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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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The activation of STAT6 is mediated by the formation of STAT2: STAT6 heterodimer. (A) STAT6 formed a heterodimer with STAT2. HuH7 cells either were unstimulated (PBS) or were stimulated with IFNα (400 IU/ml), IFNβ (400 IU/ml) and IL-4 (10 ng/ml) for 30 min. Extracts were then immunoprecipitated (IP) with either a STAT2 antibody or a STAT6 antibody, resolved by 7.5% SDS-PAGE, and analysed by Western blotting using either an anti-STAT6 (top panel) or an anti-STAT2 antibody. Extracts from IL-4-stimulated HuH7 cells were included as a control for STAT6 activation. (B) SiRNA against STAT2 was introduced into the cells to down-regulate the expression level of STAT2 protein. STAT2 inhibition was checked by Western blotting using anti-STAT2 antibody. The blot was later stripped and re-probed with βactin antibody to ensure equal loading of the cell extracts. (C) STAT6 phosphorylation was inhibited by down-regulating STAT2. Extracts were prepared from HuH7 cells with or without a 30-min stimulation at 37°C with either PBS or IFNα (400 IU/ml). The activation of STAT6 after STAT2 knockdown was determined by Western blotting. The blot was later stripped and re-probed with STAT6 antibody to ensure equal loading of the cell extracts. (D) Phospho-STAT6 was quantified and the results are expressed in relative expression level over basal and represented as the mean±S.D. for three repetitions. Asterisks indicate the calculated P values for paired comparisons between IFN and PBS;all were <0.05.
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fig02: The activation of STAT6 is mediated by the formation of STAT2: STAT6 heterodimer. (A) STAT6 formed a heterodimer with STAT2. HuH7 cells either were unstimulated (PBS) or were stimulated with IFNα (400 IU/ml), IFNβ (400 IU/ml) and IL-4 (10 ng/ml) for 30 min. Extracts were then immunoprecipitated (IP) with either a STAT2 antibody or a STAT6 antibody, resolved by 7.5% SDS-PAGE, and analysed by Western blotting using either an anti-STAT6 (top panel) or an anti-STAT2 antibody. Extracts from IL-4-stimulated HuH7 cells were included as a control for STAT6 activation. (B) SiRNA against STAT2 was introduced into the cells to down-regulate the expression level of STAT2 protein. STAT2 inhibition was checked by Western blotting using anti-STAT2 antibody. The blot was later stripped and re-probed with βactin antibody to ensure equal loading of the cell extracts. (C) STAT6 phosphorylation was inhibited by down-regulating STAT2. Extracts were prepared from HuH7 cells with or without a 30-min stimulation at 37°C with either PBS or IFNα (400 IU/ml). The activation of STAT6 after STAT2 knockdown was determined by Western blotting. The blot was later stripped and re-probed with STAT6 antibody to ensure equal loading of the cell extracts. (D) Phospho-STAT6 was quantified and the results are expressed in relative expression level over basal and represented as the mean±S.D. for three repetitions. Asterisks indicate the calculated P values for paired comparisons between IFN and PBS;all were <0.05.

Mentions: Since IFNα-induced STAT6 activation in lymphocytes is accompanied by the formation of STAT2: STAT6 complex [10–12], immunoprecipitation was performed to determine whether a similar mechanism exists in hepatocytes (Fig. 2A). STAT6 protein in IFN-α- or IFN-β-treated HuH7 cells was pulled down by anti-STAT6 antibody and the protein complex was subjected to Western blotting with anti-STAT2 antibody and vice versa. It is known that STAT6 activation is the primary signalling mechanism involved in the IL-4 pathway [10, 28–30]. Hence, IL-4 treated cells in this experiment were utilized as the positive control. The data in Fig. 2A indicate that STAT6 protein directly associated with STAT2 after IFN-α or IFN-β treatments; this was not the case for cells treated with PBS control. SiRNA against STAT2 was introduced into the cells to down-regulate the expression level of STAT2 protein (Fig. 2B). This treatment significantly reduced the level of phosphorylation of STAT6 protein at Tyr 641 by IFNα as compared to control RNAi treated cells (Fig. 2C and D), indicating that the formation of the STAT2: STAT6 heterodimer is required for STAT6 activation induced by type I IFNs.


