Desmosome Disassembly Research Articles

Pemphigus vulgaris autoantibodies induce an ER stress response.

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 IRE1⍺ 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 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.

The Desmoglein‐1 Transmembrane Domain Drives Lipid Raft Association and Desmosome Assembly

The epidermis is a barrier to the outside world, but mutations in genes encoding desmosomal proteins can inhibit this function, causing human diseases of skin fragility. Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a severe homozygous recessive disorder of skin fragility caused by mutations in the gene encoding desmoglein 1 (DSG1), a component of desmosomes. Desmosomes are junctional complexes that function in mechanical stress resistance across tissues. In these junctions, desmosomal cadherins such as DSG1 interact across extracellular spaces and with intracellular adaptor proteins to link cytoskeletal elements of adjacent cells. Both assembly and disassembly of desmosomes have been shown to rely on lipid raft membrane microdomains, highly ordered cholesterol and sphingolipid‐enriched regions of the plasma membrane important in signaling and trafficking. Our current model predicts that raft association promotes clustering of desmosomal proteins to drive assembly and mediate adhesion. However, the mechanisms of raft association and desmosome assembly are not fully understood. We created a DSG‐null A431 cell line to examine the relationship between DSG raft association and desmosomal adhesion. A431s are an immortal human epidermal carcinoma cell line commonly used in desmosomal studies that express a single DSG species, DSG2. The absence of DSG2 in these cells was confirmed by western blot and immunofluorescence. Further characterization of these DSG‐null A431 cells revealed mislocalized desmoplakin, a marker of mature desmosomes, and a lack of strong cell‐cell adhesion. Exogenous stable expression of GFP‐tagged wildtype DSG1 (DSG1WT‐GFP) rescued both desmosome formation and function, suggesting that these DSG‐null cells would be suitable for probing the relationship between DSG raft association and desmosome assembly and function. Recently, we discovered a novel, heterozygous mutation in the transmembrane domain (TMD)‐encoding region of DSG1 that causes a glycine to arginine substitution and SAM syndrome. DSG1SAM is a lipid raft targeting mutant suggesting an important role for the TMD in raft association. Stable expression of DSG1SAM‐GFP could not rescue desmosome formation or function in the DSG‐null cells, suggesting that a loss of raft association also impacts desmosome assembly and adhesive function. Intriguingly, a chimeric DSG1 protein containing the TMD from E‐cadherin (DSG1Ecad‐GFP), a non‐raft associating adhesive protein, only partly rescued desmosome formation and function. Furthermore, these results correlated with the predicted lipid raft affinity of each TMD, suggesting that desmosome assembly and function is dependent on raft association. We are currently analyzing a panel of GFP‐tagged DSG1TMD variants with differential raft targeting affinities and assessing their ability to support desmosome assembly and function. In conclusion, the TMD targets desmogleins to lipid rafts and modulates desmoglein adhesive function.Support or Funding InformationNIH R01AR050501, NIH R01AR048266

Keratin Isotypes Control Desmosome Stability and Dynamics through PKCα

Expression and interaction of desmosomal components and keratins provide stable cell cohesion and protect the epidermis against various types of stress. The differentiation-specific isotype composition of the keratin cytoskeleton and desmosomes is regarded as a major determinant of adhesive strength. In support, wound healing is characterized by a transient decrease in desmosomal adhesion accompanied by increased expression of keratins K6/K16/K17 at the expense of K1/K10. The significance of altered keratin expression for desmosomal composition and adhesion remains incompletely understood at a mechanistic and functional level. Here, we investigated the respective contribution of K5/K14 or K6/K17 to desmosome adhesion, on their stable re-expression in keratinocytes lacking all keratins. This revealed that K5/K14 filaments support stable desmosomes, whereas "wound healing" keratins K6/K17 induce elevated protein kinase C alpha-mediated desmosome disassembly and subsequent destabilization of epithelial sheets. Moreover, our data suggest that K5/K14 sequester protein kinase C alpha in the cytoplasm, whereas K6/K17 or the absence of all keratins enables protein kinase C alpha translocation to the plasma membrane and induction of desmosome disassembly. Gain- and loss-of-function experiments support a major role of K5 in desmosome stability control via protein kinase C alpha. Our data show that keratin isotypes differently and specifically regulate wound healing and invasion by modulating intercellular adhesion.

