RESUMO
The connection of the coronary vasculature to the aorta is one of the last essential steps of cardiac development. However, little is known about the signaling events that promote normal coronary artery formation. The bone morphogenetic protein (BMP) signaling pathway regulates multiple aspects of endothelial cell biology but has not been specifically implicated in coronary vascular development. BMP signaling is tightly regulated by numerous factors, including BMP-binding endothelial cell precursor-derived regulator (BMPER), which can both promote and repress BMP signaling activity. In the embryonic heart, BMPER expression is limited to the endothelial cells and the endothelial-derived cushions, suggesting that BMPER may play a role in coronary vascular development. Histological analysis of BMPER(-/-) embryos at early embryonic stages demonstrates that commencement of coronary plexus differentiation is normal and that endothelial apoptosis and cell proliferation are unaffected in BMPER(-/-) embryos compared with wild-type embryos. However, analysis between embryonic days 15.5-17.5 reveals that, in BMPER(-/-) embryos, coronary arteries are either atretic or connected distal to the semilunar valves. In vitro tubulogenesis assays indicate that isolated BMPER(-/-) endothelial cells have impaired tube formation and migratory ability compared with wild-type endothelial cells, suggesting that these defects may lead to the observed coronary artery anomalies seen in BMPER(-/-) embryos. Additionally, recombinant BMPER promotes wild-type ventricular endothelial migration in a dose-dependent manner, with a low concentration promoting and high concentrations inhibiting migration. Together, these results indicate that BMPER-regulated BMP signaling is critical for coronary plexus remodeling and normal coronary artery development.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Vasos Coronários/fisiologia , Células Endoteliais/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Proteínas de Transporte/genética , Immunoblotting , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Microscopia de FluorescênciaRESUMO
RATIONALE: Among the extracellular modulators of Bmp (bone morphogenetic protein) signaling, Bmper (Bmp endothelial cell precursor-derived regulator) both enhances and inhibits Bmp signaling. Recently we found that Bmper modulates Bmp4 activity via a concentration-dependent, endocytic trap-and-sink mechanism. OBJECTIVE: To investigate the molecular mechanisms required for endocytosis of the Bmper/Bmp4 and signaling complex and determine the mechanism of Bmper's differential effects on Bmp4 signaling. METHODS AND RESULTS: Using an array of biochemical and cell biology techniques, we report that LRP1 (LDL receptor-related protein 1), a member of the LDL receptor family, acts as an endocytic receptor for Bmper and a coreceptor of Bmp4 to mediate the endocytosis of the Bmper/Bmp4 signaling complex. Furthermore, we demonstrate that LRP1-dependent Bmper/Bmp4 endocytosis is essential for Bmp4 signaling, as evidenced by the phenotype of lrp1-deficient zebrafish, which have abnormal cardiovascular development and decreased Smad1/5/8 activity in key vasculogenic structures. CONCLUSIONS: Together, these data reveal a novel role for LRP1 in the regulation of Bmp4 signaling by regulating receptor complex endocytosis. In addition, these data introduce LRP1 as a critical regulator of vascular development. These observations demonstrate Bmper's ability to fine-tune Bmp4 signaling at the single-cell level, unlike the spatial regulatory mechanisms applied by other Bmp modulators.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Transporte/metabolismo , Endocitose/fisiologia , Células Endoteliais/fisiologia , Neovascularização Fisiológica/fisiologia , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Proteína Morfogenética Óssea 4/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Movimento Celular/fisiologia , Células Endoteliais/citologia , Células HEK293 , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Fenótipo , RNA Interferente Pequeno/genética , Receptores de LDL/genética , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is a pivotal player in angiogenesis. It is capable of influencing such cellular processes as tubulogenesis and endothelial cell migration, yet very little is known about the actual signaling events that mediate SDF-1alpha-induced endothelial cell function. In this report, we describe the identification of an intricate SDF-1alpha-induced signaling cascade that involves endothelial nitric oxide synthase (eNOS), JNK3, and MAPK phosphatase 7 (MKP7). We demonstrate that the SDF-1alpha-induced activation of JNK3, critical for endothelial cell migration, depends on the prior activation of eNOS. Specifically, activation of eNOS leads to production of NO and subsequent nitrosylation of MKP7, rendering the phosphatase inactive and unable to inhibit the activation of JNK3. These observations reinforce the importance of nitric oxide and S-nitrosylation in angiogenesis and provide a mechanistic pathway for SDF-1alpha-induced endothelial cell migration. In addition, the discovery of this interactive network of pathways provides novel and unexpected therapeutic targets for angiogenesis-dependent diseases.
