Impact of KLF4 on Cell Proliferation and Epithelial Differentiation in the Context of Cystic Fibrosis

Abstract

Cystic fibrosis (CF) cells display a more cancer-like phenotype vs. non-CF cells. KLF4 overexpression has been described in CF and this transcriptional factor acts as a negative regulator of wt-CFTR. KLF4 is described as exerting its effects in a cell-context-dependent fashion, but it is generally considered a major regulator of proliferation, differentiation, and wound healing, all the processes that are also altered in CF. Therefore, it is relevant to characterize the differential role of KLF4 in these processes in CF vs. non-CF cells. To this end, we used wt- and F508del-CFTR CFBE cells and their respective KLF4 knockout (KO) counterparts to evaluate processes like cell proliferation, polarization, and wound healing, as well as to compare the expression of several epithelial differentiation markers. Our data indicate no major impact of KLF4 KO in proliferation and a differential impact of KLF4 KO in transepithelial electrical resistance (TEER) acquisition and wound healing in wt- vs. F508del-CFTR cells. In parallel, we also observed a differential impact on the levels of some differentiation markers and epithelial-mesencymal transition (EMT)-associated transcription factors. In conclusion, KLF4 impacts TEER acquisition, wound healing, and the expression of differentiation markers in a way that is partially dependent on the CFTR-status of the cell.

Pathophysiology of Lung Disease and Wound Repair in Cystic Fibrosis

“…Crespin et al, 2014 [198] Primary cell lines: non-CF and CF nasal and bronchial epithelial cells. Sousa et al, 2020 [200] α1AT: alpha-1 antitrypsin; HDMF: 4-hydroxy-2,5-dimethyl-3(2H)-Furanone; NE: neutrohil elastase.…”

“…Further work was done to assess the impact of KLF4 in TEER acquisition and wound repair, using the isogenic pair wt-CFTR and F508del-CFTR-CFBE cells and their respective KLF4 knockout (KO) counterparts (via CRISPR-Cas9) [ 200 ] in polarized conditions. CF cells showed a higher proliferation rate than non-CF cells (as evaluated by Ki-67 staining and growth curve), which, however, was not influenced by KLF4 KO.…”

“…Although a different actin cytoskeleton organization was observed by comparing wt- to F508del-cells, no alteration in lamellipodia formation was observed in the presence of KLF4 KO. Sousa and colleagues [ 200 ] showed that KLF4 KO decreases wound closure even further in F508del-CFTR cells (although not significantly), suggesting that KLF4 promotes wound repair. On the other hand, the wound closure in non-CF cells was not altered by KLF4 KO.…”

“…Sousa and colleagues [ 200 ] investigated in polarized models of bronchial epithelial cells (wt- vs. F508del-CFTR CFBE cells) the effect of KLF4 KO on proliferation and wound repair (as discussed above) as well as the modulation of differentiation markers and EMT induction. KLF4 KO decreased the levels of E-Cadherin and CK18, whilst increasing the levels of vimentin and N-cadherin in both cell lines.…”

CFTR, Cell Junctions and the Cytoskeleton

“…Interestingly, we recently found that knocking-out (KO) KLF4, a transcription factor known to regulate epithelial differentiation among other processes [ 41 , 42 ], in polarized wt-CFTR-expressing bronchial epithelial (CFBE) cells leads to a significant decrease in TEER. The opposite was seen in F508del-CFTR-expressing cells upon KLF4-KO which led to an increase in TEER levels compared to control cells, albeit not reaching wt-CFTR TEER levels [ 43 ]. This suggests that KLF4 is needed for epithelial integrity/polarization of normal cells expressing wt-CFTR.…”