The Experts below are selected from a list of 258 Experts worldwide ranked by ideXlab platform
Joo L Ong - One of the best experts on this subject based on the ideXlab platform.
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ultrasound effect on Osteoblast Precursor cells in trabecular calcium phosphate scaffolds
Biomaterials, 2007Co-Authors: Mark R Appleford, Judith A Cole, Jiri Protivinský, Joo L OngAbstract:This study investigated the in vitro effect of low-intensity pulsed ultrasound (LIPUS) on human embryonic palatal mesenchyme cells (HEPM, CRL-1486, ATCC, Manassas, VA), an Osteoblast Precursor cell line, during early adhesion to calcium phosphate scaffolds. Hydroxyapatite (HA) and β-tricalcium phosphate (TCP) ceramic scaffolds were produced by a template coating method. Phospho-specific antibody cell-based ELISA (PACE) technique was utilized on stress activation proteins, including the extracellular signal-regulated kinase (ERK1/2), P38, c-Jun N-terminal kinase (JNK) and the anti-apoptosis mediator protein kinase B (PKB/AKT). Cell-based ELISAs were also performed on the membrane anchoring protein vinculin and α6β4 integrin. LIPUS stimulated activation of PERK 1/2, PJNK, PP38 and vinculin in traditional two-dimensional (2-D) culture. Calcium release from the scaffolds was partially involved in the activation of PERK 1/2 when cell response was compared between culture on 2-D surfaces and three-dimensional (3-D) HA and TCP scaffolds. Effects of calcium extracted media from scaffolds alone could not account for the full activation of PJNK, PP38, PAKT, vinculin and α6β4 integrin. LIPUS stimulation further increased PERK activity on TCP scaffolds corresponding with an increase in both vinculin and α6β4 integrin levels. It was concluded from this study that LIPUS treatment can significantly affect stress signaling mediators and adhesion proteins in Osteoblast Precursor cells during the early cell-attachment phase to trabecular patterned scaffolds.
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antibacterial and osteogenic properties of silver containing hydroxyapatite coatings produced using a sol gel process
Journal of Biomedical Materials Research Part A, 2007Co-Authors: Mark R Appleford, Yongxing Liu, W Chen, A P Ong, Harry S Courtney, Joo L OngAbstract:Since bacterial infection is a rising complication following the wide use of implant, there is considerable attention on the effect of implant surface properties on bacterial adhesion. In this study, the effect of silver (Ag) doped hydroxyapatite (HA) coatings on initial antibacterial adhesion and Osteoblast cell proliferation and differentiation was investigated. Using a sol-gel process, HA coatings doped with 1 wt % AgNO(3) (AgHA1.0) and 1.5 wt % Ag (AgHA1.5) were prepared. Coated surfaces were characterized using X-ray diffraction (XRD) and contact angles measurements. The initial bacteria adhesion was evaluated using a RP12 strain of Staphylococcus epidermidis (ATCC 35984) and the Cowan I strain of Staphylococcus aureus, whereas Osteoblast proliferation and differentiation were evaluated using human embryonic palatal mesenchyme cells (HEPM), an Osteoblast Precursor cell line. In this study, XRD analysis of all surfaces indicated peaks corresponding to HA. Contact angles for AgHA surfaces were observed to be significantly lower when compared to HA surfaces. In vitro initial bacterial adhesion study indicated a significantly reduced number of S. epidermidis and S. aureus on AgHA surfaces when compared to HA surface. The use of HEPM cells indicated no significant difference in double-stranded DNA (dsDNA) production between all surfaces. Additionally, no differences in alkaline phosphatase specific activity were observed between HA and AgHA1.0 surfaces. Overall, it was concluded that AgHA1.0 has the similar biological activity as HA, with respect to bone cell proliferation and differentiation. In addition, the AgHA1.0 was also concluded to have the ability to minimize the initial bacteria adhesion. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.
