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Identification and molecular characterization of an Akirin2 homolog in Chinese loach (Paramisgurnus dabryanus)
Fish & Shellfish Immunology 36 (2014) 435e443
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Identification and molecular characterization of an Akirin2 homolog in Chinese loach (Paramisgurnus dabryanus) Xianli Xue a, Liwen Wang b, Yeyu Chen a, Xinshang Zhang a, Huiying Luo a, Zhongyuan Li a, Heng Zhao a, *, Bin Yao a, * a Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12, Zhongguancun South Street, Beijing 100081, PR China b National Animal Husbandry Extension Service, Beijing 100125, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 October 2013 Received in revised form 21 December 2013 Accepted 23 December 2013 Available online 3 January 2014
Akirin is a nuclear factor involved in innate immune responses of arthropods and mammals. In this study we have cloned an Akirin2 gene, pdakirin2, from freshwater Chinese loach (Paramisgurnus dabryanus) and characterized its biological functions. Phylogenetic analysis revealed deduced PdAkirin2 had high sequence identities to Akirin2 homologs from fish and mammals (7091%), it contained two conserved nuclear localization signals (NLSs) with verified sub-cellular localization. Quantitative real-time (qRT)PCR analysis indicated that PdAkirin2 was present in a wide range of loach tissues and showed upregulation with challenges of Aeromonas hydrophila NJ-1, LPS and poly I:C. PdAkirin2 as an immune factor had significant effects on the expression of cytokines (TNFa, IFN-a, IFN-g, IL-4 and IL-1b) and transcription factor NF-kB. This study provides insights into the potential role of PdAkirin2 in the innate immune system. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Chinese loach (Paramisgurnus dabryanus) Akirin2 Inducible expression Cytokines Transcription factor
1. Introduction The nuclear factor-k light chain enhancer of activated B cells (NF-kB) regulates genes related to immune responses, including innate immune cell activation, inflammation, dendritic cell maturation and lymphocyte activation [1]. Akirins, necessary for NF-kBdependent gene expression in drosophila and mice, are characterized with highly conserved N-/C-termini and one or two nuclear localization signals (NLSs) [2]. These proteins involve in the immune deficiency (Imd) pathway, leading to the synthesis of AMPs against the invasion of Gram-negative bacteria. Akirins have been attracting much attention for their biological significance, being given the name Subolesin in arthropods [3], Mighty in mice [4], FBI1 in rats [5] and Bhringi in Drosophila [6]. Based on the structures, functions and evolutionary relationships, Macqueen and Johnston proposed an eukaryotic gene family to cover both orthologs and paralogs of Akirin, in which Akirin1 (Mighty) is a critical promyogenic factor and Akirin2 is a conserved nuclear factor involved in innate immune responses [2,7]. It
* Corresponding authors. Tel.: þ86 10 82106065; fax: þ86 10 82106054. E-mail addresses: [email protected] (H. Zhao), [email protected], [email protected] (B. Yao). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.12.021
typically exists as one gene (akirin2) in birds and reptiles, two genes in mammals and amphibians (akirin1 and akirin2), and two or more genes in teleost and family Salmonidae [7,8]. These Akirin coorthologs are generated by a genomic duplication or tetraploidization event during teleost evolution [8,9]. Akirin has diverse nuclear functions related to the regulation of gene expression. For example, Akirin is involved in embryonic development as an important growth factor in arthropods, and plays an essential myogenic role in Drosophila melanogaster [2]. In Caenorhabditis elegans, Akirin is required for diakinesis bivalent structure and synaptonemal complex disassembly at meiotic prophase I [10]. Vertebrate Akirins involve in signal transduction in muscle regeneration, regulation of chemotaxis and carcinogenesis, etc [5,11]. In comparison with mammals and amphibians that have a relatively well-developed adaptive immune system, fish rely more greatly on their innate immune system. Considering the importance and insufficient information of Akirin2 in the innate immunity of fish, we performed the current work to identify the first akirin gene in Chinese loach (Paramisgurnus dabryanus), a freshwater fish widely spread in eastern Asia with commercially importance in both traditional Chinese medicine and food. The loach body is covered with a mucin-rich mucus layer, from which a polysaccharide (named MAP) with anti-proliferative and apoptotic
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effects has been isolated [11e13]. Several antimicrobial peptides including misgurin [14], hepcidin [15], and MAPP [16] have also been identified in the mud loach. Loach as an experimental organism has lots of merits, such as small size, high fecundity, yearround multiple spawning, and relatively well-established genetic techniques [15]. In this study, we cloned an akirin2 homolog from Chinese loach and evaluated its expression profile and relationship with cytokines. Our study provides insights into the potential immune functions of Akirin2 in Chinese loach. 2. Materials and methods 2.1. Fish, challenge strain, and cell line Adult Chinese loaches were obtained from a fish farm in Beijing, China, and were cultured in a rectangular tank with continuously circulated freshwater (27 C). Prior to the experiments, they were fed twice a day for two weeks and starved for three days in sterilized freshwater to clear the gut. The fish were anesthetized in a water bath containing 120 mg/l of tricaine methanesulfonate (MS222, SigmaeAldrich). The tissues of skin, brain, gill, kidney, liver, spleen, oocyte, sperm, heart, intestine and muscle were dissected from three individuals, immediately frozen in liquid nitrogen, followed by storage at 70 C or RNA isolation. Aeromonas hydrophila NJ-1 was isolated from Crucian carp (Carassius carassius) and donated by Professor Yongjie Liu (Nanjing Agricultural University, Nanjing, China). The strain was cultured in LuriaeBertani (LB) medium. Cells of strain NJ-1 were collected by centrifugation at 3000 g for 15 min after growth at 30 C for 12 h, washed three times in 0.9% saline/PBS (pH 7.4) and suspended in the same buffer. ZF4 cells were grown at 28 C in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS, 100 mg/ml of penicillin and 100 mg/ml of streptomycin in the presence of 5% CO2. All cells were reared in 25 cm2 tissue culture flasks (Nunc) for primary growth. 2.2. Total RNA extraction and cDNA synthesis Total RNAs were extracted from above-mentioned samples with the SV Total RNA Isolation System (Promega) according to the manufacturer’s protocols. DNase I from Promega was used to remove DNA. The purity of RNA was assessed by the ratios of the absorbance at 260 and 280 nm (A260/280) and at 260 and 230 nm (A260/230), and the integrity of 28S and 18S rRNA was examined by electrophoresis in 1.2% agarose gel. The RNA templates were reverse-transcribed to generate first strand cDNAs by Rever Tra Ace-a-Ô (TOYOBO). The resulting cDNAs were kept at 20 C before use.
obtained with the primers Supplementary Table S1).
