Osteogenic differentiation of C2C12 myogenic progenitor cells requires the Fos-related antigen Fra-1 – A novel target of Runx2

Bone formation is a developmental process requiring the differentiation of mesenchymal stem cells into osteoblasts. It is established that Runx2 tightly regulates osteoblast differentiation and bone formation. Fos-related antigen Fra-1 is an essential factor for bone formation. Current evidence does not support a relationship between Fra-1 and Runx2 in osteogenesis. Here, we explored the possibility that Runx2 regulates Fra-1 expression during osteogenic differentiation of C2C12 myogenic progenitor cells. Expression of Fra-1 was induced rapidly after activation of Runx2 in a Tet-on stable C2C12 cell-line (C2C12/Runx2^Dox sub-line). Transient transfection assay showed that Runx2 activates Fra-1 promoter-reporter activity, suggesting that Fra-1 may be a direct target of Runx2. To determine the minimal region of the Fra-1 promoter that was activated by Runx2, a series of Fra-1 promoter deletion constructs were made. By transient transfection assay, we defined the minimal region to the proximal 342 bp (?84 to +258). Two potential Runx2-binding sites (at positions +139 and +208) were predicted within this region. Mutation of the Runx2 motif at position +208 significantly decreased Fra-1 promoter activity compared to wild type, whereas mutation of Runx2 at position +139 had no effect. Electrophoretic mobility shift assay (EMSA) demonstrated the existence of one atypical Runx2-binding element at position +208, and chromatin immunoprecipitation (ChIP) assay revealed that Runx2 bound to the native Fra-1 promoter in vivo via this site. Finally, forced expression of Fra-1 resulted in upregulation of alkaline phosphatase (ALP), a marker of early osteoblast differentiation. Together, these results indicate that Fra-1 is a direct target of Runx2 during osteogenic differentiation of C2C12 myogenic progenitor cells.     

The Ewing's sarcoma fusion protein,EWS‐FLI,binds Runx2 and blocks osteoblast differentiation

Ewing's sarcomas are highly aggressive round cell tumors of bone and soft tissues that afflict children and young adults. The majority of these tumors harbor the t(11;22) translocation and express the fusion protein EWS‐FLI. Modern molecular profiling experiments indicate that Ewing's tumors originate from mesenchymal precursors in young individuals. EWS‐FLI alters the morphology of mesenchymal cells and prevents lineage specification; however, the molecular mechanisms for differentiation arrest are unclear. We recently showed that EWS‐FLI binds Runx2, a master regulator of osteoblast differentiation. In this report, we demonstrate that FLI sequences within EWS‐FLI are responsible for interactions with Runx2. EWS‐FLI blocks the expression of osteoblastic genes in a multipotent progenitor cell line that requires Runx2 to integrate bone morphogenic protein (Bmp)2 signaling while increasing proliferation and altering cell morphology. These results demonstrate that EWS‐FLI blocks the ability of Runx2 to induce osteoblast specification of a mesenchymal progenitor cell. Disrupting interactions between Runx2 and EWS‐FLI1 may promote differentiation of the tumor cell. J. Cell. Biochem. 111: 933–943, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Haploinsufficiency of Runx2 results in bone formation decrease and different BSP expression pattern changes in two transgenic mouse models

Runx2 has been identified as "a master gene" for the differentiation of osteoblasts and Runx2-deficient mice has demonstrated a complete absence of mature osteoblast and ossification. To further characterize the Runx2 responsive elements within the bone sialoprotein (BSP) promoter and further investigate into the role of Runx2 haploinsufficiency in osteoblast differentiation, mBSP9.0Luc mice and mBSP4.8Luc mice were crossed with Runx2-deficient mice respectively. Luciferase assay, micro CT scan, and histological analysis were performed using tissues isolated from mBSP9.0luc/Runx2+/- mice, mBSP4.8luc/Runx2+/- mice and their corresponding Runx2+/+ littermates. Alkaline phosphatase activity, mineralization assays and RT-PCR analysis using calvarial osteoblasts isolated from these transgenic mice were also performed. Luciferase assay demonstrated an early increase in luciferase expression in mBSP9.0luc/Runx2+/- mice before the expression level of luciferase dramatically decreased and turned lower than that in their control littermates in later stages. In contrast, luciferase expression in mBSP4.8luc/Runx2+/- failed to show such an early increase. Micro CT scan and histological analysis showed that BMD and trabecular bone volume were decreased and bone formation was delayed in Runx2+/- mice. Furthermore, mineralization assay and semi-quantitative RT-PCR assay demonstrated a gene-dose-dependent decrease in bone nodule formation and bone marker genes expression levels in cultured calvarial osteoblasts derived from Runx2 knockout mice. Reconstitution of Runx2-null cells with Runx2 vector partially rescued the osteoblast function defects. In conclusion, the 9.0 kb BSP promoter demonstrated a higher tissue-specific regulation of the BSP gene by Runx2 in vivo and full Runx2 gene dose is essential for osteoblast differentiation and normal bone formation.     