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effect of chemically modified titanium surfaces on protein adsorption and Osteoblast Precursor cell behavior
International Journal of Oral & Maxillofacial Implants, 2007Co-Authors: Jiri Protivinský, Mark R Appleford, Jakob Strnad, Ales Helebrant, Joo L OngAbstract:Purpose To investigate the effects of different chemically modified titanium surfaces on protein adsorption and the Osteoblastic differentiation of human embryonic palatal mesenchymal (HEPM) cells. Materials and methods Three different surfaces were evaluated. The first, a machined surface (Ti-M), was considered a control. The second surface was acid etched (Ti-AE). The third surface was prepared by exposing the Ti-AE samples to sodium hydroxide (NaOH) solution (Ti-AAE). The surface characteristics of chemically modified titanium were investigated by means of scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and profilometry. To evaluate the production of biomarkers, commercial kits were utilized. Results Surface composition and morphology affected the kinetics of protein adsorption. Ti-AE surfaces manifested a greater affinity for fibronectin adsorption compared to Ti-M or Ti-AAE surfaces. It was observed that Ti-AE and Ti-AAE surfaces promoted significantly greater cell attachment compared to Ti-M surfaces. Statistically significant differences were also observed in the expression of alkaline phosphatase (ALP) activity, osteocalcin, and osteopontin on all 3 titanium surfaces. ALP activity and osteocalcin production up to day 12 suggested that differentiation of the cells into Osteoblasts had occurred and that cells were expressing a bone-forming phenotype. Conclusions It was thus concluded from this study that surface morphology and composition play a critical role in enhancing HEPM cell proliferation and differentiation into Osteoblast cells.
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effects of trabecular calcium phosphate scaffolds on stress signaling in Osteoblast Precursor cells
Biomaterials, 2007Co-Authors: Mark R Appleford, Judith A Cole, David L Carnes, Myunghyun Lee, Joel D Bumgardner, Warren O Haggard, Joo L OngAbstract:Abstract The objective of this research was to investigate stress-signaling patterns in response to two-dimensional (2-D) and three-dimensional (3-D) calcium phosphate (CP) materials using human embryonic palatal mesenchyme cells (HEPM, CRL-1486, ATCC, Manassas, VA), an Osteoblast Precursor cell line. Control discs and scaffolds were fabricated from hydroxyapatite and β tri-CP ceramics. Phospho-specific antibody cell-based ELISA technique was utilized on members of the mitogen-activated protein kinase cascade including; the extracellular signal-regulated kinases (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and the anti-apoptosis mediator protein kinase B (AKT). Quantification of these signals was evaluated during the early attachment phase of Osteoblast Precursor cells. In this study, it was observed that 3-D CP scaffolds significantly activated the stress mediators p38 and JNK but not ERK1/2. This signal trend was matched with an up-regulation in AKT, suggesting the ability of cells to manage high stress signals in response to 3-D CP architecture and that 3-D CP scaffolds are necessary for studies simulating a natural trabecular bone organization. The absence of these signals in 2-D CP surfaces indicated the importance of local architecture conditions on cell stress response. It was concluded from this study that Osteoblast Precursor cells cultured in 3-D CP scaffolds experience greater stress-signaling patterns when compared to 2-D CP surfaces.
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in vitro anti bacterial and biological properties of magnetron co sputtered silver containing hydroxyapatite coating
Biomaterials, 2006Co-Authors: Joo L Ong, Joel D Bumgardner, Yongxing Liu, W Chen, Harry S Courtney, M Bettenga, C M AgrawalAbstract:Bacterial infection after implant placement is a significant rising complication. In order to reduce the incidence of implant-associated infections, several biomaterial surface treatments have been proposed. In this study, the effect of in vitro antibacterial activity and in vitro cytotoxicity of co-sputtered silver (Ag)-containing hydroxyapatite (HA) coating was evaluated. Deposition was achieved by a concurrent supply of 10 W to the Ag target and 300 W to the HA target. Heat treatment at 4001C for 4 h was performed after 3 h deposition. X-ray diffraction, contact angles measurements, and surface roughness were used to characterize the coating surfaces. The RP12 strain of Staphylococcus epidermidis (ATCC 35984) and the Cowan I strain of Staphylococcus aureus were used to evaluate the antibacterial activity of the Ag-HA coatings, whereas human embryonic palatal mesenchyme cells, an Osteoblast Precursor cell line, were used to evaluate the in vitro cytotoxicity of the coatings. X-ray diffraction analysis performed in this study indicated peaks corresponding to Ag and HA on the co-sputtered Ag-HA surfaces. The contact angles for HA and Ag-HA surfaces were observed to be significantly lower when compared to Ti surfaces, whereas no significant difference in surface roughness was observed for all groups. In vitro bacterial adhesion study indicated a significantly reduced number of S. epidermidis and S. aureus on Ag-HA surface when compared to titanium (Ti) and HA surfaces. In addition, no significant difference in the in vitro cytotoxicty was observed between HA and Ag-HA surfaces. Overall, it was concluded that the creation of a multifunctional surface can be achieved by co-sputtering the osteoconductive HA with antibacterial Ag. r 2006 Elsevier Ltd. All rights reserved.
