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

描述（由申请人提供）：佩吉特病（PD）是骨重建与骨吸收增加的局灶性区域伴随着旺盛的新骨形成的最夸张的例子。过多的新骨形成导致脆弱的编织骨沉积，这是PD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）驱动骨形成的增加，因为靶向OCL的治疗减少骨吸收和形成。然而，负责PD中骨形成增加的机制尚不清楚。遗传和环境因素有助于PD的发展。与PD相关的最常见突变是SQSTM 1/p62基因，特别是p62 P392 L。携带种系p62 P394 L（p62小鼠）（人p62 P392 L的鼠等效物）的小鼠表现出OCL增加，但不发生PD。来自70%的PD患者的OCL表达麻疹病毒核衣壳蛋白（MVNP）基因，并且MVNP靶向表达OCL的转基因小鼠（MVNP小鼠）发生OCL和PD特征性骨病变。重要的是，MVNP小鼠中IL-6表达的缺失阻断了Pagetic OCL形成并增加了体内骨形成。因此，环境因素的表达（例如，OCL中的MVNP）是PD中发生特征性骨异常所必需的。最近，我们发现MVNP而不是p62 P394 L增加OCL和EphB 4在成骨细胞（OB）上的偶联因子ephrinB 2的表达，这是由IL-6介导的。这些结果表明，MVNP在OCL诱导耦合因子，增加OB活性。此外，MVNP诱导OCL表达IGF 1，这可能进一步增加骨形成。该提议将通过检验MVNP通过诱导OCL上的ephrinB 2和OB上的EphB 4（部分通过MVNP上调OCL中的IL-6和IGF 1）增加PD中的OB活性的假设来评估MVNP在PD中异常OB活性中的作用。因此，我们将：1）测试MVNP诱导OCL中的ephrinB 2和IGF 1以及OB中的EphB 4增加OB活性的假设。我们将确定调节ephrinB 2/EphB 4和IGF 1水平/活性对由MVNP诱导的OB活性增加的影响。我们还将确定来自p62 P392 L PD患者的OCL中的MVNP表达是否增加OCL中的ephrinB 2和IGF 1并增强其诱导OB分化的能力。2)检验IL-6和IGF 1介导MVNP诱导ephrinB 2和EphB 4的假设。我们将确定MVNP诱导IL-6诱导ephrinB 2/EphB 4的机制，以及IGF 1是否通过表达MVNP的OCL增强ephrinB 2和/或IL-6的产生，或者仅仅通过ephrinB 2/EphB 4增强OB活性。对于这些研究，我们将使用来自MVNP、WT、IL-6-/-以及MVNP和WT小鼠的细胞，在离体OCL中敲低IL-6或IGF 1。3)产生具有OCL中ephrinB 2和/或IGF 1以及OB中EphB 4的靶向缺失的p62/MVNP小鼠，以评估ephrinB 2/EphB 4和IGF 1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.