Haizhou Wang

The transcription regulation analysis of Ctenopharyngodon idellus PKR and PKZ genes.

Protein kinase R (PKR), the double-stranded RNA-activated protein kinase, exists in mammalian and fish. PKZ, a PKR-like protein kinase containing Z-DNA binding domains, just exists in fish. PKR and PKZ work synergistically in the antiviral defense by inhibiting intracellular protein translation. The transcriptional factor IRF3 (interferon regulatory factor 3) acts as a key regulator of type I IFN (Interferon) and ISG (interferon stimulated gene). On the basis of the cloned CiIRF3 previously, CiIRF3 with His-tag was over-expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin. In this study, we have demonstrated that grass carp (Ctenopharyngodon idellus) PKR (CiPKR) and PKZ (CiPKZ) genes were inducible by Poly I:C in C. idella kidney (CIK) cells. So, they might be implicated in the intracellular antiviral activity. To understand the up regulatory mechanism of CiPKR and CiPKZ genes upon virus induction, we constructed wild type (pGL3-CiPKR-luc and pGL3-CiPKZ-luc) and the mutant (pGL3-CiPKR-nISRE-luc and pGL3-CiPKZ-nISRE-luc) reporter gene vectors according to the promoter sequences of CiPKR (KJ704845) and CiPKZ (KJ704844). In vitro, gel mobility shift assays demonstrated that CiIRF3 can combine CiPKR and CiPKZ promoters with high affinity. However, CiIRF3 bound to the mutants CiPKR-nISRE and CiPKZ-nISRE faintly. Whereafter, the recombinant plasmids of pGL3-CiPKR-luc, pGL3-CiPKZ-luc were transiently co-transfected with pcDNA3.1-CiIRF3, pcDNA3.1-CiIRF7 respectively into CIK cells. Cell transfection assays indicated that CiIRF3 and CiIRF7 up-regulated the transcriptional level of CiPKR and CiPKZ. The results also revealed that the consensus sequence of ISRE (interferon stimulated response element) is an important regulatory element for the transcriptional initiation of CiPKR and CiPKZ.

Ctenopharyngodon idella PKZ facilitates cell apoptosis through phosphorylating eIF2α.

PKZ, protein kinase containing Z-DNA binding domains, is a novel member of eIF2α kinase in fish. PKZ can be up-regulated in response to Poly I:C or heat stress, and it has a typical eIF2α kinase activity. However, the relationship between fish PKZ and apoptosis remains unclear. In the present study, effects of PKZ on apoptosis were explored. Initially, we found that PKZ and PKR were up-regulated by poly I:C in a time-dependent manner. Q-PCR analysis showed that the change of Caspase-3 mRNA was consistent with that of PKZ under the stimulation of poly I:C or transfection with PKZ siRNA in CIK cells. It suggested that PKZ might mediate the induction of apoptosis. Knockdown and overexpression assays indicated that a significant increase of apoptotic cell number was observed in the wild type PKZ (PKZ-wt) transfected PKZ-deficient cells, while mutant PKZ-K198R lost this ability. MTT also showed that overexpression of PKZ-wt in CIK cells resulted in a striking decrease of cell viability rate to about 32.5%, whereas the viability of cell with PKZ-K198R was about 88.1%. Likewise, when transfected eIF2α-wt or phosphomimetic eIF2α (S51D), CIK cells displayed higher apoptotic rate than the controls. In contrast, overexpression of phosphodeficient eIF2α (S51A) blocked induction of apoptosis. In addition, levels of eIF2α phosphorylation were significantly related to apoptosis in these CIK cells. Furthermore, when mutant eIF2α (S51A) was transiently transfected into the PKZ/PKR knockdown cells that transfected with wildtype PKZ previously, the apoptotic rates and levels of eIF2α phosphorylation were dramatically decreased. Overall, these results suggested that PKZ could facilitate apoptosis via eIF2α phosphorylation.

