Plakoglobin phosphorylation at serine 665 is capable of stabilizing cadherin-mediated adhesion in keratinocytes
Franziska Vielmuth, Anna M. Sigmund, Desalegn T. Egu, Matthias Hiermaier, Letyfee S. Steinert, Sina Moztarzadeh, Mariia Klimkina, Margarethe E.C. Schikora, Paulina M. Rion, Thomas Schmitt, Katharina Meier, Kamran Ghoreschi, Anja K.E. Horn, Mariya Y. Radeva, Daniela Kugelmann, Jens Waschke
Franziska Vielmuth, Anna M. Sigmund, Desalegn T. Egu, Matthias Hiermaier, Letyfee S. Steinert, Sina Moztarzadeh, Mariia Klimkina, Margarethe E.C. Schikora, Paulina M. Rion, Thomas Schmitt, Katharina Meier, Kamran Ghoreschi, Anja K.E. Horn, Mariya Y. Radeva, Daniela Kugelmann, Jens Waschke
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Research Article Cell biology

Plakoglobin phosphorylation at serine 665 is capable of stabilizing cadherin-mediated adhesion in keratinocytes

Abstract

In pemphigus, autoantibodies against the desmosomal cadherins desmoglein (DSG) DSG1 and DSG3 cause intraepidermal blistering. Recently, we found that increasing cAMP with the phosphodiesterase-4 inhibitor apremilast stabilizes keratinocyte cohesion in pemphigus. This effect is paralleled by phosphorylation of the desmosomal plaque protein plakoglobin (PG) at serine 665 (S665). Here, we investigated the relevance of PG phosphorylation at S665 for stabilization of keratinocyte cohesion and further characterized the underlying mechanisms. Ultrastructural analysis of a recently established PG-S665 phospho-deficient mouse model (PG-S665A) showed diminished keratin insertion. Accordingly, the protective effect of apremilast against pemphigus autoantibody-induced skin blistering was diminished, and apremilast failed to restore alterations of the keratin cytoskeleton in PG-S665A mice. Keratinocytes derived from PG-S665A mice revealed a disorganized keratin cytoskeleton and reduced single-molecule binding strength of DSG3. In line with this, in ex vivo human skin, increased cAMP augmented keratin insertion into desmosomal plaques. Additionally, PG phosphorylated at S665 colocalized with desmoplakin and keratin filaments anchoring to desmosomes and increased cAMP-accelerated assembly of desmosomes. Taken together, phosphorylation of PG at S665 was crucial for protective effects of apremilast in pemphigus and for maintenance of DSG3 binding and keratin filament anchorage to desmosomes.

Authors

Franziska Vielmuth, Anna M. Sigmund, Desalegn T. Egu, Matthias Hiermaier, Letyfee S. Steinert, Sina Moztarzadeh, Mariia Klimkina, Margarethe E.C. Schikora, Paulina M. Rion, Thomas Schmitt, Katharina Meier, Kamran Ghoreschi, Anja K.E. Horn, Mariya Y. Radeva, Daniela Kugelmann, Jens Waschke

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Figure 3

Keratin insertion into desmosomes is dependent on cAMP signaling.

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Keratin insertion into desmosomes is dependent on cAMP signaling. (A) Sc...
(A) Schematic of analysis of desmosome ultrastructure in electron micrographs; 30–50 desmosomes were analyzed. (B) Electron micrograph from human ex vivo skin model showing desmosomes connecting keratinocytes in the basal/suprabasal layer of the epidermis. Human skin was injected for 1 hour with vehicle, forskolin/rolipram (F/R), or apremilast (apr). cAMP increase led to augmented keratin insertion into desmosomes. Zoom-ins are marked with green rectangles in overview micrographs. Representative of n = 3. Scale bars: 1 μm; 250 nm. (C and D) Quantification of desmosome length (C) and keratin insertion (D) from human ex vivo skin model in B reveals that F/R did not change desmosome length but confirms that keratin insertion is significantly increased in keratin insertion index. n = 3. (E) Electron micrograph from murine skin of PG-WT and PG-S665A animals reveals altered keratin insertion in PG-S665A mice epidermis. Zoom-ins are marked with green rectangles in overview micrographs. Representative of n = 4. Scale bars: 1 μm; 250 nm. (F) Quantification of desmosome number from E with 10 cell borders/n. (G) Quantification of E reveals no difference in desmosome length between PG-WT and PG-S665A. (H) Keratin insertion index calculated from means of 30–40 desmosomes/n from E showed significant impaired keratin insertion in PG-S665A mice epidermis. (I) Quantification of desmosome symmetry in PG-WT and PG-S665A mice epidermis showed a trend toward impaired symmetry in PG-S665A mice epidermis; 30–40 desmosomes/n. (G–I) n = 4; data shown as mean ± SEM. One-way ANOVA with Tukey’s post hoc test (C and D), unpaired 2-tailed t test (G and I), Mann-Whitney U test (F and H). *P < 0.05.