MVNP, p62P392L and IL-6 in the Pathogenesis of PD

描述(由申请人提供)：Paget病(PD)是骨重建中最夸张的例子，局部区域骨吸收增加，伴随着旺盛的新骨形成。过多的新骨形成导致弱编织骨的沉积，这是导致帕金森病的许多临床后遗症的原因，包括骨畸形或骨折、颅骨增厚、骨痛和神经根压迫。破骨细胞(OCL)推动骨形成的增加，因为针对OCL的治疗减少了骨的吸收和形成。然而，帕金森病患者骨形成增加的机制尚不清楚。遗传因素和环境因素共同参与了帕金森病的发生发展。与帕金森病相关的最常见的突变是SQSTM1/p62基因，特别是p62P392L。携带胚系p62P394L的小鼠(p62小鼠)表现出OCL增加，但不会发展为帕金森病。70%的PD患者的OCL表达麻疹病毒核衣壳蛋白(MVNP)基因，靶向表达MVNP的转基因小鼠(MVNP小鼠)会出现PD特有的OCL和骨病变。重要的是，在MVNP小鼠中，IL-6的表达缺失阻止了Pagtic OCL的形成，并增加了体内的骨形成。因此，环境因素(如MVNP)在OCL中的表达是帕金森病特有的骨异常发展所必需的。最近，我们发现MVNP而不是p62P394L能促进OCL和成骨细胞(OB)表达偶联因子EphB4，这种偶联因子是由IL-6介导的。这些结果表明，OCL中的MVNP诱导偶联因子，使OB活性增加。此外，MVNP通过OCL诱导IGF1的表达，可能进一步促进骨形成。本研究将通过验证MVNP通过诱导OCL上的ewitinB2和OBS上的EphB4，部分通过MVNP上调OCL中IL-6和IGF1的表达来增加PD的OB活性的假说，来评估MVNP在PD OB活动异常中的作用。因此，我们将：1)检验MVNP在OCL中诱导ePhinB2和IGF1，在OB中诱导EphB4增加OB活性的假设。我们将确定调节ewitinB2/EphB4和IGF1水平/活性对MVNP诱导的OB活性增加的影响。我们还将确定p62P392L PD患者OCL中MVNP的表达是否增加了OCL中的ewitinB2和IGF1，并增强了其诱导OB分化的能力。2)验证IL-6和IGF1介导MVNP诱导EphB_2和EphB_4的假说。我们将确定IL-6‘S通过MVNP诱导ePhinB2/EphB4产生的机制，以及IGF1是通过表达MVNP的OCL增加ewitinB2和/或IL-6的产生，还是仅仅通过ewitinB2/EphB4增强OB的活性。在这些研究中，我们将使用MVNP、WT、IL-6-/-以及MVNP和WT小鼠的细胞，在体外OCL中下调IL-6或IGF1。3)建立p62/MVNP小鼠，靶向缺失OCL中的ePhinB2和/或IGF1，以及OBS中的EphB4，以评估ePhinB2/EphB4和IGF1在PD中OB活性增加中的作用。

DESCRIPTION (provided by applicant): Paget's Disease (PD) is the most exaggerated example of coupled bone remodeling with focal areas of increased bone resorption accompanied by exuberant new bone formation. The excessive new bone formation results in deposition of weak woven bone, which is responsible for many of the clinical sequelae of PD including bone deformity or fracture, skull thickening, bone pain and nerve root compression. Osteoclasts (OCLs) drive the increased bone formation, because treatments targeting OCLs decrease bone resorption and formation. However, the mechanisms responsible for the increased bone formation in PD are unknown. Genetic and environmental factors contribute to development of PD. The most frequent mutations linked to PD are in the SQSTM1/p62 gene, in particular p62P392L. Mice harboring germline p62P394L(p62 mice), the murine equivalent of human p62P392L, exhibit increased OCLs, but do not develop PD. OCLs from 70% of PD patients express the measles virus nucleocapsid protein (MVNP) gene, and transgenic mice with targeted expression of MVNP to OCLs (MVNP mice) develop OCLs and bone lesions characteristic of PD. Importantly, loss of IL-6 expression in MVNP mice blocked the pagetic OCL formation and increased bone formation in vivo. Thus, expression of environmental factors (e.g., MVNP) in OCLs is required for the development of characteristic bone abnormalities in PD. Recently, we found that MVNP but not p62P394L increases expression of the coupling factors ephrinB2 by OCLs and EphB4 on osteoblasts (OBs), which was mediated by IL-6. These results suggest that MVNP in OCLs induces coupling factors that increase OB activity. Further, MVNP induced expression of IGF1 by OCLs which may further increase bone formation. This proposal will assess the role of MVNP in the abnormal OB activity in PD by testing the hypothesis that MVNP increases OB activity in PD through induction of ephrinB2 on OCLs and EphB4 on OBs, in part through MVNP's up-regulation of IL-6 and IGF1 in OCLs. Thus, we will: 1) Test the hypothesis that MVNP's induction of ephrinB2 and IGF1 in OCLs and EphB4 in OBs increases OB activity. We will determine the effects of modulating ephrinB2/EphB4 and IGF1 levels/activity on the increased OB activity induced by MVNP. We will also determine if MVNP expression in OCLs from p62P392L PD patients increases ephrinB2 and IGF1 in OCLs and enhances their capacity to induce OB differentiation. 2) Test the hypothesis that IL-6 and IGF1 mediate the induction of ephrinB2 and EphB4 by MVNP. We will determine the mechanisms responsible for IL-6's induction of ephrinB2/ EphB4 by MVNP, and if IGF1 enhances ephrinB2 and/or IL-6 production by OCLs expressing MVNP or simply enhances OB activity via ephrinB2/EphB4. For these studies, we will use cells from MVNP, WT, IL-6-/- and MVNP and WT mice with knockdown of IL-6 or IGF1 in OCLs ex vivo. 3) Generate p62/MVNP mice with targeted deletion of ephrinB2 and/or IGF1 in OCLs and EphB4 in OBs to assess the roles of ephrinB2/EphB4 and IGF1 in the increased OB activity in PD.