Impaired Dual-Specificity Protein Phosphatase DUSP4 Reduces Corticosteroid Sensitivity

We have reported that phosphorylation of the glucocorticoid receptor (GR) at Ser²²⁶ reduces GR nuclear translocation, resulting in corticosteroid insensitivity in patients with severe asthmas. A serine/threonine protein phosphatase 2A, which regulates c-Jun N-terminal kinase (JNK) 1 and GR-Ser²²⁶ signaling, is involved in this mechanism. Here, we further explored protein kinase dual-specificity phosphatases (DUSPs) with the ability to dephosphorylate JNK, and identified DUSP4 as a phosphatase involved in the regulation of corticosteroid sensitivity. The effects of knocking down DUSPs (DUSP1, 4, 8, 16, and 22) by small interfering RNA (siRNA) were evaluated in a monocytic cell line (U937). Corticosteroid sensitivity was determined by dexamethasone enhancement of FK506-binding protein 51 or inhibition of tumor necrosis factor α (TNFα)–induced interferon γ and interleukin 8 expression and GR translocation from cell cytoplasm to nucleus. The nuclear/cytoplasmic GR, phosphorylation levels of GR-Ser²²⁶ and JNK1, coimmunoprecipitated GR-JNK1-DUSP4, and DUSP4 expression were analyzed by western blotting and/or imaging flow cytometry. Phosphatase activity of immunoprecipitated (IP)-DUSP4 was measured by fluorescence-based assay. Knockdown of DUSP4 enhanced phosphorylation of GR-Ser²²⁶ and JNK1 and reduced GR nuclear translocation and corticosteroid sensitivity. Coimmunoprecipitation experiments showed that DUSP4 is associated with GR and JNK1. In peripheral blood mononuclear cells from severe asthmatics, DUSP4 expression was reduced versus healthy subjects and negatively correlated with phosphorylation levels of GR-Ser²²⁶ and JNK1. Formoterol enhanced DUSP4 activity and restored corticosteroid sensitivity reduced by DUSP4 siRNA. In conclusion, DUSP4 regulates corticosteroid sensitivity via dephosphorylation of JNK1 and GR-Ser²²⁶. DUSP4 activation by formoterol restores impaired corticosteroid sensitivity, indicating that DUSP4 is crucial in regulating corticosteroid sensitivity, and therefore might be a novel therapeutic target in severe asthma.