Loss of Desmoglein Binding Is Not Sufficient for Keratinocyte Dissociation in Pemphigus

Abstract

Pemphigus vulgaris (PV) is a severe autoimmune disease in which autoantibodies against the desmosomal cell adhesion molecules desmoglein (Dsg) 1 and Dsg3 cause intraepidermal blister formation. Mechanistically, the fundamental question is still unresolved whether loss of cell cohesion is a result of (1) direct inhibition of Dsg interaction by autoantibodies or (2) intracellular signaling events, which are altered in response to antibody binding and finally cause desmosome destabilization. We used atomic force microscopy (AFM) to perform Dsg3 adhesion measurements on living keratinocytes to investigate the contributions of direct inhibition and signaling to loss of cell cohesion after autoantibody treatment. Dsg3 binding was rapidly blocked following antibody exposure under conditions where no depletion of surface Dsg3 was detectable, demonstrating direct inhibition of Dsg3 interaction. Inhibition of p38MAPK, a central signaling molecule in PV pathogenesis, abrogated loss of cell cohesion, but had a minor effect on loss of Dsg3 binding. Similarly, the cholesterol-depleting agent methyl-β-cyclodextrin (β-MCD) fully blocked cell dissociation, but did not restore Dsg3 interactions or prevent the activation of p38MAPK. These results demonstrate that inhibition of Dsg3 binding is not sufficient to cause loss of cell cohesion, but rather alters signaling events which, in lipid raft-dependent manner, induce cell dissociation.

Desmoglein 3-Dependent Signaling Regulates Keratinocyte Migration and Wound Healing

“…Binding of PV autoantibodies leads to a quick activation of p38MAPK which modulates intermediate filament uncoupling of desmosomes and in turn enhances Dsg3 internalization and depletion. It was suggested that primarily the nondesmosomal Dsg3 molecules present on the cell surface and not connected to the keratin filaments sense the initial binding of autoantibodies and relay this signal into the cell, which results in pronounced p38MAPK-dependent Dsg3 depletion (Müller et al, 2008;Spindler and Waschke, 2014;Vielmuth et al, 2015). Furthermore, PKC is activated following a rapid Ca 2+ influx after PV-IgG application (Osada et al, 1997;Rotzer et al, 2014;Seishima et al, 1995) which switches the Ca 2+ -independent, hyperadhesive desmosomes found in fully confluent keratinocytes to a Ca 2+ -dependent phenotype (Cirillo et al, 2010;Dehner et al, 2014).…”

Pemphigus Autoantibodies Induce Blistering in Human Conjunctiva

“…6,7,28 In our human conjunctiva model, p38MAPK as well as its known downstream target in PV, MK2, 8,30 were activated after PV-IgG treatment. Interestingly, this activation still was present after 12 hours of incubation in our model and is known to be rapidly activated after PV-IgG incubation.…”

“…Fetal calf serum-supplemented DMEM was chosen as culture media due to good evidence for growth of conjunctival cell lines 19,20 and the same medium is a standard medium in human keratinocytes 8 in combination with human IgG-fractions. Samples were incubated for 12 hours in 5% CO 2 at 378C under the respective conditions.…”

“…5 Here, an activation of p38MAPK appears to be central because inhibition of this kinase prevents loss of cell cohesion in cell cultures and murine PV models even under conditions in which Dsg3 interaction is blocked by autoantibody binding. [6][7][8] An ocular involvement in PV also was reported to occur and was diagnosed most frequently as conjunctivitis, 9 which was speculated to correlate with a disease severity 10 or relapse. 9 At first sight, conjunctivitis associated with PV appears to be surprising because the disease is strictly autoantibody-dependent, without any additional factors from the immune necessary.…”

Techniques to stimulate and interrogate cell–cell adhesion mechanics

“…Further, this binding event can be effectively blocked by calcium depletion and Dsg3 antibodies [178]. In a separate study, the same group identified that inhibition of Dsg3 binding, which occurs in the autoimmune disease pemphigus vulgaris, does not lead to cell–cell adhesion loss, rather it alters downstream signaling events that may contribute to that effect, such as p38 mitogen-activated protein kinase (p38 MAPK) [179]. Studies using SMFS also revealed the mechanisms of bond formation between desmosomal cadherins: Dsc forms calcium-dependent homophilic bonds and Dsg forms calcium-independent heterophilic bonds with Dsc.…”