Role of TGIF in Wnt-Induced Bone Formation

描述（由申请人提供）：骨骼发育和体内平衡依赖于一个高阶网络，该网络确保成骨细胞的骨形成和破骨细胞的骨吸收之间的有效平衡。这一网络的紊乱通常与骨骼疾病有关，最严重的是骨质疏松症，具有骨折的固有风险以及相关的发病率和死亡率。骨重建的主要调节因子是经典Wnt信号通路，其对成骨细胞分化的几个方面施加严格控制。同源结构域蛋白TGIF在转化生长因子β（TGF-β）信号通路中作为转录辅抑制因子发挥作用，已知其限制骨形成。为了进一步了解TGIF的生理功能和调节是如何控制的，我们使用酵母双杂交试验筛选了TGIF结合蛋白。一个以前未鉴定的TGIF结合蛋白引起了我们的注意是Axin 2，它通过其介导破坏体形成的潜力作为Wnt信号传导的负调节剂，破坏体是一种信号传导模块，蛋白激酶GSK 3-<$标记<$-连环蛋白进行泛素依赖性降解。在随后的功能获得和丧失研究中，发现TGIF促进Wnt诱导的转录反应。我们的初步研究还表明，TGIF是必不可少的Wnt信号的能力，诱导成骨细胞分化。引人注目的是，我们发现TGIF可以通过一种独立于其抑制TGF-β信号传导能力的机制来调节骨形成，这揭示了TGIF的另一种生理功能是确保有效的成骨细胞命运决定响应Wnt。与其在成骨细胞定向中的作用一致，小鼠中TGIF的消融损害了成骨细胞的成熟并降低了骨量。总之，这些发现使我们提出了一个工作假设，其中TGIF可能有助于调节骨中的经典Wnt信号传导，对破坏体支架复合物施加严格的控制。因此，本研究计划的具体目标是：目的1：进一步阐明TGIF影响Wnt介导的成骨细胞分化和骨形成的分子机制，特别强调其在破坏体组装/拆卸中的可能作用，从而影响连环蛋白的稳定性。目标二：分析TGIF的完全缺失和成骨细胞靶向缺失对骨稳态的影响以及Wnt诱导成骨细胞分化和骨形成的能力。 TGIF作为Wnt信号传导和骨形成调节的真正贡献者的功能表征，将加强我们对维持骨稳态的关键生理过程的了解，最终演变为针对骨质疏松症和其他低骨量综合征的安全合成代谢治疗药物的设计和实施的新概念。 公共卫生相关性：Wnt信号促进成骨细胞增殖和分化，并且该信号通路的扰动影响骨稳态，导致人类骨疾病。我们发现同源结构域蛋白TGIF的生理功能是确保响应Wnt信号传导的成骨细胞命运的正确决定。我们相信，他的新发现的骨形成调节剂的功能特性将打开一个新的研究领域，无论是在了解调节成骨细胞及其骨基质分泌活性的机制方面，还是在药物发现方面，都有望改善骨质疏松症和其他低骨量综合征的治疗。

DESCRIPTION (provided by applicant): Skeletal development and homeostasis depend on a higher-order network that ensures efficient balance between bone formation by osteoblasts and resorption by osteoclasts. Perturbations of this network are often associated with skeletal disorders, with the most severe being osteoporosis, with inherent risk of fracture and associated morbidity and mortality. A master regulator of bone remodeling is the canonical Wnt signaling pathway, which exerts a tight control over several aspects of osteoblast differentiation. The homeodomain protein TGIF functions as a transcriptional corepressor in the transforming growth factor beta (TGF-¿) signaling pathway, which is known to restrict bone formation. To advance our understanding of how TGIF's physiological functions and regulation are controlled, we have screened for TGIF-binding proteins using the yeast two-hybrid assay. A previously unidentified TGIF binding protein that attracted our attention is Axin2, which functions as a negative regulator of Wnt signaling through its potential to mediate formation of the destructosome, a signaling module where the protein kinase GSK3-¿ marks ¿-Catenin for ubiquitin-dependent degradation. In subsequent gain- and loss-of-function studies, TGIF was found to promote Wnt-induced transcriptional responses. Our preliminary investigations also suggest that TGIF is essential to the ability of Wnt signaling to induce osteoblast differentiation. Strikingly, we found that TGIF can regulate bone formation by a mechanism independent of its ability to suppress TGF-¿ signaling, revealing that an alternative physiological function of TGIF is to ensure effective osteoblast cell fate determination in response to Wnt. Consistent with its role in osteoblast commitment, ablation of TGIF in mice impaired osteoblast maturation and decreased bone mass. Taken together, these findings led us to propose a working hypothesis in which TGIF may contribute to the regulation of canonical Wnt signaling in bone, imposing a stringent control over the destructosome scaffolding complex. Accordingly, the specific aims of this research proposal are: Aim 1: Further delineate the molecular mechanisms by which TGIF affects Wnt-mediated osteoblast differentiation and bone formation, with particular emphasis on its possible role in the assembly/disassembly of the destructosome, and thereby ¿-Catenin stability. Aim 2: Analyze the effects of full and osteoblast-targeted deletion of TGIF on bone homeostasis and the ability of Wnt to induce osteoblast differentiation and bone formation in vitro and in vivo. Functional characterization of TGIF as a bona fide contributor to the regulation of Wnt signaling and bone formation, will strengthen our knowledge of key physiological processes that maintain bone homeostasis, ultimately evolving into new concepts in the design and implementation of safe anabolic therapeutic drugs against osteoporosis and other low bone mass syndromes. PUBLIC HEALTH RELEVANCE: Wnt signaling fosters osteoblast proliferation and differentiation, and perturbations of this signaling pathway affect bone homeostasis, resulting in bone disorders in human. We discovered that a physiological function of the homeodomain protein TGIF is to ensure proper osteoblast cell fate determination in response to Wnt signaling. We believe that functional characterization of his newly discovered regulator of bone formation will open a new field of investigation both in terms of understanding the mechanisms that regulate osteoblasts and their bone matrix-secreting activity and in terms of drug discovery with the hope to improve the treatment of osteoporosis and other low bone mass syndromes.