RESUMO
Bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic factors, which may be a potential candidate for bone tissue engineering. However, the osteogenic capacity of BMP9 still need to be further enhanced. In this study, we determined the effect of Wnt10b on BMP9-induced osteogenic differentiation in mesenchymal stem cell (MSCs) and the possible mechanism underlying this process. We introduced the polymerase chain reaction (PCR), Western blot analysis, histochemical stain, ectopic bone formation, and microcomputed tomography analysis to evaluate the effect of Wnt10b on BMP9-induced osteogenic differentiation. Meanwhile, PCR, Western blot analysis, chromatin immunoprecipitation, and immunoprecipitation were used to analyze the possible relationship between BMP9 and Wnt10b. We found that BMP9 upregulates Wnt10b in C3H10T1/2 cells. Wnt10b increases the osteogenic markers and bone formation induced by BMP9 in C3H10T1/2 cells, and silencing Wnt10b decreases these effects of BMP9. Meanwhile, Wnt10b enhances the level of phosphorylated Smad1/5/8 (p-Smad1/5/8) induced by BMP9, which can be reduced by silencing Wnt10b. On the contrary, Wnt10b inhibits adipogenic markers induced by BMP9, which can be decreased by silencing Wnt10b. Further analysis indicated that BMP9 upregulates cyclooxygenase-2 (COX-2) and phosphorylation of cAMP-responsive element binding (p-CREB) simultaneously. COX-2 potentiates the effect of BMP9 on increasing p-CREB and Wnt10b, while silencing COX-2 decreases these effects. p-CREB interacts with p-Smad1/5/8 to bind the promoter of Wnt10b in C3H10T1/2 cells. Our findings suggested that Wnt10b can promote BMP9-induced osteogenic differentiation in MSCs, which may be mediated through enhancing BMP/Smad signal and reducing adipogenic differentiation; BMP9 may upregulate Wnt10b via the COX-2/p-CREB-dependent manner.
Assuntos
Adipogenia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Proteínas Wnt/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular , Coristoma/patologia , Humanos , Camundongos , Camundongos Nus , Fosforilação , Transdução de Sinais , Proteínas Smad/metabolismoRESUMO
Pioglitazone/metformin adduct is a novel compound synthesized from pioglitazone and metformin combined at a molar mass ratio of 1:1. The aim of this study was to investigate the effects of pioglitazone/metformin adduct on high glucose-induced insulin secretion and apoptosis in INS-1 cells. Western blot and CCK8 analyses showed that the death rate of INS-1 cells increased in response to glucose treatment in a concentration-dependent manner. ELISA assays and Western blot analyses showed that insulin secretion peaked following treatment with glucose concentration at 33.33 mM. Treatment of INS-1 cells with 1 µM pioglitazone/metformin adduct in the presence of 33.33 mM glucose greatly improveded the levels of insulin and apoptosis rates compared to those of the control group. Analysis of mechanism underlying these effects revealed the involvement of the p21-p53-MDM2 signaling pathway. Our results indicate that pioglitazone/metformin adduct is superior to pioglitazone and/or metformin in regulating high glucose-induced insulin secretion and apoptosis in INS-1 cells.
Assuntos
Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Metformina/farmacologia , Pioglitazona/farmacologia , Animais , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Sinergismo Farmacológico , Hipoglicemiantes/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ratos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Bone morphogenetic protein 9 (BMP9), as one of the most potent osteogenic factors, is a promising cytokine for bone tissue engineering. Wnt11 can regulate the development of the skeletal system and is related to high bone mass syndrome. However, the effect of Wnt11 on BMP9-induced osteogenic differentiation remains unknown. In this study, we investigated the relationship between Wnt11- and BMP9-induced osteogenic differentiation in mesenchymal stem cells (MSCs). We recapitulated the osteogenic potential of BMP9 in C3H10T1/2 cells. The messenger RNA expression of Wnt11 is detectable in the available progenitor cells, and BMP9 can obviously increase the protein level of Wnt11 in these cells. Exogenous Wnt11 potentiates the effect of BMP9 on increasing alkaline phosphatase (ALP) activities, the expression of osteopontin (OPN), and Runt-related transcription factor 2 (Runx2), so does matrix mineralization in C3H10T1/2 cells. Although Wnt11 cannot increase the BMP9-induced ectopic bone formation, it can increase the bone density induced by BMP9 apparently. Wnt11 increases the level of p-Smad1/5/8, as well as p-p38. Meanwhile, Wnt11 promotes the effect of BMP9 on increasing the levels of p-Smad1/5/8 and p-p38. Inhibition of p38 decreases the BMP9-induced ALP activities, the expression of OPN, and the mineralization in C3H10T1/2 cells. However, all of these effects of the p38 inhibitor on BMP9-induced osteogenic markers can be almost reversed by the overexpression of Wnt11. Our findings suggested that Wnt11 can enhance the osteogenic potential of BMP9 in MSCs, and this effect may be partly mediated through enhancing BMPs/Smads and the p38 MAPK signal, which was induced by BMP9.
