RESUMO
Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity.
Assuntos
Células Endoteliais/fisiologia , Células Estreladas do Fígado/fisiologia , Hepatócitos/fisiologia , Células de Kupffer/fisiologia , Fígado/citologia , Macrófagos/fisiologia , Monócitos/fisiologia , Animais , Comunicação Celular , Diferenciação Celular , Células Cultivadas , Microambiente Celular , Feminino , Regulação da Expressão Gênica , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Notch/metabolismoRESUMO
Herpesviruses modulate immune control to secure lifelong infection. The mechanisms Human Cytomegalovirus (HCMV) employs in this regard can reveal unanticipated aspects of cellular signaling involved in antiviral immunity. Here, we describe a novel relationship between the TGF-ß family cytokine BMP9 and HCMV infection. We identify a cross-talk between BMP9-induced and IFN receptor-mediated signaling, showing that BMP9 boosts the transcriptional response to and antiviral activity of IFNß, thereby enhancing viral restriction. We also show that BMP9 is secreted by human fibroblasts upon HCMV infection. However, HCMV infection impairs BMP9-induced enhancement of the IFNß response, indicating that this signaling role of BMP9 is actively targeted by HCMV. Indeed, transmembrane proteins US18 and US20, which downregulate type I BMP receptors, are necessary and sufficient to cause inhibition of BMP9-mediated boosting of the antiviral response to IFNß. HCMV lacking US18 and US20 is more sensitive to IFNß. Thus, HCMV has a mutually antagonistic relationship with BMP9, which extends the growing body of evidence that BMP signaling is an underappreciated modulator of innate immunity in response to viral infection.
Assuntos
Fator 2 de Diferenciação de Crescimento , Imunidade Inata , Humanos , Citocinas/metabolismo , Citomegalovirus/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Transdução de SinaisRESUMO
Amputation injuries in mammals are typically non-regenerative; however, joint regeneration is stimulated by BMP9 treatment, indicating the presence of latent articular chondrocyte progenitor cells. BMP9 induces a battery of chondrogenic genes in vivo, and a similar response is observed in cultures of amputation wound cells. Extended cultures of BMP9-treated cells results in differentiation of hyaline cartilage, and single cell RNAseq analysis identified wound fibroblasts as BMP9 responsive. This culture model was used to identify a BMP9-responsive adult fibroblast cell line and a culture strategy was developed to engineer hyaline cartilage for engraftment into an acutely damaged joint. Transplanted hyaline cartilage survived engraftment and maintained a hyaline cartilage phenotype, but did not form mature articular cartilage. In addition, individual hypertrophic chondrocytes were identified in some samples, indicating that the acute joint injury site can promote osteogenic progression of engrafted hyaline cartilage. The findings identify fibroblasts as a cell source for engineering articular cartilage and establish a novel experimental strategy that bridges the gap between regeneration biology and regenerative medicine.
Assuntos
Diferenciação Celular , Fibroblastos/citologia , Cartilagem Hialina/citologia , Regeneração , Engenharia Tecidual/métodos , Animais , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrogênese , Fibroblastos/efeitos dos fármacos , Fator 2 de Diferenciação de Crescimento/farmacologia , Cartilagem Hialina/metabolismo , Cartilagem Hialina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Rationale: Hepatopulmonary syndrome (HPS) is a severe complication of liver diseases characterized by abnormal dilation of pulmonary vessels, resulting in impaired oxygenation. Recent research highlights the pivotal role of liver-produced BMP-9 (bone morphogenetic protein-9) in maintaining pulmonary vascular integrity. Objectives: This study aimed to investigate the involvement of BMP-9 in human and experimental HPS. Methods: Circulating BMP-9 levels were measured in 63 healthy control subjects and 203 patients with cirrhosis with or without HPS. Two animal models of portal hypertension were employed: common bile duct ligation with cirrhosis and long-term partial portal vein ligation without cirrhosis. Additionally, the therapeutic effect of low-dose BMP activator FK506 was investigated, and the pulmonary vascular phenotype of BMP-9-knockout rats was analyzed. Measurements and Main Results: Patients with HPS related to compensated cirrhosis exhibited lower levels of circulating BMP-9 compared with patients without HPS. Patients with severe cirrhosis exhibited consistently low levels of BMP-9. HPS characteristics were observed in animal models, including intrapulmonary vascular dilations and an increase in the alveolar-arterial gradient. HPS development in both rat models correlated with reduced intrahepatic BMP-9 expression, decreased circulating BMP-9 level and activity, and impaired pulmonary BMP-9 endothelial pathway. Daily treatment with FK506 for 2 weeks restored the BMP pathway in the lungs, alleviating intrapulmonary vascular dilations and improving gas exchange impairment. Furthermore, BMP-9-knockout rats displayed a pulmonary HPS phenotype, supporting its role in disease progression. Conclusions: The study findings suggest that portal hypertension-induced loss of BMP-9 signaling contributes to HPS development.
