RESUMO
BACKGROUND: BMPR1A-mediated signaling transduction plays an essential role in intestinal growth. Variations of BMPR1A lead to a rare autosomal dominant inherited juvenile polyposis syndrome (JPS) with high probability of developing into colorectal cancer (CRC). Nonsense and frameshift variations, generating premature termination codons (PTCs), are the most pathogenic variants in the BMPR1A gene. OBJECTIVE: This study aimed to investigate the molecular genetic etiology in a Chinese family with three generations of CRC. METHODS: Pathogenic variants of 18 known CRC susceptibility genes were examined in a Chinese CRC family through multigene panel testing using the next-generation sequencing platform. The candidate gene variant was validated in the family members by Sanger sequencing. Potential biological functions of the gene variant were further investigated in the RKO colon cancer cell line. RESULTS: A novel nonsense variant (c.1114A > T, p.Lys372*) of BMPR1A was identified in the CRC family. This variant generated a PTC at the kinase domain and caused nonsense-mediated mRNA decay. Read-through inducing reagents G418 and PTC124 partially restored BMPR1A expression and its following signaling pathway. CONCLUSION: The identification of the novel BMPR1A variant enriched the genotype-phenotype spectrum of BMPR1A. Meanwhile, our finding also provided support for future PTC-targeting therapy for BMPR1A-mediated JPS and CRC.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Códon sem Sentido , Linhagem , Humanos , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Predisposição Genética para Doença , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Povo Asiático/genética , Degradação do RNAm Mediada por Códon sem Sentido , Linhagem Celular Tumoral , População do Leste AsiáticoRESUMO
BACKGROUND: Juvenile Polyposis Syndrome (JPS) is an autosomal dominant condition with hamartomatous polyps in the gastrointestinal tract, associated with an increased risk of gastrointestinal malignancy. Disease causing variants (DCVs) in BMPR1a or SMAD4 account for 45-60% of JPS cases, with BMPR1a DCVs accounting for 17-38% of JPS cases. Within those with either a BMPR1a or SMAD4 DCV, there is phenotypic variability in location of polyps, risk of malignancy and extra-intestinal manifestations with limited published reports of gene-phenotype association or genotype-phenotype correlation. We aimed to identify any gene-phenotype association or genotype-phenotype correlation in BMPR1a to inform surveillance recommendations, and gene-specific modification to the ACMG classification of pathogenicity of DCVs. METHODS: A literature search was performed through EMBASE, MEDLINE and PubMed. Studies that were included explored BMPR1a DCV-related JPS or contiguous deletion of PTEN and BMPR1a. Data was also drawn from the BMPR1a specific databases on LOVD and ClinVar. RESULTS: There were 211 DCVs in BMPR1a identified, 82 from patients with JPS in the literature, and 17 from LOVD and 112 from ClinVar classified as pathogenic or likely pathogenic. These included missense, nonsense and frameshift variants and large deletions, occurring across all functional domains of the gene. Unlike in SMAD4 carriers, gastric polyposis and malignancy were not identified in our review in BMPR1a carriers, but colonic polyposis and malignancy occurred in carriers of either BMPR1a or SMAD4 DCVs. Those with contiguous deletion of PTEN and BMPR1a can present with JPS of infancy, with a severe phenotype of GI bleeding, diarrhoea, exudative enteropathy and rectal prolapse. No specific BMPR1a genotype-phenotype correlation could be ascertained including by variant type or functional domain. CONCLUSION: Phenotypic characteristics cannot be used to inform variant location in BMPR1a. However, the phenotypic characteristics of BMPR1a DCV carriers, being almost exclusively related to the colon and rectum, can assist in pathogenicity assessment of BMPR1a variants. Given these findings, we propose that carriers of BMPR1a DCVs should only require surveillance for colorectal polyps and malignancy, and that surveillance for gastric polyps and malignancy may be unnecessary. However variant location within BMPR1a does not support differential surveillance recommendations.
