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

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.

为阐明哮喘促炎条件下糖皮质激素受体（glucocorticoid receptor, GR）的信号调控机制，本研究将培养的人气道平滑肌（human airway smooth muscle, HASM）细胞分别或联合给予促炎细胞因子与GR配体处理，随后检测配体依赖性及非依赖性GR激活与下游信号事件的诱导变化。
单独给予可的松或地塞米松（dexamethasone, DEX）配体刺激，可快速诱导GR转位入核；与之类似，单独给予Th2型细胞因子IL-13，或联合给予多效性细胞因子组合IL-1β/TNFα的非配体依赖性刺激，同样可快速触发GR转位入核。上述非配体依赖的GR转位响应可因细胞同时暴露于GR配体与细胞因子而增强。
类似地，单独给予DEX或IL-13处理，均可诱导GR丝氨酸211位点（GRSer211）磷酸化，标志受体处于激活状态；而DEX与IL-13联合处理则可引发更强且更持久的GRSer211磷酸化。值得注意的是，上述单独给予IL-13所诱导的非配体依赖性GR响应，并未伴随GR与其共识DNA序列的下游结合或GR反式激活；然而，当存在IL-13时，DEX诱导的GR-DNA结合与转录活性均显著增强，并伴随转录辅因子MED14的招募增加。
在预先暴露于IL-13的细胞中，若通过转染丝氨酸磷酸化缺陷的GR突变体，或使用丝裂原活化蛋白激酶（mitogen-activated protein kinases, MAPKs）抑制剂ERK1/2与JNK抑制GRSer211磷酸化，则可阻断DEX诱导的GR信号响应。
综上，本研究的创新性数据揭示了HASM细胞中此前未被发现的稳态调控机制：哮喘促炎细胞因子通过MAPK介导非配体依赖性GR激活，进而增强糖皮质激素配体诱导的GR信号通路。上述发现提示，这种由细胞因子驱动的GR信号稳态机制紊乱，可能至少部分参与了重症哮喘患者普遍存在的糖皮质激素治疗抵抗现象。