Involvement of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes
Involvement of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes
复制标题
DOI：
10.1002/art.1780400207
复制
发表时间：
1997-02-01
影响因子：
--
通讯作者：
Hla, T
中科院分区：
文献类型：
--
作者：
Crofford, LJ;Tan, B;Hla, T
目标。目的探讨转录因子核因子κ B （nf - κ B）在原发性类风湿滑膜细胞中受白细胞介素-1 β (IL-1 β)刺激的环氧化酶-2 （COX-2）表达升高中的作用。我们用IL-1 β处理早期传代的类风湿滑膜细胞，并通过Western blot分析NF-kappa B易位到细胞核的时间过程，以及通过电泳迁移位移法检测NF-kappa B与COX-2启动子/增强子的结合。我们将NF-kappa B结合的时间进程与COX-2信使RNA （mRNA）和蛋白的表达联系起来。然后用取自人NF-kappa B p65 RNA转录起始位点的正义或反义硫代修饰寡核苷酸处理滑膜细胞。我们分析了这些处理后NF-kappa B与COX-2启动子的结合和COX-2蛋白水平。IL-1 β迅速刺激p65、p50和c-rel nf - κ B亚基从细胞质向细胞核的易位。电泳迁移率转移分析显示，COX-2启动子/增强子内的2个NF-kappa B位点结合，其时间过程与NF-kappa B核定位的时间过程相同。超转移分析显示，结合活性主要是由于p65-p50异源二聚体和p50同源二聚体。在NF-kappa B结合后适当的延迟时间，COX-2 mRNA和蛋白水平升高。NF-kappa B p65反义寡核苷酸预处理RA滑膜细胞可降低与COX-2启动子的结合，降低COX-2蛋白的表达。这些数据表明，nf - κ B信号通路参与了RA滑膜细胞中IL-1 β诱导的COX-2表达的调控。
Objective. To evaluate involvement of the transcription factor nuclear factor kappa B (NF-kappa B) in the increased expression of cyclooxygenase-2 (COX-2) stimulated by interleukin-1 beta (IL-1 beta) in primary rheumatoid synoviocytes.Methods. We treated early-passage rheumatoid synoviocytes with IL-1 beta and examined the time course of NF-kappa B translocation to the nucleus by Western blot analysis, as well as NF-kappa B binding to the COX-2 promoter/enhancer by electrophoretic mobility shift assay. We correlated the time course of NF-kappa B binding with expression of COX-2 messenger RNA (mRNA) and protein. Synoviocytes were then treated with either sense or antisense phosphorothioate-modified oligonucleotides derived from the transcription start site of the human NF-kappa B p65 RNA. We analyzed NF-kappa B binding to the COX-2 promoter and COX-2 protein levels after these treatments.Results. IL-1 beta rapidly stimulated the translocation of the p65, p50, and c-rel NF-kappa B subunits from the cytoplasm to the nucleus. Electrophoretic mobility shift assay demonstrated binding to 2 NF-kappa B sites within the COX-2 promoter/enhancer, with a time course identical to that of nuclear localization of NF-kappa B. Supershift analysis revealed that binding activity was due primarily to the p65-p50 heterodimer and the p50 homodimer. With appropriate lag time after NF-kappa B binding, COX-2 mRNA and protein were increased. Pretreatment of RA synoviocytes with NF-kappa B p65 antisense oligonucleotides resulted in decreased binding to the COX-2 promoter and decreased COX-2 protein expression,Conclusion. These data demonstrate that signaling via the NF-kappa B pathway is involved in regulation of COX-2 expression induced by IL-1 beta in RA synoviocytes.