Multilineage differentiation of dental follicle cells and the roles of Runx2 over‐expression in enhancing osteoblast/cementoblast‐related gene expression in dental follicle cells

Objectives: Dental follicle cells (DFCs) provide the origin of periodontal tissues, and Runx2 is essential for bone formation and tooth development. In this study, pluripotency of DFCs was evaluated and effects of Runx2 on them were investigated. Materials and methods: The DFCs were induced to differentiate towards osteoblasts, adipocytes or chondrocytes, and alizarin red staining, oil red O staining or alcian blue staining was performed to reveal the differentiated states. Bone marrow stromal cells (BMSCs) and primary mouse fibroblasts served as controls. DFCs were also infected with recombinant retroviruses encoding either full‐length Runx2 or mutant Runx2 without the VWRPY motif. Western blot analysis, real‐time real time RT‐PCR and in vitro mineralization assay were performed to evaluate the effects of full‐length Runx2 or mutant Runx2 on osteogenic/cementogenic differentiation of the cells. Results: The above‐mentioned staining methods demonstrated that DFCs were successfully induced to differentiate towards osteoblasts, adipocytes or chondrocytes respectively, confirming the existence of pluripotent mesenchymal stem cells in dental follicle tissues. However, staining intensity in DFC cultures was weaker than in BMSC cultures. Real‐time PCR analysis indicated that mutant Runx2 induced a more pronounced increase in expression levels of OC, OPN, Col I and CP23 than full‐length Runx2. Mineralization assay also showed that mutant Runx2 increased mineralization nodule formation more prominently than full‐length Runx2. Conclusions: Multipotent DFCs can be induced to differentiate towards osteoblasts, adipocytes or chondrocytes in vitro. Runx2 over‐expression up‐regulated expression levels of osteoblast/cementoblast‐related genes and in vitro enhanced osteogenic differentiation of DFCs. In addition, mutant Runx2‐induced changes in DFCs were more prominent than those induced by full‐length Runx2.     

Runx2‐mediated bcl‐2 gene expression contributes to nitric oxide protection against hydrogen peroxide‐induced osteoblast apoptosis

Nitric oxide (NO) can regulate osteoblast activities. This study was aimed to evaluate the protective effects of pretreatment with sodium nitroprusside (SNP) as a source of NO on hydrogen peroxide‐induced osteoblast insults and its possible mechanisms. Exposure of human osteosarcoma MG63 cells to hydrogen peroxide significantly increased cellular oxidative stress, but decreased ALP activity and cell viability, inducing cell apoptosis. Pretreatment with 0.3 mM SNP significantly lowered hydrogen peroxide‐induced cell insults. Treatment of human MG63 cells with hydrogen peroxide inhibited Bcl‐2 mRNA and protein production, but pretreatment with 0.3 mM SNP significantly ameliorated such inhibition. Sequentially, hydrogen peroxide decreased the mitochondrial membrane potential, but increased the levels of cytochrome c and caspase‐3 activity. Pretreatment with 0.3 mM SNP significantly lowered such alterations. Exposure to hydrogen peroxide decreased Runx2 mRNA and protein syntheses. However, pretreatment with 0.3 mM SNP significantly lowered the suppressive effects. Runx2 knockdown using RNA interference inhibited Bcl‐2 mRNA production in human MG63 cells. Protection of pretreatment with 0.3 mM SNP against hydrogen peroxide‐induced alterations in ALP activity, caspase‐3 activity, apoptotic cells, and cell viability were also alleviated after administration of Runx2 small interference RNA. Thus, this study shows that pretreatment with 0.3 mM SNP can protect human MG63 cells from hydrogen peroxide‐induced apoptotic insults possibly via Runx2‐involved regulation of bcl‐2 gene expression. J. Cell. Biochem. 108: 1084–1093, 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.