Endoplasmic Reticulum Transmembrane Proteins ZDHHC1 and STING Both Act as Direct Adaptors for IRF3 Activation in Teleost

IFN regulatory factor (IRF)3 is a central regulator for IFN-β expression in different types of pathogenic infections. Mammals have various pathogenic sensors that are involved in monitoring pathogen intrusions. These sensors can trigger IRF3-mediated antiviral responses through different pathways. Endoplasmic reticulum–associated proteins stimulator of IFN gene (STING) and zinc finger DHHC-type containing 1 (ZDHHC1) are critical mediators of IRF3 activation in response to viral DNA infections. In this study, grass carp STING and ZDHHC1 were found to have some similar molecular features and subcellular localization, and both were upregulated upon stimulation with polyinosinic:polycytidylic acid, B-DNA, or Z-DNA. Based on these results, we suggest that grass carp STING and ZDHHC1 might possess some properties similar to their mammalian counterparts. Overexpression of ZDHHC1 and STING in Ctenopharyngodon idella kidney cells upregulated IFN expression, whereas knockdown of IRF3 inhibited IFN activation. In addition, coimmunoprecipitation and GST pull-down assays demonstrated that STING and ZDHHC1 can interact separately with IRF3 and promote the dimerization and nuclear translocation of IRF3. Furthermore, we also found that small interfering RNA–mediated knockdown of STING could inhibit the expression of IFN and ZDHHC1 in fish cells. Similarly, knockdown of STING resulted in inhibition of the IFN promoter. In contrast, ZDHHC1 knockdown also inhibited IFN expression but had no apparent effect on STING, which indicates that STING is necessary for IFN activation through ZDHHC1. In conclusion, STING and ZDHHC1 in fish can respond to viral DNA or RNA molecules in cytoplasm, as well as activate IRF3 and, eventually, trigger IFN expression.

Poly I:C facilitates the phosphorylation of Ctenopharyngodon idellus type I IFN receptor subunits and JAK kinase

ABSTRACT Members of the Janus kinase (JAK) family, JAK1 and TYK2 take part in JAK‐STAT signaling pathway mediated by interferon in mammalian cells. Similar to the mammalian counterparts, fish JAK1 and TYK2 also perform their potential biological activities by phosphorylating cytokine receptors and STAT. In the present study, Ctenopharyngodon idellus JAK1 (CiJAK1) and TYK2 (CiTYK2) were cloned and identified. The full‐length cDNA of CiJAK1 (KT724352.1) is 3829 bp, with an Open Reading Frame (ORF) of 3465 bp encoding a putative protein of 1154 amino acids. The full‐length cDNA of CiTYK2 (KT724353.1) is 4337 bp, including an ORF of 3168 bp encoding 1055 amino acids. Structurally, both of them have B41, SH2, TyrKc and TyrKc common domains. CiJAK1 and CiTYK2 share a high degree of homology with their respective counterparts from Danio rerio and Cyprinus carpio by phylogenetic tree analysis. Polyinosinic‐polycytidylic acid (Poly I:C), a synthetic dsRNA analogue, can launch the JAK‐STAT antiviral signaling pathway. To elucidate the molecular mechanism of Poly I:C initiating the antiviral signaling pathway in fish, C. idellus kidney (CIK) cells were stimulated with Poly I:C and then the cell lysates were separated on 10% SDS‐PAGE. The results showed that not only Poly I:C drastically increased the expression level of CiJAK1 and CiTYK2, but also it induced the phosphorylation of CiJAK1 and CiTYK2, as well as C. idellus type I IFN receptor subunits, CiCRFB1 and CiCRFB5. In detail, the levels of p‐CiJAK1 and p‐CiTYK2 were evidently up‐regulated at 3 h post stimulation; however the phosphorylation levels of CiCRFB1 and CiCRFB5 displayed a sharp up‐regulation at 12 h post stimulation of Poly I:C. As a basic mechnism of feedback regulation of JAK‐STAT signaling pathway, overexpression of CiCRFB1 and CiCRFB5 in CIK cells facilitated the phosphorylation of CiJAK1 and CiTYK2. HIGHLIGHTSThe full‐length cDNA of Ctenopharyngodon idellus JAK1 and TYK2 were cloned, identified and analyzed by evolutionary tree.CiJAK1 and CiTYK2 were ubiquitously expressed at low level but were obviously up‐regulated under the induction of Poly I:C.Poly I:C induced phosphorylation of CiCRFB1, CiCRFB5, CiTYK2 and CiJAK1.Overexpression of CiCRFB1 and CiCRFB5 facilitated the phosphorylation of CiJAK1 and CiTYK2 in a feedback manner.

