The extent of desmoglein 3 depletion in pemphigus vulgaris is dependent on Ca(2+)-induced differentiation: a role in suprabasal epidermal skin splitting?
ABSTRACT: Pemphigus vulgaris (PV) is an autoimmune disease of the skin and mucous membranes and is characterized by development of autoantibodies against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 and formation of intraepidermal suprabasal blisters. Depletion of Dsg3 is a critical mechanism in PV pathogenesis. Because we did not detect reduced Dsg3 levels in keratinocytes cultured for longer periods under high-Ca(2+) conditions, we hypothesized that Dsg depletion depends on Ca(2+)-mediated keratinocyte differentiation. Our data indicate that depletion of Dsg3 occurs specifically in deep epidermal layers both in skin of patients with PV and in an organotypic raft model of human epidermis incubated using IgG fractions from patients with PV. In addition, Dsg3 depletion and loss of Dsg3 staining were prominent in cultured primary keratinocytes and in HaCaT cells incubated in high-Ca(2+) medium for 3 days, but were less pronounced in HaCaT cultures after 8 days. These effects were dependent on protein kinase C signaling because inhibition of protein kinase C blunted both Dsg3 depletion and loss of intercellular adhesion. Moreover, protein kinase C inhibition blocked suprabasal Dsg3 depletion in cultured human epidermis and blister formation in a neonatal mouse model. Considered together, our data indicate a contribution of Dsg depletion to PV pathogenesis dependent on Ca(2+)-induced differentiation. Furthermore, prominent depletion in basal epidermal layers may contribute to the suprabasal cleavage plane observed in PV.
Project description:Pemphigus vulgaris (PV) autoantibodies directly inhibit desmoglein (Dsg) 3-mediated transinteraction. Because cellular signaling also seems to be required for PV pathogenesis, it is important to characterize the role of direct inhibition in pemphigus acantholysis to allow establishment of new therapeutic approaches. Therefore, we modeled the Dsg1 and Dsg3 sequences into resolved cadherin structures and predicted peptides targeting the adhesive interface of both Dsg3 and Dsg1. In atomic force microscopy single molecule experiments, the self-designed cyclic single peptide specifically blocked homophilic Dsg3 and Dsg1 transinteraction, whereas a tandem peptide (TP) consisting of two combined single peptides did not. TP did not directly block binding of pemphigus IgG to their target Dsg antigens but prevented PV-IgG-induced inhibition of Dsg3 transinteraction in cell-free (atomic force microscopy) and cell-based (laser tweezer) experiments, indicating stabilization of Dsg3 bonds. Similarly, PV-IgG-mediated acantholysis and disruption of Dsg3 localization in HaCaT keratinocytes was partially blocked by TP. This is the first evidence that direct inhibition of Dsg3 binding is important for PV pathogenesis and that peptidomimetics stabilizing Dsg transinteraction may provide a novel approach for PV treatment.
Project description:The intercellular interactions of the desmosomal cadherins, desmoglein and desmocollin, are required for epidermal cell adhesion. Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering disease characterized by autoantibodies against desmoglein (Dsg) 3. During calcium-induced desmosome assembly, treatment of primary human keratinocytes with pathogenic monovalent anti-Dsg3 mAbs produced from a PV patient causes a decrease of Dsg3 and desmoplakin but not desmocollin (Dsc) 3 in the Triton-insoluble fraction of cell lysates within 2 hours. Immunofluorescence and antibody ELISA studies suggest that pathogenic mAbs cause internalization of cell-surface Dsg3 but not Dsc3 through early endosomes. Electron microscopy demonstrated a lack of well-formed desmosomes in keratinocytes treated with pathogenic compared to nonpathogenic mAbs. In contrast, pathogenic mAbs caused late depletion of Dsg3 from preformed desmosomes at 24 hours, with effects on multiple desmosomal proteins including Dsc3 and plakoglobin. Together, these studies indicate that pathogenic PV mAbs specifically cause internalization of newly synthesized Dsg3 during desmosome assembly, correlating with their pathogenic activity. Monovalent human PV anti-Dsg mAbs reproduce the effects of polyclonal PV IgG on Dsg3 and will facilitate future studies to further dissect the cellular mechanisms for the loss of cell adhesion in pemphigus.
