The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenes...The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin(SOST) that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influencea A osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or "osteokines"from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin(OCN) secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2(LCN2) is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain.We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.展开更多
YAP(yes-associated protein) is a transcriptional factor that is negatively regulated by Hippo pathway, a conserved pathway for the development and size control of multiple organs. The exact function of YAP in bone hom...YAP(yes-associated protein) is a transcriptional factor that is negatively regulated by Hippo pathway, a conserved pathway for the development and size control of multiple organs. The exact function of YAP in bone homeostasis remains controversial. Here we provide evidence for YAP's function in promoting osteogenesis, suppressing adipogenesis, and thus maintaining bone homeostasis.YAP is selectively expressed in osteoblast(OB)-lineage cells. Conditionally knocking out Yap in the OB lineage in mice reduces cell proliferation and OB differentiation and increases adipocyte formation, resulting in a trabecular bone loss. Mechanistically, YAP interacts with β-catenin and is necessary for maintenance of nuclear β-catenin level and Wnt/β-catenin signaling. Expression of β-catenin in YAP-deficient BMSCs(bone marrow stromal cells) diminishes the osteogenesis deficit. These results thus identify YAP-β-catenin as an important pathway for osteogenesis during adult bone remodeling and uncover a mechanism underlying YAP regulation of bone homeostasis.展开更多
This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes, adipocytes and neuroc...This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes, adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced into chondrocytes, adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and detected for expressions of type II collagen, triglyceride or neuron-specific enolase and nestin. The result showed that the resuscitated cells could differentiate into chondrocytes after exposure to transforming growth factor β1 (TGF-β1), insulin-like growth factor I (IGF-I) and vitamin C (VC), and uniformly changed morphologically from a spindle-like fibroblastic appearance to a polygonal shape in three weeks. The induced cells were heterochromatic to safranin O and expressed cartilage matrix-procollagenal (II) mRNA. The resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and IGF-I showed adi-pogenesis, and lipid vacuoles accumulation was detectable after 21 d. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron specific endolase (NSE) that were special surface markers associated with neural cells at different stage. This study suggested that the resuscitated hMSCs should be still a population of pluripotential cells and that it could be used for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.展开更多
Carboxyl terminus of Hsp70-interacting protein(CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that ...Carboxyl terminus of Hsp70-interacting protein(CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal(BMS) cells derived from Chip^(-/-) mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip-deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip^(-/-) mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.展开更多
Objective To evaluate the effects of retinoic acid(RA)on expression of bone morphogenetic protein 7(BMP-7)in rat fetus with cleft palate,and the effects of RA on proliferation and apoptosis of osteoblasts.Methods All-...Objective To evaluate the effects of retinoic acid(RA)on expression of bone morphogenetic protein 7(BMP-7)in rat fetus with cleft palate,and the effects of RA on proliferation and apoptosis of osteoblasts.Methods All-trans RA(ATRA)was used to induce congenital cleft palate in Wistar rat.BMP-7 mRNA expression in maxillary bone tissue of fetal rats was measured by Northern blotting analysis.Flow cytometry and MTT assay were used to measure the apoptosis and proliferation of ATRA-treated MC-3T3-E1 cells.BMP-7 mRNA and protein expressions in ATRA-treated MC-3T3-E1 cells were detected by RT-PCR and Western blotting analysis.Results ATRA could induce cleft palate of rat fetus.The incidence rate of cleft palate induced by 100 mg/kg ATRA(45.5%)was significantly higher than 50 mg/kg ATRA(12.5%,P<0.05).BMP-7 mRNA expression decreased in maxillary bone tissue of rat fetus with cleft palate.MC-3T3-E1 cells proliferation treated with 1×10-6 mol/LATRA decreased by 60%,the cell apoptosis increased by 2 times.BMP-7 mRNA and protein levels in MC-3T3-E1 cells treated with 1×10-6 mol/L ATRA decreased by 60% and 80%,respectively,compared with ATRA-untreated cells(P<0.05).Conclusions BMP-7 may play an important role in embryonic palate development.RA may possess the ability to down-regulate cell proliferation through regulation of BMP-7 gene expression.展开更多
