Pemphigus is an autoimmune blistering skin disease caused primarily by autoantibodies against desmoglein (Dsg)1 and 3. Here, we characterized the mechanisms engaged by pemphigus IgG from patients with different clinical phenotypes and autoantibody profiles. All pemphigus vulgaris (PV) and pemphigus foliaceus (PF) IgG and AK23, a monoclonal mouse antibody against Dsg3, caused loss of cell cohesion, cytokeratin retraction and p38MAPK activation. Strong alterations in Dsg3 distribution were caused by mucosal (aDsg3 antibodies), mucocutaneous (aDsg1 + aDsg3) as well as atypical (aDsg3) PV-IgG. All PV-IgG fractions and AK23 compromised Dsg3 but not Dsg1 binding and enhanced Src activity. In contrast, rapid Ca2+ influx and Erk activation were induced by mucocutaneous PV-IgG and pemphigus foliaceus (PF) IgG (aDsg1) whereas cAMP was increased by mucosal and mucocutaneous PV-IgG only. Selective inhibition of p38MAPK, Src or PKC blocked loss of keratinocyte cohesion in response to all autoantibody fractions whereas Erk inhibition was protective against mucocutaneous PV-IgG and PF-IgG only. These results demonstrate that signaling patterns parallel the clinical phenotype as some mechanisms involved in loss of cell cohesion are caused by antibodies targeting Dsg3 whereas others correlate with autoantibodies against Dsg1. The concept of key desmosome regulators may explain observations from several experimental models of pemphigus.

Our data demonstrate that blistering can be prevented by inhibition of p38MAPK in the human epidermis. Moreover, typical morphological alterations induced by mcPV-IgG such as interdesmosomal widening and the reduction of desmosome size at least in part require p38MAPK signalling.

Tightly controlled intercellular adhesion is crucial for the integrity and function of the epidermis. The keratin filament cytoskeleton anchors desmosomes, supramolecular complexes required for strong intercellular adhesion. We tested whether keratin filaments control cell adhesion by regulating the adhesive properties of desmosomal cadherins such as desmoglein (Dsg) 3. Atomic force microscopy and fluorescence recovery after photobleaching experiments showed reduced Dsg3 adhesive forces and membrane stability in murine keratinocytes lacking all keratin filaments. Impairment of the actin cytoskeleton also resulted in decreased Dsg3 immobilization but did not affect Dsg3 binding properties, indicating that the latter are exclusively controlled by keratins. Reduced binding forces were dependent on p38 mitogen-activated protein kinase activity, which was deregulated in keratin-deficient cells. In contrast, inhibition of protein kinase C signaling, which is known to be controlled by keratins, promoted and spatially stabilized Dsg3-mediated interactions in the membrane. These results show a previously unreported mechanism for how keratins stabilize intercellular adhesion on the level of single desmosomal adhesion molecules.

The desmosomal transmembrane adhesion molecules desmoglein 3 (Dsg3) and desmocollin 3 (Dsc3) are required for strong keratinocyte cohesion. Recently, we have shown that Dsg3 associates with p38MAPK and suppresses its activity. Here, we further investigated the role of Dsg3-dependent control of p38MAPK function. Dsg3-deficient mice display recurrent spontaneously healing skin erosions. In lesional and perilesional biopsies, p38MAPK activation was detectable compared to control animals. This led us to speculate that Dsg3 regulates wound repair in a p38MAPK-dependent manner. Indeed, scratch wounded keratinocyte monolayers exhibited p38MAPK activation and loss of Dsg3 in cells lining the wound edge. Human keratinocytes after silencing of Dsg3 as well as primary cells isolated from Dsg3 knockout animals exhibited accelerated migration, which was further corroborated in an ex vivo skin outgrowth assay. Importantly, migration was efficiently blocked by inhibition of p38MAPK, indicating that p38MAPK mediates the effects observed upon loss of Dsg3. In line with this, we show that levels of active p38MAPK associated with Dsc3 are increased in Dsg3-deficient cells. These data indicate that Dsg3 controls a switch from an adhesive to a migratory keratinocyte phenotype via p38MAPK inhibition. Thus, loss of Dsg3 adhesion may foster wound closure by allowing p38MAPK-dependent migration.Journal of Investigative Dermatology accepted article preview online, 30 September 2015. doi:10.1038/jid.2015.380.