RESUMO
BACKGROUND: Juvenile polyposis syndrome (JPS) is one of the hereditary polyposis syndromes caused by abnormal regulation of transforming growth factor ß signaling because of mutations in BMPR1A and SMAD4. Juvenile polyposis syndrome patients with SMAD4 mutations develop cardiovascular events, whereas those with BMPR1A usually do not. Analysis of genetic mutations in JPS patients can be helpful in devising suitable strategies for medical management. In this study, we demonstrate the pathogenicity of a novel intronic mutation in BMPR1A using mRNA extracted from colonic mucosa of a boy with JPS. METHODS: Genomic DNA extracted from peripheral blood and total RNA isolated from the colonic mucosa were used for DNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analyses, respectively. RESULTS: A 13-year-old boy, with no previous medical history, presented to the hospital complaining of bloody stools. Colonoscopy revealed multiple polyps in the colon, and the resected polyps were compatible with juvenile polyps. Sequencing analysis revealed a novel intronic mutation (c.778+5G>C) in BMPR1A. Reverse transcription polymerase chain reaction analysis of RNA extracted from the colonic mucosa showed an aberrant splicing form of BMPR1A. Trio analysis showed that his mother also had the same BMPR1A mutation. She was diagnosed with cancer of the cecum and polyposis of the colon at the age of 41. CONCLUSION: We demonstrate the presence of a novel BMPR1A intronic mutation that exhibits splicing abnormality in a family with JPS. Further research and development will help elucidate the genotype-phenotype relationship in JPS.
Assuntos
Mutação em Linhagem Germinativa , RNA , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Feminino , Humanos , Polipose Intestinal/congênito , Mutação , Síndromes Neoplásicas Hereditárias , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Bone morphogenetic proteins (BMPs) are members of the TGF-ß family that signal via the BMP receptor (BMPR) signaling cascade, distinct from canonical TGF-ß signaling. BMP downstream signaling is strongly induced within epidermal keratinocytes in cutaneous psoriatic lesions, and BMP7 instructs monocytic cells to acquire characteristics of psoriasis-associated Langerhans dendritic cells (DCs). Regulatory T (Treg)-cell numbers strongly increase during psoriatic skin inflammation and were recently shown to limit psoriatic skin inflammation. However, the factors mediating Treg-cell accumulation in psoriatic skin currently remain unknown. OBJECTIVE: We sought to investigate the role of BMP signaling in Treg-cell accumulation in psoriasis. METHODS: The following methods were used: immunohistology of patients and healthy controls; ex vivo models of Treg-cell generation in the presence or absence of Langerhans cells; analysis of BMP versus canonical TGF-ß signaling in DCs and Treg cells; and modeling of psoriatic skin inflammation in mice lacking the BMPR type 1a in CD11c+ cells. RESULTS: We here demonstrated a positive correlation between Treg-cell numbers and epidermal BMP7 expression in cutaneous psoriatic lesions and show that unlike Treg cells from healthy skin, a portion of inflammation-associated Treg cells exhibit constitutive-active BMP signaling. We further found that BMPR signaling licenses inflammation-associated Langerhans cell/DC to gain an enhanced capacity to promote Treg cells via BMPR-mediated CD25 induction and that this effect is associated with reduced skin inflammation. CONCLUSIONS: Psoriatic lesions are marked by constitutive high BMP7/BMPR signaling in keratinocytes, which instructs inflammatory DCs to gain enhanced Treg-cell-stimulatory activity. Locally secreted BMP7 can directly promote Treg-cell generation through the BMP signaling cascade.
Assuntos
Proteína Morfogenética Óssea 7/imunologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/imunologia , Células Dendríticas/imunologia , Queratinócitos/imunologia , Psoríase/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , Adulto JovemRESUMO
Genetic variation of insulin receptor substrate 1 (IRS-1) was found to modulate the insulin resistance of adipose tissues, but the underlying mechanism was not clear. To investigate how the IRS-1 was involved in the browning of white adipose tissue through miRNA, we identified a mutated Irs-1 (Irs-1-/- ) mice model and found that this mice had a reduced subcutaneous WAT (sWAT) and increased brown adipose tissue (BAT) in the interscapular region. So we isolated the bone marrow stromal cells and analyzed differentially expressed miRNAs and adipogenesis-related genes with miRNA arrays and PCR arrays. Irs-1-/- mice showed decreased miR-503 expression, but increased expression of its target, bone morphogenetic protein receptor type 1a (BMPR1a). Overexpression of miR-503 in preadipocytes downregulated BMPR1a and impaired adipogenic activity through the phosphotidylinositol 3-kinase (PI3K/Akt) pathway, while the inhibitor had the opposite effect. In both Irs-1-/- and cold-induced models, sWAT exhibited BAT features, and showed tissue-specific increased BMPR1a expression, PI3K expression, and Akt phosphorylation. Thus, our results showed that IRS-1 regulated brown preadipocyte differentiation and induced browning in sWAT through the miR-503-BMPR1a pathway, which played important roles in high-fat diet-induced obesity.
