PDF(Pubmed)
Abstract：
BACKGROUND: The epithelial-mesenchymal transition (EMT) of human bronchial epithelial cells (HBECs) is essential for airway remodeling during asthma. Wnt5a has been implicated in various lung diseases, while its role in the EMT of HBECs during asthma is yet to be determined. This study sought to define whether Wnt5a initiated EMT, leading to airway remodeling through the induction of autophagy in HBECs.
METHODS: Microarray analysis was used to investigate the expression change of WNT5A in asthma patients. In parallel, EMT models were induced using 16HBE cells by exposing them to house dust mites (HDM) or interleukin-4 (IL-4), and then the expression of Wnt5a was observed. Using in vitro gain- and loss-of-function approaches via Wnt5a mimic peptide FOXY5 and Wnt5a inhibitor BOX5, the alterations in the expression of the epithelial marker E-cadherin and the mesenchymal marker protein were observed. Mechanistically, the Ca2+/CaMKII signaling pathway and autophagy were evaluated. An autophagy inhibitor 3-MA was used to examine Wnt5a in the regulation of autophagy during EMT. Furthermore, we used a CaMKII inhibitor KN-93 to determine whether Wnt5a induced autophagy overactivation and EMT via the Ca2+/CaMKII signaling pathway.
RESULTS: Asthma patients exhibited a significant increase in the gene expression of WNT5A compared to the healthy control. Upon HDM and IL-4 treatments, we observed that Wnt5a gene and protein expression levels were significantly increased in 16HBE cells. Interestingly, Wnt5a mimic peptide FOXY5 significantly inhibited E-cadherin and upregulated α-SMA, Collagen I, and autophagy marker proteins (Beclin1 and LC3-II). Rhodamine-phalloidin staining showed that FOXY5 resulted in a rearrangement of the cytoskeleton and an increase in the quantity of stress fibers in 16HBE cells. Importantly, blocking Wnt5a with BOX5 significantly inhibited autophagy and EMT induced by IL-4 in 16HBE cells. Mechanistically, autophagy inhibitor 3-MA and CaMKII inhibitor KN-93 reduced the EMT of 16HBE cells caused by FOXY5, as well as the increase in stress fibers, cell adhesion, and autophagy.
CONCLUSIONS: This study illustrates a new link in the Wnt5a-Ca2+/CaMKII-autophagy axis to triggering airway remodeling. Our findings may provide novel strategies for the treatment of EMT-related diseases.
METHODS: Microarray analysis was used to investigate the expression change of WNT5A in asthma patients. In parallel, EMT models were induced using 16HBE cells by exposing them to house dust mites (HDM) or interleukin-4 (IL-4), and then the expression of Wnt5a was observed. Using in vitro gain- and loss-of-function approaches via Wnt5a mimic peptide FOXY5 and Wnt5a inhibitor BOX5, the alterations in the expression of the epithelial marker E-cadherin and the mesenchymal marker protein were observed. Mechanistically, the Ca2+/CaMKII signaling pathway and autophagy were evaluated. An autophagy inhibitor 3-MA was used to examine Wnt5a in the regulation of autophagy during EMT. Furthermore, we used a CaMKII inhibitor KN-93 to determine whether Wnt5a induced autophagy overactivation and EMT via the Ca2+/CaMKII signaling pathway.
RESULTS: Asthma patients exhibited a significant increase in the gene expression of WNT5A compared to the healthy control. Upon HDM and IL-4 treatments, we observed that Wnt5a gene and protein expression levels were significantly increased in 16HBE cells. Interestingly, Wnt5a mimic peptide FOXY5 significantly inhibited E-cadherin and upregulated α-SMA, Collagen I, and autophagy marker proteins (Beclin1 and LC3-II). Rhodamine-phalloidin staining showed that FOXY5 resulted in a rearrangement of the cytoskeleton and an increase in the quantity of stress fibers in 16HBE cells. Importantly, blocking Wnt5a with BOX5 significantly inhibited autophagy and EMT induced by IL-4 in 16HBE cells. Mechanistically, autophagy inhibitor 3-MA and CaMKII inhibitor KN-93 reduced the EMT of 16HBE cells caused by FOXY5, as well as the increase in stress fibers, cell adhesion, and autophagy.
