RESUMEN
BACKGROUND: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the BMPR2 (bone morphogenetic protein receptor type 2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of SIN3a (switch-independent 3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. METHODS: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells. Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific adeno-associated virus serotype 1 or a lentivirus encoding for human SIN3a in vivo. RESULTS: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. It is interesting that we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. It is interesting that SIN3a overexpression inhibited human pulmonary arterial smooth muscle cells proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA-sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 (DNA methyltransferase 1) and EZH2 (enhancer of zeste 2 polycomb repressive complex 2) while promoting the expression of the DNA demethylase TET1 (ten-eleven translocation methylcytosine dioxygenase 1). Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF (CCCTC-binding factor) binding to the BMPR2 promoter. Last, we identified intratracheal delivery of adeno-associated virus serotype human SIN3a to be a beneficial therapeutic approach in PAH by attenuating pulmonary vascular and right ventricle remodeling, decreasing right ventricle systolic pressure and mean pulmonary arterial pressure, and restoring BMPR2 expression in rodent models of PAH. CONCLUSIONS: All together, our study unveiled the protective and beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in human pulmonary arterial smooth muscle cells. Our study also identified lung-targeted SIN3a gene therapy using adeno-associated virus serotype 1 as a new promising therapeutic strategy for treating patients with PAH.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Terapia Genética/métodos , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/terapia , Complejo Correpresor Histona Desacetilasa y Sin3/biosíntesis , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Metilación , Ratones , Hipertensión Arterial Pulmonar/genética , Ratas , Ratas Sprague-Dawley , Complejo Correpresor Histona Desacetilasa y Sin3/metabolismoRESUMEN
Sensory neurons in trigeminal ganglia (TG) of calves latently infected with bovine herpesvirus 1 (BoHV-1) abundantly express latency-related (LR) gene products, including a protein (ORF2) and two micro-RNAs. Recent studies in mouse neuroblastoma cells (Neuro-2A) demonstrated ORF2 interacts with ß-catenin and a ß-catenin coactivator, high-mobility group AT-hook 1 (HMGA1) protein, which correlates with increased ß-catenin-dependent transcription and cell survival. ß-Catenin and HMGA1 are readily detected in a subset of latently infected TG neurons but not TG neurons from uninfected calves or reactivation from latency. Consequently, we hypothesized that the Wnt/ß-catenin signaling pathway is differentially expressed during the latency and reactivation cycle and an active Wnt pathway promotes latency. RNA-sequencing studies revealed that 102 genes associated with the Wnt/ß-catenin signaling pathway were differentially expressed in TG during the latency-reactivation cycle in calves. Wnt agonists were generally expressed at higher levels during latency, but these levels decreased during dexamethasone-induced reactivation. The Wnt agonist bone morphogenetic protein receptor 2 (BMPR2) was intriguing because it encodes a serine/threonine receptor kinase that promotes neuronal differentiation and inhibits cell death. Another differentially expressed gene encodes a protein kinase (Akt3), which is significant because Akt activity enhances cell survival and is linked to herpes simplex virus 1 latency and neuronal survival. Additional studies demonstrated ORF2 increased Akt3 steady-state protein levels and interacted with Akt3 in transfected Neuro-2A cells, which correlated with Akt3 activation. Conversely, expression of Wnt antagonists increased during reactivation from latency. Collectively, these studies suggest Wnt signaling cooperates with LR gene products, in particular ORF2, to promote latency.IMPORTANCE Lifelong BoHV-1 latency primarily occurs in sensory neurons. The synthetic corticosteroid dexamethasone consistently induces reactivation from latency in calves. RNA sequencing studies revealed 102 genes associated with the Wnt/ß-catenin signaling pathway are differentially regulated during the latency-reactivation cycle. Two protein kinases associated with the Wnt pathway, Akt3 and BMPR2, were expressed at higher levels during latency but were repressed during reactivation. Furthermore, five genes encoding soluble Wnt antagonists and ß-catenin-dependent transcription inhibitors were induced during reactivation from latency. These findings are important because Wnt, BMPR2, and Akt3 promote neurogenesis and cell survival, processes crucial for lifelong viral latency. In transfected neuroblastoma cells, a viral protein expressed during latency (ORF2) interacts with and enhances Akt3 protein kinase activity. These findings provide insight into how cellular factors associated with the Wnt signaling pathway cooperate with LR gene products to regulate the BoHV-1 latency-reactivation cycle.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Regulación Enzimológica de la Expresión Génica , Herpesvirus Bovino 1/fisiología , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Células Receptoras Sensoriales/inmunología , Ganglio del Trigémino/enzimología , Activación Viral/fisiología , Latencia del Virus/fisiología , Vía de Señalización Wnt , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Bovinos , Supervivencia Celular , Proteínas Proto-Oncogénicas c-akt/genética , Células Receptoras Sensoriales/patología , Células Receptoras Sensoriales/virología , Ganglio del Trigémino/patología , Ganglio del Trigémino/virologíaRESUMEN
