RESUMO
Adhesion between stem cells and their niche provides stable anchorage and signaling cues to sustain properties such as quiescence. Skeletal muscle stem cells (MuSCs) adhere to an adjacent myofiber via cadherin-catenin complexes. Previous studies on N- and M-cadherin in MuSCs revealed that although N-cadherin is required for quiescence, they are collectively dispensable for MuSC niche localization and regenerative activity. Although additional cadherins are expressed at low levels, these findings raise the possibility that cadherins are unnecessary for MuSC anchorage to the niche. To address this question, we conditionally removed from MuSCs ß- and γ-catenin, and, separately, αE- and αT-catenin, factors that are essential for cadherin-dependent adhesion. Catenin-deficient MuSCs break quiescence similarly to N-/M-cadherin-deficient MuSCs, but exit the niche and are depleted. Combined in vivo, ex vivo and single cell RNA-sequencing approaches reveal that MuSC attrition occurs via precocious differentiation, re-entry to the niche and fusion to myofibers. These findings indicate that cadherin-catenin-dependent adhesion is required for anchorage of MuSCs to their niche and for preservation of the stem cell compartment. Furthermore, separable cadherin-regulated functions govern niche localization, quiescence and MuSC maintenance.
Assuntos
Caderinas , Nicho de Células-Tronco , Nicho de Células-Tronco/genética , Caderinas/genética , Caderinas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Transdução de Sinais , Cateninas/genética , Cateninas/metabolismo , Músculo Esquelético/metabolismo , Adesão Celular/genéticaRESUMO
The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state results in the loss of regenerative potential of the mammalian heart shortly after birth. Nonmuscle myosin IIB (NM IIB)-mediated actomyosin contractility regulates cardiomyocyte cytokinesis in the embryonic heart, and NM IIB levels decline after birth, suggesting a role for cellular tension in the regulation of cardiomyocyte cell cycle activity in the postnatal heart. To investigate the role of actomyosin contractility in cardiomyocyte cell cycle arrest, we conditionally activated ROCK2 kinase domain (ROCK2:ER) in the murine postnatal heart. Here, we show that α5/ß1 integrin and fibronectin matrix increase in response to actomyosin-mediated tension. Moreover, activation of ROCK2:ER promotes nuclear translocation of Yap, a mechanosensitive transcriptional co-activator, and enhances cardiomyocyte proliferation. Finally, we show that reduction of myocardial α5 integrin rescues the myocardial proliferation phenotype in ROCK2:ER hearts. These data demonstrate that cardiomyocytes respond to increased intracellular tension by altering their intercellular contacts in favor of cell-matrix interactions, leading to Yap nuclear translocation, thus uncovering a function for nonmuscle myosin contractility in promoting cardiomyocyte proliferation in the postnatal heart.
Assuntos
Actomiosina , Integrina alfa5 , Animais , Camundongos , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Proliferação de Células , Integrina alfa5/metabolismo , Mamíferos/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The roles of cellular orientation during trabecular and ventricular wall morphogenesis are unknown, and so are the underlying mechanisms that regulate cellular orientation. Myocardial-specific Numb and Numblike double-knockout (MDKO) hearts display a variety of defects, including in cellular orientation, patterns of mitotic spindle orientation, trabeculation, and ventricular compaction. Furthermore, Numb- and Numblike-null cardiomyocytes exhibit cellular behaviors distinct from those of control cells during trabecular morphogenesis based on single-cell lineage tracing. We investigated how Numb regulates cellular orientation and behaviors and determined that N-cadherin levels and membrane localization are reduced in MDKO hearts. To determine how Numb regulates N-cadherin membrane localization, we generated an mCherry:Numb knockin line and found that Numb localized to diverse endocytic organelles but mainly to the recycling endosome. Consistent with this localization, cardiomyocytes in MDKO did not display defects in N-cadherin internalization but rather in postendocytic recycling to the plasma membrane. Furthermore, N-cadherin overexpression via a mosaic model partially rescued the defects in cellular orientation and trabeculation of MDKO hearts. Our study unravels a phenomenon that cardiomyocytes display spatiotemporal cellular orientation during ventricular wall morphogenesis, and its disruption leads to abnormal trabecular and ventricular wall morphogenesis. Furthermore, we established a mechanism by which Numb modulates cellular orientation and consequently trabecular and ventricular wall morphogenesis by regulating N-cadherin recycling to the plasma membrane.
