Smad4/B-Catenin Signaling Cross-Talk for Osteoblastogenesis

描述(由申请人提供)：Wnt/？-catenin系统对胚胎发育中的骨骼发育是必不可少的，并在成年生活中调节骨量。然而，Wnt/β-catenin信号刺激出生后骨骼成骨和骨形成的机制仍然不清楚。最近的数据表明，在成骨细胞分化过程中，β-catenin在两个点上提供了关键的调节信号；在未成熟但已确定的成骨细胞前体中，-catenin有助于成熟为分泌基质的成骨细胞并扩大成骨细胞前体池；在已分化的成骨细胞中，它抑制破骨细胞的形成，可能还抑制成骨细胞的终末分化。在以前的研究中，我们证明了β-连环蛋白与骨形态发生蛋白-2和骨形态发生蛋白-4(BMP-2/4)在新骨形成过程中相互作用。事实上，我们发现BMP信号对于连环蛋白的完全成骨刺激是必需的，而Tcf/Lef依赖的转录活性不是必需的。我们还发现β-catenin竞争性地招募到BMP或Wnt信号通路中，BMP和β-catenin信号的交集至少部分是由Smad4/β-catenin相互作用介导的。这个项目的中心假设是，β-连环蛋白的成骨作用源于它与未成熟成骨细胞中的BMP信号，特别是Smad4信号的相互作用，导致？-连环蛋白竞争性地募集到依赖于Tcf/Lef或Smad4的典型信号。因此，β-catenin可以通过Tcf/Lef转录活性刺激未成熟成骨细胞的增殖，也可以通过募集到含有Smad4的转录复合体来刺激未成熟成骨细胞的成熟。为了验证这一假说，我们建议，1：通过Smad4确定β-catenin促成骨作用对BMP信号的依赖性；2：分析Smad4在调节Wnt依赖的成骨和Tcf/Lef信号中的作用；3分析Smad4和β-catenin在成骨中的分子相互作用。我们将使用基于可诱导的、有条件的基因消融或激活模型的体内和体外方法来研究干扰Smad4的表达是否改变了连环蛋白的成骨作用和Tcf/Lef依赖的“典型”活性。考虑到？-catenin在骨细胞调节中的基础作用，了解？-catenin传递有丝分裂或分化信号的机制对于全面了解控制骨骼发育和动态平衡的分子网络至关重要。拟议的研究还将揭示分子相互作用的生物学重要性，该分子相互作用可能被用作骨合成代谢的新靶点。 公共卫生相关性：骨质疏松症等骨质脱矿患者刺激骨形成的治疗选择有限。这项研究将研究决定骨形成细胞如何复制或分化以产生新骨的两个分子之间的相互作用。研究结果将使我们更好地了解骨形成细胞在成年动物中的增殖和功能，并将揭示刺激骨形成的新方法，这些方法可能在未来用于发现治疗骨量低和骨折的新方法。

DESCRIPTION (provided by applicant): The Wnt/¿-catenin system is essential for skeletal development in embryogenesis and regulates bone mass in adult life. However, the mechanisms by which Wnt/¿-catenin signaling stimulates osteogenesis and bone formation in the post-natal skeleton remain nebulous. Recent data suggest that ¿-catenin provides critical regulatory cues at two points during the osteoblast differentiation program; in immature but committed osteoblast precursors ¿-catenin favors maturation into matrix secreting osteoblasts and expansion of the osteoblast precursor pool; in differentiated osteoblasts it inhibits osteoclastogenesis and perhaps terminal osteoblast differentiation. In previous studies, we demonstrated that ¿-catenin interacts with bone morphogenetic protein-2 and 4 (BMP-2/4) in producing new bone. Indeed, we find that BMP signaling is required for full osteogenic stimulation by ¿-catenin, whereas Tcf/Lef-dependent transcriptional activity is not. We also find that ¿-catenin is competitively recruited to either BMP or Wnt signaling pathways, and that the intersection of BMP and ¿-catenin signaling is, at least in part, mediated by Smad4/¿-catenin interactions. The central hypothesis of this project is that the pro-osteogenic action of ¿-catenin originates from its interactions with BMP signaling, and specifically Smad4, in immature osteoblasts, resulting in competitive recruitment of ¿-catenin to either canonical Tcf/Lef-dependent or Smad4-dependent signals. Thus, ¿-catenin can function as stimulator of either proliferation, via Tcf/Lef transcriptional activity, or maturation of immature osteoblasts, via recruitment into Smad4-containing transcriptional complexes. To test this hypothesis we propose to, 1: determine the dependency of ¿-catenin pro-osteogenic action on BMP signaling via Smad4; 2: analyze the role of Smad4 in modulating Wnt-dependent osteogenesis and Tcf/Lef signaling; 3 analyze the molecular interactions between Smad4 and ¿-catenin for osteogenesis. We will use in vivo and in vitro approaches based on inducible, conditional gene ablation or activation models to study whether interference with Smad4 expression alters ¿-catenin pro-osteogenic action and "canonical" Tcf/Lef-dependent activity. Considering the fundamental role of ¿-catenin in bone cell regulation, understanding the mechanisms by which ¿-catenin delivers either a mitogenic or a differentiation signal is essential to gain a full picture of the molecular network by which bone development and homeostasis are controlled. The proposed studies will also disclose the biologic importance of a molecular interaction that may be used as a new target for bone anabolism. PUBLIC HEALTH RELEVANCE: Therapeutic options for stimulating bone formation in subjects with bone demineralization, such as osteoporosis, are limited. This research will study an interaction between two molecules that determine how bone forming cells replicate or differentiate to produce new bone. Results will allow us to better understand how bone forming cells proliferate and function in adult animals, and will disclose new ways to stimulate bone formation that may be used in the future to discover new treatments for people with low bone mass and fractures.