Interaction of IRF9 and STAT2 synergistically up-regulates IFN and PKR transcription in Ctenopharyngodon idella

&NA; IRF9 is a key factor in the JAK‐STAT pathway. Under the stimulation of type I IFN, IRF9 interacts with STAT1 and STAT2 to form the IFN‐I‐stimulated gene factor 3 (ISGF3) which activates the transcription of ISG. However, many studies also showed that the dimmer IRF9/STAT2 rather than the tripolymer IRF9/STAT1/STAT2 acts as the ISGF3 in cells in response to IFN signals. In the present study, the full‐length cDNA sequence of IRF9 (termed CiIRF9, KT601055) and STAT2 (term CiSTAT2, KT781914) from grass carp were cloned and identified. A low level of constitutive expression of CiIRF9 was detected by RT‐PCR in grass carp tissues, but it was significantly up‐regulated by LPS and poly I:C stimulation. In vitro, a high‐affinity interaction between CiIRF9 and the promoter of CiIFN or CiPKR was demonstrated by gel mobility shift assay. In vivo, the promoter activities of CiIFN and CiPKR were not only increased by transient transfection of CiIRF9, but also prominently increased by co‐transfection of CiIRF9 and CiSTAT2. Moreover, the interaction of CiIRF9 and CiSTAT2 was further investigated by in vivo and in vitro protein interaction assays. Recombinant CiIRF9 and CiSTAT2, both tagged with FLAG (or HA), were expressed in HEK 293T cells by transient transfection experiment. Co‐immunoprecipitation assays showed that CiIRF9 can interact with CiSTAT2 in vivo. Soluble GST‐ST2‐936 (containing the N‐terminal and coiled‐coil domain of CiSTAT2) was expressed and purified from E. coli. A GST pull‐down assay suggested that GST‐tagged ST2‐936 efficiently bound to FLAG‐tagged IRF9. The data indicated that interaction of IRF9 and STAT2 synergistically up‐regulated the transcriptional level of IFN and ISG genes. HighlightsThe full‐length cDNA of grass carp IRF9 and STAT2 were cloned and identified.CiIRF9 was ubiquitously expressed at low level but was obviously up‐regulated under the induction of LPS and poly I:C.CiIRF9 can up‐regulate the transcription level of CiIFN and CiPKR by binding to their promoters.The co‐transfection of CiIRF9 and CiSTAT2 into cells significantly up‐regulated the transcription level of CiIFN and CiPKR..CiIRF9 can interact with CiSTAT2 in vivo and in vitro.

Identification of grass carp (Ctenopharyngodon idella) XBP1S as a primary member in ER stress

ABSTRACT X‐box binding protein 1 (XBP1), a vital basic leucine zipper transcription factor for the related gene transcription in endoplasmic reticulum (ER) stress, belongs to the CREB/ATF family. In mammals, XBP1S is the activated one of XBP1 isoform. In order to study the role of fish XBP1S, we cloned and identified the XBP1S (KU509247) from grass carp (Ctenopharyngodon idella) (named CiXBP1S) by homologous cloning and RACE technique. The full length of CiXBP1S is 1694 bp along with 124 bp of 5′ UTR, 418 bp of 3′ UTR and the longest open reading frame (1152 bp) encoding a polypeptide of 383 amino acids with a well conserved DNA binding domain (BRLZ domain). CiXBP1S shares significant homology to zebrafish XBP1S (˜90%) at amino acid level. RT‐PCR showed that the expression of CiXBP1S was ubiquitous in all tested grass carp tissues and was significantly up‐regulated under the stimulation with tunicamycin (Tm) in CIK (C. idellus kidney) cells. To study the molecular mechanism of transcriptional regulation for XBP1 signaling pathway in fish, we cloned grass carp XBP1 promoter sequence. Its promoter is 1036 bp in length and divided into two distinct regions in which an ER stress response element (ERSE) exists in the proximal region. Meanwhile, grass carp ATF6 (CiATF6N) and CiXBP1S were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni‐NTA His‐Bind resin. Gel mobility shift assay showed that CiATF6N and CiXBP1S had the high affinity with CiXBP1 promoter sequence in vitro. Co‐transfection of pcDNA3.1‐CiATF6 (or pcDNA3.1‐CiXBP1S respectively) with pGL3‐CiXBP1P2 (or pGL3‐CiXBP1P1 respectively) into epithelioma papulosum cyprini (EPC) cells showed that CiATF6 and CiXBP1S played a positive role in CiXBP1S transcription. CiXBP1S also had high affinity with CiGRP78 and CiGRP94 promoter sequences. In addition, recombinant plasmids of pGL3‐CiGRP78P and pGL3‐CiGRP94P were constructed and transiently co‐transfected with pcDNA3.1‐CiXBP1S (pcDN3.1‐CiXBP1S‐nBRLZ, respectively) into EPC cells. The result showed that CiXBP1S can activate CiGRP78 and CiGRP94 promoters. HIGHLIGHTSThe full‐length cDNA and promoter sequence of grass carp XBP1S was cloned and identified.CiXBP1S was ubiquitous expression in tested tissues and significantly up‐regulated in CIK cells after stimulation with Tm.CiATF6 and CiXBP1S can activate the transcription activity of CiXBP1 by binding to its promoter.CiXBP1S played a positive role in CiGRP78 and CiGRP94 transcription.

