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Quantitative analysis of the in vitro mineralization of preosteoblasts after exposure to ionizing radiation under standard and osteogenic conditions
S86
Abstracts
In conclusion, P2Y2 receptor overexpression in rats results in decreased bone mineral density, partially due to increased bone resorption, but also decreased formation (detected by serum bone markers and bone histomorphometry). This is consistent with previously published in vitro effects of P2Y2 receptor activation in osteoblasts. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.250
PP062 In vitro expression of bone related matrix proteins on osteogenic cells treated with TGF-β1 T.A.G. Donatoa,⁎, E.F. Martinezb, G.D.C. Ribeiro-Santosa, V.E. Arana-Chaveza a Biomaterials and Oral Biology, University of São Paulo, São Paulo, Brazil b Oral Pathology, Institute and Reseach Center São Leopoldo Mandic, Campinas, Brazil Abstract: Transforming growth factor-beta 1 (TGF-β1) is an important mediator of cell growth, but conflicting results have been reported regarding its effect on osteoblastic cell differentiation. Our previous study on calvaria-derived osteogenic primary cultures had demonstrated that TGF-β1 inhibited the differentiation of osteoblastic cells; they revealed typical fibroblasts morphology with no extracellular matrix formation and therefore with few collagen fibrils. This in vitro study aimed to evaluate the effects of TGF-β1 (5 ng/μL), with different supplementation time periods (7, 10 and 14 days) on the establishment of osteogenic phenotype of MC3T3-E1 line cell. For comparison, ascorbic acid (AA; 5 μg/mL)/β-glycerophosphate (βGP; 10 mM), dexamethasone (Dex; 10− 7 M) and TGF-β1/Dex were also added. We have performed quantitative PCR and Western blotting to evaluate the alkaline phosphatase (ALP), type I collagen (COL I) and osteocalcin (OC) expression after these treatments. The results have shown that ALP, COL I and OC expression was enhanced by AA/βGP treatment. In contrast, TGF-β1 alone or supplemented with Dex decreased the expression of these proteins in all studied period times. The present results demonstrated that the TGF-β1 pathway may interfere on the biomineralization and differentiation of osteoblastic cells by reducing the expression of key extracellular matrix proteins on bone development. Grants are from FAPESP 10/00352-6 and 10/ 18094-3. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.251
PP063 Retinoic acid inhibits osteoblastic mineralization and promotes osteocyte formation T. Linda,⁎, A. Sundqvista, L. Hua, G. Pejlerb, A. Jacobsona, H. Melhusa a Uppsala University, Sweden b Swedish University of Agricultural Sciences, Uppsala, Sweden Abstract: High levels of serum retinol have been associated with thinning of bone cortex and increased risk of fractures in humans. In an effort to clarify the direct effect of vitamin A on normal bone formation we have studied the effects of retinoic acid (RA), the active metabolite of vitamin A, in human primary osteoblasts and in murine osteoblast cell lines. We have also used a retinoic acid receptor (RAR) antagonist together with inhibitors of endogenous RA metabolism in mineralization experiments in vitro and studied mice suffering from hypervitaminosis A. We have analyzed gene and protein expression and osteoblast proliferation in vitro and measured mineralization apposition rates in vivo. We find that RAR-signaling inhibits osteoblast mineralization in vitro and in vivo. Osteoblastic differentiation induces up-regulation of the RA degrading enzyme Cyp26b1, indicating that osteoblasts are actively reducing endogenous RA levels during differentiation. In line with this, RA reduced expression of osteoblast marker genes. In contrast to osteoblast markers, RA induced expression of osteocyte markers. In addition, we found that RA inhibited osteoblast proliferation in vitro. In conclusion, we have shown that RAR signaling inhibits both osteoblast growth and function and promotes osteocyte formation. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.252