Assuntos
Movimento Celular , Quimiocina CXCL12/fisiologia , Fosfatases de Especificidade Dupla/metabolismo , Endotélio Vascular/citologia , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Bovinos , Células Cultivadas , Células Endoteliais , Humanos , Transdução de SinaisRESUMO
During vascular development, endothelial cells are exposed to a variety of rapidly changing factors, including fluctuating oxygen levels. We have previously shown that ankyrin repeat and suppressor of cytokine signalling box protein 4 (ASB4) is the most highly differentially expressed gene in the vascular lineage during early differentiation and is expressed in the embryonic vasculature at a time when oxygen tension is rising because of the onset of placental blood flow. To further our understanding of the regulation of ASB4 expression in endothelial cells, we tested the effect of various stressors for their ability to alter ASB4 expression in the immortalized murine endothelial cell lines MS1 and SVR. ASB4 expression is decreased during hypoxic insult and shear stress, whereas it is increased in response to tumour necrosis factor alpha (TNF-α). Further investigation indicated that nuclear factor kappa B (NF-κB) is the responsible transcription factor involved in the TNF-α-induced upregulation of ASB4, placing ASB4 downstream of NF-κB in the TNF-α signalling cascade and identifying it as a potential regulator for TNF-α's numerous functions associated with inflammation, angiogenesis and apoptosis.
Assuntos
Endotélio Vascular/metabolismo , Oxigênio/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular Transformada , Regulação da Expressão Gênica no Desenvolvimento , Hipóxia/metabolismo , Camundongos , NF-kappa B/metabolismo , Transdução de Sinais , Estresse Mecânico , TransfecçãoRESUMO
The molecular mechanisms of endothelial differentiation into a functional vascular network are incompletely understood. To identify novel factors in endothelial development, we used a microarray screen with differentiating embryonic stem (ES) cells that identified the gene for ankyrin repeat and SOCS box protein 4 (ASB4) as the most highly differentially expressed gene in the vascular lineage during early differentiation. Like other SOCS box-containing proteins, ASB4 is the substrate recognition molecule of an elongin B/elongin C/cullin/Roc ubiquitin ligase complex that mediates the ubiquitination and degradation of substrate protein(s). High levels of ASB4 expression in the embryonic vasculature coincide with drastic increases in oxygen tension as placental blood flow is initiated. However, as vessels mature and oxygen levels stabilize, ASB4 expression is quickly downregulated, suggesting that ASB4 may function to modulate an endothelium-specific response to increasing oxygen tension. Consistent with the hypothesis that ASB4 function is regulated by oxygen concentration, ASB4 interacts with the factor inhibiting HIF1alpha (FIH) and is a substrate for FIH-mediated hydroxylation via an oxygen-dependent mechanism. Additionally, overexpression of ASB4 in ES cells promotes differentiation into the vascular lineage in an oxygen-dependent manner. We postulate that hydroxylation of ASB4 in normoxia promotes binding to and degradation of substrate protein(s) to modulate vascular differentiation.