Assuntos
Modelos Animais de Doenças , Fator 2 de Diferenciação de Crescimento , Síndrome Hepatopulmonar , Síndrome Hepatopulmonar/metabolismo , Síndrome Hepatopulmonar/fisiopatologia , Animais , Masculino , Ratos , Humanos , Pessoa de Meia-Idade , Feminino , Transdução de Sinais , Idoso , Pulmão/metabolismo , Cirrose Hepática/complicações , Cirrose Hepática/fisiopatologia , Adulto , Estudos de Casos e Controles , Tacrolimo/farmacologia , Tacrolimo/uso terapêutico , Hipertensão Portal/fisiopatologiaRESUMO
BACKGROUND: BMP9 and BMP10 are two major regulators of vascular homeostasis. These two ligands bind with high affinity to the endothelial type I kinase receptor ALK1, together with a type II receptor, leading to the direct phosphorylation of the SMAD transcription factors. Apart from this canonical pathway, little is known. Interestingly, mutations in this signaling pathway have been identified in two rare cardiovascular diseases, hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension. METHODS: To get an overview of the signaling pathways modulated by BMP9 and BMP10 stimulation in endothelial cells, we employed an unbiased phosphoproteomic-based strategy. Identified phosphosites were validated by western blot analysis and regulated targets by RT-qPCR. Cell cycle analysis was analyzed by flow cytometry. RESULTS: Large-scale phosphoproteomics revealed that BMP9 and BMP10 treatment induced a very similar phosphoproteomic profile. These BMPs activated a non-canonical transcriptional SMAD-dependent MAPK pathway (MEKK4/P38). We were able to validate this signaling pathway and demonstrated that this activation required the expression of the protein GADD45ß. In turn, activated P38 phosphorylated the heat shock protein HSP27 and the endocytosis protein Eps15 (EGF receptor pathway substrate), and regulated the expression of specific genes (E-selectin, hyaluronan synthase 2 and cyclooxygenase 2). This study also highlighted the modulation in phosphorylation of proteins involved in transcriptional regulation (phosphorylation of the endothelial transcription factor ERG) and cell cycle inhibition (CDK4/6 pathway). Accordingly, we found that BMP10 induced a G1 cell cycle arrest and inhibited the mRNA expression of E2F2, cyclinD1 and cyclinA1. CONCLUSIONS: Overall, our phosphoproteomic screen identified numerous proteins whose phosphorylation state is impacted by BMP9 and BMP10 treatment, paving the way for a better understanding of the molecular mechanisms regulated by BMP signaling in vascular diseases.
Assuntos
Proteínas Morfogenéticas Ósseas , Células Endoteliais , Pontos de Checagem do Ciclo Celular , Fosforilação , Pontos de Checagem da Fase G1 do Ciclo CelularRESUMO
AIM: The regenerative capacity of dental pulp relies on the odonto/osteogenic differentiation of dental pulp cells (DPCs), but dynamic microenvironmental changes hinder the process. Bone morphogenetic protein 9 (BMP9) promotes differentiation of DPCs towards an odonto/osteogenic lineage, forming dentinal-like tissue. However, the molecular mechanism underlying its action remains unclear. This study investigates the role of DLX6 antisense RNA 1 (DLX6-AS1) in odonto/osteogenic differentiation induced by BMP9. METHODOLOGY: Custom RT2 profiler PCR array, quantitative Real-Time PCR (qRT-PCR) and western blots were used to investigate the expression pattern of DLX6-AS1 and its potential signal axis. Osteogenic ability was evaluated using alkaline phosphatase and alizarin red S staining. Interactions between lncRNA and miRNA, as well as miRNA and mRNA, were predicted through bioinformatic assays, which were subsequently validated via RNA immunoprecipitation and dual luciferase reporter assays. Student's t-test or one-way ANOVA with post hoc Tukey HSD tests were employed for data analysis, with a p-value of less than .05 considered statistically significant. RESULTS: DLX6-AS1 was upregulated upon BMP9 overexpression in DPCs, thereby promoting odonto/osteogenic differentiation. Additionally, miR-128-3p participated in BMP9-induced odonto/osteogenic differentiation by interacting with the downstream signal MAPK14. Modifying the expression of miR-128-3p and transfecting pcMAPK14/siMAPK14 had a rescue impact on odonto/osteogenic differentiation downstream of DLX6-AS1. Lastly, miR-128-3p directly interacted with both MAPK14 and DLX6-AS1. CONCLUSIONS: DLX6-AS1 could regulate the odonto/osteogenic differentiation of DPCs under the control of BMP9 through the miR-128-3p/MAPK14 axis.
