Targeting thrombospondin 1 in bone resorption

描述（由申请人提供）：多发性骨髓瘤（MM）最常见和最严重的两种并发症是局部骨骼破坏和全身性骨质流失。这是因为破骨细胞介导的骨吸收增加和伴随的骨形成减少。在健康和大多数疾病状态下，破骨细胞来源于单核细胞/巨噬细胞谱系中的前体。然而，骨髓瘤骨病中导致破骨细胞数量增加的细胞和分子事件以及破骨细胞前体的性质尚不确定。这一应用建立在我们最近的发现之上，即骨髓瘤细胞可以快速诱导人类树突状细胞（dc）形成破骨细胞，但有趣的是，在相同的条件下，不能诱导单核细胞或巨噬细胞形成破骨细胞。这些研究还确定了CD47-血小板spondin1 （TSP1）相互作用在这一过程中的主导作用。CD47，也被称为整合素相关蛋白，是两个配体TSP1和SIRP11的结合伙伴。CD47在骨髓瘤细胞中明显过表达，骨髓瘤细胞以接触依赖的方式诱导dc表达TSP1。我们发现，阻断TSP1与CD47的相互作用完全抑制骨髓瘤细胞刺激dc破骨细胞形成的能力，以及RANKL/CSF1诱导正常骨髓前体破骨细胞形成的能力。此外，令人意想不到的是，阻断TSP1/CD47相互作用也显著减弱了pth诱导的骨吸收。后一项发现表明，CD47和TSP1之间的相互作用可能在除骨髓瘤外的其他病理生理条件下介导破骨细胞活性方面发挥重要作用。为了研究TSP1/CD47相互作用在骨髓瘤诱导的骨溶解中的作用，我们将追求三个具体目标。在特异性目标1中，我们将在骨髓瘤动物模型中确定体内阻断TSP1/CD47相互作用对骨吸收和肿瘤生长的影响。我们假设阻断TSP1/CD47相互作用可以防止骨髓瘤诱导的骨质流失。在Specific Aim 2中，我们将确定TSP1是否在刺激破骨细胞生成中起作用。我们还将探讨RANKL介导dc诱导骨髓瘤TSP1表达的可能性。最后，我们将开始探索一氧化氮是介导TSP1/CD47在破骨细胞前体和dc中相互作用的关键细胞内信号的可能性。在Specific Aim 3中，我们将比较来自单核细胞的人类破骨细胞的表型、基因表达谱和功能特性，以及来自人类骨髓瘤细胞与人类dc相互作用的人类破骨细胞。我们期望来自dc的破骨细胞与来自单核细胞的破骨细胞具有不同的特征。这些研究将为一种针对骨髓瘤骨病和其他加速骨质流失状态的TSP1的新方法提供基础。我们发现抗体介导的TSP1阻断在体内是有效的，这表明TSP1可能是控制病理性骨溶解的一个非常接近的药物靶点。

DESCRIPTION (provided by applicant): Two of the most common and severe complications of multiple myeloma (MM) are localized skeletal destruction and generalized bone loss. These occur because of both an increase in osteoclast-mediated bone resorption and an accompanying reduction in bone formation. In health and in most disease states, osteoclasts derive from precursors in the monocyte/macrophage lineage. However, the cellular and molecular events that lead to increased numbers of osteoclasts as well as the nature of osteoclast precursors in myeloma bone disease are not known with certainty. This application builds on our recent finding that myeloma cells rapidly induce formation of osteoclasts from human dendritic cells (DCs), but interestingly, not from monocytes or macrophages under identical conditions. These studies also identified a dominant role for CD47- thrombospondin1 (TSP1) interactions in this process. CD47, also known as Integrin Associated Protein, is a binding partner for two ligands, TSP1 and SIRP11. CD47 is markedly overexpressed by myeloma cells and myeloma cells induce TSP1 expression by DCs in a contact dependent manner. We have found that blocking the interaction of TSP1 with CD47 completely inhibits the ability of myeloma cells to stimulate the formation of osteoclasts from DCs as well as RANKL/CSF1 induced osteoclastogenesis from normal marrow precursors. In addition and quite unexpectedly, blocking the TSP1/CD47 interaction also markedly attenuated PTH-induced bone resorption in vivo. This latter finding indicates that the interaction between CD47 and TSP1 may be important in mediating osteoclast activity in other pathophysiologic conditions besides myeloma. In order to examine the role of the TSP1/CD47 interaction in myeloma-induced osteolysis we will pursue three specific aims. In Specific Aim 1, we will determine the effects of blocking the TSP1/CD47 interaction in vivo on bone resorption and tumor growth in an animal model of myeloma. We hypothesize that blocking the TSP1/CD47 interaction will prevent myeloma-induced bone loss. In Specific Aim 2, we will determine whether TSP1 plays a role in stimulating osteoclastogenesis. We will also explore the possibility that RANKL mediates myeloma induction of TSP1 expression by DCs. Finally, we will begin to explore the possibility that nitric oxide is a key intracellular signal mediating the effects of the TSP1/CD47 interaction in osteoclast precursors and DCs. In Specific Aim 3, we will compare the phenotype, gene expression profile and functional properties of human osteoclasts derived from monocytes to those derived from the interaction of human myeloma cells with human DCs. We expect that osteoclasts derived from DCs will have a distinct profile compared to those derived from monocytes. These studies will provide the basis for a novel approach targeting TSP1 in myeloma bone disease and other states of accelerated bone loss. Our finding that antibody-mediated blockade of TSP1 is effective in vivo suggest that TSP1 may be a very approachable drug target for controlling pathologic osteolysis.