage is unknown. In this study, we found that Smad7 is required for both axial and appendicular skeletal development. Loss of Smad7 led to impairment of the cell cycle in chondrocytes and to defects in terminal maturation. This phenotype was attributed to upregulation of both BMP and TGFß signaling in Smad7 mutant growth plates. Moreover, Smad7-/- mice develop hypocellular cores in the medial growth plates, associated with elevated HIF1α levels, cell death, and intracellular retention of types II and X collagen. Thus, Smad7 may be required to mediate cell stress responses in the growth plate during development.
Asunto(s)
Condrocitos/citología , Placa de Crecimiento/metabolismo , Proteína smad7/genética , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Células Cultivadas , Condrocitos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Placa de Crecimiento/embriología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Noqueados , Osteogénesis , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
This study investigated interactions between the effects of mechanical stretch and thrombin on RhoA activation in rat aortic smooth muscle cells (RASMC). Equibiaxial, pulsatile stretch, or thrombin produced a significant increase in RhoA activation. Surprisingly, in combination, 30 min of stretch inhibited the ability of thrombin to activate RhoA. NO donors and 8-bromo-cGMP significantly inhibited thrombin-induced RhoA activation. Interestingly, the nitric oxide synthase (NOS) inhibitor L-NAME increased basal RhoA activity, suggesting that NOS activity exerts a tonic inhibition on RhoA. Stretching RASMC increases nitrite production, consistent with the idea that NO contributes to the inhibitory effects of stretch. Thrombin stimulates MAP kinase and NF-kappaB pathways through Rho and these responses were blocked by 8-bromo-cGMP or stretch and restored by L-NAME. These data suggest that stretch, acting through NO and cGMP, can prevent the ability of thrombin to stimulate Rho signaling pathways that contribute to pathophysiological proliferative and inflammatory responses.
Asunto(s)
Aorta/metabolismo , Miocitos del Músculo Liso/metabolismo , FN-kappa B/antagonistas & inhibidores , Trombina/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Animales , Aorta/citología , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas I-kappa B/metabolismo , FN-kappa B/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Resistencia a la Tracción , Trombina/farmacología , Trombina/fisiología , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
AIMS: The strategies that control formation of the ventricular wall during heart development are not well understood. In previous studies, we documented IGF2 as a major mitogenic signal that controls ventricular cardiomyocyte proliferation and chamber wall expansion. Our objective in this study was to define the tissue source of IGF2 in heart development and the upstream pathways that control its expression. METHODS AND RESULTS: Using a number of mouse genetic tools, we confirm that the critical source of IGF2 is the epicardium. We find that epicardial Igf2 expression is controlled in a biphasic manner, first induced by erythropoietin and then regulated by oxygen and glucose with onset of placental function. Both processes are independently controlled by retinoic acid signalling. CONCLUSIONS: Our results demonstrate that ventricular wall cardiomyocyte proliferation is subdivided into distinct regulatory phases. Each involves instructive cues that originate outside the heart and thereby act on the epicardium in an endocrine manner, a mode of regulation that is mostly unknown in embryogenesis.
