Cytokines alter glucocorticoid receptor phosphorylation in airway cells: role of phosphatases.

Corticosteroid insensitivity (CSI) represents a profound challenge in managing patients with asthma. We recently demonstrated that short exposure of airway smooth muscle cells (ASMCs) to proasthmatic cytokines drastically reduced their responsiveness to glucocorticoids (GCs), an effect that was partially mediated via interferon regulatoryfactor-1,suggestingtheinvolvementofadditionalmechanisms (Am J Respir Cell Mol Biol 2008;38:463–472). Although GC receptor (GR) can be phosphorylated at multiple serines in the Nterminalregion, the major phosphorylationsites criticalfor GR transcriptional activity are serines 211 (Ser211) and 226 (Ser226). We tested the novel hypothesis that cytokine-induced CSI in ASMCs is due to an impaired GR phosphorylation. Cells were treated with TNF-a (10 ng/ml) and IFN-g (500 UI/ml) for 6 hours and/or fluticasone (100 nm) added 2 hours before. GR was constitutively phosphorylatedatSer226 butnotat Ser211residues.Cytokines dramatically suppressed fluticasone-induced phosphorylation of GR on Ser211butnotonSer226residueswhileincreasingtheexpressionof Ser/Thr protein phosphatase (PP)5 but not that of PP1 or PP2A. Transfection studies using a reporter construct containing GC responsive elements showed that the specific small interfering RNA– induced mRNA knockdown of PP5, but not that of PP1 or PP2A, partially prevented the cytokine suppressive effects on GR-meditated transactivation activity. Similarly, cytokines failed to inhibit GC-induced GR-Ser211 phosphorylation when expression of PP5 wassuppressed.Weproposethatthenovelmechanismthatproasthmaticcytokine-inducedCSIinASMCsisdue,inpart,toPP5-mediated