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Published in final edited form as: J Invest Dermatol. 2025 Feb 3;145(9):2219–2228.e4. doi: 10.1016/j.jid.2024.12.028

Pemphigus vulgaris autoantibodies induce an ER stress response

Coryn L Hoffman 1,#, Navaneetha Krishnan Bharathan 1,#, Yoshitaka Shibata 1, William Giang 1, Johann E Gudjonsson 2, John T Seykora 3, Stephen M Prouty 3, Sara N Stahley 1,5, Aimee S Payne 4, Andrew P Kowalczyk 1,5
PMCID: PMC12317713  NIHMSID: NIHMS2086210  PMID: 39909113

Abstract

Desmosomes are intercellular junctions that mediate cell-cell adhesion and are essential for maintaining tissue integrity. Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies (IgG) targeting desmoglein 3 (Dsg3), a desmosomal cadherin. PV autoantibodies cause desmosome disassembly and loss of cell-cell adhesion, but the molecular signaling pathways that regulate these processes are not fully understood. Using high-resolution time-lapse imaging of live keratinocytes, we found that ER tubules make frequent and persistent contacts with internalizing Dsg3 puncta in keratinocytes treated with PV patient IgG. Biochemical experiments demonstrated that PV IgG activated ER stress signaling pathways, including both IRE1α and PERK pathways, in cultured keratinocytes. Further, ER stress transcripts were upregulated in PV patient skin. Pharmacological inhibition of ER stress protected against PV IgG-induced desmosome disruption and loss of keratinocyte cell-cell adhesion, suggesting that ER stress may be an important pathomechanism and a therapeutically targetable pathway for PV treatment. These data support a model in which desmosome adhesion is integrated with ER function to serve as a cell adhesion stress sensor that is activated in blistering skin disease.

Keywords: desmosomes, cadherins, pemphigus, endoplasmic reticulum, ER stress

INTRODUCTION

Desmosomes are intercellular junctions that link the intermediate filament cytoskeleton to sites of cell-cell contact (Bharathan et al., 2024, Perl et al., 2024, Zimmer and Kowalczyk, 2024). These junctions mediate robust cell-cell adhesion and are critical for integrity of tissues that experience high levels of mechanical stress, including the skin and heart (Müller et al., 2021, Toivola et al., 2024, Waschke, 2008). The desmosomal cadherins, desmogleins and desmocollins, form adhesive interactions that are coupled to the intermediate filament cytoskeleton by intracellular plaque proteins. These desmosomal plaque proteins include armadillo proteins such as plakoglobin and plakophilins, as well as plakin family proteins, including desmoplakin (Delva et al., 2009). Together, these proteins mechanically integrate cells within the epidermis (Perl et al., 2024). Desmosome disruption due to genetic defects, autoimmunity, or infection causes tissue fragility and is the hallmark of various skin diseases (Hegazy et al., 2022).

Pemphigus vulgaris (PV) is an autoimmune bullous disease in which autoantibodies (IgG) against desmoglein 3 (Dsg3) cause steric hindrance, Dsg3 internalization, desmosome disassembly, and loss of keratinocyte adhesion in mucosal epithelia and epidermal keratinocytes (Amagai and Stanley, 2012, Egu et al., 2022, Spindler et al., 2023, Stahley and Kowalczyk, 2015). Clinically, patients present with blisters on the skin and erosions on mucous membranes (Hammers and Stanley, 2020, Kridin and Schmidt, 2021). Current treatments for PV involve immunosuppression (Abeles et al., 2024, Ellebrecht et al., 2022, Ujiie et al., 2021), which puts patients at serious risk for infection. These concerns underscore the need for a deeper understanding of PV pathomechanisms that could lead to the development of more targeted therapies. While it is well-established that PV autoantibodies against Dsg3 cause desmosome disassembly and loss of keratinocyte adhesion, the signaling mechanisms by which PV IgG cause these pathogenic responses are not fully understood.

We recently reported the structure and dynamics of an endoplasmic reticulum (ER)-desmosome complex (Bharathan et al., 2023). The ER is involved in a multitude of cellular processes including protein biosynthesis, lipid synthesis and transfer between organelles, and maintenance of calcium homeostasis (Prinz et al., 2020, Schwarz and Blower, 2016, Voeltz et al., 2024). In addition, the ER is an important stress-sensing organelle that responds to various forms of cellular stress, including metabolic, chemical, and mechanical stresses (Hetz et al., 2020, Mauro, 2014, Phuyal et al., 2023). ER stress has been implicated in the pathogenesis of various skin diseases such as melanoma, Darier’s disease (DD), rosacea, vitiligo, and epidermolysis bullosa simplex (Evtushenko et al., 2021, Park et al., 2019). Interestingly, PV IgG activate p38 MAPK, a stress-activated protein kinase that is known to be involved in ER stress signaling (Cipolla et al., 2017). However, a functional relationship between ER stress and desmosome integrity has yet to be elucidated in the context of PV.

Using live-cell time-lapse confocal microscopy, we find that ER tubules localize to desmosomes and remain associated with Dsg3 puncta during PV IgG-induced internalization. PV IgG rapidly activate ER stress signaling by both the IRE1α and PERK pathways in cultured keratinocytes and ER stress transcripts are upregulated in PV patient skin. Pharmacological inhibition of ER stress prevents PV IgG-induced desmosome disruption and loss of keratinocyte adhesion. These findings indicate that desmosomal adhesion is integrated with ER function to serve as a sensor of desmosomal adhesion stress and that ER signaling pathways could be a potential therapeutic target for the treatment of PV and related disorders.

