Direct inhibition precedes p38 mitogen activated protein kinase-mediated uncoupling in desmosomes to reduce desmoglein 3 adhesion by pemphigus autoantibodies

Abstract Background Pemphigus vulgaris (PV) is an autoimmune blistering skin disease caused by impaired desmosome adhesion. Altered signalling pathways and direct inhibition of desmoglein (Dsg) binding contribute to loss of cell adhesion, but the sequence of these events is still a matter of debate. Objectives To characterize the early sequence of events following autoantibody binding to Dsg3 in the pathogenesis of pemphigus. Methods We established stimulated emission depletion imaging in combination with atomic force microscopy single-molecule force measurements to elucidate the primary events following autoantibody binding. Therefore, we measured the Dsg3 binding properties on individual desmosomes and used Triton X-100 fractionation, Western blotting, immunofluorescence and keratinocyte dissociation assays. Results We found that the primary loss of cell adhesion and Dsg3 binding occurs in human keratinocytes as soon as autoantibodies are detectable in desmosomes, which is as early as 5 min for the monoclonal anti-Dsg3 antibody AK23 and 15 min after the addition of PV IgG autoantibodies from patients. Activation of p38 mitogen-activated protein kinase (MAPK) – a central signalling mechanism in PV – was significant after 30 min but not detectable in desmosomes after 5 min of AK23 incubation. Nevertheless, p38 MAPK was required for the loss of cytoskeletal anchorage of Dsg3 molecules in desmosomes and inhibition of p38 MAPK-blunted loss of Dsg3 binding and cell adhesion. Conclusions The results show that autoantibody-induced direct inhibition of Dsg3 binding precedes p38 MAPK-mediated cytoskeletal uncoupling at desmosomes. Thus, the signalling function of Dsg3 in activating p38 MAPK is triggered by the loss of transinteraction, which is the primary target point for therapeutic strategies to stabilize keratinocyte adhesion in PV.