Assuntos
Fator 2 de Diferenciação de Crescimento/farmacologia , Células-Tronco Mesenquimais/metabolismo , Proteínas Wnt/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Feminino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Nus , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteínas Wnt/genéticaRESUMO
Vascular calcification is a notable risk factor for cardiovascular system. High phosphate can induce calcification in vascular smooth muscle cells (VSMCs), but the detail mechanism underlying this process remains unclear. In the present study, we determined the relationship between high phosphate and bone morphogenetic protein 9 (BMP9) in VSMCs, the effect of BMP9 on calcification in VSMCs and the effect of COX-2 on BMP9 induced calcification in VSMCs, as well as the possible mechanism underlying this biological process. We found that high phosphate obviously up-regulates the expression of BMP9 in VSMCs. Over-expression of BMP9 decreases the level of alpha-smooth muscle cell actin (α-SMA) apparently, but increases the level of Runx-2, Dlx-5, and ALP in VSMCs. Meanwhile, BMP9 increases the level of OPN and OCN, promotes mineralization in VSMCs and induces calcification in thoracic aorta. High phosphate and over-expression of BMP9 increases the level of COX-2. Over-expression of COX-2 enhances the inhibitory effect of BMP9 on α-SAM and increases the level of OPN and OCN induced by BMP9. However, inhibition of COX-2 decreases the BMP9-induced calcification in VSMCs and thoracic aorta. For mechanism, we found that high phosphate or BMP9 increases the level of ß-catenin and p-GSK3ß in VSMCs, but no substantial effect on GSK3ß. However, COX-2 inhibitor decreases the expression of ß-catenin induced by BMP9. Our findings indicated that BMP9 is involved in the phosphate-induced calcification in VSMCs and COX-2 partly mediates the BMP9-induced calcification in VSMCs through activating Wnt/ß-catenin pathway.
Assuntos
Calcinose/metabolismo , Ciclo-Oxigenase 2/biossíntese , Fator 2 de Diferenciação de Crescimento/biossíntese , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfatos/efeitos adversos , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Calcinose/induzido quimicamente , Calcinose/patologia , Células Cultivadas , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fosfatos/farmacologia , Ratos , beta Catenina/metabolismoRESUMO
Postmenopausal osteoporosis (PMOP)-related fractures usually result in morbidity and mortality in aging women, so it remains a global public health concern, and new effective safe treatments are urgently needed recently. Efficient osteogenesis from mesenchymal stem cells (MSCs) would have the clinical application potential in treating multiple osteal disorders. Follicle-stimulating hormone (FSH), a pituitary glycoprotein hormone highly associated with menopausal bone turnover, whose peculiar part of receptor binding is follicle-stimulating hormone ß-subunit (FSHß). Bone morphogenetic protein 9 (BMP9), a potent osteogenic factor, can up-regulate FSHß in mouse embryonic fibroblasts (MEFs). However, it is unclear, whether extrapituitary FSHß affects BMP9-induced osteogenesis in MEFs. In this study, we investigated the role of FSHß in BMP9-induced osteogenesis in MEFs. We found that exogenous expression of FSHß significantly increased BMP9-induced alkaline phosphatase activity (ALP), the expression of osteogenic transcriptional factors, Runx2 and Osx, and the established late osteogenic markers, osteopontin (OPN) and osteocalcin (OCN), so does the ectopic bone formation. Mechanistically, FSHß dramatically enhanced BMP9-induced BMP/Smad signal transduction, presenting the augment phosphorylation of Smad1/5/8, whereas treatment with anti-FSHß antibodies suppressed these effects. An adenylate cyclase inhibitor obviously suppressed ALP and BMP/Smad signal transduction induced by BMP9 or the combination of BMP9 and FSHß in MEFs. Collectively, our findings suggested that FSHß may promote BMP9-induced activation of BMP/Smad signaling through a FSH/FSH receptor (FSHR)/cAMP dependent pathway in MEFs partly. J. Cell. Biochem. 