RESULTS

ER tubules localize to desmosomes during disassembly in PV

We recently reported an association of peripheral ER tubules with keratin filaments and the desmosome plaque (Bharathan et al., 2023). In the present study, we used time-lapse spinning disk confocal imaging to determine how the ER-desmosome complex dynamically responds during PV IgG-induced desmosome disassembly. We used lentivirus to generate N/TERT human keratinocyte cell lines (Dickson et al., 2000) stably expressing KDEL-StayGold to label the ER lumen (Hirano et al., 2022). Cell surface Dsg3 was labelled with a fluorescently-conjugated non-pathogenic mAb (P2C2-CF568) (Cho et al., 2019) directed against the Dsg3 extracellular domain. In cells treated with normal human (NH) IgG, ER tubules maintained stable associations with junctional Dsg3 and remained anchored several hours after IgG addition (Figure 1a, Supplementary Video 1). PV IgG-treated cells exhibited similarly stable ER-Dsg3 associations initially (Figure 1b, Supplementary Video 2). This initial stage was followed by stereotypical Dsg3 clustering at cell-cell contacts, and then formation of linear arrays perpendicular to the cell-cell contact, as described previously (Jennings et al., 2011). Remarkably, during these dynamic reorganization events, ER tubules maintained associations with both Dsg3 clusters and linear arrays (Figure 1c, Supplementary Video 3). These data demonstrate a stable structural association between ER tubules and desmosomes during PV IgG-induced desmosome disassembly.

Figure 1. ER tubules localize to desmosomes and remain associated with Dsg3 puncta during PV IgG-induced internalization.

Figure 1.

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N/TERT keratinocytes expressing luminal ER marker KDEL-StayGold (magenta) were pre-labeled with a fluorescently-conjugated (CF568) Dsg3 antibody (orange), treated with IgG, and imaged every 10 sec for various lengths of time during the first 3.5 hours following IgG addition. (a) Dsg3 puncta formed stable associations with peripheral ER tubules in NH IgG-treated cells even 3 hours after IgG addition (white arrows). (b) Stable Dsg3-ER associations were seen within the first 30 minutes following addition of PV1a IgG (white arrows). (c) ER tubules formed stable associations with Dsg3 puncta undergoing clustering (white arrows) and wrapped around linear arrays (yellow arrows) forming perpendicular to the cell-cell contact (dashed line). Scale bar = 5 μm.

PV IgG induce a rapid ER stress response

The physical association of ER with desmosomes during PV IgG-induced desmosome disassembly raised the possibility that ER signaling pathways are activated upon keratinocyte exposure to PV IgG. To determine if PV IgG induce ER stress signaling upon binding to Dsg3, we treated primary normal human epidermal keratinocytes (NHEKs) with PV IgG from several patients (PV1, PV2, or PV3) for 15 minutes to 1 hour and probed for IRE1α and eIF2α phosphorylation as markers of ER stress pathway activation. Western blot analysis revealed that IRE1α and eIF2α phosphorylation were upregulated within 15 minutes of keratinocyte exposure to PV IgG (Figure 2a). PV IgG induced eIF2α phosphorylation in a dose-dependent manner, with significant increases beginning at 100 μg/mL (Supplementary Figure S1). While polyclonal PV IgG induced IRE1α and eIF2α phosphorylation within 15 minutes, pathogenic monoclonal antibody AK23 did not (Supplementary Figure S2). To investigate if an ER stress response is observed in PV patients, we performed qRT-PCR of PV patient epidermis to evaluate the transcriptional levels of key ER stress markers. The expression of DDIT3, XBP1s, ATF3, and HSPA5 were increased in PV patient skin biopsies from blister margins relative to healthy skin (Figure 2b). While the relative expression of these transcripts was variable, at least one of these markers was upregulated in each of four different patient samples. Collectively, these data demonstrate that ER stress pathways are activated in both in vitro cell culture models of PV and in PV patient epidermis.

Figure 2. ER stress is activated in PV IgG-treated keratinocytes and PV patient skin.

Figure 2.

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(a) NHEKs were treated with NH or PV IgG (PV1, PV2, or PV3) for 15 minutes to 1 hour, and ER stress marker phosphorylation was monitored by western blot. PV3 alone significantly increased IRE1α phosphorylation after 15 min. PV1 significantly increased eIF2α phosphorylation after 15 minutes and 1 hour, while PV2 significantly increased eIF2α phosphorylation after 1 hour. However, all PV IgG-treated NHEKs exhibited a trend towards increased IRE1α and eIF2α phosphorylation relative to NH IgG-treated NHEKs. (b) Expression of ER stress transcripts was monitored by qRT-PCR in PV patient skin biopsies from blister margins. All four patients show differential upregulation of ER stress markers relative to NH controls (dashed line). Mean value ± SEM is represented. ** P < 0.01, and *** P < 0.001 using an unpaired student’s t-test comparing PV IgG-treated to NH IgG-treated.

