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).

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.

Expression and functional characterization of vitronectin gene from Japanese flounder (Paralichthys olivaceus).

Vitronectin (Vtn) is a multifunctional protein that plays significant roles in cell adhesion, migration, spreading and survival, and in the regulation of membrane attack complex formation and the terminal pathway of complement activation in innate immune response. However, the expression and immune significance of Vtn in fish remains largely unknown. In order to understand the involvement of Vtn in fish innate immune response, here we cloned and characterized a full-length Vtn ortholog cDNA, termed PoVtn, from Japanese flounder Paralichthys olivaceus. The deduced PoVtn protein is comprised of 438 amino acids with a 19-amino-acid signal peptide sequence (1Met-19Ala) at the N-terminus. Protein domain analysis revealed that PoVtn possesses a conserved N-terminal somatomedin B domain followed by a conserved RGD motif and four haemopexin-like domains. Sequence analysis revealed that PoVtn has two potential glycosylation sites and shares 44-74% sequence identity with other teleost Vtn proteins. PoVtn mRNA was ubiquitously distributed in all examined normal tissues and showed the highest expression in Japanese flounder hepatopancreas tissue. PoVtn expression was induced by LPS and poly(I:C) challenges in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) and shows a pathogen-associated molecular pattern- and cell type-dependent manner. The expression of PoVtn was also modulated by bacterial challenge with Edwardsiella tarda in Japanese flounder immune-related tissues including head kidney, gill and spleen. Furthermore, overexpression of PoVtn in Japanese flounder FG-9307 cells significantly attenuated the LPS- and poly(I:C)-induced proinflammatory cytokines IL-1beta and TNF-alpha gene expression. Taken our findings together, we for the first time characterized Vtn gene expression in response to inflammatory stimuli in fish. Our results suggested a potential role of PoVtn in regulating fish innate immunity.

Comparative analysis of dual specificity protein phosphatase genes 1, 2 and 5 in response to immune challenges in Japanese flounder Paralichthys olivaceus.

Dual-specificity MAP kinase (MAPK) phosphatases (DUSPs) are well-established negative modulators in regulating MAPK signaling in mammalian cells and tissues. Our previous studies have shown the involvement of DUSP6 in regulating innate immunity in Japanese flounder Paralichthys olivaceus. In order to gain a better understanding of the role of DUSPs in fish innate immunity, in the present study we identified and characterized three additional DUSP genes including DUSP1, 2 and 5 in P. olivaceus. The three Japanese flounder DUSP proteins share common domain structures composed of a conserved N-terminal Rhodanase/CDC25 domain and a C-terminal catalytic phosphatase domain, while they show only less than 26% sequence identities, indicating that they may have different substrate selectivity. In addition, mRNA transcripts of all the three DUSP genes are detected in all examined Japanese flounder tissues; however, DUSP1 is dominantly expressed in spleen while DUSP2 and 5 are primarily expressed in skin. Furthermore, all the three DUSP genes are constitutively expressed in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) with unequal distribution patterns. Moreover, all the three DUSPs gene expression was induced differently in response to the LPS and double-stranded RNA mimic poly(I:C) stimulations both in the Japanese flounder HKMs and PBLs, suggesting an association of DUSPs with TLR signaling in fish. Taken together, the co-expression of various DUSPs members together with their different responses to the immune challenges indicate that the DUSP members may operate coordinately in regulating the MAPK-dependent immune responses in the Japanese flounder.

Characterization of Japanese flounder (Paralichthys olivaceus) Caspase1 involved in extracellular ATP-mediated immune signaling in fish.

