Desmosome Degradation Research Articles

Abstract MP218: Cop9 Signalosome Subunit 6 Restricts Desmosomal Proteome Degradation To Prevent Desmosomal Targeted Cardiac Disease

Dysregulated protein degradative pathways are increasingly recognized as mediators of human cardiac disease. This pathway may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication. Genetic mutations in desmosomal genes resulting in the destabilization/breakdown of the desmosomal proteome are a central hallmark of all genetic-based desmosomal-targeted diseases, including the cardiac disease arrhythmogenic right ventricular (RV) dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here we identified a desmosomal resident regulatory complex, composed of subunit 6 of the COP9 signalosome (CSN6), enzymatically restricted neddylation and targets desmosomal proteome. Pharmacological restoration of CSN enzymatic function (via neddylation inhibitors) could rescue desmosomal protein loss in CSN6 deficient cardiomyocytes. Through the generation of two novel mouse models, we showed that cardiomyocyte-restricted CSN6 loss in mice selectively accelerated desmosomal destruction to trigger classic disease features associated with ARVD/C. We further showed that disruption of CSN6-mediated (neddylation) pathways underlined ARVD/C as CSN6 binding, localization, levels and function were impacted in hearts of classic ARVD/C mouse models and ARVD/C patients impacted by desmosomal loss and mutations, respectively. We anticipate our findings have broad implications towards understanding mechanisms driving desmosome degradation in other desmosomal-based diseases, such as cancers.

Immunohistochemical expression of kallikrein 7 in oral squamous cell carcinoma.

Background and Objectives:The kallikrein (KLK) family of genes consists of 15 members, many of which are highly expressed in number of cancers compared to their normal parent tissues. KLK7 was initially characterized as an enzyme implicated in the degradation of intercellular cohesive structures in the stratum corneum of stratified squamous epithelia, preceding desquamation in the skin. It catalyzes the degradation of desmosomes in the outermost layer of skin and permits cell shedding to take place at the skin surface. Overexpression of KLK7 in tumor cells has been reported to significantly enhance the invasive potential in intracranial malignancies and ovarian cancer cells. Thus, KLK7 could contribute to the degradation of extracellular matrices in oral squamous cell carcinoma (OSCC) tissues, promoting invasion of neoplastic cells locally and facilitating metastasis to regional lymph nodes. The objectives of the present study were to compare the expression of KLK 7 in normal subjects and patients with OSCC, to correlate the expression of KLK 7 with respect to the clinical staging of OSCC and to evaluate the expression of KLK 7with respect to different histopathological grades of OSCC.Materials and Methods:Thirty cases of OSCC were staged clinically and graded histopathologically. The immunohistochemical method was used to detect the expression of KLK 7 in OSCC. The scores obtained were documented and compared statistically.Results:KLK 7 immunoreactivity was noticed in all cases of OSCC. A statistically significant difference was observed in immunoreactivity of KLK 7 between the normal and OSCC (P = 0.0001*) and in different histopathological grades (P = 0.0001*) and in different clinical stages (P = 0.0127*) of OSCC using Kruskal–Wallis analysis of variance test.Conclusion:The KLK 7 immunoexpression histopathologically increased from low grade to high grade and clinically from Stage 1 to Stage 4 in OSCC. Hence, increased expression of KLK 7 may be related to poor prognosis in patients with OSCC.

Immunoadsorption of Desmoglein-3-Specific IgG Abolishes the Blister-Inducing Capacity of Pemphigus Vulgaris IgG in Neonatal Mice.

Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disease which is associated with autoantibodies directed against two desmosomal proteins, desmoglein (Dsg) 3 and 1. Treatment of PV is rather challenging and relies on the long-term use of systemic corticosteroids and additional immunosuppressants. More recently, autoantibody-depleting therapies such as rituximab, high-dose intravenous immunoglobulins, and immunoadsorption were shown to be valuable treatment options in PV. Specific removal of pathogenic autoantibodies would further increase efficacy and usability of immunoadsorption. Here, we tested the capacity of our recently developed prototypic Dsg1- and Dsg3-specific adsorbers to remove circulating pathogenic autoantibodies from three different PV patients. The pathogenic potential of the Dsg3/1-depleted IgG fractions and the anti-Dsg3-specific IgG was explored in two different in vitro assays based on cultured human keratinocytes, the desmosome degradation assay and the dispase-based dissociation assay. In addition, the neonatal mouse model of PV was used. In both in vitro assays, no difference between the pathogenic effect of total PV IgG and anti-Dsg3-specific IgG was seen, while Dsg3/1-depleted and control IgG were not pathogenic. For the samples of all 3 PV patients, depletion of anti-Dsg3/1 IgG resulted in a complete loss of pathogenicity when injected into neonatal mice. In contrast, injection of anti-Dsg3-specific IgG, eluted from the column, induced gross blistering in the mice. Our data clearly show that anti-Dsg3-specific IgG alone is pathogenic in vitro and in vivo, whereas Dsg3/1-depletion results in a complete loss of pathogenicity. Furthermore, our data suggest that Dsg-specific adsorption may be a suitable therapeutic modality to efficiently reduce pathogenic autoantibodies in patients with severe PV.

Streptococcal Cysteine Protease-Mediated Cleavage of Desmogleins Is Involved in the Pathogenesis of Cutaneous Infection.

Streptococcus pyogenes is responsible for a wide variety of cutaneous infections ranging from superficial impetigo to fulminant invasive necrotizing fasciitis. Dysfunction of desmosomes is associated with the pathogenesis of cutaneous diseases. We identified streptococcal pyrogenic exotoxin B (SpeB) as a proteolytic factor that cleaves the extracellular domains of desmoglein 1 and 3. In an epicutaneous infection model, lesional skin infected with an speB deletion mutant were significantly smaller as compared to those caused by the wild-type strain. Furthermore, immunohistological analysis indicated cleavage of desmogleins that developed around the invasion site of the wild-type strain. In contrast, the speB mutant was preferentially found on the epidermis surface layer. Taken together, our findings provide evidence that SpeB-mediated degradation of desmosomes has a pathogenic role in development of S. pyogenes cutaneous infection.

Broad specificity alkaline proteases efficiently reduce the visual scaling associated with soap-induced xerosis

In xerotic skin, the proteolysis of desmosomes is reduced leading to the accumulation of corneocytes on the surface of the skin. The effect of proteases applied topically to soap-induced xerotic skin was evaluated using a five-point visual scale. The visual scaling associated with soap-induced xerosis could be ameliorated by the topical application of exogenous protease. Bovine pancreatic chymotrypsin, papain, and a bacterial protease from Bacillus licheniformis were all capable of facilitating the reduction in visual scaling in a short time. Alcalase and Optimase, both broad specificity alkaline bacterial proteases, were the most weight-efficient at delivering this clinical effect. The reduction in scaling could be achieved either by occluded application of an aqueous enzyme solution or by a two-step unoccluded application first of an aqueous enzyme solution followed by a commercial moisturizer. Morphological and immunological analysis of bacterial enzyme-treated skin revealed that topically applied protease specifically induced the degradation of the desmosomes thereby promoting desquamation. These results indicate that topical application of protease can significantly and rapidly reduce the visual scaling associated with soap-induced xerosis by promoting desmosome degradation within the corneocyte clumps.

Dry condition affects desquamation of stratum corneum in vivo

We examined whether a dry condition actually induces scaly skin in vivo. Hairless mice were kept in a high humidity condition or a low humidity condition and skin changes were examined. Scales appeared on the backs of mice kept for 3 days under the dry condition. The weight of stratum corneum (SC) was increased at this point, and these alterations were not accompanied with hyperproliferation of the nucleated cell layer of the epidermis. A decrease of desmosomal degradation was observed, though, desquamation-related enzyme activity was not altered. The regulation mechanism of desquamation is not yet clear, however, in vitro experiments suggest that the water content in SC is an important factor. The water content of SC was decreased in the dry condition. These results indicate that a dry environment perturbs desmosome degradation in intact SC by decreasing the water content of SC, and the consequent impairment of desquamation in normal skin in vivo may lead to the induction of a scaly skin surface.