The effects of lanthanum （Ⅲ） on the bone resorbing activity of rabbit mature osteoclasts （OCs） in the presence of osteoblasts （OBs） were studied in vitro by measuring the number and area of absorption pits. La...The effects of lanthanum （Ⅲ） on the bone resorbing activity of rabbit mature osteoclasts （OCs） in the presence of osteoblasts （OBs） were studied in vitro by measuring the number and area of absorption pits. La（Ⅲ） at concentrations ranging from 1.00×10-5 to 1.00×10-8 mol·L-1 show no effect on mature OC number （P>0.05）. In the OC-OB co-culture systems without La（Ⅲ）, osteoblasts alone did not influence the pit number and area whether the two kinds of cells were in contact or not （P>0.05）. Under the OC-OB not-in-contact condition, the effect of La（Ⅲ） on the bone-resorbing activity of OCs was similar to that of La（Ⅲ） in the absence of OBs （P>0.05）. However, while OCs were in direct contact with OBs, the inhibitory effects of La（Ⅲ） on OCs′ bone-resorbing activity decreased at the concentrations of 1.00×10-5, 1.00×10-6 and 1.00×10-7 (mol·L-1), and the promotion effects increased at 1.00×10-8 (mol·L-1) （P<0.05）. The results suggest that direct cell-cell contact between OC and OB be essential for OBs to play their role in regulating the response of OCs to La（Ⅲ）.展开更多
It is found that the drugs for nourishing yin to reduce pathogenic fire can significantly down-regulate, and the drags for tonifying the kidney to replenish essence can up-regulate mRNA expression of the hypothalamic ...It is found that the drugs for nourishing yin to reduce pathogenic fire can significantly down-regulate, and the drags for tonifying the kidney to replenish essence can up-regulate mRNA expression of the hypothalamic GnRH, pituitary FSH, LH and osteoblastic BGP, indicating that the Chinese drugs for tonifying the kidney can regulate gene expression of the hypothalamic GnRH, pituitary FSH, LH, and osteoblastic BGP, which is possibly one of the main mechanisms of the Chinese drug for tonifying the kidney, regulating ephebic development process and improving skeletal development in sexual precocity children.展开更多
Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor(OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs ...Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor(OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2(encoded by Ptpn11) affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using "Cre-lox P"-mediated gene excision.SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1, and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, q RT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.展开更多
Objective: To investigate the effects of insulin-like growth factor Ⅱ (IGF-Ⅱ) on promoting cell proliferation, regulating levels of cellular nitric oxide (NO) and mRNA transcriptions of inducible nitric oxide syntha...Objective: To investigate the effects of insulin-like growth factor Ⅱ (IGF-Ⅱ) on promoting cell proliferation, regulating levels of cellular nitric oxide (NO) and mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells. Methods: Mouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-Ⅱ. After the cells were treated with IGF-Ⅱ at different concentrations for different time duration,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used to examine cell proliferation,and nitrate reductase method was applied to detect NO concentrations in cell culture supernatants and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to determine transcription levels of cellular iNOS and eNOS mRNAs. Results: After the MC3T3-E 1 cells were treated with IGF-Ⅱ at concentration of 1 ng/ml for 72 h, 10 and 100 ng/ml for 24,48 and 72 h respectively, all the MTT values increased (P<0.05 or P<0.01) with obvious dosage-time dependent pattern. NO levels of the MC3T3-E1 cells treated with 100 ng/ml IGF-Ⅱ for 48 h, and with 1, 10 and 100 ng/ml IGF-Ⅱ for 72 h were remarkably lower than that of the normal control, respectively (P<0.05 or P<0.01). After the cells were treated with 100 ng/ml IGF-Ⅱ for 48 h cellular iNOS mRNA levels were significantly decreased (P<0.01). But the levels of eNOS mRNA in the cells treated with each of the used IGF-Ⅱ dosages for different time duration did not show any differences compared with the normal control (P>0.05).Conclusion: IGF-Ⅱ at different concentrations could promote proliferation of mouse MC3T3-E1 cell. This cell proliferation promotion was associated with the low NO levels maintained by IGF-Ⅱ. Higher concentration of IGF-Ⅱ could down-regulate iNOS gene expression at the level of transcription but not affect transcription of eNOS mRNA, which might be one of the mechanisms for IGF-Ⅱ maintenance of the low NO levels in MC3T3-E 1 cells.展开更多
