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高阶网络，该网络确保成骨细胞形成骨和破骨细胞吸收骨之间的有效平衡。该网络的扰动通常与骨骼疾病有关，最严重的是骨质疏松症，具有骨折和相关发病率和死亡率的固有风险。骨重塑的主要调控因子是典型的Wnt信号通路，它对成骨细胞分化的几个方面施加严格的控制。同源结构域蛋白TGIF在转化生长因子β （TGF-¿）信号通路中发挥转录辅助抑制因子的作用，TGF-¿信号通路已知限制骨形成。为了进一步了解TGIF的生理功能和调控是如何被控制的，我们使用酵母双杂交实验筛选了TGIF结合蛋白。先前未知的TGIF结合蛋白Axin2引起了我们的注意，它作为Wnt信号的负调节因子，通过其介导破坏体的形成，破坏体是一个信号模块，其中蛋白激酶GSK3-标记- catenin进行泛素依赖性降解。在随后的功能获得和功能丧失研究中，TGIF被发现促进wnt诱导的转录反应。我们的初步研究还表明，TGIF对Wnt信号诱导成骨细胞分化的能力至关重要。引人注目的是，我们发现TGIF可以通过一种独立于其抑制TGF-¿信号能力的机制来调节骨形成，这表明TGIF的另一种生理功能是在Wnt反应中确保有效的成骨细胞命运决定。与它在成骨细胞承诺中的作用一致，在小鼠中消融TGIF会损害成骨细胞成熟并减少骨量。综上所述，这些发现使我们提出了一个可行的假设，即TGIF可能有助于骨中典型Wnt信号的调节，对破坏性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：进一步描述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的组装/拆卸中可能发挥的作用，从而影响-Catenin的稳定性。目的2：体外和体内分析TGIF完全缺失和成骨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信号和骨形成调控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认识，最终演变为设计和实施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新概念。

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.