Desmosomes Research Articles

Development and regeneration dynamics of the Medaka notochord

The notochord is an embryonic tissue that acts as a hydrostatic skeleton until ossification begins in vertebrates. It is composed of outer sheath cells and inner vacuolated cells, which are generated from a common pool of disc-shaped precursors. Notochord extension during early embryogenesis is driven by the growth of vacuolated cells, reflecting in turn the expansion of their inner vacuole. Here we use desmogon, a novel desmosomal cadherin, to follow notochord development and regeneration in medaka (Oryzias latipes). We trace desmogon ​+ disc-shaped precursors at the single cell level to demonstrate that they operate as unipotent progenitors, giving rise to either sheath or vacuolated cells. We reveal that once specified, vacuolated cells grow asynchronously and drive notochord expansion bi-directionally. Additionally, we uncover distinct regenerative responses in the notochord, which depend on the nature of the injury sustained. By generating a desmogon CRISPR mutant we demonstrate that this cadherin is essential for proper vacuolated cell shape and therefore correct notochord and spine morphology. Our work expands the repertoire of model systems to study dynamic aspects of the notochord in vivo, and provides new insights in its development and regeneration properties.

Epidermal structure and differentiation.

In this Primer, Moreci and Lechler follow the lifetime of an epidermal cell from its birth to its ultimate death, and detail how this journey is necessary for epidermal function.

Cadherin Organization in Desmosomes Probed using Fluorescence Polarization Microscopy

Desmosomes are intercellular junctions found in epithelia and cardiac muscle that resist mechanical stress by linking the intermediate filaments of neighboring cells. Disruption of the desmosome-intermediate filament complex (DIFC) can cause a loss of tissue integrity as well as abnormalities in tissue differentiation, leading to a variety of diseases. Understanding these diseases will require expanding our knowledge of the relationship between structure and function in desmosomes. Of particular interest is the arrangement, or order, of the desmosomal cadherins. Cadherins are calcium-dependent, transmembrane adhesion proteins. In a single desmosome, many copies of these proteins engage in trans-binding with the cadherins of neighboring cells to form the adhesive interface. Large macromolecular complexes and membrane-associated proteins are difficult to study using traditional structural techniques. To overcome this, we pioneered excitation-resolved fluorescence polarization microscopy (FPM) to study the organization of desmosomes. After placing fluorescent tags in proteins of interest, FPM allows for the quantification of their order within a macromolecular assembly. Unlike methods that require sample fixation, FPM can be performed on living cells, providing insights into DIFC organization under physiologically relevant conditions, while preserving dynamic information. Through this methodology, our lab has shown that the most membrane-proximal extracellular domain of desmoglein 3 is ordered in desmosomes and that this order is rapidly lost after calcium removal. Here, we show that this behavior is conserved in another cadherin isoform, desmolgein 2. We also designed constructs to measure the order of the cadherin cytoplasmic domains, allowing correlation of order across the membrane. Finally, we use FPM to examine the effect of mutations that abrogate binding between intracellular DIFC components. This approach provides a unique tool to study desmosomal architecture and elucidate the roles of specific protein-protein interactions as they relate to desmosome structure and function.

Desmosomes Pattern Cell Mechanics to Govern Epidermal Tissue Form and Function

The epidermis is a stratified epithelium in which structural and functional features are polarized across multiple cell layers. This type of polarity is essential for establishing the epidermal barrier, but how it is created and sustained is poorly understood. Previous work identified a role for the classical cadherin/filamentous-actin network in establishment of epidermal polarity. However, little is known about potential roles of the most prominent epidermal intercellular junction, the desmosome, in establishing epidermal polarity, in spite of the fact that desmosome constituents are patterned across the apical to basal cell layers. Here, we show that desmosomes and their associated intermediate filaments (IF) are key regulators of mechanical polarization in epidermis, whereby basal and suprabasal cells experience different forces that drive layer-specific functions. Uncoupling desmosomes and IF or specific targeting of apical desmosomes through depletion of the superficial desmosomal cadherin, desmoglein 1, impedes basal (stratification) and suprabasal (tight junction barrier) functions. Surprisingly, disengaging desmosomes from IF also uncouples stratification from differentiation, accelerating the expression of differentiation markers. Our data support a model in which the desmosome-IF network supports a reciprocally organized distribution of ErbB1/EGFR activity in the basal layer and mechanosensitive kinase ErbB2 activity in the suprabasal layer to ensure the proper spatiotemporal coordination of cell mechanics and the biochemical program of differentiation.

