Characterization of ecto-5'-nucleotidase (CD73) involved in inflammatory regulation in Japanese flounder...

Ecto-5'-nucleotidase (CD73) is a plasma membrane glycoprotein that regulates inflammation by generating the anti-inflammatory signaling molecule adenosine in the extracellular milieu in mammals. However, the expression and immunological functions of CD73 in lower vertebrates remain unclear. In this study, we identified and characterized a CD73 homolog, designated PoCD73, in Japanese flounder (Paralichthys olivaceus) by RACE-PCR and genome walking. The PoCD73 gene possesses 9 exons and encodes a glycosylated protein that shares conserved structural features with its mammalian counterparts. PoCD73 expression is highly induced by both LPS challenge and Edwardsiella piscicida infections. Notably, PoCD73 expression is also regulated by the aconitate decarboxylase 1 (ACOD1)/itaconate pathway, which has been demonstrated to suppress inflammatory challenge-induced proinflammatory responses in fish. Furthermore, deletion of the proximal promoter region (-1/-215) substantially reduced PoCD73 promoter activity, indicating that this region constitutes a core promoter governing PoCD73 gene expression. Moreover, we identified a role for the transcription factor SP1 in regulating PoCD73 transcription. Finally, pharmacological inhibition of PoCD73 significantly upregulated LPS-induced IL-1β, IL-8, and TNF-α expression in flounder HKMs; conversely, overexpression of PoCD73 in flounder FG-9307 cells markedly attenuated LPS-induced TNF-α expression. Taken together, our study establishes the ACOD1/itaconate immunometabolic pathway as a novel regulator of CD73 expression and elucidates the immunomodulatory function of the ectonucleotidase CD73 in Japanese flounder inflammatory responses.