Development and characterization of a mouse floxed <Emphasis Type="Italic">Bmp2</Emphasis> osteoblast cell line that retains osteoblast genotype and phenotype

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways.     

BMP‐2 modulates β‐catenin signaling through stimulation of Lrp5 expression and inhibition of β‐TrCP expression in osteoblasts

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP‐2 acts synergistically with β‐catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross‐talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3‐E1 cells to investigate the effect of BMP‐2 on β‐catenin signaling. We found that BMP‐2 stimulates Lrp5 expression and inhibits the expression of β‐TrCP, the F‐box E3 ligase responsible for β‐catenin degradation and subsequently increases β‐catenin protein levels in osteoblasts. In vitro deletion of the β‐catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP‐2 treatment. These findings suggest that BMP‐2 may regulate osteoblast function in part through modulation of the β‐catenin signaling. J. Cell. Biochem. 108: 896–905, 2009. © 2009 Wiley‐Liss, Inc.     

Comparison between 8‐prenylnarigenin and narigenin concerning their activities on promotion of rat bone marrow stromal cells’ osteogenic differentiation in vitro

A number of recent studies have suggested that flavonols (a class of phytochemical with many biological activities), might exert protective effects against post‐menopausal bone loss. In the present study, we compared naringenin (NG) and 8‐prenylnaringenin (PNG), two major naturally occurring flavonols, on in vitro differentiation of osteoblasts and bone resorbing activity, of rat bone marrow stromal cells (BMSCs). Our results indicated that both compounds, at 10^?6 m , enhanced BMSCs’ differentiation. Then effects of the two compounds at 10^?6 m on ALP activity, osteocalcin secretion and calcium deposition, were compared over a time course. Numbers and areas of colonies stained for ALP (CFU‐F_ALP) expression, and mineralized bone nodules, were histochemically analysed after 12 days and 16 days osteogenic induction, respectively. Expression of BMP‐2, OPG, OSX, RUNX‐2 genes and p38MAPK protein were examined using real‐time PCR and western blotting, respectively. The data presented indicate that PNG, significantly enhanced the rat BMSCs’ differentiation and mineralization through the BMP‐2/p38MAPK/Runx2/Osterix signal pathway, greater than did NG. In conclusion, PNG has a more pronounced ability to enhance osteoblast differentiation and mineralization, than NG.     

Bone morphogenetic protein-7 (OP1) and transforming growth factor-beta1 modulate 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts

Bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGFbeta) are potent regulators of osteoblast differentiation and proliferation, processes that are crucial in bone remodeling. BMPs and TGFbeta act in concert with other local factors and hormones, among them 1,25(OH)2-vitamin D3 and insulin. Here we show that BMP7 inhibits 1,25(OH)2-vitamin D3-induced differentiation of human osteoblasts, whereas TGFbeta1 stimulates it, as assessed by assays for alkaline phosphatase (ALP) induction, matrix mineralization, and morphology changes. BMP7 or TGFbeta1 alone affects the differentiation of human osteoblasts. Similar results were obtained in assays for ALP induction using conditionally immortalized human osteoblasts (hFOB) and primary osteoblasts obtained from trabecular bone of the femoral head after hip replacement surgery. BMP7 stimulation led to a decrease of 1,25(OH)2-vitamin D3-induced binding of nuclear proteins to a vitamin D response element, as shown by electrophoretic mobility shift assay. Our results suggest that 1,25(OH)2-vitamin D3 modulates in opposite ways the effects of BMP7 and TGFbeta1 on osteoblast differentiation.     

Protein Palmitoylation Regulates Osteoblast Differentiation through BMP-Induced Osterix Expression

Osteoporosis is one of the most common diseases and can be treated by either anti-resorption drugs, anabolic drugs, or both. To search for anabolic drug targets for osteoporosis therapy, it is crucial to understand the biology of bone forming cells, osteoblasts, in terms of their proliferation, differentiation, and function. Here we found that protein palmitoylation participates in signaling pathways that control osterix expression and osteoblast differentiation. Mouse calvarial osteoblasts express most of the 24 palmitoyl transferases, with some being up-regulated during differentiation. Inhibition of protein palmitoylation, with a substrate-analog inhibitor, diminished osteoblast differentiation and mineralization, but not proliferation or survival. The decrease in differentiation capacity is associated with a reduction in osterix, but not Runx2 or Atf4. Inhibition of palmitoyl transferases had little effect in p53^−/− osteoblasts that show accelerated differentiation due to overexpression of osterix, suggesting that osterix, at least partially, mediated the effect of inhibition of palmitoyl transferases on osteoblast differentiation. BMPs are the major driving force of osteoblast differentiation in the differentiation assays. We found that inhibition of palmitoyl transferases also compromised BMP2-induced osteoblast differentiation through down-regulating osterix induction. However, palmitoyl transferases inhibitor did not inhibit Smad1/5/8 activation. Instead, it compromised the activation of p38 MAPK, which are known positive regulators of osterix expression and differentiation. These results indicate that protein palmitoylation plays an important role in BMP-induced MAPK activation, osterix expression, and osteoblast differentiation.     

