Study of Japanese flounder cytokine gene promoter functions

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Tumor necrosis factor (TNF) is known as a primary mediator of immune regulation and inflammatory response. However, little is known about TNF-mediated networks or signal cascades in fish. In this study, we characterized the regulation of gene expression of Japanese flounder TNF in cell lines and zebrafish embryo.A recombinant plasmid containing the 2.4 kb promoter region of Japanese flounder tumor necrosis factor (TNF) and green fluorescence protein (GFP) was introduced into YO-K cells derived from Japanese flounder kidney, HINAE cells derived Japanese flounder embryos and zebrafish fertilized. YO-K cells and HINAE cells were incubated with lipopolysaccharide (LPS) at 20℃ 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. In YO-K cells, expression level of GFP decreased gradually from 3 h to 6 h post stimulation, while a reverse trend was observed in HINAE cells. When the transgenic zebrafish embryos were exposed to LPS, GFP was expressed in the whole zebrafish embryonic surface and Western blot analysis also showed that the level of the expressed GFP protein in zebrafish embryo was higher at a LPS concentration of 140 μg/ml than at LPS concentrations of 0 and 70 μg/ml. These results indicate that transcription regulated by the Japanese flounder TNF promoter could be under the control of the LPS-recognition system in zebrafish embryos.