Mark R Appleford - One of the best experts on this subject based on the ideXlab platform.
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influence of substrate curvature on Osteoblast orientation and extracellular matrix deposition
Journal of Biological Engineering, 2013Co-Authors: Marcello Pilia, Teja Guda, Stefanie M Shiels, Mark R ApplefordAbstract:Background The effects of microchannel diameter in hydroxyapatite (HAp) substrates on Osteoblast behavior were investigated in this study. Microchannels of 100, 250 and 500 μm diameter were created on hydroxyapatite disks. The changes in Osteoblast Precursor growth, differentiation, extra cellular matrix (ECM) secretion and cell attachment/orientation were investigated as a function of microchannel diameter.
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ultrasound effect on Osteoblast Precursor cells in trabecular calcium phosphate scaffolds
Biomaterials, 2007Co-Authors: Mark R Appleford, Judith A Cole, Jiri Protivinský, Joo L OngAbstract:This study investigated the in vitro effect of low-intensity pulsed ultrasound (LIPUS) on human embryonic palatal mesenchyme cells (HEPM, CRL-1486, ATCC, Manassas, VA), an Osteoblast Precursor cell line, during early adhesion to calcium phosphate scaffolds. Hydroxyapatite (HA) and β-tricalcium phosphate (TCP) ceramic scaffolds were produced by a template coating method. Phospho-specific antibody cell-based ELISA (PACE) technique was utilized on stress activation proteins, including the extracellular signal-regulated kinase (ERK1/2), P38, c-Jun N-terminal kinase (JNK) and the anti-apoptosis mediator protein kinase B (PKB/AKT). Cell-based ELISAs were also performed on the membrane anchoring protein vinculin and α6β4 integrin. LIPUS stimulated activation of PERK 1/2, PJNK, PP38 and vinculin in traditional two-dimensional (2-D) culture. Calcium release from the scaffolds was partially involved in the activation of PERK 1/2 when cell response was compared between culture on 2-D surfaces and three-dimensional (3-D) HA and TCP scaffolds. Effects of calcium extracted media from scaffolds alone could not account for the full activation of PJNK, PP38, PAKT, vinculin and α6β4 integrin. LIPUS stimulation further increased PERK activity on TCP scaffolds corresponding with an increase in both vinculin and α6β4 integrin levels. It was concluded from this study that LIPUS treatment can significantly affect stress signaling mediators and adhesion proteins in Osteoblast Precursor cells during the early cell-attachment phase to trabecular patterned scaffolds.
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antibacterial and osteogenic properties of silver containing hydroxyapatite coatings produced using a sol gel process
Journal of Biomedical Materials Research Part A, 2007Co-Authors: Mark R Appleford, Yongxing Liu, W Chen, A P Ong, Harry S Courtney, Joo L OngAbstract:Since bacterial infection is a rising complication following the wide use of implant, there is considerable attention on the effect of implant surface properties on bacterial adhesion. In this study, the effect of silver (Ag) doped hydroxyapatite (HA) coatings on initial antibacterial adhesion and Osteoblast cell proliferation and differentiation was investigated. Using a sol-gel process, HA coatings doped with 1 wt % AgNO(3) (AgHA1.0) and 1.5 wt % Ag (AgHA1.5) were prepared. Coated surfaces were characterized using X-ray diffraction (XRD) and contact angles measurements. The initial bacteria adhesion was evaluated using a RP12 strain of Staphylococcus epidermidis (ATCC 35984) and the Cowan I strain of Staphylococcus aureus, whereas Osteoblast proliferation and differentiation were evaluated using human embryonic palatal mesenchyme cells (HEPM), an Osteoblast Precursor cell line. In this study, XRD analysis of all surfaces indicated peaks corresponding to HA. Contact angles for AgHA surfaces were observed to be significantly lower when compared to HA surfaces. In vitro initial bacterial adhesion study indicated a significantly reduced number of S. epidermidis and S. aureus on AgHA surfaces when compared to HA surface. The use of HEPM cells indicated no significant difference in double-stranded DNA (dsDNA) production between all surfaces. Additionally, no differences in alkaline phosphatase specific activity were observed between HA and AgHA1.0 surfaces. Overall, it was concluded that AgHA1.0 has the similar biological activity as HA, with respect to bone cell proliferation and differentiation. In addition, the AgHA1.0 was also concluded to have the ability to minimize the initial bacteria adhesion. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.
