NF-κB/Rel, not STAT5, regulates nitric oxide synthase transcription in Apostichopus japonicus
NF-κB/Rel, not STAT5, regulates nitric oxide synthase transcription in Apostichopus japonicus
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NF-kappa B/Rel（而非 STAT5）调节刺参中一氧化氮合酶的转录
DOI：
10.1016/j.dci.2016.03.019
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发表时间：
2016-08-01
影响因子：
2.9
通讯作者：
Duan, Xuemei
中科院分区：
文献类型：
--
作者：
Shao, Yina;Wang, Zhenhui;Duan, Xuemei
一氧化氮(NO)是所有脊椎动物和无脊椎动物免疫系统中参与病理生理过程的重要信号分子，主要由诱导型一氧化氮合酶(INOS)产生。为了揭示刺参一氧化氮合酶表达调控的关键机制，我们利用基因组步移法扩增了一段一氧化氮合酶启动子片段，并用荧光素酶分析鉴定了可能的转录因子结合基序。利用与荧光素酶报告基因连锁的5‘-缺失构建体瞬时转染EPC细胞，发现-6141+39区域对AjNOS基因的表达起重要作用，并且AjNOS基因5’-侧翼区的-614碱基对脂多糖有很好的反应。对功能启动子区域的分析表明，存在两个潜在的核因子-kappa B(-375~-366bp，-76~-67 bp)和三个STAT结合位点(-284~-276 bp，-95~87 bp，-81~-73 bp)。当荧光素酶报告载体和表达载体共转染时，发现转录因子NF-kappa B/Rel而不是STAT5激活了AjNOS启动子片段。此外，两个截短的报告载体与表达NF-kappa B/rel的载体共转染后发现，第一个NF-kappa B结合位点(-375~-366bp)是该启动子诱导AjNOS转录所必需的。此外，用SN50(核因子-kappaB抑制肽)阻断AjRel，在体内和体外分别抑制了AjNOS的表达和NO的产生，提示AjRel可能直接调控AjNOS。所有我们的发现都证实了核因子-kappaB依赖于调节AjNOS表达的机制，并提示了一种将NO产生与免疫反应联系起来的途径。(C)2016爱思唯尔有限公司。保留所有权利。
Nitric oxide (NO) is an important signaling molecular in the immune system of all vertebrates and invertebrates for pathologic and physiologic process, and it is largely produced by inducible nitric oxide synthase (iNOS). To uncover key mechanisms regulating NOS expression in sea cucumber Apostichopus japonicus, we amplified a fragment of the NOS promoter by genome walking approach and characterized putative transcription factor binding motifs using luciferase assay. Transient transfection of EPC cells using 5'-deletion constructs linked to luciferase reporter revealed that the region -6141+39 contributed importantly to expression of the AjNOS gene, and the -614 bp of the 5'-flanking region of the AjNOS gene responded well to LPS. Analysis of the functional promoter region revealed the presence of two potential NF-kappa B (-375 bp to -366 bp, -76 bp to -67 bp) and three STAT binding sites (-284 bp to -276 bp, -95 bp to 87 bp, -81 bp to -73 bp). When luciferase reporter vector and expression vector co-transfected revealed that NF-kappa B/Rel, but not STAT5, activate the AjNOS promoter fragment. Furthermore, two truncated reporter vectors co-transfected with vector expressing NF-kappa B/Rel revealed that the first NF-kappa B binding site (-375 bp to -366 bp) was essential for the ability of this promoter to induce AjNOS transcription. In addition, blocking the AjRel by SN50 (NF-kappa B inhibitory peptide) depressed the AjNOS expression and NO production both in vivo and in vitro, respectively, revealing that AjRel might directly modulate AjNOS. All our findings confirmed that NF-kappa B dependent mechanisms regulating expression of AjNOS and suggested a means of linking NO production to the immune response. (C) 2016 Elsevier Ltd. All rights reserved.