Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells

Bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta (TGF-beta) superfamily, is able to induce osteoblastic differentiation of C2C12 cells. Both Smad and mitogen-activated protein kinase (MAPK) pathways are essential components of the TGF-beta superfamily signaling machinery. Although Smads have been demonstrated to participate in the BMP-2-induced osteoblastic differentiation of C2C12 cells, the role of MAPK has not been addressed. This report shows that BMP-2 activates ERK and p38, but not JNK, in C2C12 cells. Pretreatment of cells with the p38 inhibitor, SB203580, dramatically reduced BMP-2-induced expression of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin (OC). Nevertheless, overexpression of MKK3, a protein kinase that phosphorylates and activates p38, failed to induce ALP or OC expression in the absence of BMP-2, indicating that p38 activation is necessary but not sufficient for the acquisition of the osteoblast phenotype by these cells. Although ALP induction was increased slightly in the presence of PD-98059, a selective inhibitor of the ERK cascade, this compound significantly inhibited both steady-state and BMP-2-induced OC RNA levels. Our results indicate that p38 and ERK cascades play a crucial role in the osteoblast differentiation of C2C12 cells mediated by BMP-2.     

Role of N-cadherin and protein kinase C in osteoblast gene activation induced by the S252W fibroblast growth factor receptor 2 mutation in Apert craniosynostosis.

Apert (Ap) syndrome is characterized by premature cranial suture ossification caused by fibroblast growth factor receptor 2 (FGFR-2) mutations. We studied the role of cadherins and signaling events in the phenotypic alterations induced by the Ap FGFR-2 S252W mutation in mutant immortalized fetal human calvaria osteoblasts. The FGFR-2 mutation caused increased expression of the osteoblast markers alkaline phosphatase (ALP), type 1 collagen (COLIA1), and osteocalcin (OC) in long-term culture. The mutation also increased cell-cell aggregation, which was suppressed by specific neutralizing anti-N- and anti-E-cadherin antibodies. Mutant osteoblasts showed increased N- and E-cadherin, but not N-cell adhesion molecule (N-CAM) messenger RNA (mRNA) and protein levels. This was confirmed in vivo by the abundant immunoreactive N- and E-cadherins in preosteoblasts in the Ap suture whereas N-CAM and alpha- and beta-catenins were unaffected. Neutralizing anti-N-cadherin antibody or N-cadherin antisense (AS) oligonucleotides but not anti-E-cadherin antibody or AS reduced ALP activity as well as ALP, COLIA1, and OC mRNA overexpression in mutant osteoblasts. Analysis of signal transduction revealed increased phospholipase Cgamma (PLCgamma) and protein kinase Calpha (PKCalpha) phosphorylation and increased PKC activity in mutant cells in basal conditions. Inhibition of PKC by calphostin C or the PKCalpha-specific inhibitor G?6976 suppressed the increased N-cadherin mRNA and protein levels as well as the overexpression of ALP, COLIA1, and OC mRNA in mutant cells. Thus, N-cadherin plays a role in the activation of osteoblast differentiation marker genes in mutant osteoblasts and PKCalpha signaling appears to be involved in the increased N-cadherin and osteoblast gene expression induced by the S252W FGFR-2 mutation in human osteoblasts.     

Climbing exercise enhances osteoblast differentiation and inhibits adipogenic differentiation with high expression of PTH/PTHrP receptor in bone marrow cells

