Abstract
In pemphigus vulgaris and pemphigus foliaceus (PF), autoantibodies against desmoglein-3 and desmoglein-1 induce epidermal cell detachment (acantholysis) and blistering. Activation of keratinocyte intracellular signaling pathways is emerging as an important component of pemphigus IgG-mediated acantholysis. We previously reported activation of p38 mitogen-activated protein kinase (MAPK) in response to pathogenic pemphigus vulgaris and PF IgG. Inhibition of p38MAPK blocked pemphigus IgG-induced cytoskeletal reorganization in tissue culture and blistering in pemphigus mouse models. We now extend these observations by demonstrating two peaks of p38MAPK activation in pemphigus tissue culture and mouse models. Administration of the p38MAPK inhibitor SB202190 before PF IgG injection blocked both peaks of p38MAPK phosphorylation and blister formation, consistent with our previous findings; however, administration of the inhibitor 4 h after PF IgG injection blocked only the later peak of p38MAPK activation but failed to block blistering. Examination of the temporal relationship of p38MAPK phosphorylation and apoptosis showed that apoptosis occurs at or after the second peak of p38MAPK activation. The time course of p38MAPK activation and apoptotic markers, as well as the ability of inhibitors of p38MAPK to block activation of the proapoptotic proteinase caspase-3, suggest that activation of apoptosis is downstream to, and a consequence of, p38MAPK activation in pemphigus acantholysis. Furthermore, these observations suggest that the earlier peak of p38MAPK activation is part of the mechanism leading to acantholysis, whereas the later peak of p38MAPK and apoptosis may not be essential for acantholysis.
Highlights
Pemphigus is a group of related autoimmune diseases characterized by blistering in the skin
We have demonstrated that both PV and pemphigus foliaceus (PF) IgG induce phosphorylation of p38MAPK and HSP25, the murine HSP27 homologue, in mouse models and that inhibitors of p38MAPK block blistering in both the PV [16] and the PF [17] passive transfer mouse models
In human skin biopsies from both PV and PF patients, phosphorylation of p38MAPK and HSP27 has been observed [18]. These observations suggest that activation of p38MAPK within the target keratinocyte contributes directly to loss of cell-cell adhesion induced by pemphigus autoantibodies. Both p38MAPK and HSP27 have been implicated in the regulation of the intermediate filament and actin cytoskeletons (19 –25); the ability of p38MAPK inhibitors to block both pemphigus IgG-activated cytoskeletal reorganization and pemphigus IgG-activated blistering suggests that p38MAPK may be acting upstream of the cytoskeleton in the mechanism of acantholysis; p38MAPK signaling has been implicated in other cellular responses
Summary
Materials—Rabbit polyclonal anti-p38MAPK antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), monoclonal anti-phospho-p38MAPK antibodies and anti-phosphoHSP 27 were from Cell Signaling Technology (Beverly, MA), and rabbit polyclonal anti-HSP25 antibodies were from StressGen (Victoria, BC, Canada). Data from in vivo passive transfer mouse experiments utilized IgG purified from a single PF patient whose serum was available in sufficient quantities to carry out the described studies The activity of this serum was determined by indirect IF on sectioned normal human skin with a titer of 1: 2560. Six hours after the addition of PV IgG, cells were washed with ice-cold PBS three times and fixed in 3.7% paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked for 1 h at room temperature with 5% goat serum in PBS followed by an overnight incubation at 4 °C with mouse antihuman cytokeratin 5/8 antibodies (BD Biosciences). B, antibodies to phospho-p38MAPK were used to stain frozen sections of skin biopsies of mice treated with PF IgG for the indicated times and examined by confocal immunofluorescence microscopy.
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