Bone morphogenetic protein-3b (BMP-3b) gene expression is correlated with differentiation in rat calvarial osteoblasts

BMP-3b (also called GDF-10) is a novel BMP-3-related protein recently discovered in rat femur tissue. Gene expression of BMP-3b in osteoblastic cells and its regulation by prolonged culture, BMP-2 and transforming growth factor beta1 (TGF-beta1) were examined. The BMP-3b gene was highly expressed in rat osteoblasts obtained from calvarial bones but not in the osteoblastic cell lines (MC3T3-E1 and U2-OS). BMP-3b mRNA increased during osteoblastic differentiation in prolonged culture and was associated with increased alkaline phosphatase (ALPase) activity. When BMP-2, an enhancer of ALPase activity, was added to the primary osteoblast culture, BMP-3b mRNA increased 6.9-fold after 24 h. In contrast, TGF-beta1 treatment, which suppresses ALPase activity, rapidly and completely inhibited gene expression of BMP-3b. The regulation of BMP-3 mRNA differed from that of BMP-3b, even though both proteins share 81% identity. These findings indicate that BMP-3b gene expression is regulated by osteoblastic differentiation and BMP-3b functions in highly differentiated osteoblasts.     

中胚叶叉头-1在骨成形蛋白-2诱导肌胚细胞C2C12转化成骨细胞过程中的作用

为了研究中胚叶叉头-1(MFH-1)基因在骨骼形成和细胞分化中的作用,利用基因重组、杂交瘤技术制作MFH-1单克隆抗体, 利用蛋白质印迹和RNA印迹分析观察了骨成形蛋白-2 (BMP-2)诱导小鼠肌胚细胞C2C12表达MFH-1、产生碱性磷酸酶和骨钙蛋白.小鼠肌胚细胞C2C12低水平地表达内源性MFH-1蛋白以及导入小鼠MFH-1 cDNA的人膀胱癌细胞HTB9也表达小鼠MFH-1蛋白,这种蛋白质定位于细胞核中.用BMP-2处理后, MFH-1蛋白和mRNA在C2C12细胞中的表达显著地增加.用反义MFH-1序列转染小鼠肌胚细胞C2C12可降低内源性MFH-1水平, BMP-2不能诱导导入反义MFH-1序列的肌胚细胞C2C12产生MFH-1蛋白,也不能诱导碱性磷酸酶(ALP)活性和骨钙蛋白量的增加.结果表明, BMP-2诱导的MFH-1蛋白在调节肌胚细胞C2C12向成骨细胞分化方面起关键作用.     

Metformin enhances the differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation as well as eNOS and BMP-2 expression

It is unclear whether metformin, one of the anti-hyperglycemic agents commonly used for type 2 diabetes, could affect bone formation through activation of AMP-activated protein kinase (AMPK). In order to clarify this issue, we investigated the effects of metformin on the differentiation and mineralization of osteoblastic MC3T3-E1 cells as well as intracellular signal transduction. Metformin (50 μM) significantly increased collagen-I and osteocalcin mRNA expression, stimulated alkaline phosphatase activity, and enhanced cell mineralization. Moreover, metformin significantly activated AMPK in dose- and time-dependent manners, and induced endothelial nitric oxide synthase (eNOS) and bone morphogenetic protein-2 (BMP-2) expressions. Supplementation of Ara-A (0.1 mM), a specific AMPK inhibitor, significantly reversed the metformin-induced eNOS and BMP-2 expressions. Our findings suggest that metformin can induce the differentiation and mineralization of osteoblasts via activation of the AMPK signaling pathway, and that this drug might be beneficial for not only diabetes but also osteoporosis by promoting bone formation.     

