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Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts.

Sobhkhez M, Skjesol A, Thomassen E, Tollersrud LG, Iliev DB, Sun B, Robertsen B, Jørgensen JB - FEBS Open Bio (2014)

Bottom Line: Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells.The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone.Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish.

View Article: PubMed Central - PubMed

Affiliation: The Norwegian College of Fishery Science, UiT The Arctic University of Norway, N-9037 Tromsø, Norway.

ABSTRACT
Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

No MeSH data available.


Related in: MedlinePlus

Expression kinetics of STAT2a, STAT2b and IRF9 in IFN-stimulated TO cells. TO cells were treated with 200 U/ml of IFNa1, IFNb and IFNc, and 100 ng/ml IFNγ for 4, 24 and 48 h and untreated cells were used as controls. The mRNA expression levels of STAT2 (A), IRF9 (B) and Mx (C) were measured by qPCR and transcripts levels were normalized against EF1αβ. The results are presented as fold increase relative to untreated cells in triplicate samples and are representative of two independent experiments.
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f0050: Expression kinetics of STAT2a, STAT2b and IRF9 in IFN-stimulated TO cells. TO cells were treated with 200 U/ml of IFNa1, IFNb and IFNc, and 100 ng/ml IFNγ for 4, 24 and 48 h and untreated cells were used as controls. The mRNA expression levels of STAT2 (A), IRF9 (B) and Mx (C) were measured by qPCR and transcripts levels were normalized against EF1αβ. The results are presented as fold increase relative to untreated cells in triplicate samples and are representative of two independent experiments.

Mentions: To test the time-course expression of the STAT2a/b and IRF9 transcripts induced by IFNa1, b and c and by IFNγ treatment of TO-cells, a qPCR was performed. Additionally the expression of the interferon induced gene, Mx, was quantified to ensure that the stimulations had worked. The results showed that all the IFNs increased the expression of STAT2a/b until 24 h post stimulation. STAT2a/b was expressed at higher levels (10-fold induction) in IFNa1 treated cells compared IFNb and IFNc treated cells (about 8-fold induction) at this time-point (Fig. 10A). At 48 h post stimulation STAT2a/b expression was modestly reduced for all the type I IFNs. The kinetics of Mx expression was very similar to that of STAT2a/b for all three IFNs, but the fold induction was much higher (Fig. 10C), thus demonstrating that the IFN-stimulations had worked. For the IFNγ stimulated cells STAT2a/b were 4-fold induced at 24 h and continued to rise until 48 h (5-fold induction). IRF9 expression was modestly increased (1,5-fold) after 4 h in type I IFN treated cells, at 24 h the IRF9 levels were slightly down-regulated, and then their levels slightly increased (about 2-fold) to a maximum at 48 h (Fig. 10B). Upon IFNγ treatment elevated IRF9 mRNA levels were also detected after 4 h and increased further to a maximum at 48 h of stimulation (about 3-fold induction) (Fig. 10B).


Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts.

Sobhkhez M, Skjesol A, Thomassen E, Tollersrud LG, Iliev DB, Sun B, Robertsen B, Jørgensen JB - FEBS Open Bio (2014)

Expression kinetics of STAT2a, STAT2b and IRF9 in IFN-stimulated TO cells. TO cells were treated with 200 U/ml of IFNa1, IFNb and IFNc, and 100 ng/ml IFNγ for 4, 24 and 48 h and untreated cells were used as controls. The mRNA expression levels of STAT2 (A), IRF9 (B) and Mx (C) were measured by qPCR and transcripts levels were normalized against EF1αβ. The results are presented as fold increase relative to untreated cells in triplicate samples and are representative of two independent experiments.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215117&req=5

f0050: Expression kinetics of STAT2a, STAT2b and IRF9 in IFN-stimulated TO cells. TO cells were treated with 200 U/ml of IFNa1, IFNb and IFNc, and 100 ng/ml IFNγ for 4, 24 and 48 h and untreated cells were used as controls. The mRNA expression levels of STAT2 (A), IRF9 (B) and Mx (C) were measured by qPCR and transcripts levels were normalized against EF1αβ. The results are presented as fold increase relative to untreated cells in triplicate samples and are representative of two independent experiments.
Mentions: To test the time-course expression of the STAT2a/b and IRF9 transcripts induced by IFNa1, b and c and by IFNγ treatment of TO-cells, a qPCR was performed. Additionally the expression of the interferon induced gene, Mx, was quantified to ensure that the stimulations had worked. The results showed that all the IFNs increased the expression of STAT2a/b until 24 h post stimulation. STAT2a/b was expressed at higher levels (10-fold induction) in IFNa1 treated cells compared IFNb and IFNc treated cells (about 8-fold induction) at this time-point (Fig. 10A). At 48 h post stimulation STAT2a/b expression was modestly reduced for all the type I IFNs. The kinetics of Mx expression was very similar to that of STAT2a/b for all three IFNs, but the fold induction was much higher (Fig. 10C), thus demonstrating that the IFN-stimulations had worked. For the IFNγ stimulated cells STAT2a/b were 4-fold induced at 24 h and continued to rise until 48 h (5-fold induction). IRF9 expression was modestly increased (1,5-fold) after 4 h in type I IFN treated cells, at 24 h the IRF9 levels were slightly down-regulated, and then their levels slightly increased (about 2-fold) to a maximum at 48 h (Fig. 10B). Upon IFNγ treatment elevated IRF9 mRNA levels were also detected after 4 h and increased further to a maximum at 48 h of stimulation (about 3-fold induction) (Fig. 10B).

Bottom Line: Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells.The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone.Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish.

View Article: PubMed Central - PubMed

Affiliation: The Norwegian College of Fishery Science, UiT The Arctic University of Norway, N-9037 Tromsø, Norway.

ABSTRACT
Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

No MeSH data available.


Related in: MedlinePlus