C-Src mediated tyrosine phosphorylation of plakophilin 3 as a new mechanism to control desmosome composition in cells exposed to oxidative stress

Plakophilins (PKP1 to PKP3) are essential for the structure and function of desmosomal junctions as demonstrated by the severe skin defects observed as a result of loss-of-function mutations in mice and men. PKPs play additional roles in cell signaling processes, such as those controlling the cellular stress response and cell proliferation. A key post-translational process controlling PKP function is phosphorylation. We have discovered that reactive oxygen species (ROS) trigger the c-Src kinase-mediated tyrosine (Tyr)-195 phosphorylation of PKP3. This modification is associated with a change in the subcellular distribution of the protein. Specifically, PKP3 bearing phospho-Tyr-195 is released from the desmosomes, suggesting that phospho-Tyr-195 is relevant for the control of desmosome disassembly and function, at least in cells exposed to ROS. Tyr-195 phosphorylation is transient under normal physiological conditions and seems to be strictly regulated, as the activation of particular growth factor receptors results in a modification at this site only when tyrosine phosphatases are inactivated by pervanadate. We have identified Tyr-195 of PKP3 as a phosphorylation target of epidermal growth factor receptor signaling. Interestingly, this PKP3 phosphorylation also occurs in certain poorly differentiated adenocarcinomas of the prostate, suggesting a possible role in tumor progression. Our study thus identifies a new mechanism controlling PKP3 and hence desmosome function in epithelial cells.

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.

Ultrastructure of acantholysis in pemphigus foliaceus re‐examined from the current perspective

Pemphigus foliaceus (PF) is a chronic cutaneous autoimmune blistering disease that is characterized by superficial blistering of the skin, and according to the current perspective is caused by autoantibodies directed against desmoglein (Dsg) 1. To examine early acantholysis in the skin of patients with PF at an ultrastructural level. Two Nikolsky-negative (N-), five Nikolsky-positive (N+) and two lesional skin biopsies from immunoserologically defined patients with PF were studied by light and electron microscopy. We found no abnormalities in N- PF skin, whereas all the N+ skin biopsies displayed intercellular widening between desmosomes, a decreased number of desmosomes and hypoplastic desmosomes in the lower epidermal layers. Acantholysis was present in two of five N+ biopsies, but only in the upper epidermal layers. The lesional skin biopsies displayed acantholysis in the higher epidermal layers. Hypoplastic desmosomes were partially (pseudo-half-desmosomes) or completely torn off from the opposing cell. We propose the following mechanism for acantholysis in PF: initially PF IgG causes a depletion of nonjunctional Dsg1, leading to intercellular widening between desmosomes starting in the lower layers and spreading upwards. Depletion of nonjunctional Dsg1 impairs the assembly of desmosomes, resulting in hypoplastic desmosomes and a decreased number of desmosomes. In addition, antibodies might promote disassembly of desmosomes. In the upper layers of the epidermis, where Dsg3 is not expressed and cannot compensate for Dsg1 loss, ongoing depletion of Dsg1 will finally result in a total disappearance of desmosomes and subsequent acantholysis.

