Fehrholz Markus, Speer Christian P, Kunzmann Steffen
University Children's Hospital, University of Würzburg, Würzburg, Germany.
PLoS One. 2014 May 14;9(5):e97357. doi: 10.1371/journal.pone.0097357. eCollection 2014.
Caffeine administration is an important part of the therapeutic treatment of bronchopulmonary dysplasia (BPD) in preterm infants. However, caffeine mediated effects on airway remodelling are still undefined. The TGF-β/Smad signalling pathway is one of the key pathways involved in airway remodelling. Connective tissue growth factor (CTGF), a downstream mediator of TGF-β, and transgelin, a binding and stabilising protein of the cytoskeleton, are both regulated by TGF-β1 and play an important role in airway remodelling. Both have also been implicated in the pathogenesis of BPD. The aim of the present study was to clarify whether caffeine, an unspecific phosphodiesterase (PDE) inhibitor, and rolipram, a prototypical PDE-4 selective inhibitor, were both able to affect TGF-β1-induced Smad signalling and CTGF/transgelin expression in lung epithelial cells. Furthermore, the effect of transgelin knock-down on Smad signalling was studied. The pharmacological effect of caffeine and rolipram on Smad signalling was investigated by means of a luciferase assay via transfection of a TGF-β1-inducible reporter plasmid in A549 cells. The regulation of CTGF and transgelin expression by caffeine and rolipram were studied by promoter analysis, real-time PCR and Western blot. Endogenous transgelin expression was down-regulated by lentiviral transduction mediating transgelin-specific shRNA expression. The addition of caffeine and rolipram inhibited TGF-β1 induced reporter gene activity in a concentration-related manner. They also antagonized the TGF-β1 induced up-regulation of CTGF and transgelin on the promoter-, the mRNA-, and the protein-level. Functional analysis showed that transgelin silencing reduced TGF-β1 induced Smad-signalling and CTGF induction in lung epithelial cells. The present study highlights possible new molecular mechanisms of caffeine and rolipram including an inhibition of Smad signalling and of TGF-β1 regulated genes involved in airway remodelling. An understanding of these mechanisms might help to explain the protective effects of caffeine in prevention of BPD and suggests rolipram to be a potent replacement for caffeine.
给予咖啡因是早产儿支气管肺发育不良（BPD）治疗的重要组成部分。然而，咖啡因介导的对气道重塑的影响仍不明确。转化生长因子-β（TGF-β）/Smad信号通路是参与气道重塑的关键通路之一。结缔组织生长因子（CTGF）是TGF-β的下游介质，而凝溶胶蛋白是细胞骨架的结合和稳定蛋白，二者均受TGF-β1调控，并在气道重塑中起重要作用。它们也都与BPD的发病机制有关。本研究的目的是阐明非特异性磷酸二酯酶（PDE）抑制剂咖啡因和典型的PDE-4选择性抑制剂咯利普兰是否都能影响TGF-β1诱导的肺上皮细胞中Smad信号以及CTGF/凝溶胶蛋白的表达。此外，还研究了凝溶胶蛋白敲低对Smad信号的影响。通过在A549细胞中瞬时转染TGF-β1诱导型报告质粒，利用荧光素酶测定法研究了咖啡因和咯利普兰对Smad信号的药理作用。通过启动子分析、实时定量聚合酶链反应（PCR）和蛋白质免疫印迹法研究了咖啡因和咯利普兰对CTGF和凝溶胶蛋白表达的调控。通过慢病毒转导介导凝溶胶蛋白特异性短发夹RNA（shRNA）表达，下调内源性凝溶胶蛋白表达。咖啡因和咯利普兰的添加以浓度相关的方式抑制TGF-β1诱导的报告基因活性。它们还在启动子、信使核糖核酸（mRNA）和蛋白质水平拮抗TGF-β1诱导的CTGF和凝溶胶蛋白上调。功能分析表明，凝溶胶蛋白沉默降低了TGF-β1诱导的肺上皮细胞中Smad信号和CTGF诱导。本研究突出了咖啡因和咯利普兰可能的新分子机制，包括对Smad信号以及参与气道重塑的TGF-β1调控基因的抑制作用。对这些机制的理解可能有助于解释咖啡因在预防BPD中的保护作用，并提示咯利普兰是咖啡因的有效替代品。