Dysregulated Wnt signaling is associated with the pathogenesis of cancers, fibrosis, and vascular diseases. Inhibition of Wnt signaling has shown efficacy in various pre-clinical models of these disorders. One of the ...Dysregulated Wnt signaling is associated with the pathogenesis of cancers, fibrosis, and vascular diseases. Inhibition of Wnt signaling has shown efficacy in various pre-clinical models of these disorders. One of the key challenges in developing targeted anti-cancer drugs is to balance efficacy with on-target toxicity. Given the crucial role Wnts play in the differentiation of osteoblasts and osteoclasts, acute inhibition of Wnt signaling is likely to affect bone homeostasis. In this study, we evaluated the skeletal effect of small molecule inhibitor of an o-acyl transferase porcupine(PORCN) that prevents Wnt signaling by blocking the secretion of all Wnts. Micro-computed tomography and histomorphometric evaluation revealed that the bones of mice treated with two structurally distinct PORCN inhibitors LGK974 and ETC-1922159(ETC-159) had loss-of-bone volume and density within 4 weeks of exposure. This decreased bone mass was associated with a significant increase in adipocytes within the bone marrow. Notably,simultaneous administration of a clinically approved anti-resorptive, alendronate, a member of the bisphosphonate family,mitigated loss-of-bone mass seen upon ETC-159 treatment by regulating activity of osteoclasts and blocking accumulation of bone marrow adipocytes. Our results support the addition of bone protective agents when treating patients with PORCN inhibitors.Mitigation of bone toxicity can extend the therapeutic utility of Wnt pathway inhibitors.展开更多
Objective: To study the effects of Shenggu injection (生骨注射液,SGI) on mRNA expression of vascular endothelia growth factor (VEGF) in rat osteoblasts in vitro and to explore its possible molecular mechanisms in prom...Objective: To study the effects of Shenggu injection (生骨注射液,SGI) on mRNA expression of vascular endothelia growth factor (VEGF) in rat osteoblasts in vitro and to explore its possible molecular mechanisms in promoting fracture healing. Methods: Rat osteoblasts cultured in vitro were stimulated with SGI according to the protocol. The expression levels of VEGF mRNA in the cells in every group were examined by reverse-transcriptase ploymerase chain reaction (RT-PCR). Results: When osteoblasts were stimulated with different concentrations of SGI for 5 days, the expression of VEGF mRNA peaked with 1 mg/ml SGI on the 5th day. When treated with 1 mg/ml SGI from the 1st to the 5th day, the expression of VEGF mRNA increased gradually with the increase of culturing time. Conclusion: SGI could promote significantly the expression of VEGF mRNA in rat osteoblasts in vitro. The levels of expression of VEGF mRNA changed along with different concentrations and stimulating time of SGI.展开更多
Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse lin...Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagen1α1 (Col1α1) promoter (Col1α1-Cre). Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Col1α1-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4Co/Co). Multiple tissue PCR of Col1α1-Cre;Smad4Co/+ mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Col1α1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Col1α1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.展开更多
To study the effects of XW630 on bone formation in overiectomized(OVX) rats and in human osteoblast like cell line TE85. [WT5”BX]Method.[WT5”BZ] Bone histomorphometric analysis was performed with undecalcified bone ...To study the effects of XW630 on bone formation in overiectomized(OVX) rats and in human osteoblast like cell line TE85. [WT5”BX]Method.[WT5”BZ] Bone histomorphometric analysis was performed with undecalcified bone sections and tetracycline intraperitoneally labeling. [WT5”BX]Results.[WT5”BZ] Compared with that of OVX rats, the static data of trabecular bone volume(TBV)/ total tissue volume(TTV), TBV/sponge bone volume(SBV) and mean trabecular plate density (MTPD) were enhanced while mean trabecular plate spacing(MTPS) decreased after treated with XW630 for 13w. The dynamic data of single labeled surface [Sfract(s)], double labeled surface[Sfract(d)],Sfract(d+1/2s),trabecular osteoid surface(TOS), and bone formation rate in tissue level (Svf) were increased and osteoid maturation period (OMP) shortened in XW630 group. In osteoblast like cells, both 3H thymidine incorporation and cell count increased after treated with XW630 for 48. Treated with XW630 for 12～18h,inducible nitric oxide synthase(iNOS) activity and cGMP content increased in time dependent manners. [WT5”BX]Conclusions.[WT5”BZ] XW630 enhanced bone activation frequency and increased trabecular connectivity, stability, and strength. The cellular mechanism related to effects of XW630 on bone formation in ovariectomized rats.展开更多
