Nuclear Factor-κB Plays a Positive Role in TNFα Expression in Golden Pompano, Trachinotus ovatus (Linnaeus 1758)

Abstract

Tumour necrosis factor-α (TNFα) is a multifarious mediator of lymphoid tissue growth and antimicrobial defence mechanisms, and it acts as a pro-inflammatory regulator. The function of TNFα in parasite infection and the underlying mechanism through which nuclear factor-κB (NF-κB) regulates TNFα remain largely unclear in teleosts. In the present study, TNFα (ToTNFα) from golden pompano (Trachinotus ovatus) was identified, and its sequence features and expression levels were determined. The genomic DNA sequence is composed of 1,130 bp, consists of four exons and three introns, and encodes 341 amino acid polypeptides. The putative protein sequence shares 34.7%–61.9% identity with fish TNFα and possesses a TNF family signature and two conserved cysteine residues. Moreover, the expressions of ToNF-κB and ToTNFα are constitutively expressed in all examined tissues, with higher levels observed in the immune relevant tissues. Both ToNF-κB and ToTNFα transcription was increased in the local infection sites (skin and gill) and system immune tissues (liver, spleen and head kidney) after Cryptocaryon irritans stimulation. In addition, to investigate whether ToNF-κB is a regulator of ToTNFα, promoter analysis was performed. The region from -970 to +79 bp is known as the core promoter by different truncated mutants of ToTNFα. Subsequently, the activity of the ToTNFα-p2 promoter was dramatically reduced after targeted mutation of the M6-binding site. Additionally, an electrophoretic mobile shift assay (EMSA) verified that ToNF-κB interacted with the M6-binding site in the ToTNFα promoter region to control the expression of ToTNFα. In conclusion, the present study provides the positive regulation of TNFα transcription by NF-κB and contributes to a better understanding of the transcriptional mechanism of TNFα in fish.