Dicer inactivation in osteoprogenitor cells compromises fetal survival and bone formation, while excision in differentiated osteoblasts increases bone mass in the adult mouse

MicroRNA attenuation of protein translation has emerged as an important regulator of mesenchymal cell differentiation into the osteoblast lineage. A compelling question is the extent to which miR biogenesis is obligatory for bone formation. Here we show conditional deletion of the Dicer enzyme in osteoprogenitors by Col1a1-Cre compromised fetal survival after E14.5. A mechanism was associated with the post-commitment stage of osteoblastogenesis, demonstrated by impaired ECM mineralization and reduced expression of mature osteoblast markers during differentiation of mesenchymal cells of ex vivo deleted Dicer^c/c. In contrast, in vivo excision of Dicer by Osteocalcin-Cre in mature osteoblasts generated a viable mouse with a perinatal phenotype of delayed bone mineralization which was resolved by 1 month. However, a second phenotype of significantly increased bone mass developed by 2 months, which continued up to 8 months in long bones and vertebrae, but not calvariae. Cortical bone width and trabecular thickness in Dicer^Δoc/Δoc was twice that of Dicer^c/c controls. Normal cell and tissue organization was observed. Expression of osteoblast and osteoclast markers demonstrated increased coupled activity of both cell types. We propose that Dicer generated miRs are essential for two periods of bone formation, to promote osteoblast differentiation before birth, and control bone accrual in the adult.     

Immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to BMP2

Bone morphogenetic protein 2 (Bmp2) is essential for odontogensis and dentin mineralization. Generation of floxed Bmp2 dental mesenchymal cell lines is a valuable application for studying the effects of Bmp2 on dental mesenchymal cell differentiation and its signaling pathways during dentinogenesis. Limitation of the primary culture of dental mesenchymal cells has led to the development of cell lines that serve as good surrogate models for the study of dental mesenchymal cell differentiation into odontoblasts and mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines, which were isolated from 1st mouse mandibular molars at postnatal day 1 and immortalized with pSV40 and clonally selected. These transfected cell lines were characterized by RT‐PCR, immunohistochemistry, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iBmp2‐dp, displayed a higher proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth‐specific markers as well as demonstrated the ability to differentiate and form mineralized nodules. In addition, iBmp2‐dp cells were inducible and responded to BMP2 stimulation. Thus, we for the first time described the establishment of an immortalized mouse floxed Bmp2 dental papilla mesenchyma cell line that might be used for studying the mechanisms of dental cell differentiation and dentin mineralization mediated by Bmp2 and other growth factor signaling pathways. J. Cell. Physiol. 225: 132–139, 2010. © 2010 Wiley‐Liss, Inc.     

Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.     

Sprouty2 is involved in the control of osteoblast proliferation and differentiation through the FGF and BMP signaling pathways

Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) play essential roles in bone formation and osteoblast activity through the extracellular signal‐regulated kinase 1/2 (ERK1/2) and Smad pathways. Sprouty family members are intracellular inhibitors of the FGF signaling pathway, and four orthologs of Sprouty have been identified in mammals. In vivo analyses have revealed that Sprouty2 is associated with bone formation. However, the mechanism by which the Sprouty family controls bone formation has not been clarified. In this study, we investigated the involvement of Sprouty2 in osteoblast proliferation and differentiation. We examined Sprouty2 expression in MC3T3‐E1 cells, and found that high levels of Sprouty2 expression were induced by basic FGF stimulation. Overexpression of Sprouty2 in MC3T3‐E1 cells resulted in suppressed proliferation compared with control cells. Sprouty2 negatively regulated the phosphorylation of ERK1/2 after basic FGF stimulation, and of Smad1/5/8 after BMP stimulation. Furthermore, Sprouty2 suppressed the expression of osterix, alkaline phosphatase, and osteocalcin mRNA, which are markers of osteoblast differentiation. Additionally, Sprouty2 inhibited osteoblast matrix mineralization. These results suggest that Sprouty2 is involved in the control of osteoblast proliferation and differentiation by downregulating the FGF‐ERK1/2 and BMP‐Smad pathways, and suppresses the induction of markers of osteoblast differentiation.     

Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO₂) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl₄ or Zirconium (IV) oxynitrate (ZrO(NO₃)₂) at concentrations of 0, 5, 50 and 500 µM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 µM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.     

Deficiency of Macf1 in osterix expressing cells decreases bone formation by Bmp2/Smad/Runx2 pathway

Microtubule actin cross‐linking factor 1 (Macf1) is a spectraplakin family member known to regulate cytoskeletal dynamics, cell migration, neuronal growth and cell signal transduction. We previously demonstrated that knockdown of Macf1 inhibited the differentiation of MC3T3‐E1 cell line. However, whether Macf1 could regulate bone formation in vivo is unclear. To study the function and mechanism of Macf1 in bone formation and osteogenic differentiation, we established osteoblast‐specific Osterix (Osx) promoter‐driven Macf1 conditional knockout mice (Macf1^f/fOsx‐Cre). The Macf1^f/fOsx‐Cre mice displayed delayed ossification and decreased bone mass. Morphological and mechanical studies showed deteriorated trabecular microarchitecture and impaired biomechanical strength of femur in Macf1^f/fOsx‐Cre mice. In addition, the differentiation of primary osteoblasts isolated from calvaria was inhibited in Macf1^f/fOsx‐Cre mice. Deficiency of Macf1 in primary osteoblasts inhibited the expression of osteogenic marker genes (Col1, Runx2 and Alp) and the number of mineralized nodules. Furthermore, deficiency of Macf1 attenuated Bmp2/Smad/Runx2 signalling in primary osteoblasts of Macf1^f/fOsx‐Cre mice. Together, these results indicated that Macf1 plays a significant role in bone formation and osteoblast differentiation by regulating Bmp2/Smad/Runx2 pathway, suggesting that Macf1 might be a therapeutic target for bone disease.