Discrimination of epithelium-like and fibroblast-like phenotypes derived from ethanol-treated immortalised human gingival keratinocytes in epithelial equivalents

Abstract

Ethanol treatment of immortalised human gingival keratinocytes (IHGK) yields in an epithelium-like (EPI) and fibroblast-like (FIB) phenotype. With respect to the stratified gingival epithelium, putative structural and molecular differences assigning cells to these phenotypes have not, to date, been analysed in a three-dimensional tissue/epithelial context. Therefore, we generated epithelial equivalents (EEs) in organotypic co-cultures of IHGK, EPI and FIB cells for 1 and 2 weeks and conducted protein and gene expression studies on the EEs for epithelial biomarkers including keratin K14, integrin subunits alpha6 and beta1, E-cadherin, and mesenchymal vimentin. As in the EEs of IHGK and EPI, indirect immunofluorescence revealed continuous expression of beta1 integrin in EEs of FIB cells. However, FIB cells exhibited a significant down-regulation in K14 and integrin alpha6 protein and a loss of E-cadherin at week 2, whereas vimentin was increased. FIB EEs were devoid of transcripts for E-cadherin at both time points, although transcription of the other genes remained constant in all phenotypes. Thus, the FIB phenotype exhibited a poor epithelial structure coinciding with disturbances in the expression of epithelial biomarkers and the persistence of mesenchymal vimentin. Transcription analysis revealed post-transcriptional regulation of vimentin in IHGK and EPI and of K14 and alpha6 in FIB cells. Our findings indicate that differences in the epithelial integrity and expression of molecules in EEs allow for the discrimination of EPI and FIB cells. This suggests that FIB cells share features of epithelial-mesenchymal transition and reflect a more progressive stage in epithelial cell transformation.

Transforming Growth Factor Beta Gene Signatures are Spatially Enriched in Keloid Tissue Biopsies and Ex vivo-Cultured Keloid Fibroblasts

“…In this regard, the finding that upstream regulators such as NR3C1 appear as one of the candidate regulators of the gene expression profiles in the top region of the keloid lesion is particularly interesting given the previous association of this protein in driving epithelial mesenchymal transition (EMT) (49) especially as keratinocyte differentiation to a mesenchymal phenotype has been implicated as a key pathological event contributing to keloid lesion development (50). This conclusion is supported by the significant downregulation of several keratin isoforms (KRT25, KRT27) observed in the DEGs in the top region keloid signature as would be expected if this region contains a significant proportion of keratinocytes undergoing EMT (51,52).…”

Vacuum Ultraviolet (VUV) Light Photofunctionalization to Induce Human Oral Fibroblast Transmigration on Zirconia

“…The results of the present study solely imply the biological benefits for the behaviors and responses of fibroblasts. The gingival soft tissue consists of connective tissue fibroblasts and oral epithelial cells [107][108][109][110][111][112][113]; the fibroblasts are derived from the mesenchyme, while the epithelial cells are composed of stratified squamous epithelium. The two cell types differ in shape, behavior, and reaction to external stimuli [114][115][116][117][118][119][120][121][122][123][124][125][126].…”

Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology

“…Similar cultivation times were used in previous studies for organotypic gingival cocultures. 17,31,32 These preliminary tests on the aforementioned corneal cocultures revealed an only marginal maturation before day 5, and epithelial disarrangements starting from day 10 onward. Consequently, three cultivation times were chosen to investigate the characteristics of balanced epithelial morphogenesis: (1) 5 days for premature epithelial differentiation, (2) 6 days for progression of differentiation, and (3) 9 days for completion of differentiation, including terminal differentiation.…”