Cytokines Alter Glucocorticoid Receptor Phosphorylation in Airway Cells

Abstract

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 regulatory factor-1, suggesting the involvement of additional mechanisms (Am J Respir Cell Mol Biol 2008;38:463-472). Although GC receptor (GR) can be phosphorylated at multiple serines in the Nterminal region, the major phosphorylation sites critical for 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 phosphorylated at Ser226 but not at Ser211 residues. Cytokines dramatically suppressed fluticasone-induced phosphorylation of GR on Ser211 but not on Ser226 residues while increasing the expression of 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 RNAinduced 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 was suppressed. We propose that the novel mechanism that proasthmatic cytokine-induced CSI in ASMCs is due, in part, to PP5-mediated impairment of GR-Ser211 phosphorylation.Keywords: serine/threonine protein phosphatase; airway smooth muscle; asthma; corticosteroid insensitivity; airway remodeling Although in the majority of patients with asthma symptoms are well controlled by inhaled glucocorticoids (GCs), a subgroup of patients suffering from severe asthma respond poorly to GC therapy (1, 2). This group is responsible for a disproportionate share of health care costs and morbidity associated with the disease; as a result, corticosteroid insensitivity (CSI) in patients with severe asthma represents a significant unmet clinical need (3). Recent studies performed on bronchial biopsies from patients with asthma showed a persistent expression of eotaxin (4), CCL19 (5), ADAM33, and ADAM8 (6, 7) in airway smooth muscle (ASM) bundles despite patients being treated with inhaled or oral GCs. The expression of these GC-insensitive proasthmatic proteins by ASM correlated with the severity of asthma. We and others replicated this finding in vitro using cultured human ASM cells (ASMCs) exposed to a combination of TNF-a and IFN-g (8-10). We showed that the induction of a variety of proasthmatic genes, namely CD38, CXCL10, CX3CL1, and CCL5, by TNF-a and IFN-g was surprisingly insensitive to the inhibition by GC (9, 11). This "corticosteroidinsensitive" state induced by TNF-a and IFN-g was associate...

Dynamic responses of the adrenal steroidogenic regulatory network

“…35), points to an interaction between cytokines and GR. Indeed, previous findings show that the LPSinduced proinflammatory cytokine TNF-α inhibits GR phosphorylation in Serine 211 in human airway cells (36). In light of this, we extended our model to account for cross-talk between components of the SRN and the immune pathway, and used it to test whether these interactions could account for the observed response to LPS.…”

Basal p38 Mitogen-Activated Protein Kinase Regulates Unliganded Glucocorticoid Receptor Function in Airway Smooth Muscle Cells

“…All experiments involving human tissues were approved by the Thomas Jefferson University Institutional Review Board. The culture of human ASM cells was performed as described elsewhere (8,30).…”

“…To ensure equal loading, the membranes were stripped and reprobed with anti-total GR or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (Santa Cruz Biotechnology). Scanning densitometry of three representative immunoblots with each condition normalized over the area density of the corresponding GAPDH content was then performed using a Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD) as we have previously described (8). Immunoprecipitation experiments using anti-p38 and anti-PP5 antibodies (Santa Cruz Biotechnology) were performed as described (31).…”

“…Ligand binding, which results in Hsp90 dissociation, allows kinases to interact with GR, which becomes phosphorylated. Evidence from different cell types, including airway smooth muscle (ASM) cells, suggests that GR-mediated transactivation positively correlates with the phosphorylation status of GR at S211 residues, whereas S226 decreases GR transcriptional activities (1,(5)(6)(7)(8). In contrast to GR phosphorylation at S211 and S226 residues, which has received a lot of attention, the role of GR phosphorylation at S203 residue has not been fully investigated (7,9,10).…”

“…We recently found that the protein phosphatase (PP)5 was essential in driving cytokine-induced GC insensitivity by promoting GR dephosphorylation at S211 in ASM cells (8). However, the nature of kinases responsible for GR phosphorylation in ASM cells and in other cell types is controversial.…”