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effect of chemically modified titanium surfaces on protein adsorption and Osteoblast Precursor cell behavior
International Journal of Oral & Maxillofacial Implants, 2007Co-Authors: Jiri Protivinský, Mark R Appleford, Jakob Strnad, Ales Helebrant, Joo L OngAbstract:Purpose To investigate the effects of different chemically modified titanium surfaces on protein adsorption and the Osteoblastic differentiation of human embryonic palatal mesenchymal (HEPM) cells. Materials and methods Three different surfaces were evaluated. The first, a machined surface (Ti-M), was considered a control. The second surface was acid etched (Ti-AE). The third surface was prepared by exposing the Ti-AE samples to sodium hydroxide (NaOH) solution (Ti-AAE). The surface characteristics of chemically modified titanium were investigated by means of scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and profilometry. To evaluate the production of biomarkers, commercial kits were utilized. Results Surface composition and morphology affected the kinetics of protein adsorption. Ti-AE surfaces manifested a greater affinity for fibronectin adsorption compared to Ti-M or Ti-AAE surfaces. It was observed that Ti-AE and Ti-AAE surfaces promoted significantly greater cell attachment compared to Ti-M surfaces. Statistically significant differences were also observed in the expression of alkaline phosphatase (ALP) activity, osteocalcin, and osteopontin on all 3 titanium surfaces. ALP activity and osteocalcin production up to day 12 suggested that differentiation of the cells into Osteoblasts had occurred and that cells were expressing a bone-forming phenotype. Conclusions It was thus concluded from this study that surface morphology and composition play a critical role in enhancing HEPM cell proliferation and differentiation into Osteoblast cells.
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effects of trabecular calcium phosphate scaffolds on stress signaling in Osteoblast Precursor cells
Biomaterials, 2007Co-Authors: Mark R Appleford, Judith A Cole, David L Carnes, Myunghyun Lee, Joel D Bumgardner, Warren O Haggard, Joo L OngAbstract:Abstract The objective of this research was to investigate stress-signaling patterns in response to two-dimensional (2-D) and three-dimensional (3-D) calcium phosphate (CP) materials using human embryonic palatal mesenchyme cells (HEPM, CRL-1486, ATCC, Manassas, VA), an Osteoblast Precursor cell line. Control discs and scaffolds were fabricated from hydroxyapatite and β tri-CP ceramics. Phospho-specific antibody cell-based ELISA technique was utilized on members of the mitogen-activated protein kinase cascade including; the extracellular signal-regulated kinases (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and the anti-apoptosis mediator protein kinase B (AKT). Quantification of these signals was evaluated during the early attachment phase of Osteoblast Precursor cells. In this study, it was observed that 3-D CP scaffolds significantly activated the stress mediators p38 and JNK but not ERK1/2. This signal trend was matched with an up-regulation in AKT, suggesting the ability of cells to manage high stress signals in response to 3-D CP architecture and that 3-D CP scaffolds are necessary for studies simulating a natural trabecular bone organization. The absence of these signals in 2-D CP surfaces indicated the importance of local architecture conditions on cell stress response. It was concluded from this study that Osteoblast Precursor cells cultured in 3-D CP scaffolds experience greater stress-signaling patterns when compared to 2-D CP surfaces.
S E Harris - One of the best experts on this subject based on the ideXlab platform.
