FIGURE 7.
KLF6 regulates macrophage polarization by modulating functions of PPARγ. A, wild-type BMDMs were stimulated with 10 ng/ml IL-4, and KLF6 ChIP analysis was performed on Arg1 (−1009 to −1013) and Mrc1 (−1637 to −1642) promoters. Fold changes in KLF6 enrichment over control are indicated. ChIP analysis performed using IgG was used as negative control. B, Lyz2cre and Lyz2cre:Klf6fl/fl mouse BMDMs were induced with 10 ng/ml IL-4 for 18 h. Total mRNA was analyzed for PPARγ expression by quantitative PCR analysis. C, RAW264.7 cells were overexpressed with KLF6 and stimulated with 10 ng/ml of IL-4. These cells are analyzed for PPARγ expression by quantitative PCR analysis. D and E, RAW264.7 cells were overexpressed with Klf6, and Lyz2cre/ Lyz2cre:Klf6fl/fl mouse BMDMs were induced with 10 ng/ml IL-4 for 18 h. Total cell lysate was analyzed for PPARγ protein expression by Western blot analysis. Actin was used as loading control. E and F, RAW264.7 cells overexpressed with Klf6 and Lyz2cre, Lyz2cre:Klf6fl/fl mouse BMDMs were induced with 10 ng/ml IL-4. ChIP analysis was performed on arginase1 promoter (−986 to −1003) utilizing anti-PPARγ antibody. ChIP analysis performed using IgG was used as negative control. G, Lyz2cre and Lyz2cre:Klf6fl/fl mouse BMDMs were exposed to GW9662 (2.5 μm) or rosiglitazone (5 μm). These cells were induced with 10 ng/ml IL-4 for 18 h, and Arg1 mRNA expression was analyzed by quantitative PCR analysis. H, Lyz2cre and Lyz2cre:Klf6fl/fl mouse BMDMs were transfected with control siRNA or siRNA targeting PPARγ by nucleofection. These cells were induced with IL-4 for 18 h, and Arg1 mRNA expression was analyzed by quantitative PCR analysis. - was used as a housekeeping gene. Each experiment was performed a minimum of three times. Data represent mean ± S.D., and a p value less than 0.05 between indicated groups are considered significant.