Assuntos
Caderinas/metabolismo , Ventrículos do Coração/embriologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Organogênese , Animais , Caderinas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Proteínas do Tecido Nervoso/genéticaRESUMO
Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. At the same time, the N-cadherin/catenin cell adhesion complex accumulates at the cell termini, creating a specialized type of cell-cell contact called the intercalated disc (ICD). To investigate the relationship between ICD maturation and proliferation, αE-catenin (Ctnna1) and αT-catenin (Ctnna3) genes were deleted to generate cardiac-specific α-catenin double knockout (DKO) mice. DKO mice exhibited aberrant N-cadherin expression, mislocalized actomyosin activity and increased cardiomyocyte proliferation that was dependent on Yap activity. To assess effects on tension, cardiomyocytes were cultured on deformable polyacrylamide hydrogels of varying stiffness. When grown on a stiff substrate, DKO cardiomyocytes exhibited increased cell spreading, nuclear Yap and proliferation. A low dose of either a myosin or RhoA inhibitor was sufficient to block Yap accumulation in the nucleus. Finally, activation of RhoA was sufficient to increase nuclear Yap in wild-type cardiomyocytes. These data demonstrate that α-catenins regulate ICD maturation and actomyosin contractility, which, in turn, control Yap subcellular localization, thus providing an explanation for the loss of proliferative capacity in the newborn mammalian heart.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citoesqueleto/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , alfa Catenina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Animais Recém-Nascidos , Comunicação Celular/genética , Proteínas de Ciclo Celular , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/fisiologia , Fosfoproteínas/fisiologia , Proteínas de Sinalização YAP , alfa Catenina/genéticaRESUMO
Tissue development and regeneration involve high-ordered morphogenetic processes that are governed by elements of the cytoskeleton in conjunction with cell adhesion molecules. Such processes are particularly important in the lens whose structure dictates its function. Studies of our lens-specific N-cadherin conditional knockout mouse (N-cadcKO) revealed an essential role for N-cadherin in the migration of the apical tips of differentiating lens fiber cells along the apical surfaces of the epithelium, a region termed the Epithelial Fiber Interface (EFI), that is necessary for normal fiber cell elongation and the morphogenesis. Studies of the N-cadcKO lens suggest that N-cadherin function in fiber cell morphogenesis is linked to the activation of Rac1 and myosin II, both signaling pathways central to the regulation of cell motility including determining the directionality of cellular movement. The absence of N-cadherin did not disrupt lateral contacts between fiber cells during development, and the maintenance of Aquaporin-0 and increased expression of EphA2 at cell-cell interfaces suggests that these molecules may function in this role. E-cadherin was maintained in newly differentiating fiber cells without interfering with expression of lens-specific differentiation proteins but was not able to replace N-cadherin function in these cells. The dependence of migration of the fiber cell apical domains along the EFI for lens morphogenesis on N-cadherin provides new insight into the process of tissue development.