BACKGROUND/AIMS: Endogenous parathyroid hormone (PTH) plays an important role in fracture healing. This study investigated whether endogenous PTH regulates fracture healing by bone morphogenetic protein (BMP) and/or the transforming growth factor-ß (TGF-ß) signaling pathway. METHODS: Eight-week-old wild-type (WT) and PTH-knockout (PTH KO) male mice were selected, and models of open right-femoral fracture were constructed. Fracture healing and callus characteristics of mice in the two groups were compared by X-ray, micro-computed tomography, histological, and immunohistochemical examinations. Bone marrow mesenchymal stem cells (BMMSCs) of 8-week-old WT and PTHKO male mice were obtained and induced into osteoblasts and chondrocytes. RESULTS: We found that expression levels of Runt-related transcription factor (RUNX2), bone morphogenetic protein-receptor-type â ¡ (BMPR2), phosphorylated Smad 1/5/8, and phosphorylated cyclic adenosine monophosphate-responsive element binding protein (CREB) in the callus of PTHKO mice were significantly decreased, whereas no significant difference in expression of SOX9, TGF-ßR2,or pSMAD2/3 was observed between PTHKO and WT mice. Additionally, the activity of osteoblast alkaline phosphatase was low at 7 days post-induction, and was upregulated by addition of PTH or dibutyryl cyclic adenosine monophosphate (dbcAMP) to the cell culture. Furthermore, H89 (protein kinase A inhibitor)eliminated the simulating effects of PTH and dbcAMP, and a low concentration of cyclic adenosine monophosphate (cAMP) was observed in PTHKO mouse BMMSCs. CONCLUSION: These results suggested that endogenous PTH enhanced BMPR2 expression by a cAMP/PKA/CREB pathway in osteoblasts, and increased RUNX2 expression through transduction of the BMP/pSMAD1/5/8 signaling pathway.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Curación de Fractura/genética , Fracturas Abiertas/genética , Hormona Paratiroidea/genética , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/genética , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Fracturas Abiertas/patología , Fracturas Abiertas/terapia , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Isoquinolinas/administración & dosificación , Ratones , Ratones Noqueados , Osteoblastos , Hormona Paratiroidea/biosíntesis , Transducción de Señal/genética , Proteínas Smad/genética , Sulfonamidas/administración & dosificaciónRESUMEN
BACKGROUND: Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. METHODS: We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH. RESULTS: Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses. CONCLUSIONS: Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Endotelio Vascular/metabolismo , Hipertensión Pulmonar/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Células Cultivadas , Endotelio Vascular/patología , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Pez CebraRESUMEN
OBJECTIVES: The present study evaluated the immunohistochemical expression of BMP-2 and BMP-4 and of their receptors (BMPR-IA and BMPR-II) in solid ameloblastoma (SA), unicystic ameloblastoma (UA) and adenomatoid odontogenic tumor (AOT) in order to obtain a better understanding of their role in the development and biological behavior of these tumors. DESIGN: This study analyzed these proteins in 30 cases of SA, 10 cases of UA, and 30 cases of AOT. Immunoexpression was evaluated in the parenchyma and stroma by attributing the following scores: 0, no stained cells; 1, ≤10%; 2, >10% and ≤25%; 3, >25% and ≤50%; 4, >50% and ≤75%.; 5, >75% stained cells. RESULTS: In SAs, positive correlations were observed between the stromal and parenchymal expression of BMP-2 (p<0.001) and between the stromal expression of BMP-2 and BMP-4 (p=0.020), as well as between the stromal expression of BMPR-II and BMP-4 (p=0.001) and the stromal and parenchymal expression of BMPR-II (p<0.001). In UAs, correlations were detected between the stromal and parenchymal expression of BMP-4 (p=0.035) and between the stromal expression of BMP-4 and BMPR-IA (p=0.022). In AOTs, analysis of immunoexpression in the parenchyma revealed positive correlations between all proteins. CONCLUSION: BMPs and their receptors play an important role in the differentiation and development of ameloblastomas and AOTs, but may not explain the different biological behaviors of these lesions. The positive correlation observed in AOTs might be related to the formation of mineralized material in this tumor.