Caspase1 is a member of inflammatory Caspases that play important roles in the innate immune system. Although several teleost caspase1 genes have been identified, their partner proteins and implication in extracellular ATP-mediated immune signaling in fish are still very limited. Here we identified and characterized a caspase1 gene, named JfCaspase1, from Japanese flounder Paralichthys olivaceus. JfCaspase1 mRNA was constitutively expressed in all examined normal tissues with high expression in skin and gills and moderate expression in the enriched Japanese flounder head kidney macrophages (HKMs) and peripheral blood leukocytes (PBLs). JfCaspase1 was initially down-regulated but significantly up-regulated at the later stage upon LPS and poly(I:C) challenges in the HKMs. JfCaspase1 was also up-regulated in the Japanese flounder immune-related tissues including head kidney, gill and spleen by bacterial challenge with Edwardsiella tarda. JfCaspase1 protein is comprised of 384 amino acid residues with a calculated molecular mass of 43.75 kDa and is phylogenetically close to fish Caspase1 proteins. JfCaspase1 was co-immunopercipitated with Japanese flounder apoptosis-associated speck-like protein (ASC) when co-expressed in HeLa cells, suggesting that there is a potential interaction between the two proteins. In addition, we showed that extracellular ATP, a potent signaling molecule in activating innate immune response, rapidly up-regulates JfCaspase1 expression and enhances its enzymatic activity both in the HKMs and PBLs. Our findings indicated that the inflammatory JfCaspase1 interacted with ASC protein is implicated in the extracellular ATP-mediated immune signaling in fish.

Identification and functional analysis of dual-specificity MAP kinase phosphatase 6 gene (dusp6) in response to immune challenges in Japanese flounder Paralichthys olivaceus.

Dual-specificity phosphatase 6 (Dusp6) is a member of mitogen-activated protein kinase (MAPK) phosphatases that play crucial roles in regulating MAPK signaling and immune response. The immunological relevance of Dusp6 in fish, however, remains largely uncharacterized. In the present study, a full-length Japanese flounder dusp6 cDNA ortholog, termed PoDusp6, was identified and characterized from Paralichthys olivaceus. The deduced PoDusp6 protein is comprised of 383 amino acids with a conserved N-terminal regulatory rhodanese homology domain and a C-terminal catalytic domain. Immunofluorescence microscopy revealed that PoDusp6 protein is mainly localized in cytoplasm. Sequence analysis indicates that PoDusp6 is highly conserved (>70% identity) throughout the evolution from teleost to mammals. In unstimulated conditions, PoDusp6 mRNA was present in all examined tissues and showed the highest expression in Japanese flounder head kidney macrophages (HKMs). Immune challenge experiments revealed that the expression of PoDusp6 was down-regulated at the early stage after LPS and poly(I:C) stimulations but significantly up-regulated at the later stage in the HKMs. The similar expression pattern was also observed in the Japanese flounder immune-related tissues including head kidney, gill and spleen upon bacterial challenge with Edwardsiella tarda. Overexpression of PoDusp6 in Japanese flounder FG-9307 cells led to a significant down-regulation of proinflammatory cytokine genes IL-1beta, TNF-alpha and IFN-gamma, and antiviral gene Mx. Interestingly, inhibition of Dusp6 activity also down-regulated the LPS-induced IL-beta gene expression but did not affected on the LPS-induced IFN-gamma and TNF-alpha expression in the HKMs. Our findings suggest that the expression of PoDusp6 is modulated by immune stimuli and PoDusp6 may act as an essential modulator in fish inflammatory response.

Identification and characterization of nascent polypeptide-associated complex alpha from Chinese mitten crab (Eriocheir sinensis): A novel stress and immune response gene in crustaceans.

Disease in aquatic animals is tightly linked to environmental challenges and their immune responses are greatly modified by their external environment. The chaperone protein nascent polypeptide-associated complex alpha (NACA) has been suggested to play important roles in the cellular response to stress and immune challenges, while the related biological functions remain largely unknown in invertebrates. In the present study we identified a NACA gene (termed EsNACA) from Chinese mitten crab Eriocheir sinensis and analyzed its expression changes in response to ambient (salinity and pH) stresses and immune challenges. The EsNACA protein is comprised of 209 amino acid residues with a conserved DNA binding domain, a C-Jun binding domain, a NAC domain and an ubiquitin-associated domain and shows the highest sequence identity (87%) with its counterpart in shrimp Penaeus monodon. EsNACA mRNA transcripts are presented in all tested normal tissues with predominant expression in hepatopancreas and lower expression in hemocytes. In addition, EsNACA expression was significantly altered in response to the ambient salinity (15‰ and 30‰ salinities) and pH (pH 6 and 8.5) stresses in gill, hepatopancreas, muscle, hemocytes and intestine tissues. Furthermore, EsNACA gene expression was substantially induced upon LPS and Poly(I:C) immune stimulations in E. sinensis hemocytes in vitro. Finally, EsNACA expression was up-regulated in E. sinensis hemocytes, gill, hepatopancreas, intestine and muscle tissues in response to Vibrio anguillarum challenges in vivo. Taken together, our findings for the first time show that EsNACA is an inducible gene involved in stress and immune response in crustaceans.