Atherogenic phospholipids attenuate osteogenic signaling by BMP-2 and parathyroid hormone in osteoblasts

Cardiovascular disease, such as atherosclerosis, has been associated with reduced bone mineral density and fracture risk. A major etiologic factor in atherogenesis is believed to be oxidized phospholipids. We previously found that these phospholipids inhibit spontaneous osteogenic differentiation of marrow stromal cells, suggesting that they may account for the clinical link between atherosclerosis and osteoporosis. Currently, anabolic agents that promote bone formation are increasingly used as a new treatment for osteoporosis. It is not known, however, whether atherogenic phospholipids alter the effects of bone anabolic agents, such as bone morphogenetic protein (BMP)-2 and parathyroid hormone (PTH). Therefore we investigated the effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) on osteogenic signaling induced by BMP-2 and PTH in MC3T3-E1 cells. Results showed that ox-PAPC attenuated BMP-2 induction of osteogenic markers alkaline phosphatase and osteocalcin. Ox-PAPC also inhibited both spontaneous and BMP-induced expression of PTH receptor. Consistently, pretreatment of cells with ox-PAPC inhibited PTH-induced cAMP production and expression of immediate early genes Nurr1 and IL-6. Results from immunofluorescence and Western blot analyses showed that inhibitory effects of ox-PAPC on BMP-2 signaling were associated with inhibition of SMAD 1/5/8 but not p38-MAPK activation. These effects appear to be due to ox-PAPC activation of the ERK pathway, as the ERK inhibitor PD98059 reversed ox-PAPC inhibitory effects on BMP-2-induced alkaline phosphatase activity, osteocalcin expression, and SMAD activation. These results suggest that atherogenic lipids inhibit osteogenic signaling induced by BMP-2 and PTH, raising the possibility that hyperlipidemia and atherogenic phospholipids may interfere with anabolic therapy.     

Heterodimeric BMP-2/7 Antagonizes the Inhibition of All-Trans Retinoic Acid and Promotes the Osteoblastogenesis

Objectives

Hypervitaminosis A and alcoholism can result in a low mineral density and compromised regenerative capacity of bone, thus delaying implant osteointegration. The inhibitory effect of all-trans retinoic acid on osteoblastogenesis is considered to be one of the mechanisms. We hypothesized that heterodimeric bone morphogenetic protein-2/7 could antagonize all-trans retinoic acid and enhance osteoblastogenesis, with an aim to accelerate and enhance bone regeneration and implant osteointegration.

Materials and Methods

We applied 5 ng/ml or 50 ng/ml bone morphogenetic protein-2/7 to restore the osteoblastogenesis of pre-osteoblasts (MC3T3-E1 cell line) that was inhibited by 1 µM all-trans retinoic acid. We evaluated the efficacy by assessing cell numbers (proliferation), alkaline phosphatase activity (a marker for early differentiation), osteocalcin (a marker for late differentiation), calcium deposition (a marker for final mineralization) and the expression of osteoblastogenic genes (such as Runx2, Collagen Ia, alkaline phosphatase and osteocalcin) at different time points.

Results

All-trans retinoic acid significantly inhibited the expression of all the tested osteoblastogenic genes and proteins except alkaline phosphatase activity. In the presence of ATRA, 50 ng/ml bone morphogenetic protein-2/7 not only completely restored but also significantly enhanced all the osteoblastogenic genes and proteins. On the 28^th day, mineralization was completely inhibited by all-trans retinoic acid. In contrast, 50 ng/ml BMP-2/7 could antagonize ATRA and significantly enhance the mineralization about 2.5 folds in comparison with the control treatment (no ATRA, no BMP2/7).

Conclusions

Heterodimeric bone morphogenetic protein-2/7 bears a promising application potential to significantly promote bone regeneration and implant osteointegration for the patients with hypervitaminosis A and alcoholism.     