Assuntos
Tecido Adiposo Branco/metabolismo , Dieta Hiperlipídica , Proteínas Substratos do Receptor de Insulina/fisiologia , MicroRNAs/fisiologia , Obesidade/prevenção & controle , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Diferenciação Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND: Juvenile polyposis syndrome (JPS) is a rare autosomal dominant hereditary disorder characterized by the development of multiple distinct juvenile polyps in the gastrointestinal tract with an increased risk of colorectal cancer. Germline mutations in two genes, SMAD4 and BMPR1A, have been identified to cause JPS. CASE PRESENTATION: Here, we report a germline heterozygous missense variant (c.299G > A) in exon 3 BMPR1A gene in a family with juvenile polyposis. This variant was absent from the population database, and concluded as de novo compared with the parental sequencing. Further sequencing of the proband's children confirmed the segregation of this variant with the disease, while the variant was also predicted to have damaging effect based on online prediction tools. Therefore, this variant was classified as likely pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines. CONCLUSIONS: Germline genetic testing revealed a de novo germline missense variant in BMPR1A gene in a family with juvenile polyposis. Identification of the pathogenic variant facilitates the cancer risk management of at-risk family members, and endoscopic surveillance is recommended for mutation carriers.
Assuntos
Polipose Adenomatosa do Colo/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Mutação em Linhagem Germinativa , Polipose Intestinal/congênito , Mutação de Sentido Incorreto , Síndromes Neoplásicas Hereditárias/genética , Polipose Adenomatosa do Colo/diagnóstico , Adulto , Saúde da Família , Feminino , Testes Genéticos , Heterozigoto , Humanos , Polipose Intestinal/diagnóstico , Polipose Intestinal/genética , Masculino , Síndromes Neoplásicas Hereditárias/diagnóstico , Linhagem , Proteína Smad4/genéticaRESUMO
Hereditary mixed polyposis syndrome (HMPS) is a rare autosomal dominant disorder characterized by a mixture of typical and/or atypical juvenile polyps, adenomas and hyperplastic polyps, resulting in an increased risk of colorectal cancer. In HMPS, four different germline BMPR1A variants from five unrelated families have been reported. This study is the first to report HMPS within a Japanese family. The proband underwent repeated colonoscopic polypectomies over a 5-year period, since the age of 67. Histological examination of these resected polyps revealed adenomas, juvenile-like polyps and hyperplastic changes. Genetic testing was conducted to identify the causative genes for hereditary gastrointestinal cancer syndromes, including BMPR1A. We detected a germline variant, c.72_73delGA, in BMPR1A. The proband's elder brother, younger sister and nephew have also undergone repeated colonoscopic polypectomies at other clinics. His sister and nephew underwent genetic testing, and the same BMPR1A variant was identified.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Células Germinativas/fisiologia , Síndromes Neoplásicas Hereditárias/genética , Proteína Smad4/genética , Idoso , Feminino , Humanos , Japão , Masculino , Proteína Smad4/metabolismoRESUMO
Background/aim: Drynaria fortunei (Gusuibu; GSB) is a popular traditional Chinese medicine used for bone repair. An increasing number of studies have reported that GSB induces osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). These results provide insight into the application of GSB for bone tissue engineering techniques used to repair large bone defects. However, few studies have described the molecular mechanisms of GSB. Materials and methods: In the present study, the effects of GSB and naringin, a marker compound, on the binding of BMP-2 to BMPR and BMP-2-derived signal transduction were investigated using surface plasmon resonance (SPR) and coculturing with BMPR- expressed cell line, C2C12, respectively. Furthermore, naringin was also used to prepare naringin contained scaffolds for bone tissue engineering. The physical and chemical properties of these scaffolds were analysed using scanning electron microscopy (SEM) and highperformance liquid chromatography (HPLC). These scaffolds were cocultured with rabbit BMSCs in vitro and implanted into rabbit calvarial defects for bone repair assessment. Results: The results showed that GSB and naringin affect the binding of BMP and BMPR in SPR experiments. GSB is a subtle BMP modulator that simultaneously inhibits the binding of BMP-2 to BMPR-1A and enhances its binding to BMPR-1B. In contrast, naringin inhibited BMP-2 binding to BMPR-1A. In vitro studies involving the phosphorylation of signals downstream of BMPR and Smad showed that GSB and naringin affected stem cell differentiation by inhibiting BMPR-1A signalling. When using GSB for bone tissue engineering, naringin exhibited a higher capacity for slow and gradual release from the scaffold, which promotes bone formation via osteoinduction. Moreover, control and naringin scaffolds were implanted into rabbit calvarial defects for 4 weeks, and naringin enhanced bone regeneration in vivo significantly. Conclusions: GSB and its marker compound (naringin) could inhibit the binding of BMP-2 and BMPR-1A to control cell differentiation by blocked BMPR-1A signalling and enhanced BMPR-1B signalling. GSB and naringin could be good natural BMP regulators for bone tissue engineering.
