Molecular Characterization of E-Type Prostanoid Receptor 4 (EP4) from Ayu (Plecoglossus altivelis) and Its Functional Analysis in the Monocytes/Macrophages

Prostaglandin E2 (PGE2) plays an important role in a broad spectrum of physiological and pathological processes by interacting with E-type prostanoid receptors (EPs). EP4 is one of four EP subtypes known to mediate the immune response in mammalian monocytes/macrophages. However, the precise function and characteristics of EP4 in fish remain unclear. In this study, we characterized a novel EP4-like (PaEP4L) gene from ayu, Plecoglossus altivelis. The cDNA sequence of PaEP4L is 2781 nucleotides (nts) in length, encoding a polypeptide of 459 amino acid residues with a calculated molecular weight of 51.17 kDa. Sequence comparison and phylogenetic tree analysis showed that PaEP4L shared 76% amino acid identity with that of the Atlantic salmon (Salmo salar). PaEP4L mRNA was detected by real-time quantitative PCR (QPCR) in all tested tissues and head kidney-derived monocytes/macrophages (MO/MФ). It varied greatly in liver, spleen and MO/MФ upon Vibrio anguillarum infection. Western blot analysis revealed a significant increase of PaEP4L in cell homogenates from ayu MO/MФ upon V. anguillarum infection. Moreover, anti-PaEP4L IgG reversed the down-regulation of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) mRNA expression as well as phagocytosis in ayu MO/MФ caused by PGE2. There were no significant differences in the respiratory burst response between PGE2 treated and untreated cells. We further found that cAMP mediated PGE2/PaEP4L signal in ayu MO/MФ. In conclusion, our results indicate that PaEP4L mediates PGE2 effects on ayu MO/MФ function, revealing that EP4 also plays a role in the modulation of cells of the fish’s innate immune system.前列腺素E2（Prostaglandin E2, PGE2）可通过与E型前列腺素受体（E-type prostanoid receptors, EPs）结合，广泛参与多种生理与病理过程。EP4是目前已发现的四种EP亚型之一，能够介导哺乳动物单核细胞/巨噬细胞的免疫应答。然而，鱼类中EP4的精确功能与特性仍有待阐明。本研究从香鱼（Plecoglossus altivelis）中鉴定得到一种新型EP4样基因（命名为PaEP4L）。该基因的互补DNA（complementary DNA, cDNA）序列全长2781个核苷酸（nucleotides, nts），编码一条含459个氨基酸残基的多肽，理论分子质量为51.17 kDa。序列比对与系统发育树分析结果显示，PaEP4L与大西洋鲑（Salmo salar）的EP4L氨基酸同源性高达76%。采用实时定量聚合酶链反应（real-time quantitative PCR, QPCR）检测发现，PaEP4L mRNA在所有受试组织及头肾来源的单核细胞/巨噬细胞（MO/MФ）中均有表达；经鳗弧菌（Vibrio anguillarum）感染后，其在肝脏、脾脏及MO/MФ中的表达水平发生显著变化。蛋白质印迹法（Western blot）分析显示，香鱼MO/MФ的细胞裂解液中PaEP4L的表达量在鳗弧菌感染后显著升高。此外，抗PaEP4L免疫球蛋白G（IgG）可逆转PGE2诱导的香鱼MO/MФ中白细胞介素1β（interleukin 1β, IL-1β）、肿瘤坏死因子α（tumor necrosis factor α, TNF-α）mRNA表达下调及吞噬功能抑制。PGE2处理组与对照组细胞的呼吸爆发响应无显著差异。本研究进一步证实，环磷酸腺苷（cyclic adenosine monophosphate, cAMP）介导了香鱼MO/MФ内的PGE2/PaEP4L信号通路。综上，本研究结果表明PaEP4L可介导PGE2对香鱼MO/MФ功能的调控作用，提示EP4在鱼类先天免疫系统的细胞功能调控中同样发挥关键作用。