Short communicationMitogen-activated protein kinase kinase 6 is involved in the immune response to bacterial di-/tripeptide challenge in grass carp Ctenopharyngodon idella
Introduction
Innate immunity is essential for multicellular organisms to detect and eliminate a wide range of microbial pathogens. The innate immune system provides a rapid and widespread host defense response via different cellular pattern recognition receptors (PRRs), which can specifically recognize conserved pathogen-associated molecular patterns (PAMPs) on various microorganisms [1,2]. Mitogen-activated protein kinases (MAPKs) are multifunctional signaling intermediates that can receive various immune signals triggered by upstream cellular PRRs and activate downstream nuclear transcription factors, such as nuclear factor-κB (NF-κB) and activation protein-1 (AP-1), to regulate the transcription of a subset of immune response genes to combat the invading infectious agents [[3], [4], [5]].
MAPKs are evolutionarily conserved intracellular serine/threonine kinases that exist widely from yeasts to mammals and are crucial for the regulation of stress responses, apoptosis, and host immune defense [[6], [7], [8]]. MAPKs are activated by a diverse range of stimuli, including inflammatory cytokines, growth factors, and oxidative stress, and their function is to transduce extracellular signals from cell surface receptors to the nucleus through the phosphorylation of target proteins [9,10]. MAPK activity has been shown to be regulated by upstream MAPK kinases (MKKs), and these, in turn, are phosphorylated and activated by specific MKK kinases (MKKKs) [6,11]. The mammalian MAPK family consists of extracellular signal-regulated kinase (ERK), p38MAPK and c-Jun NH2-terminal kinase (JNK) [9,12]. Among these, p38MAPK has been found to play important roles in immune responses, from the initiation phase of innate immunity to the activation of adaptive immunity [[13], [14], [15]].
MAPK kinase 6 (MKK6), also called MEK6 or MAP2K6, is an essential component of p38MAPK signaling pathway, that are reported to be involved in the regulation of inflammation and apoptosis during immune challenges [[16], [17], [18]]. Like other MKK family members, MKK6 is a dual-specificity protein that can be phosphorylated and activated by upstream MKKKs at the Ser-X-X-X-Thr motif (X is any amino acid). Activation of MKK6 results in phosphorylation of p38MAPKs through the dual phosphorylation Thr-Gly-Tyr (TGY) motif in activation loop and ultimately regulates the expression of numerous specific target genes [19]. Considering its crucial roles in immunity, MKK6 has been extensively studied in several aquatic animals up to date [[20], [21], [22], [23]]. For example, shrimp LvMKK6 was characterized from Litopenaeus vannamei and proved to be involved in regulating expression of antimicrobial peptides and host defense against Vibrio parahaemolyticus as well as white spot syndrome virus (WSSV) infection [21]. Wang et al. reported that a sea cucumbe MKK3/6 gene was proved to be involved in defense response to Vibrio splendidus infection in vivo [22]. In bony fish, three Atlantic salmon MKK6 orthologs were identified and shown to be upstream activators of salmon p38 which could be involved in cell response to stress stimuli [24]. Recently, several other fish MKK6 genes from D. rerio (NP_001299799.1), Takifugu rubripes (XP_003977287.2) and Oryzias latipes (XP_023805137.1) were reported in National Center for Biotechnology Information (NCBI) database. However, compared to studies in mammals, these reports have provided limited information of MKK6s function in aquatic animals.
Grass carp (Ctenopharyngodon idella) is one of the most highly produced and economically important freshwater fish species in China, but they often encounter numerous types of pathogens, which cause tremendous economic losses to aquaculture [25,26]. Investigation into the function of C. idella MKK6, a conserved component of innate immune system, might contribute to a better understanding of the fish immune defense mechanism against exogenous pathogens. In this regard, we cloned the MKK6 (CiMKK6) cDNA sequence from grass carp C. idella. The mRNA expression of CiMKK6 in different tissues and developmental stages were also investigated by quantitative real-time PCR (qRT-PCR) in this study. In addition, the mRNA expression profiles of CiMKK6 upon exposure to bacterial muramyl dipeptide (MDP) and L-Ala-γ-D-Glu-meso-diaminopimelic acid (tri-DAP) challenge were analyzed in intestines of grass carp. Moreover, CiMKK6 was overexpressed in human embryonic kidney 293T (HEK293T) cells to determine its intracellular localization and function in signal transduction. To our knowledge, this was the first explored the potential roles of MKK6 in innate immunity of C. idella, and the results may lay a foundation for further researches on immune function of MKK6s in teleost.
Section snippets
Experimental fish and sample collection
Healthy grass carp, averaging 30 g in weight, were purchased from the Hunan Institute of Aquatic Science. Prior to the experiment, fish were fed daily with a standard diet in a recirculating water tank system at 25 °C for 2 weeks.
After anesthetizing fish with 2-phenoxyethanol (Sigma-Aldrich), tissues, including the intestine, liver, blood, heart, kidney, gill, muscle and spleen, of healthy fish were dissected for tissue distribution analysis. For the expression analysis of different
Cloning and characterization of the CiMKK6 sequence
The cDNA sequence of grass carp MKK6 (CiMKK6) was cloned based on the sequence retrieved from the genome database and was submitted to GenBank under accession number MH491995. To the best of our knowledge, this is the first report to describe the molecular characterization of CiMKK6 in C. idella. The nucleotide and deduced amino acid sequences of CiMKK6 are shown in Fig. S1. The CiMKK6 cDNA consisted of 1393 bp containing a 5′ untranslated region (UTR) of 155 bp, a 3′-UTR of 110 bp and an open
Conclusion
In summary, a novel fish MKK6 homolog (CiMKK6) was cloned and identified for the first time from C. idella. The deduced CiMKK6 sequence shared high structural similarity and a close evolutionary relationship with its homologs from other fish. Quantitative RT-PCR analysis showed that CiMKK6 mRNA was ubiquitously expressed in all selected tissues and in different developmental stages of grass carp. Additionally, CiMKK6 mRNA transcripts were significantly upregulated by bacterial MDP and Tri-DAP
Acknowledgments
This research was supported by the National Natural Science Foundation of China (Grant Nos. 31772865 and 31702378), the Natural Science Foundation for Young Scholars of Hunan Province (Grant No. 2017JJ3344), the Project of Scientific Research of the Hunan Provincial Education Department, China (Grant Nos. 16A022 and 17B031), the Foundation of the State Key Laboratory of Developmental Biology of Freshwater Fish (Grant No. 2018KF006), the Scientific Research Project of the Science and Technology
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