Project description:In pemphigus vulgaris (PV), autoantibodies directed against the desmosomal cadherin desmoglein (Dsg) 3 cause loss of intercellular adhesion. It is known that Dsg3 interactions are directly inhibited by autoantibody binding and that Dsg2 is upregulated in epidermis of PV patients. Here, we investigated whether heterophilic Dsg2-Dsg3 interactions occur and would modulate PV pathogenesis. Dsg2 was upregulated in PV patients' biopsies and in a human ex vivo pemphigus skin model. Immunoprecipitation and cell-free atomic force microscopy (AFM) experiments demonstrated heterophilic Dsg2-Dsg3 interactions. Similarly, in Dsg3-deficient keratinocytes with severely disturbed intercellular adhesion Dsg2 was upregulated in the desmosome containing fraction. AFM revealed that Dsg2-Dsg3 heterophilic interactions showed binding frequency, strength, Ca2+-dependency and catch-bond behavior comparable to homophilic Dsg3-Dsg3 or homophilic Dsg2-Dsg2 interactions. However, heterophilic Dsg2-Dsg3 interactions had a longer lifetime compared to homophilic Dsg2-Dsg2 interactions and PV autoantibody-induced direct inhibition was significantly less pronounced for heterophilic Dsg2-Dsg3 interactions compared to homophilic Dsg3 interactions. In contrast, a monoclonal anti-Dsg2 inhibitory antibody reduced heterophilic Dsg2-Dsg3 and homophilic Dsg2-Dsg2 binding to the same degree and further impaired intercellular adhesion in Dsg3-deficient keratinocytes. Taken together, the data demonstrate that Dsg2 undergoes heterophilic interactions with Dsg3, which may attenuate autoantibody-induced loss of keratinocyte adhesion in pemphigus.
Project description:Desmosomes provide intercellular adhesive strength required for integrity of epithelial and some non-epithelial tissues. Within the epidermis, the cadherin-type adhesion molecules desmoglein (Dsg) 1-4 and desmocollin (Dsc) 1-3 build the adhesive core of desmosomes. In keratinocytes, several isoforms of these proteins are co-expressed. However, the contribution of specific isoforms to overall cell cohesion is unclear. Therefore, in this study we investigated the roles of Dsg2 and Dsg3, the latter of which is known to be essential for keratinocyte adhesion based on its autoantibody-induced loss of function in the autoimmune blistering skin disease pemphigus vulgaris (PV). The pathogenic PV antibody AK23, targeting the Dsg3 adhesive domain, led to profound loss of cell cohesion in human keratinocytes as revealed by the dispase-based dissociation assays. In contrast, an antibody against Dsg2 had no effect on cell cohesion although the Dsg2 antibody was demonstrated to interfere with Dsg2 transinteraction by single molecule atomic force microscopy and was effective to reduce cell cohesion in intestinal epithelial Caco-2 cells which express Dsg2 as the only Dsg isoform. To substantiate these findings, siRNA-mediated silencing of Dsg2 or Dsg3 was performed in keratinocytes. In contrast to Dsg3-depleted cells, Dsg2 knockdown reduced cell cohesion only under conditions of increased shear. These experiments indicate that specific desmosomal cadherins contribute differently to keratinocyte cohesion and that Dsg2 compared to Dsg3 is less important in this context.
Project description:BACKGROUND AND PURPOSE:Pemphigus is caused by autoantibodies against desmoglein (Dsg) 1, Dsg3, and/or non-Dsg antigens. Pemphigus vulgaris (PV) is the most common manifestation of pemphigus, with painful erosions on mucous membranes. In most cases, blistering also occurs on the skin, leading to areas of extensive denudation. Despite improvements in pemphigus treatment, time to achieve remission is long, severe adverse events are frequent and 20% of patients do not respond adequately. Current clinical developments focus exclusively on modulating B cell function or autoantibody half-life. However, topical modulation of PV autoantibody-induced blistering is an attractive target because it could promptly relieve symptoms. EXPERIMENTAL APPROACH:To address this issue, we performed an unbiased screening in a complex biological system using 141 low MW inhibitors from a chemical library. Specifically, we evaluated PV IgG-induced Dsg3 internalization in HaCaT keratinocytes. Validation of the 20 identified compounds was performed using keratinocyte fragmentation assays, as well as a human skin organ culture (HSOC) model. KEY RESULTS:Overall, this approach led to the identification of four molecules involved in PV IgG-induced skin pathology: MEK1, TrkA, PI3K?, and VEGFR2. CONCLUSION AND IMPLICATIONS:This unbiased screening revealed novel mechanisms by which PV autoantibodies induce blistering in keratinocytes and identified new treatment targets for this severe and potentially life-threatening skin disease.