PP064 High bone mass phenotype is associated with impaired interferon signaling in skeletal (mesenchymal) stem cells: Potential mechanism for enhanced bone formation by Wnt singalling W. Qiua,⁎, T.E. Andersena, K. Drewsb, L. Chena, B. Abdallaha, J. Adjayeb, M. Kassema a Laboratory for Molecular Endocrinology (KMEB), Department of Endocrinology and Metabolism, University Hospital of Odense, Odense, Denmark b Molecular Embryology and Aging Group, Max-Planck Institute for Molecular Genetics, Berlin, Germany Abstract: High bone mass phenotype (HBM) is an autosomal dominant disease caused by enhanced Wnt singalling due to an activating mutation in canonical Wnt co-receptor LRP5. The molecular mechanisms mediating enhancing osteoblastic functions in these patients are highly debated. In this study, we observed that sera from HBM patients promoted human skeletal (mesenchymal) stem cell (hMSC) proliferation, osteoblast differentiation and Wnt signaling, but inhibited adipocyte differentiation. DNA microarray analysis revealed that HBM sera significantly inhibited the expression of several interferon (IFN) inducible genes. Type I and type II IFN luciferase reporter cell assays confirmed that HBM sera inhibited type I IFN signaling possibly due to the presence of IFN inhibitor. In addition, we observed that IFNβ stimulated the expression of IFN inducible genes, inhibited cell proliferation, osteoblast differentiation and enhanced adipocyte differentiation of hMSC compared to another type I IFN (IFNα2a) and type II IFN (IFNγ) ligands. Furthermore, IFNβ inhibited Wnt signaling as demonstrated by TCF-luciferase reporter assay and gene expression analysis of Wnt regulated genes. Immune staining and Western blot analysis demonstrated that IFNβ reduced β-catenin nuclear translocation through inhibition of AKT phosphorylation which decreased the phosphorylation of β-catenin at Ser552. We also observed that IFNβ up-regulated the expression of DKK1 and DKK2 independent of β-catenin activity. Finally, we observed that canonical Wnt signaling also inhibited type I IFN signaling. Our data demonstrate a novel mechanism underlying HBM phenotype where Wnt signaling inhibits IFNβ signaling in hMSC as a mechanism for enhancing osteoblast and inhibition of adipocyte differentiation of hMSC. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.253
PP065 Quantitative analysis of the in vitro mineralization of preosteoblasts after exposure to ionizing radiation under standard and osteogenic conditions Y. Hua,⁎, P. Laua, C. Hellwegb, C. Baumstark-Khanb, G. Reitzb a German Aerospace Center (DLR), Cologne, Germany b Radiation Biology, German Aerospace Center (DLR), Cologne, Germany Bone loss can be seen in astronauts traveling in space as well as patients who receive therapeutic radiation. However, until now little is known about the molecular effects of radiation regarding bone cells, especially concerning the bone forming osteoblasts. The main characteristic of mature bone cells is their ability to deposit extracellular matrix that mineralize under in vitro culture conditions. The deposited calcium co-precipitates with phosphate to form bone nodules, which was assessed using histochemical alizarin red-staining after radiation. Furthermore, bone cell mineralization was detected and quantified based on the specific binding of the fluorescent OsteoImage™ staining reagent to the hydroxyapatite portion of the bonelike nodules deposited by cells. To access the effect of ionizing radiation on cellular differentiation and mineralization status, preosteoblasts were incubated under both standard and osteogenic conditions. Osteoblast differentiation was evaluated based on alkaline phosphatase activity (ALP) and expression of mRNA encoding ALP, osteocalcin (OCN), as well as transcription factors Runx2 and Osterix which are essential for osteoblastic differentiation. Our results indicate that the gene expression levers of marker genes involved in the differentiation and the mineral content of the ECM have dose dependent manners when cells are treated with IR under osteogenic conditions. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.254
