RESUMO
BACKGROUND: Cleft palate occurs in up to 1:1,000 live births and is associated with mutations in multiple genes. Palatogenesis involves a complex choreography of palatal shelf elongation, elevation, and fusion. Transforming growth factor ß (TGFß) and bone morphogenetic protein 2 (BMP2) canonical signaling is required during each stage of palate development. The type III TGFß receptor (TGFßR3) binds all three TGFß ligands and BMP2, but its contribution to palatogenesis is unknown. RESULTS: The role of TGFßR3 during palate formation was found to be during palatal shelf elongation and elevation. Tgfbr3(-) (/) (-) embryos displayed reduced palatal shelf width and height, changes in proliferation and apoptosis, and reduced vascular and osteoblast differentiation. Abnormal vascular plexus organization as well as aberrant expression of arterial (Notch1, Alk1), venous (EphB4), and lymphatic (Lyve1) markers was also observed. Decreased osteoblast differentiation factors (Runx2, alk phos, osteocalcin, col1A1, and col1A2) demonstrated poor mesenchymal cell commitment to the osteoblast lineage within the maxilla and palatal shelves in Tgfbr3(-) (/) (-) embryos. Additionally, in vitro bone mineralization induced by osteogenic medium (OM+BMP2) was insufficient in Tgfbr3(-) (/) (-) palatal mesenchyme, but mineralization was rescued by overexpression of TGFßR3. CONCLUSIONS: These data reveal a critical, previously unrecognized role for TGFßR3 in vascular and osteoblast development during palatogenesis.
Assuntos
Calcificação Fisiológica/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neovascularização Fisiológica/fisiologia , Organogênese/fisiologia , Osteoblastos/metabolismo , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Animais , Antígenos de Diferenciação/biossíntese , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Palato Duro/irrigação sanguínea , Palato Duro/citologia , Palato Duro/embriologia , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/genéticaRESUMO
The epicardium is a major contributor of the cells that are required for the formation of coronary vessels. Mice lacking both copies of the gene encoding the Type III Transforming Growth Factor ß Receptor (TGFßR3) fail to form the coronary vasculature, but the molecular mechanism by which TGFßR3 signals coronary vessel formation is unknown. We used intact embryos and epicardial cells from E11.5 mouse embryos to reveal the mechanisms by which TGFßR3 signals and regulates epicardial cell behavior. Analysis of E13.5 embryos reveals a lower rate of epicardial cell proliferation and decreased epicardially derived cell invasion in Tgfbr3(-/-) hearts. Tgfbr3(-/-) epicardial cells in vitro show decreased proliferation and decreased invasion in response to TGFß1 and TGFß2. Unexpectedly, loss of TGFßR3 also decreases responsiveness to two other important regulators of epicardial cell behavior, FGF2 and HMW-HA. Restoring full length TGFßR3 in Tgfbr3(-/-) cells rescued deficits in invasion in vitro in response TGFß1 and TGFß2 as well as FGF2 and HMW-HA. Expression of TGFßR3 missing the 3 C-terminal amino acids that are required to interact with the scaffolding protein GIPC1 did not rescue any of the deficits. Overexpression of GIPC1 alone in Tgfbr3(-/-) cells did not rescue invasion whereas knockdown of GIPC1 in Tgfbr3(+/+) cells decreased invasion in response to TGFß2, FGF2, and HMW-HA. We conclude that TGFßR3 interaction with GIPC1 is critical for regulating invasion and growth factor responsiveness in epicardial cells and that dysregulation of epicardial cell proliferation and invasion contributes to failed coronary vessel development in Tgfbr3(-/-) mice.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Pericárdio/citologia , Pericárdio/metabolismo , Proteoglicanas/química , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/química , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sequência de Bases , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Anomalias dos Vasos Coronários/embriologia , Anomalias dos Vasos Coronários/genética , Anomalias dos Vasos Coronários/metabolismo , Primers do DNA/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Cardiovasculares , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/genética , Pericárdio/embriologia , Gravidez , Domínios e Motivos de Interação entre Proteínas , Proteoglicanas/deficiência , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/deficiência , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais , Imagem com Lapso de Tempo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta2/farmacologiaRESUMO