We developed previously a mouse voluntary climbing exercise model as a physiological mechanical loading model and reported that climbing exercise increased bone formation, but its effect on adipogenesis is unknown. We assessed the effects of loading and PTH/PTHrP receptor (PTHR1) on bone marrow adipocyte differentiation in relation with osteoblast differentiation. 8-week-old C57BL/6J male mice were divided into ground control (GC) and climbing exercise (EX) group. Mice were housed in 100-cm towers and climbed up toward a bottle placed at the top of the cage to drink water. The values of bone volume and osteoblast number were significantly higher while those of marrow adipocyte volume and number were significantly lower in the 28dayEX group than 28dayGC group. The mRNA expression levels of adipocyte differentiation genes CCAAT/enhancer-binding proteins (C/EBP) beta and delta were lower in 4dayEX mice, while the adipocyte specific genes fatty acid binding protein (aP2) and phosphoenolpyruvate carboxykinase (PEPCK) expressions were lower in 7dayEX mice. In primary bone marrow cell cultures, the number of alkaline phosphatase-positive colony forming units-fibroblastic (ALP+ CFU-f) and Oil-red-O-positive cells were both increased in the 4dayEX group. Climbing exercise transiently increases both osteogenic and adipogenic potential in bone marrow stromal cells, and inhibits terminal adipocyte differentiation and promotes osteoblast differentiation. Immunoreactivity for the PTHR1 was intense on osteoblastic cell lineage in the endosteal tibial metaphysis. PTHR1 mRNA expression was increased in 4dayEX mice and PTHR1-positive cells were increased after 7 days in the experimental group. Ex vivo addition of PTHR1 antibody decreased and that of PTHrP(1-34) increased the number of ALP+ CFU-f in bone marrow cell cultures obtained at 4 days after the exercise, while the addition of PTHR1 antibody increased and PTHrP(1-34) decreased the number of Oil-red-O-positive cells. Our results indicate that climbing exercise enhanced osteoblast differentiation and inhibited terminal differentiation of adipocyte progenitors with high expression of PTHR1 in bone marrow cells.     

BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop.

Wnt/beta-catenin signaling has recently been suggested to be involved in bone biology. The precise role of this cascade in osteoblast differentiation was examined. We show that a Wnt autocrine loop mediates the induction of alkaline phosphatase and mineralization by BMP-2 in pre-osteoblastic cells. INTRODUCTION: Loss of function of LRP5 leads to osteoporosis (OPPG syndrome), and a specific point mutation in this same receptor results in high bone mass (HBM). Because LRP5 acts as a coreceptor for Wnt proteins, these findings suggest a crucial role for Wnt signaling in bone biology. MATERIALS AND METHODS: We have investigated the involvement of the Wnt/LRP5 cascade in osteoblast function by using the pluripotent mesenchymal cell lines C3H10T1/2, C2C12, and ST2 and the osteoblast cell line MC3T3-E1. Transfection experiments were carried out with a number of elements of the Wnt/LRP5 pathway. Measuring osteoblast and adipocyte differentiation markers addressed the effect of this cascade on osteoblast differentiation. RESULTS: In mesenchymal cells, only Wnt's capable of stabilizing beta-catenin induced the expression of alkaline phosphatase (ALP). Wnt3a-mediated ALP induction was inhibited by overexpression of either Xddl, dickkopf 1 (dkk1), or LRP5deltaC, indicating that canonical beta-catenin signaling is responsible for this activity. The use of Noggin, a bone morphogenic protein (BMP) inhibitor, or cyclopamine, a Hedgehog inhibitor, revealed that the induction of ALP by Wnt is independent of these morphogenetic proteins and does not require de novo protein synthesis. In contrast, blocking Wnt/LRP5 signaling or protein synthesis inhibited the ability of both BMP-2 and Shh to induce ALP in mesenchymal cells. Moreover, BMP-2 enhanced Wntl and Wnt3a expression in our cells. In MC3T3-E1 cells, where endogenous ALP levels are maximal, antagonizing the Wnt/LRP5 pathway led to a decrease of ALP activity. In addition, overexpression of dkkl reduced extracellular matrix mineralization in a BMP-2-dependent assay. CONCLUSIONS: Our data strongly suggest that the capacity of BMP-2 and Shh to induce ALP relies on Wnt expression and the Wnt/LRP5 signaling cascade. Moreover the effects of BMP-2 on extracellular matrix mineralization by osteoblasts are mediated, at least in part, by the induction of a Wnt autocrine/paracrine loop. These results may help to explain the phenotype of OPPG patients and HBM.     

