Molecular Mechanism Of Rgdig Regulating TAK1-mediated Immune Response In Miiuy Croaker(Miichthys Miiuy)

As one of the important marine economic fish,Miichthys miiuy（miiuy croaker）not only has delicious flesh but also have high medicinal value,so farming them can bring great economic benefits.With the gradual expansion of miiuy croakers farming scale,the problem of fish diseases has become more and more serious,which directly hinders the development of miiuy croakers farming and causes huge economic losses.Therefore,in order to explore ways and methods to enhance the resistance of miiuy croakers and effectively solve such problems,it is necessary to study the immune control mechanism of miiuy croakers in depth to reduce the harm caused by pathogenic microorganisms to the aquaculture industry from the root.Among the numerous innate immune molecules in scleractinians,transforming growth factor-β-activated kinase 1（TAK1）plays an important role as a downstream molecule of the nuclear transcription factorκB（NF-κB）pathway.When receiving signals from upstream,TAK1 undergoes ubiquitination or deubiquitination,and then transmits the signals to regulate the NF-κB pathway,therefore,in the innate immune response,TAK1 is an essential member of the NF-κB pathway.In order to avoid excessive immune responses caused by pathogenic microbial infections,we investigated TAK1 and its related regulatory protein,Rho guanosine diphosphate dissociation inhibitor-γ（Rgdig）is of great importance.In this paper,we investigated the mechanism of inhibition of TAK1 expression in miiuy croakers,which relies on Rgdig to negatively regulate the signal transduction of NF-κB pathway by targeting TAK1 to undergo degradation.The findings of this study are as follows:1.Using lipopolysaccharide（LPS）to stimulate carp epithelioma papulosum cyprini（EPC）cells,and dual-luciferase reporter gene analysis revealed that after LPS stimulation,reporter genes NF-κB and interleukin-8（IL-8）were significantly activated;and overexpression of Rgdig could effectively inhibit this activation.To further verify the function of Rgdig,the Rgdig plasmid was transfected into Miichthys miiuy kidney cells（MKC）,and the cells were still stimulated with LPS.The results of quantitative real-time PCR（q RT-PCR）showed that Rgdig overexpression inhibited the transcription of downstream inflammatory cytokines in the NF-κB signaling pathway,such as tumor necrosis factor-α（TNF-α）and interleukin-1β（IL-1β）.Next,si-Rgdig was transfected into MKC cells,and the expression of Rgdig was disrupted at the m RNA level.In further experiments,si-Rgdig was transfected into MKC cells,followed by q RT-PCR assays to assess the expression of cytokines（TNF-αand IL-1β）after knockdown of Rgdig.2.Because the activation of NF-κB is mediated by a complex cascade including TAK1,this article also verified whether Rgdig targets and regulates TAK1-mediated NF-κB signaling.The results of dual-luciferase reporter gene experiments showed that Rgdig attenuated the activation of the TAK1-mediated NF-κB pathway.Subsequently,it was further characterized whether the activation of TAK1-mediated NF-κB pathway was affected by the concentration of Rgdig and the duration of action,and Rgdig was found to inhibit the activation of TAK1-mediated NF-κB pathway in the results of concentration and time gradient experiments.Taken together,Rgdig inhibited NF-κB signaling pathway by targeting and regulating TAK1.3.After the preliminary determination that Rgdig can inhibit TAK1-mediated NF-κB signaling activation,the relationship between Rgdig and TAK1 was explored in this paper.Using western blot experiments,we found that overexpression of Rgdig decreased the protein expression level of TAK1,and demonstrated by co-Immunoprecipitation（co-IP）experiments that Rgdig could interact with TAK1 and decrease TAK1 expression in a dose-and time-dependent manner.4.By treating EPC cells with inhibitors,such as proteasome inhibitor（MG132）,autophagy inhibitor（3-MA）and lysosome inhibitor（NH₄Cl）,and by western blot experiments,it was tentatively deduced that Rgdig promotes TAK1 degradation through the proteasome pathway.Meanwhile,the results of dual-luciferase reporter gene assay demonstrated that MG132 could weaken the inhibitory effect of Rgdig on TAK1-mediated NF-κB signaling pathway.To further investigate the molecular mechanism of TAK1 degradation,TAK1,Rgdig and ubiquitinated plasmid（Ubiquitin,Ub）were cotransfected into HEK293 cells,and TAK1 was analyzed by immunoprecipitation and protein blotting to show that it underwent ubiquitinated degradation.the experimental results in this paper suggest that Rgdig is involved in regulating the immune response induced by bacterial infection as a negative regulator.In this study,the findings show a mechanism of action for the suppression of TAK1 expression in otoliths that relies on Rgdig to negatively regulate NF-κB signaling by targeting TAK1 for degradation.It was determined that TAK1 can directly interact with Rgdig and can promote TAK1 degradation through the ubiquitin-proteasome pathway.Rgdig was then found to negatively regulate the TAK1-mediated NF-κB signaling pathway and may also inhibit the expression of target genes and their downstream inflammatory cytokines.This study provides a new theoretical basis for the innate immune signaling pathway in scleractinian fish,which not only provides evidence for the regulatory mechanism of TAK1 signaling by Rgdig,but also enriches the NF-κB signaling pathway in teleost fish,which may also provide new insights into the regulatory mechanism in mammals.