CONCLUSIONS: This study illustrates a new link in the Wnt5a-Ca2+/CaMKII-autophagy axis to triggering airway remodeling. Our findings may provide novel strategies for the treatment of EMT-related diseases.
摘要：
背景：人支气管上皮细胞（HBECs）的上皮-间质转化（EMT）对于哮喘期间的气道重塑至关重要。Wnt5a与各种肺部疾病有关，虽然其在哮喘期间HBECs的EMT中的作用尚待确定。这项研究试图确定Wnt5a是否启动了EMT，通过诱导HBECs中的自噬导致气道重塑。
方法：采用微阵列分析研究WNT5A在哮喘患者中的表达变化。并行,EMT模型使用16HBE细胞通过暴露于室内尘螨（HDM）或白介素-4（IL-4）来诱导，然后观察到Wnt5a的表达。通过Wnt5a模拟肽FOXY5和Wnt5a抑制剂BOX5使用体外功能增益和功能丧失方法，观察到上皮标记E-cadherin和间充质标记蛋白表达的变化。机械上,评价Ca2+/CaMKII信号通路和自噬。自噬抑制剂3-MA用于检测Wnt5a在EMT期间对自噬的调节。此外，我们使用CaMKII抑制剂KN-93来确定Wnt5a是否通过Ca2+/CaMKII信号通路诱导自噬过度激活和EMT.
结果：与健康对照相比，哮喘患者的WNT5A基因表达显着增加。在HDM和IL-4治疗后，我们观察到Wnt5a基因和蛋白表达水平在16HBE细胞中显著升高。有趣的是,Wnt5a模拟肽FOXY5显著抑制E-cadherin并上调α-SMA,胶原蛋白I,和自噬标记蛋白（Beclin1和LC3-II）。罗丹明-phalloidin染色显示FOXY5导致16HBE细胞中细胞骨架的重排和应力纤维的数量增加。重要的是,用BOX5阻断Wnt5a可显著抑制IL-4诱导的16HBE细胞自噬和EMT。机械上,自噬抑制剂3-MA和CaMKII抑制剂KN-93降低了FOXY5引起的16HBE细胞的EMT，并增加了应激纤维，细胞粘附，和自噬。
结论：本研究阐明了Wnt5a-Ca2+/CaMKII-自噬轴与触发气道重塑的新联系。我们的发现可能为EMT相关疾病的治疗提供新的策略。
方法：采用微阵列分析研究WNT5A在哮喘患者中的表达变化。并行,EMT模型使用16HBE细胞通过暴露于室内尘螨（HDM）或白介素-4（IL-4）来诱导，然后观察到Wnt5a的表达。通过Wnt5a模拟肽FOXY5和Wnt5a抑制剂BOX5使用体外功能增益和功能丧失方法，观察到上皮标记E-cadherin和间充质标记蛋白表达的变化。机械上,评价Ca2+/CaMKII信号通路和自噬。自噬抑制剂3-MA用于检测Wnt5a在EMT期间对自噬的调节。此外，我们使用CaMKII抑制剂KN-93来确定Wnt5a是否通过Ca2+/CaMKII信号通路诱导自噬过度激活和EMT.
结果：与健康对照相比，哮喘患者的WNT5A基因表达显着增加。在HDM和IL-4治疗后，我们观察到Wnt5a基因和蛋白表达水平在16HBE细胞中显著升高。有趣的是,Wnt5a模拟肽FOXY5显著抑制E-cadherin并上调α-SMA,胶原蛋白I,和自噬标记蛋白（Beclin1和LC3-II）。罗丹明-phalloidin染色显示FOXY5导致16HBE细胞中细胞骨架的重排和应力纤维的数量增加。重要的是,用BOX5阻断Wnt5a可显著抑制IL-4诱导的16HBE细胞自噬和EMT。机械上,自噬抑制剂3-MA和CaMKII抑制剂KN-93降低了FOXY5引起的16HBE细胞的EMT，并增加了应激纤维，细胞粘附，和自噬。
结论：本研究阐明了Wnt5a-Ca2+/CaMKII-自噬轴与触发气道重塑的新联系。我们的发现可能为EMT相关疾病的治疗提供新的策略。