BMP-6-induced osteogenic differentiation of mesenchymal cell lines is not modulated by sex steroids and resveratrol

Bone morphogenetic protein-6 (BMP-6) is a potent inducer of osteogenic differentiation and its expression is stimulated by 17beta-estradiol. The existence of a regulatory loop between sex steroids and BMP-6 is therefore reasonable to hypothesize. Here we determined whether the sex steroids 17beta-estradiol and dihydrotestosterone, and the phytoestrogen resveratrol can modulate BMP-6-induced alkaline phosphatase activity and osteocalcin expression. Mesenchymal cells of murine (osteoblastic MC3T3-E1 cells, preadipogenic ST2 cells, prechondrogenic ATDC5 cell) and human origin (osteosarcoma SaOS and HOS cells, primary bone marrow stromal cells) were cultured in the presence of recombinant BMP-6 under serum-free conditions. BMP-6 dose-, and time-dependently increased alkaline phosphatase activity in murine cell lines, but not in human cells. Osteocalcin expression was also increased upon stimulation with BMP-6. The presence of 17beta-estradiol, dihydrotestosterone, and resveratrol had no effect on BMP-6-induced alkaline phosphatase activity and osteocalcin expression. These data suggest that osteogenic differentiation in response to BMP-6 occurs independent of steroid hormones and resveratrol in mesenchymal cells that express basal receptor levels.     

尿酸影响人骨髓间充质干细胞成骨分化过程中BMP-2 表达

目的:探讨在人骨髓间充质干细胞(h BMSCs)成骨分化过程中,不同浓度尿酸(UA)对骨形态形成蛋白-2(BMP-2)表达的影响。方法:以全骨髓贴壁培养法分离h BMSCs,将生长状态良好的第3代h BMSCs分为5组,分别为空白对照组(加入完全培养基)和成骨诱导组(加入成骨诱导液及含0 mmol/L、0.2 mmol/L、0.4 mmol/L、0.8 mmol/L尿酸的完全培养基)。连续干预诱导14d后,用倒置显微镜观察细胞形态的变化,通过观察茜素红染色情况及检测碱性磷酸酶(ALP)活性进行成骨情况的检测。RT-PCR技术检测各组细胞BMP-2 mR NA的表达情况。结果:第3代h BMSCs大多为形态单一的长梭形,呈旋涡状生长;干预诱导后的细胞逐渐变成不规则的立方形,局部形成团块状结节,以含尿酸浓度为0.8 mmol/L的成骨诱导培养基最为显著。连续干预14d后,空白对照组茜素红染色为阴性,而各成骨诱导组细胞茜素红染色结果为阳性,提示干预诱导后的细胞为成骨细胞。碱性磷酸酶活性随尿酸浓度的增加和干预时间的延长而增强(P<0.05)。RT-PCR检测结果显示,空白对照组无BMP-2 mR NA的表达。成骨诱导组随培养基中尿酸浓度的增加,BMP-2 mR NA表达逐渐增强,呈浓度依赖性(P<0.05)。结论:尿酸上调h BMSCs向成骨细胞分化过程中BMP-2 mR NA的表达。     

Over expression of bone morphogenetic protein-3b (BMP-3b) using an adenoviral vector promote the osteoblastic differentiation in C2C12 cells and augment the bone formation induced by bone morphogenetic protein-2 (BMP-2) in rats

BMP-3b is a novel BMP-3-related protein and its biological functions are unknown. In order to investigate the biological actions of BMP-3b, we constructed a BMP-3b-expressing recombinant adenoviral vector (AxCAKBMP-3b). We show that over expression of BMP-3b stimulated the induction of differentiation and the osteoinduction activity of a human BMP-2-expressing recombinant adenoviral vector (AxCAOBMP-2). C2C12 cells were infected in vitro with AxCAKBMP-3b, AxCAOBMP-2 and a control vector containing no foreign genes (AxCAwt). Cells infected with AxCAOBMP-2 and AxCAKBMP-3b produced more alkaline phosphatase and secreted more osteocalcin into the culture medium than cells infected with AxCAOBMP-2 and AxCAwt. When AxCAOBMP-2, AxCAKBMP-3b, and AxCAwt were injected into the calf muscles of nude rats (F 344/N Jcl-rnu), the osteoinduction seen with AxCAOBMP-2 and AxCAKBMP-3b was greater than that seen with AxCAOBMP-2 and AxCAwt.     

