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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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IFN-α- or IFN-β-induced phosphorylation of STAT6 is mediated by the JAK-STAT pathway. (A) JAK1 and Tyk2 were signal transducers for IFNα or IFNβ signalling in hepatocytes. A phosphorylation antibody array detecting relative levels of phosphorylation of 71 different receptor tyrosine kinases was incubated with extracts prepared from HuH7 cells; the extracts were either unstimulated (PBS), or stimulated with IFNα (400 IU/ml) or IFNβ (400 IU/ml) for 30 min. (B) The normalized activation level of JAK kinases (JAK1 and Tyk2) is presented. (C) SiRNAs against JAK1 and Tyk2 were introduced into the cells to down-regulate their expression levels. JAK1 and Tyk2 inhibition were checked by Western blotting using anti-JAK1 and TYK2 antibodies. The blot was later stripped and re-probed with β-actin antibody to ensure equal loading of the cell extracts. (D and E) Neither JAK1 (D) nor Tyk2 (E) inhibition completely inhibited the phosphorylation levels of STAT2 and STAT6. Extracts were prepared from HuH7 cells with or without a 30-min stimulation at 37°C with PBS, IFNα (400 IU/ml), and IFNβ (400 IU/ml). The activation of STAT2 and STAT6 by IFNα and IFNβ after JAK1 and Tyk2 knockdown were determined by Western blotting. The blot was later stripped and re-probed with STAT2 or STAT6 antibodies to ensure equal loading of the cell extracts. (F) STAT2 and STAT6 activation was inhibited by both JAK1 and Tyk2 knockdown. (G) STAT6 activation was not inhibited by JAK3 inhibitor. HuH7 cells were pretreated with 50 μM Pyridone 6, WHI-p131 or with DMSO as control for 6 hrs. The medium was replaced with fresh medium containing 400 IU/ml IFNα, IFNβ or PBS as control for 30 min. STAT6 inhibition were checked by Western blotting using antiphospho-STAT6 antibod-ies. The blot was later stripped and re-probed with STAT6 antibody to ensure equal loading of the cell extracts.
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fig03: IFN-α- or IFN-β-induced phosphorylation of STAT6 is mediated by the JAK-STAT pathway. (A) JAK1 and Tyk2 were signal transducers for IFNα or IFNβ signalling in hepatocytes. A phosphorylation antibody array detecting relative levels of phosphorylation of 71 different receptor tyrosine kinases was incubated with extracts prepared from HuH7 cells; the extracts were either unstimulated (PBS), or stimulated with IFNα (400 IU/ml) or IFNβ (400 IU/ml) for 30 min. (B) The normalized activation level of JAK kinases (JAK1 and Tyk2) is presented. (C) SiRNAs against JAK1 and Tyk2 were introduced into the cells to down-regulate their expression levels. JAK1 and Tyk2 inhibition were checked by Western blotting using anti-JAK1 and TYK2 antibodies. The blot was later stripped and re-probed with β-actin antibody to ensure equal loading of the cell extracts. (D and E) Neither JAK1 (D) nor Tyk2 (E) inhibition completely inhibited the phosphorylation levels of STAT2 and STAT6. Extracts were prepared from HuH7 cells with or without a 30-min stimulation at 37°C with PBS, IFNα (400 IU/ml), and IFNβ (400 IU/ml). The activation of STAT2 and STAT6 by IFNα and IFNβ after JAK1 and Tyk2 knockdown were determined by Western blotting. The blot was later stripped and re-probed with STAT2 or STAT6 antibodies to ensure equal loading of the cell extracts. (F) STAT2 and STAT6 activation was inhibited by both JAK1 and Tyk2 knockdown. (G) STAT6 activation was not inhibited by JAK3 inhibitor. HuH7 cells were pretreated with 50 μM Pyridone 6, WHI-p131 or with DMSO as control for 6 hrs. The medium was replaced with fresh medium containing 400 IU/ml IFNα, IFNβ or PBS as control for 30 min. STAT6 inhibition were checked by Western blotting using antiphospho-STAT6 antibod-ies. The blot was later stripped and re-probed with STAT6 antibody to ensure equal loading of the cell extracts.

Mentions: To determine the signalling cascade of STAT6 activation after type I IFN binding, a phosphorylation antibody array was performed to screen for receptor tyrosine kinases. Consistent with previous studies [2, 14], JAK1 and Tyk2 were signal transducers for IFN-α or IFN-β signalling in hepatocytes (Fig. 3A and B). Specific siRNAs were prepared and transfected into HuH7 cells to knockdown JAK1 and Tyk2 (Fig. 3B). However, neither JAK1 nor Tyk2 inhibition completely inhibited the phosphorylation levels of STAT1, STAT2 and STAT6 (Fig. 3C–E). When IFN-α- or IFN-β-treated cells were treated with a universal JAK kinase inhibitor (Pyridone 6) or with JAK1 + Tyk2 siRNAs, STAT6 phosphorylation was inhibited (Fig. 3G and F). As expected, treating cells with the JAK3 inhibitor WHI-p131 did not influence the phosphorylation levels of STAT6 as compared to the non-relevant inhibitor control. These data indicate that both JAK1 and Tyk2 kinases were involved in IFN-α-or IFN-β-induced STAT6 activation in hepatocytes. These two kinases, therefore, may play redundant biofunctional roles, since down regulation of either of them did not block the signalling cascades.


