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: 7998440
• 负责人: Meghan E. McGee-Lawrence
• 金额: $ 4.76万
• 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/7998440
• 关键词: Affect; Antiepileptic Agents; Binding; Biological Assay; Bone Development; Bone Regeneration; Bone remodeling; Cell Lineage; Development; Feedback; Femur; Galactosidase; Gene Expression; Genetic Transcription; Histones; In Vitro; Knock-out; Knowledge; Link; Maintenance; Malignant Neoplasms; Mechanics; Medical; Modeling; Molecular; Mouse Strains; Mus; Mutant Strains Mice; Osteoblasts; Osteogenesis; Pathway interactions; Phenotype; Play; Recruitment Activity; Regulator Genes; Regulatory Pathway; Reporter; Research; Role; Signal Pathway; Signal Transduction; Skeletal Development; Skeleton; Staining method; Stains; Stem cells; Testing; Work; X-Ray Computed Tomography; bone; bone loss; bone mass; chromatin immunoprecipitation; in vivo; inhibitor/antagonist; novel; osteochondral tissue; osteogenic; promoter; public health relevance; repaired; research study; skeletal; tibia; 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(HDAC)是基因表达的关键调节因子，因此是新药物治疗的靶标。HDAC3在成骨细胞中表达，并与Runx2结合，调节成骨基因的表达，从而在骨的发育和维持中发挥重要作用。在骨软骨前体细胞(HDAC3-CKO)中条件性敲除HDAC3会导致成骨细胞活性降低和骨量减少。这种骨丢失可能是由于Wnt/？-catenin信号的失调，这是骨重建的关键调控途径。重要的是，在HDAC3-CKO小鼠中，Axin2(Wnt信号的负反馈抑制物)水平增加。然而，尚不清楚是由于规范的Wnt信号上调导致Axin2水平升高，导致Axin2水平随之增加(作为负反馈环的一部分)，还是HDAC3-CKO小鼠中Axin2水平的增加是因为失去了HDAC3对Axin2启动子的直接抑制作用。这项拟议的研究的目的是确定HDAC3抑制如何影响成骨细胞系细胞中的Wnt/2-catenin信号。中心假设是HDAC3被Runx2主动招募到Axin2启动子，以抑制Axin2‘S对Wnt途径的抑制，从而形成连环蛋白依赖性的骨形成。检验这一假说的第一个具体目标是确定体内HDAC3缺失对典型的Wnt信号的影响。这将通过将HDAC3-CKO小鼠与两个可以直接量化Wnt信号的小鼠品系(即Wnt信号报告品系)杂交来实现。对这些小鼠骨骼上2-半乳糖苷酶染色的定量将揭示HDAC3缺乏对典型Wnt信号的人工报告(TOPGAL)和对规范Wnt信号的自然靶点(Axin2)的影响。第二个具体目标是确定HDAC3和Axin2在体内对骨量和骨形成的关系。对HDAC3-CKO：Axin2-KO突变小鼠(和产仔对照)的股骨和胫骨进行微型计算机断层扫描、组织形态计量学和力学测试，以确定HDAC3和Axin2的缺陷是否会导致正常(即野生型)骨骼表型。最终的具体目标是定义HDAC3调节Axin2的分子机制。体外机制实验，包括转录和染色质免疫沉淀分析，将解释HDAC3是如何与Axin2启动子结合和抑制的。这些研究是新颖而重要的，因为它们将是第一个在体内和体外将HDAC3与Axin2和Wnt途径机械联系起来的研究。这些结果对于了解HDAC如何与Runx2和规范的Wnt信号相互作用以获得和维持最佳骨量，从而增加对成骨细胞成熟、骨骼发育和骨再生/修复的知识具有重要意义。了解这种关系很重要，因为Axin2是Wnt/？-catenin信号的细胞内和自动反馈调节因子，这一途径目前正被用于开发新的合成代谢骨骼疗法。 公共卫生相关性：我们建议研究组蛋白脱乙酰酶3(HDAC3)、Axin2和典型的Wnt信号通路之间的新的相互作用，这些信号通路来自HDAC3缺乏模型的小鼠。这项拟议的项目意义重大，因为Wnt信号通路是新的成骨疗法的靶点，我们的研究将调查可能调节其效果的因素。此外，这项工作将产生集体影响，因为对HDAC耗竭的研究将加强对HDAC抑制剂(例如，唑林扎TM、丙戊酸盐，临床上用作抗癌和抗癫痫疗法)如何影响骨骼的理解。