Molecular Characterization and Expression Analysis of ATP-Gated P2X7 Receptor Involved in Japanese Flounder (<i>Paralichthys olivaceus</i>) Innate Immune Response

<div><p>ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish <i>P2RX7</i> homologue cDNA, termed <i>poP2RX7</i>, in Japanese flounder (<i>Paralichthys olivaceus</i>). PoP2RX7 protein shares about 60–88% sequence similarity and 45–78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. <i>poP2RX7</i> mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. <i>poP2RX7</i> mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. <i>In vivo</i> experiments further revealed that <i>poP2RX7</i> gene expression was substantially up-regulated by immune challenge with infectious bacteria <i>Edwardsiella tarda</i> and <i>Vibrio anguillarum</i>. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines <i>IL-1β</i> and <i>IL-6</i> in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.</p></div

Multiple sequence alignment of poP2X7 receptor with selected P2X7 receptor proteins.

<p>Sequence alignment was carried out by ClustalW program. Representative P2X7 receptor proteins from different species with GenBank accession numbers are hP2X7 (<i>Homo sapiens</i>, NP_002553), rP2X7 (<i>Rattus norvegicus</i>, NP_062129), sP2X7 (<i>Sparus aurata</i>, CAI59608), zP2X7 (<i>Danio rerio</i>, AAI63071) and poP2X7 (<i>Paralichthys olivaceus</i>, KC748421). Highly conserved (:), less conserved (.) and identical (*) amino acid residues identified in all the proteins are indicated. TM: transmembrane domain. The P2X family signature motif (²⁴⁴Gly–²⁷⁰Phe) was boxed in black. The two residues ¹²⁷Lys and ²⁸⁴Asn accounted for species difference of P2X7 receptors in ATP/BzATP agonist sensitivity were boxed in green <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096625#pone.0096625-Young1" target="_blank">[28]</a>. Five important residues for nucleotide binding were boxed in red and the predicted LPS/lipid-binding domain was boxed in yellow.</p

Sequence of primers used in this study.

<p>Sequence of primers used in this study.</p

Phylogenetic relationship of Japanese flounder poP2X7 with selected vertebrate P2X receptor family proteins.

<p>Maximum-likelihood phylogenetic tree was generated using MEGA 5.1 program. The bar indicates the distance and the number at each node indicates the percentage of bootstrapping after 1000 replications. The GenBank accession numbers of selected P2X receptor proteins are hP2X1 (AAC24494.1), rP2X1 (NP_037129.1), mP2X1 (NP_032797.3), bP2X1 (NP_001192729.1), zP2X1 (NP_945333.1), tP2X1 (XP_003458088.1), fP2X1 (XP_003977018.1), hP2X2 (NP_733782.1), rP2X2 (NP_446108.2), mP2X2 (AAK95327.2), bP2X2 (NP_001179572.1), zP2X2 (NP_945334.1), tP2X2 (XP_003451709.1), fP2X2 (XP_003974964.1), hP2X3 (NP_002550.2), rP2X3 (NP_112337.2), mP2X3 (NP_663501.2), bP2X3 (XP_608941.3), zP2X3 (NP_945337.2), tP2X3 (XP_003456590.1), fP2X3 (XP_003972130.1), hP2X4 (NP_001243725.1), rP2X4 (NP_113782.1), mP2X4 (NP_035156.2), bP2X4 (NP_001029221.1), zP2X4 (NP_705939.1), tP2X4 (XP_003448602.1), fP2X4 (XP_003974770.1), hP2X5 (NP_002552.2), rP2X5 (NP_542958.2), mP2X5 (NP_201578.2), bP2X5 (XP_005195684.1), zP2X5 (NP_919394.1), fP2X5 (XP_003976410.1), tP2X5 (XP_003456207.1), hP2X6 (AAF13303.1), rP2X6 (CAA66044.1), bP2X6 (XP_005195206.1), hP2X7 (NP_002553), rP2X7 (NP_062129), bP2X7 (NP_001193445), mP2X7 (CAD33539), zP2X7 (NP_945335), fP2X7 (XP_003974725), tP2X7 (XP_003444500.1) and poP2X7 (KC748421). h, Human (<i>Homo sapiens</i>); r, rat (<i>Rattus norvegicus</i>); m, mouse (<i>Mus musculus</i>); b, cattle (<i>Bos Taurus</i>); z, zebrafish (<i>Danio rerio</i>); f, fugu (<i>Takifugu rubripes</i>); t, Nile tilapia (<i>Oreochromis niloticus</i>); po, Japanese flounder (<i>Paralichthys olivaceus</i>).</p

Characterization of poP2RX7 permeability to NMDG⁺.

<p>A. Representative −100/+40 mV ramp experiment was applied to an oocyte expressing the poP2RX7 and bathed with NMDG⁺ media. For clarity only two ramps are shown, corresponding to the beginning and after 3 min of continuous application of 1 mM ATP. The values of reversal potential are shown by arrows. B. The same protocol was applied to an oocyte expressing the rP2RX7 and bathed with NMDG⁺ media. C. Representative ramp protocol applied to an oocyte expressing the poP2RX7 and bathed with LD media, no change in reversal potential is observed under these conditions. D. Summary of the changes in reversal potential (ΔE_rev) after 3 min of ATP application in oocytes expressing the poP2RX7 or the rP2RX7 bathed in NMDG⁺ or LD media. *<i>p</i><0.05, estimated by Mann-Whitney test. n = 3–7.</p

LPS and Poly(I:C)-induced gene expression of <i>poP2RX7</i> in <i>P. olivaceus</i> head kidney primary cells.

