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Different protein kinase C isozymes exert opposing effects on osteogenesis of human mesenchymal stem cells
ABSTRACTS / Bone 42 (2008) S17–S110
acute PTH treatment, real-time PCR demonstrated no significant difference in MKP-1 in less differentiated 2-week-old ectopic ossicles (implanted BMSCs) generated in mice in vivo. Preliminary immunohistochemical analysis on 4-week-old ossicles showed an increase in MKP-1 expression following an anabolic PTH treatment compared to vehicle control. These findings suggest MKP-1 as a critical regulator of the PTH and PTHrP response in osteoblasts and may be an important target gene in the anabolic action of PTH in bone. doi:10.1016/j.bone.2007.12.028
19 Runx2 and HDAC3 mediated-repression is relieved in differentiating human osteoblast cells to allow high BSP expression Virginie Lamour, Cédric Detry, Vincent Castronovo, Akeila Bellahcène Metastasis Research Laboratory, Liège, Belgium Bone sialoprotein (BSP) is a bone matrix glycoprotein whose expression coincides with terminal osteoblastic differentiation and the onset of mineralization. In this study we show that BSP expression is considerably increased in confluent Saos-2 human osteosarcoma cells and in differentiating normal human osteoblasts, concomitantly with the decrease of Runx2, a key transcription factor controlling bone formation. Therefore, we investigated the role of Runx2 in the regulation of BSP expression in Saos-2 cells. Using a mobility shift assay, we demonstrated that Runx2 binds to the BSP promoter only in preconfluent cells. Histone deacetylase 3 (HDAC3) has been recently shown to act as a Runx2 co-repressor. Chromatin immunoprecipitation assays demonstrated that both Runx2 and HDAC3 are detectable at the BSP promoter in preconfluent Saos-2 cells but not when they are confluent and overexpress BSP. Consistently, nuclear Runx2 protein level is down-regulated, whereas Saos-2 cells became increasingly confluent. Finally, the suppression of HDAC3, Runx2, or both by RNA interference induced the expression of BSP at both mRNA and protein levels in Saos-2 cells. Our data demonstrate that Runx2 and HDAC3 repress BSP gene expression and that this repression is suspended upon osteoblastic cell differentiation. Both the nuclear disappearance of Runx2 and the non-recruitment of HDAC3 represent new means to relieve Runx2-mediated suppression of BSP expression, thus allowing the acquisition of a fully differentiated and mineralizationcompetent phenotype by osteoblast cells. doi:10.1016/j.bone.2007.12.029
20 Different protein kinase C isozymes exert opposing effects on osteogenesis of human mesenchymal stem cells Jun Liu, Clemens A. van Blitterswijk, Jan de Boer Tissue Regeneration, University of Twente, Enschede, Netherlands
S25
Introduction: Protein kinase C (PKC) signaling has been implicated in proliferation of human, rat and mouse osteoblasts and also in osteogenic differentiation of a murine mesenchymal cell line. The effects of PKC signaling on osteogenesis of human mesenchymal stem cells (hMSC) are still unknown. Here, a pharmaceutical approach was used to assess the role of PKC on osteogenic differentiation of hMSCs. Materials and methods: hMSCs were seeded at 1000 cells/ cm2 and incubated with different reagents for 4–6 days. Each experiment was performed both in basic medium and in osteogenic medium with Dexmatheson. At the end of culture period, ALP expression levels were measured by flow cytometry. Results: Activation of PKC by exposure to phorbol 12myristate 13-acetate (PMA) inhibited osteogenic differentiation of hMSCs by showing the reduced ALP expression and mineralization. Staurosporine, an inhibitor of PKC, inhibited ALP expression dose-dependently. This indicated that PKC is required for osteogenesis of hMSCs. Gö6976, an inhibitor specific for the conventional PKC, up-regulated ALP expression and calcium deposition of hMSCs, suggesting that the conventional family plays a negative role in hMSC osteogenesis. However, inhibition of the novel PKC isozyme PKCΔ by Rottlerin decreased the ALP expression to greater extent and changed the cells’ cytoskeletal pattern. Conclusion: This study demonstrated that different PKC isozymes played different roles on hMSCs osteogenesis. From a tissue engineering point of view, making use of this, by either fine-tuning the activation of conventional PKC or PKCΔ, might improve the osteogenic differentiation performance of hMSCs. doi:10.1016/j.bone.2007.12.030
21 Stage-specific subsets of perichondrial osteoblasts differentially relate to endothelium and bone and display different destinations in development Christa Maes a, Tatsuya Kobayashi a , Martin K. Selig b, Sanford I. Roth b, Henry M. Kronenberg a a Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, USA b Department of Pathology, Massachusetts General Hospital, Boston, USA During endochondral bone development, the first osteoblasts arise in the perichondrium around the cartilaginous bone model. The presumed role of these cells in trabecular and cortical bone formation raises the question of whether and how they move from the perichondrium to the primary ossification center (POC). In this study we performed in vivo cell tracing to follow stage-specific subsets of osteoblast lineage cells during embryonic bone development. For this purpose, we generated OsterixCreERt; Rosa26R and Collagen1(3.2 kb)-CreERt;Rosa26R mice in which Cre is transiently activated by tamoxifen (Tam) such that osteoblasts become genetically marked and can be detected by X-gal staining of Rosa26R LacZ activity. In both
