Functional Characterization of Dermokine in Epidermal Differentiation

The most outer layer of the skin, the epidermis, acts as a protective barrier between the organism and its surrounding environment, operating as a boundary against detrimental substances, while also obstructing water loss and withstanding mechanical stress. The epidermis can be divided into four layers, related to the differentiation status of keratinocytes, ultimately resulting in formation of the cornified envelope. This process is heavily influenced by post-translational modifications, whereby limited proteolysis is critical not only for processing of structural proteins, such as filaggrin, but also regulatory mediators involved in several signalling events. An example of such a processing event that has been recently identified in Prof. Ulrich auf dem Keller’s research group is the proteolytic cleavage of the bioactive mediator dermokine by matrix metalloproteinase 10. This cleavage event might be related to keratinocyte migration and differentiation.

As an essential member of the secreted protein gene complex family, dermokine and its various gene products play a vital role in development and stratification of suprabasal layers of the epidermis. In humans, the highly spliced dermokine exists in three major isoforms, dermokine-α, dermokine-β and dermokine-γ, whereas the C-terminal cytokine-like domain, present in dermokine-β and dermokine-α, substantially shapes the biological function of this secreted protein within the extracellular space. This cytokine-like domain is likely responsible for dermokine’s bioactivity, acting as a ligand to control intracellular phosphorylation events, including the modulation of extracellular signal-regulated kinase 1/2 phosphorylation. However, the intricate mechanism through which dermokine regulates keratinocyte differentiation remains elusive.

In this work, we addressed this issue by employing optimized CRISPR/-Cas9 gene editing strategies. We engaged two main gene editing methods, a transfection-based technique and an atomic force microscopy procedure utilizing FluidFM® technology. The transfection-based approach comprises an improved protocol for virus-independent delivery of CRISPR/Cas9 plasmids and the selection of single-cell mutants. The FluidFM® method enables the direct nuclear introduction of multiple gRNA CRISPR/Cas9 ribonucleoprotein complexes. Applying these designs, we generated Dmkn βγ^-/- and Dmkn αβ^-/- keratinocytes with high gene-editing efficiency and minimal off-target effects. This study provides novel and optimized reliable gene editing techniques to generate keratinocyte CRISPR-based knockouts.

After establishment of the Dmkn βγ^-/- and Dmkn αβ^-/- keratinocytes, we aimed to investigate the role of dermokine in the course of epidermal differentiation in a human model. Thereby, we took advantage of skin organoids, which were applied to explore dermokine-β’s and dermokine-α’s role in epidermal stratification. Histological assessments observed a non-stratified epidermis along the dermokine-specific expression gradient, which was confirmed by immunohistochemical staining. Mass spectrometry-based proteomics techniques further monitored several molecular changes due to isoform-specific dermokine truncations that we validated at the protein level by parallel reaction monitoring. A subsequent unbiased proteomics screen on Dmkn βγ^-/- skin organoids unveiled changes in proteins linked to cornified envelope assembly, filaggrin processing, and desquamation in comparison to the wildtype. These changes were validated in Dmkn αβ^-/- using internal standard triggered parallel monitoring, which corroborated significant reductions in filaggrin modulating proteases and proteins governing cornified envelope assembly upon cytokine-like domain depletion. With help of targeted degradomics and heavy reference peptides, we confirmed dermokine cleavage by matrix metalloproteinase 10 and for the first time mapped the cleavage site in the human protein.

Next, we analyzed how the extracellular dermokine isoforms modulate intracellular phosphorylation events. Applying automation-assisted sample preparation and cutting-edge LC-MS/MS instrumentation together with advanced data analysis strategies, we investigated phosphorylation events in Dmkn βγ^-/- and Dmkn αβ^-/- in comparison to wildtype keratinocytes. This system-wide comparison enabled us to confidently link differentially phospho- rylated serines, threonines, or tyrosines to their specific kinases. In addition, we recorded the to our knowledge deepest phosphoproteome of keratinocytes established as of now, comprising 15468 quantified phosphosites. We could observe enhanced phosphorylation levels of various crucial receptors, such as proto-oncogene, receptor tyrosine kinase Met at S990, erb-b2 receptor tyrosine kinase 2 at T701, and insulin like growth factor 2 receptor at S2409, under endogenous dermokine levels in comparison to αβ^-/- keratinocytes. These changes illustrate dermokine’s proposed role in main physiological mechanisms in keratinocytes, including receptor activity and adhesion dynamics. Concomitantly, we identified differential dermokine-associated phosphorylation events in both the proto-oncogene tyrosine-protein kinase Src at S17, which has been detected as a key factor in cellular migration, as well as protein tyrosine phosphatase receptor type κ. Subsequently, an increase in phosphorylation of catenin δ 1 was observed when dermokine was ablated. This alteration suggests its impact on the associative state with cadherin 1 and therefore, the adhesive strength of the cells, which is known to increase upon the dephosphorylation of catenin δ 1, which might be mediated by differentially phosphorylated protein tyrosine phosphatase receptor type κ.

Overall, our findings indicate that the modulated phosphorylation of catenin δ 1 upon ablation of dermokine has potential impact on keratinocyte adhesion mediated by dermokine activity.