FIGURE 4.
IL-17A/DEX synergistically induces the expression of Lcn2 and Saa3 through upregulating CEBPB. (A–C) Schematic diagrams showing the putative binding sites for multiple transcription factors (including NF-κB, NR3C1, and C/EBP) in the mouse and human LCN2 and SAA promoters. (B and C) ChIP-qPCR of RELA, CEBPB, and NR3C1 occupancy at the promoter of mouse Lcn2 and Saa3. Nucleic extracts from untreated (CTRL) and treated mASMCs were immunoprecipitated with IgG, anti-RELA, anti-CEBPB, or anti-GR, followed by DNA purification and RT-PCR quantitation with primers spanning the putative RELA, CEBPB, or NR3C1 binding sites of the Saa3 and Lcn2 promoter. (D and E) mASMCs were untreated (CTRL) or treated with IL-17A (100 ng/ml), DEX (1 μM), or IL-17A+DEX for 24 h. mRNA expression of Cebpb was measured by RT-PCR analysis (D). Protein levels were examined by Western blotting (E). (F and G) mASMCs were transfected with pooled small interfering RNAs (siRNAs) targeting CEBPB or scrambled control and treated as indicated for 24 h. The mRNA and protein levels were then analyzed by RT-PCR (F) and ELISA (G), respectively. (H) Western blot analysis of CEBPB in mASMCs transfected with pooled siRNAs targeting Cebpb or scrambled control and treated as indicated for 24 h. (I) mASMCs were pretreated with DEX for 4 h and then treated with ActD (5 μM) either alone (CTRL) or in the presence of IL-17A (100 ng/ml) for 25, 50, 70, and 100 min, then subjected to RT-PCR analysis (n = 3 independent plates of cells). The indicated mRNA levels were normalized to those of Gapdh and are presented as half-life. Data represent mean ± SEM (n = 3 biological replicates). (J) Primary mASMCs isolated from UBC-Cre-ERT2/LSL-HA-Act1f/+ mice were treated with IL-17A or sham control (CTRL) for 3 h and subjected to RIP with IgG or anti-HA. Cebpb mRNA was analyzed by qRT-PCR. Relative values normalized against IgG control are shown. (K) ChIP analysis of IL-17A/DEX–induced binding of CEBPB and NR3C1 to Cebpb promoter as described in (B) and (C). (L) REMSA for the binding of purified recombinant Act1 SEFIR to the serial-deletion mutants of Cebpb240 (142–382 nt). For (D), data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. For (B), (C), and (K), data represent mean ± SEM. Two-way ANOVA was performed to compare each treated group with the control group, followed by Dunnett's multiple-comparisons test. For (F) and (G), statistical analysis was performed using two-tailed Student t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For (I) and (J), data represent mean ± SEM. Statistical analysis was performed using a two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. All data are representative of three independent experiments. AU, fold induction relative to unchallenged control mice; TS, transcription start site.