Pre-osteoclast fusion

描述（由申请人提供）：破骨细胞（OC）的骨吸收超过成骨细胞（OB）的骨形成是一个生物学问题，在广泛和昂贵的疾病中发挥核心作用，包括骨质疏松症、骨关节炎和类风湿性关节炎、牙周病和假体/植入物松动。单核前体融合成多核OC的机制和调节的描述是重要的，因为正常的骨吸收需要融合。在我们对OC分化过程中基因表达变化的研究中，我们发现了OC-STAMP（破骨细胞刺激跨膜蛋白），并注意到与其他人所示的融合所必需的因子（称为DC-STAMP）有一系列惊人的相似之处。相似性包括1）RANKL刺激OC前体后mRNA和蛋白质的上调; 2）预测的跨膜（TM）螺旋拓扑结构; 3）在OC-STAMP的C-末端一半中存在DC-STAMP家族共有序列; 4）通过敲除、敲低或抗体抑制体内和体外多核破骨细胞的形成; 5）过表达后刺激融合。这两种蛋白质在陆生脊椎动物中高度保守。在急性模型中，诱导OC分化的骨硬化大鼠模型，我们提出了初步的证据，抗OC-STAMP抗体抑制体内破骨细胞的形成。[最近的一份报告验证了我们的假设，即OC-STAMP-/-小鼠会像DC-STAMP -/-小鼠一样具有单核OC。该提案将根据3个特定目的研究前OC融合，以阐明其在OC细胞生物学和体内活性中的作用，作为控制骨丢失的潜在治疗靶点，以及与作为融合因子的DC-STAMP的关系。SA 1将建立OC-和DC-STAMP两者的膜拓扑结构（已报道DC-STAMP是7-TM超家族成员，但不同的算法预测不同的拓扑结构）。所获得的信息应有助于“下一代”抗体和/或抑制性肽针对这些融合蛋白的已证实的细胞外环，并为有关活动和调节的进一步假设提供基础。在SA 2下，将产生OC-和DC-STAMP敲除小鼠（现有品系的请求被拒绝）。我们将在我们手中确认两种菌株的骨骼和OC表型，并且我们将使用源自它们的细胞进行融合实验以确定OC-和DC-STAMP的相互作用或相互依赖性以用于融合。将进行研究以鉴定OC-和DC- STAMP的潜在配体和调节因子。在SA 3中，我们将增加我们的初步研究的规模，即用抗C-STAMP抗体在t1/t1大鼠中进行急性体内OC抑制。我们还将使用无胸腺大鼠研究抗[融合蛋白]抗体抑制在经证实的卵巢切除大鼠骨质疏松症模型中的作用。总之，这些研究应该提供关于OC-STAMP和DC-STAMP的拓扑结构和细胞生物学、关于前OC融合、关于OC-STAMP损失对骨骼的表型影响以及原则上抑制前OC融合是否是阻止体内骨损失的有效方法的重要新信息。

DESCRIPTION (provided by applicant): Bone resorption by osteoclasts (OCs) exceeding formation by osteoblasts (OBs) is a biological problem with a central role in widespread and costly disorders, including osteoporosis, osteo- and rheumatoid arthritis, periodontal disease, and prosthesis/implant loosening. Delineation of mechanisms and regulation of the fusion of mononuclear precursors into multinucleated OCs is important since fusion is needed for normal bone resorption. In our studies of gene expression changes during OC differentiation, we discovered OC-STAMP (osteoclast-stimulatory transmembrane protein) and noted a series of striking similarities to a factor shown by others to be essential to fusion, called DC-STAMP. Similarities include 1) up- regulation of mRNA and protein upon stimulation of OC precursors by RANKL; 2) predicted topology of transmembrane (TM) helices; 3) presence of a DC-STAMP family consensus sequence in the C-terminal half of OC-STAMP; 4) suppression of formation of multinucleated osteoclasts in vivo and in vitro by either knockout, knockdown, or antibody; 5) stimulation of fusion upon overexpression. Both proteins are highly conserved in terrestrial vertebrates. In a model of acute, induced OC differentiation in an osteopetrotic rat model, we present preliminary evidence that anti-OC-STAMP antibody suppresses osteoclastogenesis in vivo. [A recent report validated our hypothesis that OC-STAMP -/- mice would, like DC-STAMP -/- mice, have mononuclear OCs.] This proposal will investigate pre-OC fusion under 3 Specific Aims to clarify its role in OC cell biology and activity in vivo, as a potential therapeutic targetto control bone loss, and in relation to DC- STAMP as a fusion factor. SA1 will establish the membrane topology of both OC- and DC-STAMP (DC- STAMP has been reported to be a 7-TM superfamily member, yet different algorithms predict different topologies). Information gained should facilitate "next-generation" antibodies and/or inhibitory peptides directed at proven extracellular loops of these fusigens and provide a basis for further hypotheses about activities and regulation. Under SA2, OC- and DC-STAMP knockout mice will be produced (requests for existing lines were refused). We will confirm the skeletal and OC phenotypes of both strains in our hands, and we will use cells derived from them to perform fusion experiments to determine interaction or interdependence of OC- and DC- STAMP for fusion. Studies will be done to identify potential ligands and regulatory factors for OC- and DC- STAMP. Under SA3, we will increase the size of our preliminary study of acute, in vivo OC inhibition in tl/tl rats with anti-C-STAMP antibody. We will also investigate the effects of anti-[fusigens] antibody inhibition in the proven, ovariectomized rat model of osteoporosis using athymic rats. Together, these studies should provide significant new information about the topology and cell biology of both OC-STAMP and DC-STAMP, about pre- OC fusion, about the phenotypic impact of loss of OC-STAMP on the skeleton, and whether in principle, inhibition of pre-OC fusion is a valid approach to stemming bone loss in vivo.