Desmosome Disassembly in Response to Pemphigus Vulgaris IgG Occurs in Distinct Phases and Can Be Reversed by Expression of Exogenous Dsg3

Pemphigus vulgaris (PV) is an epidermal blistering disorder caused by antibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). The mechanism by which PV IgG disrupts adhesion is not fully understood. To address this issue, primary human keratinocytes (KCs) and patient IgG were used to define the morphological, biochemical, and functional changes triggered by PV IgG. Three phases of desmosome disassembly were distinguished. Analysis of fixed and living KCs demonstrated that PV IgG cause rapid Dsg3 internalization, which likely originates from a non-junctional pool of Dsg3. Subsequently, Dsg3 and other desmosomal components rearrange into linear arrays that run perpendicular to cell contacts. Dsg3 complexes localized at the cell surface are transported in a retrograde manner along with these arrays before being released into cytoplasmic vesicular compartments. These changes in Dsg3 distribution are followed by depletion of detergent-insoluble Dsg3 pools and by the loss of cell adhesion strength. Importantly, this process of disassembly can be prevented by expressing exogenous Dsg3, thereby driving Dsg3 biosynthesis and desmosome assembly. These data support a model in which PV IgG cause the loss of cell adhesion by altering the dynamics of Dsg3 assembly into desmosomes and the turnover of cell surface pools of Dsg3 through endocytic pathways.

Autoantibodies in the Autoimmune Disease Pemphigus Foliaceus Induce Blistering via p38 Mitogen-Activated Protein Kinase-Dependent Signaling in the Skin

Pemphigus foliaceus (PF) is a human autoimmune blistering disease in which a humoral immune response targeting the skin results in a loss of keratinocyte cell-cell adhesion in the superficial layers of the epidermal epithelium. In PF, desmoglein-1-specific autoantibodies induce blistering. Evidence is beginning to accumulate that activation of signaling may have an important role in the ability of pathogenic pemphigus IgGs to induce blistering and that both p38 mitogen-activated protein kinase (MAPK) and heat shock protein (HSP) 27 are part of this signaling pathway. This study was undertaken to investigate the ability of PF IgGs to activate signaling as well as the contribution of this signaling pathway to blister induction in an in vivo model of PF. Phosphorylation of both p38 MAPK and HSP25, the murine HSP27 homolog, was observed in the skin of PF IgG-treated mice. Furthermore, inhibition of p38 MAPK blocked the ability of PF IgGs to induce blistering in vivo. These results indicate that PF IgG-induced blistering is dependent on activation of p38 MAPK in the target keratinocyte. Rather than influencing the immune system, limiting the autoantibody-induced intracellular signaling response that leads to target end-organ damage may be a more viable therapeutic strategy for the treatment of autoimmune diseases. Inhibition of p38 MAPK may be an effective strategy for the treatment of PF.

Structural studies on desmosomes

Desmosomes are cadherin-based intercellular junctions that primarily provide mechanical stability to tissues such as epithelia and cardiac muscle. Desmosomal cadherins, which are Ca(2+)-dependent adhesion molecules, are of central importance in mediating direct intercellular interaction. The close association of these proteins, with intracellular components of desmosomes ultimately linked to the cytoskeleton, is believed to play an important role in tissue morphogenesis during development and wound healing. Elucidation of the binding mechanism of adhesive interfaces between the extracellular domains of cadherins has been approached by structural, biophysical and biochemical methods. X-ray crystal structures of isolated extracellular domains of cadherins have provided compelling evidence of the mutual binding of the highly conserved N-terminal residue, Trp(2), from opposing proteins. This binding interface was also implicated by biochemical and cell-adhesion assays and mutagenesis data to be the primary adhesive interface between cells. Recent results based on electron tomography of epidermal desmosomes were consistent with this view, showing cadherin molecules interacting at their N-terminal tips. An integrative structural approach involving X-ray crystallography, cryo-electron tomography and immuno-electron microscopy should give the complete picture of the architecture of this important junction; identifying its various proteins and showing their arrangements and binding interfaces under native conditions. Together with these 'static' approaches, live-cell imaging of cultured keratinocytes should provide important insights into the dynamic property of the assembly and disassembly of desmosomes.

