The focal adhesion(FA)is the structural basis of the cell-extracellular matrix crosstalk and plays important roles in control of organ formation and function.Here we show that expression of FA protein vinculin is dram...The focal adhesion(FA)is the structural basis of the cell-extracellular matrix crosstalk and plays important roles in control of organ formation and function.Here we show that expression of FA protein vinculin is dramatically reduced in osteocytes in patients with aging-related osteoporosis.Vinculin loss severely impaired osteocyte adhesion and dendrite formation.Deleting vinculin using the mouse 10-kb Dmp1-Cre transgenic mice causes dramatic bone loss in the weight-bearing long bones and spine,but not in the skull,in both young and aged mice by impairing osteoblast formation and function without markedly affecting bone resorption.Vinculin loss impairs the anabolic response of skeleton to mechanical loading in mice.Vinculin knockdown increases,while vinculin overexpression decreases,sclerostin expression in osteocytes without impacting expression of Mef2c,a major transcriptional regulator of the Sost gene,which encodes sclerostin.Vinculin interacts with Mef2c and retains the latter in the cytoplasm.Thus,vinculin loss enhances Mef2c nuclear translocation and binding to the Sost enhancer ECR5 to promote sclerostin expression in osteocytes and reduces bone formation.Consistent with this notion,deleting Sost expression in osteocytes reverses the osteopenic phenotypes caused by vinculin loss in mice.Finally,we find that estrogen is a novel regulator of vinculin expression in osteocytes and that vinculin-deficient mice are resistant to ovariectomy-induced bone loss.Thus,we demonstrate a novel mechanism through which vinculin inhibits the Mef2c-driven sclerostin expression in osteocytes to promote bone formation.展开更多
BACKGROUND Steroid-induced avascular necrosis of the femoral head(SANFH)involves bone metabolism imbalance and lacks effective therapies.Mesenchymal stem cells(MSCs),particularly human umbilical cord MSCs(hUCMSCs),off...BACKGROUND Steroid-induced avascular necrosis of the femoral head(SANFH)involves bone metabolism imbalance and lacks effective therapies.Mesenchymal stem cells(MSCs),particularly human umbilical cord MSCs(hUCMSCs),offer promise due to their osteogenic and immunomodulatory potential.Sclerostin(SOST)inhibits bone formation,so we developed a multi-target gene silencing strategy against SOST using RNA interference.We created hUCMSCs with SOST-silenced(sh-hUCMSCs)and compared their therapeutic efficacy with unmodified hUCMSCs in SANFH mice.This study explores a novel approach to enhance osteogenesis and mitigate SANFH progression.AIM To assess the effects of sh-hUCMSCs on bone metabolism in SANFH.METHODS hUCMSCs were isolated from placental tissue and transfected with SOST-targeting short hairpin RNA plasmids.A SANFH mouse model was established through intraperitoneal injection of lipopolysaccharide(20μg/kg)followed by intramuscular methylprednisolone administration(40 mg/kg).Mice were randomized into four experimental groups(n=10/group):Sham control,SANFH(untreated),hUCMSCs-treated,and sh-hUCMSCs-treated.Micro-computed tomography was used to measure bone volume(BV),bone surface area,bone surface/BV ratio,tra-becular number,trabecular thickness,and trabecular separation.Quantification of adipocyte area by hematoxylin and eosin staining.Collagen fiber volume was assessed by Masson’s trichrome staining.Serum levels of osteopro-tegerin(OPG),receptor activator of nuclear factor kappa B(RANK),RANK ligand(RANKL),tartrate-resistant acid phosphatase,and the OPG/RANKL ratio were measured by enzyme-linked immunosorbent assay.The expression levels of alkaline phosphatase,OPG,SOST,β-catenin,peroxisome proliferator-activated receptor gamma,and CCAAT/enhancer-binding protein in bone tissue were determined by western blot analysis.RESULTS hUCMSCs and sh-hUCMSCs exhibited typical fibroblast-like morphology and high expression of MSC surface markers(CD90,CD73,CD105>98%).These cells demonstrated tri-lineage differentiation potential,confirmed by positive Alizarin