Background Although various systemic and local factors such as abnormal carbohydrate or calcium metabolism, aging, and hormonal disturbances have been suggested as causes of ossification of the posterior longitudinal ...Background Although various systemic and local factors such as abnormal carbohydrate or calcium metabolism, aging, and hormonal disturbances have been suggested as causes of ossification of the posterior longitudinal ligament （OPLL）, the etiology of OPLL is not fully understood. The purpose of this study was to investigate whether bone morphogenetic protein （BMP）-2 is a candidate gene to modify the susceptibility of OPLL and the mechanism of signal transduction in ossification. Methods A total of 420 OPLL patients and 506 age- and sex-matched controls were studied. The complete coding sequence of the human BMP-2 gene was analyzed using polymerase chain reaction （PCR） and direct sequencing. All single nucleotide polymorphisms （SNPs） were detected and genotyped. BMP-2 expression vectors containing positive polymorphisms were constructed and transfected into the C3H10T1/2 cells. The expression of BMP-2 and the Smad signal pathway in positive cell clones were detected by Western blotting. The alkaline phosphatase （ALP） activity was determined using quantitative detection kits. Results The frequencies for the 109T〉G and 570A〉T polymorphisms were different between the case and control groups. The ＂TG＂ genotype in 109T〉G polymorphism is associated with the occurrence of OPLL, the frequency of the ＂G＂ allele is significantly higher in patients with OPLL than in control subjects （P 〈0.001）. The ＂AT＂ genotype in 570A〉T polymorphism is associated with the occurrence of OPLL, the frequency of the ＂T＂ allele is significantly higher in patients with OPLL than in control subjects （P=0.005）. Western blotting analysis revealed that the expression of P-Smadl/5/8 protein transfected by wild-type or mutant expression vectors were significantly higher than control groups （P 〈0.05）, but there was no statistical difference in each experimental group （P 〉0.05）. The expression of Smad4 protein transfected by wild-type or mutant expression vectors was significantly higher than control groups （P 〈0.05）. The expression of Smad4 protein transfected by pcDNA3.1-BMP2 （109G） and pcDNA3.1-BMP2 （109G, 570T） was significantly higher than the other experimental groups （P 〈0.05）. The increase in ALP activity has been detected in pcDNA3.1-BMP2 （109G） and pcDNA3.1-BMP2 （109G, 570T） transfected cells up to 4 weeks after stable transfection. Activity of ALP was （30.56±0.46） nmol.min^-1.mg^-1 protein and （29.62±0.68） nmol.min^-1.mg^-1 protein, respectively. This was statistically different compared with the other experimental groups （P 〈0.05）. Conclusions BMP-2 is the predisposing gene of OPLL. The ＂TG＂ genotype in the 109T〉G and the ＂AT＂ genotype in the 570A〉T polymorphisms are associated with the occurrence of OPLL. The 109T〉G polymorphism in exon-2 of the BMP-2 gene is positively associated with the level of Smad4 protein expression and the activity of ALP. The Smad mediated sicjnaling pathway plays an important role durincl the Datholoqical process of OPLL induced by SNPs of BMP-2 aene.展开更多
Peptides from Pilose antler aqueous extract(PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs). However, the underlying molecular mechanisms are not w...Peptides from Pilose antler aqueous extract(PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE’s effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction(Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase(ALP), runt-related transcription factor 2(Runx2), osteocalcin(OCN), bone morphogenetic protein-2(BMP-2), and collagen I(COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200μg·mL^-1 showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.展开更多
Mesenchymal stem cells(MSCs)are ubiquitously-existing multipotent progenitors that can self-renew and differentiate into multiple lineages including osteocytes,chondrocytes,adipocytes,tenocytes and myocytes.MSCs repre...Mesenchymal stem cells(MSCs)are ubiquitously-existing multipotent progenitors that can self-renew and differentiate into multiple lineages including osteocytes,chondrocytes,adipocytes,tenocytes and myocytes.MSCs represent one of the most commonly-used adult progenitors and serve as excellent progenitor cell models for investigating lineagespecific differentiation regulated by various cellular signaling pathways,such as bone morphogenetic proteins(BMPs).As members of TGFb superfamily,BMPs play diverse and important roles in development and adult tissues.At least 14 BMPs have been identified in mammals.Different BMPs exert distinct but overlapping biological functions.Through a comprehensive analysis of 14 BMPs in MSCs,we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of MSCs.Nonetheless,a global mechanistic view of BMP signaling in regulating the proliferation and differentiation of MSCs remains to be fully elucidated.Here,we conducted a comprehensive transcriptomic profiling in the MSCs stimulated by 14 types of BMPs.Hierarchical clustering analysis classifies 14 BMPs into three subclusters:an osteo/chondrogenic/adipogenic cluster,a tenogenic cluster,and BMP3 cluster.We also demonstrate that six BMPs(e.g.,BMP2,BMP3,BMP4,BMP7,BMP8,and BMP9)can induce ISmads effectively,while BMP2,BMP3,BMP4,BMP7,and BMP11 up-regulate Smad-independent MAP kinase pathway.Furthermore,we show that many BMPs can upregulate the expression of the signal mediators of Wnt,Notch and PI3K/AKT/mTOR pathways.While the reported transcriptomic changes need to be further validated,our expression profiling represents the first-of-its-kind to interrogate a comprehensive transcriptomic landscape regulated by the 14 types of BMPs in MSCs.展开更多
