RESUMEN
BACKGROUND: The purpose of present study was to identify the differentially expressed genes (DEGs) associated with BMP-9-induced osteogenic differentiation of mesenchymal stem cells (MSCs) by using bioinformatics methods. METHODS: Gene expression profiles of BMP-9-induced MSCs were compared between with GFP-induced MSCs and BMP-9-induced MSCs. GSE48882 containing two groups of gene expression profiles, 3 GFP-induced MSC samples and 3 from BMP-9-induced MSCs, was downloaded from the Gene Expression Omnibus (GEO) database. Then, DEGs were clustered based on functions and signaling pathways with significant enrichment analysis. Pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) demonstrated that the identified DEGs were potentially involved in cytoplasm, nucleus, and extracellular exosome signaling pathway. RESULTS: A total of 1967 DEGs (1029 upregulated and 938 downregulated) were identified from GSE48882 datasets. R/Bioconductor package limma was used to identify the DEGs. Further analysis revealed that there were 35 common DEGs observed between the samples. GO function and KEGG pathway enrichment analysis, among which endoplasmic reticulum, protein export, RNA transport, and apoptosis was the most significant dysregulated pathway. The result of protein-protein interaction (PPI) network modules demonstrated that the Hspa5, P4hb, Sec61a1, Smarca2, Pdia3, Dnajc3, Hyou1, Smad7, Derl1, and Surf4 were the high-degree hub nodes. CONCLUSION: Taken above, using integrated bioinformatical analysis, we have identified DEGs candidate genes and pathways in BMP-9 induced MSCs, which could improve our understanding of the key genes and pathways for BMP-9-induced osteogenic of MSCs.
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Diferenciación Celular/genética , Biología Computacional/métodos , Bases de Datos Genéticas , Factor 2 de Diferenciación de Crecimiento/genética , Factor 2 de Diferenciación de Crecimiento/fisiología , Células Madre Mesenquimatosas/fisiología , Osteogénesis/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Transcriptoma , Chaperón BiP del Retículo Endoplásmico , Estudios de Asociación Genética/métodos , Factor 2 de Diferenciación de Crecimiento/metabolismo , Proteínas de Choque Térmico , Humanos , Procolágeno-Prolina Dioxigenasa , Proteína Disulfuro Isomerasas , Canales de Translocación SECRESUMEN
Bone morphogenetic protein-9 (BMP-9) is a novel cytokine which is cloned from the fetal mouse liver cDNA library and belongs to the member of the transforming growth factor-ß (TGF-ß) superfamily. BMP-9 is mainly secreted by the liver and exerts a variety of physiological functions. In this review, we present the latest knowledge on the biochemistry of BMP-9 and its role in glucose metabolism and lipid homeostasis. We introduced the expression site, structure, synthesis, and secretion of BMP-9, as well as BMP-9 signaling pathway. We also discuss the effects of BMP-9 on glucose metabolism and lipid metabolism in different organs. BMP-9 can regulate glucose and lipid homeostasis in the body by inhibiting liver gluconeogenesis, transforming white adipose tissue to brown adipose tissue, promoting muscle glycogen synthesis, increasing the uptake and utilization of glucose by muscle tissue, increasing liver and adipose tissue insulin sensitivity, promoting insulin synthesis and secretion, inhibiting liver lipid deposition, and playing a leptin-like role. Finally, through the results of animal intervention studies and human clinical studies in the review, we deeply understand the association of BMP-9 with obesity, insulin resistance (IR), type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD), which provides new ideas for the prevention and treatment of diseases.
