Desmosomal Adhesion Research Articles

Atomic force microscopy identifies regions of distinct desmoglein 3 adhesive properties on living keratinocytes

Desmosomes provide strong cell–cell adhesion which is crucial for the integrity of tissues such as the epidermis. However, nothing is known about the distribution and binding properties of desmosomal adhesion molecules on keratinocytes. Here we used atomic force microscopy (AFM) to simultaneously visualize the topography of living human keratinocytes and the distribution and binding properties of the desmosomal adhesion molecule desmoglein 3 (Dsg3). Using recombinant Dsg3 as sensor, binding events were detectable diffusely and in clusters on the cell surface and at areas of cell–cell contact. This was blocked by removing Ca2+ and by addition of Dsg3-specific antibodies indicating homophilic Dsg3 binding. Binding forces of Dsg3 molecules were lower on the cell surface compared to areas of cell–cell contact. Our data for the first time directly demonstrate the occurrence of Dsg3 molecules outside of desmosomes and show that Dsg3 adhesive properties differ depending on their localization. From the Clinical EditorUsing atomic force microscopy in the study of keratinocytes, this study directly demonstrates the occurrence of desmoglein 3 molecules outside of desmosomes and reveales that the adhesive properties of these molecules do differ depending on their localization.

Desmosomal defects in acantholytic squamous cell carcinomas.

Acantholytic squamous cell carcinoma (Acantholytic SCC) are epithelial tumors characterized by a loss of cell adhesion between neoplastic keratinocytes. The mechanism underlying loss of cell-cell adhesion in these tumors is not understood. A retrospective analysis of acantholytic SCC (n = 17) and conventional SCC (n = 16, controls not showing acantholysis) was conducted using a set of desmosomal and adherens junction protein antibodies. Immunofluorescence microscopy was used to identify tumors with loss of adhesion protein expression. The vast majority of acantholytic SCC (89%) showed focal loss of at least one desmosomal cell adhesion protein. Most interestingly, 65% of these tumors lost expression of two or more desmosomal proteins. Loss of cell adhesion in acantholytic SCC is most likely linked to the focal loss of desmosomal protein expression, thus providing potential mechanistic insight into the patho-mechanism underlying this malignancy.

Nanorobotic investigation identifies novel visual, structural and functional correlates of autoimmune pathology in a blistering skin disease model.

There remain major gaps in our knowledge regarding the detailed mechanisms by which autoantibodies mediate damage at the tissue level. We have undertaken novel strategies at the interface of engineering and clinical medicine to integrate nanoscale visual and structural data using nanorobotic atomic force microscopy with cell functional analyses to reveal previously unattainable details of autoimmune processes in real-time. Pemphigus vulgaris is a life-threatening autoimmune blistering skin condition in which there is disruption of desmosomal cell-cell adhesion structures that are associated with the presence of antibodies directed against specific epithelial proteins including Desmoglein (Dsg) 3. We demonstrate that pathogenic (blister-forming) anti-Dsg3 antibodies, distinct from non-pathogenic (non-blister forming) anti-Dsg3 antibodies, alter the structural and functional properties of keratinocytes in two sequential steps - an initial loss of cell adhesion and a later induction of apoptosis-related signaling pathways, but not full apoptotic cell death. We propose a “2-Hit” model for autoimmune disruption associated with skin-specific pathogenic autoantibodies. These data provide unprecedented details of autoimmune processes at the tissue level and offer a novel conceptual framework for understanding the action of self-reactive antibodies.