acute PTH treatment, real-time PCR demonstrated no significant difference in MKP-1 in less differentiated 2-week-old ectopic ossicles (implanted BMSCs) generated in mice in vivo. Preliminary immunohistochemical analysis on 4-week-old ossicles showed an increase in MKP-1 expression following an anabolic PTH treatment compared to vehicle control. These findings suggest MKP-1 as a critical regulator of the PTH and PTHrP response in osteoblasts and may be an important target gene in the anabolic action of PTH in bone. doi:10.1016/j.bone.2007.12.028
19 Runx2 and HDAC3 mediated-repression is relieved in differentiating human osteoblast cells to allow high BSP expression Virginie Lamour, Cédric Detry, Vincent Castronovo, Akeila Bellahcène Metastasis Research Laboratory, Liège, Belgium Bone sialoprotein (BSP) is a bone matrix glycoprotein whose expression coincides with terminal osteoblastic differentiation and the onset of mineralization. In this study we show that BSP expression is considerably increased in confluent Saos-2 human osteosarcoma cells and in differentiating normal human osteoblasts, concomitantly with the decrease of Runx2, a key transcription factor controlling bone formation. Therefore, we investigated the role of Runx2 in the regulation of BSP expression in Saos-2 cells. Using a mobility shift assay, we demonstrated that Runx2 binds to the BSP promoter only in preconfluent cells. Histone deacetylase 3 (HDAC3) has been recently shown to act as a Runx2 co-repressor. Chromatin immunoprecipitation assays demonstrated that both Runx2 and HDAC3 are detectable at the BSP promoter in preconfluent Saos-2 cells but not when they are confluent and overexpress BSP. Consistently, nuclear Runx2 protein level is down-regulated, whereas Saos-2 cells became increasingly confluent. Finally, the suppression of HDAC3, Runx2, or both by RNA interference induced the expression of BSP at both mRNA and protein levels in Saos-2 cells. Our data demonstrate that Runx2 and HDAC3 repress BSP gene expression and that this repression is suspended upon osteoblastic cell differentiation. Both the nuclear disappearance of Runx2 and the non-recruitment of HDAC3 represent new means to relieve Runx2-mediated suppression of BSP expression, thus allowing the acquisition of a fully differentiated and mineralizationcompetent phenotype by osteoblast cells. doi:10.1016/j.bone.2007.12.029
20 Different protein kinase C isozymes exert opposing effects on osteogenesis of human mesenchymal stem cells Jun Liu, Clemens A. van Blitterswijk, Jan de Boer Tissue Regeneration, University of Twente, Enschede, Netherlands
S25
Introduction: Protein kinase C (PKC) signaling has been implicated in proliferation of human, rat and mouse osteoblasts and also in osteogenic differentiation of a murine mesenchymal cell line. The effects of PKC signaling on osteogenesis of human mesenchymal stem cells (hMSC) are still unknown. Here, a pharmaceutical approach was used to assess the role of PKC on osteogenic differentiation of hMSCs. Materials and methods: hMSCs were seeded at 1000 cells/ cm2 and incubated with different reagents for 4–6 days. Each experiment was performed both in basic medium and in osteogenic medium with Dexmatheson. At the end of culture period, ALP expression levels were measured by flow cytometry. Results: Activation of PKC by exposure to phorbol 12myristate 13-acetate (PMA) inhibited osteogenic differentiation of hMSCs by showing the reduced ALP expression and mineralization. Staurosporine, an inhibitor of PKC, inhibited ALP expression dose-dependently. This indicated that PKC is required for osteogenesis of hMSCs. Gö6976, an inhibitor specific for the conventional PKC, up-regulated ALP expression and calcium deposition of hMSCs, suggesting that the conventional family plays a negative role in hMSC osteogenesis. However, inhibition of the novel PKC isozyme PKCΔ by Rottlerin decreased the ALP expression to greater extent and changed the cells’ cytoskeletal pattern. Conclusion: This study demonstrated that different PKC isozymes played different roles on hMSCs osteogenesis. From a tissue engineering point of view, making use of this, by either fine-tuning the activation of conventional PKC or PKCΔ, might improve the osteogenic differentiation performance of hMSCs. doi:10.1016/j.bone.2007.12.030
21 Stage-specific subsets of perichondrial osteoblasts differentially relate to endothelium and bone and display different destinations in development Christa Maes a, Tatsuya Kobayashi a , Martin K. Selig b, Sanford I. Roth b, Henry M. Kronenberg a a Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, USA b Department of Pathology, Massachusetts General Hospital, Boston, USA During endochondral bone development, the first osteoblasts arise in the perichondrium around the cartilaginous bone model. The presumed role of these cells in trabecular and cortical bone formation raises the question of whether and how they move from the perichondrium to the primary ossification center (POC). In this study we performed in vivo cell tracing to follow stage-specific subsets of osteoblast lineage cells during embryonic bone development. For this purpose, we generated OsterixCreERt; Rosa26R and Collagen1(3.2 kb)-CreERt;Rosa26R mice in which Cre is transiently activated by tamoxifen (Tam) such that osteoblasts become genetically marked and can be detected by X-gal staining of Rosa26R LacZ activity. In both