Expression and role of gap junction protein connexin43 in immune challenge-induced extracellular ATP release in Japanese flounder (Paralichthys olivaceus).

Connexin43 (Cx43) is the best characterized gap junction protein that allows the direct exchange of signaling molecules during cell-to-cell communications. The immunological functions and ATP permeable properties of Cx43 have been insensitively examined in mammals. The similar biological significance of Cx43 in lower vertebrates, however, is not yet understood. In the present study we identified and characterized a Cx43 ortholog (termed PoCx43) from Japanese flounder (Paralichthys olivaceus) and investigated its role in immune challenge-induced extracellular ATP release. PoCx43 mRNA transcripts are widely distributed in all tested normal tissues and cells with predominant expression in the brain, and are significantly up-regulated by LPS, poly(I:C) and zymosan challenges and Edwardsiella tarda infections as well, suggesting that PoCx43 expression was modulated by the inflammatory stresses. In addition, cyclic AMP (cAMP), an essential second messenger, also plays an important role in regulating PoCx43 gene expression, by which the PoCx43-mediated gap junctional communication may be regulated. Furthermore, overexpression of PoCx43 in Japanese flounder FG-9307 cells significantly potentiates the LPS- and poly(I:C)-induced extracellular ATP release and this enhanced ATP release was attenuated by pre-incubation with Cx43 inhibitor carbenoxolone. In a complementary experiment, down-regulation of PoCx43 endogenous expression in FG-9307 cells with small interfering RNA also significantly reduced the PAMP-induced extracellular ATP release, suggesting that PoCx43 is an important ATP release conduit under the immune challenge conditions. Finally, we showed that extracellular ATP stimulation led to an increased PoCx43 expression which probably provides a feedback mechanism in regulating PoCx43 expression at the transcriptional level. These findings suggest that PoCx43 is an inducible immune response gene and an important conduit for immune challenge-induced extracellular ATP release in fish.

Cloning and characterization of apoptosis-associated speck-like protein containing a CARD domain (ASC) gene from Japanese flounder Paralichthys olivaceus.

Apoptosis-associated speck-like protein containing a CARD domain (ASC) is a critical adaptor molecule in multiple inflammasome protein complexes that mediate inflammation and host defense. However, few studies have been performed in lower vertebrates such as in teleost. Here we identified and characterized a novel ASC gene (namely PoASC) from Japanese flounder Paralichthys olivaceus. The complete cDNA sequence of PoASC contains a 22 bp 5'-untranslated sequence, a 612 bp open reading frame, and a 438 bp 3'-untranslated sequence. The deduced PoASC protein is comprised of 203 amino acids with a conserved N-terminal PYD domain and a C-terminal CARD domain and shows 35-62% sequence identity with other vertebrate ASC proteins. PoASC mRNA transcripts was detected in various Japanese flounder tissues and is dominantly expressed in hepatopancreas. Oligomeric speck-like structures were observed when PoASC was exogenously expressed in Japanese flounder FG-9307 cells. Immune challenge experiments revealed that PoASC gene expression was significantly induced in the Japanese flounder head kidney macrophages and peripheral blood leukocytes by the canonical TLR ligands LPS, Poly(I:C) and zymosan stimulations. In addition, the induction of PoASC was also observed in Edwardsiella tarda challenged head kidney and gill tissues. Furthermore, we for the first time showed that extracellular ATP, an important signaling molecule in triggering innate immune response and activation of NLR inflammasome, significantly up-regulates PoASC expression in the Japanese flounder head kidney macrophages in a dose-dependent manner. Together, these findings addressed the involvement of PoASC in TLR and extracellular ATP-mediated innate immune signaling in the Japanese flounders.