Relationship between Hdac3 suppression and Wnt signaling in osteoblasts

成骨细胞中 Hdac3 抑制与 Wnt 信号传导的关系

• 批准号: 8139233
• 负责人: Meghan E. McGee-Lawrence
• 金额: $ 5.13万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-05 至 2012-08-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8139233
• 关键词: Affect; Age-Months; Antiepileptic Agents; Binding; Biological; Biological Assay; Bone Development; Bone Marrow; Bone Regeneration; Bone remodeling; Calvaria; Cell Lineage; Cells; Complex; Data; Deacetylase; Development; Embryo; Epigenetic Process; Exons; Feedback; Femur; Galactosidase; Gene Expression; Gene Expression Regulation; Genetic Models; Genetic Transcription; Histones; Immunohistochemistry; In Vitro; Knock-out; Knowledge; Laboratories; LacZ Genes; Link; Maintenance; Malignant Neoplasms; Mechanics; Mediating; Medical; Mentors; Messenger RNA; Modeling; Molecular; Molecular Biology; Mouse Strains; Mus; Mutant Strains Mice; Osteoblasts; Osteogenesis; Pathway interactions; Phenotype; Play; Proteins; Recruitment Activity; Regulator Genes; Regulatory Pathway; Reporter; Repression; Research; Role; Signal Pathway; Signal Transduction; Site; Skeletal Development; Skeletal bone; Skeleton; Staining method; Stains; Stem cells; Techniques; Testing; Training; Transcript; Trichostatin A; Vorinostat; Work; X-Ray Computed Tomography; bone; bone loss; bone mass; career; career development; chromatin immunoprecipitation; in vivo; inhibitor/antagonist; interest; meetings; novel; osteochondral tissue; osteogenic; promoter; public health relevance; repaired; research study; skeletal; skeletal tissue; skills; tibia; tool; transcription factor; valproate

DESCRIPTION (provided by applicant): Histone deacetylases (Hdacs) are crucial regulators of gene expression and are therefore targets for new medical therapies. Hdac3 is expressed in osteoblasts and binds to Runx2 to regulate osteoblastic gene expression, thus playing an important role in bone development and maintenance. Conditional knockout of Hdac3 in osteochondral progenitor cells (Hdac3-CKO) leads to decreased osteoblast activity and a low bone mass phenotype. This bone loss may be due to disregulation of Wnt/¿-catenin signaling, a key regulatory pathway for bone remodeling. Importantly, levels of Axin2 (a negative feedback inhibitor of Wnt signaling) are increased in Hdac3-CKO mice. However, it is not clear whether Axin2 levels are increased as a result of upregulated canonical Wnt signaling, leading to a concomitant increase in Axin2 (as part of a negative feedback loop) or if the increased Axin2 levels in Hdac3-CKO mice result from losing a direct repressive effect of Hdac3 on the Axin2 promoter. The objective of the proposed research is to determine how Hdac3 suppression affects Wnt/2-catenin signaling in osteoblast lineage cells. The central hypothesis is that Hdac3 is actively recruited to the Axin2 promoter by Runx2 to repress Axin2's inhibition of the Wnt pathway, thus ¿-catenin-dependent bone formation. The first specific aim to test this hypothesis is to determine the consequences of Hdac3-depletion on canonical Wnt signaling in vivo. This will be accomplished by crossing Hdac3-CKO mice with two mouse strains in which Wnt signaling can be directly quantified (i.e., Wnt signaling reporter strains). Quantification of 2-galactosidase staining on bones from these mice will reveal the effects of Hdac3 deficiency on an artificial reporter of canonical Wnt signaling (TOPGAL) and on a natural target of canonical Wnt signaling (Axin2). The second specific aim is to determine relationships between Hdac3 and Axin2 on bone mass and bone formation in vivo. Femurs and tibias will be analyzed in Hdac3- CKO:Axin2-KO mutant mice (and littermate controls) with micro-computed tomography, histomorphometry, and mechanical testing to determine if deficiencies in both Hdac3 and Axin2 result in a normal (i.e., wild-type) skeletal phenotype. The final specific aim is to define the molecular mechanisms by which Hdac3 regulates Axin2. Mechanistic in vitro experiments including transcription and chromatin immunoprecipitation assays will explain how Hdac3 associates with and represses the Axin2 promoter. These studies are novel and important because they will be the first to mechanistically link Hdac3 with Axin2 and the Wnt pathway in vivo and in vitro. The results will be important for understanding how Hdacs interact with Runx2 and canonical Wnt signaling to obtain and maintain optimal bone mass, thereby increasing knowledge of osteoblast maturation, skeletal development and bone regeneration/repair. Understanding this relationship is important because Axin2 is an intracellular and auto-feedback regulator of Wnt/¿-catenin signaling, a pathway that is currently being targeted in the development of new anabolic skeletal therapies. PUBLIC HEALTH RELEVANCE: We propose to study novel interactions between histone deacetylases 3 (Hdac3), Axin2, and the canonical Wnt signaling pathway in mice derived from an Hdac3-deficient model. The proposed project is significant because the Wnt signaling pathway is a target for new osteogenic therapies, and our studies will investigate factors that may modulate its effects. Furthermore, the work will have collective impact because studies of Hdac depletion will enhance understanding of how Hdac inhibitors (e.g., ZolinzaTM, Valproate, used clinically as anti-cancer and anti-epileptic therapies) affect the skeleton.