ER stress inhibitors prevent PV IgG-induced desmosome disruption and loss of adhesion in cultured keratinocytes

Previous studies indicate that desmosome disassembly and disruption of keratinocyte adhesion occur several hours after PV IgG binding (Calkins et al., 2006, Jennings et al., 2011). Since the ER stress response appears to be temporally upstream of desmosome disruption and loss of adhesion in PV, we investigated whether ER stress inhibition is protective against PV IgG-induced desmosome disruption. We inhibited ER stress with miglustat, a pharmacological chaperone (Abian et al., 2011, Alfonso et al., 2005, Savignac et al., 2014), or GSK2606414, a PERK inhibitor (Axten et al., 2012). To determine if miglustat or GSK2606414 prevent the PV IgG-induced ER stress response, we treated NHEKs with either miglustat or GSK2606414, followed by exposure to either NH or PV IgG for 15 minutes to 1 hour and probed for phosphorylated ER stress markers. Western blot analysis revealed that treatment of keratinocytes with PV1, PV2, or PV3 significantly increased IRE1α and eIF2α phosphorylation within 15 minutes. Miglustat pre-treatment abrogated IRE1α phosphorylation by these three PV autoantibodies (Figure 3a) but did not reduce PV IgG-induced eIF2α phosphorylation (Figure 3b). Further, GSK2606414 significantly reduced IRE1α phosphorylation induced by the PV3 antibody, but not by PV1a or PV2 (Figure 3c). However, GSK2606414 reduced eIF2α phosphorylation induced by all three PV antibodies, albeit only after 1 hour of PV exposure (Figure 3d). These findings demonstrate that miglustat and GSK2606414 prevent the PV IgG-induced ER stress response by targeting distinct pathways.

Figure 3. Miglustat and GSK2606414 prevent PV IgG-induced ER stress activation.

Figure 3.

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NHEKs were pre-treated with vehicle control (VEH), miglustat (MIG), DMSO, or GSK2606414 (GSK’414) and then treated with IgG from NH or PV patients (PV1, PV1a, PV2, or PV3) for 15 minutes or 1 hour, and ER stress marker phosphorylation was monitored by western blot. (a) At 15 minutes, MIG significantly prevented PV IgG-induced IRE1α phosphorylation but (b) did not have a significant effect on eIF2α phosphorylation. (c) GSK’414 significantly prevented PV IgG-induced phosphorylation of IRE1α at 15 minutes and (d) eIF2α at 1 hour. Mean value ± SEM is represented. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 using a two-way ANOVA.

To evaluate the effect of ER stress inhibition on PV IgG-induced desmosome disruption, we immunolabeled Dsg3 in NHEKs pre-treated with either miglustat or GSK2606414. Treatment with PV1a or PV2 dramatically disrupted Dsg3 border localization compared to cells treated with NH IgG. However, pre-treatment with either miglustat or GSK2606414 prevented PV IgG-induced disruption of Dsg3 border localization (Figure 4a). To assess the effect of ER stress inhibition on PV IgG-induced loss of cell-cell adhesion, we performed a dispase fragmentation assay (Hudson et al., 2004, Huen et al., 2002) in NHEKs pretreated with either miglustat or GSK2606414. PV IgG treatment caused extensive monolayer fragmentation compared to NH IgG. However, pre-treatment with either miglustat or GSK2606414 significantly reduced monolayer fragmentation in PV IgG-treated cultures (Figure 4b). Together, these results indicate that inhibition of ER stress protects against PV IgG-induced desmosome disruption and loss of cell-cell adhesion.

Figure 4. ER stress inhibitors prevent pathogenic PV IgG responses.

Figure 4.

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NHEKs were pre-treated with vehicle control (VEH), miglustat (MIG), DMSO, or GSK2606414 (GSK’414) and then treated with IgG from NH or PV patients (PV1a or PV2). (a) Localization of Dsg3 was monitored by immunofluorescence microscopy. Dsg3 border localization was decreased by both PV IgG in the VEH- and DMSO-treated cells but not in MIG- or GSK’414-treated cells. Scale bar = 20 μm. (b) Loss of adhesion was assessed by dispase fragmentation assay. PV IgG treatment significantly increased fragmentation relative to NH IgG-treated cells. MIG or GSK’414 treatment significantly reduced PV IgG-mediated fragmentation. Mean value ± SEM is represented. ns = not significant, * P < 0.05, *** P < 0.001, and **** P < 0.0001 using a two-way ANOVA.

DISCUSSION

The results presented here demonstrate that the ER mediates a keratinocyte stress response to PV IgG. Live-cell confocal microscopy revealed that ER tubules associate with Dsg3 puncta as they internalize during desmosome disassembly in response to PV IgG. These findings are consistent with our previous work demonstrating that ER tubules form extensive contacts with both desmosomes and the keratin intermediate filament cytoskeleton (Figure 5) (Bharathan et al., 2023). ER stress signaling was observed in both cultured keratinocytes treated with PV IgG and in PV patient skin biopsies. Further, we demonstrate that pharmacological inhibition of ER stress alleviates the pathogenic effects of PV IgG on desmosome disruption and keratinocyte adhesion. Collectively, these findings suggest that the ER plays a role in the pathogenic mechanisms that underlie PV IgG-induced loss of adhesion.

Figure 5. Proposed model of ER stress signaling in response to PV IgG.

Figure 5.

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ER tubules (purple) form close associations with the desmosome junction (dark blue) and keratin filaments (light blue) on either side of a cell-cell contact. ER-desmosome associations are maintained following PV IgG-mediated Dsg3 clustering and linear array formation. Exposure to PV IgG leads to activation of ER stress through the IRE1α and PERK pathways, leading to transcriptional activation of the unfolded protein response and subsequent downstream signaling.

