Coffer P, Lutticken C, van Puijenbroek A, Klop-de Jonge M, Horn F, Kruijer W
Hubrecht Laboratory for Developmental Biology, Utrecht, The Netherlands.
Oncogene. 1995 Mar 2;10(5):985-94.
The product of the junB gene is a member of the AP-1 family of transcription factors that activate transcription by binding to TPA-responsive elements (TREs) within the promoters of target genes. Components of AP-1 are immediate-early genes whose expression is upregulated by a plethora of extracellular stimuli and are important in mediating cellular proliferation and differentiation. Such stimuli include the pleiotropic cytokine interleukin-6 (IL-6) which plays a role in immune and inflammatory responses and ciliary neurotrophic factor (CNTF) which enhances survival and differentiation of neurons and glia. We have analysed expression from junB promoter-CAT reporter constructs in HepG2 cells and found that a region between -196 and -91 can mediate response to IL-6 and CNTF and was able to confer responsiveness to a heterologous promoter. We further show by gel retardation analysis that distinct nuclear factors induced by IL-6 specifically bind to this interleukin-6 response element (IRE). This region contains both a putative ETS- and a STAT-transcription factor binding site. We show by mutational analysis and supershift data that the IL-6 induced complex indeed contains the transcription factor APRF/Stat3 that is both necessary and sufficient for activation. Interestingly this site does not appear to bind Stat1 itself, as shown by supershift analysis and a lack of response to IFN-gamma both at the DNA-binding and transcriptional level. Furthermore, we demonstrate that the junB IRE-binding activity induced by IL-6 requires tyrosine kinase activity, whereas induced transactivation of IRE-constructs additionally occurs through an H7-sensitive pathway that is p21ras-independent, implicating serine/threonine kinases in the transactivation of IRE-binding factors.
junB基因的产物是转录因子AP-1家族的成员，该家族通过与靶基因启动子内的佛波酯反应元件（TREs）结合来激活转录。AP-1的组成部分是即刻早期基因，其表达受到多种细胞外刺激的上调，并且在介导细胞增殖和分化中起重要作用。此类刺激包括在免疫和炎症反应中起作用的多效性细胞因子白细胞介素-6（IL-6）以及增强神经元和神经胶质细胞存活与分化的睫状神经营养因子（CNTF）。我们分析了HepG2细胞中junB启动子-CAT报告基因构建体的表达，发现-196至-91之间的区域可介导对IL-6和CNTF的反应，并能够赋予异源启动子反应性。我们通过凝胶阻滞分析进一步表明，IL-6诱导的不同核因子特异性结合该白细胞介素-6反应元件（IRE）。该区域同时包含一个假定的ETS转录因子结合位点和一个STAT转录因子结合位点。我们通过突变分析和超迁移数据表明，IL-6诱导的复合物确实包含激活所必需且充分的转录因子APRF/Stat3。有趣的是，如超迁移分析以及在DNA结合和转录水平上对IFN-γ缺乏反应所示，该位点似乎不结合Stat1本身。此外，我们证明IL-6诱导的junB IRE结合活性需要酪氨酸激酶活性，而IRE构建体的诱导反式激活另外通过一条对H7敏感且不依赖p21ras的途径发生，这表明丝氨酸/苏氨酸激酶参与了IRE结合因子的反式激活。
