| Runt-related transcription factor(Runx) is a transcription factor family, including three members: Runx1, Runx2 and Runx3 in mammals, which play critical roles in various biological processes. Among them, Runx1 and Runx3 exert more potent functions in the immune system, which are mainly involved in the regulation of proliferation, differentiation and functions of immune cells. At present, three Runx family members and their cofactor CBFβ, have been cloned and identified in zebrafish and pufferfish. However, the roles of Runx1 and Runx3 in fish immunity have not been defined. In this study, the molecular characteristics of grass carp Runx1, Runx3 and CBFβ were identified. Subsquently, evidence suggested their involvement in the immune response. Furthermore, their immune functions, especially the roles in the immunoregulation of the transforming growth factor β(TGF-β) were characterized. The results in this study are shown as follows:1. Molecular identification and inductive expression analysis of grass carp Runx1 and Runx3:(1) cDNA sequences of five splice variants of grass carp Runx1(Runx1 v1-5) and Runx3 were cloned by using degenerate PCR and RACE PCR. It was suggested that they are homologs of mammalian Runx1 and Runx3 according to domain analysis, multiple alignment of amino acid sequences and phylogenetic tree analysis.(2) Among the five splice variants of Runx1, Runx1 v1-3 possessed intact functional domains, including transcription activation domain(TAD), inhibitory domain(ID), PY motif and the C-terminal VWRPY motif, while Runx1 v4-5 lacked TAD, ID, PY and VWRPY. Tissue distribution assay showed that Runx1 v1-3 and v4-5 shared similar tissue distribution patterns. They were expressed with the highest abundance of transcripts in thymus, lower in spleen, kidney and headkidney, and relatively high levels in peripheral blood leukocytes(PBLs). In parallel, grass carp Runx3 was also expressed at high level in thymus, and relatively less extent in spleen and PBLs. The mRNA levels of Runx1 v1-3, Runx1 v4-5 and Runx3 were upregulated by both LPS and PHA, but only Runx1 v1-3 and Runx3 mRNA levels were promoted by poly I:C.(4) After Aeromonas Hydrophila(A.Hydr) challenge in grass carp, expression levels of Runx1 v1-3 and Runx3 increased at varying degrees in headkidney, spleen and thymus, while enhancement of Runx1 v4-5 expression levels was observed only in spleen and thymus. In particular, Runx1 v1-3 and Runx1 v4-5 presented different expression patterns at time kinetics level in both in vitro and in vivo experiments, suggesting a possibility that Runx1 v4-5 may serve as naturally inhibitors, which regulates excessive activation of Runx1 signaling pathway in bony fish.2. Isolation, identification and expression characteristics of grass carp Runx cofactor CBFβ:(1) cDNA sequence of grass carp CBFβ was isolated through a variety of PCR technologies; multiple alignment of the amino acid sequences and phylogenetic tree analysis suggested that it is a homolog of mammalian CBFβ.(2) 3D homology modeling showed that grass carp CBFβ shares high similarity with its mammalian counterparts in spatial structures; such highly conserved structures suggesting its conserved functions during evolution. CBFβ has similar tissue distribution pattern with Runx1 and Runx3. The highest expression of CBFβ expression was detected in thymus, and relatively lower expression in spleen and headkidney, and high expression was observed in peripheral blood leukocytes.(4) CBFβ mainly localized in the nucleus, proving its basic features that function as a transcription factor.(5) The mRNA expression of CBFβ could be stimulated by LPS, PHA and poly I:C in PBLs.(6) In A.hydr infections, CBFβ expression levels were enhanced in headkidney, spleen and thymus, further confirming that CBFβ is involved in the piscine immunity.3. Functional identification of grass carp Runx1 and Runx3:(1) Five variants of Runx1 and Runx3 could directly transport into the nucleus without stimulation, which proved their properties as the transcription factors.