Akirin2-F and
Akirin2-R
(see
2.4. Sequence analysis and phylogenetic tree construction The nucleotide and deduced amino acid sequences of pdakirin2 were analyzed with the BLASTx and BLASTp at the NCBI (http:// www.ncbi.nlm.gov/blast), respectively. The isoelectric point and molecular weight were predicted using the compute pI/Mw program (http://cn.expasy.org/tools/pi_tool.html). NLS was predicted by PSORT II (http://psort.ims.u-tokyo.ac.jp) [18]. Besides, the protein domains, families, and functional sites were predicted using PROSITE program (http://kr.expasy.org/prosite/). Multiple protein sequence alignments of PdAkirin2 with other vertebrate Akirin2 homologs were performed using ClustalW [17]. Phylogenetic analysis of PdAkirin2 and other homologs was performed with MEGA 5.05 with neighbor-joining (NJ) algorithm and 1000 bootstrap resampling iterations [18]. Data were analyzed by P-distance, and gaps were removed by Pairwise deletion [18e20]. 2.5. Sub-cellular localization of PdAkirin2 The expression plasmid pDsRed2-c1 was purchased from Invitrogen. Four gene fragments coding for mature Pdakirin2 and mature Pdakirin2 without the first NSL (pdakirin2-DNLS1), without the second NSL (pdakirin2-DNLS2) and without both NSLs (pdakirin2-DNLS1&2) were obtained using overlap PCR with specific primers (Supplementary Table S1), confirmed by sequencing, and individually subcloned into the BamHI-HindIII site of vector pDsRed2-c1 with T4 DNA ligase (Fermantas) to construct four recombined plasmids. Positive selection of recombinant plasmids based on sequencing was performed, and those coding for noncytotoxic proteins were extracted by Qiagen Plasmid Midi and Maxi Kits and quantified by using the ultraviolet spectrophotometric method. ZF4 cells were seeded in 12-well plates at 90e95% confluency (2 106 cells/well). Transfections were performed in triplicates. Recombinant plasmids and Lipofectamine 2000 (Invitrogen) were mixed at the ratio of 1:0.5 to 1:5 (w/v) in 250 ml of Opti-MEM reduced serum medium (Invitrogen), and incubated for 20 min at room temperature. The empty plasmid was used as the control. After 5-h incubation in DMEM/F12 medium, the cells were collected and grown in fresh DMEM/F12 medium containing 15% FBS, 100 mg/ml penicillin and 100 mg/ml streptomycin for 48 h. Transfected cells were stained with 1 mg/ml 2-(4-Amidinophenyl)6-indolecarbamidine dihydrochloride (DAPI) for 20 min. Then cells were observed under an Olympus fluorescence microscope. The pictures were merged by ImageJ2x program. 2.6. Expression of PdAkirin2
2.3. Cloning of the akirine2 gene (pdakirin2) Specific primers AK-F and AK-R (see Supplementary Table S1) were designed based on the conserved sequences (CG/AATLKR and FVKFTH/QDQ) of Akirin2 homologs from Salvelinus alpinus (ACV49698.1), Salmo salar (NP_001165956.1), Oncorhynchus mykiss (NP_001182094.1), Danio rerio (AAH65319.1), Oryzias latipes (XP_004083464.1 and ACG55699.1), Siniperca chuatsi (ACO88907.1), and Epinephelus coioides (AFA41485.1) and used for touchdown PCR. The PCR products were purified and cloned into pEASY-T3 vector (Transgen) for sequencing. The 50 and 30 flanking regions were amplified by thermal asymmetric interlaced (TAIL)PCR and rapid amplification of cDNA ends (RACE), respectively, with specific primers as shown in Supplementary Table S1, and assembled with the known sequence. The full-length Pdakirin2 was
Quantitative real-time (qRT)-PCR was used to examine the distribution of PdAkirin2 mRNAs in skin, muscle, gill, brain, kidney, liver, spleen, intestine, oocyte, heart and sperm following the MIQE guideline [21]. The expression levels of PdAkirin2 were determined with the IQÔ Multicolor Detection System (Bio-Rad) using SYBR Green Real-time PCR Master Mix (TOYOBO) and a pair of PdAkirin2specific primers (Q-AK-F and Q-AK-R, Supplementary Table S1). The eukaryotic elongation factor-1a coding gene (eef-1a) was chosen as the reference and used for subsequent normalization [22]. The cDNA samples were diluted serially to construct standard curves. The 25-ml reactions each contained 1 ml of diluted cDNA as the template, 12.5 ml of 2 SYBR Green Real time PCR Master Mix, 1 ml of each primer (0.4 mM), and 10.5 ml of PCR-sterile water. Equal volume of water instead of cDNA was prepared as a negative control. The
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qRT-PCR was performed as follows: 94 C for 30 s, 40 cycles of 94 C for 30 s, 60 C for 30 s, and 72 C for 30 s. To ensure the primer specificity, a melt curve analysis was performed at the end of each run from 55 C to 95 C with a ramp speed of 0.5 C. The data were analyzed using Bio-Rad iQ5 2.1 Standard Edition Optical System Software, and the yields of qRT-PCR were analyzed by electrophoresis in 2.0% agarose gels. The 2DDCT method was used to analyze the expression level of akirin2 [23]. All data were shown as mean S.D. (N ¼ 3). 2.7. Expression profile of PdAkirin2 in normal and challenged tissues Thirty-six Chinese loaches (about 15 g) were intraperitoneally injected with 1 ml of freshly prepared A. hydrophila NJ-1 (approximately 5 107 cfu/ml) as described above or 0.9% saline as controls. Gill, liver and kidney tissues of bacterium-injected fish were collected at time points of 6, 12 and 24 h post injection. The tissues
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of un-injected fish at time point of 0 h and saline-injected fish at time point of 12 h were used as controls. RNA extraction and determination of PdAkirin2 expression levels were performed as described above. Spleen cells were prepared following the method of Pärt et al. [24] for the assessment of defense responses of PdAkirin2. The cells at a concentration of 1 106 cells/ml were incubated in 6-well plates with 50 mg/ml of poly I:C, 25 mg/ml LPS, or 2 106 cfu/ml of A. hydrophila NJ-1. An equivalent volume of saline buffer was treated as controls. Spleen cells were collected at time points of 4 and 18 h post injection. Triplicate of each treatment was set. All cells were harvested for RNA isolation and cDNA synthesis, and qRT-PCR was performed as described above. 2.8. Effects of PdAkirin2 on NF-kB and cytokine expression To analyze the relationship of PdAkirin2 and cytokines, the fulllength Pdakirin2 was inserted to EcoRI-NotI site of pcDNA3.1(þ)
Fig. 1. Multiple sequence alignments of PdAkirin2 from Paramisgurnus dabryanus (KC688278) with other Akirin2 homologes from Homo sapiens (BAD96421.1), Mus musculus (NP_001007590.2), Danio rerio (AAH65319.1), Salmo Salar (NP_001165956.1) and Scophthalmus maximus (ADK27484). The two nuclear location signal sequences (NLSs) were boxed by gray box and underlined, respectively. The consensus sequence was indicated by asterisks, and the similar sequence was indicated by dots.
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Fig. 2. Phylogenetic tree of PdAkirin2 and its homologs from invertebrates and vertebrates. The tree was established via Neighbor-joining (NJ) method in Mega 5.05 version. The scale bar corresponds to 0.05 estimated amino acid substitutions per site.
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Fig. 3. Sub-cellular localization of PdAkirin2 and its mutants without the first/second NLSs in ZF4 cells. (A) Schematic diagram of four recombinant plasmids harboring both RFP-coding gene and gene fragments coding for pDRA, pDRA-DNLS1, pDRA-DNLS2 and pDRA-DNLS1&2. (B) ZF4 cells transfected with four reconstructed vectors. DAPI staining indicated the nuclei of cells.