Assuntos
Diferenciação Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Oxigênio/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Repetição de Anquirina , Asparagina/metabolismo , Sítios de Ligação , Linhagem Celular , Linhagem da Célula , Células Cultivadas , Sequência Conservada , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Deleção de Genes , Humanos , Hidroxilação , Hibridização In Situ , Rim/citologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Mutação Puntual , Testes de Precipitina , Ligação Proteica , Conformação Proteica , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/genética , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
The mechanisms that regulate the differentiation program of multipotential stem cells remain poorly understood. In order to define the cues that delineate endothelial commitment from precursors, we screened for candidate regulatory genes in differentiating mouse embryoid bodies. We found that the PR/SET domain protein, PRDM6, is enriched in flk1(+) hematovascular precursor cells using a microarray-based approach. As determined by 5' RACE, full-length PRDM6 protein contains a PR domain and four Krüppel-like zinc fingers. In situ hybridization in mouse embryos demonstrates staining of the primitive streak, allantois, heart, outflow tract, paraaortic splanchnopleura (P-Sp)/aorto-gonadal-mesonephric (AGM) region and yolk sac, all sites known to be enriched in vascular precursor cells. PRDM6 is also detected in embryonic and adult-derived endothelial cell lines. PRDM6 is co-localized with histone H4 and methylates H4-K20 (but not H3) in vitro and in vivo, which is consistent with the known participation of PR domains in histone methyltransferase activity. Overexpression of PRDM6 in mouse embryonic endothelial cells induces apoptosis by activating caspase-3 and inducing G1 arrest. PRDM6 inhibits cell proliferation as determined by BrdU incorporation in endothelial cells, but not in rat aortic smooth muscle cells. Overexpression of PRDM6 also results in reduced tube formation in cultured endothelial cells grown in Matrigel. Taken together, our data indicate that PRDM6 is expressed by vascular precursors, has differential effects in endothelial cells and smooth muscle cells, and may play a role in vascular precursor differentiation and survival by modulating local chromatin-remodeling activity within hematovascular subpopulations during development.
Assuntos
Vasos Sanguíneos/citologia , Vasos Sanguíneos/metabolismo , Diferenciação Celular , Células Endoteliais/citologia , Proteínas Repressoras/metabolismo , Células-Tronco/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Ciclo Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Células Endoteliais/enzimologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas Metiltransferases , Ratos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Células-Tronco/citologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
We have used global gene expression analysis to establish a comprehensive list of candidate genes in the developing vasculature during embryonic (ES) cell differentiation in vitro. A large set of genes, including growth factors, cell surface molecules, transcriptional factors, and members of several signal transduction pathways that are known to be involved in vasculogenesis or angiogenesis, were found to have expression patterns as expected. Some unknown or functionally uncharacterized genes were differentially regulated in flk1+ cells compared with flk1- cells, suggesting possible roles for these genes in vascular commitment. Particularly, multiple components of the Wnt signaling pathway were differentially regulated in flk1+ cells, including Wnt proteins, their receptors, downstream transcriptional factors, and other components belonging to this pathway. Activation of the Wnt signal was able to expand vascular progenitor populations whereas suppression of Wnt activity reduced flk1+ populations. Suppression of Wnt signaling also inhibited the formation of matured vascular capillary-like structures during late stages of embryoid body differentiation. These data indicate a requisite and ongoing role for Wnt activity during vascular development, and the gene expression profiles identify candidate components of this pathway that participate in vascular cell differentiation.
Assuntos
Diferenciação Celular/fisiologia , Embrião de Mamíferos/citologia , Endotélio Vascular/citologia , Perfilação da Expressão Gênica , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Animais , Linhagem da Célula , Células Cultivadas , Camundongos , Células-Tronco/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0139209.].
RESUMO
Endothelial cells differentiate from mesoderm-derived precursors to initiate the earliest events in vascular development. Although the signaling events that regulate the successive steps of vascular development are known in some detail, the transcriptional processes that regulate the first steps in vasculogenesis are not well defined. We have studied the regulatory mechanisms of flk1 expression as a model to understand the upstream events in endothelial cell differentiation, since flk1 is the earliest marker of endothelial precursors. Using a variety of biochemical approaches, we identified a cis-acting element in the first intron of the flk1 gene that is required for endothelium-dependent expression in transgenic reporter gene assays. Using the yeast one-hybrid system, we identified HoxB5 as the transcription factor that binds this cis-acting element, the HoxB5-binding element (HBE). HoxB5 mRNA colocalized with flk1 expression in differentiating embryoid bodies, and HoxB5 potently transactivated the flk1 promoter in an HBE-dependent fashion in transient-transfection assays. Overexpression of HoxB5 led to expansion of flk1(+) angioblasts in differentiating embryoid bodies and increased the number of PECAM (platelet-endothelial cell adhesion molecule)-positive primitive blood vessels. HoxB5 is necessary and sufficient to activate the cell-intrinsic events that regulate the differentiation of angioblasts and mature endothelial cells from their mesoderm-derived precursors.
