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...

Like many steroid receptors, the glucocorticoid (GC) receptor (GR) is a phosphoprotein. Although there are multiple phosphorylation sites critical for GR transcriptional activity (i.e., serine [S]203, S211, and S226), their respective role in driving GR functions is highly cell specific. We have recently identified protein phosphatase 5 as an essential Ser/Thr phosphatase responsible for impairing GR function via S211 dephosphorylation in airway smooth muscle (ASM) cells. Because p38 mitogen-activated protein kinase (MAPK) directly phosphorylates GR in different cell types in a stimulus-and celldependent manner, we investigated the role of p38 MAPK on GR phosphorylation and function in ASM cells. Cells were transfected with 100 nM p38 MAPK small interfering RNA or 2 mg MAPK kinase 3 expression vector (a specific kinase that directly activates p38 MAPK) in the presence or absence of fluticasone (100 nM) and/or p38 MAPK pharmacological inhibitor SB203580. We found that p38 MAPK blockade positively regulates GR nuclear translocation and GR-dependent induction of the steroid-target gene GC-induced leucine zipper in a hormone-independent manner. We also found that p38 MAPK-dependent regulation of GR functions was associated with a differential action on GR phosphorylation at S203 and S211 residues. This study demonstrated that the inactive state of GR in resting conditions is not only ensured by the absence of the GC ligand but also by p38 MAPK-dependent phosphorylation of unliganded GR at specific residues, which appears to be important in determining the overall GC responsiveness of ASM cells.

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The use of the complete DNA sequence for the Marek's disease virus (MDV) serotype 1 vaccine strain CVI988 Rispens in comparative genomic studies with virulent strains of MDV has revealed the presence of a number of insertions, deletions and single-nucleotide polymorphisms. In this study, we investigated a SNP in the H/ACA box of the viral RNA subunit of telomerase (vTR). We sequenced vTR from four different batches of CVI988 vaccine originating from a single commercial company. The A-to-G mutation defining the SNP in the H/ACA box of CVI988 vTR was present in only some of the batches. Thus, although this mutation affects CVI988 vTR function, it is not shared by all CVI988 isolates and may be a stochastic rather than causative event in CVI988 attenuation.

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