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Proteína Morfogenética Óssea 2/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Flavanonas/farmacologia , Polypodiaceae/química , Engenharia Tecidual/métodos , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Células Cultivadas , Masculino , Osteogênese/efeitos dos fármacos , Coelhos , Transdução de Sinais/efeitos dos fármacosRESUMO
Two of the most problematic orthopedic and neurosurgeon visits are associated with spine and craniofacial fractures. Therefore, more attention needs to be paid to finding a medicine to repair these fractures. Amongst the most mysterious herbs, Aloe vera stands out. In the present study, the ameliorating function of A. vera on osteogenesis was studied in vitro and in vivo. Osteoblast-like cells were exposed to A. vera, followed by analysis of cell viability, lactate dehydrogenase release, and intracellular reactive oxygen species (ROS) production. The results showed an enhanced cell biocompatibility in a dose-dependent manner due to attenuated intracellular ROS production. Furthermore, a docking study indicated that the strong affinity of A. vera constituents to type I bone morphogenic protein receptor (BMPR1A) without the involvement of the BMPR1A chain B. The induction of osteogenesis prompts extracellular calcium deposition by osteoblasts, which affirms successful in vitro bone regeneration. However, injection of A. vera in rats with critical size calvarial defects induced Runx2, alkaline phosphatase (ALP), OCN, and BMP2 genes overexpression, which led to the formation of victorious bone with enhanced bone density and ALP activity. It is worthy to note that Aloin has the highest affinity to BMPR1A, whereas there are no reports regarding the impact of Aloenin, Aloesin, and γ-sitosterol on osteogenesis. Furthermore, some of them have antitumor potency, and it might be proposed that they are considered as a bone substitute in the osteotomy site of osteosarcoma with the aim of bone recovery and suppression of osteosarcoma. The whole consequences of this investigation manifests the plausibility of using A. vera as an antioxidant and osteoconductive substitute.
Assuntos
Aloe/química , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Osteogênese/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Animais , Antioxidantes , Proteína Morfogenética Óssea 2/genética , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/genética , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cromonas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glucosídeos , Humanos , L-Lactato Desidrogenase/genética , Osteoblastos/efeitos dos fármacos , Osteogênese/genética , Compostos Fitoquímicos/química , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Exogenous bone morphogenetic proteins (Bmp) are well known to induce ectopic bone formation, but the physiological effect of Bmp signaling on normal bone is not completely understood. By deleting the receptor Bmpr1a in osteoblast lineage cells with Dmp1-Cre, we observed a dramatic increase in trabecular bone mass in postnatal mice, which was due to a marked increase in osteoblast number that was likely to be driven by hyperproliferation of Sp7(+) preosteoblasts. Similarly, inducible deletion of Bmpr1a in Sp7(+) cells specifically in postnatal mice increased trabecular bone mass. However, deletion of Smad4 by the same approaches had only a minor effect, indicating that Bmpr1a signaling suppresses trabecular bone formation through effectors beyond Smad4. Besides increasing osteoblast number in the trabecular bone, deletion of Bmpr1a by Dmp1-Cre also notably reduced osteoblast activity, resulting in attenuation of periosteal bone growth. The impairment in osteoblast activity correlated with reduced mTORC1 signaling in vivo, whereas inhibition of mTORC1 activity abolished the induction of protein anabolism genes by BMP2 treatment in vitro. Thus, physiological Bmpr1a signaling in bone exerts a dual function in both restricting preosteoblast proliferation and promoting osteoblast activity.
