Molecular characterization and expression analysis of the putative interleukin 6 receptor (IL-6Rα and glycoprotein-130) in rainbow trout (Oncorhynchus mykiss): salmonid IL-6Rα possesses a polymorphic N-terminal Ig-domain with variable numbers of two repeats Maria M. Costa1,2, Tiehui Wang1, Milena M. Monte1 and Christopher J. Secombes1*. 1 Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen AB24 2TZ, UK. Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), Eduardo Cabello 6, Vigo, Spain. 2 * Author for correspondence: Prof. Christopher J. Secombes Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK. Tel:0044-1224-272872 Fax:0044-1224-272396 Email: c.secombes@abdn.ac.uk The nucleotide sequence data will appear in the EMBL/DDBJ/GenBank nucleotide sequence database under the following accession numbers: FN824530 (IL-6Rα) and FN824531 (gp130). 1 Abstract Interleukin (IL)-6, the founding member of IL-6 family cytokines, plays non-redundant roles in hematopoiesis and acute phase responses. IL-6 signals via a specific private IL-6Rα and a common beta chain gp130. In this study, we sequence analysed both IL-6Rα and gp130 in rainbow trout. The trout gp130 cDNA encodes 906 aa and is similar in size, extracellular domain structure (D1-6) and presence of intracellular motifs important for signal transduction to tetrapod gp130. The trout IL-6Rα cDNA encodes for 834 aa and is larger compared to tetrapod IL-6Rαs, as are other fish IL-6Rα molecules due to a large D1 domain. However, the cytokine binding domain is well conserved across vertebrates, with four conserved cysteine residues in the N-terminal FNIII domain and a WSXWS motif in the C-terminal FNIII domain. Furthermore, phylogenetic tree analysis confirmed that the reported fish IL-6Rα and gp130 molecules are orthologues to their tetrapod counterparts. The extra-large D1 domain of the salmonid IL-6Rα molecules results from the insertions of two repetitive sequences of [TS]-[TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]-S-[KD]-N-[GR], respectively. Furthermore the numbers of repetitions of the two motifs were variable in different individuals and cell lines, and even in the same fish allelic polymorphism exists. Trout IL-6Rα was expressed at higher levels than gp130 in a number of tissues examined and the expression of both IL-6Rα and gp130 could be modulated by LPS and Poly I:C in four trout cell lines studied. The expression patterns of the receptors suggest that high level expression of IL-6Rα is critical for IL-6 responsiveness. 2 3 Introduction Interleukin (IL)-6 is the founding cytokine of the IL-6 family that comprises in addition to IL-6, IL-11, IL-27, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), CT-1, CT-2 and cardiotrophin-like cytokine (CLC) and neuropoietin (NPN) (Huising et al. 2005; Wang et al. 2005; Silver and Hunter 2010; Hirano 2010). Generally, they are involved in inflammatory and immunologic processes as well as in hematopoiesis, liver and neuronal regeneration, embryonic development and cardiovascular physiology. Through activation of target genes involved in growth, differentiation, survival, apoptosis, and proliferation, IL-6 family cytokines play a key role in cellular and tissue homeostasis (Fischer and Hilfiker-Kleiner 2007). In mammals, all the IL-6 family cytokines signal via a common glycoprotein, known as gp130, regardless of the contribution played by the cytokine-specific α chains, e.g. IL-6Rα (Gerez et al. 2007). Engagement of gp130 with cytokine-specific α chains triggers the activation of at least three different downstream signalling pathways, the signal transducers and activators of transcription (STAT) 1 and 3, the Src-homology tyrosine phosphatase 2 (SHP2)-Ras-ERK, and the PI3K-Akt system (Silver and Hunter 2010; Hirano 2010). Due to the common use of gp130 as a signal chain, the IL-6 family cytokines are highly redundant in their capacity to mediate biological functions. They also show member specific activity. For example, IL-6 deficient mice exhibit defects in hematopoiesis, acute phase protein synthesis, antigen-specific antibody production, chemokine induction, leukocyte recruitment, hepatocyte regeneration and susceptibility to infections (Fasnacht and Müller 2008). IL-6 can induce cell signalling not only via the classic pathway involving the transmembrane receptor IL-6Rα associated with gp130, but also via the soluble receptor IL-6Rα, which binds to IL-6 and induces a signal mediated by the ubiquitous gp130 molecule (trans-signalling) (Assier et al. 2010). This second pathway widens the scope of IL-6 signalling since cells expressing no membrane bound IL-6R can also be stimulated by the trans-signal pathway. Both IL-6Rα and gp130 are type-I cytokine receptors, also known as hematopoietin receptors, that share a modular architecture with an N-terminal extracellular and C-terminal intracellular orientation. Every type I cytokine receptor has a characteristic ~200 aa cytokine binding homology domain (CBD) consisting of two fibronectin type-III (FNIII) domains from the N-terminal to Cterminal. The N-terminal domain contains four conserved cysteine residues that form two intrachain disulfide bonds, whilst the C-terminal domain has a conserved WSXWS motif that has been proposed to be required for maintaining the receptor in a conformation favourable to cytokine binding (Bazan 1990; Boulay et al. 2003). In addition, both receptors have an immunoglobulin (Ig)-like domain N-terminal to the CBD and gp130, that belongs to the class of “tall cytokine receptors”, also has three FNIII-like domains between the CBD and the transmembrane domain (Waetzig et al. 2010). Thus, IL-6Rα and gp130 have 3 (D1-3) and 6 (D1-D6) domains within their extracellular regions, respectively. 4 The type I cytokine receptors have no intrinsic enzyme activity but are constitutively associated with tyrosine kinases of the Janus kinase (JAK) family through the box 1 and/or box 2 motifs in the intracellular region of the signalling chains. Cytokine binding induces receptor oligomerization and activation of associated JAKs that phosphorylate themselves and the intracellular domains of the receptors. The phosphorylated tyrosine residues in the receptors then serve as docking sites for a second family of proteins, the STAT molecules. Binding of STATs to intracellular domains of receptors leads to their tyrosine phosphorylation and subsequent dissociation from the receptors. The phosphorylated STATs form dimers and translocate into the nucleus, where they bind to specific regulatory elements in the promoters of responsive genes and act as transcription factors to modulate gene expression (Murray 2007; Hershey 2003). The intracellular domain of IL-6Rα lacks STAT binding sites and is devoid of signalling (Silver and Hunter 2010). Mammalian gp130 possesses conserved boxes (1-3), and multiple docking sites, mainly for STAT3 but also STAT1 and STAT5, in the intracellular domain (Heinrinch et al. 1998). Gp130 is expressed ubiquitously. Therefore it has so far been a generally accepted idea that the responsiveness of individual cell types to IL-6-type cytokines is mainly determined by the expression of a specific α receptor (i.e. IL-6Rα) or by the expression of a second specific signal-transducing subunit. It is known that mammalian IL-6Rα expression is limited to particular cell types such as neutrophils, monocytes/macrophages and some lymphocytes (Waetzig et al. 2010). A few IL-6 family members have been successfully identified recently in several fish species. The orthologues of mammalian IL-6 have been cloned in fugu (Tagifugu rubripes) (Bird et