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Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Factor 2 de Diferenciación de Crecimiento/fisiología , Resistencia a la Insulina , Metabolismo de los Lípidos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Animales , HumanosRESUMEN
The aim of the present work was to address the role of BMP9 in different genetic backgrounds (C57BL/6, BALB/c, and 129/Ola) of mice deleted for Bmp9. We found that Bmp9 deletion led to premature mortality only in the 129/Ola strain. We have previously shown that Bmp9 deletion led to liver sinusoidal endothelial cells (LSEC) capillarization and liver fibrosis in the 129/Ola background. Here, we showed that this is not the case in the C57BL/6 background. Analysis of LSEC from Wild-type (WT) versus Bmp9-KO mice in the C57BL/6 background showed no difference in LSEC fenestration and in the expression of differentiation markers. Comparison of the mRNA expression of LSEC differentiation markers between WT C57BL/6 and 129/Ola mice showed a significant decrease in Stabilin2, Plvap, and CD209b, suggesting a more capillary-like phenotype in WT C57BL/6 LSECs. C57BL/6 mice also had lower BMP9 circulating concentrations and hepatic Vegfr2 mRNA levels, compared to the 129/Ola mice. Taken together, our observations support a role for BMP9 in liver endothelial cell fenestration and prevention of fibrosis that is dependent on genetic background. It also suggests that 129/Ola mice are a more suitable model than C57BL/6 for the study of liver fibrosis subsequent to LSEC capillarization.
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Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Factor 2 de Diferenciación de Crecimiento/fisiología , Cirrosis Hepática , Hígado/metabolismo , Animales , Biomarcadores/metabolismo , Capilares/metabolismo , Capilares/patología , Diferenciación Celular , Células Endoteliales/patología , Factor 2 de Diferenciación de Crecimiento/genética , Hígado/patología , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Bone morphogenetic protein (BMP)-9 has been reported to regulate energy balance in vivo. However, the mechanisms underlying BMP9-mediated regulation of energy balance remain incompletely understood. Here, we investigated the role of BMP9 in energy metabolism. In the current study, we found that hepatic BMP9 expression was down-regulated in insulin resistance (IR) mice and in patients who are diabetic. In mice fed a high-fat diet (HFD), the overexpression of hepatic BMP9 improved glucose tolerance and IR. The expression of gluconeogenic genes was down-regulated, whereas the level of insulin signaling molecule phosphorylation was increased in the livers of Adenovirus-BMP9-treated mice and glucosamine-treated hepatocytes. Furthermore, BMP9 overexpression ameliorated triglyceride accumulation and inhibited the expression of lipogenic genes in both human hepatocellular carcinoma HepG2 cells treated with a fatty acid mixture as well as the livers of HFD-fed mice. In hepatocytes isolated from sterol regulatory element-binding protein (SREBP)-1c knockout mice, the effects of BMP9 were ablated. Mechanistically, BMP9 inhibited SREBP-1c expression through the inhibition of liver X receptor response element 1 activity in the SREBP-1c promoter. Taken together, our results show that BMP9 is an important regulator of hepatic glucose and lipid metabolism.-Yang, M., Liang, Z., Yang, M., Jia, Y., Yang, G., He, Y., Li, X., Gu, H. F., Zheng, H., Zhu, Z., Li, L. Role of bone morphogenetic protein-9 in the regulation of glucose and lipid metabolism.
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Glucosa/metabolismo , Factor 2 de Diferenciación de Crecimiento/fisiología , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas/fisiología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Células Cultivadas , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos/farmacología , Regulación de la Expresión Génica , Factor 2 de Diferenciación de Crecimiento/biosíntesis , Factor 2 de Diferenciación de Crecimiento/genética , Hepatocitos/metabolismo , Humanos , Resistencia a la Insulina , Metabolismo de los Lípidos/genética , Lipogénesis/genética , Hígado/efectos de los fármacos , Neoplasias Hepáticas/patología , Receptores X del Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cultivo Primario de Células , Regiones Promotoras Genéticas/genética , ARN Mensajero/biosíntesis , Receptores de Leptina/deficiencia , Proteínas Recombinantes/metabolismo , Elementos de Respuesta/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/deficiencia , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/metabolismoRESUMEN