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Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The PTEN hamartoma tumor syndrome (PHTS) is caused by heterozygous germline variants in PTEN. Here, we report two unrelated patients with juvenile polyposis, macrocephaly, intellectual disability, and hyperpigmented skin macules. Both patients were clinically suspected for the Bannayan-Riley-Ruvalcaba syndrome (BRRS), a PHTS subentity. By array-CGH analysis, we identified an interstitial 10q23.1q23.3 deletion in a buccal mucosa sample of Patient 1 that encompassed PTEN, BMPR1A, and KLLN, among others. In contrast, neither sequencing nor array-CGH analysis identified a pathogenic variant in PTEN or BMPR1A in a blood sample of Patient 2. However, in a surgical specimen of the thyroid gland high-level mosaicism for a 10q23.2q23.3 deletion was observed. Additionally, the pathogenic PTEN variant c.956_959delCTTT p.(Thr319LysfsTer24) was detected in his thyroid tissue. The frame shift variant was neither detected in the patient's blood nor in his buccal mucosa sample. Low-level mosaicism for the microdeletion was identified in a buccal swap sample, and reanalysis of the blood sample suggested marginal-level mosaicism for deletion. The 10q23.2q23.3 deletion mosaicism was also identified in a subsequently resected colonic polyp. Thus, in both cases, the diagnosis of a 10q23 deletion syndrome, which clinically presented as BRRS, was established. Overall, the study expands the BRRS spectrum and highlights the relevance of considering mosaicism in PHTS. We conclude that in all patients with a clear clinical suspicion of PHTS, in which genetic analyses of DNA from blood and buccal swap samples fail to identify causative genetic variants, genetic analyses of additional tissues are recommended.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Deleção Cromossômica , Cromossomos Humanos Par 10 , Síndrome do Hamartoma Múltiplo/genética , Mosaicismo , Mutação , PTEN Fosfo-Hidrolase/genética , Adolescente , Feminino , Humanos , MasculinoRESUMO
Craniosynostosis is caused by premature fusion of one or more sutures in an infant skull, resulting in abnormal facial features. The molecular and cellular mechanisms by which genetic mutations cause craniosynostosis are incompletely characterized, and many of the causative genes for diverse types of syndromic craniosynostosis have not yet been identified. We previously demonstrated that augmentation of BMP signaling mediated by a constitutively active BMP type IA receptor (ca-BmpR1A) in neural crest cells (ca1A hereafter) causes craniosynostosis and superimposition of heterozygous null mutation of Bmpr1a rescues premature suture fusion (ca1A;1aH hereafter). In this study, we superimposed heterozygous null mutations of the other two BMP type I receptors, Bmpr1b and Acvr1 (ca1A;1bH and ca1A;AcH respectively hereafter) to further dissect involvement of BMP-Smad signaling. Unlike caA1;1aH, ca1A;1bH and ca1A;AcH did not restore the craniosynostosis phenotypes. In our in vivo study, Smad-dependent BMP signaling was decreased to normal levels in mut;1aH mice. However, BMP receptor-regulated Smads (R-Smads; pSmad1/5/9 hereafter) levels were comparable between ca1A, ca1A;1bH and ca1A;AcH mice, and elevated compared to control mice. Bmpr1a, Bmpr1b and Acvr1 null cells were used to examine potential mechanisms underlying the differences in ability of heterozygosity for Bmpr1a vs. Bmpr1b or Acvr1 to rescue the mut phenotype. pSmad1/5/9 level was undetectable in Bmpr1a homozygous null cells while pSmad1/5/9 levels did not decrease in Bmpr1b or Acvr1 homozygous null cells. Taken together, our study indicates that different levels of expression and subsequent activation of Smad signaling differentially contribute each BMP type I receptor to BMP-Smad signaling and craniofacial development. These results also suggest differential involvement of each type 1 receptor in pathogenesis of syndromic craniosynostoses.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Transdução de Sinais , Crânio/embriologia , Crânio/metabolismo , Proteínas Smad/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Craniossinostoses/genética , Craniossinostoses/patologia , Regulação da Expressão Gênica no Desenvolvimento , Heterozigoto , Camundongos Endogâmicos C57BL , Mutação/genética , Osteoblastos/metabolismo , Fenótipo , Transdução de Sinais/genética , Crânio/anormalidades , Crânio/patologiaRESUMO