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vector, which was further transfected into ZF4 cells. The empty pcDNA3.1(þ) was used as control. The CMV promoter was used for Pdakirin2 overexpression in ZF4 cells. After 72-h incubation, cells were collected for RNA extraction, cDNA synthesis and qPCR analysis as described above. The relative expression levels of NF-kB and cytokines like tumor necrosis factor (TNFa), interleukin (IL-1b and IL-4), and interferon (IFN-a and IFN-g) were examined in ZF4 cells by qPCR, with the Rpl13a gene as reference [25]. All the primers were shown in Supplementary Table S1, and the data were analyzed by using the 2DDCT method. 2.9. Statistical analysis All data were analyzed with One-way analysis of variance (ANOVA) after the logarithmic, reciprocal and square-root transformation of 2DDCT values. The differences among groups were identified by ANOVA of SPSS 16.0 with DUNCAN’S test. Statistical significance was set at p < 0.05 or p < 0.01.
four recombinant plasmids harboring the RFP-coding gene and gene fragments coding for pDRA, pDRA-DNLS1 (removal of PKRRRC), pDRA-DNLS2 (removal of KRRH) and pDRA-DNLS1&2 (removal of PKRRRC and KRRH) were constructed and transfected into ZF4 cells to test the localization of PdAkirin2 (Fig. 3). ZF4 cells transfected with the recombinant full-length of pdakirin2 gene exhibited a positive nuclear signal, while cells transfected with single NLS-truncated mutants displayed a diffuse red fluorescence in the cytoplasm and nucleus, and those with double NLSs truncation displayed a diffuse red fluorescence in the cytoplasm only. 3.4. Expression profile of PdAkirin2 in normal tissues qRT-PCR was conducted to identify the expression profile of PdAkirin2 in adult Chinese loach. As shown in Fig. 4, Pdakirin2 was detected in all tested tissues, with higher level of expression in kidney, sperm and muscle, moderate in intestine, skin, brain, heart, oocyte and liver, and low in gill and spleen.
3. Results 3.5. Expression profile of PdAkirin2 in challenged tissues 3.1. Gene cloning and sequence analysis of PdAkirin2 Using the primers AK-F and AK-R specific for Akirin2, a gene fragment of 504 bp was cloned from the cDNA of P. dabryanus muscle. The flanking regions were then obtained by 50 -TAIL-PCR and 30 -RACE techniques, and assembled with the known sequence to give the 1475-bp Pdakirin2 (KC688278). The open reading frame (ORF) contains 555 bp, encoding a protein of 184 amino acids. The 50 and 30 terminal untranslated regions (UTR) were 310 and 610 bp in length, respectively. The theoretical molecular mass and pI value of mature PdAkirin2 were 20.85 kDa and 9.01, respectively. Two NLSs were identified at residues 2227 (PKRRRC) and 7578 (KRRH) of deduce PdAkirin2, respectively (Fig. 1). BLASTp analysis showed that PdAkirin2 was highly conserved at the terminal regions (residues 130 and 130183), which were separated by less conserved residues. The amino acid sequence alignments (Fig. 1) demonstrated that PdAkirin2 was similar to Akirin2 from mammals and fish, with 91% identity to D. rerio (AAH65319.1), 86% to S. salar (NP_001165956.1), 85% to Scophthalmus maximus (ADK27484.1), 72% to Mus musculus (NP_001007590.2) and 71% to Homo sapiens (BAD96421.1). 3.2. Phylogenetic analysis of PdAkirin2 The evolutionary relationship of PdAkirin2 with other Akirins was examined by a phylogenetic tree using deduced amino acid sequences (Fig. 2). PdAkirin2 represented a true ortholog of fish Akirin paralogs, and clustered with teleost Akirin2 sequences from D. rerio (AAH65319.1). The tree was consistent with the species phylogeny, and was divided into two major clustersdvertebrate and invertebrate. Vertebrate Akirins were split into two subgroups, i.e. Akirin2 and Akirin1. Compared with subgroups that contained only fish or mammal homologs, members of subgroup Akirin2 were much diverse, consisting of homologs from fish, mammals and chicken. Furthermore, Akirin1 and 2 were split into four subgroups with Akirin1(1a), Akirin1(2a), Akirin1(1b), Akirin1(2b) and Akirin2(1a), Akirin2(2a), Akirin2(1b), Akirin2(2b), separately, in S. salar, S. alpinus and O. mykiss. Invertebrate both subolesin of arachnida and Bhringi of insect in arthropod were other aliases of Akirin.
Gill is the first line of immune defense in Chinese loach, and kidney and liver are the main immune organs [26]. These three tissues were selected for PdAkirin2 expression profile analysis by using qRT-PCR. When loach were challenged with A. hydrophila NJ1, PdAkirin2 transcripts were up-regulated in all tested tissues, showing the highest level at 6 h in kidney (10.8 fold; Fig. 5(A)) and at 12 h in liver (3.5 fold; Fig. 5(B)) and gill (11.7 fold; Fig. 5(C)) (p < 0.01). The time course of PdAkirin2 expression in the primary spleen cells upon challenges of LPS, Poly I:C and A. hydrophila NJ-1 was also studied. Post stimulation of strain NJ-1, LPS and poly I:C, the expression of PdAkirin2 was up-regulated at 4 h (2.2e2.4 fold) and 18 h (4.0e9.5 fold) (Fig. 6), respectively. The impact of LPS was more notable than that of poly I:C but less than that of A. hydrophila. 3.6. Expression of cytokines and NF-kB post PdAkirin2 overexpression The expression profiles of NF-kB and six cytokines (TNFa, IL-1b, IL-4, IFN-a and IFN-g) were examined in ZF4 cells upon the overexpression of PdAkirin2. Except for IFN-a and IFN-g that retained the control level, NF-kB and all other cytokines showed up-regulation with PdAkirin2 overexpression, 2.1 fold for NF-kB, 3.8 fold for TNFa, 2.6 fold for IL-1b and 2.2 fold for IL-4 (Fig. 7).
3.3. Sub-cellular localization of PdAkirin2 Sequence analysis showed that PdAkirin2 was a typical Akirin with two NLSs at the N-terminus (PKRRRC and KRRH). Therefore,
Fig. 4. qRT-PCR analysis of the expression profile of PdAkirin2 in different tissues of Paramisgurnus dabryanus. The eef-1a gene was used as an internal control. Vertical bars represented the mean S.D (n ¼ 3).
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Fig. 6. qRT-PCR analysis of the time course of PdAkirin2 expression in primary spleen cells with challenges of LPS, poly(I:C) and A. hydrophila (NJ-1), respectively. PBStreated cells were used as control. All data relative to eef-1a were shown as mean S.D (n ¼ 3). Significant differences were indicated by one (*) and two asterisks (**) at p < 0.05 and p < 0.01, respectively.
Fig. 5. qRT-PCR analysis of the time course of PdAkirin2 expression in kidney (A), liver (B) and gill (C) with A. hydrophila (NJ-1) challenge, and primary spleen cells, respectively. All data relative to eef-1a are shown as mean S.D (n ¼ 3). Non-treated (0 h) and saline-treated fish (12 h) were used as controls. Significant differences were indicated by one (*) and two asterisks (**) at p < 0.05 and p < 0.01, respectively.
