Xiaodong Li

Runx2 (Cbfa1, AML-3) Interacts with Histone Deacetylase 6 and Represses the p21 CIP1/WAF1 Promoter

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.

Histone Deacetylase 3 Depletion in Osteo/Chondroprogenitor Cells Decreases Bone Density and Increases Marrow Fat

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.

The formin-homology-domain-containing protein FHOD1 enhances cell migration

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 (ΔC, 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 ΔC (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.

Suberoylanilide hydroxamic acid (SAHA; vorinostat) causes bone loss by inhibiting immature osteoblasts

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.

The histone deacetylase inhibitor, vorinostat, reduces tumor growth at the metastatic bone site and associated osteolysis, but promotes normal bone loss

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. Mol Cancer Ther; 9(12); 3210–20. ©2010 AACR.

Runx2 and Bone Morphogenic Protein 2 Regulate the Expression of an Alternative Lef1 Transcript During Osteoblast Maturation

Lymphoid Enhancer Binding Factor (Lef) 1 is a transcriptional effector of the Wnt/Lrp5/β‐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. Lef1ΔN is a naturally occurring isoform driven by a promoter (p2) within the intron between exons 3 and 4 of Lef1. Lef1ΔN 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 Lef1ΔN expression. BMP2 stimulated Lef1ΔN expression, whereas Wnt3a blocked basal and BMP2‐induced expression of Lef1ΔN transcripts during osteoblast differentiation. We determined that the Lef1ΔN p2 promoter is active in osteoblasts and Runx2 regulates its activity. Stable overexpression of Lef1ΔN in differentiating osteoblasts induced the expression of osteoblast differentiation genes, osteocalcin and type 1 collagen. Taken together, our results suggest Lef1ΔN is a crucial regulator of terminal differentiation in osseous cells. J. Cell. Physiol. 221: 480–489, 2009.

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

Ewings 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 Ewings 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.

Runx2 Protein Represses Axin2 Expression in Osteoblasts and Is Required for Craniosynostosis in Axin2-deficient Mice

Background: Runx2 and Axin2 are required for proper skeletal development. Results: Runx2 and Hdac3 repress Axin2 transcription in osteoblasts. Runx2 insufficiency prevents craniosynostosis in Axin2-deficient mice. Conclusion: A Runx2-Axin2 regulatory mechanism controls the pace of calvarial bone formation. Significance: The molecular and functional interplay between Runx2 and Axin2 controls the rate of cranial suture closure. Similar interactions may occur during skeletal development and carcinogenesis. 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 because of 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 whether 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.

TEThered to Runx: Novel Binding Partners for Runx Factors

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 Ewings 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.

Induction of Fracture Repair by Mesenchymal Cells Derived from Human Embryonic Stem Cells or Bone Marrow

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.