The role of desmosomes in the ear plug formation in the bowhead whale (Balaena mysticetus).

The external acoustic meatus (EAM) of most baleen whales accumulates cellular debris annually in the lumen as whales age, forming a lamellated ear plug. The bowhead whale ear plug is formed from annually molting lining of the EAM as the entire epithelium releases at the level of the stratum basale during the spring migration. Epithelial regeneration is mostly completed by the fall migration, remaining intact for 6-7 months before being torn off the following spring. Desmosomes are integral to cell-cell adhesion with connecting desmosomal cadherins desmoglein (dsg) and desmocollin (dsc). Paraffin sections of the oral cavity and EAM lining of spring and fall adult bowhead whales, as well as the EAM of spring-caught juvenile, were immunohistochemically examined for the presence of these cadherins. In all fall specimens, both cadherins occurred in all layers except the superficial keratinous layer of the oral cavity. In spring, three different conditions existed: (a) oral cavity of spring-caught adults had reduced cadherins, with superficial fissuring in its keratinized layer and vacuolation in the upper stratum spinosum; (b) EAM of juvenile spring-caught whales displayed fissuring with accompanying reduction of both cadherins in its superficial lining; and (c) EAM lining of spring-caught adults displayed deep fissures, reduced cadherins, and absence of dsc1 in the fissuring zone. These results suggest that shedding of skin layers in mammals, whether normal molting, pathological, or the result of injury and wound repair all revolve around desmosome function. The specific role, structure, and location of these two cadherins need to be further addressed.

Desmoglein-2 mutations in propeptide cleavage-site causes arrhythmogenic right ventricular cardiomyopathy/dysplasia by impairing extracellular 1-dependent desmosomal interactions upon cellular stress

Desmoglein-2 (DSG2) mutations, which encode a heart-specific cadherin crucial for desmosomal adhesion, are frequent in arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D). DSG2 mutations have been associated with higher risk of biventricular involvement. Among DSG2 mutations, mutations of the inhibitory propeptide consensus cleavage-site (Arg-X-Arg/Lys-Arg), are particularly frequent. In the present work, we explored the functional consequences of DSG2 propeptide cleavage site mutations p.Arg49His, p.Arg46Trp, and p.Arg46Gln on localization, adhesive properties, and desmosome incorporation of DSG2. We studied the expression of mutant-DSG2 in human heart and in epithelial and cardiac cellular models expressing wild-type or mutant (p.Arg49His, p.Arg46Trp, and p.Arg46Gln) proDSG2-GFP fusion proteins. The consequences of the p.Arg46Trp mutation on DSG2 adhesiveness were studied by surface plasmon resonance. Incorporation of mutant p.Arg46Trp DSG2 into desmosomes was studied under low-calcium culture conditions and cyclic mechanical stress. We demonstrated in human heart and cellular models that all three mutations prevented N-terminal propeptide cleavage, but did not modify intercellular junction targeting. Surface plasmon resonance experiments showed a propeptide-dependent loss of interaction between the cadherin N-terminal extracellular 1 (EC1) domains. Additionally, proDSG2 mutant proteins were abnormally incorporated into desmosomes under low-calcium culture conditions or following mechanical stress. This was accompanied by an epidermal growth factor receptor-dependent internalization of proDSG2, suggesting increased turnover of unprocessed proDSG2. Our results strongly suggest weakened desmosomal adhesiveness due to abnormal incorporation of uncleaved mutant proDSG2 in cellular stress conditions. These results provide new insights into desmosomal cadherin regulation and ARVC/D pathophysiology, in particular, the potential role of mechanical stress on desmosomal dysfunction.

Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces.