4. Discussion Akirin has recently been found as one of most essential components of host defense. In the present study, we have described the molecular characterization of an Akirin2 gene, Pdakirin2, in
Chinese loach. The possible biological functions of PdAkirin2 were studied in loach tissues with and without challenges of A. hydrophila, which was a Gram-negative bacterium and chosen as the causative agent in this study. Upon its challenge, the mRNA levels of PdAkirin2 were significantly up-regulated in kidney and gill at 6 h and in liver at 12 h. In comparison with gill and kidney, liver showed a 6-h postponed expression of PdAkirin2. The expression level of PdAkirin2 in gill retained high even at 24 h, which might be associated with immune regulation and control. These results were similar to the data in Drosophila and in S. maximus that Akirin was up-regulated post bacterial challenge [2,28]. The mRNA levels of PdAkirin2 in primary spleen cells were also increased with the challenges of LPS, poly I:C and A. hydrophila, the similar results were reported in spleen cells of rock bream (Oplegnathus fasciatus) [27]. By contrast, the induced expression of Akirin1 was not found in S. maximus treated with poly I:C [28]. Multiple sequence alignment indicated that two highly conserved termini of deduced PdAkirin2 were separated by the middle sequences. Removal of both NLSs resulted in the diffuse distribution of PdAkirin2 in the cytoplasm of transfected ZF4 cells, and the intact PdAkirin2 was strictly localized to nuclei. These results confirmed that the motifs PKRRRC and KRRH of PdAkirin2 play an important role in the nuclear localization of Chinese loach as previously reported in insect and mammals [4]. Interestingly, mammals and invertebrate deuterostomes also have two NLSs, but they are localized in the front and middle regions of Akirins [8]. Both functional and genetic evidence demonstrate that fishes have a network of signaling molecules, cytokines and chemokines, which can control and coordinate the innate and acquired immune responses [26]. Recombinant trout tumor necrosis factors (TNFa) protein could enhance leucocyte migration and phagocytic activity of trout macrophages, and increase the expression of proinflammatory cytokines such as IL-6, IL-1b as well. Besides, as mediators of inflammatory responses, IL-1b had a significant role to play in phagocytosis, lymphocyte proliferation and superoxide production. IL-4 were associated with elimination of extracellular parasites and with allergic responses [29]. NF-kB was involved in cytokines production as a transcription factor in its immunological function [30]. Akirin2 had been verified to be a new NF-kB regulator in mice [2]. The relationship between PdAkirin2 with cytokines and NF-kB was studied in ZF4 cells, NF-kB and some cytokines (TNFa, IL1b, and IL-4) were up-regulated, while IFN-a and IFN-g were not. Similar results had been reported in mouse and porcine that overexpression of Akirin2 led to up-regulation of IL-6 [2,31]. It
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Fig. 7. Effect of PdAkirin2 on the expression of NF-kB, TNFa, IL-1b, IL-4, IFN-a and IFN-g. All data relative to eef-1a were shown as mean S.D (n ¼ 3). Significant differences were indicated by one and two asterisks (**) at p < 0.05 and p < 0.01, respectively.
suggests that PdAkirin2 has an important role in both physiological immunity and pathological inflammation. However, no study has demonstrated a direct interaction of Akirin and NF-kB, suggesting that Akirins may interact with some inter-mediary components. 143-3 proteins were potential candidates since they were identified to regulate the nuclear localization of transcriptional factors [8], and might play a role in the transcriptional regulation of promoter targets by forming a complex with Akirin2 [5]. In this paper, we have cloned Pdakirin2 from Chinese loach P. dabryanus. It was expressed in a wide range of loach tissues and was up-regulated in response to challenges of A. hydrophila NJ-1, LPS and poly I:C. PdAkirin2 overexpression in vitro had significant effects on the expression of cytokines and NF-KB. These data indicated that PdAkirin2 might be an important effective component involved in the innate immunity of P. dabryanus. Acknowledgments This work was supported by the National High-Tech Research and Development Program (863 Program, 2013AA102803), the National Science and Technology Support Program (2011BADB02), the National Science Fund for Distinguished Young Scholars (31225026), and the National “948” Project (2011-G7-4). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2013.12.021. References [1] Oeckinghaus A, Ghosh S. The NF-kB family of transcription factors and its regulation. Cold Spring Harb Perspect Biol 2009:1. [2] Goto A, Matsushita K, Gesellchen V, Chamy LE, Kuttenkeuler D, Takeuchi O, et al. Akirins are highly conserved nuclear proteins required for NF-[kappa]Bdependent gene expression in drosophila and mice. Nat Immunol 2008;9:97e 104. [3] Galindo RC, Doncel-Pérez E, Zivkovic Z, Naranjo V, Gortazar C, Mangold AJ, et al. Tick subolesin is an ortholog of the akirins described in insects and vertebrates. Dev Comp Immunol 2009;33:612e7. [4] Marshall A, Salerno MS, Thomas M, Davies T, Berry C, Dyer K, et al. Mighty is a novel promyogenic factor in skeletal myogenesis. Exp Cell Res 2008;314: 1013e29. [5] Komiya Y, Kurabe N, Katagiri K, Ogawa M, Sugiyama A, Kawasaki Y, et al. A novel binding factor of 14-3-3b functions as a transcriptional repressor and promotes anchorage-independent growth, tumorigenicity, and metastasis. J Biol Chem 2008;283:18753e64. [6] Nowak SJ, Aihara H, Gonzalez K, Nibu Y, Baylies MK. Akirin links twistregulated transcription with the brahma chromatin remodeling complex during embryogenesis. PLoS Genet 2012;8:e1002547.