Assuntos
Caderinas/metabolismo , Diferenciação Celular/fisiologia , Células Epiteliais/citologia , Cristalino/embriologia , Morfogênese/fisiologia , Animais , Aquaporinas/metabolismo , Caderinas/genética , Movimento Celular/genética , Ativação Enzimática , Epitélio/fisiologia , Proteínas do Olho/metabolismo , Cristalino/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miosina Tipo II/metabolismo , Neuropeptídeos/metabolismo , Receptor EphA2/biossíntese , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
RATIONALE: Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. Thus, they are unable to effectively replace dying cells in the injured heart. The recent discovery that the transcriptional coactivator Yes-associated protein (Yap) is necessary and sufficient for cardiomyocyte proliferation has gained considerable attention. However, the upstream regulators and signaling pathways that control Yap activity in the heart are poorly understood. OBJECTIVE: To investigate the role of α-catenins in the heart using cardiac-specific αE- and αT-catenin double knockout mice. METHODS AND RESULTS: We used 2 cardiac-specific Cre transgenes to delete both αE-catenin (Ctnna1) and αT-catenin (Ctnna3) genes either in the perinatal or in the adult heart. Perinatal depletion of α-catenins increased cardiomyocyte number in the postnatal heart. Increased nuclear Yap and the cell cycle regulator cyclin D1 accompanied cardiomyocyte proliferation in the α-catenin double knockout hearts. Fetal genes were increased in the α-catenin double knockout hearts indicating a less mature cardiac gene expression profile. Knockdown of α-catenins in neonatal rat cardiomyocytes also resulted in increased proliferation, which could be blocked by knockdown of Yap. Finally, inactivation of α-catenins in the adult heart using an inducible Cre led to increased nuclear Yap and cardiomyocyte proliferation and improved contractility after myocardial infarction. CONCLUSIONS: These studies demonstrate that α-catenins are critical regulators of Yap, a transcriptional coactivator essential for cardiomyocyte proliferation. Furthermore, we provide proof of concept that inhibiting α-catenins might be a useful strategy to promote myocardial regeneration after injury.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células/fisiologia , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , alfa Catenina/fisiologia , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular , Células Cultivadas , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Proteínas de Sinalização YAPRESUMO
Strong cell-cell adhesion mediated by adherens junctions is dependent on anchoring the transmembrane cadherin molecule to the underlying actin cytoskeleton. To do this, the cadherin cytoplasmic domain interacts with catenin proteins, which include α-catenin that binds directly to filamentous actin. Originally thought to be a static structure, the connection between the cadherin/catenin adhesion complex and the actin cytoskeleton is now considered to be dynamic and responsive to both intercellular and intracellular signals. Alpha-catenins are mechanosensing proteins that undergo conformational change in response to cytoskeletal tension thus modifying the linkage between the cadherin and the actin cytoskeleton. There are three α-catenin isoforms expressed in mouse and human: αE-catenin (CTNNA1), αN-catenin (CTNNA2) and αT-catenin (CTNNA3). This review summarizes recent progress in understanding the in vivo function(s) of α-catenins in tissue morphogenesis, homeostasis and disease. The role of α-catenin in the regulation of cellular proliferation will be discussed in the context of cancer and regeneration.
Assuntos
Saúde , Coração/fisiologia , Neoplasias/metabolismo , Regeneração , alfa Catenina/metabolismo , Animais , Humanos , Modelos BiológicosRESUMO
It is generally accepted that the intercalated disc (ICD) required for mechano-electrical coupling in the heart consists of three distinct junctional complexes: adherens junctions, desmosomes and gap junctions. However, recent morphological and molecular data indicate a mixing of adherens junctional and desmosomal components, resulting in a 'hybrid adhering junction' or 'area composita'. The α-catenin family member αT-catenin, part of the N-cadherin-catenin adhesion complex in the heart, is the only α-catenin that interacts with the desmosomal protein plakophilin-2 (PKP2). Thus, it has been postulated that αT-catenin might serve as a molecular integrator of the two adhesion complexes in the area composita. To investigate the role of αT-catenin in the heart, gene targeting technology was used to delete the Ctnna3 gene, encoding αT-catenin, in the mouse. The αT-catenin-null mice are viable and fertile; however, the animals exhibit progressive cardiomyopathy. Adherens junctional and desmosomal proteins were unaffected by loss of αT-catenin, with the exception of the desmosomal protein PKP2. Immunogold labeling at the ICD demonstrated in the αT-catenin-null heart a preferential reduction of PKP2 at the area composita compared with the desmosome. Furthermore, gap junction protein Cx43 was reduced at the ICD, including its colocalization with N-cadherin. Gap junction remodeling in αT-catenin-knockout hearts was associated with an increased incidence of ventricular arrhythmias after acute ischemia. This novel animal model demonstrates for the first time how perturbation in αT-catenin can affect both PKP2 and Cx43 and thereby highlights the importance of understanding the crosstalk between the junctional proteins of the ICD and its implications for arrhythmogenic cardiomyopathy.