黄芩素通过BMP-2/Smad通路促进MC3T3-E1成骨作用的实验研究

目的 探讨黄芩素（BAI）对小鼠胚胎成骨细胞前体细胞（MC3T3-E1）成骨分化的作用及其分子机制。方法 将MC3T3-E1分为对照组（正常培养）和BAI组（以Baicalein处理）,在成骨分化条件培养下采用CCK-8检测BAI对MC3T3-E1细胞增殖的影响;分别以碱性磷酸酶染色（ALP）、茜素红染色（ARS）检测MC3T3-E1细胞成骨分化水平与矿化能力,实时荧光定量PCR检测成骨标志基因ALP、COL1A1、RUNX2、OSX的mRNA表达水平,通过免疫印迹法（Western-blot）检测MC3T3-E1细胞中BMP-2、Smad1、p-Smad1蛋白表达水平,通过免疫荧光技术（IF）检测RUNX2、COL1A1表达水平。结果 与对照组比较,BAI干预1 d后发现,BAI组COL1A1（P<0.001）、RUNX2（P <0.05）、OSX（P <0.05） mRNA表达水平在成骨分化中表达上升;干预3 d后发现,与对照组比较,BAI组ALP（P <0.05）、RUNX2（P <0.001）mRNA表达上升;干预7 d后发现,与对照组比较,BAI组COL1A1（P <0.05）mRNA表达水平较对照组上升,BMP-2、p-Smad1/Smad1蛋白表达水平上升（P <0.05）。免疫荧光中成骨标志蛋白RUNX2、COL1A1表达增多（P <0.05）。结论 BAI可通过激活BMP-2/Smad通路促进MC3T3-E1成骨分化。     

BMP-2基因转染的人骨髓基质干细胞复合PLA/PCL支架体外构建组织工程骨

目的　用人骨形态发生蛋白 2腺病毒表达载体 (Ad -BMP - 2 )转染的人骨髓基质干细胞 (hBMSC) ,复合PLA/PCL(聚乳酸 /聚己内酯 )生物降解支架体外构建组织工程骨。方法　用Ad -BMP - 2转染体外培养的成人BMSC ,免疫组化、原位杂交染色和蛋白印迹方法检测细胞BMP - 2的表达 ,并通过流式细胞仪和ALP活性检测分析其对细胞增殖、分化的影响。然后将转染后细胞接种到PLA/PCL支架上 ,扫描电镜观察细胞贴附、生长状况。结果　转染后 ,hBMP - 2基因在mRNA水平和蛋白水平均有表达 ;S期细胞比例和ALP活性明显增高。扫描电镜见转染细胞分布均匀 ,伸展良好。结论　Ad-BMP - 2可高效转染hBMSC ,且促进细胞增殖及成骨转化。转染后细胞在PLA/PCL上生长良好 ,BMP - 2基因治疗的组织工程骨构建成功     

Effect of simvastatin on bone morphogenetic protein-2 expression and alkaline phosphatase activity of bone marrow stromal cell]

OBJECTIVE: To study the effect of simvastatin on the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphates (ALP) activity in the primary cultured bone marrow stromal cells, and to elucidate the mechanism of the anabolic osteogenetic effect of simvastatin. METHODS: Bone marrow stromal cells in femur and tibia of adult mouse were cultured in vitro. after treated with different concentrations of simvastatin (0, 0.1, 0.2, 0.5 and 1.0 mumol/L) or recombinant human BMP-2 for 72 hours, ALP activity of bone marrow stromal cells was determined. BMP-2 expression of bone marrow stromal cells was analyzed by using immunocytochemistry and Western blotting. RESULTS: After treated with simvastatin for 72 hours, BMP-2 expression increased, while little BMP-2 expression could be observed in the control group. ALP activity also increased in a dose-dependent manner; t-test showed that ALP activity in the group which concentrations of simvastatin were 0.5 mumol/L (t = 2.35, P = 0.041), 1.0 mumol/L (t = 2.348, P = 0.041) had significant difference when compared with control group. CONCLUSION: Simvastatin lead to high expression of BMP-2 in bone marrow stromal cells, via the increased auto- or para-crine of BMP-2, and ALP activity increased. These may be parts of the mechanism on the anabolic osteogenetic effect of simvastatin.     