RATIONALE: Although many familial cases of pulmonary arterial hypertension exhibit an autosomal dominant mode of inheritance with the majority having mutations in essential constituents of the BMP (bone morphogenetic protein) signaling, the specific contribution of the long-term loss of signal transduction triggered by the BMPR2 (type 2 BMP receptor) remains poorly characterized. OBJECTIVE: To investigate the role of BMP9, the main ligand of ALK1 (Activin receptor-like kinase 1)/BMPR2 heterocomplexes, in pulmonary hypertension. METHOD AND RESULTS: The absence of BMP9 in Bmp9-/- mice and its inhibition in C57BL/6 mice using neutralizing anti-BMP9 antibodies substantially prevent against chronic hypoxia-induced pulmonary hypertension judged by right ventricular systolic pressure measurement, right ventricular hypertrophy, and pulmonary distal arterial muscularization. In agreement with these observations, we found that the BMP9/BMP10 ligand trap ALK1ECD administered in monocrotaline or Sugen/hypoxia (SuHx) rats substantially attenuate proliferation of pulmonary vascular cells, inflammatory cell infiltration, and regresses established pulmonary hypertension in rats. Our data obtained in human pulmonary endothelial cells derived from controls and pulmonary arterial hypertension patients indicate that BMP9 can affect the balance between endothelin-1, apelin, and adrenomedullin. We reproduced these in vitro observations in mice chronically exposed to hypoxia, with Bmp9-/- mice exhibiting lower mRNA levels of the vasoconstrictor peptide ET-1 (endothelin-1) and higher levels of the 2 potent vasodilator factors apelin and ADM (adrenomedullin) compared with Bmp9+/+ littermates. CONCLUSIONS: Taken together, our data indicate that the loss of BMP9, by deletion or inhibition, has beneficial effects against pulmonary hypertension onset and progression.
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Factor 2 de Diferenciación de Crecimiento/antagonistas & inhibidores , Hipertensión Pulmonar/prevención & control , Receptores de Activinas Tipo II/farmacología , Animales , Células Cultivadas , Endotelina-1/genética , Factor 2 de Diferenciación de Crecimiento/fisiología , Humanos , Hipoxia/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas WistarRESUMEN
MicroRNAs are identified as negative regulators in gene expression through silencing gene expression at the post-transcriptional and translational levels. Bone morphogenetic protein 9 (BMP9) is the most effective in inducing osteogenesis in the BMP family, the members of which were originally identified as osteoinductive cytokines. In the current study, the role of miR23b in the progression of BMP9induced C2C12 myoblasts was investigated. The results indicated that miR23b was significantly downregulated in C2C12 myoblasts induced by BMP9. Overexpression of miR23b significantly inhibited osteogenesis in the C2C12 myoblasts. In addition, it was observed that Runx2 was negatively regulated by miR23b at the posttranscriptional level, via a specific target site within the 3'UTR of Runx2. Knockdown of Runx2 promoted miR23binduced inhibition of osteogenesis in C2C12 myoblasts. The expression of Runx2 was observed to be frequently upregulated in osteoblast cell lines and inversely correlated with miR23b expression. Thus, the results of the present study suggest that miR23b inhibits BMP9induced C2C12 myoblast osteogenesis via targeting of the Runx2 gene, acting as a suppressor. The current study contributes to the understanding of the functions of BMP9 in ossification.
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Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Factor 2 de Diferenciación de Crecimiento/fisiología , MicroARNs/genética , Osteoblastos/fisiología , Animales , Diferenciación Celular , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación hacia Abajo , Células HCT116 , Células HEK293 , Humanos , Ratones , MicroARNs/metabolismo , Osteogénesis , Interferencia de ARNRESUMEN
The transition to pulmonary respiration after birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. Two members of the TGFß family, bone morphogenetic protein 9 (BMP9) and BMP10, have been recently involved in postnatal angiogenesis, both being necessary for remodeling of newly formed microvascular beds. The aim of the present work was to study whether BMP9 and BMP10 could be involved in closure of the DA. We found that Bmp9 knockout in mice led to an imperfect closure of the DA. Further, addition of a neutralizing anti-BMP10 antibody at postnatal day 1 (P1) and P3 in these pups exacerbated the remodeling defect and led to a reopening of the DA at P4. Transmission electron microscopy images and immunofluorescence stainings suggested that this effect could be due to a defect in intimal cell differentiation from endothelial to mesenchymal cells, associated with a lack of extracellular matrix deposition within the center of the DA. This result was supported by the identification of the regulation by BMP9 and BMP10 of several genes known to be involved in this process. The involvement of these BMPs was further supported by human genomic data because we could define a critical region in chromosome 2 encoding eight genes including BMP10 that correlated with the presence of a patent DA. Together, these data establish roles for BMP9 and BMP10 in DA closure.