al. 2005), rainbow trout (Oncorhynchus mykiss) (Iliev et al. 2007), flounder (Paralichthys olivaceus) (Nam et al. 2007) and seabream (Sparus aurata) (Castellana et al. 2008), as well as IL-11 in rainbow trout (Wang et al. 2005), carp (Huising et al. 2005) and flounder (Santos et al. 2008). In addition, novel fish IL-6 family members have also been discovered including a fish IL-11 like molecule (Huising et al. 2005; Santos et al. 2008), the M17 molecule that has homology to mammalian LIF/OSM (Fujiki et al. 2003; Hwang et al. 2007; Wang and Secombes 2009) and a CNTF-like gene, that may be homologous to an ancestral gene that gave rise to mammalian CNTF/CLC/CT-1/CT-2 (Wang and Secombes 2009). The functional characterisation of these fish molecules has been started. For example, goldfish M17 induces differentiation of and nitric oxide production by macrophages (Hanington and Belosevic 2007) and trout IL-6 promotes macrophage growth and induces antimicrobial peptide gene expression (Costa et al. 2011). The characterisation of the receptor subunits of fish IL-6 family members is scarce (Hannington and Belosevic 2005; Santos et al. 2007), despite the large number of putative receptors that have been annotated after the sequencing of several model fish genomes (Aparicio et al. 2002; Jaillon et al. 2004; Liongue and Ward 2007) and various large expressed sequence tag (EST) projects (Leong et al. 2010). In the current paper, we have sequence analysed IL-6Rα and gp130 in rainbow trout, and compared them with other predicted fish 5 homologues. We found that the fish IL-6Rα possesses a large Ig-like D1 domain and in salmonids this D1domain was further enlarged by an insertion of two types of 13 aa repeat sequences that exist at different frequencies. We examined the expression of the two receptors in healthy fish and found that the expression of trout IL-6Rα was generally higher than that of gp130. We further analysed their expression and modulation in four trout cell lines after stimulation with lipopolysaccharide (LPS) and polyinosinic: polycytidylic acid (Poly I:C), as two common pathogen-associated molecular patterns (PAMPs). 6 Materials and methods Fish Rainbow trout were obtained from a commercial Scottish fish farm (Almond bank. Perthshire, UK). The animals were maintained in recirculating filtered freshwater tanks (1 m diameter fibreglass/350 L) at 15 ± 1°C with aeration. They were fed twice daily with a standard commercial trout diet (Ewos, Scotland). Prior to experimental work, animals were acclimatized to laboratory conditions for a minimum period of two weeks. Cell lines The rainbow trout monocyte/macrophage cell line RTS-11 (derived from spleen cells, Ganassin and Bols 1998) was maintained in Leibovitz medium (L-15; Invitrogen) containing 30% foetal calf serum (FCS; Labtech International) and antibiotics (Penicillin-Streptomycin (P/S), Invitrogen) at 20°C. The cell lines, RTG-2 (a fibroblast-like cell line derived from gonad; Wolf and Quimby 1962), RTGill (Gill-derived; Schirmer et al. 1998) and RTL (an epithelial cell line derived from liver; Lee et al. 1993), were maintained in the same medium as for RTS-11 cells except with reduced FCS (10%) as described previously (Wang et al. 2011b). Cells were maintained in 75 cm2 flasks at 20 °C until confluent and then sub-cultured into new media. Cloning of IL-6Rα and gp130 Two Atlantic salmon Salmo salar IL-6Rα like sequences (ACN10857 and ACN11013) were found in the NCBI database with multiple numbers of two repeats within the Ig-like domain that is crucial for ligand receptor binding. To verify if these repeats also exist in other salmonids we sequence analyzed the IL-6Rα in rainbow trout. Blast search of the EST database at NCBI (http://blast.ncbi.nlm.nih.gov/Blast) using the salmon sequences identified multiple trout ESTs encoding the C-terminal of a potential trout IL-6Rα homologue. Primers F1 and F2 (Table 1) were designed against the ESTs BX883008 and GE837037 and used for 3’RACE using SMART cDNA prepared from head kidney (HK) as described previously (Wang et al. 2008). A 1 Kb 3’-RACE product was obtained that contained the sequence encoding for the C-terminal and the 3’untranslated region (UTR) of the trout IL-6Rα. A 2.8 Kb product was amplified using primers F3/R3 (Table 1) designed to the 5’- and 3’-UTR and that contained the complete coding region of trout IL-6Rα. The primer positions are marked in supplementary Figure 1. Blast search of the NCBI EST database using the mammalian gp130 protein sequence identified multiple salmonid ESTs that were potentially derived from a salmonid gp130. To obtain the complete coding region, primers F1 and R1 (Table 1 and supplementary Figure 2) were designed to the 5’- and 3’-UTR of ESTs 7 EG758643 and CU065376, respectively, and used for PCR. A 3 Kb band was amplified from a HK cDNA sample that contained the complete coding region of gp130. Analysis of the frequency polymorphism of the two repeats in IL-6Rα The expression of IL-6Rα was detected in different cell lines and tissues at different levels. To ensure the amplified bands of the repeated cDNA sequences could be revealed on an agarose gel, two rounds of PCR were performed. The first PCR was carried out using primer pair repF1/R1 (Table 1 and supplementary Figure 1) and the resultant product was diluted 100 fold and used as template for the second nested PCR using primer pair repF2/R2 (Table 1). The amplified products were run on a 1% agarose gel containing ethidium bromide and selected PCR products were cloned and sequence analyzed as above. Sequence and phylogenetic analysis The obtained nucleotide sequences were analyzed and assembled using the AlignIR program (LI-COR). A multiple sequence alignment was generated using ClustalW2 (Chenna et al. 2003) and the translation was performed using ExPaSy tools (http://us.expasy.org/tools). The domain distribution was analysed with Simple Modular Architecture Research Tool (SMART, http://smart.embl-heidelberg.de/), Scan Prosite (http://expasy.org/prosite/) (Gasteiger et al. 2005) and Pfam (http://pfam.sanger.ac.uk/) (Finn et al. 2010). The signal peptide was predicted used SignalP 3.0 (Emanuelsson et al. 2007) and secondary structure predicted using SOPM (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopm.html), YASPIN http://www.ibi.vu.nl/programs/yaspinwww/) and homology with other species. Phylogenetic trees were created by the Maximum Likelihood method based on the JTT matrix-based model using the MEGA program (version 5) (Tamura et al. 2011), and were bootstrapped 2,000 times. Tissue distribution Tail fins, gills, thymus, brain, scales, skin, muscle, liver, spleen, gonad, head and caudal kidney, intestine and heart from six healthy animals were harvested in order to analyze the expression level in different tissues. Samples were homogenised and kept in TRI reagent (Sigma) until their processing. RNA isolation, cDNA production and real-time PCR analysis of gene expression RNA isolation from TRI lysate was performed following the manufacturer’s protocol. 