A functional link between ER and desmosome regulation first came from studies on DD, a desmosomal skin disease that is caused by mutations in the ER calcium pump, sarcoendoplasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) (Harmon et al., 2024, Sakuntabhai et al., 1999). SERCA2 dysfunction in DD has been shown to cause dysregulation of desmoplakin dynamics, impaired adhesion strength, and constitutive ER stress (Hobbs et al., 2011, Savignac et al., 2014, Sugiura, 2013). Further, inhibition of ER stress with chemical chaperones protects against keratin aggregation in epidermolysis bullosa simplex, a blistering skin disease (Chamcheu et al., 2011, Chamcheu et al., 2016). ER stress has also been implicated in the pathology of arrhythmogenic cardiomyopathy, a condition that can be caused by defective desmosomal adhesion (Pitsch et al., 2021). The activation of ER stress in these other diseases that display adhesion defects is similar to what we observe in PV. In addition, direct mechanical stimulation of ER tubules using optogenetic tools induces rapid ER calcium release and increases levels of binding-immunoglobulin protein (BiP, encoded by HSPA5) and phosphorylated eIF2α (Song et al., 2024). Together, these studies suggest that mechanics and desmosomal adhesion are integrated with ER function and that skin disease models may be used to elucidate the functions of the ER-desmosome complex and its potential role in stress-sensing (Angulo-Urarte et al., 2020).

ER stress signaling intersects with various pathways that are known to be activated in PV, including p38 MAPK, PKC, and EGFR (Sharma et al., 2007, Spindler and Waschke, 2014, Takayanagi et al., 2015). p38 MAPK is a stress-activated protein kinase that is known to be involved in signaling both upstream and downstream of ER stress (Kim et al., 2008, Koeberle et al., 2015, Yang et al., 2007). p38 MAPK is phosphorylated as early as 15-30 min after PV IgG treatment in cultured keratinocytes (Berkowitz et al., 2005) and is also activated in PV patient skin (Berkowitz P. et al., 2008). These findings are consistent with our results demonstrating that ER stress is rapidly activated by PV IgG (Figure 2a). Likewise, p38 MAPK knockdown prevents PV IgG-induced loss of desmosomal Dsg3 in cultured keratinocytes (Mao et al., 2011) and p38 MAPK inhibition prevents blistering in mouse models of pemphigus (Berkowitz P. et al., 2008, Berkowitz et al., 2006). It remains unclear whether abrogating p38 MAPK activity leads to reduced ER stress in PV patient skin. Interestingly, we observe variability in the ER stress signatures between different PV patient biopsies (Figure 2b), which could reflect differences in stages of blister formation, progression of disease state, treatment regimens, or patient-specific antibody repertoires. Together with our observations, these findings suggest that ER stress may be a targetable pathomechanism in PV.

We used miglustat, an FDA-approved inhibitor of ceramide-specific glucosyltransferase that is thought to have molecular chaperone activities (Abian et al., 2011, Alfonso et al., 2005). Savignac and colleagues had previously shown that miglustat restored desmosome formation and improved adhesion strength in a keratinocyte model of DD, a disease that exhibits constitutive ER stress (Savignac et al., 2014). Consistent with their findings, we show that miglustat does not affect eIF2α phosphorylation. However, we find that miglustat does reduce IRE1α phosphorylation by PV IgG (Figure 3a). Interestingly, p38 MAPK, whose activation has been well-characterized in PV, is downstream of IRE1α, so it is possible that the IRE1α pathway is the more prominent ER stress pathway that is activated in PV (Figure 5). Consistent with this hypothesis, miglustat had a significant protective effect against PV IgG-induced desmosome disruption and loss of adhesion (Figure 4). Overall, these findings suggest that ER stress pathways are activated in several types of desmosome disorders and may represent a therapeutically relevant pathway for the treatment of desmosomal disease.

METHODS

Cell line generation, transfection, culture, and reagents

All cells were cultured at 37°C and 5% CO2. N/TERT keratinocytes were generated as previously described (Dickson et al., 2000). N/TERTs were cultured in Keratinocyte Serum-Free Media (K-SFM) supplemented with 30 μg/mL bovine pituitary extract and 0.2 ng/mL human recombinant epidermal growth factor (37010022, Gibco, Waltham, MA). For daily maintenance and subculturing of N/TERTs, the final calcium concentration was adjusted to 400 μM. The cells were switched to medium containing 550 μM calcium (high calcium media) 18-24 hours before experimentation. N/TERTs used for live-cell imaging were lentivirally-transduced with a KDEL-StayGold fluorescent protein as a soluble marker of the ER lumen (er-(n2)oxStayGold(c4)v2.0) (Hirano et al., 2022) by incubating cells with 8 μg/mL polybrene (TR-1003-G, EMD Millipore, Burlington, MA) in cell culture medium for 24 hours. Cells stably infected with the KDEL-StayGold lentivirus were selected using puromycin (4 μg/mL) (ABT-440, Boston BioProducts, Milford, MA). Bulk sorting of cell lines expressing the lentiviral constructs was performed by fluorescence-activated cell sorting to obtain populations with similar expression levels.

NHEKs were isolated from neonatal foreskin as previously described (Calkins et al., 2006, Schell et al., 2023). NHEKs (no later than passage 4) were cultured in KGM Gold Keratinocyte Growth Medium BulletKit (00192060, Lonza, Basel, Switzerland). For daily maintenance and subculturing of NHEKs, the final calcium concentration was adjusted to 50 μM to prevent desmosome formation. Cells were switched to high calcium media (550 μM) for 5-24 hours before experimentation to induce desmosome formation.

