THE UBIQUITIN-PROTEOSOME PATHWAY AND BMP-2 EXPRESSION

描述(由申请人提供)：在体外和体内，对蛋白酶体的药物抑制导致啮齿类动物骨骼中骨形成的急剧增加(Garrett.et al.，2003)。我们的初步数据表明，这种作用是由骨生长调节因子骨形态发生蛋白-2(BMP-2)的表达增加所介导的(至少在很大程度上)。我们最近的初步数据表明，Gli家族的转录调控因子对BMP-2的影响负有责任。这项应用的目的是(1)确定抑制泛素-蛋白酶体途径促进成骨细胞分化和骨形成的分子机制，(2)在体内确定BMP-2表达增加在这些作用中的作用，以及(3)阐明Gli转录因子家族在BMP-2表达增加中的作用。我们最新的初步数据表明，在体外的骨细胞中，Gli2(以及较小程度的Gli1和Gli3)刺激BMP-2的转录，而在蛋白酶体中产生的截短形式的Gli3是一个强大的抑制因子(Gli2完全被蛋白酶体降解，但Gli1不被蛋白酶体处理)。因此，我们的假设是，BMP-2的转录通常由Gli2(刺激它)和截短的Gli3(与Gli2的刺激作用相反)调控。蛋白酶体抑制剂促进Gli2的积累，并抑制截短的Gli3的形成。因此，这些抑制剂选择性地刺激BMP-2转录和骨形成，主要是因为Gli2对BMP-2启动子的影响，而不是被截断的Gli3所对抗。在这一应用中，我们的目标是通过一系列体内和体外生物学实验来验证这一假说，这些实验旨在确定这些单个Gli家族成员在BMP-2转录和骨形成中的确切作用，以及它们与蛋白酶体抑制的关系。我们还计划通过过度表达Gli3的野生型和显性负突变的转基因小鼠来表征负责将Gli3靶向蛋白酶体，即(-TrCP)的E3泛素连接酶在骨形成中的作用。这些实验将澄清我们对泛素-蛋白酶体途径抑制对骨形成的调控作用的理解，并确定调控成骨细胞分化和骨形成的重要潜在分子靶点。

DESCRIPTION (provided by applicant): Pharmacologic inhibition of the proteasome causes dramatic increases in bone formation in rodent bones in vitro and in vivo (Garrett.et al., 2003). Our preliminary data suggests that this effect is mediated (at least in major part) by increased expression of the bone growth regulatory factor bone morphogenetic protein-2 (BMP-2). Our recent Preliminary Data suggest that the Gli family of transcriptional regulators is responsible for the effects on BMP-2. It is the purpose of this application to (1) determine the molecular mechanisms by which inhibition of the ubiquitin-proteasome pathway increases osteoblast differentiation and bone formation, (2) determine the role of increased BMP-2 expression in these effects in vivo, and (3) clarify the role of the Gli family of transcription factors in the increases in BMP-2 expression. Our most recent Preliminary Data suggest that in bone cells in vitro, Gli2 (and to a lesser extent, Gli1 and 3) stimulates BMP-2 transcription, while a truncated form of Gli3 produced in the proteasome is a powerful repressor (Gli2 is degraded completely by the proteasome, but Gli1 is not processed by the proteasome). Our hypothesis therefore is that BMP-2 transcription is normally regulated by Gli2 (which stimulates it) and truncated Gli3 (which opposes the stimulatory effects of Gli2). Proteasome inhibitors enhance accumulation of Gli2 and impair formation of truncated Gli3. These inhibitors thereby selectively stimulate BMP-2 transcription and bone formation primarily because of the effects of Gli2 on the BMP-2 promoter when unopposed by truncated Gli3. In this application, our goal is to test this hypothesis by a series of in vivo and in vitro biological experiments designed to determine the precise roles of these individual Gli family members on BMP-2 transcription and bone formation, as well as their relationship to proteasome inhibition. We also plan to characterize the role of the E3 ubiquitin ligase responsible for targeting Gli3 to the proteasome, namely (-TrCP, in bone formation by the use of transgenic mice overexpressing wild-type and dominant-negative mutants of this enzyme. These experiments should clarify our understanding of the regulatory effects of the ubiquitin-proteasome pathway inhibition on bone formation, and identify important potential molecular targets for regulation of osteoblast differentiation and bone formation.