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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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STAT6 is tyrosine phosphorylated in response to IFNα and IFNβ in HuH7 and Hep3B cells. (A and B) IFN-α and IFN-β were able to activate STAT1, STAT2, STAT3, STAT5 and STAT6 in HuH7 (A) and Hep3B (B) cells. HuH7 and Hep3B cells were either unstimulated or stimulated with 400 IU/ml IFNα or IFNβ for the indicated time. 20μg cell extracts were resolved by 7.5% SDS-PAGE, and analysed by Western blotting using phosphoprotein specific antibodies (p-STAT1, p-STAT2, p-STAT3, p-STAT5, p-STAT6). The blot was later stripped and re-probed with STAT1, STAT2, STAT3, STAT5, STAT6 and βactin antibodies to ensure equal loading of the cell extracts. (C and D) STAT6 activation exhibited similar kinetic patterns as STAT1 in IFN-α or β treated HuH7 (C) and Hep3B (D) cells. The amount of activated STAT1 and STAT6 of HuH7 (C) and Hep3B (D) cells treated with 400 IU/ml IFNα or IFNβ was quantified and the results are expressed in relative expression level over basal; the results are represented as the mean±S.D. for three repetitions. Asterisks indicate the calculated P values for paired comparisons between IFNα and IFNβ at various times; all were < 0.05.
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fig01: STAT6 is tyrosine phosphorylated in response to IFNα and IFNβ in HuH7 and Hep3B cells. (A and B) IFN-α and IFN-β were able to activate STAT1, STAT2, STAT3, STAT5 and STAT6 in HuH7 (A) and Hep3B (B) cells. HuH7 and Hep3B cells were either unstimulated or stimulated with 400 IU/ml IFNα or IFNβ for the indicated time. 20μg cell extracts were resolved by 7.5% SDS-PAGE, and analysed by Western blotting using phosphoprotein specific antibodies (p-STAT1, p-STAT2, p-STAT3, p-STAT5, p-STAT6). The blot was later stripped and re-probed with STAT1, STAT2, STAT3, STAT5, STAT6 and βactin antibodies to ensure equal loading of the cell extracts. (C and D) STAT6 activation exhibited similar kinetic patterns as STAT1 in IFN-α or β treated HuH7 (C) and Hep3B (D) cells. The amount of activated STAT1 and STAT6 of HuH7 (C) and Hep3B (D) cells treated with 400 IU/ml IFNα or IFNβ was quantified and the results are expressed in relative expression level over basal; the results are represented as the mean±S.D. for three repetitions. Asterisks indicate the calculated P values for paired comparisons between IFNα and IFNβ at various times; all were < 0.05.

Mentions: To study the signalling pathways induced by type I IFN in hepatocytes, HuH7 and Hep3B cells were treated with 400 IU/ml IFNα or IFNβ. We cannot detect any growth inhibition activities in HuH7 and Hep3B cells when treated with up to 3000 IU/ml IFN-α or IFN-β (data not shown). Cells were then harvested at different time points and Western blotted to study the activation of STAT proteins after treatments. As shown in Fig. 1, both IFN-α and IFN-β were able to activate STAT1, STAT2 and STAT3 in HuH7 (Fig. 1A) and Hep3B (Fig. 1B) cells, which are common pathways involved in type I IFN signalling. Interestingly, STAT5 and STAT6 were also activated in response to IFN-α and IFN-β, although STAT5 activation was much weaker than STAT6. IFNβ induced stronger STAT5 activation than IFNα in both cell lines tested (Fig. 1). The time course study revealed that STAT2 and STAT3 activation prolonged longer time than STAT1 and STAT6 activation by detecting the content of phosphorylated tyrosine. In general, IFNβ treatment induced more acute and stronger effects on STAT proteins than IFNα (Fig. 1A and B). It is notable that STAT6 activation exhibited kinetic patterns similar to those of STAT1 in IFN-α or IFN-β treated HuH7 (Fig. 1A and C) and Hep3B (Fig. 1B and D) cells, indicating that STAT6 plays a significant role in hepatocytes in response to 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)

