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Molecular cloning
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Short communication
Molecular cloning Libing Xua, Yuhong Chena, Qiuhua Lia,c, Tianliang Hea, Xinhua Chena,b,∗ a b c
Key Laboratory of Marine Biotechnology of Fujian Province, Institute of Oceanology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, China College of Ocean and Earth Sciences, Xiamen University, Xiamen, 361102, China
ARTICLE INFO
ABSTRACT
Keywords: c-Jun Large yellow croaker (Larimichthys crocea) Expression modulation Overexpression Th1/Th2 cell development
Transcription factor c-Jun is a member of AP-1 transcription complex that can be induced by various pathogens and plays a broad regulatory role in vertebrate immune response. In this study, the complete c-Jun cDNA of large yellow croaker Larimichthys crocea (Lcc-Jun) was cloned, whose open reading frame (ORF) is 984 bp long and encodes a protein of 327 amino acids (aa). The deduced Lcc-Jun protein contains three highly conserved domains, a transactivation domain (TAD, Met1-His118), a DNA binding domain (DBD, Lys218-Arg243), and a Leucine zipper domain (LZD, Leu271-Leu299), as found in other specie c-Jun. Lcc-Jun was constitutively expressed in all examined tissues, with the higher levels in blood, heart, and head kidney. Its transcripts were not only induced in spleen and head kidney by poly (I: C) or LPS, but also up-regulated in primary head kidney leukocytes (PKL), macrophages (PKM), and granulocytes (PKG), suggesting that Lcc-Jun may be involved in immune responses induced by poly (I: C), a viral mimic, and LPS, a Gram-negative bacterial component. Overexpression of Lcc-Jun in PKL increased the expression of cytokines and transcription factors involved in T helper 1 (Th1: TNF-α, IFN-γ, and T-bet) and Th2 (IL-4/13 A/B, IL-6, and GATA3) cell development and differentiation, suggesting that LccJun may play a role in regulation of Th1/Th2 cell response. These results therefore led us to suggest that the cJun-mediated signaling pathways may have an important immune-modulatory function in teleost fish.
1. Introduction Activator protein-1 (AP-1) complex is a transcription factor that controls a number of cellular processes including cell transformation, differentiation, proliferation, and apoptosis [1]. Its structure is a dimer composed of members of the Jun family (c-Jun, JunB, and JunD) and the Fos family (c-Fos, FosB, Fra-1, and Fra-2) [2,3]. The c-Jun, a central component of AP-1 complex, was composed of three extremely important domains, including a transactivation domain (TAD), a DNA binding domain (DBD), and a C-terminal basic region-Leucine zipper domain (LZD). Phosphorylation of TAD would enhance c-Jun transcription activation and increase its stability in some extent, while DBD is responsible for DNA binding of c-Jun in a sequence-specific manner. Additionally, LZD has been found to play a role in the dimerization of cJun and its subsequent binding to the promoter regions of target genes [4]. Therefore, all these three domains are necessary for c-Jun-induced transcriptional activation and c-Jun-induced transformation [3,5]. Mammalian c-Jun was constitutively expressed in most resting cells and was rapidly induced by diverse extracellular stimulators, such as UV irradiation, bacteria or virus infection, and LPS treatment [6–9]. Once c-Jun was phosphorylated, it would take part in several signaling ∗
pathways, such as extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and JAK-STAT [10–12]. As a member of AP-1 complex, c-Jun binds to the TPA (12-O-tetradecanoylphorbol-13acetate) response elements (TRE) in the promoters of target genes and induces the expression of these genes [3,5]. During T helper (Th) cell differentiation, c-Jun has been demonstrated to regulate the expression of Th-related cytokines (Th1: IFN-γ and TNF-α; Th2: IL-4 and IL-6) by JNK or ERK signaling pathway [10,13,14]. Furthermore, the activated c-Jun induces the expression of Th-related cytokine genes by binding to the TRE in their promoters [3,5,15]. These data showed that c-Jun plays a role in the control of Th1/Th2 cell differentiation. Although the functions of c-Jun in mammals are extensively investigated, few reports focus on c-Jun in lower vertebrates [16]. In teleost fish, c-Jun gene has been cloned in several fish species, such as silver carp Hypophthalmichthys molitrix, half-smooth tongue Cynoglossus semilaevis, orange-spotted grouper Epinephelus coioides, and redlip mullet Liza haematocheila [17–20]. Their tissue expression profile and expression modulation upon viral or bacterial infections were analyzed. The transcripts of orange-spotted grouper c-Jun were significantly increased by Singapore grouper iridovirus (SGIV) infection [19]. In halfsmooth tongue, the expression levels of c-Jun were obviously up-
Corresponding author. 15 Shangxiadian Road, Fuzhou, Fujian, 350002, China. E-mail address: [email protected] (X. Chen).
https://doi.org/10.1016/j.fsi.2019.10.064 Received 24 September 2019; Received in revised form 28 October 2019; Accepted 29 October 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Libing Xu, et al., Fish and Shellfish Immunology, https://doi.org/10.1016/j.fsi.2019.10.064
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Fig. 1. Multiple amino acid sequence alignment of c-Jun from different species. Multiple amino acid sequence alignment of Lcc-Jun with other known vertebrate c-Jun was generated using DNAMAN V6.0 and shaded using BOXSHADE (version 3.21). The same amino acids were labeled with a black-colored background (homology = 100%) and conserved sequences were labeled with a gray-colored background (homology > 80%). Four phosphorylation sites (Ser58, Ser68, Thr86, and Thr88) in TAD were indicated with “♦”; three phosphorylation sites (Thr231, Ser235, and Ser241) in DBD with “•”, and five leucine sites (Leu271, 278, 285, 292, 299) in LZD with “▼”. The NCBI GenBank accession numbers of c-Jun amino acid sequences used here are listed in Table 1.
regulated in spleen and head kidney after Vibrio angillarum infection [18]. The expression levels of c-Jun in redlip mullet were changed by Lactococcus garvieae infection, LPS and poly (I: C) treatments [20]. However, information on the immune functions of c-Jun in fish is still limited. In this study, we cloned and characterized the c-Jun (Lcc-Jun) gene in large yellow croaker Larimichthys crocea, an economically important marine fish in China [21,22]. Its expression in normal and immune-stimulated tissues or primary immune-related cells was investigated. Additionally, the expression of cytokines and transcription factors involved in Th1/Th2 cell development and differentiation was also analyzed in the Lcc-Jun-overexpressed PKL. Our results revealed that Lcc-Jun may be involved in immune responses induced by poly (I: C) or LPS, and may play a role in regulation of Th1/Th2 cell response.
