Full Length ArticleMolecular characterization and functional analysis of duck IKKα
Introduction
The nuclear factor-κB (NF-κB) signaling pathway is critical in regulating not only immune and inflammatory response, but also cell death and proliferation (Guttridge et al., 1999; Hayden et al., 2006). In mammals, five NF-κB family proteins have been identified: p65 (Rel-A), p50, p52, c-Rel, and RelB, which usually form homo- or heterodimers and are sequestered in the cytoplasm bound to the inhibitor of κB (IκB) in unstimulated cells (Chen and Ghosh, 1999; Oeckinghaus and Ghosh, 2009; Wan and Lenardo, 2010). The NF-κB pathway is activated by a wide variety of agents including cytokines, oxidative stresses, bacterial lipopolysaccharides (LPS), and virus infection (Baeuerle and Henkel, 1994; Baldwin, 1996). These stimulators result in the activation of the IκB kinase (IKK) complex that phosphorylates IκB inducing its degradation and subsequent nuclear translocation of active IκB-free NF-κB (Karin, 1999). The NF-κB proteins accumulate in the nucleus, bind to specific DNA sequences in the genome, and play critical roles in the transcriptional regulation of numerous cytokines, chemokines, growth factors, adhesion molecules, and cell-cycle proteins (Oeckinghaus and Ghosh, 2009).
The IKK complex is the core element of the NF-κB signaling pathway and is composed of two catalytic subunits, IKKα and IKKβ, and one regulatory subunit IKKγ (also known as NF-κB essential modifier, NEMO) (Li et al., 1999). Human IKKα and IKKβ exhibit molecular masses of 85 and 87 kDa, respectively, and display 50% of amino acid identity and 70% of structural similarity (Margalef et al., 2012). Human IKKα and IKKβ contain an amino-terminal kinase domain (KD), a leucine zipper (LZ) region responsible for homo- or heterodimerization of the kinases, a helix-loop-helix (HLH) which is involved in modulating IKK kinase activity, and a NEMO binding domain (NBD) (DiDonato et al., 1997; Israël, 2010; Zandi et al., 1997). Human IKKα activation requires phosphorylation of Ser176 and Ser180 in its KD domain, while phosphorylation of Ser177 and Ser181 in KD domain of human IKKβ triggers its activation (Fatima et al., 2015). In mammals, IKKα not only participates in the regulation of NF-κB activity, but also contributes to TLR7/9-induced IFN-α expression through phosphorylating IRF7 (Hoshino et al., 2006). Moreover, IKKα controls epidermal differentiation independently of its kinase activity and the NF-κB pathway (Hu et al., 2001).
Considerable studies have revealed the functions of IKKα in mammals, however, to the best of our knowledge, there is no report on the function of IKKα in avian innate immunity so far. Herein, we cloned the full length of duck IKKα (duIKKα) coding sequence for the first time and investigated the basal tissue expression profiles of IKKα in duck. Our study demonstrated that duIKKα is a critical cytosolic kinase involved in NF-κB activation and proinflammatory cytokine expression in duck embryo fibroblasts (DEFs). Moreover, duIKKα expression could significantly inhibit Duck Tembusu virus (DTMUV) replication. These results in this study provide insights into the function of IKKα in avian innate immunity.
Section snippets
Cell culture, tissues, virus, and reagents
DEFs were obtained from the American Type Culture Collection (ATCC Number: CCL-141) and cultured in Minimum Essential Medium with 10% fetal bovine serum (FBS, Gibco). The duck tissues used for IKKα expression analysis, including muscular stomach, spleen, heart, liver, lung, kidney, cerebellum, cerebrum, windpipe, muscle, glandular stomach, thymus, duodenum, cecum, pancreas, and bursa of Fabricius, were obtained from three 10-days-old healthy or DTMUV-infected Cherry Valley Ducks. After three
Cloning and sequence analysis of duIKKα
To characterize the duIKKα, the full-length mRNA sequence of duIKKα was determined by 5’/3’ RACE-PCR (Fig. 1A). After ORF analysis, the duIKKα ORF was composed of 2274 bp nucleotides and coded for 757 amino acid long polypeptides which was uploaded to GenBank (Accession number: MT210098). According to the NCBI annotation, the duIKKα gene contained 32 exons located on an Unplaced Scaffold (Accession number: NW_004676822.1) (Fig. 1B).
Multiple alignments of the amino acid residues of duIKKα
Discussion
NF-κB is a family of transcription factors that regulates the expression of many genes involved in immune and inflammatory responses, cell survival and proliferation (Baeuerle and Henkel, 1994; Tak and Firestein, 2001). The core element of all NF-κB cascades is the IKK complex which is composed of two kinases, IKKα and IKKβ, and one regulatory subunit IKKγ (NEMO) (Israël, 2010). Previous studies revealed that the activation of NF-κB involves two major signaling pathways, named as the NEMO- and
Acknowledgements
This work was supported by grants from National Natural Science Foundation of China (31772737) and the Fundamental Research Funds for the Central Universities (Grant No. 2662019PY078).
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