Investigation of HE4 expression concerning epithelial-mesenchymal transition (EMT) in cystic fibrosis epithelial cells

BACKGROUND: Elevated levels of human epididymis protein 4 (HE4) have been observed in cystic fibrosis (CF), where its expression is directly affected by impaired CFTR through the NF-κB pathway in p.Phe508del-CFTR CFBE 41o- cells in vitro. Dysfunctional CFTR is also linked to epithelial-mesenchymal transition (EMT) in CF. However, no data is available whether HE4 expression changes and is associated with CF-related EMT. METHODS: The level of EMT was compared between CFBE 41o- cells with the p.Phe508del-CFTR mutation and wt-CFTR by measuring epithelial and mesenchymal markers using fluorescence microscopy and RT-qPCR. EMT was also induced by TGFβ1 in p.Phe508del-CFTR CFBE cells from 24 to 120 h to explore relationship between EMT development and the expression of HE4 and MMP9. VX-445/VX-661/VX-770 treatment restored CFTR function to observe changes in EMT phenotype. The direct effects of HE4 on EMT and MMP9 levels were examined in CFBE cells where HE4 expression was reduced through transfection with HE4-specific siRNA. Cell proliferation was assessed by Ki67 positivity. RESULTS: CFBE cells expressing p.Phe508del-CFTR were more prone to be mesenchymal than wt-CFTR CFBE cells and showed higher basal HE4 and MMP9 levels. In response to TGFβ1, even lower E-cadherin with higher N-cadherin and MMP9 levels were observed in p.Phe508del-CFTR CFBE cells compared to untreated cells. In contrast, HE4 expression decreased after 48 h and continued to decline up to 120 h. CFTR modulator treatment could reverse the EMT phenotype, normalizing HE4 and MMP9 levels. Finally, downregulated HE4 promoted EMT, resulting in higher MMP9 expression and reduced cell proliferation in p.Phe508del-CFTR CFBE cells. CONCLUSION: EMT is accompanied by decreasing HE4 expression and upregulated MMP9 level in the airway epithelial cells of CF.