FIGURE 5.

Blocking Act1 binding to Cebpb transcript reduces expression of Lcn2 and Saa3 in mice on type 17 severe asthma model. (A) SBE RNA aptamers (Cxcl1-SBE and SBE mutant) were used to compete with Cebpb probe (203–382 nt) for binding to Act1 SEFIR. (B) Confocal imaging of RFP-Act1 and fluorescein amidite–labeled SBE aptamer (SBE-FAM) in transfected mouse ASMCs. Blue, DAPI nuclear staining. Scale bars, 20 μm. (C–F) mASMCs transfected with 100 pmol SBE RNA aptamers or no oligonucleotide control were subjected to stimulation with DEX+IL-17A for 0, 6, or 24 h. mRNA expression of Cebpb (C) was then analyzed by RT-PCR. Protein levels were measured by Western blot (D). The mRNA and protein levels of Lcn2 and Saa3 were analyzed by RT-PCR (E) and ELISA (F). (G–I) Eight-week-old WT C57BL/6 female mice were sensitized and challenged with HDM, then subjected to treatment as indicated (n = 5 mice/group). Total and neutrophil cell numbers in the BAL of the indicated mice (n = 5 mice/group) were quantified (G). mRNA expression of lung tissues was quantified by RT-PCR (H). The levels of IL-17A, SAA3, and LCN2 in the serum were measured using ELISA (I). For (C), (E), and (F), data represent mean ± SEM. Two-way ANOVA was performed to compare each treated group with no oligo group, followed by Dunnett's multiple-comparisons test. For (G)–(I), data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. All data are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (J) Model of IL-17A and glucocorticoid signaling for the induction of steroid-resistant IL-17A target genes (CEBPB, LCN2, and SAA). On stimulation with glucocorticoid, IL-17A–induced NF-κB was inhibited, and instead GR cooperates with CEBPB to amplify the expression of SR genes. In addition, IL-17A induces Act1 binding and stabilization of CEBPB mRNA to enhance gene expression.