Objective: To investigate the effect of glucocorticoids on cell viability of osteoblasts and explore the cytological and molecular mechanisms of osteoblasts apoptosis induced by Glucocorticoids. Methods: Dexamethasone...Objective: To investigate the effect of glucocorticoids on cell viability of osteoblasts and explore the cytological and molecular mechanisms of osteoblasts apoptosis induced by Glucocorticoids. Methods: Dexamethasone was used to induce apoptosis of primary cultured osteoblast-like cells from skulls of suckling mouse. Surviving rate of cells, apoptosis rate, activity of Caspase-3 and DNA binding activity of nuclear factor-κB were assessed respectively. Results: It was found that Dexamethasone increased apoptosis rate of osteoblast-like cells, activated Caspase-3 while inhibited the activity of NF-κB, in a concentration-dependent and time-dependent manner. Conclusion: These results suggested that Dexamethasone induced apoptosis of primary cultured and non-transformed osteoblasts, which was Caspase-3 dependent, while NF-κB might play a protective role in the process through inhibition of Caspase-3.展开更多
基金supported in part by grants from 973 Program from the Chinese Ministry of Science and Technology (MOST) (2014CB964704 and 2015CB964503)the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB19000000)the National Natural Science Foundation of China (NSFC) (31371463, 81672119, and 81725010)
文摘The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin(SOST) that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influencea A osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or "osteokines"from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin(OCN) secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2(LCN2) is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain.We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.
基金supported in part by grants from the National Institutes of Health(AG051773)and VA(BX000838)
文摘YAP(yes-associated protein) is a transcriptional factor that is negatively regulated by Hippo pathway, a conserved pathway for the development and size control of multiple organs. The exact function of YAP in bone homeostasis remains controversial. Here we provide evidence for YAP's function in promoting osteogenesis, suppressing adipogenesis, and thus maintaining bone homeostasis.YAP is selectively expressed in osteoblast(OB)-lineage cells. Conditionally knocking out Yap in the OB lineage in mice reduces cell proliferation and OB differentiation and increases adipocyte formation, resulting in a trabecular bone loss. Mechanistically, YAP interacts with β-catenin and is necessary for maintenance of nuclear β-catenin level and Wnt/β-catenin signaling. Expression of β-catenin in YAP-deficient BMSCs(bone marrow stromal cells) diminishes the osteogenesis deficit. These results thus identify YAP-β-catenin as an important pathway for osteogenesis during adult bone remodeling and uncover a mechanism underlying YAP regulation of bone homeostasis.
基金Project supported by the Science Foundation (No. 2003C23015) and the Natural Science Foundation (No. Y204139) of Zhejiang Province, China
文摘This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes, adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced into chondrocytes, adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and detected for expressions of type II collagen, triglyceride or neuron-specific enolase and nestin. The result showed that the resuscitated cells could differentiate into chondrocytes after exposure to transforming growth factor β1 (TGF-β1), insulin-like growth factor I (IGF-I) and vitamin C (VC), and uniformly changed morphologically from a spindle-like fibroblastic appearance to a polygonal shape in three weeks. The induced cells were heterochromatic to safranin O and expressed cartilage matrix-procollagenal (II) mRNA. The resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and IGF-I showed adi-pogenesis, and lipid vacuoles accumulation was detectable after 21 d. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron specific endolase (NSE) that were special surface markers associated with neural cells at different stage. This study suggested that the resuscitated hMSCs should be still a population of pluripotential cells and that it could be used for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.