118: 1792-1802, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Subunidade beta do Hormônio Folículoestimulante/farmacologia , Fatores de Diferenciação de Crescimento/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fator 2 de Diferenciação de Crescimento , Fatores de Diferenciação de Crescimento/genética , Células HEK293 , Humanos , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Receptores do FSH/genética , Receptores do FSH/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Mesenchymal stem cells (MSCs) are suitable seed cells for bone tissue engineering because they can self-renew and undergo differentiation into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Vascular endothelial growth factor-a (VEGF-a), an angiogenic factor, is also involved in osteogenesis and bone repair. However, the effects of VEGF-a on osteogenic MSCs differentiation remain unknown. It was previously reported that bone morphogenetic protein9 (BMP9) is one of the most important osteogenic BMPs. Here, we investigated the effects of VEGF-a on BMP9-induced osteogenesis with mouse embryo fibroblasts (MEFs). We found that endogenous VEGF-a expression was undetectable in MSCs. Adenovirus-mediated expression of VEGF-a in MEFs potentiated BMP9-induced early and late osteogenic markers, including alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In stem cell implantation assays, VEGF-a augmented BMP9-induced ectopic bone formation. VEGF-a in combination with BMP9 effectively increased the bone volume and osteogenic activity. However, the synergistic effect was efficiently abolished by the phosphoinositide 3-kinase (PI3K)/AKT inhibitor LY294002. These results demonstrated that BMP9 may crosstalk with VEGF-a through the PI3K/AKT signaling pathway to induce osteogenic differentiation in MEFs. Thus, our findings demonstrate the effects of VEGF-a on BMP9-induced bone formation and provide a new potential strategy for treating nonunion fractures, large segmental bony defects, and/or osteoporotic fractures.
Assuntos
Adenoviridae/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Osteogênese , Engenharia Tecidual , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fator 2 de Diferenciação de Crescimento/antagonistas & inibidores , Humanos , Camundongos , Morfolinas/farmacologia , Osteogênese/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BMP9 has demonstrated significant osteogenic potential. In this study, we investigated the effect of Leptin on BMP9-induced osteogenic differentiation. Firstly, we found Leptin was decreased during BMP9-induced osteogenic differentiation and serum Leptin concentrations were increased in the ovariectomized (OVX) rats. Both in vitro and in vivo, exogenous expression of Leptin inhibited the process of osteogenic differentiation, whereas silencing Leptin enhanced. Exogenous Leptin could increase the malonylation of ß-catenin. However, BMP9 could increase the level of Sirt5 and subsequently decrease the malonylation of ß-catenin; the BMP9-induced osteogenic differentiation was inhibited by silencing Sirt5. These data suggested that Leptin can inhibit the BMP9-induced osteogenic differentiation, which may be mediated through reducing the activity of Wnt/ß-catenin signalling via down-regulating Sirt5 to increase the malonylation level of ß-catenin partly.
Assuntos
Regulação para Baixo , Fator 2 de Diferenciação de Crescimento , Leptina , Osteogênese , Sirtuínas , Via de Sinalização Wnt , beta Catenina , Animais , beta Catenina/metabolismo , beta Catenina/genética , Sirtuínas/metabolismo , Sirtuínas/genética , Feminino , Ratos , Osteogênese/efeitos dos fármacos , Leptina/metabolismo , Leptina/farmacologia , Fator 2 de Diferenciação de Crescimento/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Ovariectomia , Diferenciação Celular/efeitos dos fármacos , Ratos Sprague-DawleyRESUMO
Following the publication of the above article, a concerned reader drew to the authors' attention that, among Figs. 1D, 2A and 4B, certain of the control western blots had been reused in different blots. The authors have retrieved and reexamined their original data, and were able to identify the correct control western blots where the data had been inadvertently duplicated in the affected original figures. The revised versions of Figs. 2 and 4, now featuring the correct control western blots, are shown in the subsequent two pages. The authors regret that the data in question featured in the original article had been reused, and thank the Editor of International Journal of Oncology for granting them the opportunity to publish this corrigendum. All the authors agree with the publication of this corrigendum; furthermore, they apologize to the readership of the journal for any inconvenience caused. [International Journal of Oncology 46: 12051213, 2015; DOI: 10.3892/ijo.2014.2800].