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wnt β catenin signaling activates bone morphogenetic protein 2 expression in Osteoblasts
Bone, 2013Co-Authors: Rongrong Zhang, Di Chen, Babatunde O Oyajobi, S E Harris, Christopher Tsao, Hongwen Deng, Ming ZhaoAbstract:The BMP and Wnt/β-catenin signaling pathways cooperatively regulate Osteoblast differentiation and bone formation. Although BMP signaling regulates gene expression of the Wnt pathway, much less is known about whether Wnt signaling modulates BMP expression in Osteoblasts. Given the presence of putative Tcf/Lef response elements that bind β-catenin/TCF transcription complex in the BMP2 promoter, we hypothesized that the Wnt/β-catenin pathway stimulates BMP2 expression in osteogenic cells. In this study, we showed that Wnt/β-catenin signaling is active in various Osteoblast or Osteoblast Precursor cell lines, including MC3T3-E1, 2T3, C2C12, and C3H10T1/2 cells. Furthermore, crosstalk between the BMP and Wnt pathways affected BMP signaling activity, Osteoblast differentiation, and bone formation, suggesting Wnt signaling is an upstream regulator of BMP signaling. Activation of Wnt signaling by Wnt3a or overexpression of β-catenin/TCF4 both stimulated BMP2 transcription at promoter and mRNA levels. In contrast, transcription of BMP2 in osteogenic cells was decreased by either blocking the Wnt pathway with DKK1 and sFRP4, or inhibiting β-catenin/TCF4 activity with FWD1/β-TrCP, ICAT, or ΔTCF4. Using a site-directed mutagenesis approach, we confirmed that Wnt/β-catenin transactivation of BMP2 transcription is directly mediated through the Tcf/Lef response elements in the BMP2 promoter. These results, which demonstrate that the Wnt/β-catenin signaling pathway is an upstream activator of BMP2 expression in Osteoblasts, provide novel insights into the nature of functional cross talk integrating the BMP and Wnt/β-catenin pathways in Osteoblastic differentiation and maintenance of skeletal homeostasis.
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reactive oxygen species derived from nox4 mediate bmp2 gene transcription and Osteoblast differentiation
Biochemical Journal, 2011Co-Authors: Chandi C Mandal, S E Harris, Suthakar Ganapathy, Yves Gorin, Kalyankar Mahadev, Karen Block, Hanna E Abboud, Goutam Ghoshchoudhury, Nandini GhoshchoudhuryAbstract:BMP-2 (bone morphogenetic protein-2) promotes differentiation of Osteoblast Precursor cells to mature Osteoblasts that form healthy bone. In the present study, we demonstrate a novel mechanism of BMP-2-induced Osteoblast differentiation. The antioxidant NAC ( N -acetyl-L-cysteine) and the flavoprotein enzyme NAD(P)H oxidase inhibitor DPI (diphenyleneiodonium) prevented BMP-2-stimulated alkaline phosphatase expression and mineralized bone nodule formation in mouse 2T3 pre-Osteoblasts. BMP-2 elicited a rapid generation of ROS (reactive oxygen species) concomitant with increased activation of NAD(P)H oxidase. NAC and DPI inhibited BMP-2-induced ROS production and NAD(P)H oxidase activity respectively. NAD(P)H oxidases display structurally similar catalytic subunits (Nox1–5) with differential expression in various cells. We demonstrate that 2T3 pre-Osteoblasts predominantly express the Nox4 isotype of NAD(P)H oxidase. To extend this finding, we tested the functional effects of Nox4. Adenovirus-mediated expression of dominant-negative Nox4 inhibited BMP-2-induced alkaline phosphatase expression. BMP-2 promotes expression of BMP-2 for maintenance of the Osteoblast phenotype. NAC and DPI significantly blocked BMP-2-stimulated expression of BMP2 mRNA and protein due to a decrease in BMP2 gene transcription. Dominant-negative Nox4 also mimicked this effect of NAC and DPI. Our results provide the first evidence for a new signalling pathway linking BMP-2-stimulated Nox4-derived physiological ROS to BMP-2 expression and Osteoblast differentiation.
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bmp 2 modulates β catenin signaling through stimulation of lrp5 expression and inhibition of β trcp expression in Osteoblasts
Journal of Cellular Biochemistry, 2009Co-Authors: Ming Zhang, S E Harris, Rong Xie, Ying Yan, Yong Bin Lim, Dezhi Tang, Ann Chen, Peter Tai, Lianping XingAbstract: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.
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the zinc finger transcription factor gli2 mediates bone morphogenetic protein 2 expression in Osteoblasts in response to hedgehog signaling
Molecular and Cellular Biology, 2006Co-Authors: Ming Zhao, Di Chen, Mei Qiao, Babatunde O Oyajobi, S E Harris, Gregory R MundyAbstract:Bone morphogenetic protein 2 (BMP-2) plays a critical role in Osteoblast function. In Drosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression of dpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. We hypothesize that Gli2 regulates BMP-2 gene transcription and thus Osteoblast differentiation. In the present study, we show that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in Osteoblast Precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in Osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for Osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and Osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in Osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal Osteoblast differentiation.
