title: Differentiation of organotypic epidermis in the presence of skin disease-linked dominant-negative Cx26 mutants and knockdown Cx26.
abstract: In this study, we chose a differentiation-competent rat epidermal keratinocyte (REK) cell line to examine the role of Cx26 and disease-linked Cx26 mutants in organotypic epidermal differentiation. First, we generated stable REK cell lines expressing three skin disease-linked mutants (G59A, D66H and R75W). Second, we used an RNAi approach to knock down the expression of Cx26 in REKs. Interestingly, the three-dimensional (3D) architecture of the organotypic epidermis altered the intracellular spatial distribution of the mutants in comparison to 2D cultured REKs, highlighting the importance of using organotypic cultures. Unexpectedly, the presence of disease-linked mutants or the overexpression of wild-type Cx26 had little effect on the differentiation of the organotypic epidermis as determined by the architecture of the epidermis, expression of molecular markers indicative of epidermis differentiation (keratin 10, keratin 14, involucrin, loricrin) and stratification/cornification of the epidermis. Likewise, organotypic epidermis continued to differentiate normally upon Cx26 knockdown. While Cx26 has been reported to be upregulated during wound healing, no reduction in wound closure was observed in 2D REK cultures that expressed loss-of-function, dominant Cx26 mutants. In conclusion, we demonstrate that gain or loss of Cx26 function does not disrupt organotypic epidermal differentiation and offer insights into why patients harboring Cx26 mutations do not frequently present with more severe disease that encompasses thin skin.
authors.affiliation: Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, Canada.
authors.forename: Tamsin
authors.initials: T
authors.lastname: Thomas
authors.forename: Qing
authors.initials: Q
authors.lastname: Shao
authors.forename: Dale W
authors.initials: DW
authors.lastname: Laird
mesh_terms: D000818:Animals; D002450:Cell Communication; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D000072259:Connexin 26; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D000078404:Epidermal Cells; D017629:Gap Junctions / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D020868:Gene Silencing / Q000502:physiology; D015603:Keratinocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012871:Skin Diseases / Q000378:metabolism* / Q000473:pathology; D014945:Wound Healing
chemical_list: D018031:Connexin 43; D017630:Connexins; C000607044:Gjb2 protein, rat; D012333:RNA, Messenger; D000072259:Connexin 26