and granulocytes (PKG) were prepared as reported previously [22,25]. Briefly, head kidney was aseptically collected from large yellow croaker and gently pushed through a 70-μm nylon mesh (BD, USA). The cell suspension was then washed with iced-cold L-15 medium containing 5% FBS, 10 IU/g heparin, 200 IU/mL penicillin (Gibco, USA), and 200 mg/mL streptomycin (Sigma, USA). To isolate the PKL, the cell suspension was layered on freshly prepared 34%/51% Percoll (GE, USA) density gradients and centrifuged at 650×g for 30 min at 4 °C. The PKL at gradient interface were collected and re-suspended with completed L-15 medium containing 10% FBS, 100 IU/mL penicillin (Gibco, USA), and 100 mg/mL streptomycin (Sigma, USA), and then seeded into a 6-well plate and cultured at 25 °C. To isolate the PKM, the PKL suspension was added to culture dish and incubated at 25 °C for 3 h, and then the attached PKM were cultured with completed L-15 medium at 25 °C. For PKG isolation, the single cell suspension from head kidney was layered over 51% Percoll and centrifuged at 650×g for 30 min at 4 °C. The pellets containing PKG were collected. After the erythrocytes in pellets were cracked by Red Cell Lysis Buffer (TIANGEN, China), the PKG in supernatants were washed twice with L-15 medium, and cultured at 25 °C. For Lcc-Jun induction, PKL/PKM/PKG were stimulated with poly (I: C) or LPS at a final concentration of 50 μg/mL. Then cells were harvested at 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h posttreatment for total RNA extraction. The control group was treated with an equal volume of L-15 medium.
2. Materials and methods 2.1. Experimental animals and sample collection Large yellow croakers (length: 22.3 ± 1.1 cm; weight: 105 ± 15.3 g) were obtained from a mariculture farm in Ningde City, Fujian Province, China. Fish were temporarily reared with a flowthrough seawater supply at 25 °C. After 5 days of acclimation, healthy fish were divided into three groups of 20 fish each for the challenge experiments. Two experimental groups were intraperitoneally injected with poly (I: C) or LPS at a dose of 0.2 mg/100 g fish. The control group was intraperitoneally injected with sterilized phosphate buffered saline (PBS, pH 7.4) at a dose of 0.2 mL/100 g fish. Head kidney and spleen tissues were collected from 5 fish in each group at different time points (3, 6, 12, and 24 h) post-injection, frozen immediately in liquid nitrogen, and stored at −80 °C for next use.
2.4. Expression analysis of Lcc-Jun by real-time PCR To determine the tissue expression profile of Lcc-Jun, total RNA was extracted from various tissues, including blood, heart, head kidney, muscle, gill, brain, stomach, intestine, liver, spleen, and skin, from 5 healthy fish, and reverse-transcribed into the first-strand cDNA (0.1 mg of total RNA). Real-time PCR was performed with the primer set of cJun-RT-F1 and -R1 (Supplementary Table 1). PCR cycles were performed on the QuantStudio5(Thermo Fisher Scientific, USA) with SYBR Premix Ex Taq™ kit (Takara, China). The cycling conditions were 5 min at 95 °C, followed by 40 cycles of 95 °C for 15 s, 56 °C for 20 s, and 72 °C for 20 s. The fluorescence output for each cycle was analyzed upon the completion of the entire run. The relative expression levels of Lcc-Jun gene were normalized by β-actin using the 2−ΔΔCT method and expressed as the ratio of the Lcc-Jun expression levels in the skin [26,27]. To understand the modulation of Lcc-Jun expression upon poly (I: C) or LPS, total RNA was extracted from head kidney and spleen tissues collected above. Real-time PCR was performed using the conditions described above to detect the expression levels of Lcc-Jun at each time point post-injection. The relative expression levels of Lcc-Jun gene were normalized by β-actin. Fold change of gene expression levels was expressed as the ratio of the normalized gene expression levels in fish injected with poly (I: C) or LPS versus those in fish injected with PBS (defined as 1) at the same time point. The data obtained from three independent PCR assays with three replicates in each assay were subjected to statistical analysis as previously described [28]. To analyze the Lcc-Jun expression in three immune-related cells, total RNA was extracted from 2.0 × 106 of PKL, PKM or PKG isolated above using the ReliaPrep RNA Cell Miniprep System (Promega, USA). Real-time PCR was performed as described above. The relative expression levels of Lcc-Jun gene were normalized by β-actin using the 2−ΔΔCT method and expressed as the ratio of the Lcc-Jun expression
2.2. Cloning and sequence analysis of Lcc-Jun gene The cDNA sequence of Lcc-Jun gene was predicted and assembled from the large yellow croaker genome data [23]. Reverse transcriptionPCR (RT-PCR) was then performed to amplify the open reading frame (ORF) of Lcc-Jun gene with the primer sets of c-Jun F/R (Supplementary Table 1). The resulting PCR products were then cloned into the pMD18-T simple vector (TaKaRa, China) and three clones were randomly selected for sequencing. The complete Lcc-Jun cDNA sequence was obtained by aligning the cDNA sequences of these three clones. Sequences similarity analysis was performed using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The potential N-glycosylation site was predicted by NetNGlyc 1.0 Server (http://www.cbs. dtu.dk/services/NetNGlyc/) and potential O-glycosylation site by NetOGlyc 4.0 Server (http://www.cbs.dtu.dk/services/NetOGlyc/). Sequence alignment was performed using DNAMAN version 6.0 and shaded using BOXSHADE (version 3.21; https://embnet.vital-it.ch/ software/BOX_form.html). Protein domains were predicted by Simple Modular Architecture Research Tool (SMART) (http://smart.emblheidelberg.de/). Phylogenetic tree was constructed with Molecular Evolution Genetics Analysis (MEGA) software version 7.0 using the neighbor-joining method [24]. 2.3. Preparation and culture of primary immune-related cells The primary head kidney leukocytes (PKL), macrophages (PKM), 3
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pcDNA3.1-Lcc-Jun or pcDNA3.1 (as control) by Lonza Amaxa® Cell Line Optimization Nucleofector® Kit (Cologne, Germany) according to the manufacturer's instruction. The PKL samples were collected at different time points (6, 12, 18, 24, and 36 h) after electroporation for next uses. For western-blotting experiments, the PKL samples were collected and lysed in RIPA lysis buffer (Sangon Biotech, China) with protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO). Then 20 μL of the prepared protein sample was separated by electrophoresis on 12% SDSPAGE and transferred to nitrocellulose membranes. Overexpression of Lcc-Jun in the pcDNA3.1-Lcc-Jun-transfected PKL was detected by western-blotting analysis with mouse anti-His6 monoclonal antibody as primary antibody (Thermo Fisher Scientific, USA) according to the standard method [29]. For the expression analysis of selective genes (Th1: IFN-γ, TNF-α, and T-bet; Th2: IL-4/13 A/B, IL-6, and GATA3), total RNA was extracted from PKL collected above, and reverse-transcribed into the firststrand cDNA. Real-time PCR was performed with gene-specific primer sets (Supplementary Table 1) as described above. The relative expression levels of these selective genes were calculated by normalization to β-actin using the 2−ΔΔCT method and expressed as the ratio of the normalized gene expression levels in PKLs transfected with pcDNA3.1Lcc-Jun versus those in PKL transfected with pcDNA3.1 (defined as 1) at the same time point. The data obtained from three independent PCR assays with three replicates in each assay were subjected to statistical analysis as above.