Treatment of cells with PV IgG

Desmosome formation was induced by switching cells to high calcium media (550 μM) for 5-24 hours. Cells were treated with either NH IgG or PV patient IgG (400 μg/mL) for indicated time periods at 37°C. PV sera were kind gifts from Dr. Masayuki Amagai (Keio University, Tokyo) and Dr. Aimee Payne (Columbia University, New York City, NY) (Saito et al., 2012, Stahley et al., 2016). PV IgG used in this study have been assessed for recognition of Dsg3 and Dsg1 using a commercial ELISA (Supplementary Table S1). The reference standard for PV3 was standardized to 1.5. PV IgG used in this study cause loss of junctional Dsg3 as assessed by immunofluorescence, and loss of cell-cell adhesion as assessed by dispase fragmentation assay as shown previously for PV1 (Saito et al., 2012) or in this manuscript for PV1a, PV2, and PV3 (Figure 4, Supplementary Figure S3).

Drug treatments

For miglustat (N-Butyldeoxynojirimycin) (B8299, Millipore-Sigma, Burlington, MA) experiments, cells were simultaneously switched to high calcium media (550 μM) and treated with either vehicle (sterile water) or 500 μM miglustat for indicated time periods. For GSK2606414 (HY-18072, MedChemExpress, Monmouth Junction, NJ) experiments, cells were switched to high calcium media (550 μM) for indicated time periods, then treated with either DMSO or 1 μM GSK2606414 for 1 hour.

qRT-PCR analysis

RNA was isolated from previously-collected and deidentified paraffin-embedded PV patient biopsies using the Quick-RNA FFPE MiniPrep kit (ZR1008, Zymo, Irvine, CA). RNA concentration and purity were assessed using spectrophotometry. A total of 400 ng of RNA was reverse transcribed using the iScript cDNA Synthesis Kit (1708891, Bio-Rad, Hercules, CA).

For expression analysis of cells treated with NH IgG or PV IgG, qRT-PCR was performed using TaqMan Fast Advanced Master Mix (4444557, Applied Biosystems, Waltham, MA) and Light Cycler 480 (Roche, Basel, Switzerland). TaqMan MGB probes labelled with fluorescent dyes were used. Reaction was performed according to the following protocol: 50°C for 2 min, 95°C for 20 sec, and (50 cycles of 95°C for 3 sec and 60°C for 30 sec). Probes against target genes of interest were labelled with the FAM dye. TBP was labelled with the VIC dye and used as the reference gene within the same well as the target gene of interest. Normalized expression was calculated using Microsoft Excel (version 2023) by subtracting the cycle threshold (Ct) value of the internal control gene from the Ct value of the gene of interest, followed by averaging this value across all technical replicates. Fold change relative to healthy patient tissue was then calculated by the 2−ΔΔCt method. More details about TaqMan probes are provided in Supplementary Table S2.

Immunofluorescence

Cells were grown to 80% confluence on #1.5 glass coverslips coated with 0.1% gelatin solution (PCS-999-027, ATCC, Manassas, VA). For miglustat experiments, cells were simultaneously switched to high calcium media (550 μM) and treated with either vehicle (sterile water) or miglustat for 18 hours. For GSK2606414 experiments, cells were switched to high calcium media (550 μM) for 17 hours, and then treated with either DMSO or GSK2606414 for 1 hour. Drug-treated cells were then pre-labeled with P2C2, a non-pathogenic anti-Dsg3 monoclonal antibody conjugated to CF568 (P2C2-CF568) for 30 minutes at 4°C on a rocker, and then treated with NH or PV IgG in drug-containing high calcium media (550 μM) for 6 hours. Cell borders were labeled with 2 μg/mL WGA-640R (29026, Biotium, Fremont, CA) in appropriate cell culture media at 37°C for 15 minutes. Cells were then washed three times with 1X PBS + 550 μM calcium for 5 minutes each, fixed with 100% methanol at −20°C for 2 minutes, followed by 3 more washes, and mounted with ProLong Glass Antifade Mountant (P36980, Invitrogen, Waltham, MA).

Dispase fragmentation assay

Cells were grown to 100% confluence in 24-well plates. For miglustat experiments, cells were simultaneously switched to high calcium media (550 μM) and treated with either vehicle (sterile water) or miglustat for 24 hours. For GSK2606414 experiments, cells were switched to high calcium media (550 μM) for 5 hours, and then treated with either DMSO or GSK2606414 for 1 hour. Drug-treated cells were then treated with NH or PV IgG in drug-containing high calcium media (550 μM) for 18 hours. Monolayers were dissociated from the plate with dispase (354235, Corning, Corning, NY), using 40 μL dispase + 200 μL cell culture media per well in a 24-well plate. Monolayers were rinsed with 1X PBS + 550 μM calcium, transferred to 5 mL tubes, and subjected to mechanical stress on a tube rotator at 55 rpm for 1-3 min. Fragments transferred to 4-well plates were fixed and stained with 1% paraformaldehyde and methylene blue. Fragments imaged on a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA) were counted with Fiji.