Desmoglein 3 (Dsg3) plays a crucial role in cell-cell adhesion and tissue integrity. Increasing evidence suggests that Dsg3 acts as a regulator of cellular mechanotransduction, but little is known about its direct role in mechanical force transmission. The present study investigated the impact of cyclic strain and substrate stiffness on Dsg3 expression and its role in mechanotransduction in keratinocytes. A direct comparison was made with E-cadherin, a well-characterized mechanosensor. Exposure of oral and skin keratinocytes to equiaxial cyclic strain promoted changes in the expression and localization of junction assembly proteins. The knockdown of Dsg3 by siRNA blocked strain-induced junctional remodeling of E-cadherin and Myosin IIa. Importantly, the study demonstrated that Dsg3 regulates the expression and localization of yes-associated protein (YAP), a mechanosensory, and an effector of the Hippo pathway. Furthermore, we showed that Dsg3 formed a complex with phospho-YAP and sequestered it to the plasma membrane, while Dsg3 depletion had an impact on both YAP and phospho-YAP in their response to mechanical forces, increasing the sensitivity of keratinocytes to the strain or substrate rigidity-induced nuclear relocation of YAP and phospho-YAP. Plakophilin 1 (PKP1) seemed to be crucial in recruiting the complex containing Dsg3/phospho-YAP to the cell surface since its silencing affected Dsg3 junctional assembly with concomitant loss of phospho-YAP at the cell periphery. Finally, we demonstrated that this Dsg3/YAP pathway has an influence on the expression of YAP1 target genes and cell proliferation. Together, these findings provide evidence of a novel role for Dsg3 in keratinocyte mechanotransduction.

Complement Receptor 1 (CR1, CD35) Polymorphisms and Soluble CR1: A Proposed Anti-inflammatory Role to Quench the Fire of "Fogo Selvagem" Pemphigus Foliaceus.

Pemphigus foliaceus is an autoimmune disease that is sporadic around the world but endemic in Brazil, where it is known as fogo selvagem (FS). Characterized by autoantibodies against the desmosomal cadherin desmoglein 1, FS causes painful erosions, and crusts that may be widespread. The recognition of antigens, including exposed sugar moieties, activates the complement system. Complement receptor 1 (CR1, CD35), which is responsible for the Knops blood group on erythrocytes (York and McCoy antigens), is also expressed by antigen-presenting cells. This regulates the complement system by removing opsonized antigens, blocking the final steps of the complement cascade. Membrane-bound CR1 also fosters antigen presentation to B cells, whereas soluble CR1 has anti-inflammatory properties. CR1 gene polymorphisms have been associated with susceptibility to complex diseases. In order to investigate the association of CR1 polymorphisms with FS susceptibility, we developed a multiplex sequence-specific assay to haplotype eleven polymorphisms in up to 367 FS patients and 242 controls from an endemic area and 289 from a non-endemic area. We also measured soluble CR1 (sCR1) in the serum of 53 FS patients and 27 controls and mRNA levels in the peripheral blood mononuclear cells of 63 genotyped controls. The haplotypes CR1*3B2B (with the York antigen–encoded by p.1408Met) and CR1*3A2A (with p.1208Arg) were associated with protection against FS (OR = 0.57, P = 0.027, and OR = 0.46, P = 0.014, respectively). In contrast, the CR1*1 haplotype (with the McCoy antigen – encoded by p.1590Glu) was associated with FS susceptibility (OR = 4.97, P < 0.001). Heterozygote rs12034383*A/G individuals presented higher mRNA expression than homozygotes with the G allele (P = 0.04). The lowest sCR1 levels occurred in patients with active disease before treatment (P = 0.036). Patients in remission had higher levels of sCR1 than did healthy controls (P = 0.013). Among those under treatment, patients with localized lesions also presented higher sCR1 levels than those with generalized lesions (P = 0.0073). In conclusion, the Knops blood group seems to modulate susceptibility to the disease. Furthermore, corticosteroid treatment might increase sCR1 serum levels, and higher levels may play an anti-inflammatory role in patients with FS, limiting the distribution of lesions. Based on these results, we suggest CR1 as a potential new therapeutic target for the treatment of FS.

Endocytosis of IgG, Desmoglein 1, and Plakoglobin in Pemphigus Foliaceus Patient Skin.

