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

描述（由申请人提供）：佩吉特病（PD）是第二常见的骨疾病，在美国影响1-2百万患者。PD是正常骨重建的最夸张的例子之一，是骨重建研究的范例。遗传和环境因素都与PD的发病机制有关，我们目前的RO 1资助正在研究这两种成分在体内PD发展中的相互作用。破骨细胞（OCL）是参与PD发病机制的主要细胞。来自PD患者的OCL具有不同的表型。它们表达麻疹病毒核衣壳蛋白（MVNP），对1，25-（OH）2D 3和RANKL高度反应，产生高水平的IL-6和TAPII-17（VDR的共激活因子），并具有增加的骨吸收能力/OCL。重要的是，在使用TRAP启动子的转基因小鼠（TRAP-MVNP小鼠）中MVNP向OCL谱系的靶向表达导致在这些小鼠中发生pagetic骨病变和OCL。然而，维生素D受体（VDR）过度活跃在PD发展中的作用仍不清楚。我们先前报道了TAFII-17是由MVNP诱导的，并且在PD患者中存在增加的TAFII-17水平。增加的TAFII-17水平导致OCL前体对1，25-（OH）2D 3的高响应性。然而，TAFII-17自身表达的增加不足以在体外诱导pagetic OCL，而是需要向培养物中加入IL-6。重要的是，在没有VDR的情况下，MVNP不能诱导pagetic OCL。我们发现，在表达MVNP的OCL前体中，1，25-（OH）2D 3与VDR的结合增加了不同信号传导途径（例如p38和PLC 32）的激活，这增加了IL-6的产生、OCL的形成或每个OCL的核数。目的/假设：我们的假设是，与PD发病机制有关的环境因素，如麻疹病毒（MV），增加OCL前体中的IL-6水平，从而增加TAFII 17的表达。在生理浓度的1，25-（OH）2D 3下，TADII-17表达的增加增强VDR活性，这反过来激活p38 MAPK和PLC 32/CREB途径以增加IL-6产生、OCL数量、每个OCL的核和OCL骨吸收能力。此外，IL-6的升高的局部水平诱导正常OCL前体变得对维生素D高度响应，并在体内维生素D存在下形成具有增加的骨再吸收能力的超多核OCL。为了检验这一假设，我们将追求以下具体目标。具体目标1：确定VDR活性增加与OCL前体中局部高水平的IL-6结合是否会导致体内超多核OCL的形成和骨吸收增加。具体目标二：确定TRAP-IL-6小鼠给予维生素D3是否导致超多核OCL形成和骨吸收增加。研究设计：目标1：我们将通过分析TRAP-TADII-17、TRAP-IL-6和TRAP-TADII-17/IL-6，来表征在OCL中存在或不存在升高的IL-6的情况下TADII-17表达增加对体内pagetic OCL形成的影响。（通过繁殖前两个品系产生）小鼠（随访超过18个月）观察TRAP-MVNP小鼠中骨病变、异常OCL活性、过度新骨形成和我们报告的PD的其他特征。我们将这些结果与从TRAP-MVNP和WT小鼠获得的结果进行比较。目标二：我们将每天向TRAP-IL-6小鼠施用1，25-（OH）2D 3（0.1 μ g/kg IP/天）或媒介物，持续4周，并通过组织形态计量学分析以及全血离子钙、磷、碱性磷酸酶和Pyd水平的分析来确定这些小鼠是否在体内发展出pagetic OCL和PD特征性的骨病变。影响：本提案将测试VDR活性增加在体内PD-OCL和骨病变发展中的作用，以及当小鼠接受维生素D3治疗时，正常OCL前体中TAPII-17和IL-6水平增加的共表达是否足以诱导PD特征性的超多核OCL。

DESCRIPTION (provided by applicant): Paget's disease (PD) is the second most common bone disease, affecting 1-2 million patients in the U.S. PD is one of the most exaggerated examples of normal bone remodeling and is a paradigm for studies of bone remodeling. Both genetic and environmental factors have been implicated in the pathogenesis of PD, and our current RO1 grant is examining the interaction of these two components in the development of PD in vivo. The primary cell involved in the pathogenesis of PD is the osteoclast (OCL). OCLs from PD patients have a distinct phenotype. They express measles virus nucleocapsid protein (MVNP), are hyper-responsive to 1,25-(OH)2D3 and RANKL, produce high levels of IL-6 and TAFII-17, a coactivator of VDR, and have an increased bone resorbing capacity per OCL. Importantly, targeted expression of MVNP to the OCL lineage in transgenic mice using the TRAP promoter (TRAP-MVNP mice) results in development of pagetic bone lesions and OCLs in these mice. However, the role that vitamin D receptor (VDR) hyperactivity plays in the development of PD is still unclear. We previously reported that TAFII-17 is induced by MVNP, and increased TAFII-17 levels are present in PD patients. Increased TAFII-17 levels result in hyper- responsivity of OCL precursors to 1,25-(OH)2D3. However, increased expression of TAFII-17 by itself is not sufficient to induce pagetic OCL in vitro but requires addition of IL-6 to the cultures. Importantly, MVNP cannot induce pagetic OCL in the absence of VDR. We found that binding of 1,25-(OH)2D3 to VDR in OCL precursors expressing MVNP increases activation of distinct signaling pathways (e.g. p38 and PLC32), which either increase IL-6 production, OCL formation or nuclear number per OCL. Objective/Hypothesis: It is our hypothesis that environmental factors implicated in the pathogeneses of PD, such as measles virus (MV), increase IL-6 levels in OCL precursors, which in turn increase TAFII17 expression. The increased expression of TAFII-17 enhances VDR activity at physiologic concentration of 1,25-(OH)2D3, that in turn activates both the p38 MAPK and PLC32/CREB pathways to increase IL-6 production, OCL numbers, nuclei per OCL and OCL bone resorption capacity. Further, elevated local levels of IL-6 induce normal OCL precursors to become hyper-responsive to vitamin D and form hyper- multinucleated OCL with increased bone resorbing capacity in the presence of vitamin D in vivo. To test this hypothesis we will pursue the following specific aims. Specific Aim 1: Determine if increased VDR activity in combination with high local levels of IL-6 in OCL precursors results in formation of hyper-multinucleated OCLs and increased bone resorption in vivo. Specific Aim 2: Determine if administration of vitamin D3 to TRAP-IL-6 mice results in formation of hyper- multinucleated OCLs and increased bone resorption. Study Design: Aim 1: We will characterize the effects of increased expression of TAFII-17 in the absence or presence of elevated IL-6 in OCL on pagetic OCL formation in vivo by analyzing TRAP-TAFII-17, TRAP-IL-6, and TRAP- TAFII-17/IL-6 (generated by breeding the first two lines) mice (followed over 18 months) for development of bone lesions, abnormal OCL activity, excessive new bone formation and other features of PD that we have reported in our TRAP-MVNP mice. We will compare these results to those obtained from TRAP-MVNP and WT mice. Aim 2: We will administer 1,25-(OH)2D3 (0.1 <g/kg IP per day) or vehicle daily to TRAP-IL-6 mice for 4 weeks, and determine if these mice develop pagetic OCL and bone lesions characteristic of PD in vivo by histomorphometry analysis as well as analysis of whole blood ionized calcium, phosphorus, alkaline phosphatase, and Pyd levels. Impact: This proposal will test for the role of increased VDR activity in the development of PD-OCL and bone lesions in vivo and if co-expression of increased levels of TAFII-17 and IL-6 in normal OCL precursors is sufficient to induce hyper-multinucleated OCLs characteristic of PD when mice are treated with vitamin D3.