THE PHYSIOLOGY OF IGFBP DEGRADING PROTEINASES IN BONE

IGFBP 降解骨中蛋白酶的生理学

• 批准号: 7462272
• 负责人: John L Fowlkes
• 金额: $ 24.36万
• 依托单位: ARKANSAS CHILDREN'S HOSPITAL RES INST
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-15 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7462272
• 关键词: Affect; Affinity; Anabolic Agents; Binding; Binding Proteins; Biological; Biological Availability; Cell Line; Cell surface; Complex; Data; Differentiation and Growth; Distraction Osteogenesis; Endopeptidases; Eosinophil Major Basic Protein; Equilibrium; Family; Gene Expression; Hormones; In Vitro; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Insulin-Like Growth Factor Receptor; Insulin-Like Growth-Factor-Binding Proteins; Insulin-Like-Growth Factor I Receptor; Intervention; Laboratories; Lead; Matrix Metalloproteinases; Mediating; Mediator of activation protein; Metalloproteases; Modeling; Osteoblasts; Osteogenesis; Parathyroid Hormones; Peptide Hydrolases; Physiology; Play; Pregnancy-Associated Plasma Protein-A; Process; Production; Protein Overexpression; Proteolysis; Proteolytic Processing; Reagent; Recombinants; Resistance; Role; Signal Transduction; Small Interfering RNA; Somatomedins; Stream; System; Technology; Therapeutic Intervention; Transcriptional Activation; Transgenic Organisms; Up-Regulation; bone; bone cell; bone growth factor; bone loss; collagenase 3; human PTH protein; in vivo; inhibitor/antagonist; insight; member; mutant; proteinase In

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.

描述（由申请人提供）：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（IGFBPs 1 - 6）拮抗其与细胞表面1型IGF受体的结合。骨细胞产生的蛋白酶可以将IGFBPs降解为低亲和力片段，从而使IGF-IGF受体相互作用发生。我们已经确定妊娠相关血浆蛋白-A（PAPP-A）作为IGFBP-4蛋白酶，MMP-13（胶原酶-3）作为IGFBP-5蛋白酶，两者都是由成骨细胞系MC3T3-E1产生的。因此，这些IGFBPs的蛋白水解加工可能在成骨细胞生成过程中调节IGF作用。我们已经创建了抗蛋白水解的IGFBP-4和IGFBP-5的重组突变体形式，并使用这些构建体建立了稳定转染的MC3T3-E1细胞系，该细胞系过表达抗蛋白酶IGFBP突变体。在此，我们建议使用这些试剂，以探讨IGFBP蛋白水解如何影响成骨细胞的生长和分化。我们将比较IGFBP-4或-5的蛋白酶抗性形式与可裂解形式相比如何影响成骨细胞生成，以及如果抑制或下调IGFBP降解蛋白酶（即，PAPP-A和MMP-13）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。为了探讨MMP-13如何介导PTH对骨细胞的合成代谢作用，我们将确定PTH介导的MMP-13上调的钝化如何改变体外成骨细胞中IGF的作用。我们还将探讨IGFBP-5的MMP-13抗性形式如何影响PTH对成骨细胞培养的作用。体内研究将确定全身性PTH给药是否改变骨中的IGF-IGFBP-IGF蛋白酶轴，以及MMP-13的缺失是否影响PTH和IGF介导的新骨形成。总之，这些研究将为控制骨中IGF生物利用度的机制提供新的见解，以及骨形成的上游介质（如PTH）如何通过调节IGF作用对成骨细胞发生产生影响。