Molecular characterization and functional analysis of duck IKKα

Highlights

• DuIKKα is expressed in various tissues of ducks.

• Overexpression of duIKKα increases NF-κB activity in DEFs.

• The kinase domain of duIKKα is essential for NF-κB activation.

• DuIKKα exhibits antiviral activity against DTMUV infection.

Abstract

IκB kinase α (IKKα) is a vital component of the IKK complex, which is involved in innate immune response, inflammation, cell death and proliferation. Although the functional characteristics of IKKα have been extensively studied in mammals and fish, the roles of IKKα in avian remain largely unknown. In this study, we cloned and characterized the duck IKKα (duIKKα) gene for the first time. DuIKKα encoded a protein of 757 amino acid residues and showed high sequence identities with the goose IKKα. The duIKKα was expressed in all tested tissues, and a relatively high expression of duIKKα mRNA was detected in liver and heart. Overexpression of duIKKα dramatically increased NF-κB activity and induced the expression of duck cytokines IFN-β, IL-1β, IL-6, IL-8 and RANTES in DEFs. Knockdown of duIKKα by small interfering RNA significantly decreased LPS-, poly(I:C)-, poly(dA:dT)-, duck enteritis virus (DEV)-, or duck Tembusu virus (DTMUV)-induced NF-κB activation. Moreover, duIKKα exhibited antiviral activity against DTMUV infection. These findings provide important insights into the roles of duIKKα in avian innate immunity.

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.