Mode of action of glycolic acid on human stratum corneum: ultrastructural and functional evaluation of the epidermal barrier.

Alpha-hydroxy acids (AHA) such as glycolic acid have recently been used extensively in cosmetic and dermatological formulas. In low concentration (2-5%) glycolic acid is believed to facilitate progressive weakening of cohesion of the intercellular material of the stratum corneum (SC), resulting in uniform exfoliation of its outermost layers (the stratum disjunctum). Since thinning of the SC as well as changes of intercellular lipids could theoretically compromise the barrier functions of the skin, we investigated the mode of AHA action on the SC to determine whether enhanced desquamation compromises the barrier structures of the SC and changes transepidermal water loss (TEWL) values. Electron microscopy of the epidermis biopsied from the volar forearm of human volunteers after 3 weeks of treatment with a 4% glycolic acid formulation twice daily was employed to evaluate 1) epidermal morphology and thickness of the SC, (2) the lamellar body and SC lipid bilayer organization, and (3) desquamative events based on degradation of desmosomes. TEWL values and SC hydration were recorded prior to and at the end of the study. Electron microscopy revealed no ultrastructural changes in the nucleated layers of the epidermis. The lamellar body (LB) secretory system in the stratum granulosum (SG), and intercellular lipid lamellae in the SC in both vehicle- and glycolic acid-treated samples were comparable to normal human SC. Within the SC, enhanced desmosomal breakdown, promoting loss of cohesion and desquamation, was restricted to the stratum disjunctum while desmosomes of the stratum compactum were unaffected. Treated areas displayed histologically, a more compact appearing SC. TEWL values remained unchanged in glycolic acid- and vehicle-treated skin. Our findings indicate that the barrier structures of the SC are not disrupted by glycolic acid formulations at the concentration used. One of the mechanism of action of AHA on the SC seemed to be a "targeted" desmosomal (corneosomal) action without compromising the barrier structures of the skin.

The effect of glycerol and humidity on desmosome degradation in stratum corneum.

Moisturizers are known to have occlusive, emollient and humectant properties, all of which help to alleviate the symptoms of skin xerosis. Although the biological mode of action of moisturizers is poorly understood, the recent observation that skin xerosis is associated with incomplete desmosome digestion suggests that moisturizers improve the desquamation process in such conditions. To examine the possibility that certain moisturizers act by facilitating desmosomal digestion, we investigated the ability of glycerol, a common humectant, to influence this process in stratum corneum in vitro. Examining desmosome morphology in isolated stratum corneum by electron microscopy, it was observed that the desmosomes were in more advanced stages of degradation in glycerol-treated tissue compared with control tissue. This enhanced desmosomal degradation in glycerol-treated tissue was confirmed by significant decreases in the levels of immunoreactive desmoglein 1, a marker of desmosome integrity. Desmosomal degradation was also shown to be a humidity-dependent event, being significantly reduced at low relative humidity. The effect of glycerol on desmosome digestion was emphasized further in two in vitro model systems. Firstly, glycerol increased the rate of corneocyte loss from the superficial surface of human skin biopsies in a simple desquamation assay. Secondly, measurement of the mechanical strength of sheets of stratum corneum, using an extensiometer, indicated a dramatic reduction in the intercorneocyte forces following glycerol treatment. These studies demonstrated the ability of glycerol to facilitate desmosome digestion in vitro. Extrapolating from these results, we believe that one of the major actions of moisturizers in vivo is to aid the digestion of desmosomes which are abnormally retained in the superficial layers of xerotic stratum corneum.

