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Bone defects and non-union are prevalent in clinical orthopedy, and the outcomes of current treatments are often suboptimal. Bone tissue engineering offers a promising approach to treating these conditions effectively. Bone morphogenetic protein 9 (BMP9) can commit mesenchymal stem cells to osteogenic lineage, and a knowledge of the underlying mechanisms may help advance the field of bone tissue engineering. Leucine-rich repeats containing G protein-coupled receptor 4 (LGR4), a member of G protein-coupled receptors, is essential for modulating bone development. This study is aimed at investigating the impact of LGR4 on BMP9-induced osteogenesis in mesenchymal stem cells as well as the underlying mechanisms. Bone marrow stromal cells from BMP9-knockout mice exhibited diminished LGR4 expression, and exogenous LGR4 clearly restored the impaired osteogenic potency of the bone marrow stromal cells. Furthermore, LGR4 expression was increased by BMP9 in C3H10T1/2 cells. LGR4 augmented the benefits of BMP9-induced osteogenic markers and bone formation, whereas LGR4 inhibition restricted these effects. Meanwhile, the BMP9-induced lipogenic markers were increased by LGR4 inhibition. The protein levels of Raptor and p-Stat3 were elevated by BMP9. Raptor knockdown or p-Stat3 suppression attenuated the osteoblastic markers and LGR4 expression brought on by BMP9. LGR4 significantly reversed the blocking effect of Raptor knockdown or p-Stat3 suppression on the BMP9-induced osteoblastic markers. Raptor interacts with p-Stat3, and p-Stat3 activates the LGR4 promoter activity. In conclusion, LGR4 boosts BMP9 osteoblastic potency in mesenchymal stem cells, and BMP9 may up-regulate LGR4 via the mTORC1/Stat3 signal activation.
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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
Osteoporosis is a widespread bone metabolic disease characterized by reduced bone mass and bone microstructure deterioration. Ribonucleotide reductase M2 (RRM2) is a key enzyme in DNA synthesis and repair. The present study investigated the effect of RRM2 on osteogenesis of mouse embryo fibroblasts (MEFs) and its molecular mechanism. Bioinformatics analysis revealed that RRM2 expression was increased during osteogenesis of MEFs triggered by bone morphogenetic protein 9. Subsequently, MEFs were used as a mesenchymal stem cell model and osteogenic inducing medium was used to induce osteogenic differentiation. RRM2 protein expression was measured by western blotting during osteogenic differentiation induction of MEFs. RRM2 levels in MEFs were upregulated and downregulated by RRM2-overexpressing recombinant adenovirus and small interfering RNA-RRM2, respectively. Bone formation markers (RUNX family transcription factor 2, osterix, distal-less homeobox 5, collagen type I α1 chain, osteopontin and osteocalcin) were detected by reverse transcription-quantitative (RT-q) PCR and alkaline phosphatase (ALP) and Alizarin Red S staining were examined. The protein expression levels of ß-catenin and the ratio of phosphorylated (p-)GSK-3ß to GSK-3ß were detected by western blotting and the RNA expression of downstream related target genes (ß-catenin, axis inhibition protein 2 (AXIN2), transcription factor 7 like 2, lymphoid enhancer binding factor 1, c-MYC and Cyclin D1) in the Wnt/ß-catenin signaling pathway was measured by RT-qPCR. RRM2 protein expression increased as the osteogenic differentiation induction period was extended. RRM2 overexpression increased osteogenic marker RNA expression, ALP activity, bone mineralization, the protein expression levels of ß-catenin, the ratio of p-GSK-3ß to GSK-3ß and the RNA expression of downstream related target genes in the Wnt/ß-catenin signaling pathway, whereas RRM2 knockdown had the opposite effect. The findings of the present study revealed that RRM2 overexpression enhanced osteogenic differentiation, while RRM2 knockdown reduced osteogenic differentiation. RRM2 may regulate osteogenic differentiation of MEFs via the canonical Wnt/ß-catenin signaling pathway, providing a possible therapeutic target for osteoporosis.
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Tetradentate N2S2 coordination platforms are widespread in biological systems and have endowed metalloenzymes and metalloproteins with abundant reactivities and functions. However, there are only three types of N2S2 scaffolds respectively based on the bipyridine, aryl and alkyl amine derivatives, which are significantly underdeveloped for coordination chemistry. With the objective of developing a new N2S2 coordination platform to assemble a series of first-row transition metal complexes, we have designed a novel tetradentate N2S2 ligand containing a central dipyrrin donor functionalized with two thioether-substituted aryl units. Interestingly, complexation of the N2S2 ligand with the chloride salts of Ni(II), Cu(II) and Zn(II) yields various geometries with various coordination numbers. The reaction between the ligand and NiCl2 readily forms two chloride-bridged centrosymmetric dinickel complexes in which the nickel centers are hexacoordinated by an N2S2Cl2 coordination environment in distorted octahedron geometry. In contrast, metalation of the ligand with CuCl2 gives a mononuclear copper complex consisting of a pentacoordinated copper center in a trigonal bipyramidal geometry with an N2S2Cl coordination sphere. Unexpectedly, the complexation of the ligand with ZnCl2 forms a homoleptic zinc complex in which the zinc center is surrounded by an N4 coordination sphere from two dipyrrin units in a non-planar pseudo-tetrahedral geometry despite the steric hindrance of two bulk thioether-substituted aryl units. These various geometries illustrate the potential structural flexibility of this new ligand. In addition, the optical properties of these compounds were also examined. This work thus provides a new N2S2 coordination platform with geometric flexibility.
Assuntos
Cobre , Metaloproteínas , Cloretos , Cobre/química , Cristalografia por Raios X , Ligantes , Níquel/química , Sulfetos/química , Zinco/químicaRESUMO
Osteoporosis is a common systemic bone disease caused by the imbalance between osteogenic activity and osteoclastic activity. Aged women are at higher risk of osteoporosis, partly because of estrogen deficiency. However, the underlying mechanism of how estrogen deficiency affects osteoclast activity has not yet been well elucidated. In this study, GSE2208 and GSE56815 datasets were downloaded from GEO database with 25 PreH BMD women and 25 PostL BMD women in total. The RRA algorithm determined 38 downregulated DEGs and 30 upregulated DEGs. Through GO analysis, we found that downregulated DEGs were mainly enriched in myeloid cell differentiation, cytokine-related functions while upregulated DEGs enriched in immune-related biological processes; pathways like Notch signaling and MAPK activation were found in KEGG/Rectome pathway database; a PPI network which contains 66 nodes and 91 edges was constructed and three Modules were obtained by Mcode; Correlation analysis helped us to find highly correlated genes in each module. Moreover, three hub genes FOS, PTPN6, and CTSD were captured by Cytohubba. Finally, the hub genes were further confirmed in blood monocytes of ovariectomy (OVX) rats by real-time PCR assay. In conclusion, the integrative bioinformatics analysis and real-time PCR analysis were utilized to offer fresh light into the role of monocytes in premenopausal osteoporosis and identified FOS, PTPN6, and CTSD as potential biomarkers for postmenopausal osteoporosis.