[7] Macqueen DJ, Bower NI, Johnston IA. Positioning the expanded akirin gene family of Atlantic salmon within the transcriptional networks of myogenesis. Biochem Biophys Res Commun 2010;400:599e605. [8] Macqueen D, Johnston I. Evolution of the multifaceted eukaryotic akirin gene family. BMC Evol Biol 2009;9:34. [9] Macqueen DJ, Kristjánsson BK, Johnston IA. Salmonid genomes have a remarkably expanded akirin family, coexpressed with genes from conserved pathways governing skeletal muscle growth and catabolism. Physiol Genomics 2010;42:134e48. [10] Clemons AM, Brockway HM, Yin Y, Kasinathan B, Butterfield YS, Jones SJM, et al. Akirin is required for diakinesis bivalent structure and synaptonemal complex disassembly at meiotic prophase I. Mol Biol Cell 2013;24:1053e67. [11] Qin C, Ding Y, Huang K, Xu H. Protective effect of Misgurnus anguillicaudatus polysaccharide on immunological liver injury in mice. Int Immunopharmacol 2008;8:607e12. [12] Zhang C-X, Huang K-X. Apoptosis induction on HL-60 cells of a novel polysaccharide from the mucus of the loach, Misgurnus anguillicaudatus. J Ethnopharmacol 2005;99:385e90. [13] Zhang C-X, Huang K-X. Mechanism of apoptosis induced by a polysaccharide, from the loach Misgurnus anguillicaudatus (MAP) in human hepatocellular carcinoma cells. Toxicol Appl Pharmacol 2006;210:236e45. [14] Park CB, Lee JH, Park IY, Kim MS, Kim SC. A novel antimicrobial peptide from the loach, Misgurnus anguillicaudatus. FEBS Lett 1997;411:173e8. [15] Nam YK, Cho YS, Lee SY, Kim BS, Kim DS. Molecular characterization of hepcidin gene from mud loach (Misgurnus mizolepis; Cypriniformes). Fish Shellfish Immunol 2011;31:1251e8. [16] Dong XZ, Xu HB, Huang KX, Liou Q, Zhou J. The preparation and characterization of an antimicrobial polypeptide from the loach, Misgurnus anguillicaudatus. Protein Expr Purif 2002;26:235e42. [17] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673e80. [18] Hall BG. Building phylogenetic trees from molecular data with MEGA. Mol Biol Evol 2013;30:1229e35. [19] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406e25. [20] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596e9. [21] Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative realtime pcr experiments. Clin Chem 2009;55:611e22. [22] Zhang Y, Wang H, Qin F, Liu S, Wu T, Li M, et al. Molecular characterization of estrogen receptor genes in loach Paramisgurnus dabryanus and their expression upon 17a-ethinylestradiol exposure in juveniles. Gen Comp Endocrinol 2012;178:194e205. [23] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protocols 2008;3:1101e8. [24] Part P, Norrgren L, Bergstrom E, Sjoberg P. Primary cultures of epithelial cells from rainbow trout gills. J Exp Biol 1993;175:219e32. [25] Zhang X, Zhao H, Chen Y, Liu C, Meng K, Yang P, et al. Characterization and biological function analysis of the trim3a gene from zebrafish (Danio rerio). Fish Shellfish Immunol 2012;32:621e8. [26] Magnadóttir B. Innate immunity of fish (overview). Fish Shellfish Immunol 2006;20:137e51. [27] Kasthuri SR, Umasuthan N, Whang I, Wan Q, Lim B-S, Jung H-B, et al. Akirin2 homologues from rock bream, Oplegnathus fasciatus: genomic and molecular characterization and transcriptional expression analysis. Fish Shellfish Immunol 2013;35:740e7.
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Identification and molecular characterization of an Akirin2 homolog in Chinese loach (Paramisgurnus dabryanus) Xianli Xue a, Liwen Wang b, Yeyu Chen a, Xinshang Zhang a, Huiying Luo a, Zhongyuan Li a, Heng Zhao a, *, Bin Yao a, * a Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12, Zhongguancun South Street, Beijing 100081, PR China b National Animal Husbandry Extension Service, Beijing 100125, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 October 2013 Received in revised form 21 December 2013 Accepted 23 December 2013 Available online 3 January 2014
Akirin is a nuclear factor involved in innate immune responses of arthropods and mammals. In this study we have cloned an Akirin2 gene, pdakirin2, from freshwater Chinese loach (Paramisgurnus dabryanus) and characterized its biological functions. Phylogenetic analysis revealed deduced PdAkirin2 had high sequence identities to Akirin2 homologs from fish and mammals (7091%), it contained two conserved nuclear localization signals (NLSs) with verified sub-cellular localization. Quantitative real-time (qRT)PCR analysis indicated that PdAkirin2 was present in a wide range of loach tissues and showed upregulation with challenges of Aeromonas hydrophila NJ-1, LPS and poly I:C. PdAkirin2 as an immune factor had significant effects on the expression of cytokines (TNFa, IFN-a, IFN-g, IL-4 and IL-1b) and transcription factor NF-kB. This study provides insights into the potential role of PdAkirin2 in the innate immune system. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Chinese loach (Paramisgurnus dabryanus) Akirin2 Inducible expression Cytokines Transcription factor
1. Introduction The nuclear factor-k light chain enhancer of activated B cells (NF-kB) regulates genes related to immune responses, including innate immune cell activation, inflammation, dendritic cell maturation and lymphocyte activation [1]. Akirins, necessary for NF-kBdependent gene expression in drosophila and mice, are characterized with highly conserved N-/C-termini and one or two nuclear localization signals (NLSs) [2]. These proteins involve in the immune deficiency (Imd) pathway, leading to the synthesis of AMPs against the invasion of Gram-negative bacteria. Akirins have been attracting much attention for their biological significance, being given the name Subolesin in arthropods [3], Mighty in mice [4], FBI1 in rats [5] and Bhringi in Drosophila [6]. Based on the structures, functions and evolutionary relationships, Macqueen and Johnston proposed an eukaryotic gene family to cover both orthologs and paralogs of Akirin, in which Akirin1 (Mighty) is a critical promyogenic factor and Akirin2 is a conserved nuclear factor involved in innate immune responses [2,7]. It
* Corresponding authors. Tel.: þ86 10 82106065; fax: þ86 10 82106054. E-mail addresses: [email protected] (H. Zhao), [email protected], [email protected] (B. Yao). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.12.021
typically exists as one gene (akirin2) in birds and reptiles, two genes in mammals and amphibians (akirin1 and akirin2), and two or more genes in teleost and family Salmonidae [7,8]. These Akirin coorthologs are generated by a genomic duplication or tetraploidization event during teleost evolution [8,9]. Akirin has diverse nuclear functions related to the regulation of gene expression. For example, Akirin is involved in embryonic development as an important growth factor in arthropods, and plays an essential myogenic role in Drosophila melanogaster [2]. In Caenorhabditis elegans, Akirin is required for diakinesis bivalent structure and synaptonemal complex disassembly at meiotic prophase I [10]. Vertebrate Akirins involve in signal transduction in muscle regeneration, regulation of chemotaxis and carcinogenesis, etc [5,11]. In comparison with mammals and amphibians that have a relatively well-developed adaptive immune system, fish rely more greatly on their innate immune system. Considering the importance and insufficient information of Akirin2 in the innate immunity of fish, we performed the current work to identify the first akirin gene in Chinese loach (Paramisgurnus dabryanus), a freshwater fish widely spread in eastern Asia with commercially importance in both traditional Chinese medicine and food. The loach body is covered with a mucin-rich mucus layer, from which a polysaccharide (named MAP) with anti-proliferative and apoptotic
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effects has been isolated [11e13]. Several antimicrobial peptides including misgurin [14], hepcidin [15], and MAPP [16] have also been identified in the mud loach. Loach as an experimental organism has lots of merits, such as small size, high fecundity, yearround multiple spawning, and relatively well-established genetic techniques [15]. In this study, we cloned an akirin2 homolog from Chinese loach and evaluated its expression profile and relationship with cytokines. Our study provides insights into the potential immune functions of Akirin2 in Chinese loach. 2. Materials and methods 2.1. Fish, challenge strain, and cell line Adult Chinese loaches were obtained from a fish farm in Beijing, China, and were cultured in a rectangular tank with continuously circulated freshwater (27 C). Prior to the experiments, they were fed twice a day for two weeks and starved for three days in sterilized freshwater to clear the gut. The fish were anesthetized in a water bath containing 120 mg/l of tricaine methanesulfonate (MS222, SigmaeAldrich). The tissues of skin, brain, gill, kidney, liver, spleen, oocyte, sperm, heart, intestine and muscle were dissected from three individuals, immediately frozen in liquid nitrogen, followed by storage at 70 C or RNA isolation. Aeromonas hydrophila NJ-1 was isolated from Crucian carp (Carassius carassius) and donated by Professor Yongjie Liu (Nanjing Agricultural University, Nanjing, China). The strain was cultured in LuriaeBertani (LB) medium. Cells of strain NJ-1 were collected by centrifugation at 3000 g for 15 min after growth at 30 C for 12 h, washed three times in 0.9% saline/PBS (pH 7.4) and suspended in the same buffer. ZF4 cells were grown at 28 C in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS, 100 mg/ml of penicillin and 100 mg/ml of streptomycin in the presence of 5% CO2. All cells were reared in 25 cm2 tissue culture flasks (Nunc) for primary growth. 2.2. Total RNA extraction and cDNA synthesis Total RNAs were extracted from above-mentioned samples with the SV Total RNA Isolation System (Promega) according to the manufacturer’s protocols. DNase I from Promega was used to remove DNA. The purity of RNA was assessed by the ratios of the absorbance at 260 and 280 nm (A260/280) and at 260 and 230 nm (A260/230), and the integrity of 28S and 18S rRNA was examined by electrophoresis in 1.2% agarose gel. The RNA templates were reverse-transcribed to generate first strand cDNAs by Rever Tra Ace-a-Ô (TOYOBO). The resulting cDNAs were kept at 20 C before use.