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Proteínas Morfogenéticas Óseas/fisiología , Conducto Arterial/fisiología , Factor 2 de Diferenciación de Crecimiento/fisiología , Animales , Proteínas Morfogenéticas Óseas/genética , Conducto Arterial/patología , Factor 2 de Diferenciación de Crecimiento/genética , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Osteosarcoma (OS) is the most common primary malignancy of bone and is usually associated with poor prognosis due to its high incidence of metastasis and chemoresistance. Molecular pathogenesis of OS is poorly understood. We previously showed that OS cells are refractory to BMP9-induced osteogenesis and respond favorably to proliferation and tumor growth. Here we investigate if Notch signaling mediates the BMP9-promoted cell proliferation and tumor growth of human osteosarcoma (OS). We find that the expression of Notch1, Notch2, Notch3, DLL1, JAG1 and JAG2 is readily detected in most of the tested OS cell lines. BMP9-promoted OS cell proliferation, migration, and cell cycle S/G2 progression are effectively inhibited by a dominant-negative mutant of Notch1 (dnNotch1) or the γ-secretase inhibitor Compound E (ComE). Furthermore, BMP9-promoted tumor growth and osteolytic lesions in vivo are significantly inhibited by dnNotch1. BMP9 up-regulates the expression of Notch1, Notch3, DLL1, and JAG1 in OS cells. Accordingly, BMP9 stimulation induces a nuclear accumulation of NICD, which is blocked by ComE. Our results demonstrate that BMP9-promoted OS proliferation and tumor growth is at least in part mediated by Notch signaling, suggesting that osteogenic BMPs may function as upstream regulators of Notch signaling in OS tumorigenesis. Thus, pharmacologic intervention of Notch signaling may be explored as a new therapeutic strategy for human OS tumors.
Asunto(s)
División Celular/fisiología , Factor 2 de Diferenciación de Crecimiento/fisiología , Osteosarcoma/patología , Línea Celular Tumoral , Humanos , Osteosarcoma/fisiopatologíaRESUMEN
BMP-9, whose expression is highest in liver cells, has been demonstrated to regulate expression of enzymes involved in glucose homeostasis. However, the underlying mechanism of this effect has yet to be elucidated. We observed that MB109, a recombinant BMP-9 derivative, enhanced brown adipogenesis of human adipose tissue derived stem cells. With this observation of the cell culture system, we hypothesized that MB109 may be able to improve glucose metabolism by regulating expression of brown adipogenic genes. Systemic intraperitoneal injection of MB109 (200 µg/kg/wk) suppressed weight gaining of high fat diet-induced obese mice by reducing sizes of white adipocytes and decreased 16 h fasting blood glucose levels without changing food consumption or apparent behavioral performances. MB109 induced expression of brown adipogenic genes in the subcutaneous but not in the visceral fat tissues from the mice fed with high fat diet. In addition, systematic injection of MB109 enhanced fatty acid synthase expression in the liver of obese mice, which may help attenuate an obesity-associated increase of blood glucose levels. Our results demonstrate a role of BMP-9 in brown adipogenesis and suppressing pathophysiology of high fat diet-induced obesity, presumably through the activin receptor like kinase 1 signaling pathway.
