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Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer.

Wan L, Lin CW, Lin YJ, Sheu JJ, Chen BH, Liao CC, Tsai Y, Lin WY, Lai CH, Tsai FJ - J. Cell. Mol. Med. (2008)

Bottom Line: The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signalling pathway.In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra.Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan. leiwan@www.cmuh.org.tw

ABSTRACT
The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signalling pathway. Although several studies have indicated that IFN-alpha and IFN-beta can activate complexes containing STAT6, the biological role of this activation is still unknown. We found that exposure of hepatoma cells (HuH7 and Hep3B) to IFN-alpha or IFN-beta led to the activation of STAT6. Activated STAT6 in turn induced the formation of STAT2: STAT6 complexes, which led to the secretion of IL-1Ra. The activation of STAT6 by type I IFN in hepatocytes was mediated by JAK1 and Tyk2. In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra. The present study suggests a novel function of IFN-alpha and IFN-beta signalling in human hepatocytes. Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra.

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Type I IFN-induced activation of STAT6 mediated the production of IL-1Ra. (A) SiRNA against STAT6 was introduced into the cells to down-regulate the expression level of STAT6 protein. STAT6 inhibition was checked by Western blotting using anti-STAT6 antibody. The blot was later stripped and re-probed with β-actin antibody to ensure equal loading of the cell extracts. (B) STAT6 is required for secreted IL-1Ra production in HuH7 cells activated by IFNα or IFNβ. Culture supernatant were prepared from HuH7 cells with or without a 48 hour stimulation period at 37°C with phosphate buffered saline (control), IFNα (400 IU/ml), IFNβ (400 IU/ml), IL-4 (10 ng/ml), and IL-1 (1 ng/ml) as indicated. Secreted IL-1Ra concentration was assessed in supernatants of triplicate independent experiments and is presented as mean ± S.D. Asterisks indicate the calculated P values for paired comparisons between STAT6 siRNA and siRNA negative control; all P values were <0.05. (C) Secreted IL-1Ra production is down-regulated by inhibiting the expression of STAT2 in HuH7 cells activated by 400IU/ml IFNα. Assay procedures are the same as above.
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fig05: Type I IFN-induced activation of STAT6 mediated the production of IL-1Ra. (A) SiRNA against STAT6 was introduced into the cells to down-regulate the expression level of STAT6 protein. STAT6 inhibition was checked by Western blotting using anti-STAT6 antibody. The blot was later stripped and re-probed with β-actin antibody to ensure equal loading of the cell extracts. (B) STAT6 is required for secreted IL-1Ra production in HuH7 cells activated by IFNα or IFNβ. Culture supernatant were prepared from HuH7 cells with or without a 48 hour stimulation period at 37°C with phosphate buffered saline (control), IFNα (400 IU/ml), IFNβ (400 IU/ml), IL-4 (10 ng/ml), and IL-1 (1 ng/ml) as indicated. Secreted IL-1Ra concentration was assessed in supernatants of triplicate independent experiments and is presented as mean ± S.D. Asterisks indicate the calculated P values for paired comparisons between STAT6 siRNA and siRNA negative control; all P values were <0.05. (C) Secreted IL-1Ra production is down-regulated by inhibiting the expression of STAT2 in HuH7 cells activated by 400IU/ml IFNα. Assay procedures are the same as above.

Mentions: To further confirm whether the binding of STAT6 to the promoter leads to activation of IL-1Ra gene transcription, the protein levels of IL-1Ra were measured from the culture media of the cells treated with IFNα, IFNβ and IL-4, respectively. Compared with the PBS-treated control cells, IFNα, IFNβ and IL-4 treatments of hepatocytes increased the production of IL-1Ra from 20 pg/ml to 92 pg/ml (IFNα), 260 pg/ml (IFNβ) and 43 pg/ml (IL-4) (Fig. 5B).


Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer.

