Knabe Lucie, Varilh Jessica, Bergougnoux Anne, Gamez Anne-Sophie, Bonini Jennifer, Pommier Alexandra, Petit Aurélie, Molinari Nicolas, Vachier Isabelle, Taulan-Cadars Magali, Bourdin Arnaud
PhyMedExp, University of Montpellier, INSERM U1046, CNRS UMR 9214, Montpellier, France; Department of Respiratory Diseases, Montpellier University Hospital, Montpellier, France.
EA7402, Montpellier University, Montpellier, France.
Am J Physiol Lung Cell Mol Physiol. 2016 Oct 1;311(4):L696-L703. doi: 10.1152/ajplung.00280.2016. Epub 2016 Aug 5.
Impaired airway homeostasis in chronic obstructive pulmonary disease (COPD) could be partly related to club cell secretory protein (CCSP) deficiency. We hypothesize that CCSP G38A polymorphism is involved and aim to examine the influence of the CCSP G38A polymorphism on CCSP transcription levels and its regulatory mechanisms. CCSP genotype and CCSP levels in serum and sputum were assessed in 66 subjects with stable COPD included in a 1-yr observational study. Forty-nine of them had an exacerbation. In an in vitro study, the impact on the CCSP promoter of 38G wild-type or 38A variant was assessed. BEAS-2B cells were transfected by either the 38G or 38A construct, in the presence/absence of cigarette smoke extract (CSE) or lipopolysaccharides (LPS). Cotransfections with modulating transcription factors, p53 and Nkx2.1, identified by in silico analysis by using ConSite and TFSEARCH were conducted. A allele carrier COPD patients had lower serum and sputum CCSP levels, especially among active smokers, and a decreased body mass index, airflow obstruction, dyspnea, and exercise capacity (BODE) score. In vitro, baseline CCSP transcription levels were similar between the wild and variant constructs. CSE decreased more profoundly the CCSP transcription level of 38A transfected cells. The opposite effect was observed with p53 cotransfection. LPS stimulation induced CCSP repression in 38A promoter transfected cells. Cotransfection with Nkx2.1 significantly activated the CCSP promoters irrespective of the polymorphism. Circulating CCSP levels are associated with smoking and the CCSP G38A polymorphism. CSE, LPS, and the Nkx2.1 and p53 transcription factors modulated the CCSP promoter efficiency. The 38A polymorphism exaggerated the CCSP repression in response to p53 and CSE.
慢性阻塞性肺疾病（COPD）中气道稳态受损可能部分与克拉拉细胞分泌蛋白（CCSP）缺乏有关。我们推测CCSP G38A多态性与之相关，并旨在研究CCSP G38A多态性对CCSP转录水平及其调控机制的影响。在一项为期1年的观察性研究中，对66例稳定期COPD患者的CCSP基因型以及血清和痰液中的CCSP水平进行了评估。其中49例患者出现了病情加重。在一项体外研究中，评估了38G野生型或38A变体对CCSP启动子的影响。BEAS-2B细胞用38G或38A构建体转染，同时存在/不存在香烟烟雾提取物（CSE）或脂多糖（LPS）。通过使用ConSite和TFSEARCH进行计算机分析鉴定出与转录调节因子p53和Nkx2.1进行共转染。携带A等位基因的COPD患者血清和痰液中的CCSP水平较低，尤其是在现吸烟者中，且体重指数、气流阻塞、呼吸困难和运动能力（BODE）评分降低。在体外，野生型和变体构建体之间的基线CCSP转录水平相似。CSE更显著地降低了38A转染细胞的CCSP转录水平。p53共转染时观察到相反的效果。LPS刺激在38A启动子转染细胞中诱导CCSP表达受抑制。与Nkx2.1共转染可显著激活CCSP启动子，无论其多态性如何。循环CCSP水平与吸烟及CCSP G38A多态性相关。CSE、LPS以及Nkx2.1和p53转录因子调节了CCSP启动子效率。38A多态性增强了对p53和CSE的CCSP抑制反应。