Table 1 Amino acid sequence comparison of c-Jun from large yellow croaker and other vertebrates. c-Jun homologue from other species
NCBI Genbank Accession No.
Lcc-Jun Identity
Collichthys lucidus c-Jun Epinephelus coioides c-Jun Lates calcarifer c-Jun Oreochromis niloticus c-Jun Oryzias latipes c-Jun Takifugu rubripes c-Jun Danio rerio c-Jun Hypophthalmichthys molitrix c-Jun Nile crocodile c-Jun Mus musculus c-Jun Homo sapiens c-Jun Xenopus laevis c-Jun Xiphophorus maculatus c-Jun
TKS74789.1 AHE93338.1 XP_018525847.1 XP_005478994.1 XP_004068223.1 XP_003974079.1 AAY21257.1 AHH92791.1 BAD98541.1 NP_034721.1 NP_002219.1 CAB51637.1 ABB89082.1
99.69% 95.98% 95.05% 93.19% 92.88% 92.26% 81.40% 79.88% 72.23% 69.64% 69.37% 68.69% 58.88%
2.6. Statistical analysis All data were analyzed using GraphPad Prism 6 software and expressed as the mean ± standard error of the mean ( ± SEM) of three repeated experiments. The data were subjected to analysis of one way ANOVA by using IBM SPSS Statistics 19. The p < 0.05 stands for statistically significant and p < 0.01 for significantly different. 3. Results 3.1. Characterization of c-Jun in large yellow croaker
Fig. 2. Expression analysis of Lcc-Jun in various tissues. Expression levels of Lcc-Jun were examined in 11 organs/tissues of healthy fish (N = 5) by real-Time PCR. The expression levels of Lcc-Jun were normalized by β-actin and expressed as the ratio to the gene expression levels in the skin. The tissues were ordered according to the relative expression levels from the highest to the lowest. The data were obtained from three independent PCR assays with three replicates in each assay. Deviation bars represent the standard errors of the mean ( ± SEM).
The complete ORF of Lcc-Jun gene (MN496155) is 984 nucleotides (nt) in length, encoding a protein of 327 amino acids (aa), with a theoretical molecular weight of 35.9 kDa. The deduced Lcc-Jun protein contained three conserved domains, transactivation domain (TAD, Met1-His118), DNA binding domain (DBD, Lys218-Arg243), and Leucine zipper domain (LZD, Leu271-Leu299). Five N-glycosylation sites (Asn32, 36, 97, 126, 183 ) and three O-glycosylation sites (Gly22, 28, and Pro26) were predicted in Lcc-Jun sequence (Supplementary Fig. 1). Four conserved amino acids in the TAD of Lcc-Jun, Ser58, Ser68, Thr86, and Thr88, corresponded to Ser63, Ser73, Thr91, and Thr93 in the TAD of human cJun (Fig. 1), which were reported to be major phosphorylation sites [30]. Thr231, Ser235, and Ser241 in the DBD of Lcc-Jun were also conserved when compared with other species c-Jun sequences. The LZD of Lcc-Jun contained five leucines (Leu271, 278, 285, 292, 299) separated by the intervals of six amino acid residues, which was similar with leucine arrangement found in the human c-Jun (residues 280 to 308) (Fig. 1). Homology analysis showed that Lcc-Jun shared the highest sequence identity of 99.69% with lucidheadtopsping croaker c-Jun, followed by orange-spotted grouper (95.98%) and silver sea perch c-Jun (95.05%), and the lowest identity with platyfish 58.88% (Table 1). Phylogenetic tree based on the deduced amino acid sequences of c-Jun showed that Lcc-Jun formed a major clade with other fish c-Jun sequences, and presented the closest relationship with lucidheadtopsping croaker c-Jun (Supplementary Fig. 2).
levels in the PKG. For Lcc-Jun expression modulation upon poly (I: C) or LPS stimulation, total RNA was extracted from 2.0 × 106 of PKL, PKM or PKG harvested at different stimulation time points using the ReliaPrep RNA Cell Miniprep System (Promega, USA). The relative expression levels of Lcc-Jun were calculated by normalization to β-actin using the 2−ΔΔCT method and expressed as the ratio of the normalized gene expression levels in PKL/PKM/PKG stimulated with poly (I: C) or LPS versus those in PKL/PKM/PKG treated with L-15 medium (defined as 1) at the same time point. The data obtained from three independent PCR assays with three replicates in each assay were subjected to statistical analysis as above. 2.5. Effects of Lcc-Jun overexpression on expression of selective genes in PKL The ORF of Lcc-Jun gene was amplified with primer set of Lcc-JunF1 and -R1 (Supplementary Table 1) and inserted into the EcoR I/Kpn I digested expression vector pcDNA 3.1(Invitrogen, USA). The recombinant plasmid pcDNA3.1-Lcc-Jun, with Lcc-Jun ORF and 6 × His tag in the same expression box, was obtained. Then the PKL isolated above (5.0 × 106 cells/well) were electroporated with 2 μg of purified
3.2. Expression analysis of Lcc-Jun Tissue expression profile analysis showed that the Lcc-Jun 4
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Fig. 3. Expression modulation of Lcc-Jun upon poly (I: C) or LPS induction. Lcc-Jun gene expression in head kidney and spleen during 24 h of stimulation with poly (I: C) or LPS was examined by real-time PCR. Total RNA was extracted from head kidney and spleen of large yellow croakers collected at 3, 6, 12, and 24 h post-stimulation, and used for real-time PCR detection. Expression levels of β-actin were set as internal control. Fold change of gene expression levels was expressed as the ratio of the normalized gene expression levels in fish injected with poly (I: C) or LPS versus those in fish injected with PBS (defined as 1) at the same time point. The data were obtained from three independent PCR assays with three replicates in each assay. Deviation bars represents the standard errors of the mean ( ± SEM) at each time point. *P < 0.05, **P < 0.01.