Assuntos
Endotélio Vascular/metabolismo , Proteínas de Homeodomínio/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas , Desoxirribonuclease I/metabolismo , Endotélio Vascular/citologia , Citometria de Fluxo , Humanos , Íntrons , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Dados de Sequência Molecular , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Regiões Promotoras Genéticas , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Transgenes , Técnicas do Sistema de Duplo-Híbrido , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
The development of endothelial cell precursors is essential for vasculogenesis. We screened for differentially expressed transcripts in endothelial cell precursors in developing mouse embryoid bodies. We cloned a complete cDNA encoding a protein that contains an amino-terminal signal peptide, five cysteine-rich domains, a von Willebrand D domain, and a trypsin inhibitor domain. We termed this protein BMPER (bone morphogenetic protein [BMP]-binding endothelial cell precursor-derived regulator). BMPER is specifically expressed in flk-1-positive cells and parallels the time course of flk-1 induction in these cells. In situ hybridization in mouse embryos demonstrates dorsal midline staining and staining of the aorto-gonadal-mesonephric region, which is known to host vascular precursor cells. BMPER is a secreted protein that directly interacts with BMP2, BMP4, and BMP6 and antagonizes BMP4-dependent Smad5 activation. In Xenopus embryos, ventral injection of BMPER mRNA results in axis duplication and downregulation of the expression of Xvent-1 (downstream target of Smad signaling). In an embryoid body differentiation assay, BMP4-dependent differentiation of endothelial cells in embryoid bodies is also antagonized by BMPER. Taken together, our data indicate that BMPER is a novel BMP-binding protein that is expressed by endothelial cell precursors, has BMP-antagonizing activity, and may play a role in endothelial cell differentiation by modulating local BMP activity.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/farmacologia , Fator de Crescimento Transformador beta , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteína Morfogenética Óssea 6 , Células COS , Diferenciação Celular , Linhagem Celular , Clonagem Molecular , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/metabolismo , Citometria de Fluxo , Biblioteca Gênica , Genes Reporter , Humanos , Hibridização In Situ , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Reação em Cadeia da Polimerase , Testes de Precipitina , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad5 , Fatores de Tempo , Distribuição Tecidual , Transativadores/metabolismo , Transfecção , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Xenopus , Xenopus laevisRESUMO
Chronic Hepatitis B Virus (HBV) infection is generally not curable with current anti-viral drugs. Virus rebounds after stopping treatment from the stable HBV covalently-closed-circular DNA (cccDNA). The development of drugs that directly target cccDNA is hampered by the lack of robust HBV cccDNA models. We report here a novel HBV cccDNA technology that will meet the need. We engineered a minicircle HBV cccDNA with a Gaussia Luciferase reporter (mcHBV-GLuc cccDNA), which serves as a surrogate to measure cccDNA activity. The mcHBV-GLuc cccDNA was easily produced in bacteria, and it formed minichromosomes as HBV cccDNA episome DNA does when it was transfected into human hepatocytes. Compared to non-HBV minicircle plasmids, mcHBV-GLuc cccDNA showed persistent HBV-GLuc activity and HBx-dependent gene expression. Importantly, the mcHBV-GLuc cccDNA showed resistance to interferons (IFN) treatment, indicating its unique similarity to HBV cccDNA that is usually resistant to long-term IFN treatment in chronic HBV patients. Most importantly, GLuc illuminates cccDNA as a surrogate of cccDNA activity, providing a very sensitive and quick method to detect trace amount of cccDNA. The mcHBV-GLuc cccDNA model is independent of HBV infection, and will be valuable for investigating HBV cccDNA biology and for developing cccDNA-targeting drugs.