Assuntos
Arritmias Cardíacas/fisiopatologia , Cardiomiopatia Dilatada/patologia , Junções Comunicantes/metabolismo , Ventrículos do Coração/fisiopatologia , Isquemia Miocárdica/complicações , Miócitos Cardíacos/metabolismo , alfa Catenina/deficiência , Junções Aderentes/metabolismo , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/patologia , Caderinas/metabolismo , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/fisiopatologia , Conexina 43/deficiência , Conexina 43/metabolismo , Desmossomos/metabolismo , Modelos Animais de Doenças , Eletrocardiografia , Junções Comunicantes/patologia , Ventrículos do Coração/patologia , Camundongos , Camundongos Knockout , Mutação , Traumatismo por Reperfusão Miocárdica , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/patologia , Placofilinas/deficiência , Placofilinas/metabolismo , alfa Catenina/genética , alfa Catenina/metabolismoRESUMO
Cell adhesion controls various embryonic morphogenetic processes, including the development of the enteric nervous system (ENS). Ablation of ß1-integrin (ß1-/-) expression in enteric neural crest cells (ENCC) in mice leads to major alterations in the ENS structure caused by reduced migration and increased aggregation properties of ENCC during gut colonization, which gives rise to a Hirschsprung's disease-like phenotype. In the present study, we examined the role of N-cadherin in ENS development and the interplay with ß1 integrins during this process. The Ht-PA-Cre mouse model was used to target gene disruption of N-cadherin and ß1 integrin in migratory NCC and to produce single- and double-conditional mutants for these two types of adhesion receptors. Double mutation of N-cadherin and ß1 integrin led to embryonic lethality with severe defects in ENS development. N-cadherin-null (Ncad-/-) ENCC exhibited a delayed colonization in the developing gut at E12.5, although this was to a lesser extent than in ß1-/- mutants. This delay of Ncad-/- ENCC migration was recovered at later stages of development. The double Ncad-/-; ß1-/- mutant ENCC failed to colonize the distal part of the gut and there was more severe aganglionosis in the proximal hindgut than in the single mutants for N-cadherin or ß1-integrin. This was due to an altered speed of locomotion and directionality in the gut wall. The abnormal aggregation defect of ENCC and the disorganized ganglia network in the ß1-/- mutant was not observed in the double mutant. This indicates that N-cadherin enhances the effect of the ß1-integrin mutation and demonstrates cooperation between these two adhesion receptors during ENS ontogenesis. In conclusion, our data reveal that N-cadherin is not essential for ENS development but it does modulate the modes of ENCC migration and acts in concert with ß1-integrin to control the proper development of the ENS.
Assuntos
Caderinas/metabolismo , Sistema Nervoso Entérico/crescimento & desenvolvimento , Integrina beta1/metabolismo , Animais , Caderinas/genética , Caderinas/fisiologia , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/fisiologia , Feminino , Integrina beta1/genética , Integrina beta1/fisiologia , Masculino , Camundongos , Crista Neural/embriologia , Crista Neural/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Mutations in the human Jup gene cause arrhythmogenic right ventricular cardiomyopathy (ARVC), a heart muscle disease that often leads to sudden cardiac death. Inactivation of the murine Jup gene (also known as plakoglobin) results in embryonic lethality due to cardiac rupture. In an effort to generate a conditional knockout allele, a neomycin cassette was introduced into the murine plakoglobin (PG) gene. This allele (PG F(N)) functions as a hypomorph when combined with a null allele (PG Δ). About half of the PG F(N)/Δ animals were smaller than their littermates and died before weaning age, whereas the remaining PG F(N)/Δ animals survived. Despite the reduced levels of PG in the heart, there were no signs of cardiomyopathy or cardiac dysfunction as determined by echocardiography. Importantly, the PG homolog, ß-catenin (CTNNB1), was increased in the PG F(N)/Δ hearts. In addition to its structural role as part of the N-cadherin/catenin adhesion complex, ß-catenin is a downstream effector of Wnt signaling. However, no change in ß-catenin/TCF reporter activity was observed in PG F(N)/Δ embryos suggesting that excess ß-catenin was not likely causing increased transcription of Wnt/ß-catenin target genes. These data suggest novel function(s) for PG beyond the heart and define a critical threshold of PG expression that is necessary for postnatal survival.