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Ameloblastoma/metabolismo , Proteína Morfogenética Ósea 2/biosíntesis , Proteína Morfogenética Ósea 4/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/biosíntesis , Neoplasias Maxilomandibulares/metabolismo , Ameloblastoma/inmunología , Ameloblastoma/patología , Biomarcadores de Tumor/biosíntesis , Proteína Morfogenética Ósea 2/inmunología , Proteína Morfogenética Ósea 4/inmunología , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/inmunología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/inmunología , Diferenciación Celular/fisiología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inmunohistoquímica , Neoplasias Maxilomandibulares/inmunología , Neoplasias Maxilomandibulares/patología , Tejido Parenquimatoso/metabolismo , Tejido Parenquimatoso/patología , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
Bone morphogenetic proteins (BMPs) are broadly involved in normal embryo development and abnormal pathological process such as cancer. The complexity and diversity of BMPs and their signaling pathways impose quite different or even conflicting effects on clinical traits of tumors. The aim of the present study was to investigate whether different BMPs, including BMP2, BMP4, BMP6 and BMP7, influence esophageal squamous cancer cell (ESCC) growth, invasion and metastasis. BMP6 and type I receptor ALK2 and type II receptor BMPRII, ActRIIA and ActRIIB were expressed in all ESCC cell lines. In addition, adenovirus-mediated BMP overexpression did not affect ECA-109 cell growth. BMP6/7 overexpression increased ECA-109 cell invasion and metastasis, activated SMAD1/5/8 signal pathway and induced downstream gene ID1 expression. While BMP2/4 overexpression reduced ECA-109 cell invasion and metastasis and obviously promoted ERK1/2, P-38 and JNK activation with weak SMAD1/5/8 phosphorylation. When BMP6/7 favorite type I receptor ALK2 or type II receptor BMPRII was interfered with by dominant-negative mutation, BMP6/7-induced invasion and metastasis augmentation disappeared. Further investigation on clinical ESCC samples and non-tumorous adjacent tissue found that tumors with triple-positive BMP6, ALK2 and BMPRII had deeper growth than tumors with only BMP6 expression. These results suggested that different BMPs distinctly affected esophageal squamous cancer cell invasion and metastasis by employing different signal pathways.
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Receptores de Activinas Tipo I/biosíntesis , Proteína Morfogenética Ósea 6/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Receptores de Activinas Tipo I/genética , Adenoviridae/genética , Proteína Morfogenética Ósea 2/biosíntesis , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 4/biosíntesis , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 6/biosíntesis , Proteína Morfogenética Ósea 7/biosíntesis , Proteína Morfogenética Ósea 7/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Metástasis de la Neoplasia , Transducción de Señal/genética , Proteína Smad1/biosíntesis , Proteína Smad1/genéticaRESUMEN
BACKGROUNDS: Bone morphogenetic protein receptor type 2 (BMPR2) signaling is anti-inflammatory. Decreased BMPR2 expression was seen in lung tissue from chronic obstructive pulmonary disease (COPD) patients. METHODS: The selected single nucleotide polymorphisms (SNPs) in BMPR2 were genotyped with polymerase chain reaction (PCR) ligase detection reaction. The effects of SNPs on gene expression were analyzed with luciferase assays. The mRNA and protein expression levels of BMPR2 in peripheral blood mononuclear cells (PBMCs) from COPD patients were determined by quantitative PCR and western blotting, respectively. FINDINGS: Two SNPs, rs6435156C > T and rs1048829G > T in the 3'-untranslated region (3'UTR) of BMPR2 were selected and genotyped in COPD case and healthy control subjects from southern Chinese population. Both of them were found associated with significantly increased COPD risk (adjusted odds ratio [OR] = 1.58 with 95% confidence interval [CI] = 1.14-2.15, P = 0.0056 for rs6435156C > T; adjusted OR = 1.47 and 95% CI = 1.10-1.97, P = 0.0092 for rs1048829G > T). Older age, cigarette smoking, family history of cancer and COPD were all factors that interacted with rs6435156C > T and rs1048829G > T causing increased COPD risk. Cigarette smokers with rs6435156 (CT + TT) or rs1048829 (GT + TT) were more susceptible to COPD than that with the rs6435156CC or rs1048829GG genotypes. In A549 human alveolar epithelial cells, luciferase reporter assays revealed that introduction of 3'UTR of BMPR2 plasmids carrying rs6435156T allele but not rs1048829T led to lower luciferase activity than the wild-type C or G alleles. Comparing to rs6435156CC, treatment with hsa-miR-20a mimics deceased whereas hsa-miR-20a inhibitor restored the luciferase reporter activity in cells transfected with constructs carrying rs6435156TT. BMPR2 mRNA and protein expressions were significantly lower in PBMCs from COPD smokers than that in non-smokers. COPD patients carrying rs6435156T allele had less BMPR2 expression in PBMCs. INTERPRETATION: This study demonstrated that both rs6435156C > T and rs1048829G > T variants in BMPR2 contributed to increased susceptibility to COPD. The T variants of rs6435156 increased COPD risk likely by binding with hsa-miR-20a, thus leading to downregulated BMPR2 expression in lung epithelial and immune cells.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Enfermedad Pulmonar Obstructiva Crónica/genética , Anciano , Alelos , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , China , Femenino , Genotipo , Haplotipos , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Enfermedad Pulmonar Obstructiva Crónica/patología , Factores de RiesgoRESUMEN