Objective： To study the mechanism of Huogu I Formula （活骨I方） in treating osteonecrosis of femoral head. Methods： Forty-eight healthy female Leghorn chickens were randomly divided into control group, model group ...Objective： To study the mechanism of Huogu I Formula （活骨I方） in treating osteonecrosis of femoral head. Methods： Forty-eight healthy female Leghorn chickens were randomly divided into control group, model group and Huogu I group, and each group consisted of 16 chickens. At the meantime of model establishment, chickens of the Huogu I group were administrated with decoction, while the model and control group with distilled water by gavage. At the 8th and 16th week after medication, blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations. Specifically, expressions of bone morphogenetic protein-2 （BMP2）, transforming growth factor beta1 （TGFβ1）, Smad4 and Smad7 were evaluated by immunohistochemistry, while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand （OPG/RANKL） mRNA was detected by in situ hybridization. Results： Compared with the control group, serum levels of total cholesterol （TG）, triglyceride （TG） and low-density lipoprotein cholesterol （LDL-C） in the model group rose significantly. Positive cell counting of BMP2, TGF 131, Smad4 and OPG in femoral head of the model group dropped prominently. Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group, levels of TC, TG and LDL-C in Huogu I group reduced significantly. Positive cell counting of BMP2, TGFβ1, Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly. Especially at the 8th week, these variations were more significant. Conclusion： Huogu I Formula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2, TGFβ1, Smads and OPG/RANKL of osteoclast in femoral head.展开更多
基金This research was supported by a grant from the National Natural Science Foundation of China （No. 81071486）.Acknowledgments： The authors thank the DNA donors for making this study possible.
文摘Background Although various systemic and local factors such as abnormal carbohydrate or calcium metabolism, aging, and hormonal disturbances have been suggested as causes of ossification of the posterior longitudinal ligament （OPLL）, the etiology of OPLL is not fully understood. The purpose of this study was to investigate whether bone morphogenetic protein （BMP）-2 is a candidate gene to modify the susceptibility of OPLL and the mechanism of signal transduction in ossification. Methods A total of 420 OPLL patients and 506 age- and sex-matched controls were studied. The complete coding sequence of the human BMP-2 gene was analyzed using polymerase chain reaction （PCR） and direct sequencing. All single nucleotide polymorphisms （SNPs） were detected and genotyped. BMP-2 expression vectors containing positive polymorphisms were constructed and transfected into the C3H10T1/2 cells. The expression of BMP-2 and the Smad signal pathway in positive cell clones were detected by Western blotting. The alkaline phosphatase （ALP） activity was determined using quantitative detection kits. Results The frequencies for the 109T〉G and 570A〉T polymorphisms were different between the case and control groups. The ＂TG＂ genotype in 109T〉G polymorphism is associated with the occurrence of OPLL, the frequency of the ＂G＂ allele is significantly higher in patients with OPLL than in control subjects （P 〈0.001）. The ＂AT＂ genotype in 570A〉T polymorphism is associated with the occurrence of OPLL, the frequency of the ＂T＂ allele is significantly higher in patients with OPLL than in control subjects （P=0.005）. Western blotting analysis revealed that the expression of P-Smadl/5/8 protein transfected by wild-type or mutant expression vectors were significantly higher than control groups （P 〈0.05）, but there was no statistical difference in each experimental group （P 〉0.05）. The expression of Smad4 protein transfected by wild-type or mutant expression vectors was significantly higher than control groups （P 〈0.05）. The expression of Smad4 protein transfected by pcDNA3.1-BMP2 （109G） and pcDNA3.1-BMP2 （109G, 570T） was significantly higher than the other experimental groups （P 〈0.05）. The increase in ALP activity has been detected in pcDNA3.1-BMP2 （109G） and pcDNA3.1-BMP2 （109G, 570T） transfected cells up to 4 weeks after stable transfection. Activity of ALP was （30.56±0.46） nmol.min^-1.mg^-1 protein and （29.62±0.68） nmol.min^-1.mg^-1 protein, respectively. This was statistically different compared with the other experimental groups （P 〈0.05）. Conclusions BMP-2 is the predisposing gene of OPLL. The ＂TG＂ genotype in the 109T〉G and the ＂AT＂ genotype in the 570A〉T polymorphisms are associated with the occurrence of OPLL. The 109T〉G polymorphism in exon-2 of the BMP-2 gene is positively associated with the level of Smad4 protein expression and the activity of ALP. The Smad mediated sicjnaling pathway plays an important role durincl the Datholoqical process of OPLL induced by SNPs of BMP-2 aene.