Project description:Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by autoantibodies to the keratinocyte adhesion protein desmoglein 3 (Dsg3). Previous studies suggest that PV pathogenesis involves p38 mitogen-activated protein kinase-dependent and -independent pathways. However, p38 is a difficult protein to study and therapeutically target because it has four isoforms and multiple downstream effectors. In this study, we identify MAPKAP (mitogen-activated protein kinase-activated protein) kinase 2 (MK2) as a downstream effector of p38 signaling in PV and describe MK2-dependent and -independent mechanisms of blister formation using passive transfer of human anti-Dsg IgG4 mAbs to neonatal mice. In human keratinocytes, PV mAbs activate MK2 in a dose-dependent manner. MK2 is also activated in human pemphigus skin blisters, causing translocation of MK2 from the nucleus to the cytosol. Small-molecule inhibition of MK2 and silencing of MK2 expression block PV mAb-induced Dsg3 endocytosis in human keratinocytes. In addition, small-molecule inhibition and genetic deletion of p38? and MK2 inhibit spontaneous but not induced suprabasal blisters by PV mAbs in mouse passive transfer models. Collectively, these data suggest that MK2 is a key downstream effector of p38 that can modulate PV autoantibody pathogenicity. MK2 inhibition may be a valuable adjunctive therapy for control of pemphigus blistering.
Project description:Pemphigus vulgaris (PV) is a potentially fatal blistering disease characterized by autoantibodies against the desmosomal adhesion protein desmoglein (Dsg) 3. Whether autoantibody steric hindrance or signaling through pathways such as p38 MAPK is primary in disease pathogenesis is controversial. PV mAbs that cause endocytosis of Dsg3 but do not dissociate keratinocytes because of compensatory adhesion by Dsg1 do not activate p38. The same mAbs plus exfoliative toxin to inactivate Dsg1 but not exfoliative toxin alone activate p38, suggesting that p38 activation is secondary to loss of adhesion. Mice with epidermal p38? deficiency blister after passive transfer of PV mAbs; however, acantholytic cells retain cell surface Dsg3 compared with wild-type mice. In cultured keratinocytes, p38 knockdown prevents loss of desmosomal Dsg3 by PV mAbs, and exogenous p38 activation causes internalization of Dsg3, desmocollin 3, and desmoplakin. p38? MAPK is therefore not required for the loss of intercellular adhesion in PV, but may function downstream to augment blistering via Dsg3 endocytosis. Treatments aimed at increasing keratinocyte adhesion could be used in conjunction with immunosuppressive agents, potentially leading to safer and more effective combination therapy regimens.
Project description:Pemphigus is an autoimmune blistering disease targeting the desmosomal proteins desmoglein (Dsg) 1 and Dsg3. Recently, a genetic variant of the Suppression of tumorigenicity 18 (ST18) promoter was reported to cause ST18 up-regulation, associated with pemphigus vulgaris (PV)-IgG-mediated increase in cytokine secretion and more prominent loss of keratinocyte cohesion. Here we tested the effects of PV-IgG and the pathogenic pemphigus mouse anti-Dsg3 antibody AK23 on cytokine secretion and ERK activity in human keratinocytes dependent on ST18 expression. Without ST18 overexpression, both PV-IgG and AK23 induced loss of keratinocyte cohesion which was accompanied by prominent fragmentation of Dsg3 immunostaining along cell borders. In contrast, release of pro-inflammatory cytokines such as IL-1?, IL-6, TNF?, and IFN-? was not altered significantly in both HaCaT and primary NHEK cells. These experiments indicate that cytokine expression is not strictly required for loss of keratinocyte cohesion. Upon ST18 overexpression, fragmentation of cell monolayers increased significantly in response to autoantibody incubation. Furthermore, production of IL-1? and IL-6 was enhanced in some experiments but not in others whereas release of TNF-? dropped significantly upon PV-IgG application in both EV- and ST18-transfected HaCaT cells. Additionally, in NHEK, application of PV-IgG but not of AK23 significantly increased ERK activity. In contrast, ST18 overexpression in HaCaT cells augmented ERK activation in response to both c-IgG and AK23 but not PV-IgG. Because inhibition of ERK by U0126 abolished PV-IgG- and AK23-induced loss of cell cohesion in ST18-expressing cells, we conclude that autoantibody-induced ERK activation was relevant in this scenario. In summary, similar to the situation in PV patients carrying ST18 polymorphism, overexpression of ST18 enhanced keratinocyte susceptibility to autoantibody-induced loss of cell adhesion, which may be caused in part by enhanced ERK signaling.