5-10 µg total RNA was converted into cDNA using Bioscript (Bioline). The cDNA generated was diluted with 400 μl of TE buffer (10 mM Tris HCl; 1 mM EDTA, pH 7.5) and 4 μl were used as template for quantitative PCRs using the primers described in Table 1. Real time PCR analysis was performed as previously (Wang et al. 2007; 2011), using a Light Cycler real-time PCR system (Roche). The expression of the candidate genes was normalized to 8 the housekeeping gene EF-1α. Fold change was calculated by dividing the normalized expression values of stimulated cells by that of the controls. Modulation of gene expression of cells (5 ml at 5×105 cells/ml) were seeded into 25 cm2 cell culture flasks and incubated at 20°C overnight. The cells were then stimulated with E. coli LPS (Sigma-Aldrich) and Poly I:C (Sigma-Aldrich) at a final concentration of 25 µg/ml and 50 µg/ml, respectively. The stimulation was terminated at 4, 8 and 12 h by dissolving the cells in TRI reagent. Statistics The SPSS package 17.0 (SPSS Inc. Chicago, Illinois) was used for statistic analysis of changes of gene expression levels. To improve the normality of real-time quantitative PCR measurements, a log2 transformation of the arbitrary units after normalization to the expression level of EF-1α was performed as described previously (Wang et al. 2011a; b). The relative expression levels of IL-6Rα and gp130 in tissues were analysed by paired samples T tests. One way-ANOVA and the LSD post hoc test was used to analyze the expression data in the cell lines after stimulation, with P <0.05 between treatment groups and control groups considered significant. 9 Results Sequence analysis of rainbow trout IL-6Rα The cDNA sequence of the rainbow trout IL-6Rα chain (rtIL-6Rα), isolated from a HK cDNA sample (EMBL acc. no. FN824530), was 2956 nucleotides with a 5’-UTR of 182 bp, a long open reading frame (ORF) encoding for 834 aa and a 3’-UTR of 244 bp followed by a poly A tail (Supplementary Fig. 1). There were four upstream ATGs before the main ORF in the 5’-UTR and a polyadenylation signal (AATAAA) 13 bp upstream of the poly A tail in the 3’-UTR. The translation from the main ORF had a predicted signal peptide of 19 aa, an extracellular region of 707 aa, a transmembrane domain of 23 aa and an intracellular region of 85 aa. The extracellular region of mammalian IL-6Rα contains an immunoglobulin-like (Ig-like) domain (D1), and a cytokine binding domain (CBD) composed of two FNIII domains (D2 and D3) that each contain seven β-strands. Whilst the D2 and D3 of CBD were identifiable in trout IL-6Rα and were conserved among IL-6Rα molecules from mammals, birds and fish, the trout Ig-like D1 domain was relatively large as also seen in salmon IL-6Rα molecules. This was due to the presence of multiple copies of two different motifs corresponding to the 13 aa peptide sequences [TS]-[TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]S-[KD]-N-[GR] respectively. Eight repetitions of the first motif and 7 of the second were found in the translation of the cDNA sequence from HK (Supplementary Fig. 1). Multiple N-glycosylation sites (NXT/S) were also present in the extracellular region of the translation, with each repeated motif containing a potential N-glycosylation site. The trout IL-6Rα shared 84% aa identity to the two Atlantic salmon IL-6Rα EST entries, and even higher identities (90.3% and 90.9%) when the amino acid repeat sequence was removed (Table 2). It shared 27.5% and 24.4% identities to IL-6Rα molecules from tetraodon and zebrafish, respectively, but only marginal (16.118.9%) identities to tetrapod IL-6Rα molecules. In contrast, the tetraodon and zebrafish IL-6Rα molecules shared higher identities (23.0-26.8%) to their tetrapod orthologues. However, when the aa repeat sequences in the salmonid IL-6Rα molecules were removed for analysis, comparable identities (21.0-24.7%) to tetrapod orthologues were seen as with tetraodon and zebrafish (Table 2). It is worth noting that the fish IL-6Rα molecules shared the highest identities to tetrapod IL-6Rα relative to other IL-6Rα related tetrapod sequences including IL-11Rα and CNTFR (Table 2). To compare the aa sequence of salmonid IL-6Rα and their orthologues from other species, a multiple alignment was constructed using IL-6Rα molecules from tetraodon and representative tetrapods. Only one Atlantic salmon sequence (acc. no. ACN10857) was used as the two salmon sequences shared 96.1% identity when the sequence repeats were removed (Table 2). The zebrafish IL-6Rα sequence was also excluded as the N-terminal of this sequence was likely incomplete. The alignment showed that the extracellular D2 and D3 domains, and to a lesser extent the D1 domain, are relatively conserved except for an insertion in the D1 10 domain in fish IL-6Rα molecules (Table 2). Thus, all the fish IL-6Rα molecules are larger than the tetrapod ones. The salmonid IL-6Rα molecules were further enlarged by the two aa repeat sequences (Fig. 1). The conserved features included 10 cysteine residues (C1-10), a proline rich hinge region between the D1-D2 and D2-3 domains, and the type I cytokine receptor signature (WSXWS). Of the 10 conserved cysteine residues, 8 of them (C1-5, 7, 9 and 10) are completely conserved across vertebrates, whilst C6 and C8 were conserved only in fish (Fig.1). It is known that C2-3 in D1, C4-5 in D2 and C7-9 in D2 form disulphide bonds in the human IL-6Rα (Cole et al. 1999). In addition, the N-terminal D1 is disulphide-bonded to D2 through C1 and C10 (Varghese et al. 2002). The conserved C6 and C8 in fish IL-6Rα may form an additional disulphide bond to stabilise their tertiary structure. Each IL-6Rα molecule from different species has multiple putative Nglycosylation sites, but none of them are completely conserved across all vertebrates. Sequence analysis of rainbow trout gp130 A 2,968 bp cDNA fragment was amplified using primers F1 and R1 that contained a 5’-UTR of 169 bp, an ORF of 2721 bp, and part of the 3’-UTR of the trout gp130 gene (Supplementary Fig. 2, EMBL acc. no. FN824531). There were three upstream ATGs before the main ORF that could be potentially translated into three short peptides, and at least 6 mRNA instability motifs (ATTTA) in the 3’-UTR. The main ORF encodes for a 906 aa protein with a predicted signal peptide of 26 aa, an extracellular region of 609 aa, a transmembrane domain of 21 aa and an intracellular region of 250 aa (Supplementary Fig. 2). Structurally, trout gp130 is predicted to be composed of one C2-type Ig-like domain (D1) and five FNIII domains (D2-D6) in its extracellular portion. As with other type I cytokine receptors, rtgp130 possesses a WSXWS motif in the D3 domain (Supplementary Fig. 2). The trout translation had 14 putative glycosylation sites in the extracellular domain. The trout gp130 had a similar size (906 aa) to tetrapod gp130 molecules but shared higher aa identities of 42.5% (zebrafish) and 40.7% (tetraodon) to fish gp130s relative to tetrapod gp130 molecules (33.7-35.7%, Table 3). The gp130 molecules from fish, amphibians and birds shared highest aa identities to mammalian gp130 compared to any related mammalian sequences including GCSFR, LIFR, OSMR and IL-31Rα. It is worth noting that mammalian gp130 molecules share high aa identities (76.2-90.8%), whilst only 38.5-42.5% identities were shared by fish gp130 molecules. The amphibian and bird gp130 also had low identities to gp130 molecules from fish and mammals (Table 3). To compare the aa sequence of gp130 from trout and other species, a multiple alignment was constructed using all the known gp130 molecules from fish and selected tetrapod gp130 molecules. Good conservation was seen across the whole molecule, including a signal peptide, a six domain (D1-D6) extracellular region, a transmembrane domain and an intracellular domain. There were 4 conserved cysteine residues in the extracellular region, with an additional 7 conserved in tetrapods (Fig. 2). In human gp130 C1-C3 and C2-C4 in the D1 domain, C5-C6 and C7-C8 in the D2 domain, and C9-C10 in the D5 domain form disulphide bonds 11 (Moritz et al. 2001). Other conserved extracellular features were proline rich hinge regions between the (D16) domains, the type I cytokine receptor signature (WSXWS) in the D3 domain, and two N-glycosylation sites, one in the middle of the D1 domain and the other at the N-terminal of D2. As the signal chain of the IL-6R, gp130 contains several conserved critical intracellular regions named boxes 1-3 (Murakami et al. 1991; Baumann et al. 1994; Lai et al. 1995) that are required for different signalling mechanisms. All the three boxes were well conserved across vertebrates (Fig. 2). The YXXQ motif in the intracellular region of mammalian gp130 is a known docking site for STAT3, and critical for the phosphorylation of STAT3 on Ser727 in human gp130 (Abe et al. 2001). Four YXXQ motifs (YXXQ1-4) are also conserved in tetrapod gp130, and YXXQ1 and 3 are also conserved in fish gp130 with YXXQ4 also conserved in some fish but not trout (Fig. 2). The YSTV motif, a known docking site for SHP-2 and important for activation of the Ras/Raf/MEK/Erk pathway, is also conserved across vertebrates with YSTV in tetrapods and YSSV in fish (Fig. 2). The cell surface expression of gp130 is tightly regulated by internalization via a dileucine dependent internalization motif (Radtke et al. 2010). The internalization motif, as well as a serine rich region, were well conserved in gp130 molecules across vertebrates. Phylogenetic tree analysis To confirm the identities of trout IL-6Rα and gp130, un-rooted Maximum Likelihood (ML) trees (Fig. 3) were inferred using MEGA5 software (Tamura et al. 2011) with the Jones-Thornton-Taylor (JTT) aa matrix and all sites. In the IL-6 receptor alpha chain tree, the known fish and selected tetrapod IL-6Rα, CNTFR and IL-11Rα were included in the analysis. The fish IL-6Rα molecules formed an independent clade and grouped with IL6Rα molecules from tetrapods with high bootstrap support (100%). They were separate from the CNTFR and IL-11Rα groups, further supporting the identities of fish IL-6Rα molecules (Fig. 3A). In the gp130 and related gene phylogenetic tree analysis, all the known gp130 molecules from fish and from selected tetrapods, as well as mammalian molecules closely related to gp130, including GCSFR, LIFR, OSMR and IL-31Rα, were analysed. All the related receptors formed independent clades with high bootstrap support (100%) and were separate from the gp130 group. The fish gp130 molecules grouped within the gp130 clade with good bootstrap support (80%) (Fig. 3B). Repeat polymorphism Using nested PCR with primers covering the whole repeat region in the Ig-like D1 domain of the IL-6Rα chain, amplicons with similar size could be amplified from all the 14 tissues examined from the same fish (Fig. 4A). However, the size of the amplicons could vary between fish, and it was possible to obtain more than one. Furthermore, the amplicons from 4 different cell lines showed great differences in size, from 0.7 Kb to 1.3 Kb (Fig. 4B), probably because they originated from different fish. To further investigate the nature of the 12 repeat polymorphism, amplicons from different fish and cell lines were cloned and sequence analyzed. We found that individual fish or cell lines could possess more than one pattern of repeats, as shown in Figure 4C. The numbers of repeats found in the analysed samples varied between 3 to 13 for the first 13 aa repeat and between 4 to 17 for the second repeat. Since the repeats were associated with potential glycosylation sites, this meant that the trout IL-6Rα from different individuals may also have different N-glycosylation potential. Tissue distribution of expression of IL-6Rα and gp130 The expression of IL-6Rα and gp130 was detectable by real-time PCR in all the 14 tissues examined. The lowest transcript expression level of IL-6Rα was in the scales (4 units), whilst for gp130 it was in the intestine (1 unit, Fig. 5A). The highest expression of IL-6Rα was in spleen (448 units), followed by head kidney, liver, and caudal kidney. IL-6Rα was also moderately expressed in gills, brain, heart, thymus and intestine (20-55 units). The highest expression of gp130 was in muscle (60 units), followed by brain, gonad and skin. Gp130 expression in other tissues was low (1-9 units, Fig. 5A). In general, the expression level of IL-6Rα was higher than that of gp130 in most of the tissues examined, except in brain, scales, skin, muscle and gonad where the expression of IL-6Rα and gp130 was not significantly different (Fig. 5B). For example, the expression of IL6Rα was 102, 77, 51 and 37 fold higher than that of gp130 in head kidney, liver, spleen and gills, respectively (Fig. 5B). Modulation of IL-6Rα and gp130 expression in four trout cell lines Both IL-6Rα and gp130 were highly expressed in the monocyte/macrophage cell line RTS-11, at comparable levels (Fig. 6A). The epithelial RTL cell line and fibroid cell lines RTG-2 and RTGill also expressed moderate levels of gp130 but only expressed low levels of IL-6Rα (Fig. 6A). The expression of IL-6Rα and gp130 could be modulated to some degree in a cell type and time dependent manner by LPS and Poly I:C (Fig. 6B and 6C). A moderate increase of IL-6Rα expression was seen at 8 h and 24 h in RTL and RTG-2 cells, and at 24 h in RTS-11 cells after LPS stimulation. However, IL-6Rα expression was decreased at 4 h in RTL and at 24 h in RTGill after LPS stimulation (Fig. 6B). IL-6Rα expression was refractory to Poly I:C stimulation in RTG-2, RTGill and RTS-11 cells, but showed a small increase at 8 h in RTL. In contrast, gp130 expression was refractory to LPS stimulation in RTL, RTG-2 and RTGill cells, whilst in RTS-11 cells the high level constitutive expression of gp130 was down regulated at 8 h by LPS (Fig. 6C). Poly I:C was able to increase gp130 expression at 8 h and 24 h in RTG-2 and at 4 h and 8 h in RTGill, and like LPS it decreased gp130 expression at 8 h in RTS-11 cells (Fig. 6C). 13 Discussion In this report, the rainbow trout IL-6Rα and gp130 were cloned and sequence analysed. We found that salmonid IL-6Rα had a large D1 domain mainly due to an insertion of two amino acid repeat sequences. We discovered that the number of each of the two repeats was variable in four trout cell lines studied and in different fish. Finally, we examined the expression of IL-6Rα and gp130 in tissues from healthy fish and their modulation by LPS and Poly I:C in the cell lines. Although the fish IL-6Rα and gp130 molecules were quite divergent (Tables 2 and 3), they showed similar domain structure, i.e. 3 and 6 domains, respectively, in the extracellular regions, with a WSXWS motif at the C-terminal (D3) of the cytokine binding domain (CBD), as seen in mammals. Key intracellular motifs were also conserved in gp130 and included the box-1-3, critical for Jak association and signalling, the docking sites for other downstream molecules, such as the YXXQ motif for STAT3 and the YSTV motif for SHP-2 (Fig. 2). Phylogenetic tree analysis (Fig. 3) grouped the fish molecules with their tetrapod counterparts with high bootstrap support, adding further confirmation that the molecules found are the orthologues to mammalian IL6Rα and gp130. However, important differences were observed, including a large D1 domain in fish IL-6Rα, and a different potential disulphide bond pattern in both IL-6Rα and gp130, that will be discussed below. Domain structure prediction and multiple alignment (Fig. 1) suggested the Ig-like D1 domain in all fish IL6Rα molecules is bigger than in tetrapods. The salmonid IL-6Rα sequences were further enlarged in this domain by the insertion of two different repeat motifs corresponding to the 13 aa peptide sequences [TS][TF]-VSTTT-[ND]-TTSNG and TTVS-[AT]-IKD-[DG]-S-[KD]-N-[GR] respectively. The