Pro-Asthmatic Cytokines Regulate Unliganded and Ligand-Dependent Glucocorticoid Receptor Signaling in Airway Smooth Muscle

Abstract

To elucidate the regulation of glucocorticoid receptor (GR) signaling under pro-asthmatic conditions, cultured human airway smooth muscle (HASM) cells were treated with proinflammatory cytokines or GR ligands alone and in combination, and then examined for induced changes in ligand-dependent and -independent GR activation and downstream signaling events. Ligand stimulation with either cortisone or dexamethsone (DEX) acutely elicited GR translocation to the nucleus and, comparably, ligand-independent stimulation either with the Th2 cytokine, IL-13, or the pleiotropic cytokine combination, IL-1β/TNFα, also acutely evoked GR translocation. The latter response was potentiated by combined exposure of cells to GR ligand and cytokine. Similarly, treatment with either DEX or IL-13 alone induced GR phosphorylation at its serine-211 residue (GRSer211), denoting its activated state, and combined treatment with DEX+IL-13 elicited heightened and sustained GRSer211 phosphorylation. Interestingly, the above ligand-independent GR responses to IL-13 alone were not associated with downstream GR binding to its consensus DNA sequence or GR transactivation, whereas both DEX-induced GR:DNA binding and transcriptional activity were significantly heightened in the presence of IL-13, coupled to increased recruitment of the transcriptional co-factor, MED14. The stimulated GR signaling responses to DEX were prevented in IL-13-exposed cells wherein GRSer211 phosphorylation was suppressed either by transfection with specific serine phosphorylation-deficient mutant GRs or treatment with inhibitors of the MAPKs, ERK1/2 and JNK. Collectively, these novel data highlight a heretofore-unidentified homeostatic mechanism in HASM cells that involves pro-asthmatic cytokine-driven, MAPK-mediated, non-ligand-dependent GR activation that confers heightened glucocorticoid ligand-stimulated GR signaling. These findings raise the consideration that perturbations in this homeostatic cytokine-driven GR signaling mechanism may be responsible, at least in part, for the insensirtivity to glucocorticoid therapy that is commonly seen in individuals with severe asthma.

Glucocorticoid and TNF signaling converge at A20 (TNFAIP3) to repress airway smooth muscle cytokine expression

“…1). Moreover, aligned with prior ASM studies (8,26), this process appears to be enhanced at certain loci by TNF. For example, GR occupancy levels were markedly increased by TNF treatment compared with vehicle at several tested loci, including IL1A and CXCL8 (see Fig.…”

“…2, C and D, top). In some cases there was detectable GR occupancy with vehicle or TNF treatment alone, consistent with other recent reports of nuclear localization of so-called unliganded GR in ASM (8,26). Regardless of the magnitude of inductive effects of TNF or dex alone on GR occupancy, however, TNF ϩ dex cotreatment resulted in generally similar patterns and levels of GR binding at interro- gated regions within the pro-and anti-inflammatory gene sets.…”

Cistrome-based Cooperation between Airway Epithelial Glucocorticoid Receptor and NF-κB Orchestrates Anti-inflammatory Effects

“…occupancy in the absence of supplemental dex treatment)? We previously attributed this phenomenon, which has been observed in earlier studies by us and other groups (36,53,55,78,79), to the activity of glucocorticoid-like molecules presumed to be present at low levels within the culture medium. However, several aspects of our ChIP-seq data are incompatible with this notion.…”

Lipid Raft- and Protein Kinase C-mediated Synergism between Glucocorticoid- and Gonadotropin-releasing Hormone Signaling Results in Decreased Cell Proliferation

“…The association of the GR with the membrane could provide a potential mechanism allowing the reciprocal modulation of downstream signaling pathways between the GR and other membrane-associated receptors. Interestingly, synergistic responses involving GCs and other ligands such as gonadotropin-releasing hormone (GnRH), interleukin-2 (IL-2), IL-13, activin, and tumor necrosis factor ␣ (TNF-␣) have been reported (10,19,(32)(33)(34). However, the mechanisms of these synergistic responses and whether they involve membrane-associated GR in complex with other receptors has not been previously reported.…”