ROLE OF NOVEL TRANSCRIPTION FACTORS IN THE PATHOGENESIS OF RHEUMATOID ARTHRITIS

新型转录因子在类风湿关节炎发病机制中的作用

• 批准号: 3747720
• 负责人: MATTHEW J FENTON
• 金额: --
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3747720
• 关键词: affinity chromatography; antirheumatic agents; drug screening /evaluation; gene expression; genetic regulation; human subject; in situ hybridization; interleukin 1; molecular cloning; nucleic acid probes; pathologic process; polymerase chain reaction; protein structure function; rheumatoid arthritis; synovial fluid; transcription factor

In patients with rheumatoid arthritis (RA), resident synovial cells of the pannus, as well as infiltrating mononuclear cells, produce a wide variety of bioactive molecules that contribute to the development and severity of the disease. Interleukin 1alpha(IL-1alpha) and interleukin 1beta (IL- 1beta) are proinflammatory cytokines that play a central role in the immune imbalance and tissue destruction associated with RA. Expression of the IL- 1alpha and beta genes is ultimately controlled by transcriptional regulatory proteins that bind to specific sequences within the genes and transduce intracellular second messenger signals. We recently identified a novel family of nuclear proteins, collectively termed NFIL-1betaA (betaA), that recognize the same highly conserved sequence within the IL- 1beta promotor and appear to be required for IL-1beta gene expression. The beta family may represent several distinct proteins that contain at least one DNA-binding polypeptide. One specific isoform of betaA that is exclusively expressed in fibroblasts and endothelial cells contains both a 36 kDa (p36) and a 90 kDa (p90) DNA-binding polypeptide. The broad goals of this proposal are to study the specific betaA isoform(s) expressed by synovial fibroblasts, assess their functional role in directing IL-1beta gene expression, and to examine the regulation of p36 and p90 expression in rheumatoid tissues. Specifically, we will obtain molecular probes for p36 and p90 by cloning the cDNAs for these proteins, using two distinct approaches; (a) to screen a lambdagt-11 expression library using radiolabelled double stranded DNA oligonucleotides containing the betaA binding sequence, and (b) to purify p36 and p90 crude nuclear extracts by DNA affinity chromatography in order to obtain partial amino acid sequence data. The partial amino acid sequence information will then be used to generate oligonucleotide probes for cDNA library screening or polymerase chain reaction cloning. Subsequently, we will examine the regulation of p36 and p90 expression in normal and rheumatoid synovial fibroblasts. The ability of several selected antirheumatic drugs (that are known to suppress IL-1 production) to modulate betaA expression will also be evaluated. Lastly, synovial tissue samples will be examined for betaA expression by in situ hybridization using antisense mRNA probes, and by immunohistochemical staining using the betaA antisera, in order to determine the relative levels of betaA produced by the distinct cell types within the diseased joint that can concurrently express IL-1. These studies should greatly contribute to our understanding of how IL-1 production is regulated at the molecular level in synovial fibroblasts.

在类风湿关节炎(RA)患者中，常驻滑膜细胞 血管疙瘩和渗入的单个核细胞一样，产生各种各样的 这些生物活性分子对疾病的发展和严重程度做出了贡献。 这种疾病。白介素1α(IL-1α)和白介素1β(IL-1β) 1β)是在免疫中起中心作用的促炎细胞因子 与RA相关的失衡和组织破坏。白介素2的表达 1α和β基因最终由转录调控 与基因内特定序列结合的调节蛋白和 转导细胞内的第二信使信号。我们最近确认了 一个新的核蛋白家族，统称为NFIL-1betaA (BetaA)，在IL-1中识别相同的高度保守的序列 1β启动子，似乎是IL-1β基因表达所必需的。这个 β家族可能代表几种不同的蛋白质，它们至少包含 一种DNA结合多肽。一种特定的βA亚型，即 仅在成纤维细胞和内皮细胞中表达的 36 kDa(P36)和90 kDa(P90)DNA结合多肽。宏伟目标 这项建议的目的是研究S表达的特定的βA亚型 滑膜成纤维细胞，评估它们在引导IL-1β中的功能作用 基因表达，并检测p36和p90表达的调节。 类风湿组织。具体来说，我们将获得p36的分子探针。 和p90通过克隆这些蛋白质的cDNA，使用两个不同的 方法；(A)使用以下方法筛选lambdagt-11表达文库 含βA的放射性标记双链DNA寡核苷酸 结合序列，以及(B)纯化p36和p90粗核提取物 DNA亲和层析获得部分氨基酸序列 数据。然后，部分氨基酸序列信息将用于 为筛选cDNA文库或聚合酶生成寡核苷酸探针 链式反应克隆。随后，我们将研究监管 P36和P90在正常和类风湿滑膜成纤维细胞中的表达。这个 几种选定的抗风湿药物(已知可抑制 IL-1的产生)来调节βA的表达也将被评估。 最后，滑膜组织样本将通过在 反义mRNA探针原位杂交及免疫组织化学方法 使用BetaA抗血清进行染色，以确定相对 患者体内不同细胞类型产生的βA水平 可同时表达IL-1的关节。这些研究应该很大程度上 有助于我们理解IL-1的产生是如何在 滑膜成纤维细胞的分子水平。