Acral peeling in Nagashima type palmo-plantar keratosis patients reveals the role of serine protease inhibitor B 7 in keratinocyte adhesion.

Acral peeling skin syndrome (APSS) is a heterogenous group of genodermatoses, manifested by peeling of palmo-plantar skin and occasionally associated with erythema and epidermal thickening. A subset of APSS is caused by mutations in protease inhibitor encoding genes, resulting in unopposed protease activity and desmosomal degradation and/or mis-localization, leading to enhanced epidermal desquamation. We investigated two Arab-Muslim siblings with mild keratoderma and prominent APSS since infancy. Genetic analysis disclosed a homozygous mutation in SERPINB7, c.796C > T, which is the founder mutation in Nagashima type palmo-plantar keratosis (NPPK). Although not previously formally reported, APSS was found in other patients with NPPK. We hypothesized that loss of SERPINB7 function might contribute to the peeling phenotype through impairment of keratinocyte adhesion, similar to other protease inhibitor mutations that cause APSS. Mis-localization of desmosomal components was observed in a patient plantar biopsy compared with a biopsy from an age- and gender-matched healthy control. Silencing of SERPINB7 in normal human epidermal keratinocytes led to increased cell sheet fragmentation upon mechanical stress. Immunostaining showed reduced expression of desmoglein 1 and desmocollin 1. This study shows that in addition to stratum corneum perturbation, loss of SERPINB7 disrupts desmosomal components, which could lead to desquamation, manifested by skin peeling.

The Role of Desmoglein 1 in Gap Junction Turnover Revealed through the Study of SAM Syndrome.

An effective epidermal barrier requires structural and functional integration of adherens junctions, tight junctions, gap junctions (GJ), and desmosomes. Desmosomes govern epidermal integrity while GJs facilitate small molecule transfer across cell membranes. Some patients with severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome, caused by biallelic desmoglein 1 (DSG1) mutations, exhibit skin lesions reminiscent of erythrokeratodermia variabilis, caused by mutations in connexin (Cx) genes. We, therefore, examined whether SAM syndrome-causing DSG1 mutations interfere with Cx expression and GJ function. Lesional skin biopsies from SAM syndrome patients (n = 7) revealed decreased Dsg1 and Cx43 plasma membrane localization compared with control and nonlesional skin. Cultured keratinocytes and organotypic skin equivalents depleted of Dsg1 exhibited reduced Cx43 expression, rescued upon re-introduction of wild-type Dsg1, but not Dsg1 constructs modeling SAM syndrome-causing mutations. Ectopic Dsg1 expression increased cell-cell dye transfer, which Cx43 silencing inhibited, suggesting that Dsg1 promotes GJ function through Cx43. As GJA1 gene expression was not decreased upon Dsg1 loss, we hypothesized that Cx43 reduction was due to enhanced protein degradation. Supporting this, PKC-dependent Cx43 S368 phosphorylation, which signals Cx43 turnover, increased after Dsg1 depletion, while lysosomal inhibition restored Cx43 levels. These data reveal a role for Dsg1 in regulating epidermal Cx43 turnover.

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.

Filaggrin 2 Deficiency Results in Abnormal Cell-Cell Adhesion in the Cornified Cell Layers and Causes Peeling Skin Syndrome Type A.

Peeling skin syndromes form a large and heterogeneous group of inherited disorders characterized by superficial detachment of the epidermal cornified cell layers, often associated with inflammatory features. Here we report on a consanguineous family featuring noninflammatory peeling of the skin exacerbated by exposure to heat and mechanical stress. Whole exome sequencing revealed a homozygous nonsense mutation in FLG2, encoding filaggrin 2, which cosegregated with the disease phenotype in the family. The mutation was found to result in decreased FLG2 RNA levels as well as almost total absence of filaggrin 2 in the patient epidermis. Filaggrin 2 was found to be expressed throughout the cornified cell layers and to colocalize with corneodesmosin that plays a crucial role in maintaining cell-cell adhesion in this region of the epidermis. The absence of filaggrin 2 in the patient skin was associated with markedly decreased corneodesmosin expression, which may contribute to the peeling phenotype displayed by the patients. Accordingly, using the dispase dissociation assay, we showed that FLG2 downregulation interferes with keratinocyte cell-cell adhesion. Of particular interest, this effect was aggravated by temperature elevation, consistent with the clinical phenotype. Restoration of corneodesmosin levels by ectopic expression rescued cell-cell adhesion. Taken together, the present data suggest that filaggrin 2 is essential for normal cell-cell adhesion in the cornified cell layers.