RESUMO
Pulmonary arterial hypertension (PAH) is a chronic disease characterized by a progressive increase in mean pulmonary arterial pressure. Mutations in the BMPR2 and AQP1 genes have been described in familial PAH. The bone morphogenetic proteins BMP9 and BMP10 bind with high affinity to BMPR2. Administration of BMP9 has been proposed as a potential therapeutic strategy against PAH, although recent conflicting evidence dispute the effect of such a practice. Considering the involvement of the above molecules in PAH onset, progression, and therapeutic value, we examined the effects of modulation of BMP9, BMPR2, and AQP1 on BMP9, BMP10, BMPR2, AQP1, and TGFB1 expression in human pulmonary microvascular endothelial cells in vitro. Our results demonstrated that silencing the BMPR2 gene resulted in increased expression of its two main ligands, namely BMP9 and BMP10. Exogenous administration of BMP9 caused the return of BMP10 to basal levels, while it restored the decreased AQP1 protein levels and the decreased TGFB1 mRNA and protein expression levels caused by BMPR2 silencing. Moreover, AQP1 gene silencing also resulted in increased expression of BMP9 and BMP10. Our results might possibly imply that the effect of exogenously administered BMP9 on molecules participating in the BMP signaling pathway could depend on the expression levels of BMPR2. Taken together, these results may provide insight into the highly complex interactions of the BMP signaling pathway.
Assuntos
Aquaporina 1 , Receptores de Proteínas Morfogenéticas Ósseas Tipo II , Células Endoteliais , Fator 2 de Diferenciação de Crescimento , Transdução de Sinais , Fator de Crescimento Transformador beta1 , Humanos , Aquaporina 1/metabolismo , Aquaporina 1/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Fator 2 de Diferenciação de Crescimento/genética , Células Endoteliais/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Fator de Crescimento Transformador beta1/metabolismo , Pulmão/metabolismo , Pulmão/irrigação sanguínea , Microvasos/metabolismo , Microvasos/citologia , Células Cultivadas , Inativação Gênica , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/genética , Proteínas Morfogenéticas ÓsseasRESUMO
Hepatocellular carcinoma (HCC) is a highly lethal malignant neoplasm, and the involvement of bone morphogenetic protein 9 (BMP9) has been implicated in the pathogenesis of liver diseases and HCC. Our goal was to investigate the role of BMP9 signaling in regulating N6-methyladenosine (m6A) methylation and cell cycle progression, and evaluate the therapeutic potential of BMP receptor inhibitors for HCC treatment. We observed that elevated levels of BMP9 expression in tumor tissues or serum samples from HCC patients were associated with a poorer prognosis. Through in vitro experiments utilizing the m6A dot blotting assay, we ascertained that BMP9 reduced the global RNA m6A methylation level in Huh7 and Hep3B cells, thereby facilitating their cell cycle progression. This effect was mediated by an increase in the expression of the inhibitor of DNA-binding protein 1 (ID1). Additionally, using methylated RNA immunoprecipitation qPCR(MeRIP-qPCR), we showed that the BMP9-ID1 pathway promoted CyclinD1 expression by decreasing the m6A methylation level in the 5' UTR of mRNA. This occurred through the upregulation of the fat mass and obesity-associated protein (FTO) in Huh7 and Hep3B cells. In our in vivo mouse xenograft models, we demonstrated that blocking the BMP receptor with LDN-212854 effectively suppressed HCC growth and induced global RNA m6A methylation. Overall, our findings indicate that the BMP9-ID1 pathway promotes HCC cell proliferation by down-regulating the m6A methylation level in the 5' UTR of CyclinD1 mRNA. Targeting the BMP9-ID1 pathway holds promise as a potential therapeutic strategy for treating HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Camundongos , Regiões 5' não Traduzidas , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Receptores de Proteínas Morfogenéticas Ósseas , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Fator 2 de Diferenciação de Crescimento/genética , Proteína 1 Inibidora de Diferenciação , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismoRESUMO
