Two novel MKKs (MKK4 and MKK7) from Ctenopharyngodon idella are involved in the intestinal immune response to bacterial muramyl dipeptide challenge. (April 2019) - Qu, Fufa
Abstract: 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 ( Ci MKK4 and Ci MKK7) were first identified and characterized from grass carp ( Ctenopharyngodon idella ). Similar to other reported MKKs, the present Ci MKK4 and Ci MKK7 contained a conserved serine/threonine protein kinase (S_TKc) domain and a canonical dual phosphorylation motif. Quantitative real-time PCR results showed that Ci MKK4 and Ci MKK7 were broadly transcribed in all selected tissues and developmental stages of grass carp. The mRNA expression levels of Ci MKK4 and Ci MKK7 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 Ci MKK4 and Ci MKK7 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 Ci MKK4 and Ci MKK7 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 Ci MKK4 and Ci MKK7 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. *Highlights: The first experimental evidence for presence of MKK4 and MKK7 in Ctenopharyngodon idella. Ci MKK4 and Ci MKK7 mRNA were broadly expressed in all tested tissues and developmental stages. Transcript levels of Ci MKK4 and Ci MKK7 could be significantly induced by bacterial muramyl dipeptide challenge. The stimulating effects of MDP on Ci MKK4 and Ci MKK7 were inhibited by dipeptide carnosine and Ala-Gln. Ci MKK4 and Ci MKK7 were cytoplasm- and nucleus-localized proteins which could enhance AP-1-Luc activation.