RESUMO
Bone morphogenetic protein 9 (BMP9) is an effective osteogenic factor and a promising candidate for bone tissue engineering. The osteoblastic potential of BMP9 needs to be further increased to overcome its shortcomings. However, the details of how BMP9 triggers osteogenic differentiation in mesenchymal stem cells (MSCs) are unclear. In this study, we used real-time PCR, western blot, histochemical staining, mouse ectopic bone formation model, immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation to investigate the role of pyruvate dehydrogenase kinase 4 (PDK4) in BMP9-induced osteogenic differentiation of C3H10T1/2 cells, as well as the underlying mechanism. We found that PDK4 was upregulated by BMP9 in C3H10T1/2 cells. BMP9-induced osteogenic markers and bone mass were increased by PDK4 overexpression, but decreased by PDK4 silencing. ß-catenin protein level was increased by BMP9, which was enhanced by PDK overexpression and decreased by PDK4 silencing. BMP9-induced osteogenic markers were reduced by PDK4 silencing, which was almost reversed by ß-catenin overexpression. PDK4 increased the BMP9-induced osteogenic markers, which was almost eliminated by ß-catenin silencing. Sclerostin was mildly decreased by BMP9 or PDK4, and significantly decreased by combined BMP9 and PDK4. In contrast, sclerostin increased significantly when BMP9 was combined with PDK4 silencing. BMP9-induced p-SMAD1/5/9 was increased by PDK4 overexpression, but was reduced by PDK4 silencing. PDK4 interacts with p-SMAD1/5/9 and regulates the sclerostin promoter. These findings suggest that PDK4 can increase the osteogenic potential of BMP9 by enhancing Wnt/ß-catenin signaling via the downregulation of sclerostin. PDK4 may be an effective target to strengthen BMP9-induced osteogenesis.
Assuntos
Fator 2 de Diferenciação de Crescimento , Células-Tronco Mesenquimais , Osteogênese , Piruvato Desidrogenase Quinase de Transferência de Acetil , Via de Sinalização Wnt , Animais , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Diferenciação Celular , Fator 2 de Diferenciação de Crescimento/genética , Fator 2 de Diferenciação de Crescimento/metabolismo , Células-Tronco Mesenquimais/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismoRESUMO
Silk as a natural biomaterial is considered as a promising bone substitute in tissue regeneration. Sericin and fibroin are the main components of silk and display unique features for their programmable mechanical properties, biocompatibility, biodegradability and morphological plasticity. It has been reported that sericin recombinant growth factors (GFs) can support cell proliferation and induce stem cell differentiation through cross-talk of signaling pathways during tissue regeneration. The transgenic technology allows the productions of bioactive heterologous GFs as fusion proteins with sericin, which are then fabricated into solid matrix or hydrogel format. Herein, using an injectable hydrogel derived from transgenic platelet-derived GF (PDGF)-BB silk sericin, we demonstrated that the PDGF-BB sericin hydrogel effectively augmented osteogenesis induced by bone morphogenetic protein (BMP9)-stimulated mesenchymal stem cells (MSCs) in vivo and in vitro, while inhibiting adipogenic differentiation. Further gene expression and protein-protein interactions studies demonstrated that BMP9 and PDGF-BB synergistically induced osteogenic differentiation through the cross-talk between Smad and Stat3 pathways in MSCs. Thus, our results provide a novel strategy to encapsulate osteogenic factors and osteoblastic progenitors in transgenic sericin-based hydrogel for robust bone tissue engineering.