A preliminary study on clinical outcome of corticosteroid therapy in pemphigus patients

Introduction: Pemphigus is life threating blistering of autoimmune disease of the skin and mucous membrane characterised by autoantibodies (auto ABs) against desmoglein-3 (DSG-3). Desmosomal adhesions, protein expressed on the surface of epidermal keratinocytes. Aim: The present study was to assess the incidence rate of pemphigus, to evaluate the clinical course along with clinical manifestations, Complications and Metabolic factors of patients with pemphigus and to investigate the disease severity and induction of remission during the clinical course and whereas to assess the oxidative stress and antioxidant status in pemphigus patients in our hospital. Material and Methods: A prospective study was conducted over a period of January 2012 to December 2012 at dermatology department, MGM Hospital, Warangal. The data was collected from 32 cases of Pemphigus on the basis of Age, Sex, Social Habits, BMI, Histopathological forms, Clinical manifestations and Drug therapy. This hospital is 1200 bedded government hospital provided Outpatient and Inpatient care for Indian citizen especially in Telangana region free of cost. Results: Of the 32 pemphigus patients, 75% were diagnosed with Pemphigus Vulgaris (PV), 12.5% with Pemphigus Foliaceus (PF) and 12.5% with Bullous Pemphigoid (BP). The male to female ratio was approximately 1:1.3. The mean age of onset was 40.8 years in Pemphigus patients. The Onset of disease was 29.85. 34% of patients with Pemphigus had both the mucosal and skin involvement during the clinical course while 25% at the onset of disease. The most common complication was found to be increase blood sugar (40%). Most commonly prescribed adjuvant is dapsone. Majority attained the complete remission and remaining of them attains partial remission. Oxidative stress levels were higher and antioxidant status levels were lower in study subjects when compared to controls. Conclusions: PV is the most common subtype of Pemphigus in our Hospital and usually affects females more than males. Our study reveals that mucosal and skin involvement is common. Corticosteroids and dapsone as adjuvant is majorly prescribed. Most of patients attain complete remission and remaining of them achieves partial remission. Oxidative stress levels were higher and antioxidant status levels were lower in our study subjects when compared to controls.

A Desmoplakin Point Mutation with Enhanced Keratin Association Ameliorates Pemphigus Vulgaris Autoantibody-Mediated Loss of Cell Cohesion

Desmoplakin (DP) serves to anchor intermediate filaments in desmosomal complexes. Recent data suggest that a specific DP point mutation (S2849G) exhibits increased keratin filament association and fosters Ca(2+) insensitivity of desmosomes in keratinocytes, presumably by rendering DP inaccessible for protein kinase C (PKC) phosphorylation. Previously, we have reported that depletion of the desmosomal adhesion molecule desmoglein (Dsg)3 induced by autoantibodies from patients with the blistering skin disease pemphigus vulgaris (PV) IgG is reduced in maturated desmosomes and dependent on PKC signaling. We investigated the role of DP-S2849G for loss of cell cohesion mediated by PV-IgG. In cell dissociation assays, expression of green fluorescent protein-tagged DP-S2849G (DP-S2849G-GFP) increased cell cohesion in two different human keratinocyte cell lines and ameliorated loss of cell adhesion induced by pemphigus autoantibodies. Depletion of Dsg3 was inhibited by DP-S2849G-GFP in the cytoskeletal (Triton X-100 insoluble) fraction, and keratin filament retraction, a hallmark of PV, was efficiently blocked similar to treatment with the PKC inhibitor Bim-X. We found that DP is phosphorylated after incubation with PV-IgG in a PKC-dependent manner and that DP-S2849G-GFP expression prevents DP phosphorylation and increases association of PKC-α with PKC scaffold receptor for activated C-kinase 1. Taken together, our data indicate that DP phosphorylation at S2849 represents an important mechanism in pemphigus pathogenesis, which, by reversing Ca(2+) insensitivity, promotes Dsg3 depletion.

Hyper-adhesion: a unique property of desmosomes.

Hyper-adhesion is a unique, strongly adhesive form of desmosomal adhesion that functions to maintain tissue integrity. In this short review, we define hyper-adhesion, summarise the evidence for it in culture and in vivo, discuss its role in development, wound healing, and skin disease, and speculate about its molecular and cellular basis.

