Ctenopharyngodon idella NF-κB subunit p65 modulates the transcription of IκBα in CIK cells

NF-kappa B is an important transcription factor for regulating the multiple inflammatory and immune related gene transcription. It can bind with the nuclear factor kappa B site within the promoter of target genes to regulate their transcriptions. p65, the all-important subunit of NF-kappa B, is ubiquitously expressed in cells. In the present study, we cloned and identified the p65 subunit from grass carp (Ctenopharyngodon idella) (named Cip65) by homologous cloning and RACE technique. The full length of Cip65 cDNA is 2481 bp along with 9 bp 5' UTR, 639 bp 3' UTR and the largest open reading frame (1833 bp) encoding a poly peptide of 610 amino acids with a well conserved Rel-homology domain (RHD) in N-terminal and a putative transcription activation domain (TAD) in C-terminal. Cip65 gathers with other teleost p65 proteins to form a fish-specific Glade clearly distinct from those of mammalian and amphibian counterparts on the phylogenetic tree. In CIK (C. idellus kidney) cells, the expression of Cip65 was significantly up-regulated under the stimulation with Poly I:C. As one member of the NF-kappa B inhibitor protein (I kappa B) family, I kappa B alpha can dominate the activity of NF-kappa B by interacting with it. To study the molecular mechanisms of negative feedback loop of NF-kappa B signaling in fish, we cloned grass carp I kappa B alpha (Cilidia) promoter sequence. Cilidla promoter is 414 bp in length containing two RelA binding sites and a putative atypical TATA-box. Meanwhile, Cip65 and its mutant proteins including C-terminus deletion mutant of Cip65 (Cip65-AC) and N-terminus deletion mutant of Cip65 (Cip65-AN) were expressed in Escherichia coil BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, Cip65 rather than Cip65-AC and Cip65-AN showed high affinity with Max promoter sequence by gel mobility shift assays. In vivo, the cotransfection of pcDNA3.1-Cip65 (or pcDNA3.1-Cip65-41C, pcDNA3.1-Cip65-AN respectively) with pGL3-CiI kappa B alpha and pRL-TK renilla luciferase plasmid into CIK cells showed that pcDNA3.1-Cip65 rather than pcDNA3.1-Cip65-AC and pcDNA3.1-Cip65-AN, can increase the luciferase activity. Taken together, these results suggested that Cip65 can regulate the expression of CiI kappa B alpha and works as a negative feedback loop in NF-kappa B pathway. (C) 2016 Elsevier Ltd. All rights reserved.