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Adipogénesis , Factor 2 de Diferenciación de Crecimiento/fisiología , Obesidad/prevención & control , Adipogénesis/genética , Animales , Secuencia de Bases , Peso Corporal , Cartilla de ADN , Dieta Alta en Grasa , Ratones , Obesidad/etiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Mesenchymal stem cells (MSCs) are multipotent progenitors that can undergo osteogenic differentiation under proper stimuli. We demonstrated that BMP9 is one of the most osteogenic BMPs. However, the molecular mechanism underlying BMP9-initiated osteogenic signaling in MSCs remains unclear. Through gene expression profiling analysis we identified several candidate mediators of BMP9 osteogenic signaling. Here, we focus on one such signaling mediator and investigate the functional role of cysteine-rich with EGF-like domains 2 (Creld2) in BMP9-initiated osteogenic signaling. Creld2 was originally identified as an ER stress-inducible factor localized in the ER-Golgi apparatus. Our genomewide expression profiling analysis indicates that Creld2 is among the top up-regulated genes in BMP9-stimulated MSCs. We confirm that Creld2 is up-regulated by BMP9 in MSCs. ChIP analysis indicates that Smad1/5/8 directly binds to the Creld2 promoter in a BMP9-dependent fashion. Exogenous expression of Creld2 in MSCs potentiates BMP9-induced early and late osteogenic markers, and matrix mineralization. Conversely, silencing Creld2 expression inhibits BMP9-induced osteogenic differentiation. In vivo stem cell implantation assay reveals that exogenous Creld2 promotes BMP9-induced ectopic bone formation and matrix mineralization, whereas silencing Creld2 expression diminishes BMP9-induced bone formation and matrix mineralization. We further show that Creld2 is localized in ER and the ER stress inducers potentiate BMP9-induced osteogenic differentiation. Our results strongly suggest that Creld2 may be directly regulated by BMP9 and ER stress response may play an important role in regulating osteogenic differentiation.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Diferenciación Celular/fisiología , Retículo Endoplásmico/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Factor 2 de Diferenciación de Crecimiento/fisiología , Células Madre Mesenquimatosas/citología , Animales , Moléculas de Adhesión Celular/genética , Línea Celular , Proteínas de la Matriz Extracelular/genética , Silenciador del Gen , Humanos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Lymphatic vessels are critical for the maintenance of tissue fluid homeostasis and their dysfunction contributes to several human diseases. The activin receptor-like kinase 1 (ALK1) is a transforming growth factor-ß family type 1 receptor that is expressed on both blood and lymphatic endothelial cells (LECs). Its high-affinity ligand, bone morphogenetic protein 9 (BMP9), has been shown to be critical for retinal angiogenesis. The aim of this work was to investigate whether BMP9 could play a role in lymphatic development. We found that Bmp9 deficiency in mice causes abnormal lymphatic development. Bmp9-knockout (KO) pups presented hyperplastic mesenteric collecting vessels that maintained LYVE-1 expression. In accordance with this result, we found that BMP9 inhibited LYVE-1 expression in LECs in an ALK1-dependent manner. Bmp9-KO pups also presented a significant reduction in the number and in the maturation of mesenteric lymphatic valves at embryonic day 18.5 and at postnatal days 0 and 4. Interestingly, the expression of several genes known to be involved in valve formation (Foxc2, Connexin37, EphrinB2, and Neuropilin1) was upregulated by BMP9 in LECS. Finally, we demonstrated that Bmp9-KO neonates and adult mice had decreased lymphatic draining efficiency. These data identify BMP9 as an important extracellular regulator in the maturation of the lymphatic vascular network affecting valve development and lymphatic vessel function.
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Factor 2 de Diferenciación de Crecimiento/fisiología , Linfangiogénesis/genética , Vasos Linfáticos/fisiología , Mesenterio/embriología , Animales , Animales Recién Nacidos , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Factor 2 de Diferenciación de Crecimiento/genética , Factor 2 de Diferenciación de Crecimiento/metabolismo , Humanos , Linfangiogénesis/fisiología , Vasos Linfáticos/metabolismo , Proteínas de Transporte de Membrana , Mesenterio/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
BMP9 signaling has been implicated in hereditary hemorrhagic telangiectasia (HHT) and vascular remodeling, acting via the HHT target genes, endoglin and ALK1. This study sought to identify endothelial BMP9-regulated proteins that could affect the HHT phenotype. Gene ontology analysis of cDNA microarray data obtained after BMP9 treatment of primary human endothelial cells indicated regulation of chemokine, adhesion, and inflammation pathways. These responses included the up-regulation of the chemokine CXCL12/SDF1 and down-regulation of its receptor CXCR4. Quantitative mass spectrometry identified additional secreted proteins, including the chemokine CXCL10/IP10. RNA knockdown of endoglin and ALK1 impaired SDF1/CXCR4 regulation by BMP9. Because of the association of SDF1 with ischemia, we analyzed its expression under hypoxia in response to BMP9 in vitro, and during the response to hindlimb ischemia, in endoglin-deficient mice. BMP9 and hypoxia were additive inducers of SDF1 expression. Moreover, the data suggest that endoglin deficiency impaired SDF1 expression in endothelial cells in vivo. Our data implicate BMP9 in regulation of the SDF1/CXCR4 chemokine axis in endothelial cells and point to a role for BMP9 signaling via endoglin in a switch from an SDF1-responsive autocrine phenotype to an SDF1 nonresponsive paracrine state that represses endothelial cell migration and may promote vessel maturation.