transcripts could be detected in all tissues tested, with the higher abundance in blood, heart, and head kidney, and the lowest abundance in the skin (Fig. 2). To further understand the modulation of Lcc-Jun expression in head kidney and spleen tissues, two main immune tissues in fish [31], upon poly (I: C) or LPS induction, its expression levels in these two tissues post-induction were analyzed by real-time PCR. As shown in Fig. 3A and C, Lcc-Jun was significantly up-regulated by poly
(I: C), with 1.7-, 2.1-fold increases at 3 and 6 h post-induction in head kidney, and 2.2- and 3.4-fold increases in spleen. Lcc-Jun expression was also induced by LPS in these two immune tissues, with 2.5- and 3.4fold increases at 3 and 6 h post-induction in head kidney, and 5.6- and 1.3-fold increases in spleen (Fig. 3B and D). Additionally, Lcc-Jun expression was also detected in three primary immune-related cells, PKL, PKM, and PKG, with a high transcript abundance in PKM (Fig. 4A), and
Fig. 4. Expression analysis of Lcc-Jun gene in PKL, PKM, and PKG. A: Expression levels of Lcc-Jun gene in PKL, PKM, and PKG were investigated. The relative expression levels of Lcc-Jun were normalized by β-actin and expressed as the ratio of the Lcc-Jun expression levels in the PKG. Expression levels of Lcc-Jun in PKL, PKM, and PKG were detected by real-time PCR at 1, 2, 4, 8, 16 and 24 h post stimulation (B, C, D, E, F, and G). The relative expression levels of Lcc-Jun were calculated by normalization to β-actin and expressed as the ratio of the normalized gene expression levels in PKL/PKM/PKG stimulated with poly (I: C) or LPS versus those in PKL/PKM/PKG treated with L-15 medium (defined as 1) at the same time point. The data were obtained from three independent PCR assays with three replicates in each assay. Deviation bars represents the standard errors of the mean ( ± SEM) at each time point. *P < 0.05, **P < 0.01. 5
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Fig. 5. Expression analysis of genes involved in Th1/Th2 cell differentiation and development in the pcDNA3.1-Lcc-Jun- transfected PKL. The expression changes of IFN-γ, TNF-α, IL 4/13 A/B, IL 6, T-bet, and GATA 3 in pcDNA3.1-Lcc-Jun-transfected PKL were analyzed by real-time PCR at 6, 12, 18, 24, and 36 h post-electroporation. The relative expression levels of these selective genes were calculated by normalization to β-actin and expressed as the ratio of the normalized gene expression levels in the PKLs transfected with pcDNA3.1-Lcc-Jun versus those in PKL transfected with pcDNA3.1 (defined as 1) at the same time point. The data were obtained from three independent PCR assays with three replicates in each assay. Deviation bars represents the standard errors of the mean ( ± SEM) at each time point. *P < 0.05, **P < 0.01.
was quickly up-regulated by both poly (I: C) or LPS (Fig. 4). These results suggested that Lcc-Jun may participate in both poly (I: C) - and LPS -induced immune responses.
with the higher expression levels in blood, heart, and head kidney, and the lowest in skin (Fig. 2). The similar results were also observed in silver carp, orange-spotted grouper, and redlip mullet, in which c-Jun was broadly expressed in various tissues tested and highly expressed in heart and head kidney [17,19,20]. In orange-spotted grouper, c-Jun expression was significantly up-regulated by SGIV infection [19]. In half-smooth tongue, increased expression of c-Jun was found upon challenge with Vibrio angillarum [18]. A similar expression pattern of cJun was also observed in redlip mullet, where the expression of redlip mullet c-Jun was induced by Lactococcus garvieae, LPS, and poly (I: C) [20]. Here, Lcc-Jun expression was significantly up-regulated in the immune-related tissues and cells by poly (I: C), a viral mimic, and LPS, a Gram-negative bacterial endotoxin (Figs. 3 and 4). These results therefore suggested that fish c-Jun may be involved in the immune responses against virus and bacteria. In mammals, c-Jun controlled Th1/Th2 cell differentiation and development by inducing the expression of Th1- and Th2- related cytokines or transcription factors [6,8,33]. In this study, Lcc-Jun overexpression not only induced the expression of Th1-and Th2-related cytokines (IFN-γ and TNF-α; IL-4/13 A/B and IL-6) in PKL, but also increased the expression of T-bet and GATA3 (Fig. 5), which are master transcription factors of Th1 and Th2, respectively [34], suggesting that teleost c-Jun may play a role in regulation of Th1 and Th2 responses. Mammalian c-Jun played a role in both anti-viral and anti-bacterial immune responses by JNK signaling pathway [35,36]. When Toll-like receptor 3 (TLR3) recognized the viral nucleic acid or poly (I: C), c-Jun was phosphorylated through the JNK pathway and induced the expression of downstream effectors [35]. In LPS-induced JNK pathway, LPS signal mediated by TLR4 caused the activation of c-Jun, thus increasing the production of inflammatory cytokines [36]. Meanwhile, cJun activation was found to promote the expression of IFN-γ and TNF-α in Th 1 cells by JNK pathway [12,14], and c-Jun up-regulated the expression of IL-4 and IL-6 through ERK and JNK signaling pathways, respectively [6,10]. Our results revealed that Lcc-Jun may be involved in immune responses induced by poly (I: C) or LPS, and may play a role in regulation of Th1/Th2 cell responses (Figs. 3, 4, and 5). These results therefore led us to suggest that the c-Jun-mediated signaling pathways
3.3. Expression analysis of genes involved in Th1/Th2 cell differentiation and development in Lcc-Jun- overexpressed PKL To know whether Lcc-Jun has an effect on the differentiation and development of Th1/Th2 cells, recombinant plasmid pcDNA3.1-Lcc-Jun was transfected into the isolated PKL by electroporation. Both real-time PCR and Western blotting analysis showed that Lcc-Jun was overexpressed in the PKL transfected with pcDNA3.1-Lcc-Jun (Supplementary Fig. 3). We further detected the expression levels of genes involved in Th1/Th2 cell differentiation and development in the Lcc-Jun-overexpressed PKL at different time points. The results showed that the overexpression of Lcc-Jun significantly up-regulated the expression levels of Th1-related cytokines IFN-γ and TNF-α and transcription factor T-bet and Th2-related cytokines IL-4/13A/B and IL-6 and transcription factor GATA3 (Fig. 5). 4. Discussion In the present study, we cloned the c-Jun (Lcc-Jun) gene from large yellow croaker. Lcc-Jun possesses three conserved domains, TAD (Met1His118), DBD (Lys218-Arg243), and LZD (Leu271-Leu299), as found in other species c-Jun (Fig. 1). Four amino acids Ser58, Ser68, Thr86, and Thr88 in the Lcc-Jun TAD, conserved in c-Jun sequences from various species, were considered to be major phosphorylation sites for c-Jun transactivation [30]. Three residues Thr231, Ser235, and Ser241 in the Lcc-Jun DBD were corresponding to Thr239, Ser243, and Ser249 in human c-Jun, whose phosphorylation contributed to the binding of c-Jun and DNA elements [32]. The leucine repeats (Leu271, 278, 285, 292, 299) in the Lcc-Jun LZD were also conserved in the known c-Jun sequences, and this domain was found to facilitate c-Jun dimerization [4]. Therefore, structural similarity between teleost and mammal c-Jun suggested their functional conservation. Lcc-Jun mRNA was constitutively expressed in all tissues examined, 6
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may have an important immune-modulatory function in teleost fish. However, the exact signaling pathways in which fish c-Jun is involved still need further investigations.
[16]
Notes
[17]
The authors declare no competing financial interest.
[18]
Acknowledgements The work was supported by grants from the National Key R&D Program of China (2018YFD0900503), National Natural Science Foundation of China (U1605211 and 31602194), China Agricultural Research System (CARS-47), and Marine Economic Development Subsidy Fund of Fujian Province (FJHJF-L-2019-2).
[19] [20]
[21]
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fsi.2019.10.064.