Pemphigus foliaceus (PF) is one of the two main forms of pemphigus and is characterized by circulating IgG to the desmosomal cadherin desmoglein 1 (DSG1) and by subcorneal blistering of the skin. The pathomechanism of blister formation in PF is unknown. Previously we have shown that PF IgG induces aggregation of DSG1, plakoglobin (PG), and IgG outside of desmosomes, what in immunofluorescence of PF patient skin visualizes as a granular IgG deposition pattern with a limited number of coarse IgG aggregates between cells. Here we have investigated the fate of these aggregates in skin and found that these are cleared by endocytosis. We performed double immunofluorescence staining on snap-frozen skin biopsies of six PF patients for the following molecules: IgG, the desmosomal proteins DSG1 and DSG3, desmocollins 1 and 3, PG, desmoplakin and plakophilin 3, and for the endosomal marker early endosomal antigen 1 and the lysosomal markers cathepsin D and lysosomal-associated membrane protein 1. Endosomes were present in all cells but did not make contact with the aggregates in the basal and suprabasal layers. In the higher layers they moored to the aggregates, often symmetrically from two adjacent cells, and IgG, DSG1, and PG were taken up. Finally these endosomes became localized perinuclear. Endocytosis was only observed in perilesional or lesional skin but not in non-lesional skin. Older immunoelectron microscopic studies have suggested that in PF skin endocytosis of detached desmosomes takes place but we found no other desmosomal proteins to be present in these endosomes. Double staining with cathepsin D and LAMP-1 revealed no overlap with IgG, DSG1, or PG suggesting that lysosomes have no role in the clearing process. Collectively, our results show that endocytosis is part of the pathogenic process in PF but that no detached desmosomes are taken up but instead the deposited IgG is taken up together with DSG1 and PG.

The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell–cell adhesion and regulates various signaling pathways involved in the progression and metastasis of cancer where it is upregulated. We show here that expression of Dsg3 impacts on the expression and function of p53, a key transcription factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect enhanced by treating cells with UVB, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 was Dsg3 specific since neither E-cadherin nor desmoplakin knockdown caused similar effects. Analysis of Dsg3−/− mouse skin also indicated an increase of p53/p21WAF1/CIP1 and cleaved caspase-3 relative to Dsg3+/− controls. Finally, we evaluated whether this pathway was operational in the autoimmune disease PV in which Dsg3 serves as a major antigen involved in blistering pathogenesis. We uncovered increased p53 with diffuse cytoplasmic and/or nuclear staining in the oral mucosa of patients, including cells surrounding blisters and the pre-lesional regions. This finding was verified by in vitro studies where treatment of keratinocytes with PV sera, as well as a characterized pathogenic antibody specifically targeting Dsg3, evoked pronounced p53 expression and activity accompanied by disruption of cell–cell adhesion. Collectively, our findings suggest a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, and this pathway is disrupted in PV.

239 Desmosomes pattern cell mechanics to govern epidermal tissue form and function

The epidermis exhibits polarization of structural and functional features across multiple cell layers, but how this polarity is created and preserved is poorly understood. Desmosomes (DSMs) are cell-cell adhesive organelles linking to intermediate filaments (IF) and are critical for signaling and tissue integrity. The DSM cadherin desmoglein 1 (Dsg1) is first expressed as cells commit to differentiate and stratify, with expression peaking in the granular layer (SG). We hypothesize the pattern of Dsg1 and associated IF is crucial for establishing proper epidermal polarity, especially mechanical polarity. To test this, we used laser ablation and whole mount imaging to assess layer-specific mechanical properties of 3D epidermal cultures. These data support an epidermal mechanical gradient: keratinocytes are under compressive forces in the basal layer and high tension in the suprabasal layers. We previously showed Dsg1 regulates basal cell forces to promote stratification. Here, a DSM/IF uncoupling mutant recapitulates Dsg1 loss, suggesting a requirement of IF attachment. High apical tension in simple epithelia helps generate barrier-critical tight junctions (TJs). TJs localize to the SG in epidermis, where tension is high and Dsg1 expression peaks. Thus, we assessed the ability of Dsg1 to polarize tension and TJs in the SG. Dsg1 loss disrupts the polarized SG distribution of the tension sensitive TJ component ZO1. Our data suggest Dsg1 modulates TJs through the mechanosensitive kinase ErbB2, which localizes to the TJ forming layer. Dsg1 loss disrupts this localization and decreases total and phosphorylated ErbB2. Uncoupling the DSM/IF linkage or inhibiting ErbB2 significantly decreases epidermal electrical barrier function. As mechanical forces regulate cell behavior, our data suggest a model in which patterning of DSM components regulates epidermal polarity by orchestrating the distribution of mechanical inputs to drive morphogenesis and development of a life-essential barrier.