BACKGROUND: Pulmonary arterial hypertension (PAH) is a proliferative disease of the pulmonary vasculature that preferentially affects women. Estrogens such as the metabolite 16α-hydroxyestrone (16αOHE) may contribute to PAH pathogenesis, and alterations in cellular energy metabolism associate with PAH. We hypothesized that 16αOHE promotes heritable PAH (HPAH) via microRNA-29 (miR-29) family upregulation and that antagonism of miR-29 would attenuate pulmonary hypertension in transgenic mouse models of Bmpr2 mutation. METHODS AND RESULTS: MicroRNA array profiling of human lung tissue found elevation of microRNAs associated with energy metabolism, including the miR-29 family, among HPAH patients. miR-29 expression was 2-fold higher in Bmpr2 mutant mice lungs at baseline compared with controls and 4 to 8-fold higher in Bmpr2 mice exposed to 16αOHE 1.25 µg/h for 4 weeks. Blot analyses of Bmpr2 mouse lung protein showed significant reductions in peroxisome proliferator-activated receptor-γ and CD36 in those mice exposed to 16αOHE and protein derived from HPAH lungs compared with controls. Bmpr2 mice treated with anti-miR-29 (20-mg/kg injections for 6 weeks) had improvements in hemodynamic profile, histology, and markers of dysregulated energy metabolism compared with controls. Pulmonary artery smooth muscle cells derived from Bmpr2 murine lungs demonstrated mitochondrial abnormalities, which improved with anti-miR-29 transfection in vitro; endothelial-like cells derived from HPAH patient induced pluripotent stem cell lines were similar and improved with anti-miR-29 treatment. CONCLUSIONS: 16αOHE promotes the development of HPAH via upregulation of miR-29, which alters molecular and functional indexes of energy metabolism. Antagonism of miR-29 improves in vivo and in vitro features of HPAH and reveals a possible novel therapeutic target.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Microambiente Celular/fisiología , Hidroxiestronas/metabolismo , Hipertensión Pulmonar/metabolismo , MicroARNs/biosíntesis , Animales , Microambiente Celular/efectos de los fármacos , Femenino , Humanos , Hidroxiestronas/toxicidad , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/diagnóstico , Masculino , Ratones , Ratones Transgénicos , MicroARNs/antagonistas & inhibidoresRESUMEN
The present study was designed to evaluate the role of growth differentiation factor-5 (GDF-5) and bone morphogenetic protein type II receptor (BMPR-II) in the development of lumbar intervertebral disc degeneration (IDD). A total of 24 patients with lumbar IDD (experiment group) and 6 patients with lumbar vertebral fracture (control group) were enrolled in the study. Tissue samples of IVD from the experiment group and control group were obtained during lumbar fusion operation, respectively. Fixation and decalcification of IVD tissue were performed, and then HE staining was carried out to observe the morphological changes of the lumbar IVD tissues. The expression of GDF-5 and BMPRII in human lumbar IVD was detected by immunohistochemical staining. HE staining results showed that non- and minimal degeneration was found in 11 cases (score range, 0-3), moderate degeneration in 12 cases (score range, 4-8), and severe degeneration in 7 cases (score range, 9-12). According to the immunohistochemical results, the positive expression rates of GDF-5 and BMPRII in NP were higher than those in AF of the non- and minimal degeneration group, moderate degeneration group and severe degeneration group (all P < 0.05). However, no significant difference in GDF-5 or BMPRII positive expression was observed among the normal, non- and minimal, moderate and severe degeneration groups in neither NP area nor AF area (all P > 0.05). In conclusion, our results showed that GDF-5 and BMPRII expressed both in normal and degenerated IVD tissues, and GDF-5 might have an inhibition effect on degenerated lumbar IVD, suggesting that gene therapy may be a useful approach in producing physiological effects during early- and late-phase of lumbar IDD.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Factor 5 de Diferenciación de Crecimiento/biosíntesis , Degeneración del Disco Intervertebral/metabolismo , Humanos , Inmunohistoquímica , Vértebras LumbaresRESUMEN
BACKGROUND: The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells. METHODS AND RESULTS: In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2(Δ140Ex1/+) rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. CONCLUSIONS: EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.