[22]
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Short communication
Molecular cloning Libing Xua, Yuhong Chena, Qiuhua Lia,c, Tianliang Hea, Xinhua Chena,b,∗ a b c
Key Laboratory of Marine Biotechnology of Fujian Province, Institute of Oceanology, Fujian Agriculture and Forestry University, Fuzhou, 350002, China Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, China College of Ocean and Earth Sciences, Xiamen University, Xiamen, 361102, China
ARTICLE INFO
ABSTRACT
Keywords: c-Jun Large yellow croaker (Larimichthys crocea) Expression modulation Overexpression Th1/Th2 cell development
Transcription factor c-Jun is a member of AP-1 transcription complex that can be induced by various pathogens and plays a broad regulatory role in vertebrate immune response. In this study, the complete c-Jun cDNA of large yellow croaker Larimichthys crocea (Lcc-Jun) was cloned, whose open reading frame (ORF) is 984 bp long and encodes a protein of 327 amino acids (aa). The deduced Lcc-Jun protein contains three highly conserved domains, a transactivation domain (TAD, Met1-His118), a DNA binding domain (DBD, Lys218-Arg243), and a Leucine zipper domain (LZD, Leu271-Leu299), as found in other specie c-Jun. Lcc-Jun was constitutively expressed in all examined tissues, with the higher levels in blood, heart, and head kidney. Its transcripts were not only induced in spleen and head kidney by poly (I: C) or LPS, but also up-regulated in primary head kidney leukocytes (PKL), macrophages (PKM), and granulocytes (PKG), suggesting that Lcc-Jun may be involved in immune responses induced by poly (I: C), a viral mimic, and LPS, a Gram-negative bacterial component. Overexpression of Lcc-Jun in PKL increased the expression of cytokines and transcription factors involved in T helper 1 (Th1: TNF-α, IFN-γ, and T-bet) and Th2 (IL-4/13 A/B, IL-6, and GATA3) cell development and differentiation, suggesting that LccJun may play a role in regulation of Th1/Th2 cell response. These results therefore led us to suggest that the cJun-mediated signaling pathways may have an important immune-modulatory function in teleost fish.
1. Introduction Activator protein-1 (AP-1) complex is a transcription factor that controls a number of cellular processes including cell transformation, differentiation, proliferation, and apoptosis [1]. Its structure is a dimer composed of members of the Jun family (c-Jun, JunB, and JunD) and the Fos family (c-Fos, FosB, Fra-1, and Fra-2) [2,3]. The c-Jun, a central component of AP-1 complex, was composed of three extremely important domains, including a transactivation domain (TAD), a DNA binding domain (DBD), and a C-terminal basic region-Leucine zipper domain (LZD). Phosphorylation of TAD would enhance c-Jun transcription activation and increase its stability in some extent, while DBD is responsible for DNA binding of c-Jun in a sequence-specific manner. Additionally, LZD has been found to play a role in the dimerization of cJun and its subsequent binding to the promoter regions of target genes [4]. Therefore, all these three domains are necessary for c-Jun-induced transcriptional activation and c-Jun-induced transformation [3,5]. Mammalian c-Jun was constitutively expressed in most resting cells and was rapidly induced by diverse extracellular stimulators, such as UV irradiation, bacteria or virus infection, and LPS treatment [6–9]. Once c-Jun was phosphorylated, it would take part in several signaling ∗
pathways, such as extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and JAK-STAT [10–12]. As a member of AP-1 complex, c-Jun binds to the TPA (12-O-tetradecanoylphorbol-13acetate) response elements (TRE) in the promoters of target genes and induces the expression of these genes [3,5]. During T helper (Th) cell differentiation, c-Jun has been demonstrated to regulate the expression of Th-related cytokines (Th1: IFN-γ and TNF-α; Th2: IL-4 and IL-6) by JNK or ERK signaling pathway [10,13,14]. Furthermore, the activated c-Jun induces the expression of Th-related cytokine genes by binding to the TRE in their promoters [3,5,15]. These data showed that c-Jun plays a role in the control of Th1/Th2 cell differentiation. Although the functions of c-Jun in mammals are extensively investigated, few reports focus on c-Jun in lower vertebrates [16]. In teleost fish, c-Jun gene has been cloned in several fish species, such as silver carp Hypophthalmichthys molitrix, half-smooth tongue Cynoglossus semilaevis, orange-spotted grouper Epinephelus coioides, and redlip mullet Liza haematocheila [17–20]. Their tissue expression profile and expression modulation upon viral or bacterial infections were analyzed. The transcripts of orange-spotted grouper c-Jun were significantly increased by Singapore grouper iridovirus (SGIV) infection [19]. In halfsmooth tongue, the expression levels of c-Jun were obviously up-
Corresponding author. 15 Shangxiadian Road, Fuzhou, Fujian, 350002, China. E-mail address: [email protected] (X. Chen).
https://doi.org/10.1016/j.fsi.2019.10.064 Received 24 September 2019; Received in revised form 28 October 2019; Accepted 29 October 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Libing Xu, et al., Fish and Shellfish Immunology, https://doi.org/10.1016/j.fsi.2019.10.064
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Fig. 1. Multiple amino acid sequence alignment of c-Jun from different species. Multiple amino acid sequence alignment of Lcc-Jun with other known vertebrate c-Jun was generated using DNAMAN V6.0 and shaded using BOXSHADE (version 3.21). The same amino acids were labeled with a black-colored background (homology = 100%) and conserved sequences were labeled with a gray-colored background (homology > 80%). Four phosphorylation sites (Ser58, Ser68, Thr86, and Thr88) in TAD were indicated with “♦”; three phosphorylation sites (Thr231, Ser235, and Ser241) in DBD with “•”, and five leucine sites (Leu271, 278, 285, 292, 299) in LZD with “▼”. The NCBI GenBank accession numbers of c-Jun amino acid sequences used here are listed in Table 1.
regulated in spleen and head kidney after Vibrio angillarum infection [18]. The expression levels of c-Jun in redlip mullet were changed by Lactococcus garvieae infection, LPS and poly (I: C) treatments [20]. However, information on the immune functions of c-Jun in fish is still limited. In this study, we cloned and characterized the c-Jun (Lcc-Jun) gene in large yellow croaker Larimichthys crocea, an economically important marine fish in China [21,22]. Its expression in normal and immune-stimulated tissues or primary immune-related cells was investigated. Additionally, the expression of cytokines and transcription factors involved in Th1/Th2 cell development and differentiation was also analyzed in the Lcc-Jun-overexpressed PKL. Our results revealed that Lcc-Jun may be involved in immune responses induced by poly (I: C) or LPS, and may play a role in regulation of Th1/Th2 cell response.
