Indian Hedgehog Signaling in Osteoblast Differentiation

印度刺猬信号在成骨细胞分化中的作用

• 批准号: 6705948
• 负责人: Fanxin Long
• 金额: $ 32.36万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6705948
• 关键词: biological signal transduction; bone development; bone fracture; cadherins; cell differentiation; chondrocytes; gene expression; gene targeting; genetically modified animals; histology; in situ hybridization; laboratory mouse; osteoblasts; osteoporosis; polymerase chain reaction; transcription factor; western blottings

DESCRIPTION (provided by applicant): A fundamental understanding of the molecular mechanisms underlying osteoblast differentiation is critical for developing novel therapeutics to treat osteoporosis and to enhance bone fracture healing. Indian hedgehog (Ihh), one of the three mammalian homologs of the Drosophila Hedgehog (Hh) gene and the only known Hh gene expressed in the vertebrate skeleton, is indispensable for osteoblast differentiation in the endochondral skeleton. Because Ihh deficient mice arrest early in the development of the osteoblast lineage, it is therefore not known whether Ihh is also important for later stages of osteoblast differentiation. Moreover, the molecular mechanisms underlying the early roles of Ihh in osteoblast differentiation are not known. This proposal sets out to expand our knowledge about the potential roles of Ihh in osteoblast differentiation and to determine whether Ihh functions by interacting with other molecules important for this process. Three Specific Aims are proposed. Aim 1: to examine potential roles of hedgehog signaling in preosteoblasts and osteoblasts. Specifically, the Cre-loxP technology will be used to remove the gene Smoothened, which encodes an obligatory component of the Hh pathway, from preosteoblasts and osteoblasts; Aim 2: to assess relationship between Ihh and Cbfa1 in osteoblast differentiation. In the Ihh null mutant, Cbfa1 expression is abolished in the perichondrium of the long bones, where osteoblasts first arise during development. In order to determine whether Cbfa1 deficiency is solely responsible for the osteoblast defect, Cbfa1 wilI be forced-expressed by a genetic means in the perichondrium to examine whether osteoblast differentiation is restored in Ihh null mice; Aim 3: to examine interactions between lhh and Wnt/b-catenin signaling pathways in osteoblast differentiation. Although the Wnt canonical signaling pathway has been implicated in osteoblast proliferation and function in postnatal life in both mice and men, genetic evidence linking Wnt signaling to osteoblast differentiation is lacking. Therefore Cre-loxP technology will be used to remove b-catenin from the progenitor cells of the osteoblast lineage. Biochemical analyses of the potential interaction between Ihh and b-catenin signaling pathways will also be performed in a cell culture system. Finally, genetic experiments will be performed to determine whether artificial activation of b-catenin signaling will rescue the osteogenic defect in the absence of Hh signaling.

描述（由申请人提供）：对成骨细胞分化的分子机制的基本理解对于开发新型疗法以治疗骨质疏松症和增强骨断裂愈合至关重要。印度刺猬（IHH）是果蝇刺猬（HH）基因的三个哺乳动物同源物之一，也是脊椎动物骨骼中唯一已知的HH基因，对于内骨骨骼中的成骨分布是必不可少的。由于IHH缺乏小鼠在成骨细胞谱系的发展早期停滞不前，因此尚不清楚IHH对于成骨细胞分化的以后阶段是否也很重要。此外，尚不清楚IHH在成骨细胞分化中发挥早期作用的分子机制。该提案旨在扩大我们对IHH在成骨细胞分化中潜在作用的知识，并确定IHH是否通过与对此过程重要的其他分子相互作用来函数。提出了三个具体目标。目的1：检查刺猬信号传导在骨细胞和成骨细胞中的潜在作用。具体而言，将使用CRE-LOXP技术从前骨细胞和成骨细胞中删除基因平滑，该基因平滑的基因平滑。目标2：评估成骨细胞分化中IHH和CBFA1之间的关系。在IHH无效突变体中，CBFA1表达在长骨的peri骨中被取消，在发育过程中首次出现成骨细胞。为了确定CBFA1缺乏症是否完全负责成骨细胞缺陷，CBFA1 Wili被perichondrium中的遗传手段表达表达，以检查IHH无效小鼠中是否还原成骨细胞分化。目标3：检查成骨细胞分化中LHH和Wnt/B-catenin信号通路之间的相互作用。尽管Wnt规范信号通路与小鼠和男性的成骨细胞增殖和功能有关，但缺乏将Wnt信号与成骨细胞分化联系起来的遗传证据。因此，将使用Cre-LoxP技术从成骨细胞谱系的祖细胞中去除B-catenin。 IHH和B-catenin信号通路之间潜在相互作用的生化分析也将在细胞培养系统中进行。最后，将进行遗传实验，以确定在没有HH信号传导的情况下，B-catenin信号传导的人工激活是否会挽救成骨缺陷。