Valvular heart disease is a major cause of mortality and morbidity. Revealing the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. A key step in valve formation during heart development is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endocardial cells in the atrioventricular cushion (AVC). The type III transforming growth factor-ß receptor (TGFßR3) regulates AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Little is known concerning the signaling mechanisms downstream of TGFßR3. Here we use endocardial cell EMT in vitro to determine the role of 2 well-characterized downstream TGFß signaling pathways in TGFßR3-dependent endocardial cell EMT. Targeting of Smad4, the common mediator Smad, demonstrated that Smad signaling is required for EMT in the AVC and TGFßR3-dependent EMT stimulated by TGFß2 or BMP-2. Although we show that Smads 1, 2, 3, and 5 are required for AVC EMT, overexpression of Smad1 or Smad3 is not sufficient to induce EMT. Consistent with the activation of the Par6/Smurf1 pathway downstream of TGFßR3, targeting ALK5, Par6, or Smurf1 significantly inhibited EMT in response to either TGFß2 or BMP-2. The requirement for ALK5 activity, Par6, and Smurf1 for TGFßR3-dependent endocardial cell EMT is consistent with the documented role of this pathway in the dissolution of tight junctions. Taken together, our data demonstrate that TGFßR3-dependent endocardial cell EMT stimulated by either TGFß2 or BMP-2 requires Smad4 and the activation of the Par6/Smurf1 pathway.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Endocárdio/citologia , Transdução de Sinais , Fator de Crescimento Transformador beta2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Embrião de Galinha/metabolismo , Endocárdio/metabolismo , Humanos , Proteína Smad4/genética , Proteína Smad4/metabolismo , Transfecção , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismoAssuntos
Reanimação Cardiopulmonar/educação , Competência Clínica , Educação Continuada em Enfermagem/organização & administração , Recursos Humanos de Enfermagem Hospitalar/educação , Desempenho de Papéis , Doença Aguda/enfermagem , Atitude do Pessoal de Saúde , Equipe de Respostas Rápidas de Hospitais/organização & administração , Humanos , Kansas , Pesquisa em Educação em Enfermagem , Recursos Humanos de Enfermagem Hospitalar/psicologia , Objetivos Organizacionais , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , AutoeficáciaRESUMO
Maxillary hypoplasia occurs due to insufficient maxillary intramembranous ossification, leading to poor dental occlusion, respiratory obstruction and cosmetic deformities. Conditional deletion of Jagged1 (Jag1) in cranial neural crest (CNC) cells using Wnt1-cre; Jagged1(f/f) (Jag1CKO) led to maxillary hypoplasia characterized by intrinsic differences in bone morphology and density using µCT evaluation. Jag1CKO maxillas revealed altered collagen deposition, delayed ossification, and reduced expression of early and late determinants of osteoblast development during maxillary ossification. In vitro bone cultures on Jag1CKO mouse embryonic maxillary mesenchymal (MEMM) cells demonstrated decreased mineralization that was also associated with diminished induction of osteoblast determinants. BMP receptor expression was dysregulated in the Jag1CKO MEMM cells suggesting that these cells were unable to respond to BMP-induced differentiation. JAG1-Fc rescued in vitro mineralization and osteoblast gene expression changes. These data suggest that JAG1 signaling in CNC-derived MEMM cells is required for osteoblast development and differentiation during maxillary ossification.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Maxila/metabolismo , Maxila/fisiologia , Proteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Osteogênese , Animais , Densidade Óssea , Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica , Cálcio/metabolismo , Diferenciação Celular , Embrião de Mamíferos/patologia , Proteína Jagged-1 , Maxila/diagnóstico por imagem , Maxila/embriologia , Mesoderma/citologia , Mesoderma/embriologia , Camundongos Knockout , Tamanho do Órgão , Palato/metabolismo , Receptores Fc/metabolismo , Receptores Notch/metabolismo , Proteínas Serrate-Jagged , Transdução de Sinais , Microtomografia por Raio-XRESUMO
Coronary vessel development depends on a subpopulation of epicardial cells that undergo epithelial to mesenchymal transformation (EMT) and invade the subepicardial space and myocardium. These cells form the smooth muscle of the vessels and fibroblasts, but the mechanisms that regulate these processes are poorly understood. Mice lacking the Type III Transforming Growth Factor ß Receptor (TGFßR3) die by E14.5 due to failed coronary vessel development accompanied by reduced epicardial cell invasion. BMP2 signals via TGFßR3 emphasizing the importance of determining the relative contributions of the canonical BMP signaling pathway and TGFßR3-dependent signaling to BMP2 responsiveness. Here we examined the role of TGFßR3 in BMP2 signaling in epicardial cells. Whereas TGFß induced loss of epithelial character and smooth muscle differentiation, BMP2 induced an ALK3-dependent loss of epithelial character and modestly inhibited TGFß-stimulated differentiation. Tgfbr3(-/-) cells respond to BMP2 indicating that TGFßR3 is not required. However, Tgfbr3(-/-) cells show decreased invasion in response to BMP2 and overexpression of TGFßR3 in Tgfbr3(-/-) cells rescued invasion. Invasion was dependent on ALK5, ALK2, ALK3, and Smad4. Expression of TGFßR3 lacking the 3 C-terminal amino acids required to interact with the scaffolding protein GIPC (GAIP-interacting protein, C terminus) did not rescue. Knockdown of GIPC in Tgfbr3(+/+) or Tgfbr3(-/-) cells rescued with TGFßR3 decreased BMP2-stimulated invasion confirming a requirement for TGFßR3/GIPC interaction. Our results reveal the relative roles of TGFßR3-dependent and TGFßR3-independent signaling in the actions of BMP2 on epicardial cell behavior and demonstrate the critical role of TGFßR3 in mediating BMP2-stimulated invasion.