Histone deacetylase 7 associates with Runx2 and represses its activity during osteoblast maturation in a deacetylation-independent manner.

UNLABELLED: HDAC7 associates with Runx2 and represses Runx2 transcriptional activity in a deacetylase-independent manner. HDAC7 suppression accelerates osteoblast maturation. Thus, HDAC7 is a novel Runx2 co-repressor that regulates osteoblast differentiation. INTRODUCTION: Runx2 is a key regulator of gene expression in osteoblasts and can activate or repress transcription depending on interactions with various co-factors. Based on previous observations that several histone deacetylases (HDACs) repress Runx2 activity and that HDAC inhibitors accelerate osteoblast differentiation in vitro, we hypothesized that additional HDACs may also affect Runx2 activity. MATERIALS AND METHODS: A panel of HDACs was screened for repressors of Runx2 activity. Immunofluorescence, co-immunoprecipitation, GST-pulldowns, and chromatin immunoprecipitations were used to characterize the interactions between Runx2 and HDAC7. Expression of osteoblast markers was examined in a C2C12 cell osteoblast differentiation model in which HDAC7 levels were reduced by RNAi. RESULTS: Runx2 activity was repressed by HDAC7 but not by HDAC9, HDRP, HDAC10, or HDAC11. HDAC7 and Runx2 were found co-localized in nuclei and associated with Runx2-responsive promoter elements in osseous cells. A carboxy-terminal domain of Runx2 associated with multiple regions of HDAC7. Although direct interactions with Runx2 were confined to the carboxy terminus of HDAC7, this region was dispensable for repression. In contrast, the amino terminus of HDAC7 bound Runx2 indirectly and was necessary and sufficient for transcriptional repression. Treatment with HDAC inhibitors did not decrease inhibition by HDAC7, indicating that HDAC7 repressed Runx2 by deacetylation-independent mechanism(s). Suppression of HDAC7 expression in C2C12 multipotent cells by RNAi accelerated their BMP2-dependent osteoblast differentiation program. Consistent with this observation, BMP2 decreased nuclear localization of HDAC7. CONCLUSIONS: These results establish HDAC7 as a regulator of Runx2's transcriptional activity and suggest that HDAC7 may be an important regulator of the timing and/or rate of osteoblast maturation.

Gene profile analysis of osteoblast genes differentially regulated by histone deacetylase inhibitors.

BACKGROUND: Osteoblast differentiation requires the coordinated stepwise expression of multiple genes. Histone deacetylase inhibitors (HDIs) accelerate the osteoblast differentiation process by blocking the activity of histone deacetylases (HDACs), which alter gene expression by modifying chromatin structure. We previously demonstrated that HDIs and HDAC3 shRNAs accelerate matrix mineralization and the expression of osteoblast maturation genes (e.g. alkaline phosphatase, osteocalcin). Identifying other genes that are differentially regulated by HDIs might identify new pathways that contribute to osteoblast differentiation. RESULTS: To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid) for 18 hours in osteogenic conditions. The promotion of osteoblast differentiation by HDIs in this experiment was confirmed by osteogenic assays. Gene expression profiles relative to vehicle-treated cells were assessed by microarray analysis with Affymetrix GeneChip 430 2.0 arrays. The regulation of several genes by HDIs in MC3T3-E1 cells and primary osteoblasts was verified by quantitative real-time PCR. Nine genes were differentially regulated by at least two-fold after exposure to each of the three HDIs and six were verified by PCR in osteoblasts. Four of the verified genes (solute carrier family 9 isoform 3 regulator 1 (Slc9a3r1), sorbitol dehydrogenase 1, a kinase anchor protein, and glutathione S-transferase alpha 4) were induced. Two genes (proteasome subunit, beta type 10 and adaptor-related protein complex AP-4 sigma 1) were suppressed. We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway. CONCLUSION: This study identifies osteoblast genes that are regulated early by HDIs and indicates pathways that might promote osteoblast maturation following HDI exposure. One gene whose upregulation following HDI treatment is consistent with this notion is Slc9a3r1. Also known as NHERF1, Slc9a3r1 is required for optimal bone density. Similarly, the regulation of Wnt receptor genes indicates that this crucial pathway in osteoblast development is also affected by HDIs. These data support the hypothesis that HDIs regulate the expression of genes that promote osteoblast differentiation and maturation.

Sarcoma derived from cultured mesenchymal stem cells.

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 transposon-related 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.

