Airways Inflammation Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N4N1.
Biochem J. 2010 Mar 15;427(1):113-24. doi: 10.1042/BJ20091038.
In the present study, IL (interleukin)-1beta increased GM-CSF (granulocyte/macrophage colony-stimulating factor) expression from pulmonary A549 cells and primary HBE (human bronchial epithelial) cells. These responses were repressed by the glucocorticoid dexamethasone, allowing the use of A549 cells as a relevant model. IL-1beta induced GM-CSF release into the culture medium by 6 h and in cell lysates (cytosolic) at 2 h. These effects were profoundly inhibited by dexamethasone, yet IL-1beta-induced GM-CSF mRNA and unspliced nRNA (nuclear RNA; a surrogate of transcription rate) were modestly inhibited by dexamethasone at times up to 2 h. Although this indicates an effect on protein synthesis, actinomycin D chase experiments also indicated post-transcriptional repression by dexamethasone. Dexamethasone-dependent mRNA repression increased with time and was prevented by translational blockade. In addition, dexamethasone and the dissociated steroid RU24858 repressed GM-CSF release in an actinomycin D-sensitive manner, thereby implicating glucocorticoid-induced gene expression. At 2 h, IL-1beta-induced expression of GM-CSF protein, but not mRNA, was sensitive to the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] inhibitors PD098059 and U0126. Although this indicates a role for the MEK/ERK pathway in GM-CSF translation, PD098059 subsequently destabilized GM-CSF mRNA. Dexamethasone and RU24858 both reduced IL-1beta-induced ERK phosphorylation and increased MKP-1 (MAPK phosphatase-1) expression. Inhibition of ERK phosphorylation was reproduced by MKP-1 overexpression and prevented by MKP-1-targeting siRNA (small interfering RNA). Since MKP-1 prevented GM-CSF expression by transcriptional, post-transcriptional and translational processes, we propose that glucocorticoids induce MKP-1 expression to reduce both MEK/ERK activation and GM-CSF protein synthesis. Thus de novo gene expression, particularly of MKP-1, is involved in the repressive effects of glucocorticoids.
在本研究中，白细胞介素-1β（IL-1β）可增加肺 A549 细胞和原代人支气管上皮细胞（HBE）中的粒细胞/巨噬细胞集落刺激因子（GM-CSF）的表达。这些反应可被糖皮质激素地塞米松所抑制，因此可将 A549 细胞用作相关模型。IL-1β可在 6 小时时诱导 GM-CSF 释放到培养基中，在 2 小时时诱导细胞裂解物（细胞质）中的 GM-CSF 释放。这些作用可被地塞米松显著抑制，但地塞米松在长达 2 小时的时间内仅能适度抑制 IL-1β诱导的 GM-CSF mRNA 和未剪接的 nRNA（核 RNA；转录率的替代物）。虽然这表明其对蛋白质合成有影响，但放线菌素 D 追踪实验也表明地塞米松通过转录后抑制来抑制 GM-CSF。地塞米松依赖性 mRNA 抑制作用随时间增加，并且可被翻译阻断所阻止。此外，地塞米松和分离的类固醇 RU24858 以放线菌素 D 敏感的方式抑制 GM-CSF 释放，从而暗示糖皮质激素诱导的基因表达。在 2 小时时，IL-1β诱导的 GM-CSF 蛋白表达，但不是 mRNA 表达，对地塞米松和 RU24858 敏感。尽管这表明 MEK[丝裂原激活蛋白激酶/细胞外信号调节激酶（ERK）激酶]MAPK（mitogen-activated protein kinase）激酶]抑制剂 PD098059 和 U0126 在 GM-CSF 翻译中起作用，但 PD098059 随后使 GM-CSF mRNA 不稳定。地塞米松和 RU24858 均可降低 IL-1β诱导的 ERK 磷酸化，并增加 MAPK 磷酸酶-1（MKP-1）的表达。通过过表达 MKP-1 可复制 ERK 磷酸化的抑制作用，并通过 MKP-1 靶向 siRNA（小干扰 RNA）来阻止 ERK 磷酸化的抑制作用。由于 MKP-1 通过转录、转录后和翻译过程来抑制 GM-CSF 表达，因此我们提出糖皮质激素诱导 MKP-1 表达以减少 MEK/ERK 激活和 GM-CSF 蛋白合成。因此，新基因表达，特别是 MKP-1 的表达，参与了糖皮质激素的抑制作用。