BACKGROUND/AIMS: Bone morphogenetic proteins (BMPs) and BMP receptors widely participate in osteolytic metastasis of breast cancer, while their role in tumor-stromal interaction is largely unknown. In this study, we investigated whether BMP receptor type 1a (BMPR1a) can alter the interaction between metastatic cancer cells and osteoclast precursors. METHODS: Adenovirus-mediated RNA interference was used to interrupt target genes of human breast cancer cell lines and nude mice were injected intratibially with the cancer cells. Tumor-bearing mice were examined by bioluminescence imaging and microCT. Sections of metastatic legs were measured by a series of staining methods. Murine bone marrow mononuclear cells or RAW264.7 cells were cultured with conditioned media of breast cancer cells. RT-PCR, Western blotting and ELISA were used to test mRNA and protein expressions of target molecules. RESULTS: Expression of BMPR1a of MDA-MB-231-luc cells at tumor-bone interface was apparently stronger than that of cancer cells distant from the interface. Mice injected with BMPR1a-knockdown MDA-MB-231-luc cells showed reduced tumor growth and bone destruction compared with control groups. Knockdown (KD) of BMPR1a of MDA-MB-231-luc cells or MCF-7 cells decreased the level of receptor activator for NF-κB ligand (RANKL). Level of RANKL in MDA-MB-231-luc cells or MCF-7 cells was reduced by p38 inhibitor. Compared with control group, knockdown of p38 of breast cancer cells decreased cancer-induced osteoclastogenesis. CONCLUSION: Knockdown of BMPR1a of breast cancer cells suppresses their production of RANKL via p38 pathway and inhibits cancer-induced osteoclastogenesis, which indicates that BMPR1a might be a possible target in breast cancer-induced osteolytic metastasis.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Neoplasias da Mama/patologia , Ligante RANK/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Imidazóis/farmacologia , Células MCF-7 , Camundongos , Camundongos Nus , Osteogênese/efeitos dos fármacos , Piridinas/farmacologia , Células RAW 264.7 , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Tíbia/diagnóstico por imagem , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In recent years, several studies have found that the disruption of type IA receptor of bone morphogenetic proteins (BMPR1A) could increase bone mass. However, whether disruption of BMPR1A could have an effect on bone quality and bone strength is currently unknown. Osteoblast-targeted conditional knockout (cKO) of BMPRIA by crossing 3.2-kb Col1-CreER™ mice with BMPR1A fx +/+ mice was conducted. Then, in vitro and in vivo studies were employed to examine the effect of BMPR1A knockout on bone quality and bone strength. It was found that the ultimate force and stiffness of the femora decreased significantly in cKO mice when compared to control mice. The content of collagen and mineralization level decreased as the structure of the collagen became disorganized. The morphology of osteocytes in cKO mice was abnormal as well. The expression level of osteocalcin, a marker for the terminal differentiation of osteoblasts, decreased in cKO mice. This data indicated that the differentiation of osteoblasts in cKO mice was impaired. Immunohistochemistry examination revealed deregulated expression of dickkopf 1(DKK1) in osteocytes in cKO mice. Adding DKK1 to the culture medium reversed these effects. In conclusion, even though disruption of BMPR1A could increase bone mass, it also impairs bone quality and bone strength.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Osso e Ossos/fisiologia , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Animais , Biomarcadores/metabolismo , Fenômenos Biomecânicos , Contagem de Células , Diferenciação Celular , Colágeno/metabolismo , Masculino , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/ultraestrutura , Osteócitos/metabolismo , Osteócitos/ultraestrutura , Tíbia/fisiologiaRESUMO