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)

The activation of STAT6 is mediated by the formation of STAT2: STAT6 heterodimer. (A) STAT6 formed a heterodimer with STAT2. HuH7 cells either were unstimulated (PBS) or were stimulated with IFNα (400 IU/ml), IFNβ (400 IU/ml) and IL-4 (10 ng/ml) for 30 min. Extracts were then immunoprecipitated (IP) with either a STAT2 antibody or a STAT6 antibody, resolved by 7.5% SDS-PAGE, and analysed by Western blotting using either an anti-STAT6 (top panel) or an anti-STAT2 antibody. Extracts from IL-4-stimulated HuH7 cells were included as a control for STAT6 activation. (B) SiRNA against STAT2 was introduced into the cells to down-regulate the expression level of STAT2 protein. STAT2 inhibition was checked by Western blotting using anti-STAT2 antibody. The blot was later stripped and re-probed with βactin antibody to ensure equal loading of the cell extracts. (C) STAT6 phosphorylation was inhibited by down-regulating STAT2. Extracts were prepared from HuH7 cells with or without a 30-min stimulation at 37°C with either PBS or IFNα (400 IU/ml). The activation of STAT6 after STAT2 knockdown was determined by Western blotting. The blot was later stripped and re-probed with STAT6 antibody to ensure equal loading of the cell extracts. (D) Phospho-STAT6 was quantified and the results are expressed in relative expression level over basal and represented as the mean±S.D. for three repetitions. Asterisks indicate the calculated P values for paired comparisons between IFN and PBS;all were <0.05.
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Related In: Results  -  Collection

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fig02: The activation of STAT6 is mediated by the formation of STAT2: STAT6 heterodimer. (A) STAT6 formed a heterodimer with STAT2. HuH7 cells either were unstimulated (PBS) or were stimulated with IFNα (400 IU/ml), IFNβ (400 IU/ml) and IL-4 (10 ng/ml) for 30 min. Extracts were then immunoprecipitated (IP) with either a STAT2 antibody or a STAT6 antibody, resolved by 7.5% SDS-PAGE, and analysed by Western blotting using either an anti-STAT6 (top panel) or an anti-STAT2 antibody. Extracts from IL-4-stimulated HuH7 cells were included as a control for STAT6 activation. (B) SiRNA against STAT2 was introduced into the cells to down-regulate the expression level of STAT2 protein. STAT2 inhibition was checked by Western blotting using anti-STAT2 antibody. The blot was later stripped and re-probed with βactin antibody to ensure equal loading of the cell extracts. (C) STAT6 phosphorylation was inhibited by down-regulating STAT2. Extracts were prepared from HuH7 cells with or without a 30-min stimulation at 37°C with either PBS or IFNα (400 IU/ml). The activation of STAT6 after STAT2 knockdown was determined by Western blotting. The blot was later stripped and re-probed with STAT6 antibody to ensure equal loading of the cell extracts. (D) Phospho-STAT6 was quantified and the results are expressed in relative expression level over basal and represented as the mean±S.D. for three repetitions. Asterisks indicate the calculated P values for paired comparisons between IFN and PBS;all were <0.05.
Mentions: Since IFNα-induced STAT6 activation in lymphocytes is accompanied by the formation of STAT2: STAT6 complex [10–12], immunoprecipitation was performed to determine whether a similar mechanism exists in hepatocytes (Fig. 2A). STAT6 protein in IFN-α- or IFN-β-treated HuH7 cells was pulled down by anti-STAT6 antibody and the protein complex was subjected to Western blotting with anti-STAT2 antibody and vice versa. It is known that STAT6 activation is the primary signalling mechanism involved in the IL-4 pathway [10, 28–30]. Hence, IL-4 treated cells in this experiment were utilized as the positive control. The data in Fig. 2A indicate that STAT6 protein directly associated with STAT2 after IFN-α or IFN-β treatments; this was not the case for cells treated with PBS control. SiRNA against STAT2 was introduced into the cells to down-regulate the expression level of STAT2 protein (Fig. 2B). This treatment significantly reduced the level of phosphorylation of STAT6 protein at Tyr 641 by IFNα as compared to control RNAi treated cells (Fig. 2C and D), indicating that the formation of the STAT2: STAT6 heterodimer is required for STAT6 activation induced by type I IFNs.

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