Publications (18)48.24 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Runx2 and Axin2 regulate craniofacial development and skeletal maintenance. Runx2 is essential for calvarial bone development, as Runx2 haploinsufficiency causes cleidocranial dysplasia. In contrast, Axin2-deficient mice develop craniosynostosis due to high β-catenin-activity. Axin2 levels are elevated in Runx2-/- calvarial cells and Runx2 represses transcription of Axin2 mRNA, suggesting a direct relationship between these factors in vivo. Here we demonstrate that Runx2 binds several regions of the Axin2 promoter and that Runx2-mediated repression of Axin2 transcription depends on Hdac3. To determine if Runx2 contributes to the etiology of Axin2-deficiency induced craniosynostosis, we generated Axin2-/- : Runx2+/- mice. These double mutant mice had longer skulls than Axin2-/- mice, indicating that Runx2 haploinsufficiency rescued the craniosynostosis phenotype of Axin2-/- mice. Together, these studies identify a key mechanistic pathway for regulating intramembranous bone development within the skull that involves Runx2 and Hdac3-mediated suppression of Axin2 to prevent the untimely closure of the calvarial sutures.
- [Show abstract] [Hide abstract] ABSTRACT: Development of novel therapeutic approaches to repair fracture non-unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)-derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non-union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM-MSCs) or a no cell control group (n = 10-12 per group). Preliminary studies demonstrated that both for hESC-derived MSCs and hBM-MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque, and stiffness were significantly greater in the hBM-MSC as compared to the control group that received no cells; values for these parameters in the hESC-derived MSC group were intermediate between the hBM-MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X-ray in the hESC-derived MSC group. Our results thus indicate that while hESC-derived MSCs may have potential to induce fracture healing in non-unions, hBM-MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells.
- [Show abstract] [Hide abstract] ABSTRACT: Histone deacetylase (Hdac) inhibitors are used clinically to treat cancer and epilepsy. Although Hdac inhibition accelerates osteoblast maturation and suppresses osteoclast maturation in vitro, the effects of Hdac inhibitors on the skeleton are not understood. The purpose of this study was to determine how the pan-Hdac inhibitor, suberoylanilide hydroxamic acid (SAHA; a.k.a. vorinostat or Zolinza(TM)) affects bone mass and remodeling in vivo. Male C57BL/6J mice received daily SAHA (100mg/kg) or vehicle injections for 3 to 4weeks. SAHA decreased trabecular bone volume fraction and trabecular number in the distal femur. Cortical bone at the femoral midshaft was not affected. SAHA reduced serum levels of P1NP, a bone formation marker, and also suppressed tibial mRNA levels of type I collagen, osteocalcin and osteopontin, but did not alter Runx2 or osterix transcripts. SAHA decreased histological measures of osteoblast number but interestingly increased indices of osteoblast activity including mineral apposition rate and bone formation rate. Neither serum (TRAcP 5b) nor histological markers of bone resorption were affected by SAHA. P1NP levels returned to baseline in animals which were allowed to recover for 4weeks after 4weeks of daily SAHA injections, but bone density remained low. In vitro, SAHA suppressed osteogenic colony formation, decreased osteoblastic gene expression, induced cell cycle arrest, and caused DNA damage in bone marrow-derived adherent cells. Collectively, these data demonstrate that bone loss following treatment with SAHA is primarily due to a reduction in osteoblast number. Moreover, these decreases in osteoblast number can be attributed to the deleterious effects of SAHA on immature osteoblasts, even while mature osteoblasts are resistant to the harmful effects and demonstrate increased activity in vivo, indicating that the response of osteoblasts to SAHA is dependent upon their differentiation state. These studies suggest that clinical use of SAHA and other Hdac inhibitors to treat cancer, epilepsy or other conditions may potentially compromise skeletal structure and function.