Osteoblasts and osteoclasts are well orchestrated through different mechanisms of communication during bone remodeling. Previously, we found that osteoclast-specific disruption of one of the BMP receptors, Bmpr1a, results in increased osteoblastic bone formation in mice. We hypothesized that BMPR1A signaling in osteoclasts regulates production of either membrane bound proteins or secreted molecules that regulated osteoblast differentiation. In our current study, we co-cultured wild-type osteoblasts with either control osteoclasts or osteoclasts lacking BMPR1A signaling activity. We found that loss of Bmpr1a in osteoclasts promoted osteoblast mineralization in vitro. Further, we found that the expression of Cx43/Gja1 in the mutant osteoclasts was increased, which encoded for one of the gap junction proteins connexin 43/gap junction alpha 1. Knockdown of Gja1 in the mutant osteoclasts for Bmpr1a reduced osteoblastic mineralization when co-cultured. Our findings suggest that GJA1 may be one of the downstream targets of BMPR1A signaling in osteoclasts that mediates osteoclast-osteoblast communication during bone remodeling. J. Cell. Biochem. 118: 605-614, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica/fisiologia , Conexina 43/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Transdução de Sinais/fisiologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Proteínas Morfogenéticas Ósseas/genética , Comunicação Celular/fisiologia , Conexina 43/genética , Camundongos , Camundongos Knockout , Osteoblastos/citologiaRESUMO
Bone morphogenetic proteins (BMPs) regulate multiple aspects of skeletal development in vertebrates. Although exogenously applied BMPs can induce chondrogenesis de novo, the role and mechanism of physiologic BMP signaling during precartilaginous mesenchymal condensation is not well understood. By deleting the type I BMP receptors or the transcription factor Smad4 in the limb bud mesenchyme, we find that loss of BMP-Smad signaling abolishes skeletal development due to a failure in mesenchymal condensation. In the absence of Smad4, expression of Sox9, an essential transcription factor for chondrogenesis, initiates normally in the proximal mesenchyme of the limb bud, but fails to maintain its level or expand to the more distal territory at the later stages. However, forced-expression of Sox9 does not restore cartilage formation in the Smad4-deficeint embryo. In vitro micromass cultures show that the Smad4-deficient cells fail to condense in a cell-autonomous manner, even though they express several cell adhesion molecules either normally or even at a higher level. Thus, BMP-Smad signaling critically controls mesenchymal condensation to initiate skeletal development likely through a Sox9-independent mechanism.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Extremidades/embriologia , Botões de Extremidades/metabolismo , Mesoderma/embriologia , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , Proteína Smad4/metabolismo , Animais , Primers do DNA/genética , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Botões de Extremidades/embriologia , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOX9/metabolismoRESUMO
Deficiency in pulmonary surfactant results in neonatal respiratory distress, and the known genetic mutations in key components of surfactant only account for a small number of cases. Therefore, determining the regulatory mechanisms of surfactant production and secretion, particularly during the transition from prenatal to neonatal stages, is essential for better understanding of the pathogenesis of human neonatal respiratory distress. We have observed significant increase of bone morphogenetic protein (BMP) signaling in neonatal mouse lungs immediately after birth. Using genetically manipulated mice, we then studied the relationship between BMP signaling and surfactant production in neonates. Blockade of endogenous BMP signaling by deleting Bmpr1a (Alk3) or Smad1 in embryonic day 18.5 in perinatal lung epithelial cells resulted in severe neonatal respiratory distress and death, accompanied by atelectasis in histopathology and significant reductions of surfactant protein B and C, as well as Abca3, whereas prenatal lung development was not significantly affected. We then identified a new BMP-Smad1 downstream target, Nfatc3, which is known as an important transcription activator for surfactant proteins and Abca3. Furthermore, activation of BMP signaling in cultured lung epithelial cells was able to promote endogenous Nfatc3 expression and also stimulate the activity of an Nfatc3 promoter that contains a Smad1-binding site. Therefore, our study suggests that the BMP-Alk3-Smad1-Nfatc3 regulatory loop plays an important role in enhancing surfactant production in neonates, possibly helping neonatal respiratory adaptation from prenatal amniotic fluid environment to neonatal air breathing.