Grass carp ( Ctenopharyngodon idella ) STAT3 regulates the eIF2α phosphorylation through interaction with PKR

ABSTRACT In mammals, STAT3 (Signal transducer and activator of transcription 3) plays an important role in growth, multiplication, differentiation and participates in inflammation, tumorigenesis, metabolic disorders and immune response. STAT3 is a protein that shuttles between the nucleus and cytoplasm. Compared to the STAT3 in cell nucleus, we did not know the function of STAT3 in cytoplasm for a long time. Some recent studies have shown that cytoplasmic STAT3 regulates autophagy through the interaction with the double‐stranded RNA‐activated protein kinase (PKR), which plays an important role in cellular antiviral response. Fish is a good target for developmental and comparative immunology. In the present study, we found that the expression of grass carp (Ctenopharyngodon idella) STAT3 (CiSTAT3) was ubiquitous and significantly up‐regulated under the stimulation of poly I:C. To explore the potential function of fish cytoplasmic STAT3 in the antiviral signaling pathways, in this paper we analyzed the relationship between cytoplasmic CiSTAT3 and CiPKR. We demonstrated that the CiSTAT3 can combine with CiPKR in vivo and in vitro. The SH2 domain of CiSTAT3 and the C‐terminus of CiPKR play an important role in this process. Moreover, the dimer of CiSTAT3 and CiPKR was formed under normal circumstances, however, it was dissociated under the induction of poly I:C. So, we guessed the binding of CiSTAT3 and CiPKR may regulate cell viability. It has also been shown that overexpression of CiSTAT3 in CIK cells can significantly reduce the level of p‐eIF2&agr;. On the contrary, the siRNA‐mediated knockdown of CiSTAT3 and Stattic induction in CIK cells can up‐regulate the p‐eIF2&agr; level. To further understand the relationship between CiSTAT3 and p‐eIF2&agr; level, we carried out the CiPKR‐knockdown experiment. The result indicated that CiSTAT3 regulated the level of p‐eIF2&agr; through binding to CiPKR. In addition, overexpression of CiSTAT3 in CIK cells was able to improve the cell viability. These results above unraveled the molecular mechanism of fish cytoplasmic STAT3 regulating the eIF2&agr; phosphorylation and cell viability. Therefore, the function of fish cytoplasmic STAT3 is similar to those of mammals. HighlightsCiSTAT3 responded to the stimulation of poly I:C.Physical and functional interaction between cytoplasmic CiSTAT3 and CiPKR was showed in vivo and in vitro.The phosphorylation level of eIF2&agr; was significantly decreased with overexpression of CiSTAT3 in CIK cells.Poly I:C up‐regulated the phosphorylation level of eIF2&agr; through dissociating CiSTAT3/CiPKR complex.Overexpression of CiSTAT3 in CIK cells was able to improve the cell viability.