obtained with the primers Supplementary Table S1).
Akirin2-F and
Akirin2-R
(see
2.4. Sequence analysis and phylogenetic tree construction The nucleotide and deduced amino acid sequences of pdakirin2 were analyzed with the BLASTx and BLASTp at the NCBI (http:// www.ncbi.nlm.gov/blast), respectively. The isoelectric point and molecular weight were predicted using the compute pI/Mw program (http://cn.expasy.org/tools/pi_tool.html). NLS was predicted by PSORT II (http://psort.ims.u-tokyo.ac.jp) [18]. Besides, the protein domains, families, and functional sites were predicted using PROSITE program (http://kr.expasy.org/prosite/). Multiple protein sequence alignments of PdAkirin2 with other vertebrate Akirin2 homologs were performed using ClustalW [17]. Phylogenetic analysis of PdAkirin2 and other homologs was performed with MEGA 5.05 with neighbor-joining (NJ) algorithm and 1000 bootstrap resampling iterations [18]. Data were analyzed by P-distance, and gaps were removed by Pairwise deletion [18e20]. 2.5. Sub-cellular localization of PdAkirin2 The expression plasmid pDsRed2-c1 was purchased from Invitrogen. Four gene fragments coding for mature Pdakirin2 and mature Pdakirin2 without the first NSL (pdakirin2-DNLS1), without the second NSL (pdakirin2-DNLS2) and without both NSLs (pdakirin2-DNLS1&2) were obtained using overlap PCR with specific primers (Supplementary Table S1), confirmed by sequencing, and individually subcloned into the BamHI-HindIII site of vector pDsRed2-c1 with T4 DNA ligase (Fermantas) to construct four recombined plasmids. Positive selection of recombinant plasmids based on sequencing was performed, and those coding for noncytotoxic proteins were extracted by Qiagen Plasmid Midi and Maxi Kits and quantified by using the ultraviolet spectrophotometric method. ZF4 cells were seeded in 12-well plates at 90e95% confluency (2 106 cells/well). Transfections were performed in triplicates. Recombinant plasmids and Lipofectamine 2000 (Invitrogen) were mixed at the ratio of 1:0.5 to 1:5 (w/v) in 250 ml of Opti-MEM reduced serum medium (Invitrogen), and incubated for 20 min at room temperature. The empty plasmid was used as the control. After 5-h incubation in DMEM/F12 medium, the cells were collected and grown in fresh DMEM/F12 medium containing 15% FBS, 100 mg/ml penicillin and 100 mg/ml streptomycin for 48 h. Transfected cells were stained with 1 mg/ml 2-(4-Amidinophenyl)6-indolecarbamidine dihydrochloride (DAPI) for 20 min. Then cells were observed under an Olympus fluorescence microscope. The pictures were merged by ImageJ2x program. 2.6. Expression of PdAkirin2
2.3. Cloning of the akirine2 gene (pdakirin2) Specific primers AK-F and AK-R (see Supplementary Table S1) were designed based on the conserved sequences (CG/AATLKR and FVKFTH/QDQ) of Akirin2 homologs from Salvelinus alpinus (ACV49698.1), Salmo salar (NP_001165956.1), Oncorhynchus mykiss (NP_001182094.1), Danio rerio (AAH65319.1), Oryzias latipes (XP_004083464.1 and ACG55699.1), Siniperca chuatsi (ACO88907.1), and Epinephelus coioides (AFA41485.1) and used for touchdown PCR. The PCR products were purified and cloned into pEASY-T3 vector (Transgen) for sequencing. The 50 and 30 flanking regions were amplified by thermal asymmetric interlaced (TAIL)PCR and rapid amplification of cDNA ends (RACE), respectively, with specific primers as shown in Supplementary Table S1, and assembled with the known sequence. The full-length Pdakirin2 was
Quantitative real-time (qRT)-PCR was used to examine the distribution of PdAkirin2 mRNAs in skin, muscle, gill, brain, kidney, liver, spleen, intestine, oocyte, heart and sperm following the MIQE guideline [21]. The expression levels of PdAkirin2 were determined with the IQÔ Multicolor Detection System (Bio-Rad) using SYBR Green Real-time PCR Master Mix (TOYOBO) and a pair of PdAkirin2specific primers (Q-AK-F and Q-AK-R, Supplementary Table S1). The eukaryotic elongation factor-1a coding gene (eef-1a) was chosen as the reference and used for subsequent normalization [22]. The cDNA samples were diluted serially to construct standard curves. The 25-ml reactions each contained 1 ml of diluted cDNA as the template, 12.5 ml of 2 SYBR Green Real time PCR Master Mix, 1 ml of each primer (0.4 mM), and 10.5 ml of PCR-sterile water. Equal volume of water instead of cDNA was prepared as a negative control. The
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qRT-PCR was performed as follows: 94 C for 30 s, 40 cycles of 94 C for 30 s, 60 C for 30 s, and 72 C for 30 s. To ensure the primer specificity, a melt curve analysis was performed at the end of each run from 55 C to 95 C with a ramp speed of 0.5 C. The data were analyzed using Bio-Rad iQ5 2.1 Standard Edition Optical System Software, and the yields of qRT-PCR were analyzed by electrophoresis in 2.0% agarose gels. The 2DDCT method was used to analyze the expression level of akirin2 [23]. All data were shown as mean S.D. (N ¼ 3). 2.7. Expression profile of PdAkirin2 in normal and challenged tissues Thirty-six Chinese loaches (about 15 g) were intraperitoneally injected with 1 ml of freshly prepared A. hydrophila NJ-1 (approximately 5 107 cfu/ml) as described above or 0.9% saline as controls. Gill, liver and kidney tissues of bacterium-injected fish were collected at time points of 6, 12 and 24 h post injection. The tissues
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of un-injected fish at time point of 0 h and saline-injected fish at time point of 12 h were used as controls. RNA extraction and determination of PdAkirin2 expression levels were performed as described above. Spleen cells were prepared following the method of Pärt et al. [24] for the assessment of defense responses of PdAkirin2. The cells at a concentration of 1 106 cells/ml were incubated in 6-well plates with 50 mg/ml of poly I:C, 25 mg/ml LPS, or 2 106 cfu/ml of A. hydrophila NJ-1. An equivalent volume of saline buffer was treated as controls. Spleen cells were collected at time points of 4 and 18 h post injection. Triplicate of each treatment was set. All cells were harvested for RNA isolation and cDNA synthesis, and qRT-PCR was performed as described above. 2.8. Effects of PdAkirin2 on NF-kB and cytokine expression To analyze the relationship of PdAkirin2 and cytokines, the fulllength Pdakirin2 was inserted to EcoRI-NotI site of pcDNA3.1(þ)
Fig. 1. Multiple sequence alignments of PdAkirin2 from Paramisgurnus dabryanus (KC688278) with other Akirin2 homologes from Homo sapiens (BAD96421.1), Mus musculus (NP_001007590.2), Danio rerio (AAH65319.1), Salmo Salar (NP_001165956.1) and Scophthalmus maximus (ADK27484). The two nuclear location signal sequences (NLSs) were boxed by gray box and underlined, respectively. The consensus sequence was indicated by asterisks, and the similar sequence was indicated by dots.