RESUMO
Bone morphogenetic protein 9 (BMP9) has been validated as one of the most potent osteoinduction factors, but its underlying mechanism remains unclear. As a member of the matrix metalloproteinase (MMP) family, MMP13 may be involved in regulating the lineage-specific differentiation of mouse embryonic fibroblasts (MEFs). The goal of this study was to determine whether MMP13 regulates the osteoinduction potential of BMP9 in MEFs, which are multipotent progenitor cells widely used for stem cell biology research. In vitro and in vivo experiments showed that BMP9-induced osteogenic markers and/or bone were enhanced by exogenous MMP13 in MEFs, but were reduced by MMP13 knockdown or inhibition. The expression of hypoxia inducible factor 1 alpha (HIF-1α) was induced by BMP9, which was enhanced by MMP13. The protein expression of ß-catenin and phosphorylation level of glycogen synthase kinase-3 beta (GSK-3ß) were increased by BMP9 in MEFs, as was the translocation of ß-catenin from the cytoplasm to the nucleus; all these effects of BMP9 were enhanced by MMP13. Furthermore, the MMP13 effects of increasing BMP9-induced ß-catenin protein expression and GSK-3ß phosphorylation level were partially reversed by HIF-1α knockdown. These results suggest that MMP13 can enhance the osteoinduction potential of BMP9, which may be mediated, at least in part, through the HIF-1α/ß-catenin axis. Our findings demonstrate a novel role of MMP13 in the lineage decision of progenitor cells and provide a promising strategy to speed up bone regeneration.
Assuntos
Fator 2 de Diferenciação de Crescimento , beta Catenina , Animais , Camundongos , beta Catenina/metabolismo , Diferenciação Celular , Fibroblastos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Fator 2 de Diferenciação de Crescimento/farmacologia , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/farmacologia , Osteogênese , Regulação para CimaRESUMO
Mesenchymal stem cells (MSCs) can self-renew and differentiate into multiple lineages, making MSC transplantation a promising option for bone regeneration. Both matricellular proteins and growth factors play an important role in regulating stem cell fate. In this study, we investigated the effects of matricellular protein SMOC2 (secreted modular calcium-binding protein 2) on bone morphogenetic protein 9 (BMP9) in mouse embryonic fibroblasts (MEFs) and revealed a possible molecular mechanism underlying this process. We found that SMOC2 was detectable in MEFs and that exogenous SMOC2 expression potentiated BMP9-induced osteogenic markers, matrix mineralization, and ectopic bone formation, whereas SMOC2 knockdown inhibited these effects. BMP9 increased the levels of p-FAK and p-AKT, which were either enhanced or reduced by SMOC2 and FAK silencing, respectively. BMP9-induced osteogenic markers were increased by SMOC2, and this increase was partially abolished by silencing FAK or LY290042. Furthermore, we found that general transcription factor 2I (GTF2I) was enriched at the promoter region of SMOC2 and that integrin ß1 interacted with SMOC2 in BMP9-treated MEFs. Our findings demonstrate that SMOC2 can promote BMP9-induced osteogenic differentiation by enhancing the FAK/PI3K/AKT pathway, which may be triggered by facilitating the interaction between SMOC2 and integrin ß1.
RESUMO
Studies reported the positive and negative osteogenic effects of MEG3 in mesenchymal stem cells (MSCs). This study aims at clarifying the osteogenic potential of MEG3 and the underlying mechanism. Bone morphogenetic protein 9- (BMP9-) transfected MSCs were recruited as an osteogenic model in vitro, and ectopic bone formation were used in vivo to explore the effect of MEG3 on osteogenesis. We found that overexpression of MEG3 facilitated BMP9-induced osteogenic markers, ALP activities, and matrix mineralization. However, knockdown of MEG3 attenuated BMP9-induced osteogenic markers. MEG3 increased the phosphorylation of GSK-3ß and the protein level of ß-catenin. Pyruvate dehydrogenase kinase 4 (PDK4) can also combine with GSK-3ß and increase the latter phosphorylation. Moreover, MEG3 increased the mRNA level of PDK4. The ceRNA analysis showed that MEG3 may regulate the expression of PDK4 via microRNA 532-5p (miR-532-5p). The MEG3-enhanced GSK-3ß/ß-catenin axis can be attenuated by miR-532-5p, and miR-532-5p inhibitor partly rescued endogenous PDK4 and MEG3-mediated expression of PDK4. MEG3 may potentiate PDK4 and GSK-3ß/ß-catenin by inhibiting miR-532-5p.