Assuntos
Antivirais/metabolismo , DNA Circular/metabolismo , DNA Viral/metabolismo , Vírus da Hepatite B/genética , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Imunoprecipitação da Cromatina , DNA Circular/química , DNA Viral/análise , Genes Reporter , Células Hep G2 , Humanos , Interferons/química , Interferons/metabolismo , Interferons/farmacologia , Luciferases/genética , Luciferases/metabolismo , Microscopia de Fluorescência , Plasmídeos/genética , Plasmídeos/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
The hepatitis B virus (HBV) regulatory protein X (HBx) activates gene expression from the HBV covalently closed circular DNA (cccDNA) genome. Interaction of HBx with the DDB1-CUL4-ROC1 (CRL4) E3 ligase is critical for this function. Using substrate-trapping proteomics, we identified the structural maintenance of chromosomes (SMC) complex proteins SMC5 and SMC6 as CRL4(HBx) substrates. HBx expression and HBV infection degraded the SMC5/6 complex in human hepatocytes in vitro and in humanized mice in vivo. HBx targets SMC5/6 for ubiquitylation by the CRL4(HBx) E3 ligase and subsequent degradation by the proteasome. Using a minicircle HBV (mcHBV) reporter system with HBx-dependent activity, we demonstrate that SMC5/6 knockdown, or inhibition with a dominant-negative SMC6, enhance HBx null mcHBV-Gluc gene expression. Furthermore, SMC5/6 knockdown rescued HBx-deficient HBV replication in human hepatocytes. These results indicate that a primary function of HBx is to degrade SMC5/6, which restricts HBV replication by inhibiting HBV gene expression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Vírus da Hepatite B/fisiologia , Proteólise , Transativadores/metabolismo , Replicação Viral/fisiologia , Animais , Proteínas Cromossômicas não Histona , Regulação Viral da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/patologia , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fígado/patologia , Fígado/virologia , Camundongos , Ligação Proteica , Proteômica , Especificidade por Substrato , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Virais Reguladoras e AcessóriasRESUMO
Formation of the cardiac valves is an essential component of cardiovascular development. Consistent with the role of the bone morphogenetic protein (BMP) signaling pathway in cardiac valve formation, embryos that are deficient for the BMP regulator BMPER (BMP-binding endothelial regulator) display the cardiac valve anomaly mitral valve prolapse. However, how BMPER deficiency leads to this defect is unknown. Based on its expression pattern in the developing cardiac cushions, we hypothesized that BMPER regulates BMP2-mediated signaling, leading to fine-tuned epithelial-mesenchymal transition (EMT) and extracellular matrix deposition. In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation. However, proliferation is not impaired in the developing BMPER-/- valves. In vitro data show that BMPER directly binds BMP2. In cultured endothelial cells, BMPER blocks BMP2-induced Smad activation in a dose-dependent manner. In addition, BMP2 increases the Sox9 protein level, and this increase is inhibited by co-treatment with BMPER. Consistently, in the BMPER-/- embryos, semi-quantitative analysis of Smad activation shows that the canonical BMP pathway is significantly more active in the atrioventricular cushions during EMT. These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development. Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteínas de Transporte/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Valvas Cardíacas/embriologia , Fatores de Transcrição SOX9/metabolismo , Animais , Proteínas de Transporte/biossíntese , Linhagem Celular , Proliferação de Células , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Transdução de SinaisRESUMO
Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 µg of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.
