STAT1 regulates interferon-γ-induced angiotensinogen and MCP-1 expression in a bidirectional manner in primary cultured mesangial cells

Figures

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  Figure 1.. Regulation of AGT expression by IFN-γ in MCs. MCs were treated with 0–20 ng/ml IFN-γ for 2 hours ((a) open column, N=4 replicates per condition from four independent experiments), 8 hours (grey column, N=4 replicates per condition from four independent experiments) and 24 hours (dark grey column, N=4 replicates per condition from four independent experiments). After the treatment, AGT mRNA was measured by qRT–PCR. Expression levels of AGT mRNA were normalised based on rat β-actin mRNA levels. In addition, AGT protein levels in IFN-γ-treated MCs were evaluated by western blot analysis (b). Data are expressed as relative values compared with the control group at each time point and represent the mean ± SE. Asterisk (P<0.05) indicates a significant difference compared with the control group. AGT: angiotensinogen; IFN-γ: interferon-γ; MCs: mesangial cells; qRT–PCR: quantitative real-time reverse transcriptase polymerase chain reaction.

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  Figure 2.. Regulation of MCP-1 expression by IFN-γ in MCs. MCs were treated with 0–20 ng/ml IFN-γ for 2 hours ((a) open column, N=4 replicates per condition from four independent experiments), 8 hours (grey column, N=4 replicates per condition from four independent experiments) and 24 hours (dark grey column, N=4 replicates per condition from four independent experiments). Data are expressed as relative values compared with the control group at each time point and represent the mean ± SE. Asterisk (P<0.05) indicates a significant difference compared with the control group. MCP-1: monocyte chemoattractant protein 1; IFN-γ: interferon-γ; MCs: mesangial cells.

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  Figure 3.. Activation STAT1 and STAT3 by IFN-γ in MCs. MCs were treated with 0–20 ng/ml IFN-γ for 8 hours. After treatment, STAT1 ((a) N=3 replicates per condition from three independent experiments) and STAT3 phosphorylation ((b) N=3 replicates per condition from three independent experiments) were evaluated by western blot analysis. These phosphorylation levels were normalised based on β-actin levels. Whether IFN-γ induces translocalisation of STAT1 and STAT3 into nucleus was evaluated. MCs were treated with 5 ng/mL IFN-γ for 8 hours. Thereafter, localisations of STAT1 and STAT3 were determined by immunofluorescence staining ((c) and (d)). Data are expressed as mean ± SE. Asterisk indicates a significant difference compared with the control group at each time point (P<0.05). IFN-γ: interferon-γ; MCs: mesangial cells.

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  Figure 4.. Contribution of STAT1 to the IFN-γ-induced AGT and MCP-1 augmentations. To elucidate further the mechanisms underlying the regulation of AGT and MCP-1 by IFN-γ, basal STAT1 expression was suppressed using STAT1-siRNA ((a) N=3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 8 hours. AGT mRNA ((b) N=3 replicates per condition from three independent experiments) and MCP-1 mRNA ((c) N=3 replicates per condition from three independent experiments) levels were measured by qRT–PCR. AGT and MCP-1 mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk (P<0.05) indicates a significant difference. IFN-γ: interferon-γ; AGT: angiotensinogen; MCP-1: monocyte chemoattractant protein 1; MCs: mesangial cells; siRNA: small interference RNA; qRT–PCR: quantitative real-time reverse transcriptase polymerase chain reaction.

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  Figure 5.. Regulation of SOCS1 expression by IFN-γ in MCs. MCs were treated with 0–20 ng/ml IFN-γ for 2 hours ((a) open column, N=4 replicates per condition from four independent experiments), 8 hours (grey column, N=4 replicates per condition from four independent experiments) and 24 hours (dark grey column, N=4 replicates per condition from 4 independent experiments). After the treatment, SOCS1 mRNA was measured by qRT–PCR. SOCS1 protein levels in IFN-γ-treated MCs (8 hours) were also determined by western blot analysis (b). Expression levels of SOCS1 were normalised based on rat β-actin mRNA levels. Data are expressed as relative values compared with the control group at each time point and represent the mean ± SE. Asterisk (P<0.05) indicates a significant difference compared with the control group. SOCS1: suppressor of cytokine signaling 1; IFN-γ: interferon-γ; MCs: mesangial cells; qRT–PCR: quantitative real-time reverse transcriptase polymerase chain reaction.

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  Figure 6.. Participation of SOCS1 in IFN-γ-induced AGT augmentations. Involvement of STAT1 in SOCS1 regulation was tested using STAT1-siRNA as shown in Figure 4 (a). Furthermore, MCs were treated with SOCS1-siRNA ((b) N=3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 24 hours. Then, AGT mRNA levels were measured by qRT–PCR (c). AGT mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk (P<0.05) indicates a significant difference. SOCS1: suppressor of cytokine signaling 1; IFN-γ: interferon-γ; AGT: angiotensinogen; siRNA: small interference RNA; MCs: mesangial cells; qRT–PCR: quantitative real-time reverse transcriptase polymerase chain reaction.

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  Figure 7.. Regulation of the IFN-γ-induced AGT augmentation by STAT3. Whether STAT3 is involved in the IFN-γ-induced AGT regulation in MCs was tested using STAT3-siRNA ((a) N=3 replicates per condition from three independent experiments). Thereafter, the cells were treated with 20 ng/ml IFN-γ for 8 hours. Then, AGT mRNA ((b) N=3 replicates per condition from three independent experiments) levels were measured by qRT–PCR. AGT mRNA levels were normalised based on β-actin mRNA levels. Data are expressed as relative values compared with each control group and represent the mean ± SE. Asterisk (P<0.05) indicates a significant difference. IFN-γ: interferon-γ; AGT: angiotensinogen; MCs: mesangial cells; siRNA: small interference RNA; qRT–PCR: quantitative real-time reverse transcriptase polymerase chain reaction.

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  Figure 8.. Schematic summary of proposed MCP-1, AGT regulation by IFN-γ-STAT1 in rat MCs. IFN-γ induces MCP-1 and SOCS1 expression directly via STAT1 in rat MCs. Conversely, STAT1-induced expression of SOCS1 attenuates IFN-γ-STAT3-mediated expression of AGT, providing feedback regulation of IFN-γ-AGT signaling in rat MCs. These results demonstrate that while IFN-γ increases both AGT and MCP-1 expression, STAT1 plays an opposing role in the regulation of each factor in MCs. MCP-1: monocyte chemoattractant protein 1; AGT: angiotensinogen; IFN-γ: interferon-γ; SOCS1: suppressor of cytokine signaling 1; MCs: mesangial cells.