PP066/NIOP06 Regulation of osteoclast formation by toll-like receptors 2 and 5 A. Kassema,⁎, P. Lunberga, C. Lindholmb, P. Souzac, U. Lernera
Abstracts
In conclusion, P2Y2 receptor overexpression in rats results in decreased bone mineral density, partially due to increased bone resorption, but also decreased formation (detected by serum bone markers and bone histomorphometry). This is consistent with previously published in vitro effects of P2Y2 receptor activation in osteoblasts. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.250
PP062 In vitro expression of bone related matrix proteins on osteogenic cells treated with TGF-β1 T.A.G. Donatoa,⁎, E.F. Martinezb, G.D.C. Ribeiro-Santosa, V.E. Arana-Chaveza a Biomaterials and Oral Biology, University of São Paulo, São Paulo, Brazil b Oral Pathology, Institute and Reseach Center São Leopoldo Mandic, Campinas, Brazil Abstract: Transforming growth factor-beta 1 (TGF-β1) is an important mediator of cell growth, but conflicting results have been reported regarding its effect on osteoblastic cell differentiation. Our previous study on calvaria-derived osteogenic primary cultures had demonstrated that TGF-β1 inhibited the differentiation of osteoblastic cells; they revealed typical fibroblasts morphology with no extracellular matrix formation and therefore with few collagen fibrils. This in vitro study aimed to evaluate the effects of TGF-β1 (5 ng/μL), with different supplementation time periods (7, 10 and 14 days) on the establishment of osteogenic phenotype of MC3T3-E1 line cell. For comparison, ascorbic acid (AA; 5 μg/mL)/β-glycerophosphate (βGP; 10 mM), dexamethasone (Dex; 10− 7 M) and TGF-β1/Dex were also added. We have performed quantitative PCR and Western blotting to evaluate the alkaline phosphatase (ALP), type I collagen (COL I) and osteocalcin (OC) expression after these treatments. The results have shown that ALP, COL I and OC expression was enhanced by AA/βGP treatment. In contrast, TGF-β1 alone or supplemented with Dex decreased the expression of these proteins in all studied period times. The present results demonstrated that the TGF-β1 pathway may interfere on the biomineralization and differentiation of osteoblastic cells by reducing the expression of key extracellular matrix proteins on bone development. Grants are from FAPESP 10/00352-6 and 10/ 18094-3. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.251
PP063 Retinoic acid inhibits osteoblastic mineralization and promotes osteocyte formation T. Linda,⁎, A. Sundqvista, L. Hua, G. Pejlerb, A. Jacobsona, H. Melhusa a Uppsala University, Sweden b Swedish University of Agricultural Sciences, Uppsala, Sweden Abstract: High levels of serum retinol have been associated with thinning of bone cortex and increased risk of fractures in humans. In an effort to clarify the direct effect of vitamin A on normal bone formation we have studied the effects of retinoic acid (RA), the active metabolite of vitamin A, in human primary osteoblasts and in murine osteoblast cell lines. We have also used a retinoic acid receptor (RAR) antagonist together with inhibitors of endogenous RA metabolism in mineralization experiments in vitro and studied mice suffering from hypervitaminosis A. We have analyzed gene and protein expression and osteoblast proliferation in vitro and measured mineralization apposition rates in vivo. We find that RAR-signaling inhibits osteoblast mineralization in vitro and in vivo. Osteoblastic differentiation induces up-regulation of the RA degrading enzyme Cyp26b1, indicating that osteoblasts are actively reducing endogenous RA levels during differentiation. In line with this, RA reduced expression of osteoblast marker genes. In contrast to osteoblast markers, RA induced expression of osteocyte markers. In addition, we found that RA inhibited osteoblast proliferation in vitro. In conclusion, we have shown that RAR signaling inhibits both osteoblast growth and function and promotes osteocyte formation. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.