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Células Endoteliales/citología , Factores de Diferenciación de Crecimiento/fisiología , Neovascularización Fisiológica/fisiología , Receptores de Activinas Tipo I/fisiología , Receptores de Activinas Tipo II/fisiología , Animales , Antígenos CD/fisiología , Aorta/citología , Comunicación Autocrina , Hipoxia de la Célula , Movimiento Celular , Quimiocina CXCL12/biosíntesis , Quimiocina CXCL12/metabolismo , Medios de Cultivo Condicionados , Endoglina , Células Endoteliales/efectos de los fármacos , Factor 2 de Diferenciación de Crecimiento/farmacología , Factor 2 de Diferenciación de Crecimiento/fisiología , Miembro Posterior/irrigación sanguínea , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Isquemia/fisiopatología , Ratones , Comunicación Paracrina , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Superficie Celular/fisiología , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
ALK1 (ACVRL1) is a member of the TGFß receptor family and is expressed predominantly by arterial endothelial cells (EC). Mutations in ACVRL1 are responsible for hereditary hemorrhagic telangiectasia type 2 (HHT2), a disease manifesting as fragile vessels, capillary overgrowth, and numerous arterio-venous malformations. Arterial EC also express EphrinB2, which has multiple roles in vascular development and angiogenesis and is known to be reduced in ACVRL1 knockout mice. Using an in vitro angiogenesis model we find that the Alk1 ligand BMP9 induces EphrinB2 in EC, and this is entirely dependent on expression of Alk1 and at least one of the co-receptors BMPRII or ActRII. BMP9 induces both ID1 and ID3, and both are necessary for full induction of EphrinB2. Loss of Alk1 or EphrinB2 results in increased arterial-venous anastomosis, while loss of Alk1 but not EphrinB2 results in increased VEGFR2 expression and enhanced capillary sprouting. Conversely, BMP9 blocks EC sprouting and this is dependent on Alk1, BMPRII/ActRII and ID1/ID3. Finally, notch signaling overcomes the loss of Alk1-restoring EphrinB2 expression in EC, and curbing excess sprouting. Thus, in an in vitro model of HHT2, loss of Alk1 blocks BMP9 signaling, resulting in reduced EphrinB2 expression, enhanced VEGFR2 expression, and misregulated EC sprouting and anastomosis.