Integrative prognostic subtype discovery in high-grade serous ovarian cancer.

We sought to identify novel molecular subtypes of high-grade serous ovarian cancer (HGSC) by the integration of gene expression and proteomics data and to find the underlying biological characteristics of ovarian cancer to improve the clinical outcome. The iCluster method was utilized to analysis 131 common HGSC samples between TCGA and Clinical Proteomic Tumor Analysis Consortium databases. Kaplan-Meier survival curves were used to estimate the overall survival of patients, and the differences in survival curves were assessed using the log-rank test. Two novel ovarian cancer subtypes with different overall survival (P = .00114) and different platinum status (P = .0061) were identified. Eighteen messenger RNAs and 38 proteins were selected as differential molecules between subtypes. Pathway analysis demonstrated arrhythmogenic right ventricular cardiomyopathy pathway played a critical role in the discrimination of these two subtypes and desmosomal cadherin DSG2, DSP, JUP, and PKP2 in this pathway were overexpression in subtype I compared with subtype II. Our study extended the underlying prognosis-related biological characteristics of high-grade serous ovarian cancer. Enrichment of desmosomal cadherin increased the risk for HGSC prognosis among platinum-sensitive patients, the results guided the revision of the treatment options for platinum-sensitive ovarian cancer patients to improve outcomes.

Immortalized Human hTert/KER-CT Keratinocytes a Model System for Research on Desmosomal Adhesion and Pathogenesis of Pemphigus Vulgaris.

Pemphigus Vulgaris is an autoimmune disease that results in blister formation in the epidermis and in mucosal tissues due to antibodies recognizing desmosomal cadherins, mainly desmoglein-3 and -1. Studies on the molecular mechanisms of Pemphigus have mainly been carried out using the spontaneously immortalized human keratinocyte cell line HaCaT or in primary keratinocytes. However, both cell systems have suboptimal features, with HaCaT cells exhibiting a large number of chromosomal aberrations and mutated p53 tumor suppressor, whereas primary keratinocytes are short-lived, heterogeneous and not susceptible to genetic modifications due to their restricted life-span. We have here tested the suitability of the commercially available human keratinocyte cell line hTert/KER-CT as a model system for research on epidermal cell adhesion and Pemphigus pathomechanisms. We here show that hTert cells exhibit a calcium dependent expression of desmosomal cadherins and are well suitable for typical assays used for studies on Pemphigus, such as sequential detergent extraction and Dispase-based dissociation assay. Treatment with Pemphigus auto-antibodies results in loss of monolayer integrity and altered localization of desmoglein-3, as well as loss of colocalization with flotillin-2. Our findings demonstrate that hTert cells are well suitable for studies on epidermal cell adhesion and Pemphigus pathomechanisms.

Depletion of the cellular cholesterol content reduces the dynamics of desmosomal cadherins and interferes with desmosomal strength.

Desmosomal cadherins, desmocollins, and desmogleins are cholesterol-dependent entities responsible for the stable adhesion of desmosomes in epithelial cells. Here, we investigated the influence of cellular cholesterol depletion on the dynamic properties of the desmosomal cadherin desmocollin, particularly the lateral mobility and distribution of desmocollin 2 (Dsc2-YFP) in the plasma membrane, and how these properties influence the adhesion strength of desmosomes. Depletion of cellular cholesterol decreased the lateral mobility of Dsc2-YFP and caused dispersion of Dsc2-YFP in the plasma membrane of epithelial MDCK cells. As a consequence of the altered Dsc2-YFP dynamics, the adhesive strength of desmosomes was weakened. Moreover, our study is the first to show and quantify the co-association of desmosomes with cholesterol/sphingomyelin-enriched membrane domains at the ultrastructural level. Taken together, our data emphasize a critical role for the cellular cholesterol content in regulating the lateral mobility and distribution of Dsc2 and show that cholesterol depletion reduces the strength of desmosomal adhesions.