Pemphigus is an autoimmune dermatosis in which mucocutaneous blisters are induced primarily by autoantibodies against Desmoglein (Dsg) 1 and 3. Pemphigus vulgaris (PV) usually is associated with autoantibodies against Dsg3 whereas pemphigus foliaceus (PF) patients present autoantibodies against Dsg1. Several signaling pathways were proposed to cause loss of keratinocyte adhesion. However, relevance of different signaling pathways and role of Dsg1 and 3 to trigger signaling are not fully understood. Here, we show that Ca 2+ chelation reduced PV-IgG- and PF-IgG-mediated loss of HaCaT keratinocyte cohesion whereas EGFR inhibition did not inhibit effects of PF-IgG. PV-IgG activated EGFR in a Src-dependent manner whereas both PV-IgG and PF-IgG caused Ca 2+ influx independent of EGFR. ERK activation was Src-dependent in response to PV-IgG but not PF-IgG. To delineate the roles of Dsg isoforms to trigger signaling pathways, Dsg3- and Dsg2-deficient HaCaT keratinocyte cell lines were generated using CRISPR/Cas9. Dsg3- but not Dsg2-deficient cells were protected against PV-IgG-induced loss of cell adhesion. Ca 2+ influx and ERK activation in response to PF-IgG were preserved in both cell lines.

Autoantibodies against desmoglein (Dsg) 1 and Dsg3 primarily cause blister formation in the autoimmune disease pemphigus vulgaris (PV). Src was proposed to contribute to loss of keratinocyte cohesion. However, the role and underlying mechanisms are unclear. In keratinocytes, cell cohesion in response to autoantibodies was reduced in a Src‐dependent manner by two patient‐derived PV‐IgG fractions as well as by AK23, but not by a third PV‐IgG fraction, although Src was activated by all autoantibodies. Loss of cell cohesion was progredient and AK23 similar to PV‐IgG interfered with reconstitution of cell cohesion after Ca2+‐switch, indicating that the autoantibodies also interfered with desmosome assembly. Dsg3 co‐localized along cell contacts and interacted with the Src substrate cortactin. Concomitantly, cell adhesion was impaired in keratinocytes isolated from cortactin‐deficient mice in comparison to keratinocytes isolated from wildtype (wt). AK23‐induced loss of cell cohesion was Src‐dependent only when cortactin was expressed. Similarly, AK23 impaired reconstitution of desmosomal adhesion in Src‐dependent fashion only in the presence of cortactin and AK23‐induced skin blistering was abolished by Src inhibition in wt but not cortactin‐deficient mice. However, in human epidermis, PV‐IgG‐induced skin blistering and ultrastructural alterations of desmosomes were not affected by Src inhibition. Our data suggest that Src and cortactin are involved in pemphigus skin blistering but the contribution of Src is variable. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background Pemphigus is a severe bullous autoimmune skin disease. Pemphigus foliaceus (PF) is characterized by antidesmoglein (Dsg) 1 IgG causing epidermal blistering; mucosal pemphigus vulgaris (mPV) by anti-Dsg3 IgG inducing erosions in the mucosa; and mucocutaneous pemphigus vulgaris (PV) by affecting both, with autoantibodies targeting Dsg1 and Dsg3. Objectives To characterize the Ca 2+ flux pathway and delineate its importance in pemphigus pathogenesis and clinical phenotypes caused by different antibody profiles. Methods Immunoprecipitation, Ca 2+ flux analysis, Western blotting, immunofluorescence staining, dissociation assays and a human skin ex vivo model were used. Results PV IgG and PF IgG, but neither Dsg3-specific monoclonal antibody (AK23) nor mPV IgG, caused Ca 2+ influx in primary human keratinocytes. Phosphatidylinositol 4-kinase a interacts with Dsg1 but not with Dsg3. Its downstream targetphospholipase-C-c1 (PLC)was activated by PV IgG and PF IgG but not AK23 or mPV IgG. PLC releases inositol 1,4,5-trisphosphate (IP3) causing IP3 receptor (IP3R) activation and Ca 2+ flux from the endoplasmic reticulum into the cytosol, which stimulates Ca 2+ release-activated channels (CRAC)-mediated Ca 2+ influx. Inhibitors against PLC, IP3R and CRAC effectively blocked PV IgG and PF IgG-induced Ca 2+ influx; ameliorated alterations of Dsg1 and Dsg3 localization, and reorganization of keratin and actin filaments; and inhibited loss of cell adhesion in vitro. Finally, inhibiting PLC or IP3R was protective against PV IgG-induced blister formation and redistribution of Dsg1 and Dsg3 in human skin ex vivo. Conclusions Ca 2+ -mediated signalling is important for epidermal blistering and dependent on the autoantibody profile, which indicates different roles for signalling complexes organized by Dsg1 and Dsg3. Interfering with PLC and Ca 2+ signalling may be a promising approach to treat epidermal manifestations of pemphigus.What is already known about this topic?• Autoantibody-induced Ca 2+ signalling and activation of phospholipase C in keratinocytes has been reported as the first signalling pathway in pemphigus.• Ca 2+ influx correlates with the presence of autoantibodies targeting desmoglein (Dsg) 1.