Asunto(s)
Proliferación Celular , Ventrículos Cardíacos/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Miocitos Cardíacos/metabolismo , Pericardio/metabolismo , Transducción de Señal , Animales , Hipoxia de la Célula , Línea Celular , Eritropoyetina/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genotipo , Edad Gestacional , Glucosa/metabolismo , Ventrículos Cardíacos/embriología , Factor II del Crecimiento Similar a la Insulina/genética , Ratones Endogámicos ICR , Ratones Transgénicos , Organogénesis , Oxígeno/metabolismo , Pericardio/embriología , Fenotipo , Placenta/metabolismo , Embarazo , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptor alfa de Ácido RetinoicoRESUMEN
Bone morphogenetic protein (BMP) signaling pathways regulate multiple aspects of endochondral bone formation. The importance of extracellular antagonists as regulators of BMP signaling has been defined. In vitro studies reveal that the intracellular regulators, inhibitory Smads 6 and 7, can regulate BMP-mediated effects on chondrocytes. Although in vivo studies in which inhibitory Smads were overexpressed in cartilage have shown that inhibitory Smads have the potential to limit BMP signaling in vivo, the physiological relevance of inhibitory Smad activity in skeletal tissues is unknown. In this study, we have determined the role of Smad6 in endochondral bone formation. Loss of Smad6 in mice leads to defects in both axial and appendicular skeletal development. Specifically, Smad6-/- mice exhibit a posterior transformation of the seventh cervical vertebra, bilateral ossification centers in lumbar vertebrae, and bifid sternebrae due to incomplete sternal band fusion. Histological analysis of appendicular bones revealed delayed onset of hypertrophic differentiation and mineralization at midgestation in Smad6-/- mice. By late gestation, however, an expanded hypertrophic zone, associated with an increased pool of proliferating cells undergoing hypertrophy, was evident in Smad6 mutant growth plates. The mutant phenotype is attributed, at least in part, to increased BMP responsiveness in Smad6-deficient chondrocytes. Overall, our results show that Smad6 is required to limit BMP signaling during endochondral bone formation.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Cartílago/embriología , Transducción de Señal , Proteína smad6/fisiología , Animales , Apoptosis , Secuencia de Bases , Cartílago/citología , Proliferación Celular , Cartilla de ADN , Ratones , Ratones Noqueados , Reacción en Cadena de la PolimerasaRESUMEN
Collagen accumulation likely contributes to increased vascular and airway impedance in hypoxia-induced pulmonary hypertension (HPH). Collagen exists in multiple subtypes and can accumulate via increased synthesis or decreased degradation. To better understand the individual contributions of fibrillar (FB) and basement membrane (BM) collagen, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) to pulmonary vascular and airway remodeling in HPH, we investigated the temporal changes in gene and protein expression in the lungs of mice exposed to hypoxia for 0, 3, 6, 10 and 15 days. The earliest and largest change in gene expression was of type I FB collagen, which was significantly increased over control levels at 6, 10 and 15 days of hypoxia (p < 0.05). Type III FB and type IV BM collagen were increased at 10 and 15 days of hypoxia (p < 0.05); MMP and TIMP gene expression levels were typically higher but sometimes lower than control levels at various time points. Collagen protein content was increased in whole lungs as early as 6 days of hypoxia and increased monotonically with longer exposures. However, neither qualitative nor semi-quantitative analysis of immunohistochemistry demonstrated accumulation of type I FB collagen in compartments of the lung other than large airways, suggesting that other collagen subtypes may be important contributors to collagen protein accumulation. These results provide insight into the patterns of gene and protein expression relevant to collagen accumulation in the lung in response to chronic hypoxia, through which we can develop a better understanding of the time course of changes in matrix biology and biomechanics that occur in HPH.
Asunto(s)
Colágeno/metabolismo , Regulación de la Expresión Génica , Hipoxia , Pulmón/metabolismo , Animales , Fenómenos Biomecánicos , Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Hidroxiprolina/metabolismo , Hipertensión Pulmonar/metabolismo , Inmunohistoquímica/métodos , Pulmón/embriología , Masculino , Ratones , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
Smad proteins are intracellular molecules that mediate the canonical signaling cascade of TGFbeta superfamily growth factors. The TGFbeta superfamily comprises two groups of growth factors, BMPs and TGFbetas. Both groups can be further divided into several sub-groups based on sequence homologies and functional similarities. Ligands of the TGFbeta superfamily bind to cell surface receptors to activate Smad proteins in the cytoplasm; then the activated Smad proteins translocate into the nucleus to activate or repress specific target gene transcription. Both groups of growth factors play important roles in skeletal development and regeneration. However, whether these effects reflect signaling through canonical Smad pathways, or other non-canonical signaling pathways in vivo remains a mystery. Moreover, the mechanisms utilized by Smad proteins to initiate nuclear events and their interactions with cytoplasmic proteins are still under intensive investigation. This review will discuss the most recent progress understanding Smad signaling in the context of skeletal development and regeneration.