Red S,Oil Red O,and Alcian Blue staining,and upregulation of lineage-specific genes.After SOST-RNA interference modification,sh-hUCMSCs showed enhanced inhibition of adipogenesis and improved bone formation in a rat model of SANFH.Histological analysis revealed reduced lipid infiltration and empty lacunae in the femoral head of the sh-hUCMSC group.Western blot showed decreased CCAAT/enhancer-binding protein and peroxisome proliferator-activated receptor gamma expression(P<0.05).Masson staining and micro-computed tomography analysis confirmed significantly increased BV,trabecular number,trabecular thickness,and reduced trabecular separation in the sh-hUCMSC group compared to unmodified MSCs and SANFH groups(P<0.05).Serum enzyme-linked immunosorbent assay showed higher OPG and lower RANK,RANKL,and tartrate-resistant acid phosphatase levels in the sh-hUCMSCs group.Western blot further confirmed upregulated alkaline phosphatase,OPG,β-catenin,and downregulated SOST expression in sh-hUCMSCs compared to controls(P<0.05).These results suggest that SOST inhibition enhances the osteogenic potential and therapeutic efficacy of hUCMSCs in SANFH.CONCLUSION sh-hUCMSCs alleviate SANFH by activating the Wnt/β-catenin signaling pathway,thereby promoting osteogenic differentiation and suppressing adipogenesis to restore bone metabolic balance.展开更多
目的探讨Sclerostin对骨形态发生蛋白2(BMP-2)诱导的人骨髓间充质干细胞(BMSCs)成骨分化的影响。方法取两位男性车祸伤致截肢患者的骨髓组织进行体外人BMSCs培养。将培养细胞随机分为三组,1组未做转染(Mock),2组转染对照siRNA,3组转染Sc...目的探讨Sclerostin对骨形态发生蛋白2(BMP-2)诱导的人骨髓间充质干细胞(BMSCs)成骨分化的影响。方法取两位男性车祸伤致截肢患者的骨髓组织进行体外人BMSCs培养。将培养细胞随机分为三组,1组未做转染(Mock),2组转染对照siRNA,3组转染Sclerostin(骨形态发生蛋白抑制剂)siRNA,均采用含有0.1μg/m L BMP-2的成骨诱导培养基培养。分别在诱导培养的第0、3、7天,采用CCK-8法检测各组细胞活性,采用Cy QUANT细胞增殖检测试剂盒检测DNA含量。在培养的第14天,采用RT-PCR方法检测骨相关基因骨钙素(OCN)、骨钙蛋白(OC)及骨桥蛋白(OPN),采用碱性磷酸酶(ALP)分析试剂盒进行ALP活性分析,采用BCA蛋白检测试剂盒检测细胞蛋白含量。结果在诱导培养的第3、7天,3组细胞增殖活性、DNA含量均高于1、2组(P均<0.05)。在诱导培养的第14天,3组OCN、OC、OPN mRNA相对表达量及蛋白含量均高于1、2组(P均<0.05);1、2、3组ALP活性分别为1.00%±0.08%、1.12%±0.09%、2.50%±0.03%,3组高于1、2组(P均<0.05)。结论抑制Sclerostin表达可以促进BMP-2诱导的人BMSCs的活性及成骨分化能力。展开更多
基金supported,in part,the Shenzhen Medical Research Funds(B2402033)the Shenzhen Fundamental Research Program(JCYJ20220818100617036)+3 种基金the National Natural Science Foundation of China Grants(82250710175,82261160395,82430078,82230081)the Guangdong Provincial Science and Technology Innovation Council Grant(2017B030301018)the National Key Research and Development Program of China Grant(2019YFA0906004)the China Postdoctoral Science Foundation(2021M691435).
文摘The focal adhesion(FA)is the structural basis of the cell-extracellular matrix crosstalk and plays important roles in control of organ formation and function.Here we show that expression of FA protein vinculin is dramatically reduced in osteocytes in patients with aging-related osteoporosis.Vinculin loss severely impaired osteocyte adhesion and dendrite formation.Deleting vinculin using the mouse 10-kb Dmp1-Cre transgenic mice causes dramatic bone loss in the weight-bearing long bones and spine,but not in the skull,in both young and aged mice by impairing osteoblast formation and function without markedly affecting bone resorption.Vinculin loss impairs the anabolic response of skeleton to mechanical loading in mice.Vinculin knockdown increases,while vinculin overexpression decreases,sclerostin expression in osteocytes without impacting expression of Mef2c,a major transcriptional regulator of the Sost gene,which encodes sclerostin.Vinculin interacts with Mef2c and retains the latter in the cytoplasm.Thus,vinculin loss enhances Mef2c nuclear translocation and binding to the Sost enhancer ECR5 to promote sclerostin expression in osteocytes and reduces bone formation.Consistent with this notion,deleting Sost expression in osteocytes reverses the osteopenic phenotypes caused by vinculin loss in mice.Finally,we find that estrogen is a novel regulator of vinculin expression in osteocytes and that vinculin-deficient mice are resistant to ovariectomy-induced bone loss.Thus,we demonstrate a novel mechanism through which vinculin inhibits the Mef2c-driven sclerostin expression in osteocytes to promote bone formation.
