IGFBP 4 PROTEASE AND HUMAN OSTEOBLASTS

IGFBP 4 蛋白酶和人类成骨细胞

• 批准号: 2904795
• 负责人: Xuezhong Qin
• 金额: $ 16.41万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-04 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2904795
• 关键词: affinity chromatography; bone morphogenetic proteins; conformation; crosslink; endopeptidases; enzyme activity; gel filtration chromatography; human tissue; insulinlike growth factor; ion exchange chromatography; mass spectrometry; osteoblasts; physiologic bone resorption; polymerase chain reaction; protein binding; protein degradation; protein sequence; protein structure function; proteolysis; site directed mutagenesis; tissue /cell culture; western blottings

Insulin-like growth factors (IGFs) are important stimulators of bone formation. Past findings that IGFBP-4 is a potent inhibitor of IGF action in bone cells and that IGFBP-4 production is regulated by osteoregulatory agents support our premise that IGFBP-4 is a key component of the IGF system in bone. In addition, there is evidence that IGFBP-4 concentration is regulated at the local level by mechanisms involving both synthesis and degradation. In this study, we chose to evaluate the role of IGF-II dependent IGFBP-4 protease (IGFBP-4 protease) in regulating the availability, and thus the activity of IGFBP-4 in human osteoblasts (hOBs) for the following reasons: 1) IGFBP-4 protease produced by hOBs cleaves IGFBP-4 into forms with low IGF binding affinity and thus may control IGF-II release from the IGFBP-4/IGF-II complex, 2) The activity of IGFBP-4 protease is regulated by osteoregulatory agents such as IGF-II and TGF- and is thus relevant to bone formation. 3) IGFBP-4 protease is the newest component of the IGF system and its role in hOB metabolism has not been established. The aims of this study are to evaluate the role of IGFBP-4 protease in modulating IGF action in hOBs and elucidate the mechanism involved in IGF-II enhancement of IGFBP-4 degradation. Two hypotheses will be tested: 1) IGFBP-4 protease is an important regulatory component of the IGF system in hOBs. 2) Binding of IGF-II to IGFBP-4 alters the conformation such that the protease recognition sequence in IGFBP-4 is more accessible to IGFBP-4 protease. To test hypothesis 1, we will prepare partially purified IGFBP-4 protease to identify the primary cleavage site and the protease recognition sequence. This information together with our knowledge of the localization of the IGF binding domain will be used to design IGFBP-4 analogs which are fully protease resistant (PR) but bind to IGF with similar affinity as that of the wild type (WT). Such analogs will be used to evaluate the role of IGFBP-4 protease in releasing IGF-II from IGF-II/IGFBP-4 complex thereby increasing the local mitogenic activity of the IGFs. To test hypothesis 2, we will prepare analogs that have reduced IGF-II binding affinity but retain the proteolytic domain. If IGF-II fails to enhance degradation of such analogs, such a finding would suggest that the binding of IGF-II to IGFBP-4 is essential to IGFBP-4 proteolysis. Then whether binding of IGF-II to IGFBP-4 increases the association between IGFBP-4 and IGFBP-4 protease will be determined. We anticipate that completion of these studies will lead to our greater understanding of the role of IGFBP-4 protease in the regulation of hOB proliferation and provides a basis for the design of peptide-based IGFBP-4 protease activators for osteoporosis treatment. Moreover, PR IGFBP-4 analog (if more potent than WT IGFBP-4) may have therapeutic potential in the treatment of IGF dependent cancers.

胰岛素样生长因子(IGF)是骨形成的重要刺激因子。过去的研究结果表明，IGFBP-4是骨细胞中IGF作用的有效抑制剂，并且IGFBP-4的产生受骨调节剂的调节，这支持了我们的假设，即IGFBP-4是骨中IGF系统的关键组成部分。此外，有证据表明，IGFBP-4的浓度在地方一级受到合成和降解机制的调节。在这项研究中，我们选择评估IGF-II依赖的IGFBP-4蛋白水解酶(IGFBP-4)在调节人成骨细胞(HOBS)中的可用性以及IGFBP-4活性方面的作用，原因如下：1)HOBS产生的IGFBP-4蛋白酶将IGFBP-4裂解成与IGF结合的低亲和力的形式，从而可能控制IGFBP-4/IGF-II复合体中IGF-II的释放；2)IGFBP-4蛋白的活性受骨调节剂如IGF-II和转化生长因子-4的调节，因此与骨形成有关。3)IGFBP-4蛋白水解酶是胰岛素样生长因子系统的最新成员，其在HOB代谢中的作用尚未确定。本研究旨在探讨胰岛素样生长因子结合蛋白-4(IGFBP-4)酶在调节胰岛素样生长因子(IGF)作用中的作用，并阐明IGF-II促进IGFBP-4降解的机制。将检验两个假设：1)IGFBP-4蛋白酶是HOBS中IGF系统的重要调节成分。2)IGF-II与IGFBP-4的结合改变了IGFBP-4的构象，使IGFBP-4中的蛋白酶识别序列更容易被IGFBP-4酶所识别。为了验证假设1，我们将制备部分纯化的IGFBP-4蛋白酶来鉴定主要切割位点和蛋白酶识别序列。这些信息和我们对IGF结合域定位的了解将被用来设计完全抗蛋白酶(PR)但与IGF具有类似亲和力的IGFBP-4类似物(WT)。这些类似物将被用来评估IGFBP-4酶在从IGF-II/IGFBP-4复合体中释放IGF-II从而增加IGFS局部有丝分裂活性中的作用。为了检验假设2，我们将制备降低了IGF-II结合亲和力但保留蛋白水解域的类似物。如果IGF-II不能促进这种类似物的降解，这一发现将表明IGF-II与IGFBP-4的结合对IGFBP-4的蛋白降解是必不可少的。然后，将确定IGF-II与IGFBP-4的结合是否增加了IGFBP-4与IGFBP-4蛋白酶之间的联系。我们期望这些研究的完成将使我们更好地了解IGFBP-4蛋白酶在HOB增殖调节中的作用，并为设计用于治疗骨质疏松症的多肽IGFBP-4蛋白酶激活剂提供基础。此外，PR IGFBP-4类似物(如果比WT IGFBP-4更有效)可能在治疗IGF依赖的癌症方面具有治疗潜力。