Wnt5a Induces Epithelial-Mesenchymal Transition By Overactivating Autophagy In HBECs

Epithelial-mesenchymal transition（EMT）of airway epithelial cells is one of the critical mechanisms of airway remodeling in asthma.Revealing the endogenous driving factors is still a fundamental and clinical problem to be solved.Wnt5a is one of the members of the Wnt gene family that have been widely studied to activate the non-dependentβ-catenin signaling cascade.It can activate calmodulin-dependent protein kinase II（CaMKⅡ）through Ca²⁺to participate in various lung diseases.Autophagy is a lysosome-dependent self-degradation pathway in eukaryotic cells,which is of great significance in balancing energy sources and responding to nutritional stress at critical moments of development.Excessive autophagy can lead to EMT by degrading epithelial cell-related markers,but its mechanism in asthma airway remodeling remains elucidated.This study proposed the following hypothesis:abnormally elevated Wnt5a may induce excessive autophagy through Ca²⁺/CaMKⅡ signaling pathway to promote airway epithelial EMT.Objective:To clarify the role of Wnt5a in EMT and its mechanisms,we intend to explore（1）the effect of Wnt5a on EMT of airway epithelium in asthma;（2）the molecular mechanism of Wnt5a induced excessive autophagy in airway epithelial cells and triggered EMT.Methods:The gene expression of Wnt5a between the healthy control group and asthma patient group was observed by bioinformatics analysis.Human bronchial epithelial cell line 16HBE was treated with house dust mite（HDM,100μg/m L）and Th2 cytokine IL-4（10 ng/m L）,respectively.Real-time PCR and Western blot were used to observe the changes in genes and proteins of epithelial marker E-cadherin,mesenchymal marker proteinsα-SMA,Collagen I,and Wnt5a.Cytoskeleton morphology was observed by TRITC Phalloidin fluorescence staining.Wnt5a was blocked by Wnt5a antagonist BOX5,followed by treatment of IL-4.The effect of blocking Wnt5a on EMT was also observed.The 16HBE cells were treated with Wnt5a mimic peptide FOXY5（100μM）,and the changes in EMT were observed.In mechanism,the 16HBE cells were pretreated with an autophagy inhibitor（3-MA,5 m M）and CaMKⅡ inhibitor（KN-93,2.5μM）,respectively,followed by FOXY5 stimulation.Then EMT and autophagy-related indexes were detected.Results:The m RNA expression of WNT5A was significantly increased in asthma patients compared with the healthy group.The m RNA and protein of Wnt5a were significantly increased in HDM-or IL-4-treated 16HBE cells.Interestingly,activation of Wnt5a decreased the expression of epithelial marker E-cadherin while increased the interstitial markersα-SMA and Collagen I.In addition,16HBE cytoskeleton remodeling and stress fiber hyperplasia were induced.Beclin1 and LC3-II markers of autophagy were up-regulated,and intracellular Ca²⁺concentration was increased.By contrast,blocking Wnt5a significantly inhibited IL-4-induced expression of EMT markerα-SMA and autophagy markers LC3and Beclin1 proteins,morphological cytoskeleton changes,and migration ability of 16HBE cells.3-MA inhibited the expression of EMT markerα-SMA and autophagy markers LC3,Beclin1,and FUNDC1,cytoskeletal morphology,and migration ability of Wnt5a-induced 16HBE cells.KN-93 also reduced the autophagy of 16HBE cells induced by Wnt5a activation,reduced stress fiber proliferation,and intercellular adhesion,and inhibited EMT.Conclusion:Abnormally elevated Wnt5a in asthma induces excessive autophagy of airway epithelial cells through the Ca²⁺/CaMKⅡ signaling pathway,which promotes airway epithelial EMT and triggers airway remodeling.