Assuntos
Alelos , Miocárdio/metabolismo , gama Catenina/genética , Animais , Displasia Arritmogênica Ventricular Direita/genética , Caderinas/genética , Caderinas/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Coração/anatomia & histologia , Coração/crescimento & desenvolvimento , Coração/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transcrição Gênica , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo , gama Catenina/metabolismoRESUMO
The intercalated disc serves as an organizing center for various cell surface components at the termini of the cardiomyocyte, thus ensuring proper mechanoelectrical coupling throughout the myocardium. The cell adhesion molecule, N-cadherin, is an essential component of the intercalated disc. Cardiac-specific deletion of N-cadherin leads to abnormal electrical conduction and sudden arrhythmic death in mice. The mechanisms linking the loss of N-cadherin in the heart and spontaneous malignant ventricular arrhythmias are poorly understood. To investigate whether ion channel remodeling contributes to arrhythmogenesis in N-cadherin conditional knock-out (N-cad CKO) mice, cardiac myocyte excitability and voltage-gated potassium channel (Kv), as well as inwardly rectifying K(+) channel remodeling, were investigated in N-cad CKO cardiomyocytes by whole cell patch clamp recordings. Action potential duration was prolonged in N-cad CKO ventricle myocytes compared with wild type. Relative to wild type, I(K,slow) density was significantly reduced consistent with decreased expression of Kv1.5 and Kv accessory protein, Kcne2, in the N-cad CKO myocytes. The decreased Kv1.5/Kcne2 expression correlated with disruption of the actin cytoskeleton and reduced cortactin at the sarcolemma. Biochemical experiments revealed that cortactin co-immunoprecipitates with Kv1.5. Finally, cortactin was required for N-cadherin-mediated enhancement of Kv1.5 channel activity in a heterologous expression system. Our results demonstrate a novel mechanistic link among the cell adhesion molecule, N-cadherin, the actin-binding scaffold protein, cortactin, and Kv channel remodeling in the heart. These data suggest that in addition to gap junction remodeling, aberrant Kv1.5 channel function contributes to the arrhythmogenic phenotype in N-cad CKO mice.
Assuntos
Potenciais de Ação/fisiologia , Caderinas/metabolismo , Cortactina/metabolismo , Junções Comunicantes/metabolismo , Canal de Potássio Kv1.5/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Caderinas/genética , Cortactina/genética , Junções Comunicantes/genética , Canal de Potássio Kv1.5/genética , Camundongos , Camundongos Knockout , Especificidade de Órgãos/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismoRESUMO
We have previously shown that targeted expression of a dominant-negative truncated form of N-cadherin (Cdh2) delays acquisition of peak bone mass in mice and retards osteoblast differentiation; whereas deletion of cadherin 11 (Cdh11), another osteoblast cadherin, leads to only modest osteopenia. To determine the specific roles of these two cadherins in the adult skeleton, we generated mice with an osteoblast/osteocyte specific Cdh2 ablation (cKO) and double Cdh2(+/-);Cdh11(-/-) germline mutant mice. Age-dependent osteopenia and smaller diaphyses with decreased bone strength characterize cKO bones. By contrast, Cdh2(+/-);Cdh11(-/-) exhibit severely reduced trabecular bone mass, decreased in vivo bone formation rate, smaller diaphyses and impaired bone strength relative to single Cdh11 null mice. The number of bone marrow immature precursors and osteoprogenitor cells is reduced in both cKO and Cdh2(+/-);Cdh11(-/-) mice, suggesting that N-cadherin is involved in maintenance of the stromal cell precursor pool via the osteoblast. Although Cdh11 is dispensable for postnatal skeletal growth, it favors osteogenesis over adipogenesis. Deletion of either cadherin reduces ß-catenin abundance and ß-catenin-dependent gene expression, whereas N-cadherin loss disrupts cell-cell adhesion more severely than loss of cadherin 11. Thus, Cdh2 and Cdh11 are crucial regulators of postnatal skeletal growth and bone mass maintenance, serving overlapping, yet distinct, functions in the osteogenic lineage.