(2) The effect of grass carp Runx1 and Runx3 on the expressions of marker cytokines and transcription factors specific for various T-cell subsets were further investigated. It was found that gene expressions of grass carp of IFN-γ, IL-17A/F1 and IL-17A/F2 were augmented in grass carp PBLs by LPS, PHA and poly I:C challenge, and this stimulation was suppressed by blocking Runx1 signals with the Runx1 inhibitor. But inhibition of Runx1 signal did not affect the induction of Foxp3 by poly I:C. Due to lack of specific inhibitor of grass carp Runx3, overexpression of grass carp Runx3 were performed, showing that in grass carp Kidney(CIK) cells, overexpression of Runx3 stimulated the mRNA expression of transcription factor T-bet, but inhibited the expression of transcription factor GATA3. However, overexpression of Runx3 together with or without CBFβ had no effect on Foxp3 transcript levels.(4) The interactions between grass carp Runx1, Runx3 and cofactor CBFβ were studied, showing that the interaction between grass carp Runx1 and Runx3 was not observed, but overexpression of Runx1 variants and Runx3 significantly induced CBFβ gene expression, indicating that CBFβ may be an important target gene of Runx1 and Runx3. Notably, overexpression of CBFβ did not synergize with Runx3, a possible explanation is that endogenous CBFβ was sufficient to trigger Runx3 function, or it required other transcription factors to coordinate with Runx3-CBFβ.4. Functional characterization of grass carp Runx1 and Runx3 in the immune regulation of fish TGF-β:(1) In grass carp PBLs, recombinant grass carp TGF-β1(rgcTGF-β1) enhanced CBFβ gene expression, while exerted a dual regulation effect on mRNA expression of Runx1 v1-3 and Runx1 v4-5, which were upregulated first and then went down, suggesting their different inductive expression patterns in the immune regulation of TGF-β.(2) In particular, comparing with the induction of CBFβ and Runx1 transcripts, rgcTGF-β1 markedly increased mRNA levels of Runx3. To strengthen this result, the effect of rgcTGF-β1 on the protein level of Runx3 was examined by Western Blotting(WB), for which polyclonal antibody of grass carp Runx3 was prepared and its specificity was determined, showing that Runx3 polyclonal antibody recognized recombinant Runx3 protein with about 49 KDa molecular weight(MW) and a signal band about 46 KDa MW(endogenous Runx3) was detected in PBLs. WB assay by using this antibody revealed that rgcTGF-β1 stimulated Runx3 expression at protein level, indicating Runx3 may be the major member in Runx family in response to TGF-β1.(3) In order to verify the hypothesis that Runx1 and Runx3 mediate the regulatory effect of TGF-β, the roles of Runx1 and Runx3 were assessed in the regulation of rgcTGF-β1 on c-Myc mRNA expression in PBLs. In this case, bloking Runx1 signal amplified the inhibitory effect of rgcTGF-β1 on c-Myc gene expression, suggesting that Runx1 may serve as a negative regulator in the regulation of TGF-β1. Conversely, overexpression of Runx3 enlarged the inhibition of rgcTGF-β1 on c-Myc gene expression, suggesting Runx3 may promote such negative regulation of TGF-β1. To confirm the function of Runx3 described above, Runx3 dominant negative plasmid was constructed. Overexpression of this plasmid in CIK cells caused a significant increase of c-Myc mRNA levels, but this stimulation was inhibited by rgcTGF-β1. Combinating with the findings as described above in which rgcTGF-β1 significantly stimulated Runx3 production, these results suggested that TGF-β1 inhibited the expression of c-Myc probably by inducing Runx3 production. However, the mechanism responsible for distinct actions of Runx1 and Runx3 in rgcTGF-β-mediated c-Myc gene expression need to be further illustrated.(4) In addition, the roles of Runx1 and Runx3 in rgcTGF-β-mediated mRNA expression of a new inflammation-related molecule——novel IL-1 family member(nIL-1F)in PBLs were examined. The results showed that TGF-β1 significantly inhibited the expression of the new molecule nIL-1F, but Runx1 and Runx3 were not involved in this process, suggesting that Runx1 and Runx3 do not participate in all regulatory effects of fish TGF-β1.In summary, this study not only clarified molecular characteristics of grass carp Runx1, Runx3 and CBFβ, but also provided evidence of their involvement in the piscine immune responses. The immune functions of Runx1 and Runx3 in fish were elucidated for the first time, especially their roles in the immune regulation of TGF-β were revealed, thereby providing a better understanding of fish immunity involving key transcription factors. |
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