Supplementary Material

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ACKNOWLEDGMENTS

The Flow Cytometry Core (RRID:SCR_021134) services and instruments used in this project were funded, in part, by the Pennsylvania State University College of Medicine via the Office of the Vice Dean of Research and Graduate Students and the Pennsylvania Department of Health using Tobacco Settlement Funds (CURE). The authors thank Nate Sheaffer and Joseph Bednarczyk from Penn State College of Medicine’s Flow Cytometry Core for assistance with cell sorting. We are also grateful to the University of Pennsylvania Skin Biology and Diseases Resource-based Center (Penn SBDRC) as well as Amanda Nelson and Ryan Hobbs (Penn State College of Medicine) for instrument use, reagents, and advice. This work was supported by NIH grants R01AR081883 and R01AR048266 to APK, NIH P30-AR075043 to JEG, NIH R01-AR082675 to ASP, and NIH P30-AR-069589 to PI-Grice, Core Director-JTS.

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

Data availability statement

Datasets related to this article are hosted on ScholarSphere (https://scholarsphere.psu.edu/resources/d8faef77-b085-44db-8610-5c5a7448070e), an open-source online data repository. Supplementary Videos can be found at: https://youtube.com/playlist?list=PLRVCHiGokrM6ujJV1Twn-UREtg-U0E_RH&si=qq14sS0Xmufnf-NZ. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

