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，六种高亲和力 IGF 结合蛋白 (IGFBP 1-6) 拮抗它们与细胞表面 1 型 IGF 受体的结合。骨细胞产生的蛋白酶可以将 IGFBP 降解为低亲和力片段，从而发生 IGF-IGF 受体相互作用。我们已确定妊娠相关血浆蛋白 A (PAPP-A) 为 IGFBP-4 蛋白酶，MMP-13（胶原酶-3）为 IGFBP-5 蛋白酶，两者均由成骨细胞系 MC3T3-E1 产生。因此，这些 IGFBP 的蛋白水解过程可能会调节成骨细胞生成过程中 IGF 的作用。我们创建了抗蛋白水解的 IGFBP-4 和 IGFBP-5 重组突变体形式，并使用这些构建体建立了稳定转染的 MC3T3-E1 细胞系，过表达蛋白酶抗性 IGFBP 突变体。在此，我们建议使用这些试剂来探索 IGFBP 蛋白水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 与可裂解形式相比如何影响成骨细胞生成，以及抑制或下调 IGFBP 降解蛋白酶（即 PAPP-A 和 MMP-13）是否会影响这一过程。在体内，我们将研究IGFBP-4或-5的蛋白酶抗性或敏感形式如何改变新骨形成，以及PAPP-A是否可以克服IGFBP-4转基因过量产生对新骨形成的骨抑制作用。为了探索 MMP-13 如何介导 PTH 对骨细胞的合成代谢影响，我们将确定减弱 PTH 介导的 MMP-13 上调如何在体外改变成骨细胞中的 IGF 作用。我们还将探讨 IGFBP-5 的 MMP-13 抗性形式如何影响成骨细胞培养物中的 PTH 作用。体内研究将确定全身 PTH 给药是否会改变骨中的 IGF-IGFBP-IGF 蛋白酶轴，以及 MMP-13 的缺失是否会影响 PTH 和 IGF 介导的新骨形成。总之，这些研究将为控制骨中 IGF 生物利用度的机制，以及骨形成的上游介质（如 PTH）如何通过调节 IGF 作用对成骨细胞生成产生影响提供新的见解。

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.