Desmosomal cadherin association with Tctex-1 and cortactin-Arp2/3 drives perijunctional actin polymerization to promote keratinocyte delamination.

The epidermis is a multi-layered epithelium that serves as a barrier against water loss and environmental insults. Its morphogenesis occurs through a tightly regulated program of biochemical and architectural changes during which basal cells commit to differentiate and move towards the skin's surface. Here, we reveal an unexpected role for the vertebrate cadherin desmoglein 1 (Dsg1) in remodeling the actin cytoskeleton to promote the transit of basal cells into the suprabasal layer through a process of delamination, one mechanism of epidermal stratification. Actin remodeling requires the interaction of Dsg1 with the dynein light chain, Tctex-1 and the actin scaffolding protein, cortactin. We demonstrate that Tctex-1 ensures the correct membrane compartmentalization of Dsg1-containing desmosomes, allowing cortactin/Arp2/3-dependent perijunctional actin polymerization and decreasing tension at E-cadherin junctions to promote keratinocyte delamination. Moreover, Dsg1 is sufficient to enable simple epithelial cells to exit a monolayer to form a second layer, highlighting its morphogenetic potential.

The desmoplakin-intermediate filament linkage regulates cell mechanics.

The translation of mechanical forces into biochemical signals plays a central role in guiding normal physiological processes during tissue development and homeostasis. Interfering with this process contributes to cardiovascular disease, cancer progression, and inherited disorders. The actin-based cytoskeleton and its associated adherens junctions are well-established contributors to mechanosensing and transduction machinery; however, the role of the desmosome-intermediate filament (DSM-IF) network is poorly understood in this context. Because a force balance among different cytoskeletal systems is important to maintain normal tissue function, knowing the relative contributions of these structurally integrated systems to cell mechanics is critical. Here we modulated the interaction between DSMs and IFs using mutant forms of desmoplakin, the protein bridging these structures. Using micropillar arrays and atomic force microscopy, we demonstrate that strengthening the DSM-IF interaction increases cell-substrate and cell-cell forces and cell stiffness both in cell pairs and sheets of cells. In contrast, disrupting the interaction leads to a decrease in these forces. These alterations in cell mechanics are abrogated when the actin cytoskeleton is dismantled. These data suggest that the tissue-specific variability in DSM-IF network composition provides an opportunity to differentially regulate tissue mechanics by balancing and tuning forces among cytoskeletal systems.

Severe dermatitis, multiple allergies, and metabolic wasting syndrome caused by a novel mutation in the N-terminal plakin domain of desmoplakin.

BACKGROUND: Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a recently recognized syndrome caused by mutations in the desmoglein 1 gene (DSG1). To date, only 3 families have been reported. OBJECTIVE: We studied a new case of SAM syndrome known to have no mutations in DSG1 to detail the clinical, histopathologic, immunofluorescent, and ultrastructural phenotype and to identify the underlying molecular mechanisms in this rare genodermatosis. METHODS: Histopathologic, electron microscopy, and immunofluorescent studies were performed. Whole-exome sequencing data were interrogated for mutations in desmosomal and other skin structural genes, followed by Sanger sequencing of candidate genes in the patient and his parents. RESULTS: No mutations were identified in DSG1; however, a novel de novo heterozygous missense c.1757A>C mutation in the desmoplakin gene (DSP) was identified in the patient, predicting the amino acid substitution p.His586Pro in the desmoplakin polypeptide. CONCLUSIONS: SAM syndrome can be caused by mutations in both DSG1 and DSP. Knowledge of this genetic heterogeneity is important for both analysis of patients and genetic counseling of families. This condition and these observations reinforce the importance of heritable skin barrier defects, in this case desmosomal proteins, in the pathogenesis of atopic disease.

Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

The pathways driving desmosome and adherens junction assembly are temporally and spatially coordinated, but how they are functionally coupled is poorly understood. Here we show that the Armadillo protein plakophilin 3 (Pkp3) mediates both desmosome assembly and E-cadherin maturation through Rap1 GTPase, thus functioning in a manner distinct from the closely related plakophilin 2 (Pkp2). Whereas Pkp2 and Pkp3 share the ability to mediate the initial phase of desmoplakin (DP) accumulation at sites of cell-cell contact, they play distinct roles in later steps: Pkp3 is required for assembly of a cytoplasmic population of DP-enriched junction precursors, whereas Pkp2 is required for transfer of the precursors to the membrane. Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly. We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing. These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

Plakophilin 2 affects cell migration by modulating focal adhesion dynamics and integrin protein expression.

Plakophilin 2 (PKP2), a desmosome component, modulates the activity and localization of the small GTPase RhoA at sites of cell-cell contact. PKP2 regulates cortical actin rearrangement during junction formation, and its loss is accompanied by an increase in actin stress fibers. We hypothesized that PKP2 may regulate focal adhesion dynamics and cell migration. Here we show that PKP2-deficient cells bind efficiently to the extracellular matrix, but upon spreading display total cell areas ≈ 30% smaller than control cells. Focal adhesions in PKP2-deficient cells are ≈ 2 × larger and more stable than in control cells, and vinculin displays an increased time for fluorescence recovery after photobleaching. Furthermore, ß4 and ß1 integrin protein and mRNA expression is elevated in PKP2-silenced cells. Normal focal adhesion phenotypes can be restored in PKP2-null cells by dampening the RhoA pathway or silencing ß1 integrin. However, integrin expression levels are not restored by RhoA signaling inhibition. These data uncover a potential role for PKP2 upstream of ß1 integrin and RhoA in integrating cell-cell and cell-substrate contact signaling in basal keratinocytes necessary for the morphogenesis, homeostasis, and reepithelialization of the stratified epidermis.

The C-terminal unique region of desmoglein 2 inhibits its internalization via tail-tail interactions.

Desmosomal cadherins, desmogleins (Dsgs) and desmocollins, make up the adhesive core of intercellular junctions called desmosomes. A critical determinant of epithelial adhesive strength is the level and organization of desmosomal cadherins on the cell surface. The Dsg subclass of desmosomal cadherins contains a C-terminal unique region (Dsg unique region [DUR]) with unknown function. In this paper, we show that the DUR of Dsg2 stabilized Dsg2 at the cell surface by inhibiting its internalization and promoted strong intercellular adhesion. DUR also facilitated Dsg tail-tail interactions. Forced dimerization of a Dsg2 tail lacking the DUR led to decreased internalization, supporting the conclusion that these two functions of the DUR are mechanistically linked. We also show that a Dsg2 mutant, V977fsX1006, identified in arrhythmogenic right ventricular cardiomyopathy patients, led to a loss of Dsg2 tail self-association and underwent rapid endocytosis in cardiac muscle cells. Our observations illustrate a new mechanism desmosomal cadherins use to control their surface levels, a key factor in determining their adhesion and signaling roles.

Plakophilin 2 couples actomyosin remodeling to desmosomal plaque assembly via RhoA.

