Although many studies have implicated the effects of PV autoantibodies on DSG3 as central to the disease pathogenesis, few have examined the contributions of non-cadherin desmosomal proteins to the manifestations of PV. Here, we examine the effects of PV autoantibodies on a recently discovered desmosomal protein, Perp. By characterizing the pattern of Perp expression in keratinocytes after exposure to PV antibodies, and by examining the consequences of Perp deficiency for PV phenotypes, we gain insight into PV pathogenesis as well as Perp’s role in desmosomal adhesion.
To understand the basis for PV pathology, previous studies have characterized the process of desmosome dissolution in response to PV antibodies. Binding of PV autoantibodies leads to the formation of a DSG3-PV Ig complex, which dissociates from DP but remains associated with PG as it is internalized into endosomes (Calkins et al., 2006
). The complex eventually gets degraded in lysosomes, causing depletion of DSG3 at the cell surface and consequent intercellular adhesion defects. To determine whether Perp expression is similarly perturbed by PV autoantibodies, we tracked Perp localization relative to DSG3 and PG. Before PV autoantibody exposure, Perp colocalizes with DSG3 and PG at the plasma membrane, consistent with its role as a desmosomal protein. After PV autoantibody treatment, Perp membrane expression diminishes, and Perp localizes to cytoplasmic puncta with DSG3 and PG. By staining for EEA-1 and CD63, we show that these Perp-containing puncta are endosomes and lysosomes, respectively. These colocalization data are consistent with previous studies showing DSG3 and PG colocalization with EEA-1 and CD63 within several hours of PV autoantibody treatment (Calkins et al., 2006
). Our results suggest a model in which, on PV autoantibody binding, the desmosomal complex disassembles, with Perp separating from DP but remaining closely associated with DSG3 and PG and entering the endosomal pathway, where it can ultimately undergo lysosomal degradation. Given that Perp deficiency is sufficient to compromise desmosomal adhesion, PV-induced internalization and downregulation of Perp may contribute to the intercellular adhesion defects seen in the disease.
To clarify Perp’s contribution to PV pathology, we took advantage of keratinocytes derived from Perp−/−
mice (Ihrie et al., 2005
). We hypothesized that Perp loss could potentially either block or exacerbate the effects of PV autoantibodies. An example of the former phenomenon comes from studies demonstrating a central role for PG in the pathogenesis of PV. In the absence of PG, PV autoantibodies fail to induce keratin retraction or intercellular adhesion defects, two cellular hallmarks of the disease, supporting a signaling function for PG outside of its canonical structural role in the desmosome (Caldelari et al., 2001
). However, we determined that Perp-deficient cells do not display a defect in their response to PV autoantibodies, suggesting that Perp is not required for relaying a signal to trigger desmosome disassembly in PV. Instead, we observed a cooperative effect of Perp deficiency and PV autoantibody exposure, manifested as increased DSG3 depletion and augmented intercellular adhesion defects. These findings suggest that Perp, through its role in adhesion, can dampen the pathogenic effects of PV antibodies.
The observed cooperativity between Perp loss and PV antibodies could reflect different potential modes of action for Perp in desmosomal adhesion. For example, as a tetraspan membrane protein, Perp might act by providing intercellular structural connections through homotypic interactions of its extracellular loops. The disruption of these interactions as Perp is internalized following PV autoantibody exposure could contribute to the observed adhesion defects. Indeed, disruption of DSG homotypic and heterotypic interactions by direct binding of PV antibodies has long been proposed as an important event in PV pathogenesis (Amagai et al., 1994b
; Futei et al., 2000
; Sharma et al., 2007
). Thus, loss of both sets of contacts might result in an exacerbated adhesion defect. Alternatively, given that Perp appears to colocalize with DSG3 and PG—the two key participants in PV—and that Perp deficiency leads to increased solubility of DSG3 and PG, Perp might act to promote interactions between DSG3 and PG, or to facilitate proper incorporation of the DSG3–PG complex into the desmosome. In fact, disruption of the DSG3 and PG interaction via deletion of the armadillo-binding domain on DSG3 inhibits proper incorporation of DSG3 into desmosomes (Troyanovsky et al., 1994
; Andl and Stanley, 2001
). Thus, impaired incorporation of DSG3 into desmosomal complexes resulting from Perp deficiency may somehow enhance the ability of DSG3 to be targeted by PV autoantibodies. However, the exact mechanism by which PV IgG mediates DSG3 internalization is controversial. For example, time-lapse microscopy studies tracking desmosome assembly have shown that DSG3 is initially delivered to nondesmosomal simple membrane clusters without connections to the intermediate filament network before being incorporated into mature desmosomes, and that PV IgG induces internalization of these clusters before targeting established desmosomes (Sato et al., 2000
). In contrast, immunoelectron microscopy experiments on skin from a PV mouse model have suggested that PV autoantibodies only target desmosomal DSG3 (Shimizu et al., 2004
). Whether in simple membrane clusters or at the desmosome, if Perp deficiency renders DSG3 more susceptible to PV autoantibodies, it could translate into the enhanced intercellular adhesion defects observed in response to both Perp deficiency and PV autoantibody exposure.
Our data implicate Perp in modulating cellular responses to PV autoantibodies. Specifically, the combined effects of Perp loss and PV autoantibody exposure suggest that Perp normally plays a protective role against the pathogenic effects of PV autoantibodies, possibly through structural support, or by facilitating assembly of DSG3 and PG into desmosomes. These studies suggest the possibility of ultimately manipulating Perp to mitigate some of the clinical manifestations of PV. In future investigations we will examine this possibility as well as distinguishing the models through which Perp promotes desmosomal adhesion.