Ming Zhao - One of the best experts on this subject based on the ideXlab platform.
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wnt β catenin signaling activates bone morphogenetic protein 2 expression in Osteoblasts
Bone, 2013Co-Authors: Rongrong Zhang, Di Chen, Babatunde O Oyajobi, S E Harris, Christopher Tsao, Hongwen Deng, Ming ZhaoAbstract:The BMP and Wnt/β-catenin signaling pathways cooperatively regulate Osteoblast differentiation and bone formation. Although BMP signaling regulates gene expression of the Wnt pathway, much less is known about whether Wnt signaling modulates BMP expression in Osteoblasts. Given the presence of putative Tcf/Lef response elements that bind β-catenin/TCF transcription complex in the BMP2 promoter, we hypothesized that the Wnt/β-catenin pathway stimulates BMP2 expression in osteogenic cells. In this study, we showed that Wnt/β-catenin signaling is active in various Osteoblast or Osteoblast Precursor cell lines, including MC3T3-E1, 2T3, C2C12, and C3H10T1/2 cells. Furthermore, crosstalk between the BMP and Wnt pathways affected BMP signaling activity, Osteoblast differentiation, and bone formation, suggesting Wnt signaling is an upstream regulator of BMP signaling. Activation of Wnt signaling by Wnt3a or overexpression of β-catenin/TCF4 both stimulated BMP2 transcription at promoter and mRNA levels. In contrast, transcription of BMP2 in osteogenic cells was decreased by either blocking the Wnt pathway with DKK1 and sFRP4, or inhibiting β-catenin/TCF4 activity with FWD1/β-TrCP, ICAT, or ΔTCF4. Using a site-directed mutagenesis approach, we confirmed that Wnt/β-catenin transactivation of BMP2 transcription is directly mediated through the Tcf/Lef response elements in the BMP2 promoter. These results, which demonstrate that the Wnt/β-catenin signaling pathway is an upstream activator of BMP2 expression in Osteoblasts, provide novel insights into the nature of functional cross talk integrating the BMP and Wnt/β-catenin pathways in Osteoblastic differentiation and maintenance of skeletal homeostasis.
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the zinc finger transcription factor gli2 mediates bone morphogenetic protein 2 expression in Osteoblasts in response to hedgehog signaling
Molecular and Cellular Biology, 2006Co-Authors: Ming Zhao, Di Chen, Mei Qiao, Babatunde O Oyajobi, S E Harris, Gregory R MundyAbstract:Bone morphogenetic protein 2 (BMP-2) plays a critical role in Osteoblast function. In Drosophila, Cubitus interruptus (Ci), which mediates hedgehog signaling, regulates gene expression of dpp, the ortholog of mammalian BMP-2. Null mutation of the transcription factor Gli2, a mammalian homolog of Ci, results in severe skeletal abnormalities in mice. We hypothesize that Gli2 regulates BMP-2 gene transcription and thus Osteoblast differentiation. In the present study, we show that overexpression of Gli2 enhances BMP-2 promoter activity and mRNA expression in Osteoblast Precursor cells. In contrast, knocking down Gli2 expression by Gli2 small interfering RNA or genetic ablation of the Gli2 gene results in significant inhibition of BMP-2 gene expression in Osteoblasts. Promoter analyses, including chromatin immunoprecipitation and electrophoretic mobility shift assays, provided direct evidence that Gli2 physically interacts with the BMP-2 promoter. Functional studies showed that Gli2 is required for Osteoblast maturation in a BMP-2-dependent manner. Finally, Sonic hedgehog (Shh) stimulates BMP-2 promoter activity and Osteoblast differentiation, and the effects of Shh are mediated by Gli2. Taken together, these results indicate that Gli2 mediates hedgehog signaling in Osteoblasts and is a powerful activator of BMP-2 gene expression, which is required in turn for normal Osteoblast differentiation.
