A novel target of formoterol, a dual-specificity phosphatase DUSP4 on regulation of corticosteroid budesonide sensitivity

Introduction: We previously reported that formoterol, a long-acting β 2 -adrenoceptor agonist, activated a serine/threonine protein phosphatase PP2A which is involved in the restoration of corticosteroid (CS) sensitivity by formoterol (Kobayashi et al ERJ 2009;34:583s). We explored other phosphatases and identified dual-specificity phosphatase DUSP4 as a possible novel target of formoterol. Aims: To confirm that DUSP4 is activated by formoterol and is involved in regulation of CS sensitivity. Methods: U937 monocytic cells were incubated with IL-2 and IL-4 for 48 h to induce CS insensitivity. CS sensitivity was determined by the CS budesonide ability to inhibit TNFα-induced IL-8 production and to translocate glucocorticoid receptor (GR) from cell cytoplasm to nucleus. Phosphatase activity of immunopurified DUSP4 was measured by fluorescence-based assay. The nuclear/cytoplasmic GR ratio and phosphorylation levels of GR-Ser 226 and JNK1 kinase were analysed by western-blotting. In some experiments DUSP4 was knocked down by siRNA. Results: Formoterol increased DUSP4 activity, which was reduced under IL-2/IL-4 exposure. Knock-down of DUSP4 reduced GR nuclear translocation and CS sensitivity. Knock-down of DUSP4 also abrogated the dephosphorylation of GR-Ser 226 and JNK1 by formoterol - the effects involved in GR nuclear translocation and restoration of CS sensitivity. Conclusions: Formoterol regulates sensitivity to budesonide through activation of DUSP4 which dephosphorylates JNK1 and which in turn leads to dephosphorylation of GR-Ser 226 . This novel mechanism by formoterol may contribute to the clinical efficacy of combination of formoterol and budesonide.