Atasoy Ulus, Curry Stephanie L, López de Silanes Isabel, Shyu Ann-Bin, Casolaro Vincenzo, Gorospe Myriam, Stellato Cristiana
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
J Immunol. 2003 Oct 15;171(8):4369-78. doi: 10.4049/jimmunol.171.8.4369.
During inflammatory responses, a major posttranscriptional regulation of early response and inflammatory gene expression occurs through modulation of mRNA turnover. We report that two potent inducers of the CC chemokine eotaxin, TNF-alpha and IL-4, regulate its production in airway epithelial cells by increasing eotaxin mRNA stability. In experiments using the transcriptional inhibitor actinomycin D, eotaxin mRNA half-life was significantly prolonged by cell stimulation with TNF-alpha or IL-4, with the combination of the two cytokines being the most effective in extending the mRNA half-life. Involvement of the eotaxin 3' untranslated region in the mRNA-stabilizing effect was tested by transient transfection of a construct expressing a chimeric transcript carrying a serum-inducible beta-globin reporter linked to the eotaxin 3' untranslated region. The half-life of the chimeric mRNA was markedly increased in cells stimulated with TNF-alpha and IL-4. Evidence that the mRNA-stabilizing protein HuR participated in the cytokine effect was obtained: first, HuR presence in the cytoplasm, believed to be required for HuR-mediated mRNA stabilization, increased in both transformed (BEAS-2B cell line) and primary bronchial epithelial cells following treatment with TNF-alpha and IL-4. Second, endogenous eotaxin mRNA was found to bind to HuR in vivo, as detected by immunoprecipitation of HuR-containing messenger ribonucleoprotein complexes followed by real-time RT-PCR analysis; such association increased after cell treatment with TNF-alpha and IL-4. Third, overexpression of HuR in BEAS-2B cells significantly increased the expression of eotaxin mRNA and protein. Our findings implicate mRNA stabilization in the cytokine-mediated increase in eotaxin expression and strongly suggest a role for HuR in this effect.
在炎症反应过程中，早期反应和炎症基因表达的主要转录后调控是通过调节mRNA周转来实现的。我们报告称，CC趋化因子嗜酸性粒细胞趋化因子（eotaxin）的两种强效诱导剂——肿瘤坏死因子-α（TNF-α）和白细胞介素-4（IL-4），通过增加eotaxin mRNA的稳定性来调节其在气道上皮细胞中的产生。在使用转录抑制剂放线菌素D的实验中，TNF-α或IL-4刺激细胞可显著延长eotaxin mRNA的半衰期，两种细胞因子联合使用对延长mRNA半衰期最为有效。通过瞬时转染表达嵌合转录本的构建体来测试eotaxin 3'非翻译区在mRNA稳定作用中的参与情况，该嵌合转录本携带与eotaxin 3'非翻译区相连的血清诱导型β-珠蛋白报告基因。在用TNF-α和IL-4刺激的细胞中，嵌合mRNA的半衰期显著增加。获得了mRNA稳定蛋白HuR参与细胞因子作用的证据：首先，TNF-α和IL-4处理后，在转化的（BEAS-2B细胞系）和原代支气管上皮细胞中，被认为是HuR介导的mRNA稳定所必需的细胞质中HuR的存在增加。其次，通过对含HuR的信使核糖核蛋白复合物进行免疫沉淀，然后进行实时逆转录-聚合酶链反应（RT-PCR）分析，发现在体内内源性eotaxin mRNA与HuR结合；细胞用TNF-α和IL-4处理后，这种结合增加。第三，在BEAS-2B细胞中过表达HuR显著增加了eotaxin mRNA和蛋白的表达。我们的研究结果表明mRNA稳定在细胞因子介导的eotaxin表达增加中起作用，并强烈提示HuR在这种作用中发挥作用。