Project description:OBJECTIVES:? Desmoglein 3 (Dsg3) is a desmosomal adhesion protein expressed in basal and immediate suprabasal layers of skin. Importance of Dsg3 in cell-cell adhesion and maintenance of tissue integrity is illustrated by findings of keratinocyte dissociation in the autoimmune disease, pemphigus vulgaris, where autoantibodies target Dsg3 on keratinocyte surfaces and cause Dsg3 depletion from desmosomes. However, recognition of possible participation of involvement of Dsg3 in cell proliferation remains controversial. Currently, available evidence suggests that Dsg3 may have both anti- and pro-proliferative roles in keratinocytes. The aim of this study was to use RNA interference (RNAi) strategy to investigate effects of silencing Dsg3 in cell-cell adhesion and cell proliferation in two cell lines, HaCaT and MDCK. MATERIALS AND METHODS:? Cells were transfected with siRNA, and knockdown of Dsg3 was assessed by western blotting, fluorescence-activated cell sorting and confocal microscopy. Cell-cell adhesion was analysed using the hanging drop/fragmentation assay, and cell proliferation by colony forming efficiency, BrdU incorporation, cell counts and organotypic culture. RESULTS:? Silencing Dsg3 caused defects in cell-cell adhesion and concomitant reduction in cell proliferation in both HaCaT and MDCK cells. CONCLUSION:? These findings suggest that Dsg3 depletion by RNAi reduces cell proliferation, which is likely to be secondary to a defect in cell-cell adhesion, an essential function required for cell differentiation and morphogenesis.
Project description:Autoantibodies against desmoglein (Dsg) 1 and Dsg3 primarily cause blister formation in the autoimmune disease pemphigus vulgaris (PV). Src was proposed to contribute to loss of keratinocyte cohesion. However, the role and underlying mechanisms are unclear and were studied here. In keratinocytes, cell cohesion in response to autoantibodies was reduced in Src-dependent manner by two patient-derived PV-IgG fractions as well as by AK23 but not by a third PV-IgG fraction, although Src was activated by all autoantibodies. Loss of cell cohesion was progredient in a timeframe of 24 h and AK23, similar to PV-IgG, interfered with reconstitution of cell cohesion after Ca2+-switch, indicating that the autoantibodies also interfered with desmosome assembly. Dsg3 co-localized along cell contacts and interacted with the Src substrate cortactin. In keratinocytes isolated from cortactin-deficient mice, cell adhesion was impaired and Src-mediated inhibition of AK23-induced loss of cell cohesion for 24 h was significantly reduced compared to wild-type (wt) cells. Similarly, AK23 impaired reconstitution of cell adhesion was Src-dependent only in the presence of cortactin. Likewise, Src inhibition significantly reduced AK23-induced skin blistering in wt but not cortactin-deficient mice. These data suggest that the Src-mediated long-term effects of AK23 on loss of cell cohesion and skin blistering are dependent on cortactin-mediated desmosome assembly. However, in human epidermis PV-IgG-induced skin blistering and ultrastructural alterations of desmosomes were not affected by Src inhibition, indicating that Src may not be critical for skin blistering in intact human skin, at least when high levels of autoantibodies targeting Dsg1 are present.