基金supported by the National Natural Science Foundation of China(No.81473314)
文摘Peptides from Pilose antler aqueous extract(PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE’s effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction(Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase(ALP), runt-related transcription factor 2(Runx2), osteocalcin(OCN), bone morphogenetic protein-2(BMP-2), and collagen I(COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200μg·mL^-1 showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.
文摘目的探索成牙骨质细胞OCCM-30中骨形态发生蛋白2(BMP2)对硬化蛋白(SOST)表达的调控机制。方法用2种质量浓度的BMP2(50、100 ng·mL^(-1))处理成牙骨质OCCM-30细胞3、5、7 d,相同体积的PBS液为对照组,采用实时荧光定量聚合酶链反应(RT-PCR)、免疫印迹法检测SOST m RNA和蛋白的表达情况。将OCCM-30细胞分为5组:空白对照组、BMP2组、BMP2+dorsomorphin组、BMP2+SB202190组、BMP2+PD98059组,根据分组分别加入100 ng·mL^(-1)的BMP2和相应的试剂共培养,于3、5 d时检测SOST m RNA和蛋白的表达情况。结果 100 ng·mL^(-1)BMP2对SOST表达的上调作用强于50 ng·mL^(-1) BMP2,且有时间依赖性(P<0.05)。BMP2+dorsomorphin组、BMP2+SB202190组、BMP2+PD98059组的SOST m RNA水平和蛋白质水平均降低,其中BMP2+dorsomorphin组降低最明显(P<0.05)。结论成牙骨质细胞中BMP2主要是通过Smad信号通路介导上调SOST的表达。
文摘Mesenchymal stem cells(MSCs)are ubiquitously-existing multipotent progenitors that can self-renew and differentiate into multiple lineages including osteocytes,chondrocytes,adipocytes,tenocytes and myocytes.MSCs represent one of the most commonly-used adult progenitors and serve as excellent progenitor cell models for investigating lineagespecific differentiation regulated by various cellular signaling pathways,such as bone morphogenetic proteins(BMPs).As members of TGFb superfamily,BMPs play diverse and important roles in development and adult tissues.At least 14 BMPs have been identified in mammals.Different BMPs exert distinct but overlapping biological functions.Through a comprehensive analysis of 14 BMPs in MSCs,we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of MSCs.Nonetheless,a global mechanistic view of BMP signaling in regulating the proliferation and differentiation of MSCs remains to be fully elucidated.Here,we conducted a comprehensive transcriptomic profiling in the MSCs stimulated by 14 types of BMPs.Hierarchical clustering analysis classifies 14 BMPs into three subclusters:an osteo/chondrogenic/adipogenic cluster,a tenogenic cluster,and BMP3 cluster.We also demonstrate that six BMPs(e.g.,BMP2,BMP3,BMP4,BMP7,BMP8,and BMP9)can induce ISmads effectively,while BMP2,BMP3,BMP4,BMP7,and BMP11 up-regulate Smad-independent MAP kinase pathway.Furthermore,we show that many BMPs can upregulate the expression of the signal mediators of Wnt,Notch and PI3K/AKT/mTOR pathways.While the reported transcriptomic changes need to be further validated,our expression profiling represents the first-of-its-kind to interrogate a comprehensive transcriptomic landscape regulated by the 14 types of BMPs in MSCs.
基金Supported by the National Natural Foundation of China(No. 81173417 and 30472131)
文摘Objective： To study the mechanism of Huogu I Formula （活骨I方） in treating osteonecrosis of femoral head. Methods： Forty-eight healthy female Leghorn chickens were randomly divided into control group, model group and Huogu I group, and each group consisted of 16 chickens. At the meantime of model establishment, chickens of the Huogu I group were administrated with decoction, while the model and control group with distilled water by gavage. At the 8th and 16th week after medication, blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations. Specifically, expressions of bone morphogenetic protein-2 （BMP2）, transforming growth factor beta1 （TGFβ1）, Smad4 and Smad7 were evaluated by immunohistochemistry, while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand （OPG/RANKL） mRNA was detected by in situ hybridization. Results： Compared with the control group, serum levels of total cholesterol （TG）, triglyceride （TG） and low-density lipoprotein cholesterol （LDL-C） in the model group rose significantly. Positive cell counting of BMP2, TGF 131, Smad4 and OPG in femoral head of the model group dropped prominently. Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group, levels of TC, TG and LDL-C in Huogu I group reduced significantly. Positive cell counting of BMP2, TGFβ1, Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly. Especially at the 8th week, these variations were more significant. Conclusion： Huogu I Formula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2, TGFβ1, Smads and OPG/RANKL of osteoclast in femoral head.