and granulocytes (PKG) were prepared as reported previously [22,25]. Briefly, head kidney was aseptically collected from large yellow croaker and gently pushed through a 70-μm nylon mesh (BD, USA). The cell suspension was then washed with iced-cold L-15 medium containing 5% FBS, 10 IU/g heparin, 200 IU/mL penicillin (Gibco, USA), and 200 mg/mL streptomycin (Sigma, USA). To isolate the PKL, the cell suspension was layered on freshly prepared 34%/51% Percoll (GE, USA) density gradients and centrifuged at 650×g for 30 min at 4 °C. The PKL at gradient interface were collected and re-suspended with completed L-15 medium containing 10% FBS, 100 IU/mL penicillin (Gibco, USA), and 100 mg/mL streptomycin (Sigma, USA), and then seeded into a 6-well plate and cultured at 25 °C. To isolate the PKM, the PKL suspension was added to culture dish and incubated at 25 °C for 3 h, and then the attached PKM were cultured with completed L-15 medium at 25 °C. For PKG isolation, the single cell suspension from head kidney was layered over 51% Percoll and centrifuged at 650×g for 30 min at 4 °C. The pellets containing PKG were collected. After the erythrocytes in pellets were cracked by Red Cell Lysis Buffer (TIANGEN, China), the PKG in supernatants were washed twice with L-15 medium, and cultured at 25 °C. For Lcc-Jun induction, PKL/PKM/PKG were stimulated with poly (I: C) or LPS at a final concentration of 50 μg/mL. Then cells were harvested at 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h posttreatment for total RNA extraction. The control group was treated with an equal volume of L-15 medium.
2. Materials and methods 2.1. Experimental animals and sample collection Large yellow croakers (length: 22.3 ± 1.1 cm; weight: 105 ± 15.3 g) were obtained from a mariculture farm in Ningde City, Fujian Province, China. Fish were temporarily reared with a flowthrough seawater supply at 25 °C. After 5 days of acclimation, healthy fish were divided into three groups of 20 fish each for the challenge experiments. Two experimental groups were intraperitoneally injected with poly (I: C) or LPS at a dose of 0.2 mg/100 g fish. The control group was intraperitoneally injected with sterilized phosphate buffered saline (PBS, pH 7.4) at a dose of 0.2 mL/100 g fish. Head kidney and spleen tissues were collected from 5 fish in each group at different time points (3, 6, 12, and 24 h) post-injection, frozen immediately in liquid nitrogen, and stored at −80 °C for next use.
2.4. Expression analysis of Lcc-Jun by real-time PCR To determine the tissue expression profile of Lcc-Jun, total RNA was extracted from various tissues, including blood, heart, head kidney, muscle, gill, brain, stomach, intestine, liver, spleen, and skin, from 5 healthy fish, and reverse-transcribed into the first-strand cDNA (0.1 mg of total RNA). Real-time PCR was performed with the primer set of cJun-RT-F1 and -R1 (Supplementary Table 1). PCR cycles were performed on the QuantStudio5(Thermo Fisher Scientific, USA) with SYBR Premix Ex Taq™ kit (Takara, China). The cycling conditions were 5 min at 95 °C, followed by 40 cycles of 95 °C for 15 s, 56 °C for 20 s, and 72 °C for 20 s. The fluorescence output for each cycle was analyzed upon the completion of the entire run. The relative expression levels of Lcc-Jun gene were normalized by β-actin using the 2−ΔΔCT method and expressed as the ratio of the Lcc-Jun expression levels in the skin [26,27]. To understand the modulation of Lcc-Jun expression upon poly (I: C) or LPS, total RNA was extracted from head kidney and spleen tissues collected above. Real-time PCR was performed using the conditions described above to detect the expression levels of Lcc-Jun at each time point post-injection. The relative expression levels of Lcc-Jun gene were normalized by β-actin. Fold change of gene expression levels was expressed as the ratio of the normalized gene expression levels in fish injected with poly (I: C) or LPS versus those in fish injected with PBS (defined as 1) at the same time point. The data obtained from three independent PCR assays with three replicates in each assay were subjected to statistical analysis as previously described [28]. To analyze the Lcc-Jun expression in three immune-related cells, total RNA was extracted from 2.0 × 106 of PKL, PKM or PKG isolated above using the ReliaPrep RNA Cell Miniprep System (Promega, USA). Real-time PCR was performed as described above. The relative expression levels of Lcc-Jun gene were normalized by β-actin using the 2−ΔΔCT method and expressed as the ratio of the Lcc-Jun expression
2.2. Cloning and sequence analysis of Lcc-Jun gene The cDNA sequence of Lcc-Jun gene was predicted and assembled from the large yellow croaker genome data [23]. Reverse transcriptionPCR (RT-PCR) was then performed to amplify the open reading frame (ORF) of Lcc-Jun gene with the primer sets of c-Jun F/R (Supplementary Table 1). The resulting PCR products were then cloned into the pMD18-T simple vector (TaKaRa, China) and three clones were randomly selected for sequencing. The complete Lcc-Jun cDNA sequence was obtained by aligning the cDNA sequences of these three clones. Sequences similarity analysis was performed using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The potential N-glycosylation site was predicted by NetNGlyc 1.0 Server (http://www.cbs. dtu.dk/services/NetNGlyc/) and potential O-glycosylation site by NetOGlyc 4.0 Server (http://www.cbs.dtu.dk/services/NetOGlyc/). Sequence alignment was performed using DNAMAN version 6.0 and shaded using BOXSHADE (version 3.21; https://embnet.vital-it.ch/ software/BOX_form.html). Protein domains were predicted by Simple Modular Architecture Research Tool (SMART) (http://smart.emblheidelberg.de/). Phylogenetic tree was constructed with Molecular Evolution Genetics Analysis (MEGA) software version 7.0 using the neighbor-joining method [24]. 2.3. Preparation and culture of primary immune-related cells The primary head kidney leukocytes (PKL), macrophages (PKM), 3
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pcDNA3.1-Lcc-Jun or pcDNA3.1 (as control) by Lonza Amaxa® Cell Line Optimization Nucleofector® Kit (Cologne, Germany) according to the manufacturer's instruction. The PKL samples were collected at different time points (6, 12, 18, 24, and 36 h) after electroporation for next uses. For western-blotting experiments, the PKL samples were collected and lysed in RIPA lysis buffer (Sangon Biotech, China) with protease inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO). Then 20 μL of the prepared protein sample was separated by electrophoresis on 12% SDSPAGE and transferred to nitrocellulose membranes. Overexpression of Lcc-Jun in the pcDNA3.1-Lcc-Jun-transfected PKL was detected by western-blotting analysis with mouse anti-His6 monoclonal antibody as primary antibody (Thermo Fisher Scientific, USA) according to the standard method [29]. For the expression analysis of selective genes (Th1: IFN-γ, TNF-α, and T-bet; Th2: IL-4/13 A/B, IL-6, and GATA3), total RNA was extracted from PKL collected above, and reverse-transcribed into the firststrand cDNA. Real-time PCR was performed with gene-specific primer sets (Supplementary Table 1) as described above. The relative expression levels of these selective genes were calculated by normalization to β-actin using the 2−ΔΔCT method and expressed as the ratio of the normalized gene expression levels in PKLs transfected with pcDNA3.1Lcc-Jun versus those in PKL transfected with pcDNA3.1 (defined as 1) at the same time point. The data obtained from three independent PCR assays with three replicates in each assay were subjected to statistical analysis as above.
