We have reported that phosphorylation of glucocorticoid receptor (GR) at Ser226 reduces GR nuclear translocation resulting in corticosteroid insensitivity in patients with severe asthmas. A serine/threonine protein phosphatase, PP2A, which regulates JNK1 and GR-Ser226 signaling, is involved in this mechanism. Here, we further explored dual-specificity protein kinases (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 siRNA were evaluated in a monocytic cell line (U937). Corticosteroid sensitivity was determined by dexamethasone enhancement of FKBP51 or inhibition of TNFα-induced IFNγ and IL-8 expression and to translocate GR from cell cytoplasm to nucleus. The nuclear/cytoplasmic GR, phosphorylation levels of GR-Ser226 and JNK1, co-immunoprecipitated GR-JNK1-DUSP4, and DUSP4 expression were analyzed by Western-blotting and/or Imaging Flow Cytometry. Phosphatase activity of immunoprecipitated 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. Co-immunoprecipitation experiments showed that DUSP4 is associated with GR and JNK1. In PBMCs 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.
- ERK p38 Jun
- Serine/threonine phosphatases
- Jun Kinase
- The American Society for Pharmacology and Experimental Therapeutics