The secretome present in the conditioned medium (CM) of mesenchymal stem cells (MSCs) is a promising tool to be used in therapies to promote bone regeneration. Considering the high osteogenic potential of the bone morphogenetic protein 9 (BMP-9), we hypothesized that the secretome of MSCs overexpressing BMP-9 (MSCsBMP-9 ) enhances the osteoblast differentiation of MSCs and the bone formation in calvarial defects. CM of either MSCsBMP-9 (CM-MSCsBMP-9 ) or MSCs without BMP-9 overexpression (CM-MSCsVPR ) were obtained at different periods. As the CM-MSCsBMP-9 generated after 1 h presented the highest BMP-9 concentration, CM-MSCsBMP-9 and CM-MSCsVPR were collected at this time point and used to culture MSCs and to be injected into mouse calvarial defects. The CM-MSCsBMP-9 enhanced the osteoblast differentiation of MSC by upregulating RUNX2, alkaline phosphatase (ALP) and osteopontin protein expression, and ALP activity, compared with CM-MSCsVPR . The CM-MSCsBMP-9 also enhanced the bone repair of mouse calvarial defects, increasing bone volume, bone volume/total volume, bone surface, and trabecular number compared with untreated defects and defects treated with CM-MSCsVPR or even with MSCsBMP-9 themselves. In conclusion, the potential of the MSCBMP-9 -secretome to induce osteoblast differentiation and bone formation shed lights on novel cell-free-based therapies to promote bone regeneration of challenging defects.
Assuntos
Células-Tronco Mesenquimais , Osteogênese , Animais , Camundongos , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Células Cultivadas , Fator 2 de Diferenciação de Crescimento/genética , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , SecretomaRESUMO
Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9.
Assuntos
Fator 2 de Diferenciação de Crescimento , Células-Tronco Mesenquimais , Animais , Camundongos , Diferenciação Celular/genética , Fator 2 de Diferenciação de Crescimento/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismoRESUMO
Pulmonary arterial hypertension (PAH) is a rare and severe disorder characterized by progressive pulmonary vasculopathy. Growth differentiation factor (GDF)2 encodes the pro-protein bone morphogenetic protein (BMP) 9, activated after cleavage by endoproteases into an active mature form. BMP9, together with BMP10, are high-affinity ligands of activin receptor-like kinase 1 (ALK1) and BMP receptor type II (BMPR2). GDF2 mutations have been reported in idiopathic PAH with most patients being heterozygous carriers although rare homozygous cases have been described. The link between PAH occurrence and BMP9 or 10 expression level is still unclear. In this study, we describe a pediatric case of PAH also presenting with telangiectasias and epistaxis. The patient carries the novel homozygous GDF2 c.946A > G mutation, replacing the first arginine of BMP9's cleavage site (R316) by a glycine. We show that this mutation leads to an absence of circulating mature BMP9 and mature BMP9-10 heterodimers in the patient's plasma although pro-BMP9 is still detected at a similar level as controls. In vitro functional studies further demonstrated that the mutation R316G hampers the correct processing of BMP9, leading to the secretion of inactive pro-BMP9. The heterozygous carriers of the variant were asymptomatic, similarly to previous reports, reinforcing the hypothesis of modifiers preventing/driving PAH development in heterozygous carriers.
Assuntos
Hipertensão Arterial Pulmonar , Criança , Humanos , Proteínas Morfogenéticas Ósseas/genética , Fator 2 de Diferenciação de Crescimento/genética , Mutação , Mutação de Sentido Incorreto/genética , Hipertensão Arterial Pulmonar/genéticaRESUMO
BMP9, a member of the TGF-ß superfamily, reveals the great translational promise for it has been shown to have the strong effect of osteogenic activity in vitro and in vivo. However, the implantation of certain BMPs (bone morphogenetic proteins) into muscular tissues induces ectopic bone formation. BMPs induce osteoblastic differentiation in skeletal muscle, suggesting that myogenic stem cells, such as myoblasts, are the potential progenitors of osteoblasts during heterotopic bone differentiation. Here, we investigate the role of BMP9 during primary mouse myoblasts differentiation. We found BMP9 enhanced cell proliferation and reduced myogenic differentiation of primary mouse myoblasts. In addition, adenovirus-mediated overexpression of BMP9 delayed muscle regeneration after BaCl2-induced injury. ALK1 knockdown reversed the inhibition of myoblast differentiation induced by BMP9. Our data indicate that BMP9 inhibits myogenic differentiation in primary mouse myoblasts and delays skeletal muscle regeneration after injury.
