THE PHYSIOLOGY OF IGFBP DEGRADING PROTEINASES IN BONE

描述(申请人提供)：胰岛素样生长因子(IGF)是成骨细胞生长和分化的重要介质；然而，6个高亲和力IGF结合蛋白(IGFBPs 1-6)拮抗它们与细胞表面1型IGF受体的结合。骨细胞产生的蛋白水解酶可以将IGFBPs降解成低亲和力的片段，从而允许IGF-IGF受体相互作用。我们鉴定了妊娠相关血浆蛋白-A(PAPP-A)是一种IGFBP-4蛋白，而基质金属蛋白酶-13(胶原酶-3)是一种IGFBP-5，两者都是由成骨细胞系MC3T3-E1产生的。因此，这些IGFBPs的蛋白分解处理可能调节IGF在成骨细胞形成过程中的作用。我们已经创造了抗蛋白水解性的IGFBP-4和IGFBP-5的重组突变形式，并使用这些构建物建立了稳定表达抗蛋白酶的IGFBP突变体的MC3T3-E1细胞系。在这里，我们建议使用这些试剂来探索IGFBP蛋白分解如何影响成骨细胞的生长和分化。我们将比较蛋白酶抗性形式的IGFBP-4或-5与可切割形式相比如何影响成骨细胞的形成，以及是否抑制或下调IGFBP降解蛋白(即PAPP-A和MMP-13)影响这一过程。在体内，我们将研究蛋白酶抗性或敏感形式的IGFBP-4或-5如何改变新骨形成，以及PAPP-A是否可以克服转基因过量生产IGFBP-4对新骨形成的骨抑制作用。为了探讨基质金属蛋白酶-13如何介导甲状旁腺激素对骨细胞的合成代谢作用，我们将确定钝化甲状旁腺素介导的基质金属蛋白酶-13上调如何在体外改变成骨细胞的IGF作用。我们还将探索基质金属蛋白酶-13抵抗形式的IGFBP-5如何影响成骨细胞培养上的甲状旁腺激素作用。体内研究将确定全身应用甲状旁腺素是否改变骨中的IGF-IGFBP-IGF蛋白酶轴，以及基质金属蛋白酶-13的缺失是否影响甲状旁腺激素和胰岛素样生长因子介导的新骨形成。总之，这些研究将为控制IGF在骨中的生物利用度的机制提供新的见解，以及上游骨形成介质，如PTH，如何通过调节IGF的作用来影响成骨细胞的形成。

DESCRIPTION (provided by applicant): Insulin-like growth factors (IGFs) are important mediators of growth and differentiation of osteoblasts; however, six high-affinity IGF-binding proteins (IGFBPs 1-6) antagonize their binding to cell surface type-1 IGF receptors. Proteases produced by bone cells can degrade IGFBPs into low-affinity fragments, allowing for IGF-IGF receptor interactions to occur. We have identified pregnancy-associated plasma protein-A (PAPP-A) as an IGFBP-4 proteinase and MMP-13 (collagenase-3) as an IGFBP-5 proteinase, and both are produced by the osteoblast cell line, MC3T3-E1. Thus, proteolytic processing of these IGFBPs may modulate IGF action during osteoblastogenesis. We have created recombinant mutant forms of IGFBP-4 and IGFBP-5 that are resistant to proteolysis, and have used these constructs to establish stably transfected MC3T3-E1 cell lines overexpressing protease resistant IGFBP mutants. Herein, we propose to use these reagents in order to explore how IGFBP proteolysis influences osteoblast growth and differentiation. We will compare how protease-resistant forms of IGFBP-4 or -5 impact osteoblastogenesis compared to cleavable forms, and if inhibiting or down-regulating IGFBP-degrading proteinases (i.e., PAPP-A and MMP-13) influences this process. In vivo, we will examine how protease-resistant or sensitive forms of IGFBP-4 or -5 alter new bone formation, and if PAPP-A can overcome osteoinhibitory effects of transgenic overproduction of IGFBP-4 on new bone formation. To explore how MMP-13 may mediate anabolic affects of PTH on bone cells, we will determine how blunting of PTH-mediated up-regulation of MMP-13 may alter IGF action in osteoblasts in vitro. We will also explore how MMP-13 resistant forms of IGFBP-5 impact PTH actions on osteoblast cultures. In vivo studies will determine if systemic PTH administration alters the IGF-IGFBP-IGF protease axis in bone and if deletion of MMP-13 affects PTH and IGF-mediated new bone formation. Together, these studies will provide new insights into mechanisms controlling IGF bioavailability in bone, and how up-stream mediators of bone formation, such as PTH, may exert their affects on osteoblastogenesis through regulating IGF action.