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

描述（由申请人提供）：佩吉特病（PD）是最夸张的骨重塑，伴有骨吸收增加的病灶区域，并伴有旺盛的新骨形成。过多的新骨形成导致弱编织骨沉积，导致PD的许多临床后遗症，包括骨畸形或骨折、颅骨增厚、骨痛和神经根压迫。破骨细胞（OCLs）驱动骨形成的增加，因为针对OCLs的治疗减少骨吸收和形成。然而，PD患者骨形成增加的机制尚不清楚。遗传和环境因素影响PD的发展。与帕金森病相关的最常见突变是SQSTM1/p62基因，尤其是p62P392L。携带生殖系p62P394L的小鼠（p62小鼠），相当于人类的p62P392L，表现出增加的ocl，但不发展为PD。70% PD患者的oclc表达麻疹病毒核衣壳蛋白（MVNP）基因，靶向表达MVNP到oclc的转基因小鼠（MVNP小鼠）发生oclc和PD特征的骨病变。重要的是，在MVNP小鼠中，IL-6表达的缺失阻断了原位OCL的形成，并增加了体内骨形成。因此，环境因素（如MVNP）在ocl中的表达是PD特征性骨异常发展所必需的。最近，我们发现MVNP而非p62P394L增加了OCLs和EphB4在成骨细胞（OBs）上偶联因子ephrinB2的表达，这是由IL-6介导的。这些结果表明，OCLs中的MVNP诱导偶联因子增加OB活性。此外，MVNP诱导OCLs表达IGF1，这可能进一步促进骨形成。本提案将通过验证MVNP通过诱导OCLs上的ephrinB2和OBs上的EphB4增加PD中OB活性的假设，部分通过MVNP上调OCLs中的IL-6和IGF1，来评估MVNP在PD中OB活性异常中的作用。因此，我们将：1)验证MVNP诱导OCLs中ephrinB2和IGF1以及OBs中EphB4增加OB活性的假设。我们将确定调节ephrinB2/EphB4和IGF1水平/活性对MVNP诱导OB活性增加的影响。我们还将确定MVNP在p62P392L PD患者oclc中的表达是否会增加oclc中的ephrinB2和IGF1，并增强其诱导OB分化的能力。2)验证IL-6和IGF1介导MVNP诱导ephrinB2和EphB4的假说。我们将确定IL-6通过MVNP诱导ephrinB2/EphB4的机制，以及IGF1是通过表达MVNP的ocl增强ephrinB2和/或IL-6的产生，还是仅仅通过ephrinB2/EphB4增强OB活性。在这些研究中，我们将使用来自MVNP、WT、IL-6-/-和MVNP和WT小鼠的细胞，这些小鼠在OCLs中IL-6或IGF1在体外被敲低。3)生成靶向缺失oclc中ephrinB2和/或IGF1和OBs中EphB4的p62/MVNP小鼠，以评估ephrinB2/EphB4和IGF1在PD中OB活性升高中的作用。

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.