Assuntos
Osso e Ossos , Fator 2 de Diferenciação de Crescimento , Animais , Camundongos , Diferenciação Celular , Fator 2 de Diferenciação de Crescimento/metabolismo , Fator 2 de Diferenciação de Crescimento/farmacologia , Mioblastos/metabolismo , Osteoblastos/metabolismo , OsteogêneseRESUMO
Bone morphogenetic proteins (BMPs) have tremendous therapeutic potential regarding the treatment of bone and musculoskeletal disorders due to their osteo-inductive ability. More than twenty BMPs have been identified in the human body with various functions, such as embryonic development, skeleton genesis, hematopoiesis, and neurogenesis. BMPs can induce the differentiation of MSCs into the osteoblast lineage and promote the proliferation of osteoblasts and chondrocytes. BMP signaling is also involved in tissue remodeling and regeneration processes to maintain homeostasis in adults. In particular, growth factors, such as BMP-2 and BMP-7, have already been approved and are being used as treatments, but it is unclear as to whether they are the most potent BMPs that induce bone formation. According to recent studies, BMP-9 is known to be the most potent inducer of the osteogenic differentiation of mesenchymal stem cells, both in vitro and in vivo. However, its exact role in the skeletal system is still unclear. In addition, research results suggest that the molecular mechanism of BMP-9-mediated bone formation is also different from the previously known BMP family, suggesting that research on signaling pathways related to BMP-9-mediated bone formation is actively being conducted. In this study, we performed a phosphorylation array to investigate the signaling mechanism of BMP-9 compared with BMP-2, another influential bone-forming growth factor, and we compared the downstream signaling system. We present a mechanism for the signal transduction of BMP-9, focusing on the previously known pathway and the p53 factor, which is relatively upregulated compared with BMP-2.
Assuntos
Fator 2 de Diferenciação de Crescimento , Osteogênese , Humanos , Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 2/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Fator 2 de Diferenciação de Crescimento/metabolismo , Osteoblastos/metabolismo , Periósteo/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
Bone morphogenetic proteins (BMPs) are dimeric transforming growth factor ß (TGFß) family cytokines that were first described in bone and cartilage formation but have since been shown to be involved in many pleiotropic functions. In human, there are 15 BMP ligands, which initiate their cellular signaling by forming a complex with two copies of type I receptors and two copies of type II receptors, both of which are transmembrane receptors with an intracellular serine/threonine kinase domain. Within this receptor family, ALK1 (activin receptor-like kinase 1), which is a type I receptor mainly expressed on endothelial cells, and BMPRII (BMP Receptor type II), a type II receptor also highly expressed on endothelial cells, have been directly linked to two rare vascular diseases: hereditary hemorrhagic telangiectasia (HHT), and pulmonary arterial hypertension (PAH), respectively. BMP9 (gene name GDF2) and BMP10, two close members of the BMP family, are the only known ligands for the ALK1 receptor. This specificity gives them a unique role in physiological and pathological angiogenesis and tissue homeostasis. The aim of this current review is to present an overview of what is known about BMP9 and BMP10 on vascular regulation with a particular emphasis on recent results and the many questions that remain unanswered regarding the roles and specificities between BMP9 and BMP10.