基金supported by National Institutes of Health Grants, R01 AR054465, R01 AR070222, and R01 AR070222supported by the grants of Natural Science Foundation of China (NSFC) to TW (grants No. 81301531 and 81572104)+1 种基金supported by the grant from Shenzhen Science and Technology Innovation Committee, China (grant No. JCYJ20160331114205502)the grant from Shenzhen Development and Reform Committee, China for Shenzhen Engineering Laboratory of Orthopedic Regenerative Technologies
文摘Carboxyl terminus of Hsp70-interacting protein(CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal(BMS) cells derived from Chip^(-/-) mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip-deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip^(-/-) mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.
基金Supported by National Natural Science Foundation of China(30500414)Scientific Research Project in Department of Education of Liaoning Province(05L508,20061010)
文摘Objective To evaluate the effects of retinoic acid(RA)on expression of bone morphogenetic protein 7(BMP-7)in rat fetus with cleft palate,and the effects of RA on proliferation and apoptosis of osteoblasts.Methods All-trans RA(ATRA)was used to induce congenital cleft palate in Wistar rat.BMP-7 mRNA expression in maxillary bone tissue of fetal rats was measured by Northern blotting analysis.Flow cytometry and MTT assay were used to measure the apoptosis and proliferation of ATRA-treated MC-3T3-E1 cells.BMP-7 mRNA and protein expressions in ATRA-treated MC-3T3-E1 cells were detected by RT-PCR and Western blotting analysis.Results ATRA could induce cleft palate of rat fetus.The incidence rate of cleft palate induced by 100 mg/kg ATRA(45.5%)was significantly higher than 50 mg/kg ATRA(12.5%,P<0.05).BMP-7 mRNA expression decreased in maxillary bone tissue of rat fetus with cleft palate.MC-3T3-E1 cells proliferation treated with 1×10-6 mol/LATRA decreased by 60%,the cell apoptosis increased by 2 times.BMP-7 mRNA and protein levels in MC-3T3-E1 cells treated with 1×10-6 mol/L ATRA decreased by 60% and 80%,respectively,compared with ATRA-untreated cells(P<0.05).Conclusions BMP-7 may play an important role in embryonic palate development.RA may possess the ability to down-regulate cell proliferation through regulation of BMP-7 gene expression.
基金Project supported bythe National Natural Science Foundation of China (20031010 ,20271005)
文摘The effects of lanthanum （Ⅲ） on the bone resorbing activity of rabbit mature osteoclasts （OCs） in the presence of osteoblasts （OBs） were studied in vitro by measuring the number and area of absorption pits. La（Ⅲ） at concentrations ranging from 1.00×10-5 to 1.00×10-8 mol·L-1 show no effect on mature OC number （P>0.05）. In the OC-OB co-culture systems without La（Ⅲ）, osteoblasts alone did not influence the pit number and area whether the two kinds of cells were in contact or not （P>0.05）. Under the OC-OB not-in-contact condition, the effect of La（Ⅲ） on the bone-resorbing activity of OCs was similar to that of La（Ⅲ） in the absence of OBs （P>0.05）. However, while OCs were in direct contact with OBs, the inhibitory effects of La（Ⅲ） on OCs′ bone-resorbing activity decreased at the concentrations of 1.00×10-5, 1.00×10-6 and 1.00×10-7 (mol·L-1), and the promotion effects increased at 1.00×10-8 (mol·L-1) （P<0.05）. The results suggest that direct cell-cell contact between OC and OB be essential for OBs to play their role in regulating the response of OCs to La（Ⅲ）.
