THE UBIQUITIN-PROTEOSOME PATHWAY AND BMP-2 EXPRESSION

描述（由申请人提供）：蛋白酶体的药理学抑制引起体外和体内啮齿动物骨中骨形成的显著增加（Garrett.等，2003年）。我们的初步数据表明，这种影响是介导的（至少在主要部分）骨生长调节因子骨形态发生蛋白-2（BMP-2）的表达增加。我们最近的初步数据表明，Gli家族的转录调节因子是负责对BMP-2的影响。本申请的目的是（1）确定抑制泛素-蛋白酶体途径增加成骨细胞分化和骨形成的分子机制，（2）确定增加的BMP-2表达在体内这些作用中的作用，和（3）阐明Gli家族转录因子在BMP-2表达增加中的作用。我们最新的初步数据表明，在体外骨细胞中，Gli 2（以及在较小程度上，Gli 1和3）刺激BMP-2的转录，而在蛋白酶体中产生的截短形式的Gli 3是一个强大的阻遏物（Gli 2被蛋白酶体完全降解，但Gli 1不被蛋白酶体加工）。因此，我们的假设是，BMP-2的转录通常由Gli 2（刺激它）和截短的Gli 3（反对Gli 2的刺激作用）调节。蛋白酶体抑制剂增强Gli 2的积累并损害截短的Gli 3的形成。因此，这些抑制剂选择性地刺激BMP-2转录和骨形成，主要是因为当不被截短的Gli 3对抗时，Gli 2对BMP-2启动子的作用。在本申请中，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设，这些实验旨在确定这些单个Gli家族成员对BMP-2转录和骨形成的确切作用，以及它们与蛋白酶体抑制的关系。我们还计划表征的E3泛素连接酶的作用，负责靶向Gli 3的蛋白酶体，即（-TrCP，在骨形成中使用的转基因小鼠过度表达野生型和显性阴性突变体的这种酶。这些实验将阐明我们对泛素-蛋白酶体通路抑制对骨形成的调控作用的理解，并确定成骨细胞分化和骨形成调控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.