Abstract
BackgroundCorticosteroid insensitivity is a major barrier of treatment for some chronic inflammatory diseases, such as severe asthma, but the molecular mechanism of the insensitivity has not been fully elucidated. The object of this study is to investigate the role of protein phosphate 2A (PP2A), a serine/threonine phosphatase, on corticosteroid sensitivity in severe asthma.Methodology/Principal FindingsCorticosteroid sensitivity was determined by the dexamethasone ability to inhibit TNFα-induced IL-8 or LPS-induced TNFα production. PP2A expression, glucocorticoid receptor (GR) nuclear translocation defined as the nuclear/cytoplasmic GR ratio and phosphorylation of GR-Ser226, c-Jun N-terminal kinase 1 (JNK1) and PP2A were analysed by Western-blotting. Phosphatase activity was measured by fluorescence-based assay. Okadaic acid (OA), a PP2A inhibitor, reduced corticosteroid sensitivity with reduced GR nuclear translocation and increased GR phosphorylation in U937 monocytic cells. PP2A knockdown by RNA interference showed similar effects. IL-2/IL-4 treatment to U937 reduced corticosteroid sensitivity, and PP2A expression/activity. In peripheral blood mononuclear cells (PBMCs) from severe asthma, the PP2A expression and activity were significantly reduced with concomitant enhancement of PP2AC-Tyr307 phosphorylation compared with those in healthy volunteers. As the results, GR-Ser226 and JNK1 phosphorylation were increased. The expression and activity of PP2A were negatively correlated with phosphorylation levels of GR-Ser226. Furthermore, co-immunoprecipitation assay in U937 cells revealed that PP2A associated with GR and JNK1 and IL-2/IL-4 exposure caused dissociation of each molecule. Lastly, PP2A overexpression increased corticosteroid sensitivity in U937 cells.Conclusions/SignificancePP2A regulates GR nuclear translocation and corticosteroid sensitivity possibly by dephosphorylation of GR-Ser226 via dephosphorylation of upstream JNK1. This novel mechanism will provide new insight for the development of new therapy for severe asthma.
Highlights
Bronchial asthma has been recognized as a chronic inflammatory disease of the airways with increasing trend of its prevalence
We hypothesized that defect of phosphate 2A (PP2A) impairs steroid effects via failure of dephosphorylation of glucocorticoid receptor (GR) at Ser226 and we demonstrated this first time in peripheral blood mononuclear cells (PBMCs) obtained from severe asthmatics
Okadaic acid (OA) significantly inhibited dexamethasone (1027 M)-induced GR nuclear translocation defined as the ratio of nuclear and cytoplasmic GR band density
Summary
Bronchial asthma has been recognized as a chronic inflammatory disease of the airways with increasing trend of its prevalence. Small population (approximately 5–10%) of asthmatics develops severe asthma, and has greater morbidity with corticosteroid insensitive and a disproportionate contribution to health care spending [2]. Understanding the molecular mechanism of corticosteroid insensitivity may provide clues to improve treatment for patients with severe asthma. The impairment of corticosteroid responsiveness observed in severe asthma has been induced by decreased glucocorticoid receptor (GR) a expression, increased decoy GR receptor (GRb), defected ligand binding for GR, reduced GR nuclear translocation and GR/glucocorticoid response elements (GREs) binding [3] as well as HDAC2 reduction. In some asthmatics with corticosteroid insensitivity, nuclear translocation of GR in response to dexamethasone was impaired [4]. Corticosteroid insensitivity is a major barrier of treatment for some chronic inflammatory diseases, such as severe asthma, but the molecular mechanism of the insensitivity has not been fully elucidated. The object of this study is to investigate the role of protein phosphate 2A (PP2A), a serine/threonine phosphatase, on corticosteroid sensitivity in severe asthma
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