- [Show abstract] [Hide abstract] ABSTRACT: Vorinostat, an oral histone deacetylase inhibitor with antitumor activity, is in clinical trials for hematologic and solid tumors that metastasize and compromise bone structure. Consequently, there is a requirement to establish the effects of vorinostat on tumor growth within bone. Breast (MDA-231) and prostate (PC3) cancer cells were injected into tibias of SCID/NCr mice and the effects of vorinostat on tumor growth and osteolytic disease were assessed by radiography, micro-computed tomography, and histologic and molecular analyses. Vorinostat-treated and control mice without tumors were also examined. Tumor growth in bone was reduced ∼33% by vorinostat with inhibited osteolysis in the first few weeks of the experiment. However, osteolysis became more severe in both the vehicle and vorinostat-treated groups. Vorinostat increased the expression of tumor-derived factors promoting bone resorption, including PTHrP, IL-8, and osteopontin. After 4 weeks of vorinostat therapy, the non-tumor-bearing contralateral femurs and limbs from vorinostat-treated tumor-free SCID mice showed significant bone loss (50% volume density of controls). Thus, our studies indicate that vorinostat effectively inhibits tumor growth in bone, but has a negative systemic effect reducing normal trabecular bone mass. Vorinostat treatment reduces tumor growth in bone and accompanying osteolytic disease as a result of decreased tumor burden in bone. However, vorinostat can promote osteopenia throughout the skeleton independent of tumor cell activity.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
Dataset: Figure S2[Show abstract] [Hide abstract] ABSTRACT: MicroCT Reconstructions of skulls from 5.5 week-old male Hdac3 WT, Het and CKO mice. These are alternative angles of the microCT reconstructions shown in Figure 1E. (0.96 MB TIF)
Dataset: Figure S3[Show abstract] [Hide abstract] ABSTRACT: Hdac3-Depletion in Osx-expressing cells decreases trabecular bone volume in distal femora. A-E. Structural parameters of distal femurs from microCT reconstruction of 12 week-old male mice. n = 4-5 per group, *p<0.05. (0.10 MB TIF)
Dataset: Figure S5[Show abstract] [Hide abstract] ABSTRACT: MicroCT Reconstructions of skulls from Osx-Cre mice. Minor craniofacial defects are present in 5.5 week-old Osx-Cre mice. These mice are wildtype at the Hdac3 loci. (0.35 MB TIF)
Dataset: Table S1[Show abstract] [Hide abstract] ABSTRACT: List of the 176 genes common to two independent microarray experiments. (0.14 MB PDF)
Dataset: Figure S1[Show abstract] [Hide abstract] ABSTRACT: Photograph of representative 5.5. week-old mice used in this study. The animals' weights are listed below the photos. (0.57 MB TIF)
Dataset: Figure S4[Show abstract] [Hide abstract] ABSTRACT: HDAC3 represses the p21(CIP1) promoter. C2C12 cells were transfected with a p21-luciferase reporter and increasing concentrations of pCMV-HDAC3. Firefly luciferase activity was normalized to renilla-luciferase activity to control for transfection efficiency. Data are shown relative to cells transfected with an empty CMV vector. (0.04 MB TIF)
- [Show abstract] [Hide abstract] ABSTRACT: Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health.
- [Show abstract] [Hide abstract] ABSTRACT: RUNX transcription factors reside in the nuclear matrix where they integrate numerous signaling pathways to regulate gene expression and affect tissue development, regeneration, and tumorigenesis. An affinity purification and proteomic experiment was performed to identify novel Runx2 binding partners. The interactions between Runx2 and two nuclear factors (Ddx5 and CoAA) identified in this screen were previously described. Coactivator activator (CoAA) bound the DNA binding domain of Runx2 and prevented Runx-driven gene expression. The YxxQ motif in CoAA was required for Runx2 interactions. Members of the FET/TET family of proteins, including FUS/TLS and EWSR1, contain a similar motif and were hypothesized to interact with Runx2. Here, we provide evidence that FUS/TLS, EWSR1, and the Ewing's sarcoma t(12;21) fusion protein EWS-FLI bind Runx2 and alter its transcriptional activity. Potential roles of protein complexes containing FET/TET and RUNX family members during tumor formation and mesenchymal progenitor cell differentiation are discussed.