Assuntos
MicroRNAs , RNA Longo não Codificante , Glicogênio Sintase Quinase 3 beta/genética , Diferenciação Celular/fisiologia , RNA Longo não Codificante/genética , beta Catenina/genética , beta Catenina/metabolismo , Osteogênese , MicroRNAs/genética , MicroRNAs/metabolismo , Células CultivadasRESUMO
BACKGROUND: Due to its effective osteogenic ability, BMP9 is a promising candidate for bone regeneration medicine. Whereas, BMP9 can also induce adipogenesis simultaneously. LCN2 is a cytokine associated with osteogenesis and adipogenesis. Reducing the adipogenic potential may be a feasible measure to enhance the osteogenic capability of BMP9. OBJECTIVE: The objective of the study was to explore the role of LCN2 in regulating the BMP9-initialized osteogenic and adipogenic differentiation in mouse embryonic fibroblasts (MEFs), and clarify the possible underlying mechanism. METHODS: Histochemical stain, western blot, real-time PCR, laser confocal, immunoprecipitation, cranial defect repair, and fetal limb culture assays were used to evaluate the effects of LCN2 on BMP9-induced osteogenic and adipogenic differentiation, as well as Wnt/ß-catenin signaling. RESULTS: LCN2 was down-regulated by BMP9. The BMP9-induced osteogenic markers were inhibited by LCN2 overexpression, but the adipogenic markers were increased; LCN2 knockdown exhibited opposite effects. Similar results were found in bone defect repair and fetal limb culture tests. The level of ß-catenin nucleus translocation was found to be reduced by LCN2 overexpression, but increased by LCN2 knockdown. The inhibitory effect of LCN2 overexpression on the osteogenic capability of BMP9 was reversed by ß-catenin overexpression; whereas, the effect of LCN2 knockdown on promoting BMP9 osteogenic potential was almost eliminated by ß-catenin knockdown. LCN2 could bind with LRP6 specifically, and the inhibitory effect of LCN2 on the osteogenic potential of BMP9 could not be enhanced by LRP6 knockdown. CONCLUSION: LCN2 inhibits the BMP9-induced osteogenic differentiation but promotes its adipogenic potential in MEFs, which may be partially mediated by reducing Wnt/ß-catenin signaling via binding with LRP6.
Assuntos
Fator 2 de Diferenciação de Crescimento , Osteogênese , Animais , Camundongos , Fator 2 de Diferenciação de Crescimento/metabolismo , Fator 2 de Diferenciação de Crescimento/farmacologia , beta Catenina/metabolismo , Fibroblastos , Via de Sinalização Wnt , Diferenciação CelularRESUMO
Interleukin 6 (IL-6) plays a dual role in regulating bone metabolism, although the concrete mechanism is unclear. Bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic inducers, and a promising alternative for bone tissue engineering. The relationship between IL-6 and BMP9 in osteogenic differentiation remains to be elucidated, and the osteoblastic potential of BMP9 needs to be enhanced to overcome certain shortcomings of BMP9. In this study, we used real-time PCR, western blot, immunofluorescent stain, fetal limb culture and cranial defects repair model to explore the IL-6 role in BMP9-induced osteogenic differentiation in mouse embryonic fibroblasts (MEFs). We found that the rat serum level of IL-6 was increased in the dexamethasone-induced osteoporosis model, and IL-6 expression was detectable in several progenitor cells and MEFs. BMP9 upregulated IL-6 in MEFs, and the BMP9-induced osteoblastic markers were elevated by IL-6, but reduced by IL-6 knockdown. BMP9 and/or IL-6 both activated mTOR, and the IL-6 effect on BMP9-induced osteoblastic markers and bone formation were reduced greatly by mTOR inhibition. Raptor was up-regulated by IL-6 and/or BMP9 specifically, and the osteoblastic markers induced by IL-6 and/or BMP9 were reduced by Raptor knockdown. Meanwhile, Stat-3 was activated by IL-6 and/or BMP9, and the increase of Raptor or osteoblastic markers by IL-6 and/or BMP9 were reduced by Stat-3 inhibition. The Raptor promoter activity was regulated by p-Stat-3. Our finding suggested that IL-6 can promote the BMP9 osteoblastic potential, which may be mediated through activating Stat-3/mTORC1 pathway.