Plakoglobin but Not Desmoplakin Regulates Keratinocyte Cohesion via Modulation of p38MAPK Signaling

Plakoglobin (Pg) and desmoplakin (DP) are adapter proteins within the desmosome, providing a mechanical link between desmosomal cadherins as transmembrane adhesion molecules and the intermediate filament cytoskeleton. As in the severe skin blistering disease pemphigus, autoantibodies against desmosomal adhesion molecules induce loss of keratinocyte cohesion at least in part via p38 mitogen-activated protein kinase (p38MAPK) activation and depletion of desmosomal components, we evaluated the roles of Pg and DP in the p38MAPK-dependent loss of cell adhesion. Silencing of either Pg or DP reduced cohesion of cultured human keratinocytes in dissociation assays. However, Pg but not DP silencing caused activation of p38MAPK-dependent keratin filament collapse and cell dissociation. Interestingly, extranuclear but not nuclear Pg rescued loss of cell adhesion and keratin retraction. In line with this, Pg regulated the levels of the desmosomal adhesion molecule desmoglein 3 and tethered p38MAPK to desmosomal complexes. Our data demonstrate a role of extranuclear Pg in controlling cell adhesion via p38MAPK-dependent regulation of keratin filament organization.

Adducin Is Required for Desmosomal Cohesion in Keratinocytes

Adducin is a protein organizing the cortical actin cytoskeleton and a target of RhoA and PKC signaling. However, the role for intercellular cohesion is unknown. We found that adducin silencing induced disruption of the actin cytoskeleton, reduced intercellular adhesion of human keratinocytes, and decreased the levels of the desmosomal adhesion molecule desmoglein (Dsg)3 by reducing its membrane incorporation. Because loss of cell cohesion and Dsg3 depletion is observed in the autoantibody-mediated blistering skin disease pemphigus vulgaris (PV), we applied antibody fractions of PV patients. A rapid phosphorylation of adducin at serine 726 was detected in response to these autoantibodies. To mechanistically link autoantibody binding and adducin phosphorylation, we evaluated the role of several disease-relevant signaling molecules. Adducin phosphorylation at serine 726 was dependent on Ca(2+) influx and PKC but occurred independent of p38 MAPK and PKA. Adducin phosphorylation is protective, because phosphorylation-deficient mutants resulted in loss of cell cohesion and Dsg3 fragmentation. Thus, PKC elicits both positive and negative effects on cell adhesion, since its contribution to cell dissociation in pemphigus is well established. We additionally evaluated the effect of RhoA on adducin phosphorylation because RhoA activation was shown to block pemphigus autoantibody-induced cell dissociation. Our data demonstrate that the protective effect of RhoA activation was dependent on the presence of adducin and its phosphorylation at serine 726. These experiments provide novel mechanisms for regulation of desmosomal adhesion by RhoA- and PKC-mediated adducin phosphorylation in keratinocytes.

Plakophilins, Desmogleins, and Pemphigus: The Tail Wagging the Dog

SummaryThe importance of desmosomal cell adhesion to human health is evidenced by the autoimmune disease pemphigus vulgaris (PV), in which autoantibodies against the extracellular domain of the desmosomal cadherin desmoglein 3 cause potentially fatal blistering of the skin and mucous membranes. Tucker et al. describe how enhanced expression of a desmosomal cytoplasmic plaque protein, plakophilin-1, protects keratinocytes from PV IgG-induced loss of cell adhesion by inducing calcium-independent hyperadhesive desmosomes. This study beautifully demonstrates that desmosomal adhesion can be modulated by the molecular interactions of the desmoglein tail and suggests that these novel regulatory pathways may possibly be exploited in treating human disease.

Role of the desmosmal adhesion molecule desmoglein 2 in Crohn’s disease (60.4)