Asunto(s)
Receptores de Activinas Tipo I/genética , Endotelio Vascular/metabolismo , Efrina-B2/metabolismo , Factor 2 de Diferenciación de Crecimiento/fisiología , Telangiectasia Hemorrágica Hereditaria/metabolismo , Receptores de Activinas Tipo I/metabolismo , Receptores de Activinas Tipo II/metabolismo , Animales , Secuencia de Bases , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Cartilla de ADN , Endotelio Vascular/citología , Efrina-B2/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Regiones Promotoras Genéticas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Notch/metabolismo , Transducción de Señal , Telangiectasia Hemorrágica Hereditaria/genéticaRESUMEN
ALK1 is a type I receptor of the TGF-ß family that is involved in angiogenesis. Circulating BMP9 was identified as a specific ligand for ALK1 inducing vascular quiescence. In this work, we found that blocking BMP9 with a neutralizing antibody in newborn mice significantly increased retinal vascular density. Surprisingly, Bmp9-KO mice did not show any defect in retinal vascularization. However, injection of the extracellular domain of ALK1 impaired retinal vascularization in Bmp9-KO mice, implicating another ligand for ALK1. Interestingly, we detected a high level of circulating BMP10 in WT and Bmp9-KO pups. Further, we found that injection of a neutralizing anti-BMP10 antibody to Bmp9-KO pups reduced retinal vascular expansion and increased vascular density, whereas injection of this antibody to WT pups did not affect the retinal vasculature. These data suggested that BMP9 and BMP10 are important in postnatal vascular remodeling of the retina and that BMP10 can substitute for BMP9. In vitro stimulation of endothelial cells by BMP9 and BMP10 increased the expression of genes involved in the Notch signaling pathway (Jagged1, Dll4, Hey1, Hey2, Hes1) and decreased apelin expression, suggesting a possible cross-talk between these pathways and the BMP pathway.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Factor 2 de Diferenciación de Crecimiento/fisiología , Vasos Retinianos/fisiología , Receptores de Activinas Tipo I/química , Receptores de Activinas Tipo I/farmacología , Receptores de Activinas Tipo II , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Recuento de Células , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Factor 2 de Diferenciación de Crecimiento/antagonistas & inhibidores , Factor 2 de Diferenciación de Crecimiento/genética , Factor 2 de Diferenciación de Crecimiento/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/farmacología , Vasos Retinianos/citología , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/metabolismoRESUMEN
Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor ß (TGF-ß) type I receptor, and endoglin, a TGF-ß co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-ß to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.
Asunto(s)
Receptores de Activinas Tipo II/inmunología , Endotelio Vascular/metabolismo , Factor 2 de Diferenciación de Crecimiento/fisiología , Transducción de Señal/fisiología , Receptores de Activinas Tipo II/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Citometría de Flujo , Factor 2 de Diferenciación de Crecimiento/metabolismo , Humanos , Reacción en Cadena de la Polimerasa , Unión ProteicaRESUMEN
BACKGROUND: Bone morphogenetic proteins (BMPs) and their receptors, such as bone morphogenetic protein receptor (BMPR) II, have been implicated in a wide variety of disorders including pulmonary arterial hypertension (PAH). Similarly, endothelin-1 (ET-1), a mitogen and vasoconstrictor, is upregulated in PAH and endothelin receptor antagonists are used in its treatment. We sought to determine whether there is crosstalk between BMP signalling and the ET-1 axis in human pulmonary artery endothelial cells (HPAECs), possible mechanisms involved in such crosstalk and functional consequences thereof. METHODOLOGY/PRINCIPAL FINDING: Using western blot, real time RT-PCR, ELISA and small RNA interference methods we provide evidence that in HPAECs BMP-9, but not BMP-2, -4 and -6 significantly stimulated ET-1 release under physiological concentrations. This release is mediated by both Smad1 and p38 MAPK and is independent of the canonical Smad4 pathway. Moreover, knocking down the ALK1 receptor or BMPR II attenuates BMP-9 stimulated ET-1 release, whilst causing a significant increase in prepro ET-1 mRNA transcription and mature peptide release. Finally, BMP-9 induced ET-1 release is involved in both inhibition of endothelial cell migration and promotion of tubule formation. CONCLUSIONS/SIGNIFICANCE: Although our data does not support an important role for BMP-9 as a source of increased endothelial ET-1 production seen in human PAH, BMP-9 stimulated ET-1 production is likely to be important in angiogenesis and vascular stability. However, increased ET-1 production by endothelial cells as a consequence of BMPR II dysfunction may be clinically relevant in the pathogenesis of PAH.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Endotelina-1/biosíntesis , Factor 2 de Diferenciación de Crecimiento/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Proteína Smad1/fisiología , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Antagonistas de los Receptores de la Endotelina B , Endotelina-1/antagonistas & inhibidores , Endotelina-1/genética , Endotelina-1/metabolismo , Factor 2 de Diferenciación de Crecimiento/fisiología , Humanos , Neovascularización Fisiológica/genética , Oligopéptidos/farmacología , Piperidinas/farmacología , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Arteria Pulmonar/fisiología , Proteína Smad1/genética , Proteína Smad1/metabolismoRESUMEN
Our previous studies have demonstrated that bone morphogenetic protein 9 (BMP-9) is one of the most efficacious BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanism underlying the BMP-9-induced osteogenic differentiation of MSCs remains to be fully elucidated. In this study, dominant negative (DN) type II TGF-ß receptors were constructed and introduced into C3H10T1/2 stem cells, then in vitro and in vivo assays were carried out to analyze and identify the type II TGF-ß receptors required for BMP-9-induced osteogenesis. We found that three DN type II TGF-ß receptors, DN-BMPRII, DN-ActRII, and DN-ActRIIB, diminished BMP-9-induced alkaline phosphatase (ALP) activity, led to a decrease in BMP-9-induced Smad binding element (SBE)-controled reporter activity, reduced BMP-9-induced expressions of Smad6 and Smad7, and decreased BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, finally resulted in decreased bone masses and immature osteogenesis. These findings strongly suggested that three wild-type II TGF-ß receptors, BMPRII, ActRII and ActRIIB, may play a functional role in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells. However, C3H10T1/2 stem cells can express BMPRII and ActRII, but not ActRIIB. Using RNA interference (RNAi), we found that luciferase reporter activity and ALP activity induced by BMP-9 were accordingly inhibited along with the knockdown of BMPRII and ActRII. Taken together, our results demonstrated that BMPRII and ActRII are the functional type II TGF-ß receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells.
