Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes.

Martin Degen,Stephan Von Gunten,Eleftheria Girousi,Christos Katsaros,Lukas Muerner,Silvia Rihs,Ludovica Parisi

doi:10.3389/fcell.2021.718066

Martin Degen, Stephan Von Gunten + Show 5 more

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https://doi.org/10.3389/fcell.2021.718066

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Abstract

Variants within the gene encoding for the transcription factor Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic Cleft Lip/Palate (CLP) cases. IRF6 plays a vital role in the regulation of the proliferation/differentiation balance in keratinocytes and is involved in wound healing and migration. Since a fraction of CLP patients undergoing corrective cleft surgery experience wound healing complications, IRF6 represents an interesting candidate gene linking the two processes. However, Irf6 function has been mainly studied in mice and knowledge on IRF6 in human cells remains sparse. Here, we aimed to elucidate the role of IRF6 in human postnatal skin- and oral mucosa-derived keratinocytes. To do so, we applied CRISPR/Cas9 to ablate IRF6 in two TERT-immortalized keratinocyte cultures, which we used as model cell lines. We show that IRF6 controls the appearance of single cells and colonies, with the latter being less cohesive in its absence. Consequently, IRF6 knockout keratinocytes often moved as single cells instead of a collective epithelial sheet migration but maintained their epithelial character. Lack of IRF6 triggered severe keratinocyte differentiation defects, which were already apparent in the stratum spinosum and extended to the stratum corneum in 3D organotypic skin cultures, while it did not alter their growth rate. Finally, proteomics revealed that most of the differentially expressed proteins in the absence of IRF6 could be associated with differentiation, cell-cell adhesion as well as immune response. Our data expand the knowledge on IRF6 in human postnatal keratinocytes, which will help to better understand IRF6-related pathologies.

Highlights

Interferon regulatory factor 6 (IRF6) belongs to a family of nine transcription factors that mediate the expression of interferon following viral infections (Yanai et al, 2012)
From one additional tissue donor, we were able to isolate both skin- and mucosa-derived keratinocytes. We used these cells to prove that the observed difference in IRF6 expression between mucosal- and skin-derived keratinocytes (Figure 1A) is not due to the variability among tissue donors
After confirming tissue identity of the cells by the presence or absence of robust KRT10 levels, we observed an increased IRF6 mRNA expression in skin keratinocytes when compared to the oral mucosa counterpart (Figure 1B)

Summary

Introduction

Interferon regulatory factor 6 (IRF6) belongs to a family of nine transcription factors that mediate the expression of interferon following viral infections (Yanai et al, 2012). Epidermal keratinocytes fail to undergo terminal differentiation and lack a functional periderm, a second cell layer that covers the embryonic epithelia and protects them from pathological adhesions (Ingraham et al, 2006; Richardson et al, 2006, 2014; Hammond et al, 2019). Such premature oral adhesions are believed to hinder palatal shelf elevation during palatogenesis, resulting in orofacial clefts (Richardson et al, 2014). Lack of Irf resulted in an abnormal keratinocyte appearance in vitro with many cells being larger than controls and presenting with an increased network of stress fibers (Biggs et al, 2012, 2014)

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