numbers of these repeats were different in different cell lines and individual fish (Fig. 4), suggesting that salmonid IL-6Rα has a highly polymorphic D1 domain with the potential to affect responsiveness to IL-6. The Ig-like D1 domain of mammalian IL-6Rα is important for intracellular transport through the secretory pathway and is also involved in the noninduced shedding of the receptor to release a soluble IL-6Rα. However, deletion of the D1 domain of human IL-6Rα does not affect its bioactivity, suggesting that the D1 domain is not critical for IL-6 binding (Vollmer el al. 1999). Thus a large D1 domain in the fish IL-6Rα may be tolerable for its activity. However, the large D1 domain in fish, and particularly in salmonids, may have as yet unknown functional impacts and for example, could affect receptor secretion, membrane expression and shedding, or the stability of the IL6/IL-6R signalling complex. Structural studies have revealed that IL-6 is first engaged by IL-6Rα and then presented to gp130 in the proper geometry to facilitate a cooperative transition into the high-affinity, signalling-competent hexamer. Human IL-6 possesses a total of five points of interaction with its receptor. The site I of the protein is able to contact several residues located between the D2 and D3 domains in the IL-6Rα chain, with the D3 domain providing the majority of the contact surface. Sites IIa and IIb of IL-6 interact with amino acids present between gp130 14 domains D2 and D3, and between the D3 domains of gp130 and IL-6Rα, respectively. The IL-6 site IIIa links to the gp130 D1 domain, and the site IIIb interacts with the gp130 D1 domain and the IL-6Rα D2 domain (Boulanger et al. 2003). Thus the D2-3 of IL-6Rα and D1-3 of gp130 are critical to form a signalling complex with mammalian IL-6. The well conserved D2-3 of IL-6Rα (Fig. 1) and D1-3 of gp130 (Fig. 2) across vertebrates suggests that a similar signalling complex to that seen for mammalian IL-6/IL-6R may be conserved in fish. Soluble IL-6Rα can be produced by shedding of membrane bound receptor or by translation from alternatively spliced transcripts, as seen in humans where two transcripts occur as a result of alternative splicing, that translate into a membrane-bound form and its soluble counterpart (Oh et al. 1996). Alternative splicing variants that produce soluble IL-6Rα, or soluble IL-11Rα have not been found in chicken, as shown by RTPCR/Southern blot analysis where no transcripts shorter than the full-length chicken IL-6Rα are seen (Nishimichi et al. 2006; Kawashima et al. 2005). In trout a single band was amplified using primers located in the 5’ and 3’-UTR of the IL-6Rα sequence during our cloning procedure, indicating that alternative splicing to produce a soluble IL-6Rα is unlikely to occur in trout either. Intra-chain disulphide bonds between the thiol groups of two peptidyl-cysteine residues can contribute to protein folding and stability of cytokines and their receptors. Thus the N-terminal FNIII domain of the CBD in cytokine receptors, at least in mammals, contains two pairs of conserved cysteines arranged in a CX-(9-10)CXWX-(26-32)-CX-(10-15)-C motif linked by two disulphide bonds (Liong and Ward 2007). The tetrapod IL-6Rα have four conserved disulphide bonds, one in the D1 domain, two in the D2 domain and one that connects the D1 and D2 domains (Cole et al. 1999; Varghese et al. 2002). All the cysteine residues for the four disulphide bonds are conserved in the IL-6Rα molecule across vertebrates (Fig. 1). In addition, all the fish IL-6Rα molecules possess a fifth pair of cysteine residues in the D2 domain that may form an additional disulphide bond to stabilise their tertiary structure. Human gp130 contains two pairs of disulphide bonds in each of the D1 and D2 domains (Moritz et al., 2001), and the cysteine residues involved are conserved in gp130 molecules across tetrapods. In contrast to the fish IL-6Rαs that possess an extra cysteine pair, fish gp130 molecules lack two pairs of the cysteine residues conserved in tetrapods, one in each of the D1 and D2 domains. N-glycosylation is the most ubiquitous protein co-translational modification in the endoplasmic reticulum. This modification serves as a primary determinant for specific molecular recognition as well as protein folding and stability, and therefore is an essential and highly conserved protein modification pathway in eukaryotic cells. Multiple potential N-glycosylation sites have been predicted in both trout IL-6Rα and gp130 (Supplementary Figs. 1–2). Although we have not observed the conservation of N-glycosylation sites across vertebrates in the multiple alignment of IL-6Rα, two conserved sites, one each in the D1 and D2 domains, were identified in gp130 across vertebrates (Fig. 2). Human gp130 is N-glycosylated at 9 of 11 potential sites. 15 Mutation analysis revealed that mutein gp130 (with N-glycosylation sites removed) is not efficiently transported to the plasma membrane but is degraded in the proteasome. However, the small quantities of mutein gp130 which do reach the cell surface, are still able to transduce the IL-6 signal (Waetzig et al. 2010). Thus, N-linked glycosylation of gp130 is required for molecular stability but not the signal-transducing function, and the conservation of potential N-glycosylation sites across vertebrate gp130 molecules concurs with this. Interestingly, the salmonid IL-6Rα molecules possess polymorphic numbers of two 13 aa repeat sequences and each repeat motif contains a potential N-glycosylation site. Thus salmonid IL-6Rα from different individuals has different N-glycosylation potential as the number of repeats is variable. In mammals, gp130 is expressed ubiquitously, whilst the IL-6Rα chain is expressed only by hepatocytes, phagocytes and some lymphocytes (Saito et al. 1992). In trout a ubiquitous expression of gp130 was detected in all the 14 tissues examined (Fig. 5). Interestingly, the expression of trout IL-6Rα was comparable to or even higher than that of gp130 expression in some tissues. In contrast, the IL-6Rα expression was lower than gp130 in most of the cell lines examined (epithelial RTL and fibroid RTG-2 and RTGill), except the monocyte/macrophage like cell line (RTS-11) where comparable expression of both IL-6R subunits was seen. The relatively high level expression of IL-6Rα in tissues may be due to its expression in leucocytes, since the expression of IL-6Rα was low in the epithelial and fibroid cell lines (Fig. 6). The bioactivities of recombinant trout IL-6 have only been detected in RTS-11 cells (Costa et al. 2011) that express a high level of IL-6Rα and not in the other cell lines. Trout IL-6 is also active on leucocytes prepared from head kidney and spleen that similarly express high levels of IL-6Rα (unpublished data). Such data suggest that high level transcript expression of IL-6Rα may be critical for responsiveness to IL-6. The trout IL-6Rα was highly expressed in immune tissues such as the spleen and head kidney, and was also expressed in liver, gills, brain, heart, thymus and intestine. This is in line with the pleiotropic functions of IL-6 in the regulation of the immune response, inflammation, and hematopoiesis, as well as in the (liver) acute phase response. IL-6 was originally described as a T-cell-derived B-cell differentiation factor, essential for antibody production by activated B cells (Nishimoto and Kishimoto 2006). It also affects other immune cells, and induces T-cell activation and differentiation and macrophage differentiation. IL-6 is one of the differentiating factors for Th17 cells (Betteli et al. 2007), and switches the differentiation of monocytes from dendritic cells to macrophages (Chomarat et al. 2000). Moreover, the role of IL-6 in the production of acute phase proteins in liver, as well as its role in hepatic regeneration, has been reported (Bayne and Gerwick 2001; Drucker et al. 2010). Therefore, lymphocytes, monocytes/macrophages and hepatocytes all require the presence of the IL-6Rα chain to bind and respond to this cytokine. The highest expression level of trout gp130 was found in the muscle (Fig. 5). Mammalian gp130 is highly expressed in the sarcolemma and also in fibroblasts (Ancey et al. 2003), and is involved in myocyte hypertrophy and fibroblast proliferation (Fredj et al. 2005), suggesting an important role of IL-6 family cytokines in muscle. This result is in contrast to the expression of a related gene (JfGPH) in Japanese flounder 16 (Santos et al., 2007), thought to be an ancestral type I cytokine receptor found in teleost fish, where highest expression of JfGPH was found in immune tissues by RT-PCR. Trout gp130 was only expressed at a low level in intestine, gills, thymus and kidney, as revealed by real-time RT-PCR. This may represent a real difference between the molecules or the fish species, as also seen with the salmonid IL-6Rα chain that has an extra-large D1 domain and that is highly expressed, at least at the transcript level. Thus, in salmonids perhaps a controlled expression of gp130 helps to regulate IL-6 function. The secretion of mammalian IL-6 is induced by inflammatory stimulants such as LPS (Ghezzi et al. 2000), and IL-6 can up-regulate its own receptor expression (Thabard et al. 2001). In trout, IL-6 expression in RTS11 cells, as well as in primary macrophages is induced by LPS, Poly I:C and the proinflammatory cytokine IL1β (Costa et al. 2011). With the aim to analyse if similar mechanisms could regulate IL-6R expression in rainbow trout, we have analysed the expression of IL-6Rα and gp130 after stimulation. No change in expression level was observed after trout rIL-6 stimulation of RTS-11 cells (data not shown), suggesting that different mechanisms may be involved in the modulation of IL-6 receptor expression in fish. Despite being refractory to IL-6, the expression of both IL-6Rα and gp130 was modulated by LPS and Poly I:C in a cell line and time dependent manner. A modest increase of IL-6Rα expression was seen in RTS-11, RTL and RTG-2 cells after LPS stimulation, and a modest increase of gp130 expression was seen in RTG-2 and RTGill cells after Poly I:C stimulation (Fig. 6). However, gp130 expression was down-regulated (at 8 h) by both LPS and Poly I:C in RTS-11 cells, that express high levels of both IL-6Rα and gp130, and are known to be responsive to trout IL-6. The down-regulation of gp130 in trout macrophage may represent a control mechanism to limit their responsiveness to IL-6, perhaps in terms of autocrine signalling. It is interesting to note that Poly I:C also down-regulates the expression of JfGPH in leukocytes prepared from spleen, kidney and peripheral blood as well as in a natural embryo (HINAE) cell line (Santos et al. 2007). Clearly more work needs to be done on different cell populations and in parallel situations in different species to fully establish the mechanisms by which IL-6 receptor expression can be modulated in fish. Acknowledgments The work was supported by a European Community project IMAQUANIM (FOOD-CT-2005-007103). M.M.C. thanks the Consejo Superior de Investigaciones Científicas (CSIC, Spain) and the Xunta de Galicia for her “Ángeles Alvariño” postdoctoral contract. M.M.M. thanks the FCT (Foundation for Science and Technology, Portugal) and POPH/FSE for her PhD studentship (Grant no. SFRH/ BD/ 38236/ 2007). 17 References Abe K, Hirai M, Mizuno K, Higashi N, Sekimoto T, Miki T, Hirano T, Nakajima K (2001) The YXXQ motif in gp130 is crucial for STAT3 phosphorylation at Ser727 through an H7-sensitive kinase pathway. Oncogene 20:3464-3474. Abe T, Mikekado T, Haga S, Kisara Y, Watanabe K, Kurokawa T, Suzuki T (2007) Identification, cDNA cloning, and mRNA localization of a zebrafish ortholog of leukemia inhibitory factor. Comp Biochem Physiol B Biochem Mol Biol 147:38-44. Ancey C, Menet E, Corbi P, Fredj S, Garcia M, Rucker-Martin C, Bescond J, Morel F, Wijdenes J, Lecron JC, Potreau D. (2003) Human cardiomyocyte hypertrophy induced in vitro by gp130 stimulation. Cardiovasc Res 59:78-85. Aparicio S, Chapman J, Stupka E, Putnam N, Chia JM, Dehal P, Christoffels A, Rash S, Hoon S, Smit A, Gelpke MD, Roach J, Oh T, Ho IY, Wong M, Detter C, Verhoef F, Predki P, Tay A, Lucas S, Richardson P, Smith SF, Clark MS, Edwards YJ, Doggett N, Zharkikh A, Tavtigian SV, Pruss D, Barnstead M, Evans C, Baden H, Powell J, Glusman G, Rowen L, Hood L, Tan YH, Elgar G, Hawkins T, Venkatesh B, Rokhsar D, Brenner S (2002)Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes. Science 297:1301-1310. Assier E, Boissier MC, Dayer JM (2010) Interleukin-6: from identification of the cytokine to development of targeted treatments. Joint Bone Spine 77: 532-536. Baumann H, Symes AJ, Comeau MR, Morella KK, Wang Y, Friend D, Ziegler SF, Fink JS, Gearing DP (1994) Multiple regions within the cytoplasmic domains of the leukemia inhibitory factor receptor and gp130 cooperate in signal transduction in hepatic and neuronal cells. Mol Cell Biol 14:138-46. Bayne CJ, Gerwick L (2001) The acute phase response and innate immunity of fish. Dev Comp Immunol 25:725-743. Bazan JF (1990) Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci U S A 87:6934-6938. Bettelli E, Korn T, Kuchroo VK (2007) Th17: the third member of the effector T cell trilogy. Curr Opin Immunol 19:652-657. Boulanger MJ, Chow DC, Brevnova EE, Garcia KC (2003) Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp130 complex. Science 300:2101-2114. Chen W, Daines MO, Khurana Hershey GK (2004) Turning off signal transducer and activator of transcription (STAT): the negative regulation of STAT signaling. J Allergy Clin Immunol 114:476489. Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31: 3497-3500. Chomarat P, Banchereau J, Davoust J, Palucka AK (2000) IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat Immunol 1:510-514. Haan C, Heinrich PC, Behrmann I. (2002) Structural requirements of the interleukin-6 signal transducer gp130 for its interaction with Janus kinase 1: the receptor is crucial for kinase activation. Biochem J 361:105-111. Cole AR, Hall NE, Treutlein HR, Eddes JS, Reid GE, Moritz RL, Simpson RJ (1999) Disulfide bond structure and N-glycosylation sites of the extracellular domain of the human interleukin-6 receptor. J Biol Chem 274:7207-7215. Costa MM, Diaz-Rosales P, Secombes CJ, Wang T (2011) Bioactivity studies of rainbow trout (Oncorhynchus mykiss) interleukin-6: Effects on macrophage growth and antimicrobial peptide gene expression. Drucker C, Gewiese J, Malchow S, Scheller J, Rose-John S (2010) Impact of interleukin-6 classic- and transsignaling on liver damage and regeneration. J Autoimmun 34:29-37. Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953-971. Fasnacht N, Müller W (2008) Conditional gp130 deficient mouse mutants. Semin Cell Dev Biol 19: 379-384. 