Table 1 Amino acid sequence comparison of c-Jun from large yellow croaker and other vertebrates. c-Jun homologue from other species
NCBI Genbank Accession No.
Lcc-Jun Identity
Collichthys lucidus c-Jun Epinephelus coioides c-Jun Lates calcarifer c-Jun Oreochromis niloticus c-Jun Oryzias latipes c-Jun Takifugu rubripes c-Jun Danio rerio c-Jun Hypophthalmichthys molitrix c-Jun Nile crocodile c-Jun Mus musculus c-Jun Homo sapiens c-Jun Xenopus laevis c-Jun Xiphophorus maculatus c-Jun
TKS74789.1 AHE93338.1 XP_018525847.1 XP_005478994.1 XP_004068223.1 XP_003974079.1 AAY21257.1 AHH92791.1 BAD98541.1 NP_034721.1 NP_002219.1 CAB51637.1 ABB89082.1
99.69% 95.98% 95.05% 93.19% 92.88% 92.26% 81.40% 79.88% 72.23% 69.64% 69.37% 68.69% 58.88%
2.6. Statistical analysis All data were analyzed using GraphPad Prism 6 software and expressed as the mean ± standard error of the mean ( ± SEM) of three repeated experiments. The data were subjected to analysis of one way ANOVA by using IBM SPSS Statistics 19. The p < 0.05 stands for statistically significant and p < 0.01 for significantly different. 3. Results 3.1. Characterization of c-Jun in large yellow croaker
Fig. 2. Expression analysis of Lcc-Jun in various tissues. Expression levels of Lcc-Jun were examined in 11 organs/tissues of healthy fish (N = 5) by real-Time PCR. The expression levels of Lcc-Jun were normalized by β-actin and expressed as the ratio to the gene expression levels in the skin. The tissues were ordered according to the relative expression levels from the highest to the lowest. The data were obtained from three independent PCR assays with three replicates in each assay. Deviation bars represent the standard errors of the mean ( ± SEM).
The complete ORF of Lcc-Jun gene (MN496155) is 984 nucleotides (nt) in length, encoding a protein of 327 amino acids (aa), with a theoretical molecular weight of 35.9 kDa. The deduced Lcc-Jun protein contained three conserved domains, transactivation domain (TAD, Met1-His118), DNA binding domain (DBD, Lys218-Arg243), and Leucine zipper domain (LZD, Leu271-Leu299). Five N-glycosylation sites (Asn32, 36, 97, 126, 183 ) and three O-glycosylation sites (Gly22, 28, and Pro26) were predicted in Lcc-Jun sequence (Supplementary Fig. 1). Four conserved amino acids in the TAD of Lcc-Jun, Ser58, Ser68, Thr86, and Thr88, corresponded to Ser63, Ser73, Thr91, and Thr93 in the TAD of human cJun (Fig. 1), which were reported to be major phosphorylation sites [30]. Thr231, Ser235, and Ser241 in the DBD of Lcc-Jun were also conserved when compared with other species c-Jun sequences. The LZD of Lcc-Jun contained five leucines (Leu271, 278, 285, 292, 299) separated by the intervals of six amino acid residues, which was similar with leucine arrangement found in the human c-Jun (residues 280 to 308) (Fig. 1). Homology analysis showed that Lcc-Jun shared the highest sequence identity of 99.69% with lucidheadtopsping croaker c-Jun, followed by orange-spotted grouper (95.98%) and silver sea perch c-Jun (95.05%), and the lowest identity with platyfish 58.88% (Table 1). Phylogenetic tree based on the deduced amino acid sequences of c-Jun showed that Lcc-Jun formed a major clade with other fish c-Jun sequences, and presented the closest relationship with lucidheadtopsping croaker c-Jun (Supplementary Fig. 2).
levels in the PKG. For Lcc-Jun expression modulation upon poly (I: C) or LPS stimulation, total RNA was extracted from 2.0 × 106 of PKL, PKM or PKG harvested at different stimulation time points using the ReliaPrep RNA Cell Miniprep System (Promega, USA). The relative expression levels of Lcc-Jun were calculated by normalization to β-actin using the 2−ΔΔCT method and expressed as the ratio of the normalized gene expression levels in PKL/PKM/PKG stimulated with poly (I: C) or LPS versus those in PKL/PKM/PKG treated with L-15 medium (defined as 1) at the same time point. The data obtained from three independent PCR assays with three replicates in each assay were subjected to statistical analysis as above. 2.5. Effects of Lcc-Jun overexpression on expression of selective genes in PKL The ORF of Lcc-Jun gene was amplified with primer set of Lcc-JunF1 and -R1 (Supplementary Table 1) and inserted into the EcoR I/Kpn I digested expression vector pcDNA 3.1(Invitrogen, USA). The recombinant plasmid pcDNA3.1-Lcc-Jun, with Lcc-Jun ORF and 6 × His tag in the same expression box, was obtained. Then the PKL isolated above (5.0 × 106 cells/well) were electroporated with 2 μg of purified
3.2. Expression analysis of Lcc-Jun Tissue expression profile analysis showed that the Lcc-Jun 4
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Fig. 3. Expression modulation of Lcc-Jun upon poly (I: C) or LPS induction. Lcc-Jun gene expression in head kidney and spleen during 24 h of stimulation with poly (I: C) or LPS was examined by real-time PCR. Total RNA was extracted from head kidney and spleen of large yellow croakers collected at 3, 6, 12, and 24 h post-stimulation, and used for real-time PCR detection. Expression levels of β-actin were set as internal control. Fold change of gene expression levels was expressed as the ratio of the normalized gene expression levels in fish injected with poly (I: C) or LPS versus those in fish injected with PBS (defined as 1) at the same time point. The data were obtained from three independent PCR assays with three replicates in each assay. Deviation bars represents the standard errors of the mean ( ± SEM) at each time point. *P < 0.05, **P < 0.01.