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Transdiferenciación Celular , Células Endoteliales/patología , Hipertensión Pulmonar/patología , Mesodermo/patología , Actinas/biosíntesis , Actinas/genética , Animales , Biomarcadores , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Movimiento Celular , Células Cultivadas , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/genética , Hipoxia/complicaciones , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/patología , Monocrotalina/toxicidad , Mutación , ARN Mensajero/biosíntesis , Ratas , Sirolimus/farmacología , Remodelación Vascular , Vimentina/biosíntesis , Vimentina/genéticaRESUMEN
Body axes and germ layers evolve at gastrulation, and in mammals are driven by many genes; however, what orchestrates the genetic pathways during gastrulation remains elusive. Previously, we presented evidence that microRNA-17 (miRNA-17) family members, miR-17-5p, miR-20a, miR-93, and miR-106a were differentially expressed in mouse embryos and functioned to control differentiation of the stem cell population. Here, we identify function(s) that these miRNAs have during gastrulation. Fluorescent in situ hybridization miRNA probes reveal that these miRNAs are localized at the mid/posterior primitive streak (ps) in distinct populations of primitive ectoderm, mesendoderm, and mesoderm. Seven different miRNA prediction algorithms are identified in silico bone morphogenic protein receptor 2 (Bmpr2) as a target of these miRNAs. Bmpr2 is a member of the TGFß pathway and invokes stage-specific changes during gastrulation. Recently, Bmpr2 was shown regulating cytoskeletal dynamics, cell movement, and invasion. Our previous and current data led to a hypothesis by which members of the miR-17 family influence gastrulation by suppressing Bmpr2 expression at the primitive streak. This suppression influences fate decisions of cells by affecting genes downstream of BMPR2 as well as mesoderm invasion through regulation of actin dynamics.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Células Madre Embrionarias/citología , Gastrulación/fisiología , Regulación del Desarrollo de la Expresión Génica , MicroARNs/fisiología , Regiones no Traducidas 3' , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Linaje de la Célula/genética , Cuerpos Embrioides , Desarrollo Embrionario , Endodermo/metabolismo , Femenino , Hibridación Fluorescente in Situ , Quinasas Lim/fisiología , Masculino , Ratones , MicroARNs/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Línea Primitiva/metabolismo , Transducción de Señal , Proteínas Smad/fisiología , TransfecciónRESUMEN
Ventral midbrain (VM) dopaminergic (DA) neurons project to the dorsal striatum via the nigrostriatal pathway to regulate voluntary movements, and their loss leads to the motor dysfunction seen in Parkinson's disease (PD). Despite recent progress in the understanding of VM DA neurogenesis, the factors regulating nigrostriatal pathway development remain largely unknown. The bone morphogenetic protein (BMP) family regulates neurite growth in the developing nervous system and may contribute to nigrostriatal pathway development. Two related members of this family, BMP2 and growth differentiation factor (GDF)5, have neurotrophic effects, including promotion of neurite growth, on cultured VM DA neurons. However, the molecular mechanisms regulating their effects on DA neurons are unknown. By characterising the temporal expression profiles of endogenous BMP receptors (BMPRs) in the developing and adult rat VM and striatum, this study identified BMP2 and GDF5 as potential regulators of nigrostriatal pathway development. Furthermore, through the use of noggin, dorsomorphin and BMPR/Smad plasmids, this study demonstrated that GDF5- and BMP2-induced neurite outgrowth from cultured VM DA neurons is dependent on BMP type I receptor activation of the Smad 1/5/8 signalling pathway.
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Proteína Morfogenética Ósea 2/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/fisiología , Neuronas Dopaminérgicas/fisiología , Factor 5 de Diferenciación de Crecimiento/fisiología , Mesencéfalo/citología , Neuritas/ultraestructura , Transducción de Señal/fisiología , Proteínas Smad/fisiología , Animales , Proteína Morfogenética Ósea 2/antagonistas & inhibidores , Proteína Morfogenética Ósea 2/farmacología , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Células Cultivadas , Cuerpo Estriado/embriología , Cuerpo Estriado/crecimiento & desarrollo , Neuronas Dopaminérgicas/enzimología , Neuronas Dopaminérgicas/ultraestructura , Femenino , Regulación del Desarrollo de la Expresión Génica , Factor 5 de Diferenciación de Crecimiento/antagonistas & inhibidores , Mesencéfalo/embriología , Mesencéfalo/crecimiento & desarrollo , Neurogénesis/fisiología , Pirazoles , Pirimidinas , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Sustancia Negra/embriología , Sustancia Negra/crecimiento & desarrollo , Transfección , Tirosina 3-Monooxigenasa/biosíntesisRESUMEN
Bone morphogenetic protein (BMP) signaling is active in many tissues including the central nervous system, in which it regulates cell proliferation, differentiation and maturation. The modulation of BMP pathway is crucial since abnormality of BMP signaling may cause cellular malfunction such as apoptosis. There are evidences indicating that miR-17 family is involved in the BMP signaling. In the present study, we demonstrated that BMP2 stimulation directly increased the transcription of miR-17-92 and miR-106b-25 cluster via Smad activation, which leads to the up-regulation of mature miR-17/20a/93. In addition, we provided evidence that BMP2 activation repressed BMPRII expression through modulating miR-17 family in primary neurons. Furthermore, we proved that such negative regulation protected neurons from apoptosis induced by abnormal BMP signaling. Taken together, these results suggest a regulatory pathway of BMP-miR-17 family-BMPRII, which consist a negative feedback loop that balances BMP signaling and maintains cell homeostasis in neurons.