Ctenopharyngodon idella IRF2 and ATF4 down-regulate the transcriptional level of PRKRA

ABSTRACT PRKRA (interferon‐inducible double‐stranded RNA‐dependent protein kinase activator A) is a protective protein which regulates the adaptation of cells to ER stress and virus‐stimulated signaling pathways by activating PKR. In the present study, a grass carp (Ctenopharyngodon idella) PRKRA full‐length cDNA (named CiPRKRA, KT891991) was cloned and identified. The full‐length cDNA is comprised of a 5′ UTR (36 bp), a 3′ UTR (350 bp) and the longest ORF (882 bp) encoding a polypeptide of 293 amino acids. The deduced amino acid sequence of CiPRKRA contains three typical dsRNA binding motifs (dsRBM). Phylogenetic tree analysis revealed a closer evolutionary relationship of CiPRKRA with other fish PRKRA, especially with Danio rerio PRKRA. qRT‐PCR showed that CiPRKRA was significantly up‐regulated after stimulation with tunicamycin (Tm) and Poly I:C in C. idella kidney (CIK) cells. To further study its transcriptional regulation, the partial promoter sequence of CiPRKRA (1463 bp) containing one ISRE and one CARE was cloned by Tail‐PCR. Subsequently, grass carp IRF2 (CiIRF2) and ATF4 (CiATF4) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni‐NTA His‐Bind Resin. In vitro, both CiIRF2 and CiATF4 bound to CiPRKRA promoter with high affinity by gel mobility shift assays, revealing that IRF2 and ATF4 might be potential transcriptional regulatory factors for CiPRKRA. Dual‐luciferase reporter assays were applied to further investigate the transcriptional regulation of CiPRKRA in vivo. Recombinant plasmid of pGL3‐PRKRAPro was constructed and transiently co‐transfected into CIK cells with pcDNA3.1‐CiIRF2 and pcDNA3.1‐CiATF4, respectively. The results showed that both CiIRF2 and CiATF4 significantly decreased the luciferase activity of pGL3‐PRKRAPro, suggesting that they play a negative role in CiPRKRA transcription. HIGHLIGHTSThe full‐length cDNA of grass carp PRKRA was cloned and identified.CiPRKRA was significantly up‐regulated after stimulation with Poly I:C and TM.In vitro, CiIRF2 and CiATF4 had high affinity with CiPRKRA promoter.CiIRF2 and CiATF4 both played a negative role in CiPRKRA transcription in CIK cells.

Ctenopharyngodon idella IKKβ interacts with PKR and IκBα

Inhibitor of nuclear factor kappa-B kinase β (IKKβ) is a subunit of the IKK complex. It can activate the NF-κB pathway through phosphorylating IκB in response to a wide range of stimuli. In the present study, an IKKβ gene from grass carp (Ctenopharyngodon idella; KT282114) was cloned and identified by homologous cloning and rapid-amplification of cDNA ends (RACE) technique. The complete CiIKKβ cDNA is 3428 bp in length, with the longest open reading frame (ORF) of 2337 bp encoding a polypeptide of 778 amino acids. The deduced amino acid sequence of CiIKKβ has similar domain distribution to those of mammalian. For example, CiIKKβ consists of a serine/threonine kinase domain at the N-terminal, a basic region leucin zipper (BRLZ) domain in the middle, a homeobox associated leucin zipper (HALZ) domain and an IKKβ NEMO (NF-κB essential modulator) binding domain at the C-terminal. Phylogenetic tree analysis also showed that CiIKKβ is highly homologous to zebrafish IKKβ (DrIKKβ) and clearly distinct from the mammalian and amphibian counterparts. The expression of CiIKKβ was ubiquitously found in the liver, intestine, kidney, gill, spleen, heart, and brain tissues of grass carp and significantly up-regulated in CIK cells under the stimulation with Poly I:C and UV-inactivated grass carp hemorrhagic virus. To investigate the activation mechanism of NF-κB pathway in fish and the role of CiIKKβ in the pathway, we explored the protein interactions of protein kinase R (PKR) with IKKβ and IKKβ with IκBα by co-immunoprecipitation and GST-pull down assays. The interaction between each pair was confirmed. The results suggest that CiIKKβ may be a primary member in the activation of NF-κB pathway in fish.