Electron microscopy reveals that phospholipase C and Ca2+ signaling regulate keratin filament uncoupling from desmosomes in Pemphigus

Egu, Desalegn Tadesse; Schmitt, Thomas; Sigmund, Anna; Waschke, Jens

doi:10.1016/j.aanat.2022.151904

Cited by 6 publications

(7 citation statements)

References 30 publications

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“…In this study, we employed a combination of powerful biophysical techniques such as life cell AFM, FRAP and super resolution STED microscopy and identified PLC and therefore Ca 2+ signaling to play a crucial role in relocalization of extradesmosomal Dsg1 in response to PF-IgG. PLC signaling has long ago been shown to be involved in pemphigus IgG-induced signaling in cultured keratinocytes and most recently was demonstrated to contribute to acantholysis and keratin uncoupling from desmosomes in human skin ( 23 , 40 , 41 ).…”

Section: Discussionmentioning

confidence: 99%

Pemphigus Foliaceus Autoantibodies Induce Redistribution Primarily of Extradesmosomal Desmoglein 1 in the Cell Membrane

et al. 2022

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The autoimmune dermatosis pemphigus foliaceus (PF) is predominantly caused by IgG autoantibodies against the desmosomal cadherin desmoglein (Dsg) 1. The exact mechanisms that lead to the characteristic epidermal blistering are not yet fully understood. In the present study, we used a variety of biophysical methods to examine the fate of membrane-bound Dsg1 after incubation with PF patients’ IgG. Dispase-based dissociation assays confirmed that PF-IgG used for this study reduced intercellular adhesion in a manner dependent on phospholipase C (PLC)/Ca2+ and extracellular signal-regulated kinase (ERK) 1/2 signaling. Atomic force microscopy (AFM) revealed that Dsg1 binding on single molecule level paralleled effects on keratinocyte adhesion under the different conditions. Stimulated emission depletion (STED) super-resolution microscopy was used to investigate the localization of Dsg1 after PF-IgG incubation for 24 h. Under control conditions, Dsg1 was found to be in part co-localized with desmoplakin and thus inside of desmosomes as well as extra-desmosomal along the cell border. Incubation with PF-IgG reduced the extra-desmosomal Dsg1 fraction. In line with this, fluorescence recovery after photobleaching (FRAP) experiments demonstrated a strongly reduced mobility of Dsg1 in the cell membrane after PF-IgG treatment indicating remaining Dsg1 molecules were primarily located inside desmosomes. Mechanistically, experiments confirmed the involvement of PLC/Ca2+ since inhibition of PLC or 1,4,5-trisphosphate (IP3) receptor to reduce cytosolic Ca2+ reverted the effects of PF-IgG on Dsg1 intra-membrane mobility and localization. Taken together, our findings suggest that during the first 24 h PF-IgG induce redistribution predominantly of membrane-bound extradesmosomal Dsg1 in a PLC/Ca2+ dependent manner whereas Dsg1-containing desmosomes remain.

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Section: Discussionmentioning

confidence: 99%

Pemphigus Foliaceus Autoantibodies Induce Redistribution Primarily of Extradesmosomal Desmoglein 1 in the Cell Membrane

et al. 2022

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“…Taken together, p38MAPK and PLC/Ca 2+ /PKC were shown to be involved in depletion of Dsg1 and Dsg3 (110,112,136), as well as in keratin filament dissociation (133,135), indicating that these signaling pathways are important for disturbed desmosome assembly as well as for desmosome disassembly and internalization in pemphigus pathogenesis. For Src and ERK, it remains less clear which mechanisms are involved in loss of keratinocyte adhesion.…”

Section: Signaling Pathways Regulate Desmosome Turnovermentioning

confidence: 90%

“…These results show that desmosome disassembly and internalization may be dependent on both cell type and stimulus. For both events, uncoupling of Dsg molecules from the cytoskeleton was shown (Figure 3D) (32,133,181,194). It remains unclear to which extent mechanical destabilization of desmosomes resulting in desmosome splitting under force results from desmosome shrinkage or keratin filament dissociation.…”

