Roach Katy M, Feghali-Bostwick Carol, Wulff Heike, Amrani Yassine, Bradding Peter
Department of Infection, Immunity and Inflammation, Institute for Lung Health, University of Leicester, Glenfield Hospital, Groby Road, Leicester, LE3 9QP UK.
Department of Medicine, Division of Rheumatology and Immunology, University of South Carolina, Columbia, SC 29208 USA.
Fibrogenesis Tissue Repair. 2015 Mar 26;8:5. doi: 10.1186/s13069-015-0022-0. eCollection 2015.
Idiopathic pulmonary fibrosis (IPF) is a common and invariably lethal interstitial lung disease with poorly effective therapy. Blockade of the K(+) channel KCa3.1 reduces constitutive α-SMA and Smad2/3 nuclear translocation in IPF-derived human lung myofibroblasts (HLMFs), and inhibits several transforming growth factor beta 1 (TGFβ1)-dependent cell processes. We hypothesized that KCa3.1-dependent cell processes also regulate the TGFβ1-dependent Smad2/3 signalling pathway in HLMFs. HLMFs obtained from non-fibrotic controls (NFC) and IPF lungs were grown in vitro and examined for αSMA expression by immunofluorescence, RT-PCR, and flow cytometry. Two specific and distinct KCa3.1 blockers (TRAM-34 200 nM and ICA-17043 [Senicapoc] 100 nM) were used to determine their effects on TGFβ1-dependent signalling. Expression of phosphorylated and total Smad2/3 following TGFβ1 stimulation was determined by Western blot and Smad2/3 nuclear translocation by immunofluorescence.
KCa3.1 block attenuated TGFβ1-dependent Smad2/3 phosphorylation and nuclear translocation, and this was mimicked by lowering the extracellular Ca(2+) concentration. KCa3.1 block also inhibited Smad2/3-dependent gene transcription (αSMA, collagen type I), inhibited KCa3.1 mRNA expression, and attenuated TGFβ1-dependent αSMA protein expression.
KCa3.1 activity regulates TGFβ1-dependent effects in NFC- and IPF-derived primary HLMFs through the regulation of the TGFβ1/Smad signalling pathway, with promotion of downstream gene transcription and protein expression. KCa3.1 blockers may offer a novel approach to treating IPF.
特发性肺纤维化（IPF）是一种常见且总是致命的间质性肺病，治疗效果不佳。阻断钾离子通道KCa3.1可减少IPF来源的人肺成肌纤维细胞（HLMFs）中组成型α-平滑肌肌动蛋白（α-SMA）和Smad2/3核转位，并抑制几种转化生长因子β1（TGFβ1）依赖性细胞过程。我们推测KCa3.1依赖性细胞过程也调节HLMFs中TGFβ1依赖性Smad2/3信号通路。从非纤维化对照（NFC）和IPF肺中获得的HLMFs在体外培养，并通过免疫荧光、逆转录聚合酶链反应（RT-PCR）和流式细胞术检测αSMA表达。使用两种特异性且不同的KCa3.1阻滞剂（200 nM的TRAM-34和100 nM的ICA-17043 [司尼可保]）来确定它们对TGFβ1依赖性信号传导的影响。通过蛋白质免疫印迹法测定TGFβ1刺激后磷酸化和总Smad2/3的表达，并通过免疫荧光测定Smad2/3核转位。
KCa3.1阻断减弱了TGFβ1依赖性Smad2/3磷酸化和核转位，降低细胞外钙离子浓度可模拟这一作用。KCa3.1阻断还抑制了Smad2/3依赖性基因转录（α-SMA、I型胶原），抑制KCa3.1信使核糖核酸（mRNA）表达，并减弱TGFβ1依赖性α-SMA蛋白表达。
KCa3.1活性通过调节TGFβ1/Smad信号通路来调节NFC和IPF来源的原代HLMFs中TGFβ1依赖性效应，促进下游基因转录和蛋白表达。KCa3.1阻滞剂可能为治疗IPF提供一种新方法。