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Fig. 2. Phylogenetic tree of PdAkirin2 and its homologs from invertebrates and vertebrates. The tree was established via Neighbor-joining (NJ) method in Mega 5.05 version. The scale bar corresponds to 0.05 estimated amino acid substitutions per site.
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Fig. 3. Sub-cellular localization of PdAkirin2 and its mutants without the first/second NLSs in ZF4 cells. (A) Schematic diagram of four recombinant plasmids harboring both RFP-coding gene and gene fragments coding for pDRA, pDRA-DNLS1, pDRA-DNLS2 and pDRA-DNLS1&2. (B) ZF4 cells transfected with four reconstructed vectors. DAPI staining indicated the nuclei of cells.
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vector, which was further transfected into ZF4 cells. The empty pcDNA3.1(þ) was used as control. The CMV promoter was used for Pdakirin2 overexpression in ZF4 cells. After 72-h incubation, cells were collected for RNA extraction, cDNA synthesis and qPCR analysis as described above. The relative expression levels of NF-kB and cytokines like tumor necrosis factor (TNFa), interleukin (IL-1b and IL-4), and interferon (IFN-a and IFN-g) were examined in ZF4 cells by qPCR, with the Rpl13a gene as reference [25]. All the primers were shown in Supplementary Table S1, and the data were analyzed by using the 2DDCT method. 2.9. Statistical analysis All data were analyzed with One-way analysis of variance (ANOVA) after the logarithmic, reciprocal and square-root transformation of 2DDCT values. The differences among groups were identified by ANOVA of SPSS 16.0 with DUNCAN’S test. Statistical significance was set at p < 0.05 or p < 0.01.
four recombinant plasmids harboring the RFP-coding gene and gene fragments coding for pDRA, pDRA-DNLS1 (removal of PKRRRC), pDRA-DNLS2 (removal of KRRH) and pDRA-DNLS1&2 (removal of PKRRRC and KRRH) were constructed and transfected into ZF4 cells to test the localization of PdAkirin2 (Fig. 3). ZF4 cells transfected with the recombinant full-length of pdakirin2 gene exhibited a positive nuclear signal, while cells transfected with single NLS-truncated mutants displayed a diffuse red fluorescence in the cytoplasm and nucleus, and those with double NLSs truncation displayed a diffuse red fluorescence in the cytoplasm only. 3.4. Expression profile of PdAkirin2 in normal tissues qRT-PCR was conducted to identify the expression profile of PdAkirin2 in adult Chinese loach. As shown in Fig. 4, Pdakirin2 was detected in all tested tissues, with higher level of expression in kidney, sperm and muscle, moderate in intestine, skin, brain, heart, oocyte and liver, and low in gill and spleen.
3. Results 3.5. Expression profile of PdAkirin2 in challenged tissues 3.1. Gene cloning and sequence analysis of PdAkirin2 Using the primers AK-F and AK-R specific for Akirin2, a gene fragment of 504 bp was cloned from the cDNA of P. dabryanus muscle. The flanking regions were then obtained by 50 -TAIL-PCR and 30 -RACE techniques, and assembled with the known sequence to give the 1475-bp Pdakirin2 (KC688278). The open reading frame (ORF) contains 555 bp, encoding a protein of 184 amino acids. The 50 and 30 terminal untranslated regions (UTR) were 310 and 610 bp in length, respectively. The theoretical molecular mass and pI value of mature PdAkirin2 were 20.85 kDa and 9.01, respectively. Two NLSs were identified at residues 2227 (PKRRRC) and 7578 (KRRH) of deduce PdAkirin2, respectively (Fig. 1). BLASTp analysis showed that PdAkirin2 was highly conserved at the terminal regions (residues 130 and 130183), which were separated by less conserved residues. The amino acid sequence alignments (Fig. 1) demonstrated that PdAkirin2 was similar to Akirin2 from mammals and fish, with 91% identity to D. rerio (AAH65319.1), 86% to S. salar (NP_001165956.1), 85% to Scophthalmus maximus (ADK27484.1), 72% to Mus musculus (NP_001007590.2) and 71% to Homo sapiens (BAD96421.1). 3.2. Phylogenetic analysis of PdAkirin2 The evolutionary relationship of PdAkirin2 with other Akirins was examined by a phylogenetic tree using deduced amino acid sequences (Fig. 2). PdAkirin2 represented a true ortholog of fish Akirin paralogs, and clustered with teleost Akirin2 sequences from D. rerio (AAH65319.1). The tree was consistent with the species phylogeny, and was divided into two major clustersdvertebrate and invertebrate. Vertebrate Akirins were split into two subgroups, i.e. Akirin2 and Akirin1. Compared with subgroups that contained only fish or mammal homologs, members of subgroup Akirin2 were much diverse, consisting of homologs from fish, mammals and chicken. Furthermore, Akirin1 and 2 were split into four subgroups with Akirin1(1a), Akirin1(2a), Akirin1(1b), Akirin1(2b) and Akirin2(1a), Akirin2(2a), Akirin2(1b), Akirin2(2b), separately, in S. salar, S. alpinus and O. mykiss. Invertebrate both subolesin of arachnida and Bhringi of insect in arthropod were other aliases of Akirin.
Gill is the first line of immune defense in Chinese loach, and kidney and liver are the main immune organs [26]. These three tissues were selected for PdAkirin2 expression profile analysis by using qRT-PCR. When loach were challenged with A. hydrophila NJ1, PdAkirin2 transcripts were up-regulated in all tested tissues, showing the highest level at 6 h in kidney (10.8 fold; Fig. 5(A)) and at 12 h in liver (3.5 fold; Fig. 5(B)) and gill (11.7 fold; Fig. 5(C)) (p < 0.01). The time course of PdAkirin2 expression in the primary spleen cells upon challenges of LPS, Poly I:C and A. hydrophila NJ-1 was also studied. Post stimulation of strain NJ-1, LPS and poly I:C, the expression of PdAkirin2 was up-regulated at 4 h (2.2e2.4 fold) and 18 h (4.0e9.5 fold) (Fig. 6), respectively. The impact of LPS was more notable than that of poly I:C but less than that of A. hydrophila. 3.6. Expression of cytokines and NF-kB post PdAkirin2 overexpression The expression profiles of NF-kB and six cytokines (TNFa, IL-1b, IL-4, IFN-a and IFN-g) were examined in ZF4 cells upon the overexpression of PdAkirin2. Except for IFN-a and IFN-g that retained the control level, NF-kB and all other cytokines showed up-regulation with PdAkirin2 overexpression, 2.1 fold for NF-kB, 3.8 fold for TNFa, 2.6 fold for IL-1b and 2.2 fold for IL-4 (Fig. 7).