Assuntos
Células Endoteliais , Fator 2 de Diferenciação de Crescimento , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Células Endoteliais/metabolismo , Fator 2 de Diferenciação de Crescimento/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Humanos , Transdução de Sinais/fisiologiaRESUMO
Bone homeostasis is regulated by bone morphogenic proteins (BMPs), among which BMP9 is one of the most osteogenic. Here, we have found that BMP9 rapidly increases the protein expression of hypoxia-inducible factor-1α (HIF-1α) in osteoblasts under normoxic conditions more efficiently than BMP2 or BMP4. A combination of BMP9 and hypoxia further increased HIF-1α protein expression. HIF-1α protein induction by BMP9 is not accompanied by messenger RNA (mRNA) increase and is inhibited by the activation of prolyl hydroxylase domain (PHD)-containing protein, indicating that BMP9 induces HIF-1α protein expression by inhibiting PHD-mediated protein degradation. BMP9-induced HIF-1α protein increase was abrogated by inhibitors of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) kinase, indicating that it is mediated by PI3K-AKT signaling pathway. BMP9 increased mRNA expression of pyruvate dehydrogenase kinase 1 (PDK1), a glycolytic enzyme, and vascular endothelial growth factor-A (VEGF-A), an angiogenic factor, in osteoblasts. Notably, BMP9-induced mRNA expression of PDK1, but not that of VEGF-A, was significantly inhibited by small interference RNA-mediated knockdown of Hif-1α. BMP9-induced matrix mineralization and osteogenic marker gene expressions were significantly inhibited by chemical inhibition and gene knockdown of either Hif-1α or Pdk-1, respectively. Since increased glycolysis is an essential feature of differentiated osteoblasts, our findings indicate that HIF-1α expression is important in BMP9-mediated osteoblast differentiation through the induction of PDK1.
Assuntos
Proteínas Proto-Oncogênicas c-akt , Fator A de Crescimento do Endotélio Vascular , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Osteoblastos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Bone morphogenetic protein 9 (BMP9) and BMP10 are circulating ligands that mediate endothelial cell (EC) protection via complexes of the type I receptor ALK1 and the type II receptors activin type-IIA receptor (ACTR-IIA) and bone morphogenetic type II receptor (BMPR-II). We previously demonstrated that BMP9 induces the expression of interleukin-6, interleukin-8 and E-selectin in ECs and might influence their interactions with monocytes and neutrophils. We asked whether BMP9 and BMP10 regulate the expression of chemokine (C-C motif) ligand 2 (CCL2), a key chemokine involved in monocyte-macrophage chemoattraction. Here, we show that BMP9 and BMP10 repress basal CCL2 expression and release from human pulmonary artery ECs and aortic ECs. The repression was dependent on ALK1 and co-dependent on ACTR-IIA and BMPR-II. Assessment of canonical Smad signalling indicated a reliance of this response on Smad4. Of note, Smad1/5 signalling contributed only at BMP9 concentrations similar to those in the circulation. In the context of inflammation, BMP9 did not alter the induction of CCL2 by TNF-α. As CCL2 promotes monocyte/macrophage chemotaxis and endothelial permeability, these data support the concept that BMP9 preserves basal endothelial integrity.
Assuntos
Células Endoteliais , Fator 2 de Diferenciação de Crescimento , Receptores de Activinas Tipo II , Proteínas Morfogenéticas Ósseas , Quimiocina CCL2/genética , Fator 2 de Diferenciação de Crescimento/genética , Humanos , Fatores de ProteçãoRESUMO
Bone morphogenetic protein-9 (BMP9), a member of the transforming growth factor ß (TGFß) superfamily, plays important roles in the development and maintenance of various cell lineages via complexes of type I and type II TGFß receptors. Endoglin is a coreceptor for several TGFß family members, including BMP9, which is highly expressed in a particular stage of differentiation in erythroid cells as well as in endothelial cells. Although the importance of the interaction between BMP9 and endoglin for endothelial development has been reported, the contribution of BMP9 to endoglin-expressing erythroid cells remains to be clarified. To address this point, we prepared an anti-BMP9 antibody that blocks the BMP9-endoglin interaction. Of note, challenge with the antibody promotes erythropoiesis in wild-type mice but not in a mouse model of renal anemia in which erythropoietin (EPO) production in the kidneys is genetically ablated. While endoglin-positive erythroid progenitors are mainly maintained as progenitors when bone marrow-derived lineage-negative and cKit-positive cells are cultured in the presence of EPO and stem cell factor, the erythroid-biased accumulation of progenitors is impeded by the presence of BMP9. Our findings uncover an unrecognized role for BMP9 in attenuating erythroid differentiation via its interaction with endoglin on erythroid progenitors.