Desmoglein Versus Non-desmoglein Signaling in Pemphigus Acantholysis: CHARACTERIZATION OF NOVEL SIGNALING PATHWAYS DOWNSTREAM OF PEMPHIGUS VULGARIS ANTIGENS

Although it is accepted that pemphigus antibody binding to keratinocytes (KCs) evokes an array of intracellular biochemical events resulting in cell detachment and death, the triggering events remain obscure. It has been postulated that the binding of pemphigus vulgaris IgG (PVIgG) to KCs induces "desmosomal" signaling. Because in contrast to integrins and classical cadherins, desmoglein (Dsg) molecules are not known to elicit intracellular signaling, and because PV patients also produce non-Dsg autoantibodies, we investigated the roles of both Dsg and non-desmoglein PV antigens. The time course studies of KCs treated with PVIgG demonstrated that the activity of Src peaked at 30 min, EGF receptor kinase (EGFRK) at 60 min, and p38 MAPK at 240 min. The Src inhibitor PP2 decreased EGFRK and p38 activities by approximately 45 and 30%, respectively, indicating that in addition to Src, PVIgG evokes other triggering events. The shrinkage of KCs (cell volume reduction) became significant at 120 min, keratin aggregation at 240 min, and an increase of TUNEL positivity at 360 min. Pretreatment of KCs with PP2 blocked PVIgG-dependent cell shrinkage and keratin aggregation by approximately 50% and TUNEL positivity by approximately 25%. The p38 MAPK inhibitor PD169316 inhibited these effects by approximately 15, 20, and 70%, respectively. Transfection of KCs with small interfering RNAs that silenced expression of Dsg1 and/or Dsg3 proteins, blocked approximately 50% of p38 MAPK activity but did not significantly alter the PVIgG-dependent rise in Src and EGFRK activities. These results indicate that activation of p38 MAPK is a late signaling step associated with collapse of the cytoskeleton and disassembly of desmosomes caused by upstream events involving Src and EGFRK. Therefore, the early acantholytic events are triggered by non-Dsg antibodies.

Involvement of desmoplakin phosphorylation in the regulation of desmosomes by protein kinase C, in HeLa cells.

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, antiphosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF 109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation ofdesmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.

The pathophysiological significance of nondesmoglein targets of pemphigus autoimmunity. Development of antibodies against keratinocyte cholinergic receptors in patients with pemphigus vulgaris and pemphigus foliaceus.

To determine whether nondesmoglein (non-Dsg) autoantibodies are pathogenic and whether they recognize keratinocyte cholinergic receptors that control cell adhesion because antikeratinocyte autoimmunity in patients with pemphigus vulgaris is not limited to the development of autoantibodies to Dsg. To determine whether non-DSg autoantibodies are pathogenic, we sought to induce pemphigus in genetically engineered neonatal mice lacking Dsg 3 using pemphigus vulgaris IgGs that did not cross-react with Dsg 1. To determine whether pemphigus autoimmunity involves keratinocyte cholinergic receptors, the latter were separated from cell membranes of human keratinocytes, tagged with the covalent label [3H]propylbenzilylcholine mustard, and used as an antigen in a radioimmunoprecipitation assay of 34 pemphigus vulgaris and 6 pemphigus foliaceus serum samples. The dermatologic clinics of the University of Minnesota, Minneapolis; the Mayo Clinic, Rochester, Minn; and the University of California-Davis Medical Center, Sacramento. Serum samples were collected from 34 patients with pemphigus vulgaris and 6 patients with pemphigus foliaceus (aged 31-89 years) and from 7 age-similar patients of both sexes with nonpemphigus blistering or the following immune-mediated conditions: pemphigoid gestation, bullous drug eruption, lupus erythematosus, erythema nodosum, urticaria, acute contact dermatitis, and skin ulcers. Clinical, laboratory, and histopathologic findings. Extensive skin blistering accompanied by the Nikolsky sign and suprabasilar acantholysis was induced in the Dsg3null mice that received pemphigus, but not normal human IgGs. In the radioimmunoprecipitation assays for reactivity with cholinergic receptors, the mean radioactivity precipitated by pemphigus serum samples significantly exceeded both normal- and disease-control levels (P = .001-.02). The mean individual levels of radioactivity precipitated by 34 pemphigus vulgaris and pemphigus foliaceus serum samples (85%) exceeded control values by a mean of approximately 2.6 times. Autoantibodies to keratinocyte cell-surface molecules other than Dsg 1 and Dsg 3 can induce clinical features of pemphigus vulgaris. Patients with pemphigus vulgaris and those with pemphigus foliaceus develop IgG antibodies that precipitate radiolabeled cholinergic receptors. Because these receptors control keratinocyte adhesion and motility, their inactivation by autoantibodies may elicit intracellular signals that cause disassembly of desmosomes, leading to acantholysis and blistering.

