Oxidized Phospholipids Inhibit Glucocorticoid Receptor Nuclear Translocation and Expression of Anti-inflammatory Genes in Airway Epithelial Cells

Abstract RATIONALE: Inhaled glucocorticoids (GC) are the primary controller therapy for asthma. Upon binding GC the glucocorticoid receptor (GR) translocates to the nucleus where it binds GRE-elements leading to the activation of anti-inflammatory gene transcription. We reported that oxidized phosphatidylcholines (OxPAPC) accumulate in the airways of asthmatics to promote cytokine release, airway narrowing and airway hyperresponsiveness. OxPAPC activate intracellular kinases, and transient receptor potential ankyrin 1 (TRPA1)-mediated Ca2+ influx in airway epithelial and smooth muscle cells. We tested the hypothesis that OxPAPC inhibit GC-induced anti-inflammatory gene expression by modulating nuclear translocation of GR in lung cells. METHODS: Transcriptional trans-activation by fluticasone propionate (FP; 1μM, 5 hrs) was measured in the human bronchial epithelial cell line, 2XGRE-BEAS 2B. In some experiments, we pre-exposed the reporter cells to pathophysiologically relevant concentrations of OxPAPC (80μg/mL, up to18 hours). Luciferase transcription was measured by Firefly Luciferase assay. In some studies, we also pre-incubated cells with the TRPA1 inhibitor, HC030031 (10µM). Expression of GC-inducible genes (see below) was assayed by qRT-PCR. We measured nuclear co-localization of GR using quantitative fluorescent immunocytochemistry and a pan anti-GR antibody. All experiments included at least five replicates. Data were analyzed using one-way ANOVA and Dunnett's post hoc testing. RESULTS: In 2xGRE BEAS-2B reporter cells, OxPAPC time dependently reduced FP-induced luciferase gene transcription activity by up to 62% (eg. 3,641±29.7 vs 9,622±705.7 units, respectively, with 18hrs OxPAPC pre-exposure). Inhibition of TRPA1 did not alter FP-induced luciferase transcription, however it was sufficient to prevent in OxPAPC suppression of FP-induced gene transcription. To validate this, in the same cell lines we assayed mRNA for three GC/GR inducible genes, and observed that OxPAPC markedly attenuated FP-induced expression of KLF9 (-93.7%, p<0.01), FKBP5 (-94.8%, p<0.001) and GILZ (-79.8%, P<0.058). TRPA1 inhibition partially (∼25%) reversed these suppressive effects of OxPAPC, but this was not statistically significant. Notably OxPAPC (40 μg/mL) pre-incubation significantly inhibited nuclear co-localization of GR after FP exposure (1μM, 2 hrs) (ie. Nuclear Co-localization Coefficient 0.852 vs 0.621, for FP and OxPAPC-FP, respectively, P<0.01). CONCLUSIONS: OxPAPC is sufficient to reduce GR-dependent transcription and GC-inducible gene expression, likely by inhibiting GR nuclear translocation in an airway epithelial cell line. This suggests a mechanism for OxPAPC to contribute to steroid insensitivity and promote persistent inflammation in asthma.

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