Biotechnology Research Division, National Fisheries Research & Development Institute, 408-1 Sirang-ri, Gijang-eup, Gijang-gun, Busan 619-902, Republic of Korea.
Fish Shellfish Immunol. 2011 Dec;31(6):881-6. doi: 10.1016/j.fsi.2011.08.002. Epub 2011 Aug 16.
Toll-like receptor 5 (TLR5), which is highly conserved from lower to higher vertebrates, is an important pattern recognition receptor (PRR) for bacterial flagellin. However, a soluble form of TLR5 (TLR5S) was identified in fish that is not present in mammals. To better understand the transcriptional regulation of TLR5S gene in fish, we determined the TLR5S 5'-flanking sequence region from flounder (Paralichthys olivaceus) and assayed its promoter activity in Hirame natural embryo (HINAE) cells. The 5'-flanking region of TLR5S (715 bp) contains sequence elements for two AP-1 binding sites, two C/EBP sites, and one NF-κB site. To elucidate the functional significance of these sites, deletion clones and a site-directed mutant of NF-κB were generated. We estimated the luciferase activity in flagellin- or lipopolysaccharide-stimulated HINAE cells. The co-transfection of p65 with the wild-type TLR5S promoter greatly increased luciferase activity by more than nine-fold compared with the NF-κB mutant. Wild-type TLR5S promoter activity was increased synergistically by more than 159.5-fold in the presence of flagellin and p65. Furthermore, it was determined that the level of TRL5S mRNA was up-regulated by p65 and flagellin using a quantitative PCR. Additionally, translocation of TLR5S in the HINAE-TLR5S stable cell line after flagellin stimulation was observed by confocal microscopy. These results suggest that NF-κB and flagellin are essential components that act as a transcription factor and ligand, respectively, for maximal induction of the TLR5S promoter.
Toll 样受体 5（TLR5）在从低等到高等脊椎动物中高度保守，是细菌鞭毛蛋白的重要模式识别受体（PRR）。然而，在鱼类中发现了一种可溶性形式的 TLR5（TLR5S），而哺乳动物中则没有。为了更好地理解鱼类 TLR5S 基因的转录调控，我们从牙鲆（Paralichthys olivaceus）中确定了 TLR5S 的 5'-侧翼序列区域，并在 Hirame 天然胚胎（HINAE）细胞中测定了其启动子活性。TLR5S 的 5'-侧翼区域（715bp）包含两个 AP-1 结合位点、两个 C/EBP 位点和一个 NF-κB 位点的序列元件。为了阐明这些位点的功能意义，我们生成了缺失克隆和 NF-κB 的定点突变。我们在鞭毛蛋白或脂多糖刺激的 HINAE 细胞中估计了荧光素酶活性。与 NF-κB 突变体相比，p65 与野生型 TLR5S 启动子共转染可使荧光素酶活性大大增加超过九倍。在鞭毛蛋白和 p65 的存在下，野生型 TLR5S 启动子活性协同增加超过 159.5 倍。此外，使用定量 PCR 确定 p65 和鞭毛蛋白可上调 TRL5S mRNA 的水平。此外，在牙鲆 TLR5S 稳定细胞系中，在鞭毛蛋白刺激后观察到 TLR5S 的易位。这些结果表明，NF-κB 和鞭毛蛋白分别作为转录因子和配体，对于 TLR5S 启动子的最大诱导是必需的。
