Hunan Provincial Key Laboratory of Nutrition and Quality Control of Aquatic Animals, Department of Biological and Environmental Engineering, Changsha University, Changsha, China.
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
Front Immunol. 2022 Aug 5;13:981995. doi: 10.3389/fimmu.2022.981995. eCollection 2022.
Mitogen-activated protein kinase kinase kinase 3 (MEKK3) is an evolutionarily conserved Ser/Thr protein kinase of the MEKK family that is essential for the host immune response to pathogen challenges in mammals. However, the immune function of MEKK3s in lower vertebrate species, especially in bony fish, remains largely unknown. In this study, a fish MEKK3 (designated MEKK3) gene was cloned and identified from grass carp (). The present MEKK3 cDNA encoded a 620 amino acid polypeptide containing a conserved S-TKc domain and a typical PB1 domain. Several potential immune-related transcription factor-binding sites, including activating protein 1 (AP-1), nuclear factor kappa B (NF-κB) and signal transducer and activator of downstream transcription 3 (STAT3), were observed in the 5' upstream DNA sequence of MEKK3. A phylogenetic tree showed that MEKK3 exhibits a close evolutionary relationship with MEKK3s from and . Quantitative real-time PCR analysis revealed that MEKK3 transcripts were widely distributed in all selected tissues of healthy grass carp, with a relatively high levels observed in the gill, head kidney and intestine. Upon challenge with bacterial pathogens ( and ) and pathogen-associated molecular patterns (PAMPs) (lipopolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-mDAP (Tri-DAP) and muramyl dipeptide (MDP)), the expression levels of MEKK3 in the intestinal cells of grass carp were shown to be significantly upregulated in a time-dependent manner. injection experiments revealed that MEKK3 transcripts were significantly induced by MDP challenge in the intestine; however, these effects could be inhibited by the nutritional dipeptides carnosine and Ala-Gln. Moreover, subcellular localization analysis and luciferase reporter assays indicated that MEKK3 could act as a cytoplasmic signal-transducing activator involved in the regulation of NF-κB and MAPK/AP-1 signaling cascades in HEK293T cells. Taken together, these findings strongly suggest that MEKK3 plays vital roles in the intestinal immune response to bacterial challenges, which will aid in understanding the pathogenesis of inflammatory bowel disease in bony fish.
丝裂原活化蛋白激酶激酶激酶 3（MEKK3）是丝裂原活化蛋白激酶激酶家族中一种进化上保守的丝氨酸/苏氨酸蛋白激酶，对哺乳动物宿主对病原体挑战的免疫反应至关重要。然而，MEKK3 在低等脊椎动物物种（特别是硬骨鱼）中的免疫功能在很大程度上仍不清楚。在本研究中，从草鱼中克隆和鉴定了一种鱼类 MEKK3（命名为 MEKK3）基因。目前的 MEKK3 cDNA 编码一个含有保守 S-TKc 结构域和典型 PB1 结构域的 620 个氨基酸多肽。在 MEKK3 的 5'上游 DNA 序列中观察到几个潜在的免疫相关转录因子结合位点，包括激活蛋白 1（AP-1）、核因子 kappa B（NF-κB）和信号转导和转录激活因子 3（STAT3）。系统发育树表明，MEKK3 与 和 的 MEKK3 具有密切的进化关系。实时定量 PCR 分析显示，MEKK3 转录物广泛分布于健康草鱼所有选定组织中，在鳃、头肾和肠中表达水平较高。在受到细菌病原体（ 和 ）和病原体相关分子模式（PAMPs）（脂多糖（LPS）、肽聚糖（PGN）、L-Ala-γ-D-Glu-mDAP（Tri-DAP）和 muramyl dipeptide（MDP））挑战后，草鱼肠细胞中的 MEKK3 表达水平呈时间依赖性显著上调。注射实验表明，MDP 挑战可显著诱导肠内 MEKK3 转录物；然而，这些效应可被营养二肽肌肽和 Ala-Gln 抑制。此外，亚细胞定位分析和荧光素酶报告基因测定表明，MEKK3 可作为一种细胞质信号转导激活剂，参与调节 HEK293T 细胞中的 NF-κB 和 MAPK/AP-1 信号级联。总之，这些发现强烈表明 MEKK3 在草鱼肠道对细菌挑战的免疫反应中发挥重要作用，这将有助于理解硬骨鱼炎症性肠病的发病机制。
