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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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Treatment of HuH7 cells with IFNα, IFNβ, and IL-4 significantly enhanced STAT6 binding to CD23 and IL-1Ra promoter. Extracts were prepared from Huh7 cells with or without a 30-min stimulation at 37°C with IL-4 (10 ng/ml) (lane 1 and 5), phosphate buffered saline (lane 2 and 6;red lines for ChIP), IFNα (400 IU/ml;blue lines for ChIP) (lane 3 and 7; blue line for ChIP), and IFNβ (400 IU/ml; green lines for ChIP) (lane 4 and 8). The nuclear extracts were examined by electrophoretic mobility shift assay (EMSA) using either biotin-labelled CD23 (A) and IL-1Ra (B) GAS probes alone or together with 100-fold molar excess of non-biotin labelled probes. The antibodies against STAT6 inhibited STAT6 binding to IL-1Ra GAS probes (C). Activated STAT6 is bound to the IL-1Ra promoter in native chromatin determined by ChIP assays (D). DNA fragments recovered from ChIP assays with anti-STAT6 (solid lines) or anti-GAPDH (dashed lines) were analysed by quantitative PCR using Roche universal probe 87. The Cp values were shown in parentheses.
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fig04: Treatment of HuH7 cells with IFNα, IFNβ, and IL-4 significantly enhanced STAT6 binding to CD23 and IL-1Ra promoter. Extracts were prepared from Huh7 cells with or without a 30-min stimulation at 37°C with IL-4 (10 ng/ml) (lane 1 and 5), phosphate buffered saline (lane 2 and 6;red lines for ChIP), IFNα (400 IU/ml;blue lines for ChIP) (lane 3 and 7; blue line for ChIP), and IFNβ (400 IU/ml; green lines for ChIP) (lane 4 and 8). The nuclear extracts were examined by electrophoretic mobility shift assay (EMSA) using either biotin-labelled CD23 (A) and IL-1Ra (B) GAS probes alone or together with 100-fold molar excess of non-biotin labelled probes. The antibodies against STAT6 inhibited STAT6 binding to IL-1Ra GAS probes (C). Activated STAT6 is bound to the IL-1Ra promoter in native chromatin determined by ChIP assays (D). DNA fragments recovered from ChIP assays with anti-STAT6 (solid lines) or anti-GAPDH (dashed lines) were analysed by quantitative PCR using Roche universal probe 87. The Cp values were shown in parentheses.

Mentions: To examine whether IFN-activated-STAT6 can bind to DNA fragments containing STAT6 binding element (SBE), an electrophoretic mobility shift assay was performed using nuclear extracts from HuH7 cells cultured in the presence of IFNα, IFNβ and IL-4. Previously IL-4 was also shown to activate STAT6 in the treated cells, the nuclear extract from IL-4 treated cells was utilized as the positive control. DNA fragments containing CD23 promoter (located at bases ∼230 ∼ 214 region) and IL-Ra-SBE1 (bases ∼254 ∼–231), promoter regions previously identified as the binding elements for activated STAT6 in IL-4 treated cells, were mixed with these nuclear extracts. As shown in Fig. 4A, treatment with IFNα, IFNβ and IL-4 significantly enhanced STAT6 binding to CD23 promoter as compared to the PBS treatment control. We also found that IFN-α- or IFN-β-activated STAT6 was able to bind to IL-1Ra-SBE1 (IL-4 positive control) (Fig. 4B and C). Moreover, a ChIP experiment was also performed to confirm the binding of IFN-activated-STAT6 to IL-1Ra promoter. As shown in Fig. 4D, we can detect IL-1Ra promoter in IFNα and IFNβ treated HuH7 cells’ chromatins co-immunoprecipitated with anti-STAT6 antibody but not in chromatins co-immunoprecipitated with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. The crossing point (Cp) value for anti-STAT6 antibody co-immunoprecipitated chro-matins were 35.5, 25.55 and 24.82 of PBS-, IFNα- and IFNβ-treated HuH7 cells.


