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

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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.

Introduction

Fish have the same defense mechanisms as mammals, in that they have both innate and adaptive immunity [1]. It is essential to understand these defense mechanisms for the protection of fish against infectious diseases. Especially, innate immunity is important as a first barrier of defense against disease [2]. Cytokines play key roles in the activation of innate immunity [3]. More importantly, tumor necrosis factor (TNF) is the first trigger molecule in the activation of the innate immune network system. However, little is known about TNF-mediated networks or signal cascades in fish. In understanding the innate immune response and inflammation of fish, it is important to study the TNF transcriptional regulatory mechanism.

The first TNF gene in fish was identified in Japanese flounder (Paralichthys olivaceus) [4] and like mammalian TNF-alpha [5], [6], was shown to be regulated by various inducers, such as lipopolysaccharide (LPS), phorbol myristate acetate (PMA), and concanavalin A (Con A). It has been suggested that Japanese flounder TNF, like the mammalian TNF, works as a trigger molecule in the innate immunity as a first protection against bacterial infection. TNF is synthesized by different cell types upon stimulation with either endotoxin inflammatory mediators [7], [8] or various cytokines [9] and TNF itself [10]. LPS is a potent stimulator of monocytes and macrophages, causing secretion of TNF and other inflammatory mediators [11], [12]. LPS has been shown to activate the mammalian TNF promoter via the DNA binding protein NF-κB [13], [14]. NF-κB is thought to have a role in the constitutive high-level baseline expression of the mammalian TNF gene [15], [16]. However, it was not clear whether LPS activated only the human TNF-alpha transcription following the NF-κB-dependent pathway, since NF-κB does not seem to participate in the LPS-induced activation of the human TNF-alpha promoter [17]. We hypothesized that this inducible expression is regulated by its promoter region. In this study, two kinds of Japanese flounder cultured cells, YO-K cells derived from Japanese flounder kidney and HINAE cells derived Japanese from flounder embryos [18] were used for reporter assays. Both YO-K cells and HINAE cells consisted of fibroblastic cells. In the present study, we investigated the function and LPS-inducibility of Japanese flounder acute phase promoter in fish cell lines and evaluated the feasibility of using green fluorescence protein (GFP) and chloramphenicol acetyl transferase (CAT) as reporter genes in Japanese flounder cell lines.

Section snippets

Construction of recombinant plasmid for Japanese flounder TNF promoter assay

The 5′ flanking region of the Japanese flounder TNF gene [4] containing 2351 bp (−2151 to +201 from the transcription initiation site) (Fig. 1), was amplified by PCR using a set of primers, sense-5′-CGAGATCTGGAGCGTGGCCCATATATGA-3′ and antisens-5′-CGCTCGAGCAGTCGGTGGACGACTTCTT-3′ from a Japanese flounder BAC clone [19]. The amplified fragment was cloned into pEGFP-1 (Clontech). To confirm the sequence and direction of the insert, the recombinant plasmid was sequenced using a Thermo Sequenase II

Construction of recombinant plasmid vector, pTNF-GFP

The 5′ flanking region of the Japanese flounder TNF gene containing 2351 bp was linked to a GFP gene. This recombinant plasmid vector that is expressed under the control of the Japanese flounder TNF gene promoter was named pTNF-GFP.

LPS-inducible expression of GFP in cell lines

The GFP gene was expressed in YO-K and HINAE cells in response to a 24 h LPS stimulation (Fig. 2). In YO-K cells, basal GFP expression was not detected in unstimulated cells. Upon stimulation, the amount of GFP expression was observed to be LPS

Discussion

The Japanese flounder TNF gene promoter up-regulated the expression of the reporter gene upon LPS stimulation in both YO-K and HINAE cells. GFP mRNA expression increased immediately at 1 h after LPS treatment in both cell lines. It decreased gradually thereafter only in YO-K cells but not in HINAE cells (Fig. 3). This inducible expression pattern was similar to the expression pattern of Japanese flounder TNF gene in vivo [4], suggesting that the 2351 bp 5′ flanking region of the Japanese

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