文摘It is found that the drugs for nourishing yin to reduce pathogenic fire can significantly down-regulate, and the drags for tonifying the kidney to replenish essence can up-regulate mRNA expression of the hypothalamic GnRH, pituitary FSH, LH and osteoblastic BGP, indicating that the Chinese drugs for tonifying the kidney can regulate gene expression of the hypothalamic GnRH, pituitary FSH, LH, and osteoblastic BGP, which is possibly one of the main mechanisms of the Chinese drug for tonifying the kidney, regulating ephebic development process and improving skeletal development in sexual precocity children.
基金supported by NIH R21AR57156NIH R37 CA49152+4 种基金the Rhode Island Hospital Orthopaedic Foundationgrant from the Pediatric Orthopaedic Society of North AmericaArthritis National Research Foundationrecipient of Ryan Fellowshippilot award recipient from NIGMS1P20 GM119943
文摘Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor(OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2(encoded by Ptpn11) affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using "Cre-lox P"-mediated gene excision.SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1, and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, q RT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.
基金Project (No. 991103115) was supported by a grant from the Department of Zhejiang Science and Technology, China
文摘Objective: To investigate the effects of insulin-like growth factor Ⅱ (IGF-Ⅱ) on promoting cell proliferation, regulating levels of cellular nitric oxide (NO) and mRNA transcriptions of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) in mouse osteoblast-like cells. Methods: Mouse osteoblastic cell line MC3T3-E1 was selected as the effective cell of IGF-Ⅱ. After the cells were treated with IGF-Ⅱ at different concentrations for different time duration,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used to examine cell proliferation,and nitrate reductase method was applied to detect NO concentrations in cell culture supernatants and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to determine transcription levels of cellular iNOS and eNOS mRNAs. Results: After the MC3T3-E 1 cells were treated with IGF-Ⅱ at concentration of 1 ng/ml for 72 h, 10 and 100 ng/ml for 24,48 and 72 h respectively, all the MTT values increased (P<0.05 or P<0.01) with obvious dosage-time dependent pattern. NO levels of the MC3T3-E1 cells treated with 100 ng/ml IGF-Ⅱ for 48 h, and with 1, 10 and 100 ng/ml IGF-Ⅱ for 72 h were remarkably lower than that of the normal control, respectively (P<0.05 or P<0.01). After the cells were treated with 100 ng/ml IGF-Ⅱ for 48 h cellular iNOS mRNA levels were significantly decreased (P<0.01). But the levels of eNOS mRNA in the cells treated with each of the used IGF-Ⅱ dosages for different time duration did not show any differences compared with the normal control (P>0.05).Conclusion: IGF-Ⅱ at different concentrations could promote proliferation of mouse MC3T3-E1 cell. This cell proliferation promotion was associated with the low NO levels maintained by IGF-Ⅱ. Higher concentration of IGF-Ⅱ could down-regulate iNOS gene expression at the level of transcription but not affect transcription of eNOS mRNA, which might be one of the mechanisms for IGF-Ⅱ maintenance of the low NO levels in MC3T3-E 1 cells.
基金supported by an NIH grant to BOW (R01AR053293)the Van Andel Research Institutesupported in part by the National Research Foundation Singapore
文摘Dysregulated Wnt signaling is associated with the pathogenesis of cancers, fibrosis, and vascular diseases. Inhibition of Wnt signaling has shown efficacy in various pre-clinical models of these disorders. One of the key challenges in developing targeted anti-cancer drugs is to balance efficacy with on-target toxicity. Given the crucial role Wnts play in the differentiation of osteoblasts and osteoclasts, acute inhibition of Wnt signaling is likely to affect bone homeostasis. In this study, we evaluated the skeletal effect of small molecule inhibitor of an o-acyl transferase porcupine(PORCN) that prevents Wnt signaling by blocking the secretion of all Wnts. Micro-computed tomography and histomorphometric evaluation revealed that the bones of mice treated with two structurally distinct PORCN inhibitors LGK974 and ETC-1922159(ETC-159) had loss-of-bone volume and density within 4 weeks of exposure. This decreased bone mass was associated with a significant increase in adipocytes within the bone marrow. Notably,simultaneous administration of a clinically approved anti-resorptive, alendronate, a member of the bisphosphonate family,mitigated loss-of-bone mass seen upon ETC-159 treatment by regulating activity of osteoclasts and blocking accumulation of bone marrow adipocytes. Our results support the addition of bone protective agents when treating patients with PORCN inhibitors.Mitigation of bone toxicity can extend the therapeutic utility of Wnt pathway inhibitors.