Evidence That Stratum Corneum Chymotryptic Enzyme Is Transported to the Stratum Corneum Extracellular Space Via Lamellar Bodies

Stratum corneum chymotryptic enzyme (SCCE) is a recently discovered human serine proteinase that may be specific for keratinizing squamous epithelia. SCCE has properties compatible with a function in the degradation of intercellular cohesive structures during stratum corneum turnover and desquamation. SCCE is expressed in suprabasal keratinocytes. In this study, we demonstrate the subcellular localization of SCCE in the upper granular layer, in the stratum corneum of normal non-palmoplantar skin, and in cohesive parts of hypertrophic plantar stratum corneum, using immunoelectron microscopy of ultrathin cryosections labeled with SCCE-specific monoclonal antibodies detected with gold-labeled secondary antibodies. A narrow zone close to the transition between the granular and cornified layers showed positive SCCE staining after fixation. By means of immunoelectron microscopy, SCCE was found in association with structures resembling intracellular lamellar bodies in the uppermost granular cells and in similar structures undergoing extrusion to the extracellular space between the uppermost granular cells and the lowermost cornified cells. In the stratum corneum, the detected SCCE was confined to the extracellular space and was found in association with intact and partially degraded desmosomes, as well as in the parts of the extracellular space devoid of desmosomes. We conclude that SCCE may be stored in lamellar bodies in the stratum granulosum and transported via these structures to the stratum corneum extracellular space. The results further support the idea that the physiologic function of SCCE may be to catalyze the degradation of desmosomes in the stratum corneum during remodeling of the deeper layers of this tissue, and at a later stage serve as a prerequisite for desquamation.

Effects of protease inhibitors and Ca 2+ on degradation of desmosomes of stratum corneum sheet in vitro

Effects of protease inhibitors and Ca 2+ on degradation of desmosomes of stratum corneum sheet in vitro

Evidence that Cell Shedding from Plantar Stratum Corneum In Vitro Involves Endogenous Proteolysis of the Desmosomal Protein Desmoglein I

We have recently described a process leading to a unipolar cell shedding from pieces of plantar stratum corneum incubated in vitro, which seems to be dependent on the activity of a serine proteinase. This process has been studied further. Electron microscopy studies suggest that cell dissociation is preceded by a degradation of the intercellular parts of desmosomes. An antiserum was raised against the transmembrane protein desmoglein I (DG I) of bovine desmosomes. In extracts of layers of plantar stratum corneum with strong intercellular cohesion, this antiserum reacted with a protein of the same apparent molecular weight as bovine DG I. In dissociated cells this DG I-like protein could not be detected; instead components with molecular weights lower than DG I which reacted with the antiserum were found. During incubation of pieces of plantar stratum corneum, under conditions leading to unipolar cell shedding, there was a progressive decrease in the amounts of the DG I-like protein, and the appearance of the lower molecular weight components with DG I-like immunoreactivity. This apparent degradation of the DG I-like protein was inhibited by aprotinin, chymostatin, and zinc ion, but not by leupeptin. The results suggest that proteolytic degradation of desmosomes may be an important part of the process leading to cell dissociation in plantar stratum corneum in vitro, and that desmosomes may play an important role in plantar stratum corneum cell cohesion.

Location and nature of the epidermal permeability barrier

Synopsis The lowest region of human and pig stratum corneum has been isolated as an integral layer, referred to as the stratum compactum. This preparation is resistant to disruption by enzymes, 6M urea, Triton X-100 and organic solvents. Our evidence suggests that penetration of materials to and through the corneum depend on the state of cohesion between cells, and of the organization of intercellular lipid species. As the cells move up towards the outside of the stratum corneum, the cohesive forces are reduced due to desmosome degradation and lipid modifications, with ultimate dyshesion and sloughing of individual cells. A number of linoleate-rich lipids have been identified within human and pig epidermis which could contribute to the formation or maintenance of an epidermal permeability barrier. Our current belief is that the molecule key to barrier function is a hydroxylated linoleate derivative which appears to be formed in the stratum compactum region.

Isolation, barrier properties and lipid analysis of stratum compactum, a discrete region of the stratum corneum.

We have isolated the lowest region of human and pig stratum corneum as an integral layer which we have termed the stratum compactum. This preparation is resistant to disruption by enzymes, 6 M urea, Triton X-100 and solvents. Our evidence suggests that all cells of the stratum corneum may be equally permeable to aqueous soluble materials but that penetration of materials through the corneum depends on the state of cohesion between cells and of the organization of intercellular lipid species. As the cells move up towards the outside of the stratum corneum the cohesive forces are reduced due to desmosome degradation and lipid modifications with ultimate dyshesion and sloughing of individual cells.