Desmosome assembly and disassembly are regulated by reversible protein phosphorylation in cultured epithelial cells.

Desmosomes are one component of the intercellular junctional complex in epithelia. In cultures of epithelial cells, desmosome assembly can be regulated by modulating the calcium concentrations of the growth media. At present, very little is known about the intracellular signal transduction mechanisms that regulate desmosome assembly and disassembly in response to changing extracellular calcium concentrations. We have used inhibitors of protein kinases and phosphatases in a combined biochemical and morphological approach to analyze the role of protein phosphorylation in the assembly and disassembly of desmosomes in Madin-Darby canine kidney epithelial cells. Our results suggest that desmosomal proteins (desmoplakins I/II and desmoglein 1) are primarily phosphorylated on serine residues. Electron microscopic analyses of desmosome assembly upon induction of cell-cell contact, in the presence of protein kinase inhibitor, H-7, revealed an apparently normal assembly of desmosomes. However, complete disassembly of desmosomes was inhibited by H-7 upon removal of extracellular calcium. Under these conditions, although desmosomes split, desmosomal plaques and their associated cytokeratin filaments can not be internalized. In contrast, treatment of the cultures with okadaic acid (OA), an inhibitor of protein phosphatases, inhibited desmosome assembly but had no effect on disassembly. In addition, the inhibitory effect of okadaic acid on desmosome assembly was specific to this junction since we observed apparently normal tight junction and adherens junction in okadaic acid-treated cultures. These results suggest that assembly and disassembly of desmosomes may be regulated by extracellular Ca2+ via reversible protein phosphorylation involving both protein kinase and protein phosphatases.

Rearrangements of desmosomal and cytoskeletal proteins during the transition from epithelial to fibroblastoid organization in cultured rat bladder carcinoma cells.

Changes of cell morphology and the state of differentiation are known to play important roles in embryogenesis as well as in carcinogenesis. Examples of particularly profound changes are the conversions of epithelial to mesenchymal cells; i.e., the dissociation of some or all polygonal, polar epithelial cells and their transformation into elongate, fibroblastoid cells of high motility. As an in vitro model system for such changes in cell morphology, we have used cell cultures of the rat bladder carcinoma-derived cell line NBT-II which, on exposure to inducing medium containing a commercial serum substitute (Ultroser G), show an extensive change in their organization (epithelial-mesenchymal transition): the junctions between the epithelial cells are split, the epithelial cell organization is lost, and the resulting individual cells become motile and assume a spindle-like fibroblastoid appearance. Using immunofluorescence microscopy and biochemical protein characterization techniques, we show that this change is accompanied by a redistribution of desmosomal plaque proteins (desmoplakins, desmoglein, plakoglobin) and by a reorganization of the cytokeratin and the actin-fodrin filament systems. Moreover, intermediate-sized filaments of the vimentin type are formed in the fibroblastoid cells. We demonstrate that the modulation of desmosomal proteins, specifically an increase in soluble desmoplakins, is a relatively early event in cell dissociation and in epithelial-mesenchymal transition. In this process, a latent period of 5 h upon addition of inducing medium precedes the removal of these desmosomal components from the plasma membrane. The transition, which is reversible, is dependent on continued protein synthesis and phosphorylation but not on the presence of the inducing medium beyond the initial 2-h period. We discuss the value of this experimental system as a physiologically relevant approach for studying the regulation of the assembly and disassembly of desmosomes and other intercellular adhesion structures, and as a model of the conversion of cells from one state of differentiation into another.