Smad4/B-Catenin Signaling Cross-Talk for Osteoblastogenesis

描述（由申请人提供）：Wnt/β-连环蛋白系统对胚胎发生中的骨骼发育至关重要，并调节成年期的骨量。然而，Wnt/β-连环蛋白信号刺激出生后骨骼中骨生成和骨形成的机制仍然模糊不清。最近的数据显示，- 连环蛋白在成骨细胞分化过程中的两个点上提供了关键的调节信号;在未成熟但定向的成骨细胞前体中，-连环蛋白有利于成熟为基质分泌的成骨细胞和成骨细胞前体库的扩张;在分化的成骨细胞中，它抑制破骨细胞的发生和可能的终末成骨细胞分化。在以前的研究中，我们证明了连环蛋白与骨形态发生蛋白2和4（BMP-2/4）在产生新骨中相互作用。事实上，我们发现，BMP信号是所需的充分成骨刺激的<$-catenin，而Tcf/左依赖的转录活性不是。我们还发现，β-连环蛋白竞争性地被募集到BMP或Wnt信号通路，并且BMP和β-连环蛋白信号通路的交叉至少部分地由Smad 4/β-连环蛋白相互作用介导。该项目的中心假设是，在未成熟成骨细胞中，<$-连环蛋白的促成骨作用源于其与BMP信号传导，特别是Smad 4的相互作用，导致<$-连环蛋白竞争性募集到典型的Tcf/Lef-dependent或Smad 4-dependent信号。因此，β-连环蛋白可以通过Tcf/Lef转录活性作为增殖的刺激物，或者通过募集到含有Smad 4的转录复合物中作为未成熟成骨细胞的成熟的刺激物。为了验证这一假设，我们建议，1：确定通过Smad 4的<$-连环蛋白促成骨作用对BMP信号传导的依赖性; 2：分析Smad 4在调节Wnt依赖性成骨和Tcf/Lef信号传导中的作用; 3分析Smad 4和<$-连环蛋白之间的分子相互作用以促进成骨。我们将使用体内和体外方法的基础上诱导，条件性基因消融或激活模型，以研究是否干扰Smad 4表达改变<$-连环蛋白促成骨作用和“典型”Tcf/左依赖性活动。考虑到<$-连环蛋白在骨细胞调节中的基本作用，理解<$-连环蛋白传递促有丝分裂或分化信号的机制对于获得控制骨发育和体内平衡的分子网络的全貌至关重要。拟议的研究还将揭示分子相互作用的生物学重要性，可用作骨锚定的新靶点。 公共卫生相关性：在骨脱矿（如骨质疏松症）受试者中刺激骨形成的治疗选择有限。这项研究将研究两种分子之间的相互作用，这两种分子决定了骨形成细胞如何复制或分化以产生新骨。结果将使我们更好地了解成骨细胞如何在成年动物中增殖和发挥作用，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低骨量和骨折患者的新治疗方法。

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.