The barrier function established by intestinal epithelial cells is essential for gut homeostasis and is compromised in inflammatory bowel disease (IBD). This function is dependent on junctional complexes comprising of a set of intercellular junctions, namely tight junctions, adherens junctions, and desmosomes. Previously, we have shown that intercellular adhesion mediated by the desmosomal adhesion molecule desmoglein2 (Dsg2) is required for the integrity of tight junctions. Here, we demonstrate that Dsg2 levels are dramatically reduced in biopsies of patients with Crohn’s disease. Furthermore TNFa, a central cytokine in IBD, led to loss of cell cohesion, reduced Dsg2 expression, and increased permeability in Caco‐2 and HCT116 cells. This was paralleled by loss of Dsg2 immunostaining at cell borders with concomitant reduction of the tight junction protein claudin1. These effects were mediated at least in part by increased activity of p38MAPK, since inhibition of this kinase restored intercellular adhesion and blunted the permeability increase induced by TNFa. Importantly, stabilizing desmosomal adhesion via a Dsg‐specific peptide ameliorated loss of barrier functions. Thus, our data indicate an important role of p38MAPK‐mediated regulation of desmosomal adhesion for epithelial permeability and suggest a contribution of reduced Dsg2 expression to the pathogenesis of Crohn’s disease.

Plakophilin-1 Protects Keratinocytes from Pemphigus Vulgaris IgG by Forming Calcium-Independent Desmosomes

Plakophilin-1 (PKP-1) is an armadillo family protein critical for desmosomal adhesion and epidermal integrity. In the autoimmune skin blistering disease pemphigus vulgaris (PV), autoantibodies (IgG) target the desmosomal cadherin desmoglein 3 (Dsg3) and compromise keratinocyte cell-cell adhesion. Here, we report that enhanced expression of PKP-1 protects keratinocytes from PV IgG-induced loss of cell-cell adhesion. PKP-1 prevents loss of Dsg3 and other desmosomal proteins from cell-cell borders and prevents alterations in desmosome ultrastructure in keratinocytes treated with PV IgG. Using a series of Dsg3 chimeras and deletion constructs, we find that PKP-1 clusters Dsg3 with the desmosomal plaque protein desmoplakin in a manner dependent upon the plakoglobin binding domain of the Dsg3 tail. Furthermore, PKP-1 expression transforms desmosome adhesion from a calcium-dependent to a calcium-independent and hyper-adhesive state. These results demonstrate that manipulating the expression of a single desmosomal plaque protein can block the pathogenic effects of PV IgG on keratinocyte adhesion.

A desmoplakin point mutation insensitive to PKC phosphorylation ameliorates pemphigus vulgaris autoantibody‐mediated loss of cell cohesion (650.4)

Desmoplakin (DP) serves to anchor intermediate filaments in desmosomal complexes. Recent data suggest that a specific DP point mutation (S2849G) exhibits increased keratin filament association and fosters Ca2+‐insensitivity of desmosomes in keratinocytes, presumably by rendering DP inaccessible for PKC phosphorylation. Previously, we have shown that depletion of the desmosomal adhesion molecule desmoglein 3 (Dsg3) induced by autoantibodies from patients with the blistering skin disease pemphigus vulgaris (PV‐IgG) is reduced in maturated desmosomes and dependent on PKC signaling. Thus, we investigated the role of DP‐S2849G for loss of cell cohesion mediated by PV‐IgG. In cell dissociation assays, expression of DP‐S2849G increased cell cohesion in two different human keratinocyte cell lines and ameliorated loss of cell adhesion induced by PV‐IgG or treatment with AK23, a pathogenic Dsg3 antibody derived from a pemphigus mouse model. Depletion of Dsg3 was inhibited by DP‐S2849G in the cytoskeletal (Triton‐X 100 insoluble) fraction and keratin filament retraction, a hallmark of PV, was efficiently blocked. In line with this, DP was phosphorylated upon incubation with PV‐IgG in a PKC‐dependent manner. Taken together, our data demonstrate the relevance of keratin filament anchorage to desmosomes for cell adhesion and suggest PKC‐mediated DP phosphorylation as a central mechanism in PV pathogenesis.