Paraneoplastic Pemphigus: Paraneoplastic Autoimmune Disease of the Skin and Mucosa

Paraneoplastic pemphigus (PNP) is a rare but life-threatening mucocutaneous disease mediated by paraneoplastic autoimmunity. Various neoplasms are associated with PNP. Intractable stomatitis and polymorphous cutaneous eruptions, including blisters and lichenoid dermatitis, are characteristic clinical features caused by humoral and cell-mediated autoimmune reactions. Autoreactive T cells and IgG autoantibodies against heterogeneous antigens, including plakin family proteins and desmosomal cadherins, contribute to the pathogenesis of PNP. Several mechanisms of autoimmunity may be at play in this disease on the type of neoplasm present. Diagnosis can be made based on clinical and histopathological features, the presence of anti-plakin autoantibodies, and underlying neoplasms. Immunosuppressive agents and biologics including rituximab have been used for the treatment of PNP; however, the prognosis is poor due to underlying malignancies, severe infections during immunosuppressive treatment, and bronchiolitis obliterans mediated by autoimmunity. In this review, we overview the characteristics of PNP and focus on the immunopathology and the potential pathomechanisms of this disease.

E-Cadherin Is Important for Meibomian Gland Function as Revealed by a New Human ex Vivo Slice Culture Model

Meibomian glands within the eyelid are important for the maintenance of the integrity and health of the ocular surface. Patients with the blistering skin disease pemphigus vulgaris (PV), which is caused by autoantibodies against desmosomal cadherins, often have dry eye disease. Therefore, we studied the regulation of cell cohesion in human meibomian gland epithelial cells (HMGECs). During serum-induced differentiation for 1 to 6 days, HMGECs drastically enhanced intercellular cohesion, whereas lipid production did not change. The expression profiles of the desmosomal PV antigens desmoglein (Dsg) 3and 1 but not of the adherens junction component E-cadherin (Ecad) was dependent on the presence of serum. Surprisingly, after 1 day but not after 6 days of serum-induced differentiation, an inhibitory antibody against Ecad drastically reduced intercellular cohesion and blocked lipid production ofHMGECs. In contrast, antibodies against desmosomal cadherins, including human and mouse pemphigus autoantibodies, had no effect on monolayer integrity and lipid production. Because lipid production was unaltered in meibomian glands from Dsg3-deficient mice, we established an exvivo slice culture model of human eyelids to allow studies in a more physiologic environment. Here, the inhibitory antibody against Ecad but not a Dsg3-specific PV antibody interfered with stimulated lipid production. Together, these data demonstrate that cell cohesion is maintained differently in meibomian glandcellsand indicate that Ecad is important for meibomian gland function.

Desmoglein 1 Regulates Invadopodia by Suppressing EGFR/Erk Signaling in an Erbin-Dependent Manner.

Loss of the desmosomal cell-cell adhesion molecule, Desmoglein 1 (Dsg1), has been reported as an indicator of poor prognosis in head and neck squamous cell carcinomas (HNSCC) overexpressing epidermal growth factor receptor (EGFR). It has been well established that EGFR signaling promotes the formation of invadopodia, actin-based protrusions formed by cancer cells to facilitate invasion and metastasis, by activating pathways leading to actin polymerization and ultimately matrix degradation. We previously showed that Dsg1 downregulates EGFR/Erk signaling by interacting with the ErbB2-binding protein Erbin (ErbB2 Interacting Protein) to promote keratinocyte differentiation. Here, we provide evidence that restoring Dsg1 expression in cells derived from HNSCC suppresses invasion by decreasing the number of invadopodia and matrix degradation. Moreover, Dsg1 requires Erbin to downregulate EGFR/Erk signaling and to fully suppress invadopodia formation. Our findings indicate a novel role for Dsg1 in the regulation of invadopodia signaling and provide potential new targets for development of therapies to prevent invadopodia formation and therefore cancer invasion and metastasis. IMPLICATIONS: Our work exposes a new pathway by which a desmosomal cadherin called Dsg1, which is lost early in head and neck cancer progression, suppresses cancer cell invadopodia formation by scaffolding ErbB2 Interacting Protein and consequent attenuation of EGF/Erk signaling.

