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To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposonrelated genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures. STEM CELLS 2007;25:371-379

The Runt domain transcription factor Runx2 (AML-3, and Cbfa1) is essential for osteoblast development, differentiation, and bone formation. Runx2 positively or negatively regulates osteoblast gene expression by interacting with a variety of transcription cofactor complexes. In this study, we identified a trichostatin A-sensitive autonomous repression domain in the amino terminus of Runx2. Using a candidate approach, we found that histone deacetylase (HDAC) 3 interacts with the amino terminus of Runx2. In transient transfection assays, HDAC3 repressed Runx2-mediated activation of the osteocalcin promoter. HDAC inhibitors and HDAC3-specific short hairpin RNAs reversed this repression. In vivo, Runx2 and HDAC3 associated with the osteocalcin promoter. These data indicate that HDAC3 regulates Runx2-mediated transcription of osteoblast genes. Suppression of HDAC3 in MC3T3 preosteoblasts by RNA interference accelerated the expression of Runx2 target genes, osteocalcin, osteopontin, and bone sialoprotein but did not significantly alter Runx2 levels. Matrix mineralization also occurred earlier in HDAC3-suppressed cells, but alkaline phosphatase expression was not affected. Thus, HDAC3 regulates osteoblast differentiation and bone formation. Although HDAC3 is likely to affect the activity of multiple proteins in osteoblasts, our data show that it actively regulates the transcriptional activity of the osteoblast master protein, Runx2.Runx2 is one of three mammalian homologues of the Drosophila protein Runt. Runx2 is required for osteoblast formation and chondrocyte differentiation and thus is an essential regulator of intramembranous and endochondral bone formation (1-3). Runx2 haploinsufficiency causes the autosomal dominant bone disorder cleidocranial dysplasia (4, 5), but elevated Runx2 levels cause osteopenia (6) and contribute to murine T cell lymphomogenesis (7). Furthermore, Runx2 polymorphisms are associated with altered bone mineral density and susceptibility to osteoporotic fractures (8). The sensitivity of osteoblasts to changes in wild-type Runx2 levels provides biological proof that its activity must be tightly controlled. At the molecular level, Runx2 is regulated by transcriptional and translational mechanisms, post-translational modifications, including phosphorylation, and by interactions with other transcription factors and chromatin-modifying enzymes (9 -20). Because the Runx DNA-binding element is necessary but not sufficient to activate tissue-specific gene expression (21, 22), it was hypothesized over a decade ago that Runx proteins are promoter organizers (23). It is now apparent that Runx factors indeed interact with other transcription factors as well as recruit both co-activator and co-repressor complexes to promoters. Known co-activators of Runx2 include YAP, HES, and the histone acetyltransferases, p300, CBP, MOZ,. The list of Runx2-associating co-repressors includes just TLE, mSin3a, and histone deacetylase (HDAC) 1 6 (18, 19, 28, 29); however, among all known transcriptional co-repressors, o...

HDIs are potential therapeutic agents for cancer and neurological diseases because of their abilities to alter gene expression, induce growth arrest or apoptosis of tumors cells, and stimulate differentiation. In this report, we show that several HDIs promote osteoblast maturation in vitro and in calvarial organ cultures.Introduction: Histone deacetylase inhibitors (HDIs) are currently in phase I and II clinical trials as anticancer agents. Some HDIs are also commonly prescribed treatments for epilepsy and bipolar disorders. Although administered systemically, the effects of HDIs on osteoblasts and bone formation have not been extensively examined. In this study, we investigated the effect of histone deacetylase inhibition on osteoblast proliferation and differentiation. . The effects of these inhibitors on cell proliferation, viability, cell cycle progression, Runx2 transcriptional activity, alkaline phosphatase production, and matrix mineralization were determined. Expression levels of osteoblast maturation genes, type I collagen, osteopontin, bone sialoprotein, and osteocalcin in response to TSA were measured by quantitative PCR. Results: Concentrations of HDIs that caused hyperacetylation of histone H3 induced transient increases in osteoblast proliferation and viability but did not alter cell cycle profiles. These concentrations of HDIs also increased the transcriptional activity of Runx2. TSA accelerated alkaline phosphatase production in MC3T3-E1 cells and calvarial organ cultures. In addition, TSA accelerated matrix mineralization and the expression of osteoblast genes, type I collagen, osteopontin, bone sialoprotein, and osteocalcin in MC3T3-E1 cells. Conclusions: These studies show that histone deacetylase activity regulates osteoblast differentiation and bone formation at least in part by enhancing Runx2-dependent transcriptional activation. Therefore, HDIs are a potentially new class of bone anabolic agents that may be useful in the treatment of diseases that are associated with bone loss such as osteoporosis and cancer.

Runx2 is essential for osteoblast development and proper bone formation. A member of the Runt domain family of transcription factors, Runx2 binds specific DNA sequences to regulate transcription of numerous genes and thereby control osteoblast development from mesenchymal stem cells and maturation into osteocytes. Although necessary for gene transcription and osteoblast development, Runx2 is not sufficient for optimal gene expression or bone formation. Runx2 cooperates with numerous proteins, including transcription factors and cofactors, is posttranslationally modified, and associates with the nuclear matrix to integrate a variety of signals and organize crucial events during osteoblast development and maturation. Consistent with its role as a master organizer, alterations in Runx2 expression levels are associated with skeletal diseases. Runx2 haploinsufficiency causes cleidocranial dysplasia, while Runx2 overexpression is common in many bone‐metastatic cancers. In this review, we summarize the molecular mechanisms by which Runx2 integrates signals through coregulatory interactions, and discuss how its role as a master organizer may shift depending on promoter structure, developmental cues, and cellular context. Birth Defects Research (Part C) 75:213–225, 2005. © 2005 Wiley‐Liss, Inc.