Alterations in intranuclear localization of Runx2 affect biological activity.

The transcription factor Runx2 controls osteoblast proliferation and differentiation. Runx2 organizes and assembles gene-regulatory complexes in nuclear microenvironments where target genes are activated or suppressed in a context-dependent manner. Intranuclear localization of Runx2 is mediated by the nuclear matrix-targeting signal (NMTS), an autonomous motif with a loop (L1)-turn-loop (L2) structure that forms predicted protein-protein interaction surfaces. Here we examined the functional consequences of introducing mutations in the L1 and L2 loops of the NMTS. These mutant proteins enter the nucleus, interact with the hetero-dimeric partner Cbfbeta, and bind to DNA in vitro and in vivo. In addition, these mutants retain interaction with the carboxy-terminus interacting co-regulatory proteins that include TLE, YAP, and Smads. However, two critical mutations in the L2 domain of the NMTS decrease association of Runx2 with the nuclear matrix. These subnuclear targeting defective (STD) mutants of Runx2 compromise target gene activation or repression. The biological significance of these findings is reflected by decreased osteogenic differentiation of mesenchymal progenitors, concomitant with major changes in gene expression profiles, upon expression of the STD Runx2 mutant. Our results demonstrate that fidelity of temporal and spatial localization of Runx2 within the nucleus is functionally linked with biological activity.

Histone deacetylase inhibitors promote osteoblast maturation.

UNLABELLED: 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. MATERIALS AND METHODS: MC3T3-E1 cells, calvarial-derived primary osteoblasts, and calvarial organ cultures were treated with various commercially available HDIs (trichostatin A [TSA], sodium butyrate [NaB], valproic acid [VPA], or MS-275). 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.

Regulation of relB in dendritic cells by means of modulated association of vitamin D receptor and histone deacetylase 3 with the promoter.

The NF-kappaB component RelB is essential for dendritic cell (DC) differentiation and maturation. The vitamin D receptor (VDR) is a nuclear receptor that mediates inhibition of DC maturation and transcriptional repression of relB after engagement of its ligand, 1alpha,25-dihydroxyvitamin D(3), or related analogs (D(3) analogs). Ligand-dependent relB suppression was abolished by a histone deacetylase (HDAC) inhibitor. Constitutive association of VDR with the relB promoter was demonstrated in DCs by chromatin immunoprecipitation. Promoter binding by VDR was enhanced by ligand and reduced by LPS. Association of HDAC3 and HDAC1 with the relB VDR-binding site was observed, but only HDAC3 was reciprocally modulated by D(3) analog and LPS. Overexpression of HDAC3 caused relB promoter suppression, increased sensitivity to D(3) analog, and resistance to LPS. Depletion of HDAC3 attenuated relB suppression by D(3) analog. In vivo, D(3) analog resulted in reduced RelB in DCs from VDR WT mice but not VDR knockout mice. Other NF-lation of RelB and c-Rel in control animals. We conclude that vitamin D-regulated relB transcription in DCs is controlled by chromatin remodeling by means of recruitment of complexes including HDAC3.

Runx2: a master organizer of gene transcription in developing and maturing osteoblasts.

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.

Wnt signaling in osteoblasts and bone diseases.

Recent revelations that the canonical Wnt signaling pathway promotes postnatal bone accrual are major advances in our understanding of skeletal biology and bring tremendous promise for new therapeutic treatments for osteoporosis and other diseases of altered bone mass. Wnts are soluble glycoproteins that engage receptor complexes composed of Lrp5/6 and Frizzled proteins. A subgroup of Wnts induces a cascade of intracellular events that stabilize beta-catenin, facilitating its transport to nuclei where it binds Lef1/Tcf transcription factors and alters gene expression to promote osteoblast expansion and function. Natural extracellular Wnt antagonists, Dickkopfs and secreted frizzled-related proteins, impair osteoblast function and block bone formation. In several genetic disorders of altered skeletal mass, mutations in LRP5 create gain-of-function or loss-of-function receptors that are resistant to normal regulatory mechanisms and cause higher or lower bone density, respectively. In this review, we summarize the available molecular, cellular, and genetic data that demonstrate how Lrp5 and other components of the Wnt signaling pathway influence osteoblast proliferation, function, and survival. We also discuss regulatory mechanisms discovered in developmental and tumor models that may provide insights into novel therapies for bone diseases.

Histone deacetylase 3 interacts with runx2 to repress the osteocalcin promoter and regulate osteoblast differentiation.

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.