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 (GR(Ser211)), denoting its activated state, and combined treatment with DEX+IL-13 elicited heightened and sustained GR(Ser211) 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 GR(Ser211) 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.

Figure 1. Separate and cooperative effects of DEX-stimulated and unliganded IL-13-induced nuclear translocation of GR in HASM cells.

GR localization detected by Immunofluorescence staining (A) and its quantification (B) depicting that, relative to diffuse cytoplasmic distribution of GR in vehicle-exposed (control) HASM cells (a), treatment for 30 min with either cortisone (b) or IL-13 alone (c) elicited increased intra-nuclear localization of GR. which was further enhanced in cells co-treated with cortisone ± IL-13 (d). Note: Induced nuclear translocation of GR by IL-13 alone denotes the presence of ligand-independent GR activation, and heightened GR translocation exhibited by cells treated with cortisone ± IL-13 demonstrates cooperative effects of ligand (cortisone)-induced and IL-13-induced ligand-independent GR activation. Data represent mean±SD values based on an average of 25 HASM cells examined over 3–5 repeats (*p<0.05; **p<0.01).

Figure 2. Immunoblot analysis of separate and combined effects of ligand-stimulated and unliganded IL-13-induced GR^Ser211 phosphorylation in HASM cells.

(A) Treatment with IL-13 elicits progressive increases in p-GR^Ser211 for up to 24 h following cytokine exposure that are detected in the nuclear (N) and not the cytoplasmic (C) fraction. Induction of p-GR^Ser211 by cortisone (B) or DEX (C) is increased in HASM cells initially exposed for 24 h to IL-13; and the p-GR^Ser211 response is markedly suppressed in cells pretreated with the GR antagonist, RU486.

Figure 3. Comparison of DNA binding activity of GR in HASM cells exposed to either DEX or IL-13 alone and in combination.

(A) EMSA depicting that GR binding to its consensus DNA sequence is acutely elicited by ligand stimulation with DEX, whereas treatment with IL-13 alone does not evoke GR:DNA binding. (B) Following 24 h exposure to vehicle alone (control), HASM cells treated with DEX exhibit dose-dependent stimulation of GR:DNA binding activity, the latter quantified using a sensitive ELISA kit. GR:DNA binding activity is significantky increased at all administered doses of DEX in cells pretreated for 24 h with IL-13. Data are mean ± SE values based on 4–5 determinations at each administered dose of DEX. *p<0.05.

Figure 4. Comparison of induced temporal changes in site-specific GR phosphorylation in HASM cells treated with DEX or IL-13 alone and in combination.

Representative immunoblots from one of three experiments depicting that, nder basal conditions, relative to p-GR^Ser211, HASM cells exhibit distinctly increased p-GR^Ser203 levels. Contrasting the acute transient increase in p-GR^Ser211 elicited by DEX treatment alone (A), HASM cells treated with IL-13 alone exhibit an initial increase in p-GR^Ser203 followed by a progressive increase in p-GR^Ser211 (B), whereas combined treatment with DEX+IL-13 evokes sustained increases in p-GR^Ser203 and p-GR^Ser211 for up to 24 h (C). Note, GR^total levels are unaffected in HASM cells exposed to IL-13. By comparison, cells treated with DEX (alone or in combination with IL-13) exhibit progressive decreases in GR^total levels.

Figure 5. Interdependence of IL-13-induced GR^Ser203 and GR^Ser211 phosphorylation in HASM cells: Role in regulating GR tranactivation.