Heparin inhibits BMP-2 osteogenic bioactivity by binding to both BMP-2 and BMP receptor

Heparin demonstrates several kinds of biological activities by binding to various extracellular molecules and plays pivotal roles in bone metabolism. However, the role of heparin in the biological activity of bone morphogenetic protein (BMP) remains unclear. In the present study, we examined whether heparin has the effects on osteoblast differentiation induced by BMP-2 in vitro and also elucidated the precise mechanism by which heparin regulates bone metabolism induced by this molecule. Our results showed that heparin inhibited alkaline phosphatase (ALP) activity and mineralization in osteoblastic cells cultured with BMP-2. Heparin was found to suppress the mRNA expressions of osterix, Runx2, ALP and osteocalcin, as well as phosphorylation of Smad1/5/8 and p38 MAPK. Further, heparin bound to both BMP-2 and BMP receptor (BMPR). These results suggest that heparin suppresses BMP-2-BMPR binding, and inhibits BMP-2 osteogenic activity in vitro.     

Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage [published erratum appears in J Cell Biol 1995 Feb;128(4):following 713]

The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP- 2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)- positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.     

Differential regulation of osteogenic marker gene expression by Wnt-3a in embryonic mesenchymal multipotential progenitor cells

The Wnt family of secreted glycoproteins plays an integral role in embryonic development and differentiation. To explore the role of Wnt's in one aspect of differentiation, namely osteogenesis, we employed a retroviral gene transfer approach to express Wnt-3a in the multipotent murine embryonic mesenchymal cell line C3H10T1/2. We found that expression of Wnt-3a in these cells had a significant, positive effect on cell growth in serum-containing medium, in that the cells grew to very high densities compared to the control cells. Additionally, apoptosis was markedly inhibited by Wnt-3a. However, when the cells were grown in serum-deficient medium, the Wnt-3a-expressing cells arrested efficiently in G1 phase, indicating that serum growth factors were needed in addition to Wnt-3a for enhanced proliferation. Wnt-3a-expressing cells exhibited high levels of alkaline phosphatase gene expression and enzymatic activity, but did not show any matrix mineralization. Unexpectedly, basal expression of bone sialoprotein, osteocalcin, and osteopontin were markedly inhibited by Wnt-3a, as were other known target genes of Wnt-3a, such as Brachyury, FGF-10, and Cdx1. When Wnt-3a-expressing cells were treated with osteogenic supplements in the presence of BMP-2, alkaline phosphatase gene expression and activity were further elevated. Additionally, BMP-2 was able to reverse the inhibitory effect of Wnt-3a on osteocalcin and osteopontin gene expression. These results indicate that while Wnt-3a represses basal expression of some osteogenic genes, this repression can be partially reversed by BMP-2. Finally, the enhanced gene expression of alkaline phosphatase induced by Wnt-3a could be effectively suppressed by the combined action of dexamethasone and 1,25-dihydroxyvitamin D(3). These data show for the first time that Wnt-3a has an unusual effect on multipotential embryonic cells, in that it enhances cellular proliferation and expression of alkaline phosphatase, while it represses most other marker genes of osteogenic differentiation.     

Serial passage of MC3T3-E1 cells alters osteoblastic function and responsiveness to transforming growth factor-beta1 and bone morphogenetic protein-2.

The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage (<20) and late passage (>65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-beta1 (TGF-beta1) stimulated cell proliferation in early passage cells and induced c-fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-beta1 exerted an opposite effect. Both BMP-2 and TGF-beta1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-beta1-mediated cell proliferation, and responsiveness to TGF-beta1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.     