Assuntos
Adaptação Fisiológica , Proteínas Morfogenéticas Ósseas/fisiologia , Pulmão/metabolismo , Surfactantes Pulmonares/metabolismo , Respiração , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Animais Recém-Nascidos , Sequência de Bases , Sítios de Ligação , Expressão Gênica , Pulmão/crescimento & desenvolvimento , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas , Transdução de Sinais , Proteína Smad1/metabolismo , Ativação TranscricionalRESUMO
The aim of this study was to determine Bmpr1a and Bmp2 expression patterns during anorectal development in normal and anorectal malformation (ARM) embryos with a view to establishing the possible role of Bmpr1a and Bmp2 in ARM pathogenesis. ARM was induced with ethylenethiourea on the 10th gestational day (GD10) in rat embryos. The embryos were harvested by Cesarean deliveries. The expression of Bmpr1a and Bmp2 was evaluated in normal rat embryos (n=213) and ARM embryos (n=236) from GD14 to GD16. Immunohistochemical staining revealed, in normal embryos, that Bmpr1a and Bmp2 was mainly expressed on the epithelium of the urorectal septum (URS) and the cloacal membrane (CM) on GD14 and GD15. When the rectum separated from the urogenital sinus (UGS) on GD16, Bmpr1a- and Bmp2-immunolabeled cells were observed on the anorectal epithelium. In ARM embryos, the epithelium of the hindgut and URS demonstrated faint immunostaining for Bmpr1a and Bmp2. Analyses by Western blot and Real-time PCR revealed that Bmpr1a and Bmp2 protein and mRNA expression were significantly decreased in the ARM hindgut compared with normal hindgut on GD14 and GD15 (P<0.05). In ARM embryos, an imbalance in the spatiotemporal expression of Bmpr1a and Bmp2 was noted during anorectal morphogenesis from GD14 to GD16. Therefore, downregulation of Bmpr1a and Bmp2 at the time of cloacal separation into the primitive rectum and UGS might be related to the development of ARM.
Assuntos
Malformações Anorretais/genética , Proteína Morfogenética Óssea 2/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Sistema Digestório/embriologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Malformações Anorretais/patologia , Western Blotting , Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Sistema Digestório/metabolismo , Idade Gestacional , Imuno-Histoquímica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The importance of Bone Morphogenetic Proteins (BMPs) in the regulation of cell fate, differentiation and proliferation in the growth plate is well-known. However, in secondary cartilages (such as that in the temporomandibular joint) that grow by proliferation of prechondrocytes and differ in their pattern of growth, the role of BMPs is largely unexplored. To examine this question, we ablated Bmpr1a in the condylar cartilage of neonatal mice and assessed the consequences for mandibular condyle growth and organization at intervals over the ensuing 4 weeks. Bmpr1a deficiency caused significant chondrodysplasia and almost eliminated the chondrocytic phenotype in the TMJ. Expression of Sox9, collagen II, proteoglycan were all greatly reduced, and cell proliferation as detected by BrdU was almost non-existent in the knockout mice. Primary bone spongiosa formation was also disturbed and was accompanied by reduced Osterix expression. These findings strongly suggest that Bmpr1a is critical for the development and growth of the mandibular condyle via its effect on proliferation of prechondroblasts and chondrocyte differentiation.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Condrogênese/fisiologia , Côndilo Mandibular/citologia , Articulação Temporomandibular/citologia , Animais , Cartilagem/citologia , Cartilagem/metabolismo , Diferenciação Celular/fisiologia , Condrogênese/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Lâmina de Crescimento/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Juvenile polyposis syndrome is an autosomal dominant inherited disorder characterized by multiple juvenile polyps arising in the gastrointestinal tract and an increased risk of gastrointestinal cancers, specifically colon cancer. BMPR1A and SMAD4 germline mutations have been found in patients with juvenile polyposis syndrome. We identified a BMPR1A mutation, which involves a duplication of coding exon 3 (c.230+452_333+441dup1995), on multiple ligation dependent probe amplification in a patient with juvenile polyposis syndrome. The mutation causes a frameshift, producing a truncated protein (p.D112NfsX2). Therefore, the mutation is believed to be pathogenic. We also identified a duplication breakpoint in which Alu sequences are located. These results suggest that the duplication event resulted from recombination between Alu sequences. To our knowledge, partial duplication in the BMPR1A gene has not been reported previously. This is the first case report to document coding exon 3 duplication in the BMPR1A gene in a patient with juvenile polyposis syndrome.