18 Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer EL, Eddy SR, Bateman A (2010) The Pfam protein families database. Nucleic Acids Res 38(Database issue): D211-222. Fischer P, Hilfiker-Kleiner D (2007) Survival pathways in hypertrophy and heart failure: the gp130-STAT axis. Basic Res Cardiol 102: 393-411. Fredj S, Bescond J, Louault C, Delwail A, Lecron JC, Potreau D 2005 Role of interleukin-6 in cardiomyocyte/cardiac fibroblast interactions during myocyte hypertrophy and fibroblast proliferation. J Cell Physiol 204:428-436. Fujiki K, Nakao M, Dixon B (2003) Molecular cloning and characterisation of a carp (Cyprinus carpio) cytokine-like cDNA that shares sequence similarity with IL-6 subfamily cytokines CNTF, OSM and LIF. Dev Comp Immunol 27:127-136. Funamoto M, Hishinuma S, Fujio Y, Matsuda Y, Kunisada K, Oh H, Negoro S, Tone E, Kishimoto T, Yamauchi-Takihara K (2000) Isolation and characterization of the murine cardiotrophin-1 gene: expression and norepinephrine-induced transcriptional activation. J Mol Cell Cardiol 32:1275-1284. Ganassin RC, Bols NC (1998) Development of a monocyte/macrophage-like cell line, RTS11, from rainbow trout spleen. Fish and Shellfish Immunol 8457-8476. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. Protein Identification and Analysis Tools on the ExPASy Server: Humana Press, 2005. Gerez J, Bonfiglio J, Sosa S, Giacomini D, Acuña M, Nagashima AC, Perone MJ, Silberstein S, Renner U, Stalla GK, Arzt E. (2007) Molecular transduction mechanisms of cytokine-hormone interactions: role of gp130 cytokines. Exp Physiol 92:801-816. Ghezzi P SE, Sacco S, Foddi C, Barbaccia ML, Mennini T (2000) Neurosteroid levels are increased in vivo after LPS treatment and negatively regulate LPS-induced TNF production. Eur Cytokine Netw 11:464-469. Hanington PC, Belosevic M (2005) Characterization of the leukemia inhibitory factor receptor in the goldfish (Carassius auratus). Fish Shellfish Immunol 18:359-369. Hanington PC, Belosevic M (2007) Interleukin-6 family cytokine M17 induces differentiation and nitric oxide response of goldfish (Carassius auratus L.) macrophages. Dev Comp Immunol 31:817-829. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, Schaper F (2003) Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 374:1-20. Hirano T (2010) Interleukin 6 in autoimmune and inflammatory diseases: a personal memoir. Proc Jpn Acad Ser B Phys Biol Sci 86:717-30. Hwang JY, Santos MD, Kondo H, Hirono I, Aoki T (2007) Identification, characterization and expression of a novel cytokine M17 homologue (MSH) in fish. Fish Shellfish Immunol 23:1256-1265. Huising MO, Kruiswijk CP, van Schijndel JE., Savelkoul HF, Flik G, Verburg-van Kemenade BM (2005) Multiple and highly divergent IL-11 genes in teleost fish. Immunogenetics 57:432-443. Huising MO, Kruiswijk CP, Flik G (2006) Phylogeny and evolution of class-I helical cytokines. J Endocrinol. 189:1-25. Jaillon O, Aury JM, Brunet F, Petit JL, Stange-Thomann N, Mauceli E, Bouneau L, Fischer C, Ozouf-Costaz C, Bernot A, Nicaud S, Jaffe D, Fisher S, Lutfalla G, Dossat C, Segurens B, Dasilva C, Salanoubat M, Levy M, Boudet N, Castellano S, Anthouard V, Jubin C, Castelli V, Katinka M, Vacherie B, Biémont C, Skalli Z, Cattolico L, Poulain J, De Berardinis V, Cruaud C, Duprat S, Brottier P, Coutanceau JP, Gouzy J, Parra G, Lardier G, Chapple C, McKernan KJ, McEwan P, Bosak S, Kellis M, Volff JN, Guigó R, Zody MC, Mesirov J, Lindblad-Toh K, Birren B, Nusbaum C, Kahn D, Robinson-Rechavi M, Laudet V, Schachter V, Quétier F, Saurin W, Scarpelli C, Wincker P, Lander ES, Weissenbach J, Roest Crollius H (2004) Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431: 946-957. Kurth I, Horsten U, Pflanz S, Timmermann A, Küster A, Dahmen H, Tacken I, Heinrich PC, Müller-Newen G (2000) Importance of the membrane-proximal extracellular domains for activation of the signal transducer glycoprotein 130. J Immunol 164: 273-282. Lai CF, Ripperger J, Morella KK, Wang Y, Gearing DP, Fey GH, Baumann H (1995) Separate signaling mechanisms are involved in the control of STAT protein activation and gene regulation via the interleukin 6 response element by the box 3 motif of gp130. J Biol Chem 270:14847-14850. 19 Lee LE, Clemons JH, Bechtel DG, Caldwell SJ, Han KB, Pasitschniak-Arts M, Mosser DD, Bols NC (1993) Development and characterization of a rainbow trout liver cell line expressing cytochrome P450dependent monooxygenase activity Cell Biol Toxicol 9: 279-294. Leong JS, Jantzen SG, von Schalburg KR, Cooper GA, Messmer AM, Liao NY, Munro S, Moore R, Holt RA, Jones SJ, Davidson WS, Koop BF (2010) Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome. BMC Genomics 11: 279. Liongue C, Ward AC (2007) Evolution of Class I cytokine receptors. BMC Evol Biol 7:120. Ma Y, Streiff RJ, Liu J, Spence MJ, Vestal RE (1999) Cloning and characterization of human oncostatin M promoter. Nucleic Acids Res 27:4649-4657. Moritz RL, Hall NE, Connolly LM, Simpson RJ (2001) Determination of the disulfide structure and Nglycosylation sites of the extracellular domain of the human signal transducer gp130. J Biol Chem 276:8244-8253. Murakami M, Narazaki M, Hibi M, Yawata H, Yasukawa K, Hamaguchi M, Taga T, Kishimoto T (1991) Critical cytoplasmic region of the interleukin 6 signal transducer gp130 is conserved in the cytokine receptor family. Proc Natl Acad Sci U S 88:11349-11353. Nishimichi N, Kawashima T, Hojyo S, Horiuchi H, Furusawa S, Matsuda H (2006) Characterization and expression analysis of a chicken interleukin-6 receptor alpha. Dev Comp Immunol 30:419-429. Nishimoto N, Kishimoto T (2006) Interleukin 6: from bench to bedside. Nat Clin Pract Rheumatol 2:619-626. Heinrich PC, Behrmann I, Müller-Newen G, Schaper F, Graeve L (1998) Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem J 334: 297-314. Oh JW, Revel M, Chebath J (1996) A soluble interleukin 6 receptor isolated from conditioned medium of human breast cancer cells is encoded by a differentially spliced mRNA. Cytokine 8:401-409. Pfeffer LM, Mullersman JE, Pfeffer SR, Murti A, Shi W, Yang CH (1997) STAT3 as an Adapter to Couple Phosphatidylinositol 3-Kinase to the IFNAR1 Chain of the Type I Interferon Receptor. Science 276: 1418-1420. Radtke S, Wüller S, Yang XP, Lippok BE, Mütze B, Mais C, de Leur HS, Bode JG, Gaestel M, Heinrich PC, Behrmann I, Schaper F, Hermanns HM (2010) Cross-regulation of cytokine signalling: proinflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation. J Cell Sci1 23: 947-959. Rose TM, Bruce AG (1991) Oncostatin M is a member of a cytokine family that includes leukemia-inhibitory factor, granulocyte colony-stimulating factor, and interleukin 6. Proc Natl Acad Sci U S A88: 86418645. Saito M, Yoshida K, Hibi M, Taga T, Kishimoto T (1992) Molecular cloning of a murine IL-6 receptorassociated signal transducer, gp130, and its regulated expression in vivo. J Immunol 148:4066-4071. Santos MD, Yasuike M, Kondo H, Hirono I, Aoki T (2007) A novel type-1 cytokine receptor from fish involved in the Janus kinase/signal transducers and activators of transcription (Jak/STAT) signal pathway. Mol Immunol 44:3355-3363. Santos MD, Yasuike M, Kondo H, Hirono I, Aoki T (2008) Teleostean IL11b exhibits complementing function to IL11a and expansive involvement in antibacterial and antiviral responses. Mol Immunol 45:3494-34501. Schiemann WP, Bartoe JL, Nathanson NM (1997) Box 3-independent signaling mechanisms are involved in leukemia inhibitory factor receptor alpha- and gp130-mediated stimulation of mitogenactivatedprotein kinase. Evidence for participation of multiple signaling pathways which converge at Ras. J Biol Chem 272:16631-16636. Schirmer K, Chan AG, Greenberg BM, Dixon DG, Bols NC (1998) Ability of 16 priority PAHs to be photocytotoxic to a cell line from the rainbow trout gill. Toxicology 127:143-155. Silver JS, Hunter CA (2010) gp130 at the nexus of inflammation, autoimmunity and cancer. J Leukoc Biol 88:1145-56. Rose-John S, Waetzig GH, Scheller J, Grötzinger J, Seegert D (2007) The IL-6/sIL-6R complex as a novel target for therapeutic approaches. Expert Opinion 11:613-624. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, and Kumar S (2011) MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution doi: 10.1093/molbev/msr121. Tanimoto K, Saito Y, Hamanaka I, Kuwahara K, Harada M, Takahashi N, Kawakami R, Nakagawa Y, Nakanishi M, Adachi Y, Shirakami G, Fukuda K, Yoshimura A, Nakao K (2005) SOCS1/JAB likely 20 mediates the protective effect of cardiotrophin-1 against lipopolysaccharide-induced left ventricular dysfunction in vivo. Circ J69:1412-1417. Thabard W CM, Mellerin MP, Puthier D, Barillé S, Bataille R, Amiot M (2001) IL-6 upregulates its own receptor on some human myeloma cell lines. Cytokine 14:352-356. Timmermann A, Kuster A, Kurth I, Heinrich PC, Muller-Newen G (2002) A functional role of the membraneproximal extracellular domains of the signal transducer gp130 in heterodimerization with the leukemia inhibitory factor receptor. Eur J Biochem 269:2716-2726. Varghese JN, Moritz RL, Lou MZ, Van Donkelaar A, Ji H, Ivancic N, Branson KM, Hall NE, Simpson RJ (2002) Structure of the extracellular domains of the human interleukin-6 receptor alpha -chain. Proc Natl Acad Sci U S A99:15959-15964. Vollmer P, Oppmann B, Voltz N, Fischer M, Rose-John S (1999) A role for the immunoglobulin-like domain of the human IL-6 receptor. Intracellular protein transport and shedding. Eur J Biochem 263:438-446. Waetzig GH, Chalaris A, Rosenstiel P, Suthaus J, Holland C, Karl N, Vallés Uriarte L, Till A, Scheller J, Grötzinger J, Schreiber S, Rose-John S, Seegert D (2010) N-linked glycosylation is essential for the stability but not the signalling function of the interleukin-6 signal transducer glycoprotein 130. J Biol Chem 285:1781-1789. Wang T, Holland JW, Bols N, Secombes CJ (2005) Cloning and expression of the first nonmammalian interleukin-11 gene in rainbow trout Oncorhynchus mykiss. Febs J272:1136-1147. Wang T, Secombes CJ (2009) Identification and expression analysis of two fish-specific IL-6 cytokine family members, the ciliary neurotrophic factor (CNTF)-like and M17 genes, in rainbow trout Oncorhynchus mykiss. Mol Immunol 46:2290-2298. Weaks RL, Ramsoondar JJ, Gallagher DS Jr, Nogues C, Piedrahita JA (1997) Isolation, characterization and chromosomal localization of the porcine ciliary neurotrophic factor (CNTF) gene. Anim Genet 28:354-357. Wolf K QM (1962) Established eurythermic line of fish cells in vitro. Science. 135:1065-1066. Wrighting DM, Andrews NC (2006) Interleukin-6 induces hepcidin expression through STAT3. Blood 108:3204-3209. Xu Y KN, Luo CS, Soo P, Pocock MJ, Czabotar PE, Hilton DJ, Nicola NA, Garrett TP, Zhang JG (2010) Crystal structure of the entire ectodomain of gp130: insights into the molecular assembly of the tall cytokine receptor complexes. J Biol Chem 285:21214-21218. 21 Figure legends. Figure 1. Multiple alignment of the deduced amino acid sequences of the trout IL-6Rα chain with selected IL-6Rα molecules from other species. The multiple alignment was produced using ClustalW2 and conserved amino acids shaded using BOXSHADE (V3.21). The signal peptide (SP), and the transmembrane domain (TM) are indicated by solid lines above the alignment. The highlighted features include the ten conserved cysteine residues in D1 and D2 domains, the two proline rich hinge regions (boxed) that separate the three domains (D1-3), and the WSXWS motif in the C-terminal of D3 domain in the extracellular region. The two sets of 13 amino acids located in the Ig-like D1 domain of salmonid IL-6Rα were indicated by a black box. Figure 2. Multiple alignment of the deduced amino acid sequences of trout gp130 with selected gp130 molecules from other species. The multiple alignment was produced using ClustalW2 and conserved amino acids shaded using BOXSHADE (V3.21). The signal peptide (SP), and the transmembrane domain (TM) are indicated by solid lines above the alignment. The six modular domains (D1 to 6) separated by the five prolinerich hinge regions are indicated. The features in the extracellular region include the eight conserved cysteine residues, the two conserved N-glycosylation sites (black bar) in the D1 and D2 domains, and the WSXWS motif in the D3 domain. The conserved three boxes (box-1 to 3), the serine rich region, the di-leucine dependent receptor internalisation motif, and the YSTV and four YXXQ (YXXQ1-4) motifs in the intracellular region are also highlighted. Figure 3. Phylogenetic tree analysis of the IL-6Rα (A) and gp130 (B) chains and their relatives. The evolutionary history was inferred using the Maximum Likelihood method based on the JTT matrix-based model using the MEGA5 program. The tree with the highest log likelihood is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Node values represent percent bootstrap confidence derived from 2,000 replicates. The molecule and the common species name are followed by the accession number. The new sequences reported in this review are shaded. Figure 4. PCR (A and B) and sequence (C) analysis of the two amino acid repeat sequences in the Iglike D1 domain of rainbow trout IL-6Rα. cDNA samples were prepared from different tissues (A) and cell lines (B) and subjected to PCR amplification as described in the Materials and Methods. The resultant products were revealed on 1% agarose gels containing ethidium bromide. The results from two fish and two samples from each cell line are shown. Note the two products seen with Fish B and the different sizes seen in different cell lines. Selected PCR products were cloned and sequence analysed (C). The two repeat sequences were found to have different frequencies in different fish and cell lines, with the first 39 bp repeat encoding for the 13 aa consensus [T/S]-[T/F]-VSTTT-[D]-TTSNG, and the second 39 bp repeat encoding for the 13 aa consensus TTVS-[A/T]-IKD-[D/G]-S-[K/D]-N-[G/R]. 22 Figure 5. The expression of IL-6Rα and gp130 in vivo. The expression of IL-6Rα and gp130 was analysed in 14 tissues from healthy fish by real-time PCR. The expression of transcripts was normalized against that of the housekeeping gene EF-1α and expressed as arbitrary units (A), where 1 arbitrary unit was equal to the average expression level of gp130 in intestine, which had the lowest expression level among the tissues studied. The ratio of the expression level of IL-6Rα and gp130 in the same tissue is presented in (B), where the number over the bar is the average expression level of IL-6Rα divided by that of gp130. Data represent the average + SEM from six healthy fish. Significant results of a paired sample t-test between the expression levels of IL-6Rα and gp130 in the same tissue is shown above the bars as: *p<0.05, **p≤0. 01 and ***p≤0.001. Figure 6. Modulation of IL-6Rα and gp130 expression in four trout cell lines. The relative constitutive expression of IL-6Rα and gp130 in four trout cell lines RTL, RTG-2, RTGill and RTS-11, was examined by real-time PCR and expressed as arbitrary units (A), where 1 arbitrary unit was equal to the average expression of IL-6Rα in the RTL cell line, which had the lowest expression level among the cell lines tested. The four trout cell lines were then stimulated with lipopolysaccharide (LPS, 25 µg/ml) and polyinosinic: polycytidylic acid (Poly I:C, 50 µg/ml) for 4, 8 and 24 h and the expression of IL-6Rα (B) and gp130 (C) also examined by real-time RT-PCR and expressed as a fold change relative to time-matched controls. The p-value of a LSD post hoc test after a significant one way-analysis of variance between the stimulated and time matched controls is shown above the bars as: *p < 0.05; **p≤0.01; ***p≤0.001. 23