Assuntos
Osso e Ossos/citologia , Osso e Ossos/metabolismo , Caderinas/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Animais , Fenômenos Biomecânicos , Densidade Óssea , Caderinas/genética , Adesão Celular , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Immunoblotting , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Persistent changes in spine shape are coupled to long-lasting synaptic plasticity in hippocampus. The molecules that coordinate such persistent structural and functional plasticity are unknown. Here, we generated mice in which the cell adhesion molecule N-cadherin was conditionally ablated from postnatal, excitatory synapses in hippocampus. We applied to adult mice of either sex a combination of whole-cell recording, two-photon microscopy, and spine morphometric analysis to show that postnatal ablation of N-cadherin has profound effects on the stability of coordinated spine enlargement and long-term potentiation (LTP) at mature CA1 synapses, with no effects on baseline spine density or morphology, baseline properties of synaptic neurotransmission, or long-term depression. Thus, N-cadherin couples persistent spine structural modifications with long-lasting synaptic functional modifications associated selectively with LTP, revealing unexpectedly distinct roles at mature synapses in comparison with earlier, broader functions in synapse and spine development.
Assuntos
Região CA1 Hipocampal/citologia , Caderinas/metabolismo , Espinhas Dendríticas/fisiologia , Potenciação de Longa Duração/fisiologia , Células Piramidais/ultraestrutura , Sinapses/metabolismo , Potenciais de Ação/fisiologia , Animais , Biofísica/métodos , Caderinas/deficiência , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/genética , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Imunoeletrônica/métodos , Técnicas de Patch-Clamp/métodos , Estatísticas não Paramétricas , Sinapses/ultraestruturaRESUMO
The cadherin family of cell adhesion molecules mediates adhesive interactions that are required for the formation and maintenance of tissues. Previously, we demonstrated that N-cadherin, which is required for numerous morphogenetic processes, is expressed in the pancreatic epithelium at E9.5, but later becomes restricted to endocrine aggregates in mice. To study the role of N-cadherin during pancreas formation and function we generated a tissue-specific knockout of N-cadherin in the early pancreatic epithelium by inter-crossing N-cadherin-floxed mice with Pdx1Cre mice. Analysis of pancreas-specific ablation of N-cadherin demonstrates that N-cadherin is dispensable for pancreatic development, but required for beta-cell granule turnover. The number of insulin secretory granules is significantly reduced in N-cadherin-deficient beta-cells, and as a consequence insulin secretion is decreased.
Assuntos
Caderinas/fisiologia , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Células Secretoras de Insulina/metabolismo , Pâncreas/crescimento & desenvolvimento , Transativadores/fisiologia , Animais , Feminino , Imunofluorescência , Immunoblotting , Integrases/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pâncreas/metabolismoRESUMO
The classical cadherins are known to have both adhesive and signaling functions. It has also been proposed that localized regulation of cadherin activity may be important in cell assortment during development. In the context of eye development, it has been suggested that cadherins are important for separation of the invaginated lens vesicle from the surface ectoderm. To test this hypothesis, we conditionally deleted N-cadherin or E-cadherin from the presumptive lens ectoderm of the mouse. Conditional deletion of either cadherin alone did not produce a lens vesicle separation defect. However, these conditional mutants did exhibit common structural deficits, including microphthalmia, severe iris hyperplasia, persistent vacuolization within the fibre cell region, and eventual lens epithelial cell deterioration. To assess the co-operative roles of E-cadherin and N-cadherin within the developing lens, double conditional knockout embryos were generated. These mice displayed distinct defects in lens vesicle separation and persistent expression of another classical cadherin, P-cadherin, within the cells of the persistent lens stalk. Double mutant lenses also exhibited severe defects in lens epithelial cell adhesion and survival. Finally, the severity of the lens phenotype was shown to be sensitive to the number of wild-type E- and N-cadherin alleles. These data suggest that the co-operative expression of both E- and N-cadherin during lens development is essential for normal cell sorting and subsequent lens vesicle separation.
