Identification and characterization of two novel functional CD39 splice variants that negatively modulate the proinflammatory response in the Japanese flounder Paralichthys olivaceus

Abstract

Ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) is a key regulator of inflammatory responses by scavenging proinflammatory extracellular ATP (eATP) and promoting the generation of immunosuppressive adenosine. In addition to the previously identified CD39 homolog (designated CD39a), here we identified two novel functional CD39 splice variants (termed CD39b and CD39c) from teleost fish Japanese flounder (Paralichthys olivaceus). Transcriptional expression analysis revealed that all three CD39 splicing variants were coexpressed in Japanese flounder head kidney macrophages (HKMs) and a variety of normal tissues but with differential tissue/immune cell expression preferences. Immune challenge experiments showed that the expression of CD39 splice variants was modulated by both in vitro inflammatory stimulation and in vivo bacterium Edwardsiella piscicida infections. However, even though all CD39 splice variants were functional cell surface-expressed proteins and shared generally similar domain structures, their varied cytoplasmic N-terminal sequences resulted in a varied capability for hydrolysis of eATP and attenuation of eATP/P2XR-mediated proinflammatory cytokine expression. Pharmacological inhibition of endogenous CD39 activity in Japanese flounder HKMs or overexpression of CD39 splice variants in Japanese flounder FG-9307 cells further revealed the role of CD39 variants in down-regulating LPS-induced proinflammatory cytokine production. In addition, luciferase reporter analysis demonstrated that the CD39 splice variant promoters had varied transcriptional activities and that Sp1 was a potential transcription factor for CD39c transcription regulation. Taken together, we report for the first time that functional CD39 splice variants provide additional means to downregulate eATP- and LPS-induced proinflammatory response(s) in fish.