Proteasomal Regulation of BMP Signaling in Bone Cells

描述(申请人提供)：许多骨骼疾病是由成骨细胞和软骨细胞功能缺陷引起的或与之相关。因此，了解骨和软骨形成的控制机制对于了解这些骨骼疾病的发病机制和确定开发新的治疗方法的分子靶点至关重要。这项建议的长期目标是让首席调查员(PI)发展一项独立的职业生涯，专注于研究成骨细胞和软骨细胞功能的调节机制，并开发新的治疗策略，以改善与骨相关的病理情况的结果。在这份独立科学家奖(K02)的申请中，候选人概述了一个使他能够实现这一目标的计划。PI将使用K02获得骨组织学、组织形态计量学以及体外和体内软骨细胞研究方面的额外技能，Brendan Boyce博士和Regis O‘Keefe博士将在罗切斯特大学最先进的机构设施中为候选人提供极好的支持和培训经验。研究的重点将是泛素-蛋白酶体途径对骨和软骨形成的调控。具体地说，目标将包括研究E3泛素连接酶SMurfl对Runx2和BMP信号蛋白的调节机制。SMurf 1是E3泛素连接酶Meet结构域家族的成员，已被发现与BMP激活的Smads 1和5相互作用并诱导它们的降解(朱等，1999)。在初步研究中，我们首次证明了SMurfl通过泛素-蛋白酶体途径诱导Runx2降解(赵等人，2003年)。Smad6与Runx2相互作用，并介导SMurfl诱导的Ruhx2降解(Shenet al.，2005)。SMurfl在体外抑制成骨细胞分化，在体内抑制出生后骨形成(赵等人，2004年)。在拟议的研究中，我们将通过分析最近在Pi的实验室中产生的COL11a1-SMurf1、Col1a1-mSMurf1和Col2a1-SMurf1转基因小鼠来研究SMurfl在骨形成和软骨细胞成熟中的作用。在这些小鼠中，利用组织特异性的co1a1和co2a1启动子，SMurfl或mSMurfl转基因基因的表达被特异性地定位于成骨细胞和软骨细胞。这些研究的基本假设是，SMurfl是Runx2和Smadl功能的重要调节分子，在成骨细胞和软骨细胞分化过程中发挥着特殊的作用。该项目的具体目标是：1)确定SMurfl在骨偶联过程中对骨形成的特定作用；2)确定SMurfl在软骨细胞分化和成熟中的作用。将检验两个工作假说：1)SMurfl抑制成年小鼠骨连接过程中的骨形成；2)SMurfl是软骨细胞分化和成熟的负调控因子。这些研究的结果将为SMurfl调节成骨细胞和软骨细胞成熟程序的机制提供深入的见解。

DESCRIPTION (provided by applicant): Many bone diseases are caused by or are related to defects in osteoblast and chondrocyte function. An understanding of the mechanisms by which bone and cartilage formation is controlled is therefore critical to understanding the pathogenesis of these bone diseases and to determine the molecular targets for development of new therapies for their treatment. The long-term goal of this proposal is for the Principal Investigator (PI) to develop an independent career focused on investigating the regulatory mechanisms of osteoblast and chondrocyte function and developing novel therapeutic strategies to improve outcomes in bone-related pathologic situations. In this Independent Scientist Award (K02) application, the candidate outlines a plan that will enable him achieve this objective. The PI will use the K02 to acquire additional skills in bone histology, histomorphometry, and in vitro and in vivo chondrocyte research and Drs. Brendan Boyce and Regis O'Keefe will provide an excellent support and training experience for the candidate in the 'state of the art' institutional facilities at University of Rochester. The focus of the research will be on the regulation of bone and cartilage formation by the ubiquitin-proteasome pathway. Specifically, the aims will encompass examining the regulatory mechanisms of Runx2 and BMP signaling proteins by the E3 ubiquitin ligase Smurfl. Smurf 1 is a member of the Meet domain family of E3 ubiquitin ligases and has been found to interact with BMP-activated Smads 1 and 5 and induce their degradation (Zhu et al., 1999). In preliminary studies, we have demonstrated for the first time that Smurfl induces Runx2 degradation through an ubiquitin-proteasome pathway (Zhao et al., 2003). Smad6 interacts with Runx2 and mediates Smurfl-induced Ruhx2 degradation (Shen et al., 2005). Smurfl inhibits osteoblast differentiation in vitro and postnatal bone formation in vivo (Zhao et al., 2004). In the proposed studies, we will investigate roles of Smurfl in bone formation and chondrocyte maturation by analyzing the Col11a1-Smurfl, Col1a1-mSmurf1 and Col2a1-Smurfl transgenic mice which have been generated in the Pi's lab recently. In these mice, expression of Smurfl or mSmurfl transgene is targeted specifically to osteoblasts and chondrocytes using the tissue-specific co!1a1 and co!2a1 promoters. The underlying hypothesis for the proposed studies is that Smurfl is an important regulatory molecule for Runx2 and Smadl function and plays a specific role in osteoblast and chondrocyte differentiation. The specific aims of the project are: 1) to determine the specific effect of Smurfl on bone formation during bone coupling; and 2) to determine the role of Smurfl in chondrocyte differentiation and maturation. Two working hypotheses will be tested: 1) Smurfl inhibits bone formation during bone coupling in adult mice; and 2) Smurfl is a negative regulator in chondrocyte differentiation and maturation. The results from these studies will provide insights into the mechanisms of Smurfl in regulation of the maturational program of osteoblasts and chondrocytes.