Assuntos
Valvas Cardíacas/química , Proteínas Musculares/análise , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Camundongos , Proteínas Musculares/metabolismo , GravidezRESUMO
Vascularization of the placenta is a critical developmental process that ensures fetal viability. Although the vascular health of the placenta affects both maternal and fetal well being, relatively little is known about the early stages of placental vascular development. The ubiquitin ligase Ankyrin repeat, SOCS box-containing 4 (ASB4) promotes embryonic stem cell differentiation to vascular lineages and is highly expressed early in placental development. The transcriptional regulator Inhibitor of DNA binding 2 (ID2) negatively regulates vascular differentiation during development and is a target of many ubiquitin ligases. Due to their overlapping spatiotemporal expression pattern in the placenta and contrasting effects on vascular differentiation, we investigated whether ASB4 regulates ID2 through its ligase activity in the placenta and whether this activity mediates vascular differentiation. In mouse placentas, ASB4 expression is restricted to a subset of cells that express both stem cell and endothelial markers. Placentas that lack Asb4 display immature vascular patterning and retain expression of placental progenitor markers, including ID2 expression. Using JAR placental cells, we determined that ASB4 ubiquitinates and represses ID2 expression in a proteasome-dependent fashion. Expression of ASB4 in JAR cells and primary isolated trophoblast stem cells promotes the expression of differentiation markers. In functional endothelial co-culture assays, JAR cells ectopically expressing ASB4 increased endothelial cell turnover and stabilized endothelial tube formation, both of which are hallmarks of vascular differentiation within the placenta. Co-transfection of a degradation-resistant Id2 mutant with Asb4 inhibits both differentiation and functional responses. Lastly, deletion of Asb4 in mice induces a pathology that phenocopies human pre-eclampsia, including hypertension and proteinuria in late-stage pregnant females. These results indicate that ASB4 mediates vascular differentiation in the placenta via its degradation of ID2.
Assuntos
Diferenciação Celular/fisiologia , Proteína 2 Inibidora de Diferenciação/metabolismo , Placentação/fisiologia , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Trofoblastos/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Camundongos , Camundongos Knockout , Placenta/metabolismo , Gravidez , Proteínas Supressoras da Sinalização de Citocina/genética , Trofoblastos/citologiaRESUMO
The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging.
Assuntos
Sirtuínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Acetilação , Animais , Linhagem Celular , Deleção de Genes , Células HEK293 , Histonas/metabolismo , Humanos , Camundongos , Mutação Puntual , Mapas de Interação de Proteínas , Estabilidade Proteica , Sirtuínas/química , Sirtuínas/genética , Ativação Transcricional , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Regulação para CimaRESUMO
AIMS: Homeobox (Hox) proteins are transcriptional regulators in embryonic patterning, cell differentiation, proliferation, and migration in vertebrates and invertebrates. A growing body of evidence suggests that Hox proteins are involved in endothelial cell regulation. We have shown earlier that HoxB5 upregulates vascular endothelial growth factor receptor-2 and thereby contributes to enhanced endothelial precursor cell differentiation. Here we aim to elucidate the role of HoxB5 in angiogenesis. METHODS AND RESULTS: Endothelial cell sprouting was investigated in the human umbilical vein endothelial cell spheroid assay. We investigated in vivo angiogenesis in the chick (Gallus gallus) chorioallantoic membrane assay. Expression profiling of proangiogenic factors was done by quantitative PCR. The angiopoietin-2 (Ang2) promoter and deletion fragments thereof were cloned into the pGL3 reporter system for analysis of transcriptional activity. We observed that HoxB5 enhances endothelial cell sprouting and modulates the expression of adhesion molecules in vitro. Accordingly, we observed a modification of vascular growth by HoxB5 in vivo. The HoxB5 effect is reminiscent of the effects of angiopoietins. We demonstrate that Ang2 is upregulated upon HoxB5 overexpression and that the HoxB5 effect is abolished by the angiopoietin antagonist soluble Tie-2. CONCLUSION: HoxB5 has an activating effect on Ang2 that is essential for endothelial cell sprouting and coordinated vascular growth.
