Rider Christopher F, Shah Suharsh, Miller-Larsson Anna, Giembycz Mark A, Newton Robert
Airways Inflammation Research Group, Snyder Institute of Chronic Diseases, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
AstraZeneca R&D Mölndal, Mölndal, Sweden.
PLoS One. 2015 Jan 27;10(1):e0116773. doi: 10.1371/journal.pone.0116773. eCollection 2015.
Acting on the glucocorticoid receptor (NR3C1), glucocorticoids are widely used to treat inflammatory diseases. However, glucocorticoid resistance often leads to suboptimal asthma control. Since glucocorticoid-induced gene expression contributes to glucocorticoid activity, the aim of this study was to use a 2 × glucocorticoid response element (GRE) reporter and glucocorticoid-induced gene expression to investigate approaches to combat cytokine-induced glucocorticoid resistance. Pre-treatment with tumor necrosis factor-α (TNF) or interleukin-1β inhibited dexamethasone-induced mRNA expression of the putative anti-inflammatory genes RGS2 and TSC22D3, or just TSC22D3, in primary human airway epithelial and smooth muscle cells, respectively. Dexamethasone-induced DUSP1 mRNA was unaffected. In human bronchial epithelial BEAS-2B cells, dexamethasone-induced TSC22D3 and CDKN1C expression (at 6 h) was reduced by TNF pre-treatment, whereas DUSP1 and RGS2 mRNAs were unaffected. TNF pre-treatment also reduced dexamethasone-dependent 2×GRE reporter activation. This was partially reversed by PS-1145 and c-jun N-terminal kinase (JNK) inhibitor VIII, inhibitors of IKK2 and JNK, respectively. However, neither inhibitor affected TNF-dependent loss of dexamethasone-induced CDKN1C or TSC22D3 mRNA. Similarly, inhibitors of the extracellular signal-regulated kinase, p38, phosphoinositide 3-kinase or protein kinase C pathways failed to attenuate TNF-dependent repression of the 2×GRE reporter. Fluticasone furoate, fluticasone propionate and budesonide were full agonists relative to dexamethasone, while GSK9027, RU24858, des-ciclesonide and GW870086X were partial agonists on the 2×GRE reporter. TNF reduced reporter activity in proportion with agonist efficacy. Full and partial agonists showed various degrees of agonism on RGS2 and TSC22D3 expression, but were equally effective at inducing CDKN1C and DUSP1, and did not affect the repression of CDKN1C or TSC22D3 expression by TNF. Finally, formoterol-enhanced 2×GRE reporter activity was also proportional to agonist efficacy and functionally reversed repression by TNF. As similar effects were apparent on glucocorticoid-induced gene expression, the most effective strategy to overcome glucocorticoid resistance in this model was addition of formoterol to high efficacy NR3C1 agonists.
糖皮质激素作用于糖皮质激素受体（NR3C1），被广泛用于治疗炎症性疾病。然而，糖皮质激素抵抗常常导致哮喘控制不佳。由于糖皮质激素诱导的基因表达有助于糖皮质激素活性，本研究的目的是使用一个2×糖皮质激素反应元件（GRE）报告基因和糖皮质激素诱导的基因表达来研究对抗细胞因子诱导的糖皮质激素抵抗的方法。用肿瘤坏死因子-α（TNF）或白细胞介素-1β预处理分别抑制了地塞米松诱导的原代人气道上皮细胞和气道平滑肌细胞中假定的抗炎基因RGS2和TSC22D3或仅TSC22D3的mRNA表达。地塞米松诱导的DUSP1 mRNA不受影响。在人支气管上皮BEAS-2B细胞中，TNF预处理降低了地塞米松诱导的TSC22D3和CDKN1C表达（6小时时），而DUSP1和RGS2 mRNA不受影响。TNF预处理也降低了地塞米松依赖性的2×GRE报告基因激活。这被PS-1145和c-jun N端激酶（JNK）抑制剂VIII部分逆转，它们分别是IKK2和JNK的抑制剂。然而，两种抑制剂都不影响TNF依赖性的地塞米松诱导的CDKN1C或TSC22D3 mRNA的缺失。同样，细胞外信号调节激酶、p38、磷脂酰肌醇3激酶或蛋白激酶C途径的抑制剂未能减弱TNF依赖性的2×GRE报告基因的抑制。糠酸氟替卡松、丙酸氟替卡松和布地奈德相对于地塞米松是完全激动剂，而GSK9027、RU24858、去环索奈德和GW870086X在2×GRE报告基因上是部分激动剂。TNF按激动剂效力比例降低报告基因活性。完全激动剂和部分激动剂在RGS2和TSC22D3表达上表现出不同程度的激动作用，但在诱导CDKN1C和DUSP1方面同样有效，并且不影响TNF对CDKN1C或TSC22D3表达的抑制。最后，福莫特罗增强的2×GRE报告基因活性也与激动剂效力成比例，并且在功能上逆转了TNF的抑制作用。由于对糖皮质激素诱导的基因表达有类似影响，在该模型中克服糖皮质激素抵抗的最有效策略是将福莫特罗添加到高效能的NR3C1激动剂中。