transcripts could be detected in all tissues tested, with the higher abundance in blood, heart, and head kidney, and the lowest abundance in the skin (Fig. 2). To further understand the modulation of Lcc-Jun expression in head kidney and spleen tissues, two main immune tissues in fish [31], upon poly (I: C) or LPS induction, its expression levels in these two tissues post-induction were analyzed by real-time PCR. As shown in Fig. 3A and C, Lcc-Jun was significantly up-regulated by poly
(I: C), with 1.7-, 2.1-fold increases at 3 and 6 h post-induction in head kidney, and 2.2- and 3.4-fold increases in spleen. Lcc-Jun expression was also induced by LPS in these two immune tissues, with 2.5- and 3.4fold increases at 3 and 6 h post-induction in head kidney, and 5.6- and 1.3-fold increases in spleen (Fig. 3B and D). Additionally, Lcc-Jun expression was also detected in three primary immune-related cells, PKL, PKM, and PKG, with a high transcript abundance in PKM (Fig. 4A), and
Fig. 4. Expression analysis of Lcc-Jun gene in PKL, PKM, and PKG. A: Expression levels of Lcc-Jun gene in PKL, PKM, and PKG were investigated. The relative expression levels of Lcc-Jun were normalized by β-actin and expressed as the ratio of the Lcc-Jun expression levels in the PKG. Expression levels of Lcc-Jun in PKL, PKM, and PKG were detected by real-time PCR at 1, 2, 4, 8, 16 and 24 h post stimulation (B, C, D, E, F, and G). The relative expression levels of Lcc-Jun were calculated by normalization to β-actin and expressed as the ratio of the normalized gene expression levels in PKL/PKM/PKG stimulated with poly (I: C) or LPS versus those in PKL/PKM/PKG treated with L-15 medium (defined as 1) at the same time point. The data were obtained from three independent PCR assays with three replicates in each assay. Deviation bars represents the standard errors of the mean ( ± SEM) at each time point. *P < 0.05, **P < 0.01. 5
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Fig. 5. Expression analysis of genes involved in Th1/Th2 cell differentiation and development in the pcDNA3.1-Lcc-Jun- transfected PKL. The expression changes of IFN-γ, TNF-α, IL 4/13 A/B, IL 6, T-bet, and GATA 3 in pcDNA3.1-Lcc-Jun-transfected PKL were analyzed by real-time PCR at 6, 12, 18, 24, and 36 h post-electroporation. The relative expression levels of these selective genes were calculated by normalization to β-actin and expressed as the ratio of the normalized gene expression levels in the PKLs transfected with pcDNA3.1-Lcc-Jun versus those in PKL transfected with pcDNA3.1 (defined as 1) at the same time point. The data were obtained from three independent PCR assays with three replicates in each assay. Deviation bars represents the standard errors of the mean ( ± SEM) at each time point. *P < 0.05, **P < 0.01.
was quickly up-regulated by both poly (I: C) or LPS (Fig. 4). These results suggested that Lcc-Jun may participate in both poly (I: C) - and LPS -induced immune responses.
with the higher expression levels in blood, heart, and head kidney, and the lowest in skin (Fig. 2). The similar results were also observed in silver carp, orange-spotted grouper, and redlip mullet, in which c-Jun was broadly expressed in various tissues tested and highly expressed in heart and head kidney [17,19,20]. In orange-spotted grouper, c-Jun expression was significantly up-regulated by SGIV infection [19]. In half-smooth tongue, increased expression of c-Jun was found upon challenge with Vibrio angillarum [18]. A similar expression pattern of cJun was also observed in redlip mullet, where the expression of redlip mullet c-Jun was induced by Lactococcus garvieae, LPS, and poly (I: C) [20]. Here, Lcc-Jun expression was significantly up-regulated in the immune-related tissues and cells by poly (I: C), a viral mimic, and LPS, a Gram-negative bacterial endotoxin (Figs. 3 and 4). These results therefore suggested that fish c-Jun may be involved in the immune responses against virus and bacteria. In mammals, c-Jun controlled Th1/Th2 cell differentiation and development by inducing the expression of Th1- and Th2- related cytokines or transcription factors [6,8,33]. In this study, Lcc-Jun overexpression not only induced the expression of Th1-and Th2-related cytokines (IFN-γ and TNF-α; IL-4/13 A/B and IL-6) in PKL, but also increased the expression of T-bet and GATA3 (Fig. 5), which are master transcription factors of Th1 and Th2, respectively [34], suggesting that teleost c-Jun may play a role in regulation of Th1 and Th2 responses. Mammalian c-Jun played a role in both anti-viral and anti-bacterial immune responses by JNK signaling pathway [35,36]. When Toll-like receptor 3 (TLR3) recognized the viral nucleic acid or poly (I: C), c-Jun was phosphorylated through the JNK pathway and induced the expression of downstream effectors [35]. In LPS-induced JNK pathway, LPS signal mediated by TLR4 caused the activation of c-Jun, thus increasing the production of inflammatory cytokines [36]. Meanwhile, cJun activation was found to promote the expression of IFN-γ and TNF-α in Th 1 cells by JNK pathway [12,14], and c-Jun up-regulated the expression of IL-4 and IL-6 through ERK and JNK signaling pathways, respectively [6,10]. Our results revealed that Lcc-Jun may be involved in immune responses induced by poly (I: C) or LPS, and may play a role in regulation of Th1/Th2 cell responses (Figs. 3, 4, and 5). These results therefore led us to suggest that the c-Jun-mediated signaling pathways
3.3. Expression analysis of genes involved in Th1/Th2 cell differentiation and development in Lcc-Jun- overexpressed PKL To know whether Lcc-Jun has an effect on the differentiation and development of Th1/Th2 cells, recombinant plasmid pcDNA3.1-Lcc-Jun was transfected into the isolated PKL by electroporation. Both real-time PCR and Western blotting analysis showed that Lcc-Jun was overexpressed in the PKL transfected with pcDNA3.1-Lcc-Jun (Supplementary Fig. 3). We further detected the expression levels of genes involved in Th1/Th2 cell differentiation and development in the Lcc-Jun-overexpressed PKL at different time points. The results showed that the overexpression of Lcc-Jun significantly up-regulated the expression levels of Th1-related cytokines IFN-γ and TNF-α and transcription factor T-bet and Th2-related cytokines IL-4/13A/B and IL-6 and transcription factor GATA3 (Fig. 5). 4. Discussion In the present study, we cloned the c-Jun (Lcc-Jun) gene from large yellow croaker. Lcc-Jun possesses three conserved domains, TAD (Met1His118), DBD (Lys218-Arg243), and LZD (Leu271-Leu299), as found in other species c-Jun (Fig. 1). Four amino acids Ser58, Ser68, Thr86, and Thr88 in the Lcc-Jun TAD, conserved in c-Jun sequences from various species, were considered to be major phosphorylation sites for c-Jun transactivation [30]. Three residues Thr231, Ser235, and Ser241 in the Lcc-Jun DBD were corresponding to Thr239, Ser243, and Ser249 in human c-Jun, whose phosphorylation contributed to the binding of c-Jun and DNA elements [32]. The leucine repeats (Leu271, 278, 285, 292, 299) in the Lcc-Jun LZD were also conserved in the known c-Jun sequences, and this domain was found to facilitate c-Jun dimerization [4]. Therefore, structural similarity between teleost and mammal c-Jun suggested their functional conservation. Lcc-Jun mRNA was constitutively expressed in all tissues examined, 6
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may have an important immune-modulatory function in teleost fish. However, the exact signaling pathways in which fish c-Jun is involved still need further investigations.
[16]
Notes
[17]
The authors declare no competing financial interest.
[18]
Acknowledgements The work was supported by grants from the National Key R&D Program of China (2018YFD0900503), National Natural Science Foundation of China (U1605211 and 31602194), China Agricultural Research System (CARS-47), and Marine Economic Development Subsidy Fund of Fujian Province (FJHJF-L-2019-2).
[19] [20]
[21]
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fsi.2019.10.064.
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