Characterization of the responses of the caspase 2, 3, 6 and 8 genes to immune challenges and extracellular ATP stimulation in the Japanese flounder (Paralichthys olivaceus)

Abstract

Background: Caspases are a family of conserved intracellular cysteine-dependent aspartate-specific cysteine proteases that play important roles in regulating cell death and inflammation. Our previous study revealed the importance of the inflammatory caspase 1 gene in extracellular ATP-mediated immune signaling in Japanese flounder, Paralichthys olivaceus. To explore the potential roles of other caspases in P. olivaceus innate immunity, we extended our study by characterizing of the responses of four additional P. olivaceus caspase genes, termed JfCaspase 2, 3, 6 and 8, to inflammatory challenge and extracellular ATP stimulation.

Results: Sequence analysis revealed that the domain structures of all the Japanese flounder caspase proteins are evolutionarily conserved. Quantitative real-time PCR analysis showed that the JfCaspase 2, 3, 6 and 8 genes were expressed ubiquitously but at unequal levels in all examined Japanese flounder normal tissues. In addition, the basal gene expression levels of JfCaspase 2, 3, 6 and 8 were higher than those of JfCaspase 1 in both Japanese flounder head kidney macrophages (HKMs) and peripheral blood leukocytes (PBLs). Furthermore, immune challenge experiments showed that the inflammatory stimuli LPS and poly(I:C) significantly modulated the expression of the JfCaspase 2, 3, 6 and 8 genes in Japanese flounder immune cells. Finally, DNA fragmentation, associated with increased extracellular ATP-induced JfCaspase 2, 3, 6 and 8 gene expression and enzymatic activity, was inhibited by the caspase inhibitor Z-VAD-FMK in the HKMs.

Conclusion: Our findings demonstrate broad participation of multiple caspase genes in response to inflammatory stimulation in Japanese flounder immune cells and provide new evidence for the involvement of caspase(s) in extracellular ATP-induced apoptosis in fish.

Keywords: Apoptosis; Caspases; Extracellular ATP; Immune response; Paralichthys olivaceus; Teleost.

Fig. 1

Domain organization and sequence alignment of Japanese flounder caspase proteins. a Schematic protein domain architecture of Japanese flounder caspase proteins. CARD: caspase recruitment domain; DED: death effector domain; CASc: caspase, interleukin-1 beta converting enzyme (ICE) homologues. b Multiple alignment of the amino acid sequences of Japanese flounder caspase proteins. The CARD domain, DED domain and CASc domains are boxed in red, yellow and purple, respectively. The conserved QACR/QG pentapeptide active site motif in all the JfCaspase proteins is denoted with stars. The numbers on the right-hand and left-hand sides are the numbers of amino acids in the JfCaspase proteins. The GenBank accession numbers of the caspase proteins from Paralichths olivaceus are ARI72010.1 (caspase 1), XP_019948600.1 (caspase 2), AFC60626.1 (caspase 3), XP_019956800.1 (caspase 6), and XP_019955218.1 (caspase 8)

Fig. 2

Phylogenetic relationships of Japanese flounder caspase proteins. Phylogenetic relationships among the Japanese flounder caspase 1, 2, 3, 6 and 8 proteins and their counterparts in other teleost species were determined using the ClustalW program, and the bootstrap-consensus neighbor-joining phylogenetic tree was built using MEGA software version 5.0 with the default parameters. The GenBank accession numbers for the caspases proteins from the different species are as follows: Oreochromis niloticus caspase 1: XP_005459436.1, caspase 2: XP_005455468.1, caspase 3: NP_001269823.1, caspase 6: XP_013131214.1, caspase 8: XP_003457507.2; Maylandia zebra caspase 1: XP_004543706.1, caspase 2: XP_004550818.1, caspase 3: XP_004549328.1, caspase 6: XP_014268841.1, caspase 8: XP_012778477.1; Larimichthy crocea caspase 1: KKF14496.1, caspase 2: KKF24100.1, caspase 6: KKF09831.1, caspase 8: KKF31210.1; Takifugu ruubripes caspase 1R: XP_003979716.1, caspase 2R: XP_011615364.1, caspase 3: AAM43816.1, caspase 6: XP_003972493.2; Cynoglossus semilaevis caspase 6: XP_008315389.1; and those in the list in the legend of Fig. 1

Fig. 3

Expression of JfCaspase mRNA transcripts in Japanese flounder tissues and immune cells. a qRT-PCR analysis of the relative gene expression levels of JfCaspase 1, 2, 3, 6 and 8 in Japanese flounder normal tissues. Beta-actin served as an internal reference gene. The error bars indicate the standard deviation from triplicate experiments. b qRT-PCR analysis of the relative gene expression levels of JfCaspase 1, 2, 3, 6, 8 and 10 in Japanese flounder head kidney macrophages (HKMs) and peripheral blood leukocytes (PBLs), with beta-actin serving as an internal control gene

Fig. 4

LPS- and poly(I:C)-induced JfCaspase 1, 2, 3, 6 and 8 gene expression in Japanese flounder HKMs. Overnight-cultured Japanese flounder HKMs were stimulated with 20 μg/ml LPS (left column) or poly(I:C) (right column) for the indicated times. Total RNA from the cells was purified and transcribed into cDNA. JfCaspase 1, 2, 3, 6 and 8 gene expression changes compared with the expression in the untreated controls were determined by qRT-PCR analysis. The values are presented as the means ± standard deviation (n = 3)

Fig. 5

LPS- and poly(I:C)-induced JfCaspase 1, 2, 3, 6 and 8 gene expression in Japanese flounder PBLs. Overnight-cultured Japanese flounder PBLs were stimulated with 20 μg/ml LPS (left column) or poly(I:C) (right column) for the indicated times. PAMP-induced changes in JfCaspase 1, 2, 3, 6 and 8 gene expression were determined by qRT-PCR analysis. The values are presented as the means ± standard deviation (n = 3)

Fig. 6

Extracellular ATP-induced changes in JfCaspase gene expression, enzymatic activity and apoptosis in Japanese flounder head kidney macrophages. a JfCaspase 2, 3, 6 and 8 gene expression changes following 1 mM ATP stimulation in the HKMs were measured by qRT-PCR with beta-actin as an internal reference gene. b The eATP-induced changes in JfCaspase 2, 3, 6 and 8 enzymatic activity in Japanese flounder HKMs were measured with the respective enzyme substrates (Ac-VDQQD-pNA, Ac-DEVD-pNA, Ac-VEID-pNA and Ac-IETD-pNA, respectively), and are presented as the pNA levels calculated from a standard curve. Asterisks (*) indicate significant differences in JfCaspase activity compared with that of the untreated control group (p < 0.05). All data are presented as the means ± standard deviation of triplicate determinations from one representative experiment. The other two independent experiments showed similar results. c eATP-induced changes in DNA fragmentation in the HKMs. Untreated HKMs served as controls to show intact genomic DNA. In a parallel experiment, HKMs were treated with 1 mM ATP for 12 or 24 h. After that, genomic DNA was extracted, and DNA fragments were assessed by 2% agarose gel electrophoresis and ethidium bromide staining. M: 100 bp DNA ladder (Fermentas). d Inhibition of caspase enzymatic activity inhibited eATP-induced DNA fragmentation in HKMs. HKMs were pre-incubated with or without 100 μM pan-caspase inhibitor Z-VAD-FMK for 30 min and then treated with 1 mM ATP for 12 or 24 h in the presence or absence of 100 μM Z-VAD-FMK. DNA fragments were separated by 2% agarose gel electrophoresis and visualized with ethidium bromide staining