Rout-Pitt Nathan, Boog Bernadette, McCarron Alexandra, Reyne Nicole, Parsons David, Donnelley Martin
Robinson Research Institute, University of Adelaide, South Australia; Adelaide Medical School, University of Adelaide, South Australia; Department of Respiratory and Sleep Medicine, Women's and Children's Hospital, South Australia.
J Cyst Fibros. 2025 Jan;24(1):149-156. doi: 10.1016/j.jcf.2024.09.003. Epub 2024 Sep 12.
Molecular pathways contributing to Cystic Fibrosis pathogenesis remain poorly understood. Epithelial-mesenchymal transition (EMT) has been recently observed in CF lungs and certain CFTR mutation classes may be more susceptible than others. No investigations of EMT processes in CF animal models have been reported.
The aim of this study was to assess the expression of EMT-related markers in Phe508del and knockout (CFTR-KO) rat lung tissue and tracheal-derived basal epithelial stem cells, to determine whether CFTR dysfunction can produce an EMT state.
The expression of EMT-related markers in lung tissue and cultured tracheal basal epithelial stem cells from wildtype (WT), Phe508del, and CFTR-KO rats were assessed using qPCR and Western blots. Cell responses were evaluated in the presence of Rho-associated protein kinase (ROCK) inhibitor Y27632, which blocks EMT-pathways, or after treatment with TGFβ1 to stimulate EMT.
Different gene expression profiles were observed between Phe508del and CFTR-KO rat models compared to wild type. There was lower expression of type 1 collagen in KO lungs and primary cell cultures, while Phe508del lungs and cells had higher expression, particularly when treated with TGFβ1. The addition of Y27632 rescued changes in EMT related genes in Phe508del cells but not in KO cells.
Our findings show the first evidence of upregulated EMT pathways in the lungs and airway cells of any CF animal model. Differences in the regulation of the EMT genes and proteins in the Phe508del and CFTR-KO cells suggest that the signalling pathways underlying EMT are CFTR mutation dependent.
导致囊性纤维化发病机制的分子途径仍未被充分了解。上皮-间质转化（EMT）最近在囊性纤维化肺部中被观察到，并且某些囊性纤维化跨膜传导调节因子（CFTR）突变类型可能比其他类型更易发生。目前尚未有关于囊性纤维化动物模型中EMT过程的研究报道。
本研究旨在评估苯丙氨酸508缺失（Phe508del）和基因敲除（CFTR-KO）大鼠肺组织及气管来源的基底上皮干细胞中EMT相关标志物的表达，以确定CFTR功能障碍是否会导致EMT状态。
使用定量聚合酶链反应（qPCR）和蛋白质免疫印迹法评估野生型（WT）、Phe508del和CFTR-KO大鼠肺组织及培养的气管基底上皮干细胞中EMT相关标志物的表达。在存在阻断EMT途径的Rho相关蛋白激酶（ROCK）抑制剂Y27632的情况下，或在用转化生长因子β1（TGFβ1）刺激EMT后，评估细胞反应。
与野生型相比，Phe铲8del和CFTR-KO大鼠模型之间观察到不同的基因表达谱。在基因敲除肺组织和原代细胞培养物中，I型胶原表达较低，而Phe508del肺组织和细胞表达较高，尤其是在用TGFβ1处理时。添加Y27632可挽救Phe508del细胞中EMT相关基因的变化，但不能挽救基因敲除细胞中的变化。
我们的研究结果首次证明了在任何囊性纤维化动物模型的肺和气道细胞中EMT途径上调。Phe508del和CFTR-KO细胞中EMT基因和蛋白质调节的差异表明，EMT潜在的信号通路依赖于CFTR突变。
