REGULATORY PATHWAYS IN OSTEOCLAST FORMATION AND FUNCTION

描述（由申请方提供）：类风湿性关节炎、牙周病和一些肿瘤相关骨转移中的过度骨丢失主要是由于免疫系统的异常激活导致破骨细胞（OC）的过度刺激和激活[1-3]。确定影响骨免疫系统的常见信号分子及其对正常和病理性骨质流失的影响可能为未来治疗或影响骨和免疫系统的各种疾病奠定基础。我们已经确定了免疫调节蛋白磷脂酶C γ 2（PLC γ 2）作为RANKL（NF κ B受体激活剂配体）诱导的破骨细胞生成的中心介质的新作用，独立于PLC γ 1。OC是主要的骨吸收细胞，主要在两种主要调节剂的影响下从骨髓巨噬细胞（BMPs）发育而来：巨噬细胞集落刺激因子（M-CSF）和NF κ B配体的受体激活剂（RANKL），以及通过OC前体细胞上含有免疫受体酪氨酸激活基序（ITAM）的受体起作用的鲜为人知的共刺激因子[2，4，5]。我们的数据表明，在小鼠中靶向缺失PLC γ 2导致骨硬化表型。PLC γ 2缺陷型OC前体不能分化，这是由于AP 1和NF κ B的活化缺陷以及NFATc 1（破骨细胞生成的主要转录因子）上调失败所致。虽然NFATc 1的ITAM依赖性上调需要PLC γ 2磷酸化及其催化活性，但JNK和NF κ B通路的激活可能依赖于PLC γ 2结合和激活GAB 2（一种在RANKL介导的破骨细胞生成中显示重要的衔接蛋白[6]）并调节GAB 2募集至RANK信号复合物的能力。 PLC γ 2在OC分化中的非冗余作用表明，鉴定使PLC γ 2在其促进OC分化/功能的能力方面独特的结构域可能揭示新的OC调节机制，并为新的抗吸收疗法提供基础。根据初步数据中提供的观察结果，我们假设： 1)通过调节NFATc 1上调，OC分化需要PLC γ 2的催化活性，但不需要JNK或NFkappaB 2)通过调节GAB 2活化和随后的JNK和NF κ B活化，PLC γ 2的适配器功能是破骨细胞生成所必需的 3)PLC γ 2至少部分通过JNK/NF κ B轴和NFATc 1途径介导体内破骨细胞生成和溶骨反应。 在本提案中，我们力求： 具体目标1：a）产生PLC γ 2突变体以阐明催化活性的作用和PLC γ 2在OC分化调节过程中的衔接子功能，和B）产生表达破骨细胞生成所需的PLC γ 2的独特区域的TAT融合蛋白，目的是鉴定新的抗再吸收方法。 具体目标二：a）确定PLC γ 2在体内OC分化和炎性骨质溶解中的作用，和B）测试在携带突变形式的PLC γ 2的小鼠中的体内骨质溶解反应和包含PLC γ 2的功能结构域的TAT融合蛋白的抗再吸收作用。

DESCRIPTION (provided by applicant): Excessive bone loss in rheumatoid arthritis, periodontal disease and some tumor- associated bone metastasis is mostly due to an abnormal activation of the immune system leading to hyper-stimulation and activation of osteoclasts (OCs) [1-3]. Identifying common signaling molecules affecting the osteo-immune system and their impact on normal and pathological bone loss may lay the groundwork for future therapies or the variety of diseases that affect both bone and the immune system. We have identified a novel role for the immunomodulatory protein phospholipase Cgamma2 (PLCgamma2) as central mediator of RANKL (Receptor Activator of NFkappaB Ligand)-induced osteoclastogenesis, independent of PLCgamma1. OCs, the principal bone resorbing cells, develop from bone marrow macrophages (BMMs) primarily under the influence of two major regulators: the macrophage colony stimulating factor (M-CSF) and receptor activator of NFkappaB ligand (RANKL), and less understood costimulatory factors that act via immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors on OC precursor cells [2,4, 5]. Our data indicate that targeted deletion of PLCgamma2 in mice leads to an osteopetrotic phenotype. PLCgamma2-deficient OC precursors fail to differentiate due to defective activation of AP1 and NFkappaB and a failure in upregulation of NFATc1, a master transcription actor for osteoclastogenesis. While PLCgamma2 phosphorylation and its catalytic activity are required for ITAM - dependent upregulation of NFATc1, activation of JNK and NFkappaB pathways may be dependent on the capacity of PLCgamma2 to bind and activate GAB2, an adapter protein shown to be important in RANKL-mediated osteoclastogenesis [6], and modulate GAB2 recruitment to the RANK signaling complex. The non-redundant role of PLCgamma2 in OC differentiation suggests that identifying the structural domains which make PLCgamma2 unique in its capacity to promote OC differentiation/function may unveil novel OC regulatory mechanisms and provide the basis for new antiresorptive therapies. Based on observations presented under preliminary data we hypothesize that: 1) the catalytic activity of PLCgamma2 is required for OC differentiation by modulating NFATc1 upregulation, but not JNK or NFkappaB 2) the adapter function of PLCgamma2 is required for osteoclastogenesis by modulating GAB2 activation and subsequent activation of JNK and NFkappaB 3) PLCgamma2 mediates osteoclastogenesis and osteolytic responses in vivo, at least in part, through the JNK/NFkappaB axis and the NFATc1 pathway. In this proposal we seek to: Specific Aim 1: a) generate PLCgamma2 mutants to clarify the role of the catalytic activity and the adapter function of PLCgamma2 during regulation of OC differentiation, and b) generate TAT-fusion-proteins expressing unique regions of PLCgamma2 required for osteoclastogenesis with the intent to identify novel anti-resorptive approaches. Specific Aim 2: a) determine the role of PLCgamma2 in OC differentiation and inflammatory osteolysis, in vivo, and b) test the in vivo osteolytic response in mice harboring mutated forms of PLCgamma2 and the anti-resorptive effects of TAT-fusion proteins encompassing functional domains of PLCgamma2.