Functions of transforming growth factor-beta family type I receptors and Smad proteins in the hypertrophic maturation and osteoblastic differentiation of chondrocytes

We investigated the effects of bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta superfamily, on the regulation of the chondrocyte phenotype, and we identified signaling molecules involved in this regulation. BMP-2 triggers three concomitant responses in mouse primary chondrocytes and chondrocytic MC615 cells. First, BMP-2 stimulates expression or synthesis of type II collagen. Second, BMP-2 induces expression of molecular markers characteristic of pre- and hypertrophic chondrocytes, such as Indian hedgehog, parathyroid hormone/parathyroid hormone-related peptide receptor, type X collagen, and alkaline phosphatase. Third, BMP-2 induces osteocalcin expression, a specific trait of osteoblasts. Constitutively active forms of transforming growth factor-beta family type I receptors and Smad proteins were overexpressed to address their role in this process. Activin receptor-like kinase (ALK)-1, ALK-2, ALK-3, and ALK-6 were able to reproduce the hypertrophic maturation of chondrocytes induced by BMP-2. In addition, ALK-2 mimicked further the osteoblastic differentiation of chondrocytes induced by BMP-2. In the presence of BMP-2, Smad1, Smad5, and Smad8 potentiated the hypertrophic maturation of chondrocytes, but failed to induce osteocalcin expression. Smad6 and Smad7 impaired chondrocytic expression and osteoblastic differentiation induced by BMP-2. Thus, our results indicate that Smad-mediated pathways are essential for the regulation of the different steps of chondrocyte and osteoblast differentiation and suggest that additional Smad-independent pathways might be activated by ALK-2.     

Overexpression of BMP-2 modulates morphology, growth, and gene expression in osteoblastic cells

Bone morphogenetic proteins (BMP) play a pivotal role in growth and differentiation of osteoblastic lineage cells. BMPs are potent stimulators of bone formation in various animal models. To understand the mechanism of BMP action in bone cells, we have investigated the effects of overexpression of the BMP-2 gene on proliferation and differentiation of UMR-106 rat osteosarcoma cells. A stable UMR-106 cell line overexpressing the BMP-2 gene was established by transfection of cells using a mammalian expression vector harboring human BMP-2 cDNA followed by G418 selection. After introduction of the BMP-2 gene, UMR-106 cells appeared more spindle-shaped in morphology compared to the predominantly cuboidal appearance of the parental cells. Overexpression of BMP-2 markedly inhibited proliferation as measured by cell counting and [3H]thymidine incorporation assays. Extracellular matrix (ECM) derived from cells overexpressing BMP-2 exhibited a less supportive effect on proliferation of UMR cells than did ECM derived from parental cells. Furthermore, cell-cell communication through gap junctions was reduced more than 50% as determined by nondisruptive fluorescent dye transfer assays. Overexpression of BMP-2 significantly stimulated expression of osteocalcin and alkaline phosphatase genes, indicating its role in osteoblastic differentiation. There was little effect on osteopontin gene expression.     

RNA interference of the BMPR-IB gene blocks BMP-2-induced osteogenic gene expression in human bone cells

We have previously shown that human bone cells express bone morphogenetic protein receptor-IB (BMPR-IB). However, little is known about the precise role of this receptor in the response of osteoblastic genes to the BMP in these cells. To determine BMPR-IB-dependent osteoblastic gene expression, the present study examined the effects of BMPR-IB knockdown on BMP-induced osteoblast-associated genes. BMPR-IB mRNA and protein were markedly suppressed by transfection of cells with BMPR-IB siRNA. Using three different bone cell samples, BMP-2 stimulation of alkaline phosphatase (ALP), osteocalcin (OC), distal-less homeobox-5 (Dlx5) and core binding factor alpha-1 (Cbfa1) was found to be specifically and significantly reduced in the BMPR-IB siRNA-transfected cultures compared with that of control cultures. Our study has provided evidence that BMPR-IB-dependent signaling plays a crucial role in BMP-2 up-regulation of the ALP, OC, Dlx5 and Cbfa1 genes in bone cells, suggesting a pivotal role of this receptor in BMP-2-induced osteoblast differentiation in vitro. These findings thus suggest the possibility that BMPR-IB could be a therapeutic target for enhancing bone regeneration in vivo.