描述（由申请人提供）：组蛋白脱乙酰酶（Hdacs）是基因表达的关键调节因子，因此是新药物治疗的靶点。Hdac 3在成骨细胞中表达，并与Runx 2结合以调节成骨细胞基因表达，从而在骨发育和维持中发挥重要作用。骨软骨祖细胞中Hdac 3的条件性敲除（Hdac 3-CKO）导致成骨细胞活性降低和低骨量表型。这种骨丢失可能是由于Wnt/β-连环蛋白信号传导的失调，这是骨重建的关键调节途径。重要的是，Axin 2（Wnt信号传导的负反馈抑制剂）的水平在Hdac 3-CKO小鼠中增加。然而，尚不清楚Axin 2水平是否由于上调的经典Wnt信号传导而增加，从而导致Axin 2的伴随增加（作为负反馈环的一部分），或者Hdac 3-CKO小鼠中Axin 2水平的增加是否由于失去Hdac 3对Axin 2启动子的直接抑制作用而导致。这项研究的目的是确定Hdac 3抑制如何影响成骨细胞系细胞中的Wnt/2-catenin信号传导。核心假设是Hdac 3被Runx 2主动募集到Axin 2启动子中，以抑制Axin 2对Wnt途径的抑制，从而抑制β-连环蛋白依赖性骨形成。检验这一假设的第一个具体目的是确定Hdac 3耗尽对体内典型Wnt信号传导的后果。这将通过将Hdac 3-CKO小鼠与两种小鼠品系杂交来实现，其中可以直接定量Wnt信号传导（即，Wnt信号转导报告菌株）。对来自这些小鼠的骨上的2-半乳糖苷酶染色的定量将揭示Hdac 3缺乏对典型Wnt信号传导的人工报告子（TOPGAL）和对典型Wnt信号传导的天然靶标（Axin 2）的影响。第二个具体目的是确定Hdac 3和Axin 2对体内骨量和骨形成的关系。将在Hdac 3- CKO：Axin 2-KO突变小鼠（和同窝对照）中用显微计算机断层扫描、组织形态计量学和机械测试分析股骨和胫骨，以确定Hdac 3和Axin 2两者的缺陷是否导致正常（即，野生型）骨骼表型。最终的具体目标是确定Hdac 3调节Axin 2的分子机制。包括转录和染色质免疫沉淀测定的体外实验机制将解释Hdac 3如何与Axin 2启动子相关并抑制Axin 2启动子。这些研究是新颖和重要的，因为它们将是第一个在体内和体外将Hdac 3与Axin 2和Wnt通路机械地联系起来的研究。这些结果对于理解Hdacs如何与Runx 2和经典Wnt信号相互作用以获得和维持最佳骨量，从而增加成骨细胞成熟，骨骼发育和骨再生/修复的知识非常重要。理解这种关系很重要，因为Axin 2是Wnt/β-catenin信号传导的细胞内和自动反馈调节剂，Wnt/β-catenin信号传导是目前开发新的合成代谢骨骼疗法的目标。 公共卫生相关性：我们建议研究组蛋白去乙酰化酶3（Hdac 3），Axin 2，和经典的Wnt信号通路在小鼠中来自Hdac 3缺陷模型之间的新的相互作用。该项目具有重要意义，因为Wnt信号通路是新成骨疗法的靶点，我们的研究将调查可能调节其作用的因素。此外，这项工作将产生集体影响，因为对HSP 70耗竭的研究将增强对HSP 70抑制剂（例如，ZolinzaTM，丙戊酸盐，临床上用作抗癌和抗癫痫治疗）影响骨骼。