Expression of preosteoblast markers and Cbfa-1 and Osterix gene transcripts in stromal tumour cells of giant cell tumour of bone

In giant cell tumour of bone (GCT), mononuclear stromal cells, which represent the neoplastic component of this lesion, regulate the formation of multinucleated osteoclast-like giant cells which are the characteristic hallmark of this tumour. However, the origin of stromal tumour cells has not yet been clearly defined. In this study, we evaluated several osteoblast markers including collagen type I, bone sialoprotein (BSP), osteonectin and osteocalcin in GCT using immunohistochemical techniques. Amongst the 13 GCT specimens and 7 GCT stromal cell (GCTSC) cultures studied, majority of the GCTSC synthesized type I collagen, BSP and osteonectin proteins but did not produce the differentiated osteoblast marker, osteocalcin. We further examined the regulation of several important osteogenic genes such as Cbfa-1, osterix and osteocalcin, and regulation of ALP activity in GCTSC in culture by bone morphogenetic protein 2 (BMP-2). Real-time PCR analysis indicated that Cbfa-1, osterix and osteocalcin mRNA were present in primary cultures of GCTSC. The addition of BMP-2 upregulated Cbfa-1 and osterix gene expression within 12 h and the enhancement was still observed at 24 h. ALP activity was minimal in untreated GCTSC in cultures. The number of ALP-positive GCTSC was significantly increased following treatment with BMP-2 or combinations with beta-glycerophosphate and ascorbic acid. In contrast, BMP enhancement of osterix mRNA level and ALP activity was also seen in SaOS2 osteoblast-like cells, but not in the primary culture of normal human skin fibroblasts. In summary, our data suggest that GCT stromal tumour cells may have an osteoblastic lineage and retain the ability to differentiate into osteoblasts.     

Bone Morphogenetic Protein 2 (BMP-2) Enhances BMP-3, BMP-4, and Bone Cell Differentiation Marker Gene Expression During the Induction of Mineralized Bone Matrix Formation in Culturesof Fetal Rat Calvarial Osteoblasts

Normal bone formation is a prolonged process that is carefully regulated and involves sequential expression of growth regulatory factors by osteoblasts as they proliferate and ultimately differentiate. Since this orderly sequence of gene expression by osteoblasts suggests a cascade effect, and BMP-2 is capable of initiating and maintaining this effect, we examined the effects of BMP-2 on expression of other BMPs and compared these effects with the expression pattern of bone cell differentiation marker genes in primary cultures of fetal rat calvarial (FRC) osteoblasts. To examine the gene expression profile during bone cell differentiation and bone formation, we also examined the effects of rBMP-2 on bone formation in vivo and in vitro. rBMP-2 stimulated bone formation on the periosteal surface of mice when 500 ng/day rBMP-2 was injected subcutaneously. When rBMP-2 was added to primary cultures of FRC osteoblasts, it accelerated mineralized nodule formation in a time and concentration-dependent manner (10–40 ng/ml). rBMP-2 (40 ng/ml) enhanced BMP-3 and -4 mRNA expression during the mineralization phase of primary cultures of FRC osteoblasts. Enhancement of BMP-3 and -4 mRNA expression by rBMP-2 was associated with increased expression of bone cell differentiation marker genes, alkaline phosphatase (ALP), type I collagen, osteocalcin (OC), osteopontin (OP), and bone sialoprotein (BSP). These results suggest that BMP-2 enhances expression of other BMP genes during bone cell differentiation. BMP-2 may act in a paracrine fashion in concert with other BMPs it induces to stimulate bone cell differentiation and bone formation during remodeling. Received: 27 November 1995 / Accepted: 19 July 1996     

Dynamic expression of DKK1 protein in the process whereby Epimedium‐derived flavonoids up‐regulate osteogenic and down‐regulate adipogenic differentiation of bone marrow stromal cells in ovariectomized rats