Asunto(s)
Proteína Morfogenética Ósea 2/biosíntesis , Regulación de la Expresión Génica/fisiología , MicroARNs/metabolismo , Familia de Multigenes/fisiología , Neuronas/metabolismo , Transducción de Señal/fisiología , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Células Cultivadas , Homeostasis/fisiología , Ratones , Neuronas/citología , Proteínas Smad/metabolismoRESUMEN
Our previous studies demonstrated that BMP-2 inhibits the tumorigenicity of cancer stem cells identified as cells with high aldehyde dehydrogenase activity (ALDH(br) cells) from the human osteosarcoma cell line OS99-1. We further investigated whether BMP-2 is capable of inducing bone formation in OS99-1 cells. Flow cytometry sorting was used to isolate tumorigenic ALDH(br) and non-tumorigenic ALDH(lo) cells. qRT-PCR was used to quantify the gene expression. A xenograft model was used to verify the bone formation in vivo. There was significantly higher mRNA expression of BMPR1B and BMPR2 in ALDH(lo) cells compared with that in ALDH(br) cells and the BMPR1B expression in ALDH(lo) cells was ~8-fold higher compared to that in ALDHbr cells. BMP-2 was also found to induce higher transcription of osteogenic markers Runx-2, Osterix (Osx), alkaline phosphatase (ALP) and collagen type I in ALDH(lo) cells compared to ALDH(br) cells, which were mediated by the canonical Smad signaling pathway. In vivo, BMP-2 was identified to induce bone formation in both ALDH(br) and ALDH(lo) cells. All animals receiving 1 x 10()4 ALDH(lo) cells treated with 30 µg of BMP-2 per animal showed bone formation within 1-2 weeks after injection in mice. Bone formation induced by BMP-2 in ALDH(lo) cells showed significantly more bone mineral content compared to that in ALDH(br) cells. BMP-2 induces bone formation in heterogeneous osteosarcoma cells and BMP-2 may have a promising therapeutic role for treating human osteosarcoma by inducing differentiation along an osteogenic pathway.
Asunto(s)
Aldehído Deshidrogenasa/genética , Proteína Morfogenética Ósea 2/genética , Osteogénesis/genética , Osteosarcoma/patología , Aldehído Deshidrogenasa/biosíntesis , Animales , Densidad Ósea , Proteína Morfogenética Ósea 2/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Línea Celular Tumoral , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Osteosarcoma/genéticaRESUMEN
Constitutively activating mutations in receptor kinases recruit downstream effector pathways independently of upstream signaling, with consequences ranging from developmental syndromes to cancer. Classic fibrodysplasia ossificans progressiva (FOP) is a congenital syndrome resulting from highly conserved activating mutations of the glycine-serine-rich (GS) regulatory domain of ACVR1, encoding bone morphogenetic protein (BMP) type I receptor ALK2, which lead to inappropriate signaling and heterotopic ossification of soft tissues. It is unclear if constitutively active mutant ALK2 receptors (caALK2) can function independently of signaling complexes with type II receptors and ligands. We found that ablation of BmpRII and ActRIIa abrogated BMP ligand-mediated and caALK2-mediated signaling and transcription in cells and disrupted caALK2-induced heterotopic ossification in mice. Signaling via GS domain ALK2 mutants could be restored by the expression of either BMP type II receptor. The contribution of BMP type II receptors was independent of their ligand-binding or kinase function but was dependent upon an intact cytoplasmic domain. These data demonstrate that GS domain ALK2 mutants act independently of upstream signaling but may require a nonenzymatic scaffolding function provided by type II receptors to form functional, apparently ligand-independent signaling complexes. These findings define the minimal requirements for signaling of GS domain ALK2 mutants, with implications for the therapeutic targeting of their activity in disease.