Assuntos
Caderinas/metabolismo , Sobrevivência Celular , Células Epiteliais , Cristalino/embriologia , Cristalino/crescimento & desenvolvimento , Morfogênese , Animais , Biomarcadores/metabolismo , Caderinas/genética , Proteínas Cdh1 , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Hiperplasia/patologia , Iris/patologia , Cristalino/anormalidades , Cristalino/anatomia & histologia , Camundongos , Camundongos Knockout , Microftalmia/genética , FenótipoRESUMO
Activation of the A(2A) adenosine receptor (A(2A)R) has been shown to be cardioprotective. We hypothesized that A(2A)R overexpression could protect the heart from adriamycin-induced cardiomyopathy. Transgenic (TG) mice overexpressing the A(2A)R and wild-type mice (WT) were injected with adriamycin (5 mg.kg(-1).wk(-1) ip, 4 wk). All WT mice survived adriamycin treatment while A(2A)R TG mice suffered 100% mortality at 4 wk. Telemetry showed progressive prolongation of the QT interval, bradyarrhythmias, heart block, and sudden death in adriamycin-treated A(2A)R TG but not WT mice. Both WT and A(2A)R TG demonstrated similar decreases in heart function at 3 wk after treatment. Adriamycin significantly increased end-diastolic intracellular Ca(2+) concentration in A(2A)R TG but not in WT myocytes (P < 0.05). Compared with WT myocytes, action potential duration increased dramatically in A(2A)R TG myocytes (P < 0.05) after adriamycin treatment. Expression of connexin 43 was decreased in adriamycin treated A(2A)R TG but not WT mice. In sharp contrast, A(2A)R overexpression induced after the completion of adriamycin treatment resulted in no deaths and enhanced cardiac performance compared with WT adriamycin-treated mice. Our results indicate that the timing of A(2A)R activation is critical in terms of exacerbating or protecting adriamycin-induced cardiotoxicity. Our data have direct relevance on the clinical use of adenosine agonists or antagonists in the treatment of patients undergoing adriamycin therapy.
Assuntos
Cardiomiopatias/induzido quimicamente , Cardiomiopatias/metabolismo , Doxorrubicina/efeitos adversos , Miocárdio/metabolismo , Receptor A2A de Adenosina/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/efeitos adversos , Caderinas/metabolismo , Cálcio/metabolismo , Cardiomiopatias/mortalidade , Células Cultivadas , Conexina 43/metabolismo , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Receptor A2A de Adenosina/genéticaRESUMO
Endothelial cells express two classic cadherins, VE-cadherin and N-cadherin. The importance of VE-cadherin in vascular development is well known; however, the function of N-cadherin in endothelial cells remains poorly understood. Contrary to previous studies, we found that N-cadherin localizes to endothelial cell-cell junctions in addition to its well-known diffusive membrane expression. To investigate the role of N-cadherin in vascular development, N-cadherin was specifically deleted from endothelial cells in mice. Loss of N-cadherin in endothelial cells results in embryonic lethality at mid-gestation due to severe vascular defects. Intriguingly, loss of N-cadherin caused a significant decrease in VE-cadherin and its cytoplasmic binding partner, p120ctn. The down-regulation of both VE-cadherin and p120ctn was confirmed in cultured endothelial cells using small interfering RNA to knockdown N-cadherin. We also show that N-cadherin is important for endothelial cell proliferation and motility. These findings provide a novel paradigm by which N-cadherin regulates angiogenesis, in part, by controlling VE-cadherin expression at the cell membrane.
Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Endotélio Vascular/metabolismo , Morfogênese/fisiologia , Fosfoproteínas/metabolismo , Animais , Antígenos CD , Caderinas/genética , Cateninas , Membrana Celular/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Endotélio Vascular/citologia , Endotélio Vascular/embriologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Morfogênese/genética , Interferência de RNA , Veias Umbilicais/citologia , Veias Umbilicais/metabolismo , delta CateninaRESUMO
The study of germ cell-specific gene regulation in vitro is challenging. Here we report that the promoter of the oocyte-specific gene, Gdf9, is active in a population of cultured murine embryonic stem cells (ES) which have a phenotype resembling oocytes. The promoter region of the murine Gdf9 coupled to enhanced green fluorescent protein (eGFP) was stably transfected into XX mouse ES cells. eGFP was expressed only in oocytes of chimeric mice generated from the transfected XX ES cells. The transfected ES cells were examined when cultured on feeder layers or as embryoid bodies. Large eGFP-positive cells, surrounded by a structure resembling a zona pellucida appeared transiently in cultures of the ES cells on feeder layers. Surprisingly, they were detectable on days 1 and 2 of culture but virtually absent on day 3. Addition of leukemia inhibitory factor (LIF) to the media significantly increased the number of eGFP-positive cels resembling oocytes. Quantitative-time PCR demonstrated a parallel increase in Gdf9 and Zp3 mRNA with changes in the abundance of eGFP-positive cells. In embryoid body cultures, eGFP-positive cells appeared transiently and then re-appeared in regional clusters after 30-45 days of culture. These findings demonstrate that a population of cultured murine ES cells contain the transcriptional machinery to drive expression of an oocyte-specific gene, and that those cells phenotypically resemble oocytes.
