Viral and Genetic Regulation of Abnormal OCL Activity in Paget's Disease

佩吉特病中异常 OCL 活性的病毒和遗传调控

• 批准号: 9515641
• 负责人: DEBORAH Lynn GALSON
• 金额: $ 39.73万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-16 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9515641
• 关键词: Adult; Affect; American; Area; Autophagocytosis; Biology; Bone Marrow; Bone Resorption; Bone remodeling; Cell Lineage; Cell Nucleus; Characteristics; Complex; DNA Sequence Alteration; Data; Deformity; Degenerative polyarthritis; Development; Disease; Environmental Risk Factor; Etiology; Exhibits; Family; Family member; GTF2H1 gene; Genes; Genetic; Genetic Predisposition to Disease; Human; In Vitro; Interleukin-6; Knock-in Mouse; Knock-out; Lead; Lesion; Link; Lytic Lesion; Measles virus; Measles virus nucleocapsid protein; Measles virus nucleoprotein; Mediating; Metabolic Bone Diseases; Molecular; Mouse Protein; Mus; Mutation; Nature; Nerve compression syndrome; Osteitis Deformans; Osteoblasts; Osteoclasts; Osteogenesis; Paget' s Disease; Pain; Pathogenesis; Pathologic; Pathological fracture; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Phenocopy; Phenotype; Phosphorylation; Phosphotransferases; Play; Production; Proteins; Regulation; Reporting; Resolution; Role; Scaffolding Protein; Signal Repression; Signal Transduction; TBK1 gene; TNF gene; TNFSF11 gene; Testing; Transgenic Organisms; Viral Proteins; bone; bone metabolism; bone quality; deafness; environmental agent; genome wide association study; in vivo; insight; knock-down; member; microCT; monocyte; mutant; novel; progenitor; protein complex; protein expression; public health relevance; receptor; recruit; risk variant; small molecule inhibitor; virus genetics

 DESCRIPTION (provided by applicant): Paget's disease is a metabolic bone disease that affects up to 3% of adults over 55 in which abnormal osteoclasts (OCL) both increase bone resorption and induce formation of poor quality bone leading to bone deformity, pain, pathological fractures, deafness, and nerve compression. Strong evidence supports a role for genetic mutations in the SQSTM1 gene (encoding p62) together with environmental agents such as measles virus nucleocapsid protein (MVNP) in the development of Paget's disease. This proposal is focused on understanding how MVNP alters OCL lineage cells and cooperates with p62P392L to generate pagetic lesions. Our group has recently found that MVNP complexes with and regulates the function of two IKK-family members TBK1 (a kinase) and optineurin (OPTN, an adaptor). TBK1 has a critical positive role in MVNP induction of aberrant pagetic-like OCL with increased OCL formation, nuclei/OCL and IL-6 production, while OPTN is a novel negative regulator of OCL formation and IL-6 production. MVNP interaction with the TBK1- OPTN complex results in increased TBK1 activity and decreased OPTN levels. TBK1 phosphorylates S177- OPTN, increasing OPTN activity as autophagy cargo receptor and its degradation. Intriguingly, we have found that MVNP increases autophagic flux. Further OPTN and p62, can both recruit CYLD, a deubiquitinase, to shut down TNFa and RANKL signaling, respectively, a function lost by pagetic p62 mutations. We hypothesize that MVNP interaction with the TBK1-OPTN complex is the key mechanism by which MVNP alters OCL biology and cooperates with mutant p62 to generate the pagetic OCL phenotype. Aim 1 will determine the mechanisms by which MVNP regulates TBK1-OPTN activity. We will examine the nature of MVNP interactions with the TBK1-OPTN complex, the mechanism by which TBK1 is activated, if TBK1 phosphorylation of OPTN increases autophagy and TNFa/RANKL signaling, and if increased autophagy contributes to aspects of the pagetic OCL phenotype, in particular increased nuclei/OCL. Aim 2 will test the hypothesis that increased expression of TBK1 alone or in combination with the p62P394L mutation are sufficient to induce pagetic OCL and bone lesions in mice. We will cross our recently made TRAP-TBK1 mice to our p62KI mice and will analyze both TRAP-TBK1 and TRAP-TBK1/p62KI mice for pagetic lesion formation. We will also conduct in vitro co-expression studies transducing TBK1 into MVNP-negative peripheral blood mononuclear cells (PBMC) from p62P392L-positive Paget's patients and normals to establish if increased TBK1 activity alone or plus p62P392L induces a pagetic phenotype. Aim 3 will test the hypothesis that MVNP requires TBK1 in vivo to induce pagetic lesions. We will analyze formation of pagetic lesions in MVNP mice crossed with mice that have Tbk1 conditional knockout in OCL and evaluate if knockdown of TBK1 in MVNP+ PBMC from p62P392L-Paget's patients affects development of pagetic OCL. These studies will provide important insight into the pathogenesis of Paget's disease and may provide identification of novel targets for small molecule inhibitors for down-regulating OCL activity in Paget's disease and other pathological increases in OCL activity.