doi:10.1016/j.bone.2012.02.252
PP064 High bone mass phenotype is associated with impaired interferon signaling in skeletal (mesenchymal) stem cells: Potential mechanism for enhanced bone formation by Wnt singalling W. Qiua,⁎, T.E. Andersena, K. Drewsb, L. Chena, B. Abdallaha, J. Adjayeb, M. Kassema a Laboratory for Molecular Endocrinology (KMEB), Department of Endocrinology and Metabolism, University Hospital of Odense, Odense, Denmark b Molecular Embryology and Aging Group, Max-Planck Institute for Molecular Genetics, Berlin, Germany Abstract: High bone mass phenotype (HBM) is an autosomal dominant disease caused by enhanced Wnt singalling due to an activating mutation in canonical Wnt co-receptor LRP5. The molecular mechanisms mediating enhancing osteoblastic functions in these patients are highly debated. In this study, we observed that sera from HBM patients promoted human skeletal (mesenchymal) stem cell (hMSC) proliferation, osteoblast differentiation and Wnt signaling, but inhibited adipocyte differentiation. DNA microarray analysis revealed that HBM sera significantly inhibited the expression of several interferon (IFN) inducible genes. Type I and type II IFN luciferase reporter cell assays confirmed that HBM sera inhibited type I IFN signaling possibly due to the presence of IFN inhibitor. In addition, we observed that IFNβ stimulated the expression of IFN inducible genes, inhibited cell proliferation, osteoblast differentiation and enhanced adipocyte differentiation of hMSC compared to another type I IFN (IFNα2a) and type II IFN (IFNγ) ligands. Furthermore, IFNβ inhibited Wnt signaling as demonstrated by TCF-luciferase reporter assay and gene expression analysis of Wnt regulated genes. Immune staining and Western blot analysis demonstrated that IFNβ reduced β-catenin nuclear translocation through inhibition of AKT phosphorylation which decreased the phosphorylation of β-catenin at Ser552. We also observed that IFNβ up-regulated the expression of DKK1 and DKK2 independent of β-catenin activity. Finally, we observed that canonical Wnt signaling also inhibited type I IFN signaling. Our data demonstrate a novel mechanism underlying HBM phenotype where Wnt signaling inhibits IFNβ signaling in hMSC as a mechanism for enhancing osteoblast and inhibition of adipocyte differentiation of hMSC. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.253
PP065 Quantitative analysis of the in vitro mineralization of preosteoblasts after exposure to ionizing radiation under standard and osteogenic conditions Y. Hua,⁎, P. Laua, C. Hellwegb, C. Baumstark-Khanb, G. Reitzb a German Aerospace Center (DLR), Cologne, Germany b Radiation Biology, German Aerospace Center (DLR), Cologne, Germany Bone loss can be seen in astronauts traveling in space as well as patients who receive therapeutic radiation. However, until now little is known about the molecular effects of radiation regarding bone cells, especially concerning the bone forming osteoblasts. The main characteristic of mature bone cells is their ability to deposit extracellular matrix that mineralize under in vitro culture conditions. The deposited calcium co-precipitates with phosphate to form bone nodules, which was assessed using histochemical alizarin red-staining after radiation. Furthermore, bone cell mineralization was detected and quantified based on the specific binding of the fluorescent OsteoImage™ staining reagent to the hydroxyapatite portion of the bonelike nodules deposited by cells. To access the effect of ionizing radiation on cellular differentiation and mineralization status, preosteoblasts were incubated under both standard and osteogenic conditions. Osteoblast differentiation was evaluated based on alkaline phosphatase activity (ALP) and expression of mRNA encoding ALP, osteocalcin (OCN), as well as transcription factors Runx2 and Osterix which are essential for osteoblastic differentiation. Our results indicate that the gene expression levers of marker genes involved in the differentiation and the mineral content of the ECM have dose dependent manners when cells are treated with IR under osteogenic conditions. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.254
PP066/NIOP06 Regulation of osteoclast formation by toll-like receptors 2 and 5 A. Kassema,⁎, P. Lunberga, C. Lindholmb, P. Souzac, U. Lernera