Skin Fragility and Impaired Desmosomal Adhesion in Mice Lacking All Keratins

Keratins perform major structural and regulatory functions in epithelia. Owing to redundancy, their respective contribution to epidermal integrity, adhesion, and cell junction formation has not been addressed in full. Unexpectedly, the constitutive deletion of type II keratins in mice was embryonic lethal ∼ E9.5 without extensive tissue damage. This prompted us to analyze keratin functions in skin where keratins are best characterized. Here, we compare the mosaic and complete deletion of all type II keratins in mouse skin, with distinct consequences on epidermal integrity, adhesion, and organismal survival. Mosaic knockout (KO) mice survived ∼ 12 days while global KO mice died perinatally because of extensive epidermal damage. Coinciding with absence of keratins, epidermal fragility, inflammation, increased epidermal thickness, and increased proliferation were noted in both strains of mice, accompanied by significantly smaller desmosomes. Decreased desmosome size was due to accumulation of desmosomal proteins in the cytoplasm, causing intercellular adhesion defects resulting in intercellular splits. Mixing different ratios of wild-type and KO keratinocytes revealed that ∼ 60% of keratin-expressing cells were sufficient to maintain epithelial sheets under stress. Our data reveal a major contribution of keratins to the maintenance of desmosomal adhesion and epidermal integrity with relevance for the treatment of epidermolysis bullosa simplex and other keratinopathies.

Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV.

Plakophilins in Desmosomal Adhesion and Signaling

The regulation of adhesion and growth is important for epithelial function and dysfunction. β-catenin (armadillo in Drosophila) is the prototype of a multifunctional molecule that regulates cell adhesion via adherens junctions and cell signaling via LEF/TCF transcription factors. Desmosomal armadillo proteins comprise plakoglobin and the plakophilins 1, 2, and 3. These proteins are essential for building up the desmosome and linking the desmosomal cadherins to keratin filaments. High expression of plakophilins in desmosomes correlates with strong intercellular cohesion and is essential for tissue integrity under mechanical stress. However, like β-catenin, these proteins have diverse non-desmosomal functions, for example, in regulating actin organization, protein synthesis, and growth control. In line with these functions, their de-regulated expression with up- as well as down-regulation has been connected to cancer and metastasis. Now, recent evidence sheds light on the post-translational regulation and provides an explanation for how de-regulation of plakophilins can contribute to cancer.

Desmosomal Cadherins and Signaling: Lessons from Autoimmune Disease

Autoantibodies from patients suffering from the autoimmune blistering skin disease pemphigus can be applied as tools to study desmosomal adhesion. These autoantibodies targeting the desmosomal cadherins desmoglein (Dsg) 1 and Dsg3 cause disruption of desmosomes and loss of intercellular cohesion. Although pemphigus autoantibodies were initially proposed to sterically hinder desmosomes, many groups have shown that they activate signaling pathways which cause disruption of desmosomes and loss of intercellular cohesion by uncoupling the desmosomal plaque from the intermediate filament cytoskeleton and/or by interfering with desmosome turnover. These studies demonstrate that desmogleins serve as receptor molecules to transmit outside-in signaling and demonstrate that desmosomal cadherins have functions in addition to their adhesive properties. Two central molecules regulating cytoskeletal anchorage and desmosome turnover are p38MAPK and PKC. As cytoskeletal uncoupling in turn enhances Dsg3 depletion from desmosomes, both mechanisms reinforce one another in a vicious cycle that compromise the integrity and number of desmosomes.

Palmitoylation of plakophilin is required for desmosome assembly.

Desmosomes are prominent adhesive junctions found in various epithelial tissues. The cytoplasmic domains of desmosomal cadherins interact with a host of desmosomal plaque proteins, including plakophilins, plakoglobin and desmoplakin, which, in turn, recruit the intermediate filament cytoskeleton to sites of cell-cell contact. Although the individual components of the desmosome are known, mechanisms regulating the assembly of this junction are poorly understood. Protein palmitoylation is a posttranslational lipid modification that plays an important role in protein trafficking and function. Here, we demonstrate that multiple desmosomal components are palmitoylated in vivo. Pharmacologic inhibition of palmitoylation disrupts desmosome assembly at cell-cell borders. We mapped the site of plakophilin palmitoylation to a conserved cysteine residue present in the armadillo repeat domain. Mutation of this single cysteine residue prevents palmitoylation, disrupts plakophilin incorporation into the desmosomal plaque and prevents plakophilin-dependent desmosome assembly. Finally, plakophilin mutants unable to become palmitoylated act in a dominant-negative manner to disrupt proper localization of endogenous desmosome components and decrease desmosomal adhesion. Taken together, these data demonstrate that palmitoylation of desmosomal components is important for desmosome assembly and adhesion.

