Role of Beta-Catenin Signaling in Osteoblast Function

描述(由申请人提供)： 最近的遗传学证据表明，LRP5在支持骨形成中发挥关键作用，这为规范的WNT信号通路是成骨细胞骨形成的主要调节因子提供了强有力的推定证据。本提案是为解决这一条例的机制和范围所作的努力。这将通过在成年小鼠体内开发受控基因敲除系统，并将其应用于成骨细胞系细胞中β-连环蛋白基因的定向敲除来实现。我们假设成骨细胞中规范的WNT信号在成骨细胞分化的不同阶段通过不同的机制对骨形成产生积极作用，促进早期成骨细胞的增殖，抑制完全分化的成骨细胞的凋亡。我们还假设甲状旁腺激素(PTH)的合成代谢作用是通过PTH受体信号增强规范的WNT信号的合成代谢作用而发挥的。为了验证这些假说，我们建议首先确定规范的WNT/LRP通路在成骨前细胞和成熟成骨细胞中的不同作用，这是通过调控敲除β-连环蛋白表达来实现的。四环素调控系统将被用来在体内控制成骨细胞中cre重组酶的表达。使用3.6kb和2.3kb的I型胶原启动子将允许我们在成骨前细胞和成熟成骨细胞(3.6kb启动子)或仅在成熟成骨细胞(2.3kb启动子)中去除β-连环蛋白。我们将通过MicroCT来确定诱导的β-连环蛋白基因敲除对骨骼内环境平衡的影响以及对经历增殖和凋亡的成骨细胞数量的影响。在初步研究中，我们发现PTH受体信号与β-连环素在激活LEF/TCF转录因子方面具有协同作用，LEF/TCF转录因子是典型的WNT信号的下游靶点。因此，我们将使用β-连环蛋白基因敲除模型来确定这种汇聚信号是否是外源性PTH合成代谢效应的重要途径。这些研究的成功完成将有助于确定规范的WNT信号在骨骼功能中的作用，并将为甲状旁腺素的合成代谢效应的潜在机制提供新的见解。

DESCRIPTION (provided by applicant): The recent genetic evidence that LRP5 plays a key role in supporting bone formation provides strong presumptive evidence that the canonical wnt signaling pathway is a primary regulator of osteoblastic bone formation. The present proposal constitutes an effort to address the mechanism and scope of this regulation. This will be accomplished by developing an in vivo system of controlled gene knockout in adult mice, and applying this to the targeted knockout of the beta-catenin gene in osteoblast lineage cells. We hypothesize that canonical wnt signaling in osteoblasts produces a positive effect on bone formation via different mechanisms at different stages of osteoblast differentiation, increasing proliferation in early osteoblasts and inhibiting apoptosis in fully differentiated osteoblasts. We also hypothesize that the anabolic action of parathyroid hormone (PTH) is exerted via the ability of PTH receptor signaling to potentiate the anabolic action of canonical wnt signaling. To test these hypotheses, we propose to first determine the differential role of the canonical wnt/LRP pathway in preosteoblasts and in mature osteoblasts by regulated knockout of beta-catenin expression. The tetracycline regulatory system will be used to allow for controlled expression of the cre recombinase in osteoblasts in vivo. The use of the 3.6 kb and 2.3 kb type I collagen promoters will allow us to ablate beta-catenin in pre-osteoblasts and mature osteoblasts (3.6 kb promoter) or only in mature osteoblasts (2.3 kb promoter). We will determine the effect of induced knockout of beta-catenin on skeletal homeostasis by microCT and on the number of osteoblasts undergoing proliferation and apoptosis. In preliminary studies, we have found that PTH receptor signaling synergizes with beta-catenin in the activation of LEF/TCF transcription factors, the downstream target of canonical wnt signaling. We will therefore use the beta-catenin knockout model to determine whether this convergent signaling is an essential pathway for the anabolic effect of exogenous PTH. Successful completion of these studies will help to define the role of canonical wnt signaling in skeletal function, and will provide novel insights into the mechanism underlying the anabolic effects of PTH.