Asunto(s)
Diferenciación Celular/fisiología , Factor 2 de Diferenciación de Crecimiento/fisiología , Células Madre Mesenquimatosas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Sitios de Unión/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Diferenciación Celular/genética , Línea Celular , Trasplante de Células , Factor 2 de Diferenciación de Crecimiento/genética , Células HCT116 , Células HEK293 , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Desnudos , Osteogénesis/genética , Osteogénesis/fisiología , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Proteína smad6/genética , Proteína smad6/metabolismo , Proteína smad7/genética , Proteína smad7/metabolismo , TransfecciónRESUMEN
Endoglin, an endothelial cell-specific transforming growth factor-beta (TGF-beta) superfamily coreceptor, has an essential role in angiogenesis. Endoglin-null mice have an embryonic lethal phenotype due to defects in angiogenesis and mutations in endoglin result in the vascular disease hereditary hemorrhagic telangiectasia type I. Increased endoglin expression in the proliferating endothelium of tumors has been correlated with metastasis, tumor grade and decreased survival. Although endoglin is thought to regulate TGF-beta superfamily signaling in endothelial cells through regulating the balance between two TGF-beta-responsive pathways, the activin receptor-like kinase 5 (ALK5)/Smad2/3 pathway and the activin receptor-like kinase 1 (ALK1)/Smad1/5/8 pathway, the mechanism by which endoglin regulates angiogenesis has not been defined. Here, we investigate the role of the cytoplasmic domain of endoglin and its phosphorylation by ALK5 in regulating endoglin function in endothelial cells. We demonstrate that the cytoplasmic domain of endoglin is basally phosphorylated by ALK5, primarily on serines 646 and 649, in endothelial cells. Functionally, the loss of phosphorylation at serine 646 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to TGF-beta and endothelial cell migration, whereas loss of phosphorylation at both serines 646 and 649 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to bone morphogenetic protein-9. Taken together, these results support endoglin phosphorylation by ALK5 as an important mechanism for regulating TGF-beta superfamily signaling and migration in endothelial cells.
Asunto(s)
Células Endoteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Proteína Smad1/fisiología , Proteína Smad5/fisiología , Proteína Smad8/fisiología , Animales , Células COS , Movimiento Celular , Chlorocebus aethiops , Endoglina , Factor 2 de Diferenciación de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Visón , Fosforilación , Fosfoserina/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Receptor Tipo I de Factor de Crecimiento Transformador beta , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Bone morphogenetic protein 9 (BMP-9) is a member of the transforming growth factor (TGF)-beta/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/beta-catenin signalling plays an important role in BMP-9-induced osteogenic differentiation of MSCs. Wnt3A and BMP-9 enhanced each other's ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. beta-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers. Furthermore, beta-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP-9 induced recruitment of both Runx2 and beta-catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between beta-catenin and Runx2, plays an important role in BMP-9-induced osteogenic differentiation of MSCs.