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Receptores de Activinas Tipo II/metabolismo , Receptores de Activinas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Osificación Heterotópica/metabolismo , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo II/biosíntesis , Receptores de Activinas Tipo II/genética , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Ratones , Ratones Transgénicos , Músculo Liso Vascular , Miositis Osificante , Estructura Terciaria de Proteína , Arteria Pulmonar , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Atherosclerosis is a chronic inflammatory vascular disease driven by the subendothelial accumulation of macrophages. The mechanism regulating the inflammatory response in macrophages during atherogenesis remains unclear. Because microRNAs (miRNAs) play a crucial role in cellular signaling by posttranscriptional regulation of gene expression, we studied the miRNA expression profiles during the progression of atherosclerosis. METHODS AND RESULTS: Using an miRNA real-time polymerase chain reaction array, we found that macrophage-derived miR-342-5p and miR-155 are selectively upregulated in early atherosclerotic lesions in Apoe(-/-) mice. miR-342-5p directly targets Akt1 through its 3'-untranslated region. Akt1 suppression by miR-342-5p induces proinflammatory mediators such as Nos2 and II6 in macrophages via the upregulation of miR-155. The local application of an miR-342-5p antagomir inhibits the development of atherosclerosis in partially ligated carotid arteries. In atherosclerotic lesions, the miR-342-5p antagomir upregulated Akt1 expression and suppressed the expression of miR-155 and Nos2. This reduced Nos2 expression was associated with a diminished generation of nitrotyrosine in the plaques. Furthermore, systemic treatment with an inhibitor of miR-342-5p reduced the progression of atherosclerosis in the aorta of Apoe(-/-) mice. CONCLUSIONS: Macrophage-derived miR-342-5p promotes atherosclerosis and enhances the inflammatory stimulation of macrophages by suppressing the Akt1-mediated inhibition of miR-155 expression. Therefore, targeting miR-342-5p may offer a promising strategy to treat atherosclerotic vascular disease.
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Aterosclerosis/patología , Regulación de la Expresión Génica , Activación de Macrófagos , MicroARNs/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Vasculitis/patología , Animales , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/fisiopatología , Apolipoproteínas E/deficiencia , Aterosclerosis/genética , Aterosclerosis/fisiopatología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Estenosis Carotídea/genética , Estenosis Carotídea/patología , Estenosis Carotídea/fisiopatología , Estenosis Carotídea/prevención & control , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , Progresión de la Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-6/biosíntesis , Interleucina-6/genética , Macrófagos/metabolismo , Ratones , Ratones Noqueados , MicroARNs/antagonistas & inhibidores , MicroARNs/biosíntesis , MicroARNs/genética , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Oligonucleótidos/farmacología , Oligonucleótidos/uso terapéutico , ARN sin Sentido/farmacología , ARN sin Sentido/uso terapéutico , Ribonucleasa III/deficiencia , Ribonucleasa III/genética , Transducción de Señal/fisiología , Tirosina/análogos & derivados , Tirosina/metabolismo , Regulación hacia Arriba , Vasculitis/genética , Vasculitis/fisiopatologíaRESUMEN
Like cancer, pulmonary arterial hypertension (PAH) is characterised by a pro-proliferative and anti-apoptotic phenotype. In PAH, pulmonary artery smooth muscle cell (PASMC) proliferation is enhanced and apoptosis suppressed. The sustainability of this phenotype requires the activation of pro-survival transcription factors, such as signal transducer and activator of transcription (STAT)3 and nuclear factor of activated T-cells (NFAT). There are no drugs currently available that are able to efficiently and safely inhibit this axis. We hypothesised that plumbagin (PLB), a natural organic compound known to block STAT3 in cancer cells, would reverse experimental pulmonary hypertension. Using human PAH-PASMC, we demonstrated in vitro that PLB inhibits the activation of the STAT3/NFAT axis, increasing the voltage-gated K(+) current bone morphogenetic protein receptor type II (BMPR2), and decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), rho-associated coiled-coil containing protein kinase (ROCK)1 and interleukin (IL)-6, contributing to the inhibition of PAH-PASMC proliferation and resistance to apoptosis (proliferating cell nuclear antigen (PCNA), TUNEL, Ki67 and anexine V). In vivo, PLB oral administration decreases distal pulmonary artery remodelling, mean pulmonary artery pressure and right ventricular hypertrophy without affecting systemic circulation in both monocrotaline- and suden/chronic hypoxia-induced PAH in rats. This study demonstrates that the STAT3/NFAT axis can be therapeutically targeted by PLB in human PAH-PASMC and experimental PAH rat models. Thus, PLB could be considered a specific and attractive future therapeutic strategy for PAH.