Assuntos
Fator 2 de Diferenciação de Crescimento , Interleucina-6 , Animais , Camundongos , Ratos , Diferenciação Celular , Fibroblastos/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Interleucina-6/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Osteogênese , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: All-trans retinoic acid (ATRA) promotes the osteogenic differentiation induced by bone morphogenetic protein 9 (BMP9), but the intrinsic relationship between BMP9 and ATRA keeps unknown. Herein, we investigated the effect of Cyp26b1, a critical enzyme of ATRA degradation, on the BMP9-induced osteogenic differentiation in mesenchymal stem cells (MSCs), and unveiled possible mechanism through which BMP9 regulates the expression of Cyp26b1. METHODS: ATRA content was detected with ELISA and HPLC-MS/MS. PCR, Western blot, and histochemical staining were used to assay the osteogenic markers. Fetal limbs culture, cranial defect repair model, and micro-computed tomographic were used to evaluate the quality of bone formation. IP and ChIP assay were used to explore possible mechanism. RESULTS: We found that the protein level of Cyp26b1 was increased with age, whereas the ATRA content decreased. The osteogenic markers induced by BMP9 were increased by inhibiting or silencing Cyp26b1 but reduced by exogenous Cyp26b1. The BMP9-induced bone formation was enhanced by inhibiting Cyp26b1. The cranial defect repair was promoted by BMP9, which was strengthened by silencing Cyp26b1 and reduced by exogenous Cyp26b1. Mechanically, Cyp26b1 was reduced by BMP9, which was enhanced by activating Wnt/ß-catenin, and reduced by inhibiting this pathway. ß-catenin interacts with Smad1/5/9, and both were recruited at the promoter of Cyp26b1. CONCLUSIONS: Our findings suggested the BMP9-induced osteoblastic differentiation was mediated by activating retinoic acid signalling, viadown-regulating Cyp26b1. Meanwhile, Cyp26b1 may be a novel potential therapeutic target for the treatment of bone-related diseases or accelerating bone-tissue engineering.
Assuntos
Fator 2 de Diferenciação de Crescimento , Células-Tronco Mesenquimais , Via de Sinalização Wnt , beta Catenina/metabolismo , Fator 2 de Diferenciação de Crescimento/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Espectrometria de Massas em Tandem , Tretinoína/farmacologiaRESUMO
OBJECTIVE: Adenovirus expressing small interfering RNA (siRNA)-targeting BMPR-IB was locally administered into the air pouch of mice to improve bone resorption induced by ultra-high molecular weight polyethylene (UHMWPE) particles. METHOD: Air pouches were established on the back of BALB/c mice, followed by the surgical introduction of a section of calvaria from a syngeneic mouse donor. The bone-implanted pouches were stimulated with the UHMWPE suspension. UHMWPE-containing mice were divided into three study groups to receive injections of adenovirus expressing BMPR-IB siRNA (BMPR-IB group), adenovirus expressing missense siRNA, and virus-free culture medium (control group) into the pouches, respectively. The tissues were harvested at 14 days after the treatment for molecular and histological analyses. RESULTS: Adenovirus-mediated BMPR-IB siRNA treatment significantly improved UHMWPE particle-induced bone resorption, reduced TRAP and RANK gene and protein expression levels, and diminished the number of TRAP-positive cells. Furthermore, the BMPR-IB siRNA inhibited osteoclast differentiation by targeting osteoblast for the induction of osteoprotegerin formation and downregulation of receptor for activation of nuclear factor-κB ligand production. CONCLUSIONS: This study suggested that loss of bone morphogenetic protein signaling by BMPR-IB siRNA directs osteoblasts to decrease bone destruction in part by downregulating osteoclastogenesis through the receptor for activation of nuclear factor-κB ligand-osteoprotegerin pathway. Local administration of adenovirus expressing siRNA-targeting BMPR-IB may be a feasible and effective therapeutic candidate to treat or prevent wear debris-associated osteolysis.