 描述(申请人提供)：Paget病是一种代谢性骨骼疾病，在55岁以上的成年人中影响高达3%，其中异常破骨细胞(OCL)增加骨吸收并导致劣质骨的形成，导致骨畸形、疼痛、病理性骨折、耳聋和神经压迫。强有力的证据支持SQSTM1基因(编码p62)的基因突变与环境因素，如麻疹病毒核衣壳蛋白(MVNP)在Paget病的发展中所起的作用。这项建议的重点是了解MVNP如何改变OCL谱系细胞，并与p62P392L合作产生Pagtic病变。我们的研究组最近发现，MVNP与两个IKK家族成员TBK1(一种激酶)和optineurin(OPTN，一种接头)复合并调节其功能。Tbk1在MVNP诱导异常的Pagtic样OCL中起关键的正向作用，增加OCL的形成、核/OCL和IL-6的产生，而OPTN是一种新的OCL形成和IL-6产生的负调控因子。MVNP与TBK1-OPTN复合体的相互作用导致TBK1活性增加，OPTN水平降低。Tbk1使S177-OPTN磷酸化，增加OPTN作为自噬货物受体的活性及其降解。有趣的是，我们发现MVNP增加了自噬通量。此外，OPTN和p62都可以招募CyLD，一种脱泛素酶，分别关闭TNFa和RANKL信号，而Pagtic p62突变失去了这一功能。我们推测，MVNP与TBK1-OPTN复合体的相互作用是MVNP改变OCL生物学并与突变体p62合作产生Pagtic OCL表型的关键机制。目的1确定MVNP调节TBK1-OPTN活性的机制。我们将研究MVNP与TBK1-OPTN复合体相互作用的性质，激活TBK1的机制，如果OPTN的TBK1磷酸化增加自噬和TNFa/RANKL信号，以及增加的自噬是否有助于Pagtic OCL表型的某些方面，特别是核/OCL增加。目的2将验证这样一种假设，即单独或联合p62P394L突变增加TBK1的表达足以在小鼠中诱发寻常型OCL和骨损伤。我们将把我们最近制造的TRAP-TBK1小鼠与我们的p62KI小鼠杂交，并将分析TRAP-TBK1和TRAP-TBK1/p62KI小鼠的传递性病变形成。我们还将进行体外共表达研究，将TBK1转导到p62P392L阳性的Paget患者和正常人的MVNP阴性的外周血单核细胞(PBMC)中，以确定单独或联合p62P392L增加的TBK1活性是否会诱导Pagtic表型。目的3将验证假设，即MVNP需要在体内的TBK1诱发变态损害。我们将分析MVNP小鼠与OCL中具有TBK1条件基因敲除的小鼠杂交后Pagtic病变的形成，并评估p62P392L-Paget‘s患者MVNP+PBMC中TBK1基因敲除是否影响Pagtic OCL的发展。这些研究将为Paget病的发病机制提供重要的认识，并可能为小分子抑制剂下调Paget病的OCL活性和其他病理性OCL活性增加提供新的靶点。