Plakophilin 2 (PKP2), an armadillo family member closely related to p120 catenin (p120ctn), is a constituent of the intercellular adhesive junction, the desmosome. We previously showed that PKP2 loss prevents the incorporation of desmosome precursors enriched in the plaque protein desmoplakin (DP) into newly forming desmosomes, in part by disrupting PKC-dependent regulation of DP assembly competence. On the basis of the observation that DP incorporation into junctions is cytochalasin D-sensitive, here we ask whether PKP2 may also contribute to actin-dependent regulation of desmosome assembly. We demonstrate that PKP2 knockdown impairs cortical actin remodeling after cadherin ligation, without affecting p120ctn expression or localization. Our data suggest that these defects result from the failure of activated RhoA to localize at intercellular interfaces after cell-cell contact and an elevation of cellular RhoA, stress fibers, and other indicators of contractile signaling in squamous cell lines and atrial cardiomyocytes. Consistent with these observations, RhoA activation accelerated DP redistribution to desmosomes during the first hour of junction assembly, whereas sustained RhoA activity compromised desmosome plaque maturation. Together with our previous findings, these data suggest that PKP2 may functionally link RhoA- and PKC-dependent pathways to drive actin reorganization and regulate DP-IF interactions required for normal desmosome assembly.

Plakophilins: multifunctional scaffolds for adhesion and signaling.

Armadillo family proteins known as plakophilins have been characterized as structural components of desmosomes that stabilize and strengthen adhesion by enhancing attachments with the intermediate filament cytoskeleton. However, plakophilins and their close relatives are emerging as versatile scaffolds for multiple signaling and metabolic processes that not only facilitate junction dynamics but also more globally regulate diverse cellular activities. While perturbation of plakophilin functions contribute to inherited diseases and cancer pathogenesis, the functional significance of the multiple PKP isoforms and the mechanisms by which their behaviors are regulated remain to be elucidated.

Desmosomal dysfunction due to mutations in desmoplakin causes arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by progressive degeneration of the right ventricular myocardium, ventricular arrhythmias, fibrous-fatty replacement, and increased risk of sudden death. Mutations in 6 genes, including 4 encoding desmosomal proteins (Junctional plakoglobin (JUP), Desmoplakin (DSP), Plakophilin 2, and Desmoglein 2), have been identified in patients with ARVD/C. Mutation analysis of 66 probands identified 4 variants in DSP; V30M, Q90R, W233X, and R2834H. To establish a cause and effect relationship between those DSP missense mutations and ARVD/C, we performed in vitro and in vivo analyses of the mutated proteins. Unlike wild-type (WT) DSP, the N-terminal mutants (V30M and Q90R) failed to localize to the cell membrane in desomosome-forming cell line and failed to bind to and coimmunoprecipitate JUP. Multiple attempts to generate N-terminal DSP (V30M and Q90R) cardiac-specific transgenes have failed: analysis of embryos revealed evidence of profound ventricular dilation, which likely resulted in embryonic lethality. We were able to develop transgenic (Tg) mice with cardiac-restricted overexpression of the C-terminal mutant (R2834H) or WT DSP. Whereas mice overexpressing WT DSP had no detectable histologic, morphological, or functional cardiac changes, the R2834H-Tg mice had increased cardiomyocyte apoptosis, cardiac fibrosis, and lipid accumulation, along with ventricular enlargement and cardiac dysfunction in both ventricles. These mice also displayed interruption of DSP-desmin interaction at intercalated discs (IDs) and marked ultra-structural changes of IDs. These data suggest DSP expression in cardiomyocytes is crucial for maintaining cardiac tissue integrity, and DSP abnormalities result in ARVD/C by cardiomyocyte death, changes in lipid metabolism, and defects in cardiac development.

Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.

The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

Mechanisms of plakoglobin-dependent adhesion: desmosome-specific functions in assembly and regulation by epidermal growth factor receptor.

Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins that can be incorporated into both adherens junctions and desmosomes. Loss of PG results in defects in the mechanical integrity of heart and skin and decreased adhesive strength in keratinocyte cultures established from the skin of PG knock-out (PG-/-) mice, the latter of which cannot be compensated for by overexpressing the closely related beta-catenin. In this study, we examined the mechanisms of PG-regulated adhesion in murine keratinocytes. Biochemical and morphological analyses indicated that junctional incorporation of desmosomal, but not adherens junction, components was impaired in PG-/- cells compared with PG+/- controls. Re-expression of PG, but not beta-catenin, in PG-/- cells largely reversed these effects, indicating a key role for PG in desmosome assembly. Epidermal growth factor (EGF) receptor activation resulted in Tyr phosphorylation of PG, which was accompanied by a loss of desmoplakin from desmosomes and decreased adhesive strength following 18-h EGF treatment. Importantly, introduction of a phosphorylation-deficient PG mutant into PG null cells prevented the EGF receptor-dependent loss of desmoplakin from junctions, attenuating the effects of long term EGF treatment on cell adhesion. Therefore, PG is essential for maintaining and regulating adhesive strength in keratinocytes largely through its contributions to desmosome assembly and structure. As a target for modulation by EGF, regulation of PG-dependent adhesion may play an important role during wound healing and tumor metastasis.

Captopril prevents experimental autoimmune myocarditis.

Captopril, an angiotensin-converting enzyme inhibitor, is widely used in the treatment of a variety of cardiomyopathies, but its effect on autoimmune myocarditis has not been addressed experimentally. We investigated the effect of captopril on myosin-induced experimental autoimmune myocarditis. A/J mice, immunized with syngeneic cardiac myosin, were given 75 mg/L of captopril in their drinking water. Captopril dramatically reduced the incidence and severity of myocarditis, which was accompanied by a reduction in heart weight to body weight ratio and heart weight. Captopril specifically interfered with cell-mediated immunity as myosin delayed-type hypersensitivity (DTH) was reduced, while anti-myosin Ab production was not affected. Captopril-treated, OVA-immunized mice also exhibited a decrease in OVA DTH. In myosin-immunized, untreated mice, injection of captopril directly into the test site also suppressed myosin DTH. Interestingly, captopril did not directly affect Ag-specific T cell responsiveness because neither in vivo nor in vitro captopril treatment affected the proliferation, IFN-gamma secretion, or IL-2 secretion by Ag-stimulated cultured splenocytes. These results indicate that captopril ameliorates experimental autoimmune myocarditis and may act, at least in part, by interfering with the recruitment of cells to sites of inflammation and the local inflammatory environment.

Intermediate filament-membrane attachments function synergistically with actin-dependent contacts to regulate intercellular adhesive strength.

By tethering intermediate filaments (IFs) to sites of intercellular adhesion, desmosomes facilitate formation of a supercellular scaffold that imparts mechanical strength to a tissue. However, the role IF-membrane attachments play in strengthening adhesion has not been directly examined. To address this question, we generated Tet-On A431 cells inducibly expressing a desmoplakin (DP) mutant lacking the rod and IF-binding domains (DPNTP). DPNTP localized to the plasma membrane and led to dissociation of IFs from the junctional plaque, without altering total or cell surface distribution of adherens junction or desmosomal proteins. However, a specific decrease in the detergent-insoluble pool of desmoglein suggested a reduced association with the IF cytoskeleton. DPNTP-expressing cell aggregates in suspension or substrate-released cell sheets readily dissociated when subjected to mechanical stress whereas controls remained largely intact. Dissociation occurred without lactate dehydrogenase release, suggesting that loss of tissue integrity was due to reduced adhesion rather than increased cytolysis. JD-1 cells from a patient with a DP COOH-terminal truncation were also more weakly adherent compared with normal keratinocytes. When used in combination with DPNTP, latrunculin A, which disassembles actin filaments and disrupts adherens junctions, led to dissociation up to an order of magnitude greater than either treatment alone. These data provide direct in vitro evidence that IF-membrane attachments regulate adhesive strength and suggest furthermore that actin- and IF-based junctions act synergistically to strengthen adhesion.