STAT6 is tyrosine phosphorylated in response to IFNα and IFNβ in HuH7 and Hep3B cells. (A and B) IFN-α and IFN-β were able to activate STAT1, STAT2, STAT3, STAT5 and STAT6 in HuH7 (A) and Hep3B (B) cells. HuH7 and Hep3B cells were either unstimulated or stimulated with 400 IU/ml IFNα or IFNβ for the indicated time. 20μg cell extracts were resolved by 7.5% SDS-PAGE, and analysed by Western blotting using phosphoprotein specific antibodies (p-STAT1, p-STAT2, p-STAT3, p-STAT5, p-STAT6). The blot was later stripped and re-probed with STAT1, STAT2, STAT3, STAT5, STAT6 and βactin antibodies to ensure equal loading of the cell extracts. (C and D) STAT6 activation exhibited similar kinetic patterns as STAT1 in IFN-α or β treated HuH7 (C) and Hep3B (D) cells. The amount of activated STAT1 and STAT6 of HuH7 (C) and Hep3B (D) cells treated with 400 IU/ml IFNα or IFNβ was quantified and the results are expressed in relative expression level over basal; the results are represented as the mean±S.D. for three repetitions. Asterisks indicate the calculated P values for paired comparisons between IFNα and IFNβ at various times; all were < 0.05.
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Related In: Results  -  Collection

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fig01: STAT6 is tyrosine phosphorylated in response to IFNα and IFNβ in HuH7 and Hep3B cells. (A and B) IFN-α and IFN-β were able to activate STAT1, STAT2, STAT3, STAT5 and STAT6 in HuH7 (A) and Hep3B (B) cells. HuH7 and Hep3B cells were either unstimulated or stimulated with 400 IU/ml IFNα or IFNβ for the indicated time. 20μg cell extracts were resolved by 7.5% SDS-PAGE, and analysed by Western blotting using phosphoprotein specific antibodies (p-STAT1, p-STAT2, p-STAT3, p-STAT5, p-STAT6). The blot was later stripped and re-probed with STAT1, STAT2, STAT3, STAT5, STAT6 and βactin antibodies to ensure equal loading of the cell extracts. (C and D) STAT6 activation exhibited similar kinetic patterns as STAT1 in IFN-α or β treated HuH7 (C) and Hep3B (D) cells. The amount of activated STAT1 and STAT6 of HuH7 (C) and Hep3B (D) cells treated with 400 IU/ml IFNα or IFNβ was quantified and the results are expressed in relative expression level over basal; the results are represented as the mean±S.D. for three repetitions. Asterisks indicate the calculated P values for paired comparisons between IFNα and IFNβ at various times; all were < 0.05.
Mentions: To study the signalling pathways induced by type I IFN in hepatocytes, HuH7 and Hep3B cells were treated with 400 IU/ml IFNα or IFNβ. We cannot detect any growth inhibition activities in HuH7 and Hep3B cells when treated with up to 3000 IU/ml IFN-α or IFN-β (data not shown). Cells were then harvested at different time points and Western blotted to study the activation of STAT proteins after treatments. As shown in Fig. 1, both IFN-α and IFN-β were able to activate STAT1, STAT2 and STAT3 in HuH7 (Fig. 1A) and Hep3B (Fig. 1B) cells, which are common pathways involved in type I IFN signalling. Interestingly, STAT5 and STAT6 were also activated in response to IFN-α and IFN-β, although STAT5 activation was much weaker than STAT6. IFNβ induced stronger STAT5 activation than IFNα in both cell lines tested (Fig. 1). The time course study revealed that STAT2 and STAT3 activation prolonged longer time than STAT1 and STAT6 activation by detecting the content of phosphorylated tyrosine. In general, IFNβ treatment induced more acute and stronger effects on STAT proteins than IFNα (Fig. 1A and B). It is notable that STAT6 activation exhibited kinetic patterns similar to those of STAT1 in IFN-α or IFN-β treated HuH7 (Fig. 1A and C) and Hep3B (Fig. 1B and D) cells, indicating that STAT6 plays a significant role in hepatocytes in response to 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