Qu Fufa, Li Jialing, Zeng Xuan, She Qing, Li Yurong, Xie Wenjie, Cao Shenping, Zhou Yonghua, He Zhimin, Tang Jianzhou, Mao Zhuangwen, Wang Yuping, Fang Jiamei, Xu Wenqian, Liu Zhen
Hunan Provincial Key Laboratory of Nutrition and Quality Control of Aquatic Animals, Department of Biological and Environmental Engineering, Changsha University, Changsha 410022, China.
Hunan Provincial Key Laboratory of Nutrition and Quality Control of Aquatic Animals, Department of Biological and Environmental Engineering, Changsha University, Changsha 410022, China.
Fish Shellfish Immunol. 2022 Jan;120:82-91. doi: 10.1016/j.fsi.2021.11.016. Epub 2021 Nov 13.
Mitogen-activated protein kinase kinase kinase 4 (MAP3K4) is a multifunctional mediator of the conserved MAPK signaling pathway that plays essential roles in the regulation of immune responses in mammals. However, the function of teleost MAP3K4s in innate immunity, especially in the intestinal immune system, is still poorly understood. In the current study, we identified a fish MAP3K4 homolog (CiMAP3K4) in Ctenopharyngodon idella as well as its immune function in intestine following bacterial infection in vivo and in vitro. The open reading frame (ORF) of CiMAP3K4 encodes putative peptide of 1544 amino acids containing a predicted serine/threonine protein kinase (S_TKc) domain with high identity with other fish MAP3K4s. Phylogenetic analysis revealed the CiMAP3K4 belonged to the fish cluster and showed the closest relationship to Pimephales promelas. Quantitative real-time PCR (qRT-PCR) analysis revealed that CiMAP3K4 transcripts were widely distributed in all tested tissues, especially with high expression in the muscle and intestine of healthy grass carp. In vitro, CiMAP3K4 gene expression was upregulated by bacterial PAMPs (lipolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) and muramyl dipeptide (MDP)) and pathogens (Aeromonas hydrophila and Aeromonas veronii) in primary intestinal cells. In vivo, the mRNA expression levels of CiMAP3K4 in the intestine were significantly induced by bacterial MDP challenge in a time-dependent manner; however, this effect could be inhibited by the bioactive dipeptides β-alanyl-l-histidine (carnosine) and alanyl-glutamine (Ala-Gln). Moreover, CiMAP3K4 was located primarily in the cytoplasm, and its overexpression increased the transcriptional activity of AP-1 in HEK293T cells. Collectively, these results suggested that CiMAP3K4 might play an important role in the intestinal immune response to bacterial infections, which paves the way for a better understanding of the intestinal immune system of grass carp.
丝裂原活化蛋白激酶激酶激酶4（MAP3K4）是保守的MAPK信号通路的多功能介质，在哺乳动物免疫反应调节中起重要作用。然而，硬骨鱼MAP3K4s在先天免疫中的功能，尤其是在肠道免疫系统中的功能，仍知之甚少。在本研究中，我们在草鱼中鉴定出一种鱼类MAP3K4同源物（CiMAP3K4），并在体内和体外细菌感染后研究了其在肠道中的免疫功能。CiMAP3K4的开放阅读框（ORF）编码1544个氨基酸的推定肽，包含一个预测的丝氨酸/苏氨酸蛋白激酶（S_TKc）结构域，与其他鱼类MAP3K4s具有高度同源性。系统发育分析表明，CiMAP3K4属于鱼类聚类，与黑头软口鲦关系最为密切。定量实时PCR（qRT-PCR）分析显示，CiMAP3K4转录本广泛分布于所有测试组织中，尤其是在健康草鱼的肌肉和肠道中高表达。在体外，原代肠道细胞中的细菌PAMPs（脂多糖（LPS）、肽聚糖（PGN）、L-丙氨酸-γ-D-谷氨酸-内消旋二氨基庚二酸（Tri-DAP）和胞壁酰二肽（MDP））和病原体（嗜水气单胞菌和维氏气单胞菌）可上调CiMAP3K4基因表达。在体内，细菌MDP攻击可时间依赖性地显著诱导肠道中CiMAP3K4的mRNA表达水平；然而，生物活性二肽β-丙氨酰-L-组氨酸（肌肽）和丙氨酰-谷氨酰胺（Ala-Gln）可抑制这种作用。此外，CiMAP3K4主要位于细胞质中，其过表达增加了HEK293T细胞中AP-1的转录活性。总的来说，这些结果表明CiMAP3K4可能在肠道对细菌感染的免疫反应中起重要作用，这为更好地理解草鱼肠道免疫系统铺平了道路。
