Multiomics analysis reveals dermokine as a regulator of keratinocyte differentiation and adhesion
Vahap Canbay, Till Wüstemann, Weihua Tian, Tobias A. Beyer, Camilla Reiter Elbæk, Michael Stumpe, Gaetana Restivo, Chatpakorn Christiansen, Anabel Migenda Herranz, Susanne Mailand, Jürg Hafner, Rune Busk Damgaard, Steffen Goletz, Jörn Dengjel, Ulrich auf dem Keller, Chiara Francavilla
Vahap Canbay, Till Wüstemann, Weihua Tian, Tobias A. Beyer, Camilla Reiter Elbæk, Michael Stumpe, Gaetana Restivo, Chatpakorn Christiansen, Anabel Migenda Herranz, Susanne Mailand, Jürg Hafner, Rune Busk Damgaard, Steffen Goletz, Jörn Dengjel, Ulrich auf dem Keller, Chiara Francavilla
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Research Article Cell biology Dermatology

Multiomics analysis reveals dermokine as a regulator of keratinocyte differentiation and adhesion

Abstract

Impaired adhesion and differentiation of keratinocytes is a hallmark of several skin diseases, but only some of the factors that regulate these processes have been identified. Here, we studied the role of isoform-rich dermokine — a wound- and tumor-regulated protein — in keratinocytes using a combination of multiomics and functional approaches. CRISPR/Cas9-induced knockout of dermokine isoforms in human keratinocytes inhibited differentiation of these cells in 3-dimensional organotypic skin cultures, which was confirmed by quantitative proteomics. In 2-dimensional monocultures, dermokine deficiency affected the proteome and phosphoproteome as revealed by mass spectrometry. We found reduced abundance of differentiation-specific proteins and increased phosphorylation of the cell adhesion protein p120 (catenin δ-1). The adhesive strength of dermokine-knockout keratinocytes was impaired, which was rescued by p120 knockdown or ROCK inhibition. Finally, we verified the correlation between decreased dermokine expression and increased p120 phosphorylation in human non-healing wounds. These results identify dermokine as a regulator of keratinocyte adhesion and differentiation, involving at least in part its effect on p120 phosphorylation and ROCK. Our data point to a function of dermokine in the pathogenesis of chronic wounds.

Authors

Vahap Canbay, Till Wüstemann, Weihua Tian, Tobias A. Beyer, Camilla Reiter Elbæk, Michael Stumpe, Gaetana Restivo, Chatpakorn Christiansen, Anabel Migenda Herranz, Susanne Mailand, Jürg Hafner, Rune Busk Damgaard, Steffen Goletz, Jörn Dengjel, Ulrich auf dem Keller, Chiara Francavilla

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Figure 5

Cell-cell adhesion decreases in dermokine-depleted keratinocytes.

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Cell-cell adhesion decreases in dermokine-depleted keratinocytes. (A) Qu...
(A) Quantification of 4 or more cell clusters from the cell-cell adhesion assay using DMKN αβ–/–, DMKN βγ–/–, and WT keratinocytes. Cell clusters (≥4) were counted, and each replicate was visualized as a dot in the violin plot. N = 3 biological replicates. ***P < 0.001 (1-way ANOVA and Fisher’s least significant difference test). (B) Quantification of dispase dissociation assay data using independent clones of human DMKN αβ–/–, DMKN βγ–/–, DMKN αβγ–/–, and WT keratinocytes transfected for 72 hours with p120 or scrambled siRNA. Values are normalized to total fragment size. N = 4 biological replicates. *P < 0.05, **P < 0.01 (1-way ANOVA and Fisher’s least significant difference test). (C) Quantification of dispase dissociation assay data using DMKN αβ–/–, DMKN βγ–/–, and WT keratinocytes transfected for 72 hours with p120 (1, 2, and 3) or scrambled siRNA. N = 4 biological replicates. All conditions are significant (P < 0.05) unless indicated otherwise (ns: P > 0.05) (2-way ANOVA and Fisher’s least significant difference test). (D) Quantification of dispase dissociation assay data of DMKN αβ–/–, DMKN βγ–/–, and WT keratinocytes transfected for 48 hours with p120 or scrambled siRNA followed by transfection with expression vectors encoding p120 mutants with a siRNA-resistant mutation and either S252D or S268D mutations. N = 3 biological replicates. *P < 0.05, ****P < 0.0001 (2-way ANOVA and Fisher’s least significant difference test).