Summary Background Pemphigus vulgaris (PV) is an autoimmune disease characterized by blister formation in the epidermis and oral mucosa due to loss of keratinocyte cohesion. Autoantibodies present in patients with PV (PV‐IgG) are known to primarily target desmoglein (Dsg)1 and Dsg3 in desmosomes. The mucosal‐dominant subtype of PV (mdPV) is caused by PV‐IgG autoantibodies against the cadherin‐type adhesion molecule Dsg3. p38 mitogen‐activated protein kinase (p38MAPK) signalling has been characterized as an important pathway downstream of PV‐IgG binding and its inhibition is protective in ex vivo human skin. However, the role of p38MAPK signalling in mdPV is unknown as no experimental model has been available. Objectives To establish a human ex vivo oral mucosa culture, and evaluate the p38MAPK dependency of blister formation and of ultrastructural alterations of desmosomes induced by mdPV‐IgG. Methods Human labial mucosa was injected with mdPV‐IgG as well as AK23, a pathogenic mouse monoclonal Dsg3 antibody, in the presence or absence of p38MAPK inhibitors. Viability was evaluated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and apoptosis by terminal deoxynucleotidyl transferase dUTP nick‐end labelling assay. Blister score was determined following haematoxylin and eosin staining and Dsg3 distribution by immunostaining. Samples were processed for transmission electron microscopy to analyse desmosome ultrastructure. Results Both AK23 and mdPV‐IgG induced blisters and caused reduction in desmosome size and number in labial mucosa. Inhibition of p38MAPK was not effective in preventing these alterations. Conclusions In contrast with human epidermis, PV‐IgG and AK23 induce blisters and desmosome ultrastructural changes in labial mucosa via a mechanism not dependent on p38MAPK. What's already known about this topic? Pemphigus vulgaris IgG (PV‐IgG) induces blistering as well as a reduction in desmosome number and size mediated by p38 mitogen‐activated protein kinase (p38MAPK) signalling in ex vivo human skin. What does this study add? This study establishes a new human ex vivo mucosa model to test pathomechanisms mediated by PV‐IgG. The study demonstrates that both AK23 and mucosal‐dominant PV induce blisters and associated ultrastructural changes in labial mucosa via a mechanism not dependent on p38MAPK signalling. What is the translational message? This study highlights the respective tissue‐specific responses of oral mucosa and skin related to PV pathogenesis, similar to the patient situation.