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At Least Three Phosphorylation Events Induced by Pemphigus Vulgaris Sera are Pathogenically Involved in Keratinocyte Acantholysis

Intercellular adhesion among keratinocytes is guaranteed by desmosomes. Disruption of desmosomal integrity leads to cell-cell detachment or acantholysis, as it classically occurs in pemphigus vulgaris (PV), an autoimmune blistering disease of skin and mucous membranes. While purified PV IgG seems to trigger intracellular signaling that crucially involves p38 MAPK, keratinocyte acantholysis induced by whole PV serum may recruit a number of additional signals. In this study, the Pro-Q® Diamond Phosphoprotein Assay was used to investigate the overall changes in protein phosphorylation levels in an in vitro model of PV. We showed that keratinocytes exposed to whole PV sera underwent at least three early and transient phosphorylation events. Two bands with apparent molecular masses of ∼35 and ∼45 kDa were found to be phosphorylated within 1 min after incubation with PV sera. A third band of about 80 kDa reached the peak of phosphorylation level after 3 hours. Morphologic evidence of cell shrinkage and acantholysis were late events and did not correlate temporally with kinase activation, suggesting that cytoskeleton reorganization is a downstream phenomenon. Interestingly, pharmacological abrogation of PV-specific protein phosphorylation was able to inhibit the cell-cell detachment, rounding up, and redistribution of Dsg3 in keratinocytes. Thus, at least three phosphorylation events are pathogenically involved in pemphigus acantholysis.