Lilley Cullen M, Alarcon Andrea, Ngo My-Huyen, Araujo Jackeline S, Marrero Luis, Mix Kimberlee S
Department of Biological Sciences, Loyola University New Orleans, New Orleans, LA, United States.
Department of Orthopaedic Surgery, Louisiana State University Health Sciences Center, New Orleans, LA, United States.
Front Pharmacol. 2022 Apr 20;13:835697. doi: 10.3389/fphar.2022.835697. eCollection 2022.
Orphan nuclear receptor 4A2 (NR4A2/Nurr1) is a constitutively active transcription factor with potential roles in the onset and progression of inflammatory arthropathies. NR4A2 is overexpressed in synovium and cartilage from individuals with rheumatoid arthritis (RA), psoriatic arthritis, and osteoarthritis. This study documents the expression and tissue localization of NR4A2 and upstream regulator nuclear factor kappa B (NF-κB) in the human tumor necrosis factor-alpha (hTNF-α) transgenic mouse model of RA. Since TNF-α is a potent inducer of NR4A2 , we hypothesized that NR4A2 would also be upregulated and active during disease progression in this model. Expression levels of NR4A2, related receptors NR4A1 (Nur77) and 3 (NOR1), and NF-κB transcripts were quantified by RT-qPCR in hTNF-α and wild-type joints at three stages of disease. The protein distribution of NR4A2 and NF-κB subunit RelA (p65) was analyzed by quantitative immunohistochemistry. Global gene expression of 88 RA-related genes was also screened and compared between groups. Consistent with previous reports on the hTNF-α model, transgenic mice exhibited significant weight loss and severely swollen paws by 19 weeks of age compared to age-matched wild-type controls. NR4A1-3 and NF-κB were constitutively expressed at disease onset and in healthy joints. NF-κB transcript levels increased 2-fold in hTNF-α paws with established disease (12 weeks), followed by a 2-fold increase in NR4A2 at the late disease stage (19 weeks). NR4A2 and RelA proteins were overexpressed in inflamed synovium prior to symptoms of arthritis, suggesting that gene expression changes documented in whole paws were largely driven by elevated expression in diseased synovium. Broader screening of RA-related genes by RT-qPCR identified several differentially expressed genes in hTNF-α joints including those encoding inflammatory cytokines and chemokines, matrix-degrading enzymes and inhibitors, cell surface receptors, intracellular signaling proteins and transcription factors. Consensus binding sites for NR4A receptors and NF-κB were enriched in the promoters of differentially expressed genes suggesting central roles for these transcription factors in this model. This study is the first comprehensive analysis of NR4A2 in an animal model of RA and validates the hTNF-α model for testing of small molecules and genetic strategies targeting this transcription factor.
孤儿核受体4A2（NR4A2/Nurr1）是一种组成型活性转录因子，在炎症性关节病的发病和进展中可能发挥作用。NR4A2在类风湿性关节炎（RA）、银屑病关节炎和骨关节炎患者的滑膜和软骨中过度表达。本研究记录了NR4A2和上游调节因子核因子κB（NF-κB）在RA的人肿瘤坏死因子-α（hTNF-α）转基因小鼠模型中的表达和组织定位。由于TNF-α是NR4A2的有效诱导剂，我们假设在该模型的疾病进展过程中，NR4A2也会被上调并具有活性。通过RT-qPCR对疾病三个阶段的hTNF-α和野生型关节中NR4A2、相关受体NR4A1（Nur77）和3（NOR1）以及NF-κB转录本的表达水平进行了定量。通过定量免疫组织化学分析了NR4A2和NF-κB亚基RelA（p65）的蛋白质分布。还对88个RA相关基因的整体基因表达进行了筛选并在组间进行了比较。与之前关于hTNF-α模型的报道一致，与年龄匹配的野生型对照相比，转基因小鼠在19周龄时体重显著减轻，爪子严重肿胀。NR4A1-3和NF-κB在疾病发作时和健康关节中组成型表达。在已确诊疾病（12周）的hTNF-α爪子中，NF-κB转录水平增加了2倍，随后在疾病晚期（19周）NR4A2增加了2倍。在关节炎症状出现之前，NR4A2和RelA蛋白在发炎的滑膜中过度表达，这表明在整个爪子中记录的基因表达变化很大程度上是由患病滑膜中表达升高所驱动的。通过RT-qPCR对RA相关基因进行更广泛的筛选，在hTNF-α关节中鉴定出了几个差异表达基因，包括那些编码炎性细胞因子和趋化因子、基质降解酶和抑制剂、细胞表面受体、细胞内信号蛋白和转录因子的基因。NR-4A受体和NF-κB的共有结合位点在差异表达基因的启动子中富集，表明这些转录因子在该模型中起核心作用。本研究是对RA动物模型中NR4A2的首次全面分析，并验证了hTNF-α模型用于测试靶向该转录因子的小分子和遗传策略的有效性。