Wan L, Lin CW, Lin YJ, Sheu JJ, Chen BH, Liao CC, Tsai Y, Lin WY, Lai CH, Tsai FJ - J. Cell. Mol. Med. (2008)

Type I IFN-induced activation of STAT6 mediated the production of IL-1Ra. (A) SiRNA against STAT6 was introduced into the cells to down-regulate the expression level of STAT6 protein. STAT6 inhibition was checked by Western blotting using anti-STAT6 antibody. The blot was later stripped and re-probed with β-actin antibody to ensure equal loading of the cell extracts. (B) STAT6 is required for secreted IL-1Ra production in HuH7 cells activated by IFNα or IFNβ. Culture supernatant were prepared from HuH7 cells with or without a 48 hour stimulation period at 37°C with phosphate buffered saline (control), IFNα (400 IU/ml), IFNβ (400 IU/ml), IL-4 (10 ng/ml), and IL-1 (1 ng/ml) as indicated. Secreted IL-1Ra concentration was assessed in supernatants of triplicate independent experiments and is presented as mean ± S.D. Asterisks indicate the calculated P values for paired comparisons between STAT6 siRNA and siRNA negative control; all P values were <0.05. (C) Secreted IL-1Ra production is down-regulated by inhibiting the expression of STAT2 in HuH7 cells activated by 400IU/ml IFNα. Assay procedures are the same as above.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401122&req=5

fig05: Type I IFN-induced activation of STAT6 mediated the production of IL-1Ra. (A) SiRNA against STAT6 was introduced into the cells to down-regulate the expression level of STAT6 protein. STAT6 inhibition was checked by Western blotting using anti-STAT6 antibody. The blot was later stripped and re-probed with β-actin antibody to ensure equal loading of the cell extracts. (B) STAT6 is required for secreted IL-1Ra production in HuH7 cells activated by IFNα or IFNβ. Culture supernatant were prepared from HuH7 cells with or without a 48 hour stimulation period at 37°C with phosphate buffered saline (control), IFNα (400 IU/ml), IFNβ (400 IU/ml), IL-4 (10 ng/ml), and IL-1 (1 ng/ml) as indicated. Secreted IL-1Ra concentration was assessed in supernatants of triplicate independent experiments and is presented as mean ± S.D. Asterisks indicate the calculated P values for paired comparisons between STAT6 siRNA and siRNA negative control; all P values were <0.05. (C) Secreted IL-1Ra production is down-regulated by inhibiting the expression of STAT2 in HuH7 cells activated by 400IU/ml IFNα. Assay procedures are the same as above.
Mentions: To further confirm whether the binding of STAT6 to the promoter leads to activation of IL-1Ra gene transcription, the protein levels of IL-1Ra were measured from the culture media of the cells treated with IFNα, IFNβ and IL-4, respectively. Compared with the PBS-treated control cells, IFNα, IFNβ and IL-4 treatments of hepatocytes increased the production of IL-1Ra from 20 pg/ml to 92 pg/ml (IFNα), 260 pg/ml (IFNβ) and 43 pg/ml (IL-4) (Fig. 5B).

Bottom Line: The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signalling pathway.In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra.Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan. leiwan@www.cmuh.org.tw

ABSTRACT
The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signalling pathway. Although several studies have indicated that IFN-alpha and IFN-beta can activate complexes containing STAT6, the biological role of this activation is still unknown. We found that exposure of hepatoma cells (HuH7 and Hep3B) to IFN-alpha or IFN-beta led to the activation of STAT6. Activated STAT6 in turn induced the formation of STAT2: STAT6 complexes, which led to the secretion of IL-1Ra. The activation of STAT6 by type I IFN in hepatocytes was mediated by JAK1 and Tyk2. In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra. The present study suggests a novel function of IFN-alpha and IFN-beta signalling in human hepatocytes. Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra.

Show MeSH
Related in: MedlinePlus