Smad4/B-Catenin Signaling Cross-Talk for Osteoblastogenesis

描述（由申请人提供）：Wnt/¿-catenin系统在胚胎发育过程中对骨骼发育至关重要，并调节成人生活中的骨量。然而，Wnt/¿-catenin信号刺激出生后骨骼成骨和骨形成的机制仍不清楚。最近的数据表明，在成骨细胞分化过程中，¿-catenin在两点上提供了关键的调节线索；在未成熟但稳定的成骨细胞前体中，-连环蛋白有利于成熟成基质分泌成骨细胞和成骨细胞前体池的扩大；在分化的成骨细胞中，它抑制破骨细胞的发生，并可能抑制终末成骨细胞的分化。在之前的研究中，我们证明了¿-catenin与骨形态发生蛋白-2和4 （BMP-2/4）相互作用产生新骨。事实上，我们发现BMP信号是由¿-catenin完全刺激成骨所必需的，而Tcf/ left依赖的转录活性则不是。我们还发现-catenin竞争性地被BMP或Wnt信号通路招募，BMP和-catenin信号通路的交叉，至少部分是由Smad4/ -catenin相互作用介导的。本研究的核心假设是，在未成熟的成骨细胞中，-catenin的促成骨作用源于它与BMP信号，特别是Smad4的相互作用，导致-catenin竞争性地招募到典型的Tcf/ left依赖信号或Smad4依赖信号。因此，-catenin既可以通过Tcf/ left转录活性促进增殖，也可以通过招募到含有smad4的转录复合物中促进未成熟成骨细胞的成熟。为了验证这一假设，我们提出，1：确定¿-catenin促成骨作用对BMP信号通过Smad4的依赖性；2：分析Smad4在调节wnt依赖性成骨和Tcf/ left信号通路中的作用；分析Smad4和¿-catenin在成骨过程中的分子相互作用。我们将采用基于诱导、条件基因消融或激活模型的体内和体外方法来研究干扰Smad4表达是否会改变¿-catenin促成骨作用和“典型”Tcf/ left依赖活性。考虑到¿-catenin在骨细胞调节中的基本作用，了解¿-catenin传递有丝分裂或分化信号的机制对于全面了解骨发育和体内平衡控制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，这种相互作用可能被用作骨合成代谢的新靶点。

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.