IGFBP 4 PROTEASE AND HUMAN OSTEOBLASTS

胰岛素样生长因子（IGFs）是骨形成的重要刺激物。过去的研究发现，IGFBP-4是骨细胞中IGF作用的有效抑制剂，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浓度在局部水平受合成和降解机制的调节。在本研究中，我们选择评估IGF-II依赖性IGFBP-4蛋白酶（IGFBP-4蛋白酶）在调节人成骨细胞（hOBs）中IGFBP-4可用性从而调节其活性的作用，原因如下：1) hOBs产生的IGFBP-4蛋白酶将IGFBP-4切割成低IGF结合亲和力的形式，从而可能控制IGFBP-4/IGF- ii复合物中IGF- ii的释放。2)IGFBP-4蛋白酶的活性受IGF- ii和TGF-等骨调节因子的调节，因此与骨形成有关。3) IGFBP-4蛋白酶是IGF系统的最新组成部分，其在hOB代谢中的作用尚未确定。本研究的目的是评估IGFBP-4蛋白酶在hOBs中调节IGF作用的作用，并阐明IGF- ii增强IGFBP-4降解的机制。本文将验证两种假设：1)IGFBP-4蛋白酶是hOBs中IGF系统的重要调控组分。2) IGF-II与IGFBP-4的结合改变了构象，使IGFBP-4中的蛋白酶识别序列更容易被IGFBP-4蛋白酶获取。为了验证假设1，我们将制备部分纯化的IGFBP-4蛋白酶，以确定其初级裂解位点和蛋白酶识别序列。这些信息以及我们对IGF结合域定位的了解将用于设计IGFBP-4类似物，这些类似物具有完全蛋白酶抗性（PR），但与野生型（WT）的IGF结合具有相似的亲和力。这些类似物将用于评估IGFBP-4蛋白酶在IGF-II/IGFBP-4复合物中释放IGF-II的作用，从而增加igf的局部有丝分裂活性。为了验证假设2，我们将制备具有降低IGF-II结合亲和力但保留蛋白水解结构域的类似物。如果IGF-II不能增强这些类似物的降解，这一发现将表明IGF-II与IGFBP-4的结合对IGFBP-4蛋白水解至关重要。然后确定IGF-II与IGFBP-4结合是否会增加IGFBP-4与IGFBP-4蛋白酶之间的关联。我们预计这些研究的完成将使我们更深入地了解IGFBP-4蛋白酶在hOB增殖调控中的作用，并为设计基于肽的IGFBP-4蛋白酶激活剂用于骨质疏松症的治疗提供基础。此外，PR IGFBP-4类似物（如果比WT IGFBP-4更有效）可能在治疗IGF依赖性癌症方面具有治疗潜力。

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.