<p><i>P. olivaceus</i> head kidney primary cells were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096625#s2" target="_blank"><i>Material and methods</i></a> and stimulated with 25 µg/ml (final concentration) LPS (A) or Poly(I:C) (B). Total RNA from different time points (0, 2, 4, 6, 8, 12 and 24 h post stimulation) was extracted and the gene expression changes of <i>poP2RX7</i> were determined by qRT-PCR. <i>β-actin</i> was employed as an internal reference gene. Values labeled with different <i>lowercase</i> letters indicate significant difference (<i>p</i><0.05) among treatments. Asterisks (*) mark the significant up-regulation of <i>poP2RX7</i> mRNA compared with the untreated control group (<i>p</i><0.05). Data in this and following figures are the mean ± standard deviation of triplicate determinations from one representative experiment; similar results were obtained on two other separate experiments.</p

Electrophysiological properties of poP2RX7 expressed in <i>Xenopus</i> oocytes.

<p>A and B. Representative recordings of individual oocytes expressing poP2RX7 (A) or rP2RX7 (B), currents were gated with 1 mM ATP dissolved in Barth's or low-divalent (LD) media. (C). Summary of the peak amplitudes obtained in Barth's or LD media in oocytes expressing poP2RX7 or rP2RX7. D and E. Currents evoked by 1 mM ATP and 100 µM BzATP from individual oocytes expressing poP2RX7 (D) or rP2RX7 (E), in LD media. (F). Concentration-response curves for BzATP (open squares) and ATP (black circles) for poP2RX7 (left graph) or rP2RX7 (right graph). (G). Current facilitation of poP2RX7 (left recordings) and rP2RX7 (right recordings). In each case, three consecutive ATP pulses were applied to the same oocyte. (H). Representative recording of poP2RX7-expressing oocytes showing the currents evoked by ATP alone and the inhibition induced by pre-application with BBG for 2 min followed by an co-application with 1 µM BBG (left recordings) or 20 µM AZ10606120 (right recordings). (I). Summary of the inhibition induced by BBG or AZ10606120 (AZ) at poP2RX7 (open bars) and rP2RX7 (black bars). *<i>p</i><0.05, compared with rP2RX7 by Mann-Whitney test, n = 3–5.</p

Tissue distribution of Japanese flounder <i>poP2RX7</i> mRNA.

<p><i>poP2RX7</i> mRNA expression in healthy <i>P. olivaceus</i> tissues was analyzed by qRT-PCR with <i>β-actin</i> as an internal reference gene. Bl: blood; Br: brain; Gi: gill; HK: head kidney; TK: trunk kidney; He: heart; Hp: hepatopancreas; Sk: skin; Go: gonad; Sp: spleen; In: intestine. The identities of all PCR products were confirmed by DNA sequencing. Values are presented as means ± standard deviation from triplicate experiments.</p

Temporal expression analysis of <i>poP2RX7</i> mRNA transcripts in response to bacterial infections by real-time PCR.

<p>Animals (average 20±3 g) were injected intraperitoneally with <i>E. tarda</i> or <i>V. anguillarum</i> and sacrificed at 0, 4, 8, 12, 24, 36 and 48 h after injection. Head kidney, spleen and gill tissues from five individual animals were dissected, collected at each time point and total RNA of each tissue was pooled. The expression changes of <i>poP2RX7</i> mRNA in head kidney (A and B), gill (C and D) and spleen (E and F) upon <i>E. tarda</i> (A, C and E) and <i>V. anguillarum</i> (B, D and F) infections are relative to <i>poP2RX7</i> gene expression in control experiment of each time point (normalized to 1). <i>β-actin</i> was employed as an internal reference gene. Values marked with different <i>lowercase</i> letters indicate significant difference (<i>p</i><0.05) among treatments.</p

PoP2RX7-mediated gene expression of <i>IL-1β</i> and <i>IL-6</i> in Japanese flounder head kidney primary cells.

<p>(A–D) The involvement of poP2RX7 in ATP-evoked <i>IL-1β</i> and <i>IL-6</i> expression. Japanese flounder head kidney primary cells (1.0×10⁷/well) were pre-incubated with or without selective P2RX7 inhibitors BBG or A-740003 for 2 h and then co-treated with 100, 500 µM ATP or 100 µM BzATP for 30 min to active poP2RX7. The cells were finally incubated with normal culture medium for 2 h and used for RNA isolation. (E) The involvement of poP2RX7 in LPS-induced <i>IL-1β</i> gene expression. Japanese flounder head kidney primary cells were pre-treated with or without P2RX7 antagonist A-740003 (final concentration, 10 µM) for 2 h and then primed with 20 µg/ml LPS in the presence or absence of the P2X7R antagonist for 3 h. The gene expression changes of <i>IL-1β</i> (A, B and E) and <i>IL-6</i> (C, D) were determined by qRT-PCR with <i>β-actin</i> as an internal reference gene. Asterisks (*) mark the significant difference between experimental and untreated control groups (normalized to 1), <i>p</i><0.05. ^#, values differ at <i>p</i><0.05.</p