Section: Signaling Pathways Regulate Desmosome Turnovermentioning

confidence: 98%

“…IP3, via activation of IP3 receptor (IP3R), causes Ca 2+ release, which in turn activates PKC (187). Inhibition of PLC was protective against the dyscohesive effect of PV-IgG in cell culture (147), in vivo (157), and in human skin explant (133,136). Interestingly, specific inhibition of PLC and Ca 2+ signaling significantly ameliorated keratin dissociation and desmosome splitting (Figures 3A-D) (133).…”

Section: Plc Mediates Desmosomal Adhesion By Maintaining Keratin Anch...mentioning

confidence: 99%

“…Thus, the model is ideal to conduct experiments under controlled conditions with sizeable samples which otherwise are not feasible to apply to human subjects. Therefore, we employed a human skin organ culture model as well as a novel mucosa ex vivo model to investigate the role of various signaling molecules in PV pathogenesis (32,59,(133)(134)(135)(136). Large blisters with associated ultrastructural changes in desmosomes including reduction in desmosome density and size as well as formation of split desmosomes and keratin filament uncoupling from desmosomes was observed in samples treated with PV-IgG (Figures 2A-C) (135) which shows that the skin model reflects the ultrastructural hallmarks known from pemphigus patients' lesions (137,138).…”

Section: Ex Vivo Modelmentioning

confidence: 99%

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Mechanisms Causing Acantholysis in Pemphigus-Lessons from Human Skin

2022

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Pemphigus vulgaris (PV) is an autoimmune bullous skin disease caused primarily by autoantibodies (PV-IgG) against the desmosomal adhesion proteins desmoglein (Dsg) 1 and Dsg 3. PV patient lesions are characterized by flaccid blisters and ultrastructurally by defined hallmarks including a reduction in desmosome number and size, formation of split desmosomes, as well as uncoupling of keratin filaments from desmosomes. The pathophysiology underlying the disease is known to involve several intracellular signaling pathways downstream of PV-IgG binding. Here, we summarize our studies in which we used transmission electron microscopy to characterize the roles of signaling pathways in the pathogenic effects of PV-IgG on desmosome ultrastructure in a human ex vivo skin model. Blister scores revealed inhibition of p38MAPK, ERK and PLC/Ca2+ to be protective in human epidermis. In contrast, inhibition of Src and PKC, which were shown to be protective in cell cultures and murine models, was not effective for human skin explants. The ultrastructural analysis revealed that for preventing skin blistering at least desmosome number (as modulated by ERK) or keratin filament insertion (as modulated by PLC/Ca2+) need to be ameliorated. Other pathways such as p38MAPK regulate desmosome number, size, and keratin insertion indicating that they control desmosome assembly and disassembly on different levels. Taken together, studies in human skin delineate target mechanisms for the treatment of pemphigus patients. In addition, ultrastructural analysis supports defining the specific role of a given signaling molecule in desmosome turnover at ultrastructural level.

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EGFR Inhibition by Erlotinib Rescues Desmosome Ultrastructure and Keratin Anchorage and Protects against Pemphigus Vulgaris IgG–Induced Acantholysis in Human Epidermis

Egu,

Schmitt,

Ernst

et al. 2024

Journal of Investigative Dermatology

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Electron microscopy reveals that phospholipase C and Ca2+ signaling regulate keratin filament uncoupling from desmosomes in Pemphigus

Cited by 6 publications

References 30 publications

Pemphigus Foliaceus Autoantibodies Induce Redistribution Primarily of Extradesmosomal Desmoglein 1 in the Cell Membrane

Pemphigus Foliaceus Autoantibodies Induce Redistribution Primarily of Extradesmosomal Desmoglein 1 in the Cell Membrane

Mechanisms Causing Acantholysis in Pemphigus-Lessons from Human Skin

EGFR Inhibition by Erlotinib Rescues Desmosome Ultrastructure and Keratin Anchorage and Protects against Pemphigus Vulgaris IgG–Induced Acantholysis in Human Epidermis

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