(A) Immunoblots using anti-HA- and anti-GR phosphorylation-specific antibodies comparing the effects of IL-13 on GR^Ser203 and p-GR^Ser211 phosphorylation in HASM cells transfected with wild-type (WT) and either Ser²⁰³ or Ser²¹¹ phosphorylation-deficient mutant GR (S203A or S211A). Note: Under similar loading conditions, given by comparable levels of GR^total detected with anti-HA antibody, IL-13-induced increase in GR^Ser211 phosphorylation is prevented in HASM cells transfected with the S203A mutant GR, whereas phosphorylation of GR^Ser203 by IL-13 is unaffected in cells transfected with the S211A mutant construct (B) Comparison of effects of IL-13 on DEX-induced GR transactivation, detected using the SEAP reporter assay, in control HASM cells and cells transfected with either the S203A or S211A phosphorylation-deficient mutant GR. Note: Contrasting the lack of effect of IL-13 alone, SEAP activity is significantly increased in DEX-treated cells, and the response to DEX is significantly enhanced in the presence of IL-13. The latter stimulatory effect of IL-13 on DEX-induced GR transactivation is abrogated in cells transfected with the S203A mutant construct, and GR transactivation by DEX is prevented in cells transfected with the S211A mutant GR. Data represent mean ± SE values from 4–5 experiments (*p<0.05; **p<0.01).

Figure 6. GR phosphorylation by IL-13 and its induction of heightened DEX-stimulated GR^Ser211 phosphorylation and transactivation are suppressed by inhibition of ERK1/2 and JNK signaling.

(A) Representative immunoblots from one of three experiments demonstrating that IL-13-induced maximal GR^Ser203 and GR^Ser211 phosphorylation elicited at 3 and 12 h, respectively, are suppressed in HASM cells pretreated with either the ERK1/2 or JNK inhibitors, U0125 and SP600125, respectively, whereas pretreatment with the p38 MAPK inhiobitor, SB202190, has no appreciable effect. Comarably, heightened DEX-stimulated GR^Ser211 phosphorylation (B) and transactivation of SEAP activity (C) exhibited in IL-13-exposed HASM cells are suppressed by pretreatment with the ERK1/2 and JNK inhibitors, whereas the p38 MAPK inhiobitor has no effect. Data are mean ± SE values from 4 experiments (*p<0.05; **p<0.01).

Figure 7. Enhanced DEX-induced co-localization of p-GR^Ser211 and the transcriptional co-factor, MED14, in IL-13-exposed HASM cells is prevented by inhibition of ERK1/2 and JNK signaling.

(A) Representative co-immunoprecipitation study demonstrating increased co-localization of both phosphorylated GR^Ser211 and MED14, detected by immunoblotting (IB), with immunoprecipitated (IP) GR in nuclear extracts from HASM cells exposed to either IL-13 or DEX alone. The latter co-localiztion is further enhanced in IL-13+DEX-treated cells, and is largely suppressed by pretreating the cells with either the ERK1/2 or JNK inhibitor, whereas the p38 MAPK inhibitor has no effect. (B) Note: The above effects elicited under the different treatment conditions are detected in the absence of any corresponding change in total MED14 protein expression.

Figure 8. Schematic representation of proposed homeostatic mechanism involving cytokine-stimulated ligand-independent GR activation and its induction of heightened ligand-stimulated GR signaling and transactivation.

Cytokine (IL-13) exposure acutely elicits ERK1/2- and JNK-mediated GR^Ser203 phosphorylation that leads to p-GR^Ser203-dependent GR^Ser211 phosphorylation. The dual GR^Ser203/211 phosphorylated state is transient, as Ser203 becomes dephosphorylated (unidentified phosphatase (13)) and p-GR^Ser211 translocates to the nucleus where it associates with the transcriptional co-factor, MED14. In the absence of hormone, the unliganded p-GR^Ser211-MED14 complex is incapable of binding to DNA. With hormone exposure, ligand entering the cell can translocate to the nucleus both as free and GR-bound ligand, the latter leading to GR^Ser211 phosphorylation potentially via the kinase activities of cyclin-dependent kinase (CDK) or glycogen synthase kinase-3 (GSK-3). Note: relative to ligand treatment alone, combined cytokine+ligand exposure elicits increased intra-nuclear levels of p-GR^Ser211 coupled to MED14, leading to heightened GR transcriptional activity upon recruitment of free ligand to the cytokine-stimulated unliganded p-GR^Ser211-MED14 complex.