Elucidating the Role of Sulfatase-2 in RA Pathogenesis

项目摘要 在类风湿性关节炎（RA）中，细胞因子肿瘤坏死因子-α（TNF-α）在驱动滑膜细胞增殖中起主导作用。 软骨和骨的增生和进行性破坏。我们的初步数据表明细胞外 硫酸酯酶-2（Sulf-2）是RA滑膜成纤维细胞中TNF-α信号传导的潜在介质（或促进剂） （RASFs）。在细胞表面，Sulf-2从硫酸乙酰肝素蛋白聚糖（HSPG）上切割硫酸基团， 进而影响受体/配体结合和随后的一系列趋化因子、细胞因子 生长因子TNF-α和HSPGs之间的相互作用还没有很好的定义，Sulf-2在TNF-α中的作用 信号仍然未被探索。我们发现用siRNA或小分子抑制人RASFs中的Sulf-2， 抑制剂OKN-007可显著降低TNF-α诱导的粘附分子ICAM-1、VCAM-1表达 和钙粘蛋白-11，以及炎症介质IL-6和IL-8的产生。RNA转录组分析 RNA测序（RNAseq）显示，与阴性对照siRNA相比，Sulf-2的敲低显著降低， 减少TNF-α对粘附、细胞增殖和趋化因子相关基因的诱导，同时伴随 增加骨保护和抗炎基因的表达。RNASeq结果的途径分析 结果显示，TNF-α刺激的RASFs中Sulf-2的敲低显著改变了RASFs中48个基因的表达。 类风湿性关节炎中巨噬细胞、成纤维细胞和内皮细胞的作用这 一项提案利用了这些新的观察结果，我们研究的中心假设是Sulf-2介导了 TNF-α信号促进RA滑膜增生、侵袭和组织破坏如细胞外 酶，Sulf-2是药理学抑制的有吸引力的靶标，并且Sulf-2的小分子抑制剂具有 在人体临床试验中显示出高生物安全性。我们的目的是阐明Sulf-2参与TNF-α表达的机制。 α信号通路，并研究靶向Sulf-2依赖性通路对糖尿病的潜在治疗价值。 治疗RA。目标1中提出的研究将调查Sulf-2调节的分子机制， TNF-α信号通路和对人RASF发病机制下游介质的影响。在目标2中， 将在人TNF-α转基因小鼠模型中测试Sulf-2的药理学抑制的体内作用， RA.这些研究的成功将揭示一种新的调节TNF-α信号的分子机制， 通过调节Sulf-2来调节RASF，并验证潜在的新药物治疗的安全性和有效性 治疗这种常见的衰弱性疾病

PROJECT SUMMARY In rheumatoid arthritis (RA), the cytokine tumor necrosis factor-α (TNF-α) plays a dominant role in driving synovial hyperplasia and progressive destruction of cartilage and bone. Our preliminary data suggests the extracellular enzyme Sulfatase-2 (Sulf-2) to be a potential mediator (or facilitator) of TNF-α signaling in RA synovial fibroblasts (RASFs). At the cell surface, Sulf-2 cleaves sulfate groups from heparan sulfate proteoglycans (HSPGs), which in turn affects the receptor/ligand binding and subsequent signaling of an array of chemokines, cytokines and growth factors. Interactions between TNF-α and HSPGs are not well defined, and the role of Sulf-2 in TNF-α signaling remains unexplored. We found that inhibition of Sulf-2 in human RASFs with siRNA or a small molecule inhibitor (OKN-007) significantly reduced TNF-α-induced expression of adhesion molecules ICAM-1, VCAM-1 and Cadherin-11, and production of inflammatory mediators IL-6 and IL-8. Transcriptome analysis by RNA sequencing (RNAseq) showed that, compared to a negative control siRNA, knockdown of Sulf-2 significantly reduced TNF-α induction of genes related to adhesion, cell proliferation, and chemokines, while concomitantly increasing expression of bone-protective and anti-inflammatory genes. Pathway analysis of RNASeq results showed that Sulf-2 knockdown in TNF-α-stimulated RASFs significantly altered expression of 48 genes in the top canonical pathway Role of Macrophages, Fibroblasts and Endothelial Cells in Rheumatoid Arthritis. This proposal capitalizes on these novel observations, and the central hypothesis of our study is that Sulf-2 mediates TNF-α signaling to promote synovial hyperplasia, invasion and tissue destruction in RA. As an extracellular enzyme, Sulf-2 is an attractive target for pharmacologic inhibition, and a small molecule inhibitor of Sulf-2 has shown high biosafety in human clinical trials. We aim to elucidate the mechanism of Sulf-2 participation in TNF- α signaling and investigate the potential therapeutic value of targeting Sulf-2-dependent pathways for the treatment of RA. Studies proposed in Aim 1 will investigate the molecular mechanisms of Sulf-2 modulation of TNF-α signaling pathways and effects on downstream mediators of pathogenesis in human RASFs. In Aim 2 we will test the in vivo effects of pharmacologic inhibition of Sulf-2 in a human TNF-α transgenic mouse model of RA. The success of these studies will reveal a novel molecular mechanism of modulating TNF-α signaling in RASFs by regulating Sulf-2, and validate the safety and effectiveness of a potential new pharmacologic treatment for this common and debilitating disease.