Regulatory Pathways in Osteoclast Formation and Function

描述(申请人提供)：类风湿性关节炎、牙周病和一些肿瘤相关骨转移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(OCS)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丢失的影响，可能为未来的治疗或同时影响骨和免疫系统的各种疾病奠定基础。我们发现免疫调节蛋白磷脂酶CGamma2(PLCGamma2)是RANKL(NFkappaB配体受体激活剂)诱导的破骨细胞形成的中枢介质，不依赖于PLCGamma1。OCs是主要的骨吸收细胞，主要由骨髓巨噬细胞(BMMS)发育而来，主要受两种主要调节因子的影响：巨噬细胞集落刺激因子(M-CSF)和NFkappaB受体激活剂配体(RANKL)，以及较少了解的共刺激因子，它们通过含有免疫受体酪氨酸激活基序(ITAM)的受体作用于OC前体细胞[2，4，5]。我们的数据表明，在小鼠中，靶向缺失PLCGamma2导致了一种骨化表型。由于AP1和NFkappaB的激活缺陷，以及破骨细胞形成的主要转录因子NFATc1的上调失败，PLC-Gamma2缺陷的OC前体无法分化。虽然依赖ITAM上调NFATc1需要PLCGamma2的磷酸化及其催化活性，但JNK和NFkappaB通路的激活可能依赖于PLCGamma2结合和激活GAB2的能力，GAB2是RANKL介导的破骨细胞发生中重要的适配蛋白[6]，并调节GAB2向RANK信号复合体的募集。 PLCGamma2在OC分化中的非冗余作用表明，识别使PLCGamma2具有促进OC分化/功能的独特结构域可能揭示新的OC调节机制，并为新的抗吸收治疗提供基础。根据初步数据中提出的观察结果，我们假设： 1)通过调节NFATc1的上调，而不是JNK或NFkappaB的上调，PLCGamma2的催化活性是OC分化所必需的 2)PLCGamma2的适配功能是破骨细胞发生所必需的，它通过调节GAB2的激活以及随后JNK和NFkappaB的激活而发挥作用 3)在体内，PLCGamma2至少部分地通过JNK/NFkappaB轴和NFATc1途径介导破骨细胞的形成和骨溶解反应。 在这项建议中，我们寻求： 具体目标1：a)构建PLCGamma2突变体，以阐明其催化活性和适配功能在OC分化调控中的作用；b)构建表达破骨细胞形成所需的PLCGamma2独特区域的TAT融合蛋白，以确定新的抗吸收方法。 具体目标2：a)在体内确定PLCGamma2在OC分化和炎性骨溶解中的作用，以及b)测试携带突变形式的PLCGamma2的小鼠的体内溶骨反应以及围绕PLCGamma2功能结构域的TAT融合蛋白的抗吸收作用。

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.