Assuntos
Células-Tronco Embrionárias/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Oócitos/fisiologia , Animais , Proteína Morfogenética Óssea 15 , Técnicas de Cultura de Células , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Fator 9 de Diferenciação de Crescimento , Fator Inibidor de Leucemia/farmacologia , Camundongos , Oócitos/metabolismo , Fenótipo , Regiões Promotoras Genéticas/genética , Transfecção/métodosRESUMO
Cardiac-specific deletion of the murine gene (Cdh2) encoding the cell adhesion molecule, N-cadherin, results in disassembly of the intercalated disc (ICD) structure and sudden arrhythmic death. Connexin 43 (Cx43)-containing gap junctions are significantly reduced in the heart after depleting N-cadherin, therefore we hypothesized that animals expressing half the normal levels of N-cadherin would exhibit an intermediate phenotype. We examined the effect of N-cadherin haploinsufficiency on Cx43 expression and susceptibility to induced arrhythmias in mice either wild-type or heterozygous for the Cx43 (Gja1)-null allele. An increase in hypophosphorylated Cx43 accompanied by a modest decrease in total Cx43 protein levels was observed in the N-cadherin heterozygous mice. Consistent with these findings N-cadherin heterozygotes exhibited increased susceptibility to ventricular arrhythmias compared to wild-type mice. Quantitative immunofluorescence microscopy revealed a reduction in size of large Cx43-containing plaques in the N-cadherin heterozygous animals compared to wild-type. Gap junctions were further decreased in number and size in the N-cad/Cx43 compound heterozygous mice with increased arrhythmic susceptibility compared to the single mutants. The scaffold protein, ZO-1, was reduced at the ICD in N-cadherin heterozygous cardiomyocytes providing a possible explanation for the reduction in Cx43 plaque size. These data provide further support for the intimate relationship between N-cadherin and Cx43 in the heart, and suggest that germline mutations in the human N-cadherin (Cdh2) gene may predispose patients to increased risk of cardiac arrhythmias.
Assuntos
Arritmias Cardíacas/metabolismo , Caderinas/metabolismo , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Western Blotting , Caderinas/genética , Comunicação Celular/genética , Células Cultivadas , Conexina 43/genética , Eletrofisiologia , Imunofluorescência , Heterozigoto , Proteínas de Membrana/metabolismo , Camundongos , Células Musculares/metabolismo , Mutação , Miocárdio/metabolismo , Miocárdio/patologia , Fosfoproteínas/metabolismo , Proteína da Zônula de Oclusão-1 , beta Catenina/metabolismoRESUMO
The structural integrity of the heart is maintained by the end-to-end connection between the myocytes called the intercalated disc. The intercalated disc contains different junctional complexes that enable the myocardium to function as a syncytium. One of the junctional complexes, the zonula adherens or adherens junction, consists of the cell adhesion molecule, N-cadherin, which mediates strong homophilic cell-cell adhesion via linkage to the actin cytoskeleton. To determine the function of N-cadherin in the working myocardium, we generated a conditional knockout containing loxP sites flanking exon 1 of the N-cadherin (Cdh2) gene. Using a cardiac-specific tamoxifen-inducible Cre transgene, N-cadherin was deleted in the adult myocardium. Loss of N-cadherin resulted in disassembly of the intercalated disc structure, including adherens junctions and desmosomes. The mutant mice exhibited modest dilated cardiomyopathy and impaired cardiac function, with most animals dying within two months after tamoxifen administration. Decreased sarcomere length and increased Z-line thickness were observed in the mutant hearts consistent with loss of muscle tension because N-cadherin was no longer available to anchor myofibrils at the plasma membrane. Ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous ventricular tachycardia, confirming that the deaths were arrhythmic in nature. A significant decrease in the gap junction protein, connexin 43, was observed in the N-cadherin-depleted hearts. This animal model provides the first demonstration of the hierarchical relationship of the structural components of the intercalated disc in the working myocardium, thus establishing N-cadherin's paramount importance in maintaining the structural integrity of the heart.