- [Show abstract] [Hide abstract] ABSTRACT: Lymphoid Enhancer Binding Factor (Lef) 1 is a transcriptional effector of the Wnt/Lrp5/beta-catenin signaling cascade, which regulates osteoblast differentiation, bone density, and skeletal strength. In this study, we describe the expression and function of an alternative Lef1 isoform in osseous cells. Lef1DeltaN is a naturally occurring isoform driven by a promoter (p2) within the intron between exons 3 and 4 of Lef1. Lef1DeltaN is induced during late osteoblast differentiation. This is converse to the expression pattern of the full-length Lef1 protein, which as we previously showed, decreases during differentiation. Agonists of osteoblast maturation differentially affected Lef1DeltaN expression. BMP2 stimulated Lef1DeltaN expression, whereas Wnt3a blocked basal and BMP2-induced expression of Lef1DeltaN transcripts during osteoblast differentiation. We determined that the Lef1DeltaN p2 promoter is active in osteoblasts and Runx2 regulates its activity. Stable overexpression of Lef1DeltaN in differentiating osteoblasts induced the expression of osteoblast differentiation genes, osteocalcin and type 1 collagen. Taken together, our results suggest Lef1DeltaN is a crucial regulator of terminal differentiation in osseous cells.
- [Show abstract] [Hide abstract] ABSTRACT: Runx proteins are essential for a number of developmental processes and are aberrantly expressed in many human cancers. Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation, hematopoiesis, and neuronal development. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. In this study, we identified CoAA as a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: Formin-homology-domain-containing proteins interact with Rho-family GTPases and regulate actin cytoskeleton organization and gene transcription. FHOD1 is a member of this family, interacts with Rac1 and induces transcription from the serum response element. In this study, we examined the effects of FHOD1 expression on cytoskeletal organization and function in mammalian cells. FHOD1 proteins were stably expressed in WM35 melanoma cells and NIH-3T3 fibroblasts. Cells expressing full-length FHOD1 demonstrated an elongated phenotype compared with vector-transfected cells and cells expressing a truncated FHOD1 (1-421) that lacks the conserved FH1 and FH2 domains. Full-length FHOD1 co-localized with filamentous actin at cell peripheries. Cells transiently expressing a C-terminal FHOD1 truncation mutant (DeltaC, residues 1-1010), which lacks an autoinhibitory protein-protein interaction domain, displayed prominent stress fibers. FHOD1 (1-421) did not induce stress fibers but localized to membrane ruffles in a manner similar to the full-length protein, indicating that the FH1 and FH2 domains are required for stress fiber appearance. FHOD1 DeltaC (1-1010)-dependent stress fibers were sensitive to dominant-negative RacN17 and the RhoA and ROCK inhibitors, C3 transferase and Y-27632. Stable overexpression of full-length FHOD1 enhanced the migration of WM35 and NIH-3T3 cells to type-I collagen and fibronectin, respectively. Cells expressing FHOD1 (1-421) migrated similar to control cells. Integrin expression and activation were not affected by FHOD1 expression. Moreover, FHOD1 overexpression did not alter integrin usage during adhesion or migration. These data demonstrate that FHOD1 interacts with and regulates the structure of the cytoskeleton and stimulates cell migration in an integrin-independent manner.
- [Show abstract] [Hide abstract] ABSTRACT: Runx2 (Cbfa1, AML-3) is multifunctional transcription factor that is essential for osteoblast development. Runx2 binds specific DNA sequences and interacts with transcriptional coactivators and corepressors to either activate or repress transcription of tissue-specific genes. In this study, the p21(CIP/WAF1) promoter was identified as a repressible target of Runx2. A carboxy-terminal repression domain distinct from the well-characterized TLE/Groucho-binding domain contributed to Runx2-mediated p21 repression. This carboxy-terminal domain was sufficient to repress a heterologous GAL reporter. The repressive activity of this domain was sensitive to the histone deacetylase inhibitor trichostatin A but not to trapoxin B. HDAC6, which is insensitive to trapoxin B, specifically interacted with the carboxy terminus of Runx2. The HDAC6 interaction domain of Runx2 was mapped to a region overlapping the nuclear matrix-targeting signal. The Runx2 carboxy terminus was necessary for recruitment of HDAC6 from the cytoplasm to chromatin. HDAC6 also colocalized and coimmunoprecipitated with the nuclear matrix-associated protein Runx2 in osteoblasts. Finally, we show that HDAC6 is expressed in differentiating osteoblasts and that the Runx2 carboxy terminus is necessary for maximal repression of the p21 promoter in preosteoblasts. These data identify Runx2 as the first transcription factor to interact with HDAC6 and suggest that HDAC6 may bind to Runx2 in differentiating osteoblasts to regulate tissue-specific gene expression.
Mayo Clinic - Rochester
Рочестер, Minnesota, United States
- Division of Orthopaedic Surgery
University of Nebraska Medical Center
Omaha, NE, United States
- Department of Oral Biology