NFkB2 p100降解抑制剂治疗类风湿性关节炎的分子遗传基础

Rheumatoid arthritis (RA) is characterised by synovial inflammation induced by inflammatory cells such as T cell and B cells, and joint destruction mediated by activated osteoclasts (OC). TNF-α is the major pro-inflammatory cytokine in RA. We found that TNF-α induces OC differentiation independent of RANKL system. However, its ability to make OC is limited because it stimulates NFkB2 p100 gene expression but doesn't degrade it, which inhibit itself and RANKL induced OC formation. p100 deficiency significantly enhances synovial inflammation and joint erosion in a RA model of TNF transgenic mice by increasing TNF-induced OC formation. NCBI gene bank shows that human NFkB2 gene have two transcript variants and more than ten single-nucleotide polymorphism (SNPs)resulting in the alternation of p100 protein, which may make p100 more easily degraded. In this grant application, we will extablish RNA seq libraries.NF-kB2 gene variations in the coding region including transcript variants, SNPs,mutation,small deletion or insertion will be screened for via RNA-seq and re-varified by PCR followed by restriction enzyme cut in order to determine if the gene variations that result in alteration of p100 protein increase the susceptibility and disease severity of RA and if they are related to anti-TNF therapeutic response of RA patients. We will also verify if the gene variations result in p100 degradation automatically or induced by TNF and enhance TNF-induced OC differentiation. Finally, we will screen for the compounds that could inhibit p100 processing and OC differentiation in vitro from the possibble inhibitors of NFkB inducing kinase (NIK),required for p100 processing, and test the efficacy of the selected p100 processing inhibitor to treat collagen II induced arthritis in mice. The proposed study is a novel exploratory and developmental research, which extends our previous discoveries that deficiency of NFkB2 p100 accelerates joint destruction and synovial inflammation in animal RA model toward human translational research. Achievement of the expected results could 1)reveal a novel genetic and molecular mechanism in RA; 2)provide a novel tool to predict the responders and non-responders of RA patients to anti-TNF thearpy so as to avoid the expensive,un-necessary treatment for those patients who will not response to this drugs and 3) lead us to develop novel therapeutic approaches for RA based on p100 in future.

炎症细胞介导的滑膜炎症及破骨细胞(OC)介导的关节破坏是类风湿性关节炎(RA)的基本特征。引起RA的关键因子TNFα独立诱导OC形成，但受NFkB2p100限制。p100缺乏显著增加TNF诱导的OC形成、关节破坏及滑膜炎症，加速小鼠RA的发生和发展。人类NFkB2 有两种异构体，其中II型在p100降解的关键部位-第860位缺失一个氨基酸,并初步验证与RA的易感性相关。本研究的目标有三：1）用RA和健康人群外周血建立大样本RNA文库，通过mRNA测序筛查和验证引起p100蛋白结构改变的基因变异增加RA的易感性、病情严重性，预测其对治疗的反应性；2）阐明与RA相关的NFkB2基因变异促进p100降解及 OC形成；3）探索p100降解抑制剂治疗小鼠炎性关节炎的可能性。研究可能揭示RA全新的分子遗传机制，为预测RA对治疗的反应性提供全新的工具，并为开发新的治疗药物奠定基础。

炎症细胞介导的滑膜炎症及破骨细胞(OC)介导的关节破坏是类风湿性关节炎(RA)的基本特征。引起RA的关键因子TNF独立诱导OC形成，但受NFkB2p100限制。p100缺乏显著增加TNF诱导的OC形成、关节破坏及滑膜炎症，加速小鼠RA的发生和发展。我们的假说是，1）Nfkb2编码区基因变异或可变剪接所致的p100蛋白结构改变可使其易于降解、促进OC形成，从而增加RA的易感性、促进其病情进展；2）阻断p100降解可能成为治疗RA的新手段。我们从RA和健康人群外周血细胞提取RNA，对RNA样本进行RNA-seq，直接检测全基因mRNA序列及其变异。共检测到已知基因18,726个，预测的新基因7,556个；另检测出109,706个新转录本，其中46,393个是已知蛋白编码基因的新的可变剪接亚型，7,556 个属于新的蛋白编码基因的转录本。共检测到58个nfkb2转录本，包括6个已知的和52个新转录本。其新转录本T009150、T009162、T009169主要分布于RA病人组，其频率分别为76.1%，70%和72%，而它们在正常对照组的分布频率仅为10%，30%和20%。另检出5个nfkb2可变剪接亚型，21个SNP，4个小片段插入或缺失。通过人工诱变，我们构建了已知的I型和II型p100异构体的反转录病毒表达质粒。将其感染小鼠OC前体细胞，而后用TNF处理。我们未检测到这两种异构体对TNF诱导的p100蛋白降解和OC形成的影响。在今后的研究中，我们将根据RNA-seq获取的数据，系列研究nfkb2新转录本及其可变剪接亚型对p100降解及OC形成的影响。我们还研究了nfkb2 p100/p52伙伴蛋白RelB对OC分化的影响。TNF显著增加RelB蛋白，使M-CSF诱导的M2 向M1巨噬细胞转变，因而增加OC分化潜能、并刺激炎症因子的产生。最后，我们筛选出一个能够有效抑制RANKL诱导的p100降解和OC分化的化合物，4H-isoquinoline-1,3-dione (IQLD)，研究其治疗RA类关节炎的可能性。结果显示，IQLD能够抑制II型胶原诱导关节炎的关节破坏及OC形成。因此，IQLD可以抑制OC所致的关节破坏而用于RA的治疗。总之，我们的研究揭示了RA全新的分子遗传机制，并为开发治疗RA的新药奠定了基础。