Assuntos
Eritropoese , Fator 2 de Diferenciação de Crescimento , Animais , Endoglina/genética , Células Endoteliais , Células Precursoras Eritroides , Fator 2 de Diferenciação de Crescimento/genética , Camundongos , Transdução de SinaisRESUMO
The submandibular gland (SMG) and the sublingual gland (SLG) are two of the three major salivary glands in mammals. In mice, they are adjacent to each other and open into the oral cavity, producing saliva to lubricate the mouth and aid in food digestion. Though salivary gland dysfunction accompanied with fibrosis and metabolic disturbance is common in clinic, in-depth mechanistic research is lacking. Currently, research on how to rescue salivary function is challenging, as it must resort to using terminally differentiated acinar cells or precursor acinar cells with unknown differentiation. In this study, we established reversely immortalized mouse primary SMG cells (iSMGCs) and SLG cells (iSLGCs) on the first postnatal day (P0). The iSMGCs and iSLGCs grew well, exhibited many salivary gland characteristics, and retained the metabolism-related genes derived from the original tissue as demonstrated using transcriptome sequencing (RNA-seq) analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of these two cell lines, which overlapped with those of the SMG and SLG, were enriched in cysteine and methionine metabolism. Furthermore, we investigated the role of bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2(Gdf2), on metabolic and fibrotic functions in the SMG and SLG. We demonstrated that iSMGCs and iSLGCs presented promising adipogenic and fibrotic responses upon BMP9/Gdf2 stimulation. Thus, our findings indicate that iSMGCs and iSLGCs faithfully reproduce characteristics of SMG and SLG cells and present a promising prospect for use in future study of salivary gland metabolism and fibrosis upon BMP9/Gdf2 stimulation.
Assuntos
Fator 2 de Diferenciação de Crescimento , Glândula Sublingual , Animais , Linhagem Celular , Fibrose , Fator 2 de Diferenciação de Crescimento/metabolismo , Mamíferos , Camundongos , Glândulas Salivares/metabolismo , Glândula Sublingual/metabolismoRESUMO
Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge. Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice. Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS-induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.
Assuntos
Lesão Pulmonar Aguda/sangue , Lesão Pulmonar Aguda/patologia , Endotélio/patologia , Endotoxemia/sangue , Fator 2 de Diferenciação de Crescimento/sangue , Sepse/sangue , Lesão Pulmonar Aguda/etiologia , Animais , Estudos de Casos e Controles , Células Endoteliais/metabolismo , Endotoxemia/etiologia , Endotoxemia/patologia , Feminino , Humanos , Masculino , Camundongos , Edema Pulmonar/sangue , Edema Pulmonar/etiologia , Edema Pulmonar/patologia , Sepse/etiologia , Sepse/patologiaRESUMO
INTRODUCTION: Chondroprogenitors (CPCs) have emerged as a promising cellular therapy for cartilage-related pathologies due to their inherent primed chondrogenic potential. Studies report that the addition of growth factors such as parathyroid hormone (PTH) and Bone Morphogenic Protein (BMP) enhance the chondroinducive potential in chondrocytes and mesenchymal stem cells. This study evaluated if supplementation of the standard culture medium for cell expansion with 1-34 PTH and BMP-9 would enhance the chondrogenic potential of CPCs and reduce their hypertrophic tendency. METHODS: Human chondrocytes were isolated from patients undergoing total knee replacement for osteoarthritis (n = 3). Following fibronectin adhesion assay, passage 1 CPCs were divided and further expanded under three culture conditions (a) control, i.e., cells continued under standard culture conditions, (b) 1-34 PTH group, additional intermittent 6 h exposure with 1-34 PTH and (c) BMP-9 group, additional BMP-9 during culture expansion. All the groups were evaluated for population-doubling, cell cycle analysis, surface marker and gene expression for chondrogenesis, hypertrophy, multilineage differentiation and GAG (glycosaminoglycan)/DNA following chondrogenic differentiation. RESULTS: Concerning growth kinetics, the BMP-9 group exhibited a significantly lower S-phase and population-doubling when compared to the other two groups. Qualitative analysis for chondrogenic potential (Alcian blue, Safranin O staining and Toluidine blue for GAG) revealed that the BMP-9 group exhibited the highest uptake. The BMP-9 group also showed significantly higher COL2A1 expression than the control group, with no change in the hypertrophy marker expression. CONCLUSION: BMP-9 can potentially be used as an additive for CPCs expansion, to enhance their chondrogenic potential without affecting their low hypertrophic tendency. The mitigating effects of 1-34PTH on hypertrophy would benefit further investigation when used in combination with BMP-9 to enhance chondrogenesis whilst reducing hypertrophy.