Confocal images of control NH IgG, EGF, and PV IgG-treated keratinocytes labeled with antibody specific for dsg3 (utilizing the PV IgG passive transfer mouse model. adhesion acantholysis). Pathogenic IgG binds to the ectodomain of desmoglein (dsg)2 1 in pemphigus foliaceus (PF) (1, 2) and/or dsg3 in pemphigus vulgaris (PV) (3, 4). Accumulating evidence suggests an essential role for intracellular signaling in the mechanism by which pemphigus IgG induces acantholysis. A number of intracellular signaling events have been observed when keratinocytes are treated with pemphigus IgG (5C8). PV as well as PF autoantibodies activate p38 MAPK in keratinocyte cultures and in both the PV and PF passive transfer mouse models of pemphigus (9C11). Pretreatment with p38 inhibitors blocked PV and PF IgG-induced actin reorganization, keratin intermediate filament retraction (11, 12), dsg endocytosis (13), and blistering (9, 10), suggesting a pivotal role for p38 signaling in the mechanism of acantholysis. Unfortunately, clinical trials of p38 inhibitors for pemphigus have been hindered by the MI-3 hepatotoxicity of these compounds3. Epidermal growth factor receptor (EGFR) is usually a prominent signaling complex whose repertoire of functions is increased by cross-communication with other signaling pathways (15). In addition to its ligand-induced activity, EGFR can be transactivated by various signaling pathways in a ligand-independent manner (16C18). Activation of EGFR can lead to a variety of biological outcomes including cell growth, migration, and suppression of apoptosis. In the skin, EGFR signaling plays a major role in regulating keratinocyte proliferation, and deregulation of EGFR signaling has been observed in skin disorders such as psoriasis, squamous cell carcinoma, and melanoma (19). EGFR has been implicated in modulating cell adhesion junctions, including desmosomes (20, 21). EGFR-regulated adhesion plays an important role in modulating epithelial adhesion and motility. For example, EGFR phosphorylation of -catenin regulates adherens junction assembly/disassembly (22). An increasing number of reports implicate a role for desmoglein-EGFR interactions. Desmoglein 1 has been shown to suppress EGFR-Erk 1/2 (extracellular signal-regulated kinase 1/2) signaling in skin (23). In keratinocytes, EGF-EGFR-mediated plakoglobin phosphorylation has been shown to decrease the association of desmoplakin with the desmosome, thereby reducing cell-cell adhesion (21, 24). EGFR has been shown to regulate dsg2 endocytosis in a squamous cell carcinoma cell line. EGFR promoted dsg2 depletion from the membrane (25). Conversely, EGFR inhibition increased membrane levels of dsg2 (26) and cell adhesion in an oral squamous cell carcinoma cell line (27). Collectively, these studies predict that EGFR inhibition in normal human keratinocytes might similarly stabilize desmosome assembly and cell-cell adhesion. whereas EGFR activation might promote desmosome disassembly and reduce cell-cell adhesion. Pemphigus IgG-induced EGFR activation has been suggested to contribute to acantholysis by induction of apoptosis. EGFR inhibition blocked the observed induction of apoptosis by PV IgG in the immortalized HaCaT keratinocyte line MI-3 and A431 squamous cell carcinoma cultures (28). However, time course studies in normal human keratinocyte cultures and in mice suggest that apoptosis is not required for blistering (11, 29). Additionally, more recent studies did not detect EGFR activation in pemphigus IgG-treated keratinocyte cultures (30). Another group observed EGFR activation in keratinocytes treated with pemphigus IgG (8). However, their studies suggest that p38 activation occurs downstream of, and at time points subsequent to, EGFR activation. The EGFR inhibitor erlotinib has been used to block blistering in the passive transfer mouse model. However, the authors of this study interpreted their results to implicate a role for apoptosis in MI-3 the mechanism of acantholysis (31). Because of the role of EGFR in dsg trafficking and the seemingly conflicting reports on the role of EGFR in pemphigus acantholysis, we explored the potential for EGFR to contribute to the mechanism by MI-3 which pemphigus IgG induce loss of adhesion in keratinocytes. In this study we not only attempt to further elucidate the mechanism of PV OBSCN IgG-induced acantholysis but to clarify the involvement of EGFR in this cascade. We show that this basal activity of the EGFR contributes to desmosome stability; that EGFR is usually activated by PV IgG; that EGFR activation is usually upstream of PV IgG-induced keratin intermediate filament retraction and dsg3 endocytosis and downstream of PV IgG-induced p38 activation; and that EGFR inhibitors block PV IgG-mediated keratin intermediate filament retraction, dsg internalization, and blistering using the passive.
Confocal images of control NH IgG, EGF, and PV IgG-treated keratinocytes labeled with antibody specific for dsg3 (utilizing the PV IgG passive transfer mouse model