Chandra G, Cogswell J P, Miller L R, Godlevski M M, Stinnett S W, Noel S L, Kadwell S H, Kost T A, Gray J G
Department of Molecular Genetics, Glaxo Research Institute, Inc., Research Triangle Park, NC 27709, USA.
J Immunol. 1995 Nov 15;155(10):4535-43.
An intact cAMP response element (CRE) in the upstream regulatory sequence of IL-1 beta (-2755/-2762) has been shown to be essential for maintaining full IL-1 beta inducibility following treatment with LPS, PMA, or TNF-alpha. In the present study, using the recombinant plasmid pIL-1(4.0 kb)-chloramphenicol acetyltransferase, containing 4.0 kb of the IL-1 beta upstream regulatory sequence, we have demonstrated that dibutyryl cAMP treatment alone is capable of induction. Due to the critical nature of the CRE for the induction of IL-1 beta transcription, an effort was made to determine the importance of the cAMP signaling pathway(s) by determining whether CRE binding protein (CREB) and other CREB/activating transcription factor (ATF) family members that responded to cAMP were associated with the DNA-protein complex that forms at this site. Nuclear extracts prepared from LPS-treated THP-1 5A cells were fractionated by ammonium sulfate precipitation and heparin-Sepharose chromatography, and the resulting fractions were characterized in electrophoretic gel mobility shift assays. These purification steps resulted in an approximately 100-fold enrichment of the proteins binding to the CRE site. Western blot analysis of isolated fractions, using CREB- and ATF-1-specific Ab showed an increased level of these proteins in the enriched fractions. Tryptic digest and DNase I protection studies showed the presence of CREB protein in the complex at the CRE site. Supershift electrophoretic gel mobility shift assays and immunoprecipitation analysis provided further evidence that both CREB and ATF-1 are present in the complex. In addition, an increase in CREB phosphorylation was observed when THP-1 5A cells were treated with dibutyryl cAMP, LPS, or both. These studies confirm the importance of a cAMP signaling pathway(s) in the regulation of IL-1 beta at the transcriptional level.
白细胞介素-1β（IL-1β）上游调控序列（-2755/-2762）中完整的环磷酸腺苷反应元件（CRE）已被证明对于在脂多糖（LPS）、佛波酯（PMA）或肿瘤坏死因子-α（TNF-α）处理后维持IL-1β的完全诱导性至关重要。在本研究中，使用含有4.0 kb IL-1β上游调控序列的重组质粒pIL-1(4.0 kb)-氯霉素乙酰转移酶，我们已经证明单独使用二丁酰环磷酸腺苷（dibutyryl cAMP）处理就能够诱导。由于CRE对于IL-1β转录诱导的关键性质，我们努力通过确定CRE结合蛋白（CREB）和其他对cAMP有反应的CREB/激活转录因子（ATF）家族成员是否与在此位点形成的DNA-蛋白质复合物相关联，来确定cAMP信号通路的重要性。从经LPS处理的THP-1 5A细胞制备的核提取物通过硫酸铵沉淀和肝素-琼脂糖层析进行分级分离，并且在电泳凝胶迁移率变动分析中对所得级分进行表征。这些纯化步骤使与CRE位点结合的蛋白质富集了约100倍。使用CREB和ATF-1特异性抗体对分离级分进行的蛋白质印迹分析显示，这些蛋白质在富集级分中的水平增加。胰蛋白酶消化和DNase I保护研究表明在CRE位点的复合物中存在CREB蛋白。超迁移电泳凝胶迁移率变动分析和免疫沉淀分析提供了进一步的证据，证明CREB和ATF-1都存在于复合物中。此外，当THP-1 5A细胞用二丁酰环磷酸腺苷、LPS或两者处理时，观察到CREB磷酸化增加。这些研究证实了cAMP信号通路在转录水平上对IL-1β调节的重要性。