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)

IFN-α- or IFN-β-induced phosphorylation of STAT6 is mediated by the JAK-STAT pathway. (A) JAK1 and Tyk2 were signal transducers for IFNα or IFNβ signalling in hepatocytes. A phosphorylation antibody array detecting relative levels of phosphorylation of 71 different receptor tyrosine kinases was incubated with extracts prepared from HuH7 cells; the extracts were either unstimulated (PBS), or stimulated with IFNα (400 IU/ml) or IFNβ (400 IU/ml) for 30 min. (B) The normalized activation level of JAK kinases (JAK1 and Tyk2) is presented. (C) SiRNAs against JAK1 and Tyk2 were introduced into the cells to down-regulate their expression levels. JAK1 and Tyk2 inhibition were checked by Western blotting using anti-JAK1 and TYK2 antibodies. The blot was later stripped and re-probed with β-actin antibody to ensure equal loading of the cell extracts. (D and E) Neither JAK1 (D) nor Tyk2 (E) inhibition completely inhibited the phosphorylation levels of STAT2 and STAT6. Extracts were prepared from HuH7 cells with or without a 30-min stimulation at 37°C with PBS, IFNα (400 IU/ml), and IFNβ (400 IU/ml). The activation of STAT2 and STAT6 by IFNα and IFNβ after JAK1 and Tyk2 knockdown were determined by Western blotting. The blot was later stripped and re-probed with STAT2 or STAT6 antibodies to ensure equal loading of the cell extracts. (F) STAT2 and STAT6 activation was inhibited by both JAK1 and Tyk2 knockdown. (G) STAT6 activation was not inhibited by JAK3 inhibitor. HuH7 cells were pretreated with 50 μM Pyridone 6, WHI-p131 or with DMSO as control for 6 hrs. The medium was replaced with fresh medium containing 400 IU/ml IFNα, IFNβ or PBS as control for 30 min. STAT6 inhibition were checked by Western blotting using antiphospho-STAT6 antibod-ies. The blot was later stripped and re-probed with STAT6 antibody to ensure equal loading of the cell extracts.
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fig03: IFN-α- or IFN-β-induced phosphorylation of STAT6 is mediated by the JAK-STAT pathway. (A) JAK1 and Tyk2 were signal transducers for IFNα or IFNβ signalling in hepatocytes. A phosphorylation antibody array detecting relative levels of phosphorylation of 71 different receptor tyrosine kinases was incubated with extracts prepared from HuH7 cells; the extracts were either unstimulated (PBS), or stimulated with IFNα (400 IU/ml) or IFNβ (400 IU/ml) for 30 min. (B) The normalized activation level of JAK kinases (JAK1 and Tyk2) is presented. (C) SiRNAs against JAK1 and Tyk2 were introduced into the cells to down-regulate their expression levels. JAK1 and Tyk2 inhibition were checked by Western blotting using anti-JAK1 and TYK2 antibodies. The blot was later stripped and re-probed with β-actin antibody to ensure equal loading of the cell extracts. (D and E) Neither JAK1 (D) nor Tyk2 (E) inhibition completely inhibited the phosphorylation levels of STAT2 and STAT6. Extracts were prepared from HuH7 cells with or without a 30-min stimulation at 37°C with PBS, IFNα (400 IU/ml), and IFNβ (400 IU/ml). The activation of STAT2 and STAT6 by IFNα and IFNβ after JAK1 and Tyk2 knockdown were determined by Western blotting. The blot was later stripped and re-probed with STAT2 or STAT6 antibodies to ensure equal loading of the cell extracts. (F) STAT2 and STAT6 activation was inhibited by both JAK1 and Tyk2 knockdown. (G) STAT6 activation was not inhibited by JAK3 inhibitor. HuH7 cells were pretreated with 50 μM Pyridone 6, WHI-p131 or with DMSO as control for 6 hrs. The medium was replaced with fresh medium containing 400 IU/ml IFNα, IFNβ or PBS as control for 30 min. STAT6 inhibition were checked by Western blotting using antiphospho-STAT6 antibod-ies. The blot was later stripped and re-probed with STAT6 antibody to ensure equal loading of the cell extracts.
Mentions: To determine the signalling cascade of STAT6 activation after type I IFN binding, a phosphorylation antibody array was performed to screen for receptor tyrosine kinases. Consistent with previous studies [2, 14], JAK1 and Tyk2 were signal transducers for IFN-α or IFN-β signalling in hepatocytes (Fig. 3A and B). Specific siRNAs were prepared and transfected into HuH7 cells to knockdown JAK1 and Tyk2 (Fig. 3B). However, neither JAK1 nor Tyk2 inhibition completely inhibited the phosphorylation levels of STAT1, STAT2 and STAT6 (Fig. 3C–E). When IFN-α- or IFN-β-treated cells were treated with a universal JAK kinase inhibitor (Pyridone 6) or with JAK1 + Tyk2 siRNAs, STAT6 phosphorylation was inhibited (Fig. 3G and F). As expected, treating cells with the JAK3 inhibitor WHI-p131 did not influence the phosphorylation levels of STAT6 as compared to the non-relevant inhibitor control. These data indicate that both JAK1 and Tyk2 kinases were involved in IFN-α-or IFN-β-induced STAT6 activation in hepatocytes. These two kinases, therefore, may play redundant biofunctional roles, since down regulation of either of them did not block the signalling cascades.

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