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
View: Text | PDF
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

×

Figure 2

Apremilast abolishes PV-IgG–induced keratin alterations.

Options: View larger image (or click on image) Download as PowerPoint
Apremilast abolishes PV-IgG–induced keratin alterations. (A) Immunostain...
(A) Immunostaining of keratin 14 in PG-WT and PG-S665A mice from neonatal PV mouse model. DAPI was used to identify overall tissue morphology by nuclei staining. Basement membrane is marked with a punctuated white line and blister with an asterisk. Keratin 14, which was expressed in the basal and suprabasal epidermal layers, was compromised upon PV-IgG injection. Apr ameliorated PV-IgG–induced keratin alteration. Representative of n = 4. Scale bar: 25 μm. (B) Quantification of A. PV-IgG significantly reduced fluorescence intensity in PG-WT mice, which was absent by coincubation with apr. In contrast, baseline fluorescence levels were diminished in PG-S665A mice, and neither PV-IgG nor apr induced significant changes in keratin 14 fluorescence intensity. n = 4. Data shown as mean ± SEM. Two-way ANOVA with Tukey’s post hoc test, *P < 0.05. (C and D) STED imaging of pPG(S665) in human ex vivo skin model. Apr and the PKA inhibitor H89 were preincubated for 1 hour. Subsequently, c-IgG or PV-IgG were injected and incubated for another 1 hour. PV-IgGs and apr increased phosphorylation of PG at S665 along cell borders. Apr had an additive effect on PV-IgG–induced increase. H89 ameliorated PV-IgG–induced and apr-induced phosphorylation of PG at S665. Representative of n = 5. Scale bars: 5 μm; 10 μm. (D) Quantification of STED imaging in C. n = 5, 2-way ANOVA with Tukey’s post hoc test; *P < 0.05; apr, apremilast; c-IgG, IgG fraction of healthy control; PG, plakoglobin.