Assuntos
Adenoviridae , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/biossíntese , Reabsorção Óssea/metabolismo , Vetores Genéticos , Polietileno/efeitos adversos , RNA Interferente Pequeno , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Transplante Ósseo , Modelos Animais de Doenças , Inativação Gênica , Camundongos , Camundongos Endogâmicos BALB C , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Polietileno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transplante IsogênicoRESUMO
UNLABELLED: Craniofacial defect repair is often limited by a finite supply of available autologous tissue (ie, bone) and less than ideal alternatives. Therefore, other methods to produce bony healing must be explored. Several studies have demonstrated that low-frequency pulsed electromagnetic field (PEMF) stimulation (ie, 5-30 Hz) of osteoblasts enhances bone formation. The current study was designed to investigate whether a Food and Drug Administration-approved, high-frequency PEMF-emitting device is capable of inducing osteogenic differentiation of osteoprogenitor cells. Osteoprogenitor cells (commercially available C3H10T1/2 and mouse calvarial) in complete Dulbecco modified Eagle medium were continuously exposed to PEMF stimulation delivered by the ActiPatch at a frequency of 27.1 MHz. Markers of cellular proliferation and early, intermediate, and terminal osteogenic differentiation were measured and compared with unstimulated controls. All experiments were performed in triplicate. High-frequency PEMF stimulation increases alkaline phosphatase activity in both cell lines. In addition, high-frequency PEMF stimulation augments osteopontin and osteocalcin expression as well as mineral nodule formation in C3H10T1/2 cells, indicating late and terminal osteogenic differentiation, respectively. Cellular proliferation, however, was unaffected by high-frequency PEMF stimulation. Mechanistically, high-frequency PEMF-stimulated osteogenic differentiation is associated with elevated mRNA expression levels of osteogenic bone morphogenetic proteins in C3H10T1/2 cells. Our findings suggest that high-frequency PEMF stimulation of osteoprogenitor cells may be explored as an effective tissue engineering strategy to treat critical-size osseous defects of the craniofacial and axial skeleton. ABBREVIATIONS: ALP, alkaline phosphatase; BMP, bone morphogenetic protein; ERK-1, extracellular signal-regulated kinase 1; iCALs, immortalized calvarial cells; IHC, immunohistochemical; MAP, mitogen-activated protein; MSC, mesenchymal stem cell; OCN, osteocalcin; OPN, osteopontin; p38α, p38-reactivating kinase; PBS, phosphate-buffered saline; PEMF, pulsed electromagnetic field.
Assuntos
Diferenciação Celular/efeitos da radiação , Campos Eletromagnéticos , Osteoblastos/efeitos da radiação , Crânio/citologia , Fosfatase Alcalina/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Técnicas Imunoenzimáticas , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteopontina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
As one of the most common malignancies, colon cancer is initiated by abnormal activation of the Wnt/ß-catenin pathway. Although the treatment options have increased for some patients, overall progress has been modest. Thus, there is a great need to develop new treatments. We have found that bisbenzylisoquinoline alkaloid tetrandrine (TET) exhibits anticancer activity. TET is used as a calcium channel blocker to treat hypertensive and arrhythmic conditions in Chinese medicine. Here, we investigate the molecular basis underlying TET's anticancer activity. We compare TET with six chemotherapy drugs in eight cancer lines and find that TET exhibits comparable anticancer activities with camptothecin, vincristine, paclitaxel, and doxorubicin, and better than that of 5-fluorouracil (5-FU) and carboplatin. TET IC50 is ≤5 µM in most of the tested cancer lines. TET exhibits synergistic anticancer activity with 5-FU and reduces migration and invasion capabilities of HCT116 cells. Furthermore, TET induces apoptosis and inhibits xenograft tumor growth of colon cancer. TET treatment leads to a decrease in ß-catenin protein level in xenograft tumors, which is confirmed by T-cell factor/lymphocyte enhancer factor and c-Myc reporter assays. It is noteworthy that HCT116 cells with allelic oncogenic ß-catenin deleted are less sensitive to TET-mediated inhibition of proliferation, viability, and xenograft tumor growth. Thus, our findings strongly suggest that the anticancer effect of TET in colon cancer may be at least in part mediated by targeting ß-catenin activity. Therefore, TET may be used alone or in combination as an effective anticancer agent.
Assuntos
Benzilisoquinolinas/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Transplante Heterólogo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.