Objective: To observe the dynamic expression of DKK1 protein in the process whereby Epimedium‐derived flavonoids (EFs) regulate the balance between osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats, and to provide experimental evidence for the mechanism of EFs in the treatment of postmenopausal osteoporosis. Methods: Bone marrow stromal cells from ovariectomized rats were separated and cultivated in osteoinductive or liquid medium for 15 days in vitro. EFs (10 µg/mL) were applied to both cultures. Alkaline phosphatase (ALP) staining, ALP activity determination, Oil Red O staining and fluorescence quantitative polymerase chain reaction were used to determine the influence of EFs on osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats. Moreover, in order to explore the exact mechanism of EFs on osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats, enzyme linked immunosorbent assay was used to determine the dynamic expression of DKK1 protein in this process. Results: EFs increased activity of ALP and mRNA expression of Runx2 (early osteoblast differentiation factor) and decreased mRNA expression of PPARγ‐2 (key factor of fat generation). Importantly, EFs down‐regulated expression of DKK1 protein in an osteogenic induction medium and inhibited up‐regulation of DKK1 protein in an adipogenic induction medium. Conclusion: EFs regulate the balance between osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats by down‐regulating expression of DKK1 protein. This may be an important molecular mechanism of EFs in the context of treatment of postmenopausal osteoporosis.     

Isolation and characterization of a mesenchymal cell line that differentiates into osteoblasts in response to BMP-2 from calvariae of GFP transgenic mice

We established the clonal mesenchymal cell line, GFP-C3 (C3), which differentiates into osteoblasts in response to BMP-2 from calvariae of newborn green fluorescence protein (GFP) transgenic mice. This cell line cultured with control medium expressed low levels of alkaline phosphatase (ALP) activity and osterix mRNA and undetectable ALP and osteocalcin mRNA. Incubation of these cells with rhBMP-2 increased ALP activity dose-dependently and induced substantial levels of ALP, osteocalcin and osterix mRNA expression. C3 cells infected with adenovirus vector encoding BMP-2 (AdBMP-2) or Runx2 (AdRunx2) showed greatly increased ALP mRNA expression in a time-dependent fashion. Transduction with AdRunx2-induced expression of ALP and osteocalcin mRNA, but not osterix mRNA by day 3. Transduction with AdBMP-2 induced apparent expression of ALP and osterix mRNA by day 1 after transduction, but induced only weak expression of osteocalcin mRNA day 3 after transduction. Transplantation of C3 cells transduced with AdBMP-2 into back subfascia in wild-type mice with a complex of poly-d,l-lactic-co-glycolic acid/gelatin sponge (PGS) generated ectopic bone formation involving GFP-positive osteoblasts and osteocytes 2 weeks after transplantation. C3 cells transduced with AdRunx2 or AdLacZ failed to induce ectopic bone formation. Transplantation of C3 cells transduced with AdBMP-2 into craniotomy defects in wild-type mice using PGS as a carrier induced bone formation 2 weeks after transplantation, and replaced defects 4 weeks after transplantation. C3 cells transduced with AdRunx2 failed to induce bone repair after transplantation into craniotomy defects. These results indicate that C3 cells retain differentiation potential into osteoblasts in response to BMP-2. They are useful tools for analyzing the process of osteoblast differentiation in vivo after transplantation.     

THSD4基因在小鼠间充质干细胞及MC3T3-E1细胞成骨分化中的作用

目的 探讨THSD4基因对小鼠间充质干细胞和MC3T3-E1细胞成骨分化的影响。方法 提取绝经后骨质疏松症患者的骨髓间充质干细胞进行基因测序分析,与骨关节炎患者的骨髓间充质干细胞进行比较,分析基因表达差异。通过提取不同分化阶段的小鼠骨髓间充质干细胞（M-BMSC）及MC3T3-E1细胞的mRNA来检测THSD4 基因以及成骨分化的标志性基因（ALP、Runx2、Osx）的表达水平。通过构建慢病毒表达载体来实现对M-BMSC及MC3T3-E1细胞中THSD4的敲减及过表达,并观察其对M-BMSC及MC3T3-E1细胞成骨分化能力的影响。结果 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且通过KEGG以及GO富集分析发现THSD4基因可能与PI3K-AKT信号通路及Wnt信号通路相关。随着成骨诱导分化时间的延长,THSD4 mRNA和成骨分化标志性基因（ALP、Runx2、Osx）mRNA在MC3T3-E1以及M-BMSC中表达量均逐渐增加。过表达THSD4可以增强MC3T3-E1细胞和M-BMSC的成骨分化能力,而敲减THSD4则减弱了MC3T3-E1细胞和M-BMSC的成骨分化能力。结论 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且THSD4基因可以增强MC3T3-E1细胞以及M-BMSC的成骨分化能力。