3.3. Sub-cellular localization of PdAkirin2 Sequence analysis showed that PdAkirin2 was a typical Akirin with two NLSs at the N-terminus (PKRRRC and KRRH). Therefore,
Fig. 4. qRT-PCR analysis of the expression profile of PdAkirin2 in different tissues of Paramisgurnus dabryanus. The eef-1a gene was used as an internal control. Vertical bars represented the mean S.D (n ¼ 3).
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Fig. 6. qRT-PCR analysis of the time course of PdAkirin2 expression in primary spleen cells with challenges of LPS, poly(I:C) and A. hydrophila (NJ-1), respectively. PBStreated cells were used as control. All data relative to eef-1a were shown as mean S.D (n ¼ 3). Significant differences were indicated by one (*) and two asterisks (**) at p < 0.05 and p < 0.01, respectively.
Fig. 5. qRT-PCR analysis of the time course of PdAkirin2 expression in kidney (A), liver (B) and gill (C) with A. hydrophila (NJ-1) challenge, and primary spleen cells, respectively. All data relative to eef-1a are shown as mean S.D (n ¼ 3). Non-treated (0 h) and saline-treated fish (12 h) were used as controls. Significant differences were indicated by one (*) and two asterisks (**) at p < 0.05 and p < 0.01, respectively.
4. Discussion Akirin has recently been found as one of most essential components of host defense. In the present study, we have described the molecular characterization of an Akirin2 gene, Pdakirin2, in
Chinese loach. The possible biological functions of PdAkirin2 were studied in loach tissues with and without challenges of A. hydrophila, which was a Gram-negative bacterium and chosen as the causative agent in this study. Upon its challenge, the mRNA levels of PdAkirin2 were significantly up-regulated in kidney and gill at 6 h and in liver at 12 h. In comparison with gill and kidney, liver showed a 6-h postponed expression of PdAkirin2. The expression level of PdAkirin2 in gill retained high even at 24 h, which might be associated with immune regulation and control. These results were similar to the data in Drosophila and in S. maximus that Akirin was up-regulated post bacterial challenge [2,28]. The mRNA levels of PdAkirin2 in primary spleen cells were also increased with the challenges of LPS, poly I:C and A. hydrophila, the similar results were reported in spleen cells of rock bream (Oplegnathus fasciatus) [27]. By contrast, the induced expression of Akirin1 was not found in S. maximus treated with poly I:C [28]. Multiple sequence alignment indicated that two highly conserved termini of deduced PdAkirin2 were separated by the middle sequences. Removal of both NLSs resulted in the diffuse distribution of PdAkirin2 in the cytoplasm of transfected ZF4 cells, and the intact PdAkirin2 was strictly localized to nuclei. These results confirmed that the motifs PKRRRC and KRRH of PdAkirin2 play an important role in the nuclear localization of Chinese loach as previously reported in insect and mammals [4]. Interestingly, mammals and invertebrate deuterostomes also have two NLSs, but they are localized in the front and middle regions of Akirins [8]. Both functional and genetic evidence demonstrate that fishes have a network of signaling molecules, cytokines and chemokines, which can control and coordinate the innate and acquired immune responses [26]. Recombinant trout tumor necrosis factors (TNFa) protein could enhance leucocyte migration and phagocytic activity of trout macrophages, and increase the expression of proinflammatory cytokines such as IL-6, IL-1b as well. Besides, as mediators of inflammatory responses, IL-1b had a significant role to play in phagocytosis, lymphocyte proliferation and superoxide production. IL-4 were associated with elimination of extracellular parasites and with allergic responses [29]. NF-kB was involved in cytokines production as a transcription factor in its immunological function [30]. Akirin2 had been verified to be a new NF-kB regulator in mice [2]. The relationship between PdAkirin2 with cytokines and NF-kB was studied in ZF4 cells, NF-kB and some cytokines (TNFa, IL1b, and IL-4) were up-regulated, while IFN-a and IFN-g were not. Similar results had been reported in mouse and porcine that overexpression of Akirin2 led to up-regulation of IL-6 [2,31]. It
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Fig. 7. Effect of PdAkirin2 on the expression of NF-kB, TNFa, IL-1b, IL-4, IFN-a and IFN-g. All data relative to eef-1a were shown as mean S.D (n ¼ 3). Significant differences were indicated by one and two asterisks (**) at p < 0.05 and p < 0.01, respectively.
suggests that PdAkirin2 has an important role in both physiological immunity and pathological inflammation. However, no study has demonstrated a direct interaction of Akirin and NF-kB, suggesting that Akirins may interact with some inter-mediary components. 143-3 proteins were potential candidates since they were identified to regulate the nuclear localization of transcriptional factors [8], and might play a role in the transcriptional regulation of promoter targets by forming a complex with Akirin2 [5]. In this paper, we have cloned Pdakirin2 from Chinese loach P. dabryanus. It was expressed in a wide range of loach tissues and was up-regulated in response to challenges of A. hydrophila NJ-1, LPS and poly I:C. PdAkirin2 overexpression in vitro had significant effects on the expression of cytokines and NF-KB. These data indicated that PdAkirin2 might be an important effective component involved in the innate immunity of P. dabryanus. Acknowledgments This work was supported by the National High-Tech Research and Development Program (863 Program, 2013AA102803), the National Science and Technology Support Program (2011BADB02), the National Science Fund for Distinguished Young Scholars (31225026), and the National “948” Project (2011-G7-4). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2013.12.021. References [1] Oeckinghaus A, Ghosh S. The NF-kB family of transcription factors and its regulation. Cold Spring Harb Perspect Biol 2009:1. [2] Goto A, Matsushita K, Gesellchen V, Chamy LE, Kuttenkeuler D, Takeuchi O, et al. Akirins are highly conserved nuclear proteins required for NF-[kappa]Bdependent gene expression in drosophila and mice. Nat Immunol 2008;9:97e 104. [3] Galindo RC, Doncel-Pérez E, Zivkovic Z, Naranjo V, Gortazar C, Mangold AJ, et al. Tick subolesin is an ortholog of the akirins described in insects and vertebrates. Dev Comp Immunol 2009;33:612e7. [4] Marshall A, Salerno MS, Thomas M, Davies T, Berry C, Dyer K, et al. Mighty is a novel promyogenic factor in skeletal myogenesis. Exp Cell Res 2008;314: 1013e29. [5] Komiya Y, Kurabe N, Katagiri K, Ogawa M, Sugiyama A, Kawasaki Y, et al. A novel binding factor of 14-3-3b functions as a transcriptional repressor and promotes anchorage-independent growth, tumorigenicity, and metastasis. J Biol Chem 2008;283:18753e64. [6] Nowak SJ, Aihara H, Gonzalez K, Nibu Y, Baylies MK. Akirin links twistregulated transcription with the brahma chromatin remodeling complex during embryogenesis. PLoS Genet 2012;8:e1002547.
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