基金Supported by the National Natural Science Foundation of China,No.82260944the Key Research and Development Programs of Guangxi,No.2021AB09011。
文摘BACKGROUND Steroid-induced avascular necrosis of the femoral head(SANFH)involves bone metabolism imbalance and lacks effective therapies.Mesenchymal stem cells(MSCs),particularly human umbilical cord MSCs(hUCMSCs),offer promise due to their osteogenic and immunomodulatory potential.Sclerostin(SOST)inhibits bone formation,so we developed a multi-target gene silencing strategy against SOST using RNA interference.We created hUCMSCs with SOST-silenced(sh-hUCMSCs)and compared their therapeutic efficacy with unmodified hUCMSCs in SANFH mice.This study explores a novel approach to enhance osteogenesis and mitigate SANFH progression.AIM To assess the effects of sh-hUCMSCs on bone metabolism in SANFH.METHODS hUCMSCs were isolated from placental tissue and transfected with SOST-targeting short hairpin RNA plasmids.A SANFH mouse model was established through intraperitoneal injection of lipopolysaccharide(20μg/kg)followed by intramuscular methylprednisolone administration(40 mg/kg).Mice were randomized into four experimental groups(n=10/group):Sham control,SANFH(untreated),hUCMSCs-treated,and sh-hUCMSCs-treated.Micro-computed tomography was used to measure bone volume(BV),bone surface area,bone surface/BV ratio,tra-becular number,trabecular thickness,and trabecular separation.Quantification of adipocyte area by hematoxylin and eosin staining.Collagen fiber volume was assessed by Masson’s trichrome staining.Serum levels of osteopro-tegerin(OPG),receptor activator of nuclear factor kappa B(RANK),RANK ligand(RANKL),tartrate-resistant acid phosphatase,and the OPG/RANKL ratio were measured by enzyme-linked immunosorbent assay.The expression levels of alkaline phosphatase,OPG,SOST,β-catenin,peroxisome proliferator-activated receptor gamma,and CCAAT/enhancer-binding protein in bone tissue were determined by western blot analysis.RESULTS hUCMSCs and sh-hUCMSCs exhibited typical fibroblast-like morphology and high expression of MSC surface markers(CD90,CD73,CD105>98%).These cells demonstrated tri-lineage differentiation potential,confirmed by positive Alizarin Red S,Oil Red O,and Alcian Blue staining,and upregulation of lineage-specific genes.After SOST-RNA interference modification,sh-hUCMSCs showed enhanced inhibition of adipogenesis and improved bone formation in a rat model of SANFH.Histological analysis revealed reduced lipid infiltration and empty lacunae in the femoral head of the sh-hUCMSC group.Western blot showed decreased CCAAT/enhancer-binding protein and peroxisome proliferator-activated receptor gamma expression(P<0.05).Masson staining and micro-computed tomography analysis confirmed significantly increased BV,trabecular number,trabecular thickness,and reduced trabecular separation in the sh-hUCMSC group compared to unmodified MSCs and SANFH groups(P<0.05).Serum enzyme-linked immunosorbent assay showed higher OPG and lower RANK,RANKL,and tartrate-resistant acid phosphatase levels in the sh-hUCMSCs group.Western blot further confirmed upregulated alkaline phosphatase,OPG,β-catenin,and downregulated SOST expression in sh-hUCMSCs compared to controls(P<0.05).These results suggest that SOST inhibition enhances the osteogenic potential and therapeutic efficacy of hUCMSCs in SANFH.CONCLUSION sh-hUCMSCs alleviate SANFH by activating the Wnt/β-catenin signaling pathway,thereby promoting osteogenic differentiation and suppressing adipogenesis to restore bone metabolic balance.
文摘目的探讨Sclerostin对骨形态发生蛋白2(BMP-2)诱导的人骨髓间充质干细胞(BMSCs)成骨分化的影响。方法取两位男性车祸伤致截肢患者的骨髓组织进行体外人BMSCs培养。将培养细胞随机分为三组,1组未做转染(Mock),2组转染对照siRNA,3组转染Sclerostin(骨形态发生蛋白抑制剂)siRNA,均采用含有0.1μg/m L BMP-2的成骨诱导培养基培养。分别在诱导培养的第0、3、7天,采用CCK-8法检测各组细胞活性,采用Cy QUANT细胞增殖检测试剂盒检测DNA含量。在培养的第14天,采用RT-PCR方法检测骨相关基因骨钙素(OCN)、骨钙蛋白(OC)及骨桥蛋白(OPN),采用碱性磷酸酶(ALP)分析试剂盒进行ALP活性分析,采用BCA蛋白检测试剂盒检测细胞蛋白含量。结果在诱导培养的第3、7天,3组细胞增殖活性、DNA含量均高于1、2组(P均<0.05)。在诱导培养的第14天,3组OCN、OC、OPN mRNA相对表达量及蛋白含量均高于1、2组(P均<0.05);1、2、3组ALP活性分别为1.00%±0.08%、1.12%±0.09%、2.50%±0.03%,3组高于1、2组(P均<0.05)。结论抑制Sclerostin表达可以促进BMP-2诱导的人BMSCs的活性及成骨分化能力。