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)

Treatment of HuH7 cells with IFNα, IFNβ, and IL-4 significantly enhanced STAT6 binding to CD23 and IL-1Ra promoter. Extracts were prepared from Huh7 cells with or without a 30-min stimulation at 37°C with IL-4 (10 ng/ml) (lane 1 and 5), phosphate buffered saline (lane 2 and 6;red lines for ChIP), IFNα (400 IU/ml;blue lines for ChIP) (lane 3 and 7; blue line for ChIP), and IFNβ (400 IU/ml; green lines for ChIP) (lane 4 and 8). The nuclear extracts were examined by electrophoretic mobility shift assay (EMSA) using either biotin-labelled CD23 (A) and IL-1Ra (B) GAS probes alone or together with 100-fold molar excess of non-biotin labelled probes. The antibodies against STAT6 inhibited STAT6 binding to IL-1Ra GAS probes (C). Activated STAT6 is bound to the IL-1Ra promoter in native chromatin determined by ChIP assays (D). DNA fragments recovered from ChIP assays with anti-STAT6 (solid lines) or anti-GAPDH (dashed lines) were analysed by quantitative PCR using Roche universal probe 87. The Cp values were shown in parentheses.
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fig04: Treatment of HuH7 cells with IFNα, IFNβ, and IL-4 significantly enhanced STAT6 binding to CD23 and IL-1Ra promoter. Extracts were prepared from Huh7 cells with or without a 30-min stimulation at 37°C with IL-4 (10 ng/ml) (lane 1 and 5), phosphate buffered saline (lane 2 and 6;red lines for ChIP), IFNα (400 IU/ml;blue lines for ChIP) (lane 3 and 7; blue line for ChIP), and IFNβ (400 IU/ml; green lines for ChIP) (lane 4 and 8). The nuclear extracts were examined by electrophoretic mobility shift assay (EMSA) using either biotin-labelled CD23 (A) and IL-1Ra (B) GAS probes alone or together with 100-fold molar excess of non-biotin labelled probes. The antibodies against STAT6 inhibited STAT6 binding to IL-1Ra GAS probes (C). Activated STAT6 is bound to the IL-1Ra promoter in native chromatin determined by ChIP assays (D). DNA fragments recovered from ChIP assays with anti-STAT6 (solid lines) or anti-GAPDH (dashed lines) were analysed by quantitative PCR using Roche universal probe 87. The Cp values were shown in parentheses.
Mentions: To examine whether IFN-activated-STAT6 can bind to DNA fragments containing STAT6 binding element (SBE), an electrophoretic mobility shift assay was performed using nuclear extracts from HuH7 cells cultured in the presence of IFNα, IFNβ and IL-4. Previously IL-4 was also shown to activate STAT6 in the treated cells, the nuclear extract from IL-4 treated cells was utilized as the positive control. DNA fragments containing CD23 promoter (located at bases ∼230 ∼ 214 region) and IL-Ra-SBE1 (bases ∼254 ∼–231), promoter regions previously identified as the binding elements for activated STAT6 in IL-4 treated cells, were mixed with these nuclear extracts. As shown in Fig. 4A, treatment with IFNα, IFNβ and IL-4 significantly enhanced STAT6 binding to CD23 promoter as compared to the PBS treatment control. We also found that IFN-α- or IFN-β-activated STAT6 was able to bind to IL-1Ra-SBE1 (IL-4 positive control) (Fig. 4B and C). Moreover, a ChIP experiment was also performed to confirm the binding of IFN-activated-STAT6 to IL-1Ra promoter. As shown in Fig. 4D, we can detect IL-1Ra promoter in IFNα and IFNβ treated HuH7 cells’ chromatins co-immunoprecipitated with anti-STAT6 antibody but not in chromatins co-immunoprecipitated with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody. The crossing point (Cp) value for anti-STAT6 antibody co-immunoprecipitated chro-matins were 35.5, 25.55 and 24.82 of PBS-, IFNα- and IFNβ-treated HuH7 cells.

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