433 Pemphigus vulgaris patient disease severity correlates with in vitro assessments of desmosomal function

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease characterized by severe epidermal blistering and mucous membrane erosions. PV is caused by antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3), and in cases involving the skin, both Dsg3 and Dsg1. PV can be assessed clinically using the Pemphigus Disease Area Index (PDAI) and using an ELISA for Dsg IgG titers in patient sera. In vitro, PV IgG activity can be investigated by incubating normal human keratinocytes with PV patient IgG and monitoring changes in desmosomal protein organization and overall strength of cell-cell adhesion using a dispase-based cell-cell dissociation assay. However, the relationship between clinical PV assessments and in vitro activity of patient IgG have not been reported. In the present study, we compared the relationship between the in vitro pathogenicity of PV IgG from 23 patients to their PDAI score and ELISA titers. Overall, Dsg1 ELISA values showed a stronger correlation with PDAI scores (p<0.01) when compared to Dsg3 ELISA (p<0.02). However, the sum of Dsg3 and Dsg1 ELISA values exhibited a highly significant correlation to PDAI (p<0.0005). In vitro, treatment of keratinocytes with patient IgG resulted in a range of alterations in desmosomal protein organization that included Dsg3 clustering, the formation of linear array-like structures, and endocytosis of Dsg3-PV IgG complexes. Interestingly, the loss of cell-cell adhesion strength as assessed using in vitro dispase cell dissociation assays exhibited a positive correlation with Dsg3 ELISA titers (p<0.005), supporting the validity of the dispase assay as a measure of PV pathogenicity . These findings confirm the association of Dsg ELISA values with PDAI and establish a relationship between Dsg IgG titers in patients and loss of adhesion in cultured keratinocytes.

Plakophilin 1 but not plakophilin 3 regulates desmoglein clustering.

Plakophilins (Pkp) are desmosomal plaque proteins crucial for desmosomal adhesion and participate in the regulation of desmosomal turnover and signaling. However, direct evidence that Pkps regulate clustering and molecular binding properties of desmosomal cadherins is missing. Here, keratinocytes lacking either Pkp1 or 3 in comparison to wild type (wt) keratinocytes were characterized with regard to their desmoglein (Dsg) 1- and 3-binding properties and their capability to induce Dsg3 clustering. As revealed by atomic force microscopy (AFM), both Pkp-deficient keratinocyte cell lines showed reduced membrane availability and binding frequency of Dsg1 and 3 at cell borders. Extracellular crosslinking and AFM cluster mapping demonstrated that Pkp1 but not Pkp3 is required for Dsg3 clustering. Accordingly, Dsg3 overexpression reconstituted cluster formation in Pkp3- but not Pkp1-deficient keratinocytes as shown by AFM and STED experiments. Taken together, these data demonstrate that both Pkp1 and 3 regulate Dsg membrane availability, whereas Pkp1 but not Pkp3 is required for Dsg3 clustering.

The MAL/SRF Pathway Regulates Desmosome Protein Expression and Localization in Cancer Cells

The desmosome (DSM) is a cell‐cell adhesion complex required for the mechanical stability of tissues and regulation of other biological processes, such as proliferation and migration. Proteins from three families make up the complex: Transmembrane cadherins connect adjacent cells, plaque proteins stabilize attachment on the intracellular face, and the plakin protein desmoplakin (DSP) anchors the DSM complex to the intermediate filament cytoskeleton. Many studies have reported diverse mechanisms by which cancer progression alters DSM gene expression, but the signaling pathways that govern the expression and localization of DSM constituent proteins remain elusive. Recent work has identified the transcription factor serum response factor (SRF) as a regulator of mRNA levels and localization of DSM proteins such as the cadherin Desmoglein‐1. Here, we treated several cancer cell lines with siRNA and a pharmacological inhibitor (CCG‐1423) of SRF and its cofactor, MAL, to broadly investigate the role of these transcription factors in regulating DSM gene expression and protein localization. Our results demonstrate that abrogation of MAL/SRF signaling through both siRNA and pharmacological inhibition results in a decrease in mRNA levels of DSP. In addition, localization of DSP to borders is also reduced upon inhibition of MAL/SRF signaling. We observed similar changes for the plaque proteins Plakophilin‐2 and Plakoglobin, but not for desmosomal cadherins. As DSP is required to maintain the strength of DSM attachments, we also investigated the role of MAL/SRF signaling on the adhesion strength between cells. Our data highlight a novel link between MAL/SRF signaling and DSP expression, and contribute to a growing understanding of the variety of signaling pathways involved in mediating DSM gene expression.Support or Funding InformationThis project was funded by an NIH South Carolina IDeA Networks of Biomedical Research Excellence (SC INBRE) grant #5P20GM103499‐17 which provided a Developmental Research Project (DRP) sub‐award #22050‐ZA14 to AD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.