Qu Fufa, Tang Jianzhou, Peng Xiangyu, Zhang Hui, Shi Liping, Huang Zhenzhen, Xu Wenqian, Chen Huiqing, Shen Ying, Yan Jinpeng, Li Jianzhong, Lu Shuangqing, 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; State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Normal University, Changsha, 410081, China.
Hunan Provincial Key Laboratory of Nutrition and Quality Control of Aquatic Animals, Department of Biological and Environmental Engineering, Changsha University, Changsha, 410022, China.
Dev Comp Immunol. 2019 Apr;93:103-114. doi: 10.1016/j.dci.2019.01.001. Epub 2019 Jan 8.
Mitogen-activated protein kinase kinases (MKKs) are a class of evolutionarily conserved signalling intermediates of the MAPK signalling pathway that can be activated by a diverse range of pathogenic stimuli and are crucial for the regulation of host immune defence. In this study, two fish MKK genes (CiMKK4 and CiMKK7) were first identified and characterized from grass carp (Ctenopharyngodon idella). Similar to other reported MKKs, the present CiMKK4 and CiMKK7 contained a conserved serine/threonine protein kinase (S_TKc) domain and a canonical dual phosphorylation motif. Quantitative real-time PCR results showed that CiMKK4 and CiMKK7 were broadly transcribed in all selected tissues and developmental stages of grass carp. The mRNA expression levels of CiMKK4 and CiMKK7 in the intestine were significantly induced by bacterial muramyl dipeptide (MDP) challenge in a time-dependent manner (P < 0.01). Additionally, the stimulatory effects of bacterial MDP on CiMKK4 and CiMKK7 expression in the intestine were inhibited by the bioactive dipeptide β-alanyl-l-histidine (carnosine) and alanyl-glutamine (Ala-Gln) (P < 0.05). Moreover, overexpression analysis revealed that CiMKK4 and CiMKK7 were localized throughout the entire cell and could significantly enhance AP-1 reporter gene activation in HEK293T cells. Taken together, these results provide the first experimental demonstration that CiMKK4 and CiMKK7 are involved in the intestinal immune response to MDP challenge in C. idella, which may provide new insight into the bacterial-induced intestinal inflammation of bony fishes.
丝裂原活化蛋白激酶激酶（MKKs）是一类在进化上保守的丝裂原活化蛋白激酶（MAPK）信号通路的信号中间体，可被多种致病刺激激活，对宿主免疫防御的调节至关重要。在本研究中，首次从草鱼（Ctenopharyngodon idella）中鉴定并表征了两个鱼类MKK基因（CiMKK4和CiMKK7）。与其他已报道的MKKs相似，目前的CiMKK4和CiMKK7包含一个保守的丝氨酸/苏氨酸蛋白激酶（S_TKc）结构域和一个典型的双磷酸化基序。定量实时PCR结果表明，CiMKK4和CiMKK7在草鱼所有选定的组织和发育阶段均广泛转录。细菌胞壁酰二肽（MDP）刺激以时间依赖性方式显著诱导了CiMKK4和CiMKK7在肠道中的mRNA表达水平（P<0.01）。此外，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）抑制了细菌MDP对肠道中CiMKK4和CiMKK7表达的刺激作用（P<0.05）。此外，过表达分析表明，CiMKK4和CiMKK7定位于整个细胞，并可显著增强HEK293T细胞中AP-1报告基因的激活。综上所述，这些结果首次通过实验证明CiMKK4和CiMKK7参与了草鱼对MDP刺激的肠道免疫反应，这可能为硬骨鱼类细菌诱导的肠道炎症提供新的见解。