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Apoptosis/efectos de los fármacos , Cardiotónicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Hipertensión Pulmonar/tratamiento farmacológico , Naftoquinonas/uso terapéutico , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Calcio/metabolismo , Células Cultivadas , Hipertensión Pulmonar Primaria Familiar , Humanos , Etiquetado Corte-Fin in Situ , Interleucina-6/metabolismo , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Factores de Transcripción NFATC/biosíntesis , Canales de Potasio con Entrada de Voltaje/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Quinasas Asociadas a rho/metabolismoRESUMEN
Beyond stimulating bone formation, bone morphogenetic proteins (BMPs) are important in development, inflammation, and malignancy of the gut. We have previously shown that BMP7 has a regenerative, anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the BMP signaling pathway we monitored the effect of BMP7 therapy on the BMP signaling components in the rat colon during different stages of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed a significantly decreased BMP7 expression in the acute phase, followed by a significantly increased BMP2 and decreased BMP6 expression during the chronic phase of colitis. BMP7 therapy influenced the expression of several BMPs with the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the acute stage and significantly decreased upon BMP7 therapy. BMP receptor I expression was unchanged, whereas BMP receptor II was decreased at day 2 and increased at days 14 and 30 of TNBS inflammation. However, an opposite pattern of expression following BMP7 therapy has been observed. BMP7 increased the expression of BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy at later stages of the disease. We suggest that BMP signaling was altered during TNBS-induced colitis and was recovered with BMP7 administration, suggesting that IBD is a reversible process.
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Proteína Morfogenética Ósea 7/uso terapéutico , Colitis/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Animales , Proteína Morfogenética Ósea 2/biosíntesis , Proteína Morfogenética Ósea 4/biosíntesis , Proteína Morfogenética Ósea 6/biosíntesis , Proteína Morfogenética Ósea 7/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/biosíntesis , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Proteínas Portadoras/biosíntesis , Colitis/inducido químicamente , Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Sprague-Dawley , Proteínas Smad/biosíntesis , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
BACKGROUND: Bone morphogenetic proteins (BMPs) were first described for their roles in bone formation, but they now also are known to possess additional activities, including those relating to embryogenesis. The objectives of this work were to 1) determine if peri-attachment bovine conceptuses and bovine trophoblast cells (CT1) contain transcripts for BMP2 and 4, an innate inhibitor noggin (NOG), and BMP2/4 receptors (BMPRII, ACVR1, BMPR1A, BMPR1B), and 2) determine if BMP2 or 4 supplementation to CT1 cells affects cell proliferation, differentiation or trophoblast-specific gene expression. METHODS: RNA was isolated from day 17 bovine conceptuses and CT1 cells. After RT-PCR, amplified products were cloned and sequenced. In other studies CT1 cells were treated with BMP2 or 4 at various concentrations and effects on cell viability, cell differentiation and abundance of IFNT and CSH1 mRNA were evaluated. RESULTS: Transcripts for BMP2 and 4 were detected in bovine conceptuses and CT1 cells. Also, transcripts for each BMP receptor were detected in conceptuses and CT1 cells. Transcripts for NOG were detected in conceptuses but not CT1 cells. Cell proliferation was reduced by BMP4 but not BMP2 supplementation. Both factors reduced IFNT mRNA abundance but had no effect on CSH1 mRNA abundance in CT1 cells. CONCLUSIONS: The BMP2/4 ligand and receptor system presides within bovine trophectoderm prior to uterine attachment. BMP4 negatively impacts CT1 cell growth and both BMPs affect IFNT mRNA abundance.
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Proteína Morfogenética Ósea 2/biosíntesis , Proteína Morfogenética Ósea 4/biosíntesis , Trofoblastos/metabolismo , Receptores de Activinas Tipo I/biosíntesis , Animales , Proteína Morfogenética Ósea 2/fisiología , Proteína Morfogenética Ósea 4/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/biosíntesis , Proteínas Portadoras/biosíntesis , Bovinos , Diferenciación Celular , Proliferación Celular , Embrión de Mamíferos/metabolismo , Interferón Tipo I/biosíntesis , Proteínas Gestacionales/biosíntesisRESUMEN
Metastatic disease is the major cause of cancer deaths, and recurrent tumors at distant organs are a critical issue. However, how metastatic tumor cells become dormant and how and why tumors recur in target organs are not well understood. In this study, we demonstrate that BMP7 (bone morphogenetic protein 7) secreted from bone stromal cells induces senescence in prostate cancer stem-like cells (CSCs) by activating p38 mitogen-activated protein kinase and increasing expression of the cell cycle inhibitor, p21, and the metastasis suppressor gene, NDRG1 (N-myc downstream-regulated gene 1). This effect of BMP7 depended on BMPR2 (BMP receptor 2), and BMPR2 expression inversely correlated with recurrence and bone metastasis in prostate cancer patients. Importantly, this BMP7-induced senescence in CSCs was reversible upon withdrawal of BMP7. Furthermore, treatment of mice with BMP7 significantly suppressed the growth of CSCs in bone, whereas the withdrawal of BMP7 restarted growth of these cells. These results suggest that the BMP7-BMPR2-p38-NDRG1 axis plays a critical role in dormancy and recurrence of prostate CSCs in bone and suggest a potential therapeutic utility of BMP7 for recurrent metastatic disease.