REFERENCES

  1. Abeles I, Palma C, Meednu N, Payne AS, Looney RJ, Anolik JH. B Cell-Directed Therapy in Autoimmunity. Annu Rev Immunol 2024;42(1):103–26. [DOI] [PubMed] [Google Scholar]
  2. Abian O, Alfonso P, Velazquez-Campoy A, Giraldo P, Pocovi M, Sancho J. Therapeutic strategies for Gaucher disease: miglustat (NB-DNJ) as a pharmacological chaperone for glucocerebrosidase and the different thermostability of velaglucerase alfa and imiglucerase. Mol Pharm 2011;8(6):2390–7. [DOI] [PubMed] [Google Scholar]
  3. Alfonso P, Pampín S, Estrada J, Rodríguez-Rey JC, Giraldo P, Sancho J, et al. Miglustat (NB-DNJ) works as a chaperone for mutated acid β-glucosidase in cells transfected with several Gaucher disease mutations. Blood Cells, Molecules, and Diseases 2005;35(2):268–76. [DOI] [PubMed] [Google Scholar]
  4. Amagai M, Stanley JR. Desmoglein as a target in skin disease and beyond. J Invest Dermatol 2012;132(3 Pt 2):776–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Angulo-Urarte A, van der Wal T, Huveneers S. Cell-cell junctions as sensors and transducers of mechanical forces. Biochim Biophys Acta Biomembr 2020;1862(9):183316. [DOI] [PubMed] [Google Scholar]
  6. Axten JM, Medina JR, Feng Y, Shu A, Romeril SP, Grant SW, et al. Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). J Med Chem 2012;55(16):7193–207. [DOI] [PubMed] [Google Scholar]
  7. Berkowitz P, Chua M, Liu Z, Diaz LA, Rubenstein DS. Autoantibodies in the autoimmune disease pemphigus foliaceus induce blistering via p38 mitogen-activated protein kinase-dependent signaling in the skin. Am J Pathol 2008;173(6):1628–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Berkowitz P, Diaz LA, Hall RP, Rubenstein DS. Induction of p38MAPK and HSP27 Phosphorylation in Pemphigus Patient Skin. Journal of Investigative Dermatology 2008;128(3):738–40. [DOI] [PubMed] [Google Scholar]
  9. Berkowitz P, Hu P, Liu Z, Diaz LA, Enghild JJ, Chua MP, et al. Desmosome signaling. Inhibition of p38MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization. J Biol Chem 2005;280(25):23778–84. [DOI] [PubMed] [Google Scholar]
  10. Berkowitz P, Hu P, Warren S, Liu Z, Diaz LA, Rubenstein DS. p38MAPK inhibition prevents disease in pemphigus vulgaris mice. Proceedings of the National Academy of Sciences 2006;103(34):12855–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bharathan NK, Giang W, Hoffman CL, Aaron JS, Khuon S, Chew T-L, et al. Architecture and dynamics of a desmosome–endoplasmic reticulum complex. Nature Cell Biology 2023;25(6):823–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bharathan NK, Mattheyses AL, Kowalczyk AP. The desmosome comes into focus. J Cell Biol 2024;223:e202404120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Calkins CC, Setzer SV, Jennings JM, Summers S, Tsunoda K, Amagai M, et al. Desmoglein Endocytosis and Desmosome Disassembly Are Coordinated Responses to Pemphigus Autoantibodies *. Journal of Biological Chemistry 2006;281(11):7623–34. [DOI] [PubMed] [Google Scholar]
  14. Chamcheu JC, Navsaria H, Pihl-Lundin I, Liovic M, Vahlquist A, Törmä H. Chemical Chaperones Protect Epidermolysis Bullosa Simplex Keratinocytes from Heat Stress–Induced Keratin Aggregation: Involvement of Heat Shock Proteins and MAP Kinases. Journal of Investigative Dermatology 2011;131(8):1684–91. [DOI] [PubMed] [Google Scholar]
  15. Chamcheu JC, Siddiqui IA, Mukhtar H. Chemical chaperone therapy, a new strategy for genetic skin fragility disorders. Exp Dermatol 2016;25(3):183–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cho A, Caldara AL, Ran NA, Menne Z, Kauffman RC, Affer M, et al. Single-Cell Analysis Suggests that Ongoing Affinity Maturation Drives the Emergence of Pemphigus Vulgaris Autoimmune Disease. Cell Rep 2019;28(4):909–22.e6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cipolla GA, Park JK, Lavker RM, Petzl-Erler ML. Crosstalk between Signaling Pathways in Pemphigus: A Role for Endoplasmic Reticulum Stress in p38 Mitogen-Activated Protein Kinase Activation? Frontiers in Immunology 2017;8(1022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Delva E, Tucker DK, Kowalczyk AP. The desmosome. Cold Spring Harb Perspect Biol 2009;1(2):a002543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, et al. Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 2000;20(4):1436–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Egu DT, Schmitt T, Waschke J. Mechanisms Causing Acantholysis in Pemphigus-Lessons from Human Skin. Frontiers in Immunology 2022;13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ellebrecht CT, Maseda D, Payne AS. Pemphigus and Pemphigoid: From Disease Mechanisms to Druggable Pathways. J Invest Dermatol 2022;142(3 Pt B):907–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Evtushenko NA, Beilin AK, Kosykh AV, Vorotelyak EA, Gurskaya NG. Keratins as an Inflammation Trigger Point in Epidermolysis Bullosa Simplex. Int J Mol Sci 2021;22(22). [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hammers CM, Stanley JR. Recent Advances in Understanding Pemphigus and Bullous Pemphigoid. J Invest Dermatol 2020;140(4):733–41. [DOI] [PubMed] [Google Scholar]
  24. Harmon RM, Ayers JL, McCarthy EF, Kowalczyk AP, Green KJ, Simpson CL. Pumping the breaks on acantholytic skin disorders: targeting calcium pumps, desmosomes, and downstream signaling in Darier, Hailey-Hailey, and Grover disease. J Invest Dermatol 2024. 10.1016/j.jid.2024.06.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hegazy M, Perl AL, Svoboda SA, Green KJ. Desmosomal Cadherins in Health and Disease. Annual Review of Pathology: Mechanisms of Disease 2022;17(Volume 17, 2022):47–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hetz C, Zhang K, Kaufman RJ. Mechanisms, regulation and functions of the unfolded protein response. Nature Reviews Molecular Cell Biology 2020;21(8):421–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hirano M, Ando R, Shimozono S, Sugiyama M, Takeda N, Kurokawa H, et al. A highly photostable and bright green fluorescent protein. Nat Biotechnol 2022;40(7):1132–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hobbs RP, Amargo EV, Somasundaram A, Simpson CL, Prakriya M, Denning MF, et al. The calcium ATPase SERCA2 regulates desmoplakin dynamics and intercellular adhesive strength through modulation of PKCα signaling. The FASEB Journal 2011;25(3):990–1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hudson TY, Fontao L, Godsel LM, Choi HJ, Huen AC, Borradori L, et al. In vitro methods for investigating desmoplakin-intermediate filament interactions and their role in adhesive strength. Methods Cell Biol 2004;78:757–86. [DOI] [PubMed] [Google Scholar]
