Functional analysis of tumor necrosis factor gene promoter from Japanese flounder, Paralichthys olivaceus, using fish cell lines

Abstract

The expression vector pTNF-GFP containing the 2351 bp 5′ flanking region of Japanese flounder tumor necrosis factor (TNF) gene was linked with the green fluorescence protein (GFP) gene and was introduced into YO-K cells derived from Japanese flounder kidney and HINAE cells derived Japanese flounder embryos. YO-K cells and HINAE cells were incubated with three concentrations (250, 500, 1000 μg/ml) of lipopolysaccharide (LPS) at 20 °C for 24 h. The number of cells expressing GFP, as well as the amount of GFP protein was increased by LPS stimulation in both cell lines. GFP mRNA transcription was also induced by LPS stimulation in both YO-K cells and HINAE cells after 1 h stimulation. In YO-K cells, expression level of GFP decreased gradually from 3 to 6 h post-stimulation, while a reverse trend was observed in HINAE cells. A deletion assay of TNF gene promoter showed that the 5′ flanking region, −1783 to −1300 bp, containing cis-acting regulatory elements mediated LPS induction. An electrophoretic mobility shift assay using 2 fragments (−1783 to −1541 bp and −1540 to −1300 bp) revealed that only LPS-stimulated nuclear extracts bound to the −1540 to −1300 bp fragment. These results suggest that transcription of the TNF gene promoter in homologous cultured cells exhibited an inducible pattern and was regulated under the control of the immune system.