Assuntos
Angiopoietina-2/metabolismo , Membrana Corioalantoide/irrigação sanguínea , Células Endoteliais/metabolismo , Proteínas de Homeodomínio/metabolismo , Neovascularização Fisiológica , Angiopoietina-2/genética , Animais , Bovinos , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Embrião de Galinha , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica/métodos , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Neovascularização Fisiológica/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Receptor TIE-2/metabolismo , Transdução Genética , Regulação para CimaRESUMO
Bmper, which is orthologous to Drosophila melanogaster crossveinless 2, is a secreted factor that regulates Bmp activity in a tissue- and stage-dependent manner. Both pro- and anti-Bmp activities have been postulated for Bmper, although the molecular mechanisms through which Bmper affects Bmp signaling are unclear. In this paper, we demonstrate that as molar concentrations of Bmper exceed Bmp4, Bmper dynamically switches from an activator to an inhibitor of Bmp4 signaling. Inhibition of Bmp4 through a novel endocytic trap-and-sink mechanism leads to the efficient degradation of Bmper and Bmp4 by the lysosome. Bmper-mediated internalization of Bmp4 reduces the duration and magnitude of Bmp4-dependent Smad signaling. We also determined that Noggin and Gremlin, but not Chordin, trigger endocytosis of Bmps. This endocytic transport pathway expands the extracellular roles of selective Bmp modulators to include intracellular regulation. This dosage-dependent molecular switch resolves discordances among studies that examine how Bmper regulates Bmp activity and has broad implications for Bmp signal regulation by secreted mediators.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Transporte/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Clatrina/metabolismo , Endocitose , Fibroblastos/metabolismo , Lisossomos/metabolismo , CamundongosRESUMO
The bone morphogenetic protein (BMP) family of proteins participates in regulation of angiogenesis in physiologic and pathologic conditions. To investigate the molecular mechanisms that contribute to BMP-dependent angiogenic signaling, we performed gene expression profiling of BMP6-treated mouse endothelial cells. We detected 77 mRNAs that were differentially regulated after BMP6 stimulation. Of these, cyclooxygenase 2 (Cox2) was among the most highly up-regulated by BMP stimulation, suggesting a role for Cox2 as a downstream regulator of BMP-induced angiogenesis. Up-regulation of Cox2 by BMP6 was detected at both mRNA and protein levels in endothelial cells, and BMP6 increased production of prostaglandins in a Cox2-dependent fashion. BMP6 up-regulated Cox2 at the transcriptional level through upstream SMAD-binding sites in the Cox2 promoter. Pharmacologic inhibition of Cox2, but not Cox1, blocked BMP6-induced endothelial cell proliferation, migration, and network assembly. BMP6-dependent microvessel outgrowth was markedly attenuated in aortic rings from Cox2-/- mice or after pharmacologic inhibition of Cox2 in aortas from wild-type mice. These results support a necessary role for Cox2 in mediating proangiogenic activities of BMP6. These data indicate that Cox2 may serve as a unifying component downstream from disparate pathways to modulate angiogenic responses in diseases in which neovascularization plays an underlying pathophysiologic role.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Prostaglandinas/biossíntese , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteína Morfogenética Óssea 6 , Células Cultivadas , Inibidores de Ciclo-Oxigenase 2/farmacologia , Sondas de DNA/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Microcirculação/efeitos dos fármacos , Microcirculação/crescimento & desenvolvimento , Microcirculação/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/farmacologia , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Induction of molecular chaperones is the characteristic protective response to environmental stress, and is regulated by a transcriptional program that depends on heat shock factor 1 (HSF1), which is normally under negative regulatory control by molecular chaperones Hsp70 and Hsp90. In metazoan species, the chaperone system also provides protection against apoptosis. We demonstrate that the dual function co-chaperone/ubiquitin ligase CHIP (C-terminus of Hsp70-interacting protein) regulates activation of the stress-chaperone response through induced trimerization and transcriptional activation of HSF1, and is required for protection against stress-induced apoptosis in murine fibroblasts. The consequences of this function are demonstrated by the phenotype of mice lacking CHIP, which develop normally but are temperature-sensitive and develop apoptosis in multiple organs after environmental challenge. CHIP exerts a central and unique role in tuning the response to stress at multiple levels by regulation of protein quality control and transcriptional activation of stress response signaling.