  30. Huen AC, Park JK, Godsel LM, Chen X, Bannon LJ, Amargo EV, et al. Intermediate filament-membrane attachments function synergistically with actin-dependent contacts to regulate intercellular adhesive strength. J Cell Biol 2002;159(6):1005–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Jennings JM, Tucker DK, Kottke MD, Saito M, Delva E, Hanakawa Y, et al. Desmosome disassembly in response to pemphigus vulgaris IgG occurs in distinct phases and can be reversed by expression of exogenous Dsg3. J Invest Dermatol 2011;131(3):706–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kim I, Xu W, Reed JC. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nature Reviews Drug Discovery 2008;7(12):1013–30. [DOI] [PubMed] [Google Scholar]
  33. Koeberle A, Pergola C, Shindou H, Koeberle SC, Shimizu T, Laufer SA, et al. Role of p38 mitogen-activated protein kinase in linking stearoyl-CoA desaturase-1 activity with endoplasmic reticulum homeostasis. The FASEB Journal 2015;29(6):2439–49. [DOI] [PubMed] [Google Scholar]
  34. Kridin K, Schmidt E. Epidemiology of Pemphigus. JID Innovations 2021;1(1):100004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mao X, Sano Y, Park JM, Payne AS. p38 MAPK Activation Is Downstream of the Loss of Intercellular Adhesion in Pemphigus Vulgaris *<sup></sup>. Journal of Biological Chemistry 2011;286(2):1283–91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mauro T. Endoplasmic reticulum calcium, stress, and cell-to-cell adhesion. J Invest Dermatol 2014;134(7):1800–1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Müller L, Hatzfeld M, Keil R. Desmosomes as Signaling Hubs in the Regulation of Cell Behavior. Frontiers in Cell and Developmental Biology 2021;9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Park K, Lee SE, Shin K-O, Uchida Y. Insights into the role of endoplasmic reticulum stress in skin function and associated diseases. The FEBS Journal 2019;286(2):413–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Perl AL, Pokorny JL, Green KJ. Desmosomes at a glance. J Cell Sci 2024;137(12). [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Phuyal S, Romani P, Dupont S, Farhan H. Mechanobiology of organelles: illuminating their roles in mechanosensing and mechanotransduction. Trends Cell Biol 2023;33(12):1049–61. [DOI] [PubMed] [Google Scholar]
  41. Pitsch M, Kant S, Mytzka C, Leube RE, Krusche CA. Autophagy and Endoplasmic Reticulum Stress during Onset and Progression of Arrhythmogenic Cardiomyopathy. Cells 2021;11(1). [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Prinz WA, Toulmay A, Balla T. The functional universe of membrane contact sites. Nat Rev Mol Cell Biol 2020;21(1):7–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Saito M, Stahley SN, Caughman CY, Mao X, Tucker DK, Payne AS, et al. Signaling Dependent and Independent Mechanisms in Pemphigus Vulgaris Blister Formation. PLOS ONE 2012;7(12):e50696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sakuntabhai A, Ruiz-Perez V, Carter S, Jacobsen N, Burge S, Monk S, et al. Mutations in ATP2A2, encoding a Ca2+ pump, cause Darier disease. Nat Genet 1999;21(3):271–7. [DOI] [PubMed] [Google Scholar]
  45. Savignac M, Simon M, Edir A, Guibbal L, Hovnanian A. SERCA2 dysfunction in Darier disease causes endoplasmic reticulum stress and impaired cell-to-cell adhesion strength: rescue by Miglustat. J Invest Dermatol 2014;134(7):1961–70. [DOI] [PubMed] [Google Scholar]
  46. Schell SL, Cong Z, Sennett ML, Gettle SL, Longenecker AL, Goldberg SR, et al. Keratinocytes and immune cells in the epidermis are key drivers of inflammation in hidradenitis suppurativa providing a rationale for novel topical therapies. Br J Dermatol 2023;188(3):407–19. [DOI] [PubMed] [Google Scholar]
  47. Schwarz DS, Blower MD. The endoplasmic reticulum: structure, function and response to cellular signaling. Cell Mol Life Sci 2016;73(1):79–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sharma P, Mao X, Payne AS. Beyond steric hindrance: the role of adhesion signaling pathways in the pathogenesis of pemphigus. J Dermatol Sci 2007;48(1):1–14. [DOI] [PubMed] [Google Scholar]
  49. Song Y, Zhao Z, Xu L, Huang P, Gao J, Li J, et al. Using an ER-specific optogenetic mechanostimulator to understand the mechanosensitivity of the endoplasmic reticulum. Dev Cell 2024;59(11):1396–409.e5. [DOI] [PubMed] [Google Scholar]
  50. Spindler V, Gerull B, Green KJ, Kowalczyk AP, Leube R, Marian AJ, et al. Meeting report - Desmosome dysfunction and disease: Alpine desmosome disease meeting. J Cell Sci 2023;136(1). [DOI] [PubMed] [Google Scholar]
  51. Spindler V, Waschke J. Desmosomal Cadherins and Signaling: Lessons from Autoimmune Disease. Cell Communication & Adhesion 2014;21(1):77–84. [DOI] [PubMed] [Google Scholar]
  52. Stahley SN, Kowalczyk AP. Desmosomes in acquired disease. Cell Tissue Res 2015;360(3):439–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stahley SN, Warren MF, Feldman RJ, Swerlick RA, Mattheyses AL, Kowalczyk AP. Super-Resolution Microscopy Reveals Altered Desmosomal Protein Organization in Tissue from Patients with Pemphigus Vulgaris. Journal of Investigative Dermatology 2016;136(1):59–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sugiura K. Unfolded protein response in keratinocytes: impact on normal and abnormal keratinization. J Dermatol Sci 2013;69(3):181–6. [DOI] [PubMed] [Google Scholar]
  55. Takayanagi T, Kawai T, Forrester SJ, Obama T, Tsuji T, Fukuda Y, et al. Role of epidermal growth factor receptor and endoplasmic reticulum stress in vascular remodeling induced by angiotensin II. Hypertension 2015;65(6):1349–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Toivola DM, Polari L, Schwerd T, Schlegel N, Strnad P. The keratin-desmosome scaffold of internal epithelia in health and disease - The plot is thickening. Curr Opin Cell Biol 2024;86:102282. [DOI] [PubMed] [Google Scholar]
  57. Ujiie H, Yamagami J, Takahashi H, Izumi K, Iwata H, Wang G, et al. The pathogeneses of pemphigus and pemphigoid diseases. J Dermatol Sci 2021;104(3):154–63. [DOI] [PubMed] [Google Scholar]
  58. Voeltz GK, Sawyer EM, Hajnóczky G, Prinz WA. Making the connection: How membrane contact sites have changed our view of organelle biology. Cell 2024;187(2):257–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Waschke J. The desmosome and pemphigus. Histochemistry and Cell Biology 2008;130(1):21–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yang G, Yang W, Wu L, Wang R. H<sub>2</sub>S, Endoplasmic Reticulum Stress, and Apoptosis of Insulin-secreting Beta Cells *. Journal of Biological Chemistry 2007;282(22):16567–76. [DOI] [PubMed] [Google Scholar]
  61. Zimmer SE, Kowalczyk AP. The desmosome as a dynamic membrane domain. Curr Opin Cell Biol 2024;90:102403. [DOI] [PubMed] [Google Scholar]

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Data Availability Statement

Datasets related to this article are hosted on ScholarSphere (https://scholarsphere.psu.edu/resources/d8faef77-b085-44db-8610-5c5a7448070e), an open-source online data repository. Supplementary Videos can be found at: https://youtube.com/playlist?list=PLRVCHiGokrM6ujJV1Twn-UREtg-U0E_RH&si=qq14sS0Xmufnf-NZ. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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