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e3 ubiquitin ligase smurf1 mediates core binding factor α1 runx2 degradation and plays a specific role in Osteoblast differentiation
Journal of Biological Chemistry, 2003Co-Authors: Ming Zhao, Mei Qiao, Babatunde O Oyajobi, Gregory R Mundy, Di ChenAbstract:Abstract Osteoblast differentiation and bone formation is stimulated by bone morphogenetic protein (BMP)-2 and its downstream signaling molecules Smad1 and -5 and the Osteoblast-specific transcription factor core-binding factor α1 (Cbfa1). Proteolytic degradation of Smad1 and Cbfa1 is proteasome-dependent, and intracellular concentrations of Smad1 and Cbfa1 are enhanced by inhibition of the 26 S proteasome. Smad1 degradation is mediated by the E3 ubiquitin ligase Smurf1 (Smad ubiquitin regulatory factor 1), but the specific E3 ligase responsible for Cbfa1 degradation has not been identified. Because Cbfa1 interacts with Smad1, whose degradation is mediated by Smurf1, we examined the effect of Smurf1 on Cbfa1 degradation in Osteoblast Precursor cells. Smurf1 interacts directly with Cbfa1 and mediates Cbfa1 degradation in a ubiquitin- and proteasome-dependent manner. Because Smurf1 controls the intracellular concentrations of several key molecules in the bone formation cascade, we examined the effect of a mutant form of Smurf1 in Osteoblasts and found that expression of mutant Smurf1 markedly enhanced osteoblas tdifferentiation. Smurf1 therefore appears to be an important regulatory factor in Osteoblast differentiation and a potential molecular target for identification of bone anabolic agents.
Andrew P Mcmahon - One of the best experts on this subject based on the ideXlab platform.
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distinct roles for hedgehog and canonical wnt signaling in specification differentiation and maintenance of Osteoblast progenitors
Development, 2006Co-Authors: Stephen J Rodda, Andrew P McmahonAbstract:Hedgehog and canonical Wnt/β-catenin signaling are implicated in development of the Osteoblast, the bone matrix-secreting cell of the vertebrate skeleton. We have used genetic approaches to dissect the roles of these pathways in specification of the Osteoblast lineage. Previous studies indicate that Ihh signaling in the long bones is essential for initial specification of an Osteoblast progenitor to a Runx2 + Osteoblast Precursor. We show here that this is a transient requirement, as removal of Hh responsiveness in later Runx2 + , Osx1 + Osteoblast Precursors does not disrupt the formation of mature Osteoblasts. By contrast, the removal of canonical Wnt signaling by conditional removal of the β-catenin gene in early Osteoblast progenitors or in Runx2 + , Osx1 + Osteoblast Precursors results in a similar phenotype: Osteoblasts fail to progress to a terminal osteocalcin + fate and instead convert to a chondrocyte fate. By contrast, stabilization of β-catenin signaling in Runx2 + , Osx1 + Osteoblast Precursors leads to the premature differentiation of bone matrix secreting Osteoblasts. These data demonstrate that commitment within the Osteoblast lineage requires sequential, stage-specific, Ihh and canonical Wnt/β-catenin signaling to promote osteogenic, and block chondrogenic, programs of cell fate specification.
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distinct roles for hedgehog and canonical wnt signaling in specification differentiation and maintenance of Osteoblast progenitors
Development, 2006Co-Authors: Stephen J Rodda, Andrew P McmahonAbstract:Hedgehog and canonical Wnt/beta-catenin signaling are implicated in development of the Osteoblast, the bone matrix-secreting cell of the vertebrate skeleton. We have used genetic approaches to dissect the roles of these pathways in specification of the Osteoblast lineage. Previous studies indicate that Ihh signaling in the long bones is essential for initial specification of an Osteoblast progenitor to a Runx2+ Osteoblast Precursor. We show here that this is a transient requirement, as removal of Hh responsiveness in later Runx2+, Osx1+ Osteoblast Precursors does not disrupt the formation of mature Osteoblasts. By contrast, the removal of canonical Wnt signaling by conditional removal of the beta-catenin gene in early Osteoblast progenitors or in Runx2+, Osx1+ Osteoblast Precursors results in a similar phenotype: Osteoblasts fail to progress to a terminal osteocalcin+ fate and instead convert to a chondrocyte fate. By contrast, stabilization of beta-catenin signaling in Runx2+, Osx1+ Osteoblast Precursors leads to the premature differentiation of bone matrix secreting Osteoblasts. These data demonstrate that commitment within the Osteoblast lineage requires sequential, stage-specific, Ihh and canonical Wnt/beta-catenin signaling to promote osteogenic, and block chondrogenic, programs of cell fate specification.
