Hypoxia down-regulates glucocorticoid receptor alpha and attenuates the anti-inflammatory actions of dexamethasone in human alveolar epithelial A549 cells

Aims: Glucocorticoids (GCs) are frequently used to treat various pulmonary diseases, which are typically accompanied by hypoxia. Whether hypoxia influences the effects of GCs on human airway cells remains unclear. The aim of the present study was to characterize changes in the expression levels of two isoforms of the glucocorticoid receptor (GR) and to evaluate the anti-inflammatory actions of GCs under hypoxic conditions in A549 cells. Main methods: A549 cells were exposed to normoxic or hypoxic conditions for 24, 48 and 72 h. Morphological alterations of cells were captured using a differential interference contrast microscope (DIC), and cell cycle distribution was estimated by flow cytometry. Real-time quantitative polymerase chain reaction and western blot were used to determine the mRNA and protein expression levels of GR alpha and GR beta. Radioimmunoassay (RIA) for interleukin (IL)-8 was used to assess the anti-inflammatory actions of GCs after cells were stimulated with lipopolysaccharide (LPS). Key findings: After cells were exposed to hypoxic conditions for 48 h, visible morphological alterations in the cells were observed. Cell cycle analysis showed that the number of cells in G1 phase increased significantly under hypoxia compared to the normoxic conditions. Hypoxia caused a time-dependent decrease in both mRNA and protein expression levels for GR alpha, but not GR beta. Furthermore, when exposed to hypoxia for 48 h, the inhibitory effects of dexamethasone on LPS-stimulated IL-8 release were attenuated. Significance: These results indicate that hypoxia impairs the anti-inflammatory actions of GCs in A549 cells, which could be attributed to down-regulation of GR alpha expression under hypoxic conditions. (C) 2009 Elsevier Inc. All rights reserved.