Characterizing the Interaction of Desmosomal Cadherins at Single Molecule Level

Desmosomes are cell-cell adhesion complexes that are present in tissues that resist mechanical stress. They are mainly composed of two adhesive proteins, which are members of the cadherin superfamily of cell adhesion proteins, desmocollin (Dsc) and desmoglein (Dsg). However, the role of these proteins in desmosomal adhesion is unclear. Here, we use the single molecule force spectroscopy with an Atomic Force Microscope (AFM-FS) to characterize the interactions of type-2 isoforms of desmocollin (Dsc2) and desmoglein (Dsg2). We show that Dsc2 forms Ca2+ dependent homophilic bonds by swapping a conserved Tryptophan (Trp) residue between opposing binding partners; mutating this Trp inhibits Ca2+ dependent homophilic binding. In contrast, Dsg2 forms Ca2+ independent heterophilic bonds with Dsc2 via a mechanism that does not involve Trp strand-swapping.Previous studies suggest that desmosome formation requires the presence of classical cadherins at the site of desmosome assembly. This suggests a cross-talk between desmosomal and classical cadherins at cell-adhesion contacts. We therefore used AFM-FS to test if Dsc2 and Dsg2 interact with E-cadherin, a classical cadherin present in the epithelium. Our data shows that while Dsc2 does not bind to E-cadherin in the presence of Ca2+, Dsg2 forms Ca2+ independent complexes with E-cadherin. Using cadherin mutants we show that the interactions between Dsg2 and E-cadherin occur via a previously uncharacterized binding interface that does not involve either Trp strand-swapping or X-dimer formation (two well established classical cadherin binding mechanisms).

A New Light on an Old Disease: Adhesion Signaling in Pemphigus Vulgaris

Disruption of desmosomal cadherin adhesion leads to the activation of intracellular signaling pathways that are responsible for blister formation in pemphigus vulgaris (PV). Recent studies corroborate the implication of the p38 mitogen-activated protein kinase in PV blistering via its downstream effector mitogen-activated protein kinase activated protein kinase 2. These insights highlight the key role of cadherins in tissue homeostasis and are expected to change pemphigus management.

An alternative promoter of the human plakophilin-3 gene controls the expression of the new isoform PKP3b

The plakophilin family (PKP1 to PKP3) is an essential component of the desmosomal adhesion complex with differentiation-dependent and partially overlapping expression and possible participation of the corresponding genes in malignant transformation. Here, we describe a new protein variant of the human PKP3 gene, namely PKP3b, which differs from the published PKP3a only at the amino-terminus by the splicing in of the newly identified exon 1b. Specific antibodies have demonstrated differential expression patterns of the two variants. Whereas PKP3a is broadly expressed among epithelial cells, PKP3b is abundant in the desmosomes of stratified epithelial cells, such as HaCaT but absent or heterogeneous in simple epithelial cells such as CaCo2 or MCF7. The differential expression of the PKP3 variants has been observed in a similar manner in selected normal human tissues and carcinomas derived thereof. Both variants are localized to the desmosomes of all cells of stratified tissues, whereas the new PKP3b is heterogeneously expressed in the colon and its tumors. Therefore, we assume that both variants are controlled by alternative promoters. Reporter gene assays have confirmed that a fragment upstream of exon 1b exhibits transcriptional activity only in HaCaT cells but not in CaCo2 cells and thus has been identified as an alternative promoter driving the expression of PKP3b. Finally, by using electromobility shift assays, we found a potential binding site in the PKP3b promoter for transcription factor C/EBP regulating keratinocyte differentiation and probably also PKP3b expression. We discuss the properties of the new variant PKP3b as a possible marker protein for the analyses of differentiation and malignant transformation.