文摘Objective: To study the effects of Shenggu injection (生骨注射液,SGI) on mRNA expression of vascular endothelia growth factor (VEGF) in rat osteoblasts in vitro and to explore its possible molecular mechanisms in promoting fracture healing. Methods: Rat osteoblasts cultured in vitro were stimulated with SGI according to the protocol. The expression levels of VEGF mRNA in the cells in every group were examined by reverse-transcriptase ploymerase chain reaction (RT-PCR). Results: When osteoblasts were stimulated with different concentrations of SGI for 5 days, the expression of VEGF mRNA peaked with 1 mg/ml SGI on the 5th day. When treated with 1 mg/ml SGI from the 1st to the 5th day, the expression of VEGF mRNA increased gradually with the increase of culturing time. Conclusion: SGI could promote significantly the expression of VEGF mRNA in rat osteoblasts in vitro. The levels of expression of VEGF mRNA changed along with different concentrations and stimulating time of SGI.
基金the National Natural Sci-ence Foundation of China (No. 30430350)the National Science Supporting Program (No. 2006BAI23B01-3).
文摘Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagen1α1 (Col1α1) promoter (Col1α1-Cre). Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Col1α1-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4Co/Co). Multiple tissue PCR of Col1α1-Cre;Smad4Co/+ mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Col1α1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Col1α1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.
基金This project was supported by the National Natural ScienceFoundation of ChinaNo.39430 120
文摘To study the effects of XW630 on bone formation in overiectomized(OVX) rats and in human osteoblast like cell line TE85. [WT5”BX]Method.[WT5”BZ] Bone histomorphometric analysis was performed with undecalcified bone sections and tetracycline intraperitoneally labeling. [WT5”BX]Results.[WT5”BZ] Compared with that of OVX rats, the static data of trabecular bone volume(TBV)/ total tissue volume(TTV), TBV/sponge bone volume(SBV) and mean trabecular plate density (MTPD) were enhanced while mean trabecular plate spacing(MTPS) decreased after treated with XW630 for 13w. The dynamic data of single labeled surface [Sfract(s)], double labeled surface[Sfract(d)],Sfract(d+1/2s),trabecular osteoid surface(TOS), and bone formation rate in tissue level (Svf) were increased and osteoid maturation period (OMP) shortened in XW630 group. In osteoblast like cells, both 3H thymidine incorporation and cell count increased after treated with XW630 for 48. Treated with XW630 for 12～18h,inducible nitric oxide synthase(iNOS) activity and cGMP content increased in time dependent manners. [WT5”BX]Conclusions.[WT5”BZ] XW630 enhanced bone activation frequency and increased trabecular connectivity, stability, and strength. The cellular mechanism related to effects of XW630 on bone formation in ovariectomized rats.
文摘Objective: To investigate the effect of glucocorticoids on cell viability of osteoblasts and explore the cytological and molecular mechanisms of osteoblasts apoptosis induced by Glucocorticoids. Methods: Dexamethasone was used to induce apoptosis of primary cultured osteoblast-like cells from skulls of suckling mouse. Surviving rate of cells, apoptosis rate, activity of Caspase-3 and DNA binding activity of nuclear factor-κB were assessed respectively. Results: It was found that Dexamethasone increased apoptosis rate of osteoblast-like cells, activated Caspase-3 while inhibited the activity of NF-κB, in a concentration-dependent and time-dependent manner. Conclusion: These results suggested that Dexamethasone induced apoptosis of primary cultured and non-transformed osteoblasts, which was Caspase-3 dependent, while NF-κB might play a protective role in the process through inhibition of Caspase-3.
