1. JAK inhibitors suppress IL-17–induced expression of IκB-ζ.
JAK inhibitors suppress IL-17–induced expression of IκB-ζ. (A) HaCaT cells transfected with 20 pmol of STAT3 siRNA (siSTAT3) or control siRNA (siCtrl) were incubated with IL-17 (100 ng/ml) for 1.5 h. Transcript expression was analyzed by qPCR and shown as relative values. Data are mean ± SEM (n = 3). **p < 0.01 compared with nontreated control; #p < 0.05, ##p < 0.01 compared with siCtrl. (B) Summary of reported IC50 values of JAK inhibitors. (C) JAK inhibitors suppressed IL-17–induced IκB-ζ expression. HaCaT cells were pretreated with various concentrations of JAK inhibitors as indicated for 1 h and subsequently treated with IL-17 (100 ng/ml) for 1.5 h. The amount of IκB-ζ mRNA was determined by qPCR. Transcript expression is shown as relative values. Data are mean ± SEM (n = 3). **p < 0.01, ***p < compared with no inhibitor control; #p < 0.05 compared with cells treated with cerdulatinib. (D) JAK inhibitor treatment suppressed the level of constitutive STAT3 phosphorylation. Cell lysates were prepared from the cells treated as in (C) and analyzed by Western blotting analysis with an anti–phospho-STAT3 Ab. The experiment was repeated three times. (E) Cerdulatinib treatment compromised constitutive STAT3 binding to the IκB-ζ TSS1 promoter. HaCaT cells were treated with vehicle control or cerdulatinib (10 μM) for 1 h and subjected to ChIP with an anti-STAT3 Ab. The means of percentage input values from three independent experiments are shown. Error bars represent ±SEM. *p < 0.05 compared with IgG immunoprecipitation (control); #p < 0.05 compared with DMSO treatment. (F) Cerdulatinib suppressed IL-17–induced upregulation of IκB-ζ and β-defensin 2 proteins. HaCaT cells were pretreated with cerdulatinib (0.63 μM) for 1 h and then stimulated with IL-17 (100 ng/ml) for 3 h. The cells were lysed and analyzed by Western blotting using indicated Abs. The experiment was repeated three times. (G) A model of IL-17–driven IκB-ζ expression in cells. STAT3 activity is required for the transcriptional activation of the IκB-ζ gene. IL-17–induced inactivation of Regnase-1 allows accumulation of IκB-ζ mRNA.
Ryuta Muromoto et al. ImmunoHorizons 2019;3:
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