Loss of Desmoglein Binding Is Not Sufficient for Keratinocyte Dissociation in Pemphigus

Pemphigus vulgaris (PV) is a severe autoimmune disease in which autoantibodies against the desmosomal cell adhesion molecules desmoglein (Dsg) 1 and Dsg3 cause intraepidermal blister formation. Mechanistically, the fundamental question is still unresolved whether loss of cell cohesion is a result of (1) direct inhibition of Dsg interaction by autoantibodies or (2) intracellular signaling events, which are altered in response to antibody binding and finally cause desmosome destabilization. We used atomic force microscopy (AFM) to perform Dsg3 adhesion measurements on living keratinocytes to investigate the contributions of direct inhibition and signaling to loss of cell cohesion after autoantibody treatment. Dsg3 binding was rapidly blocked following antibody exposure under conditions where no depletion of surface Dsg3 was detectable, demonstrating direct inhibition of Dsg3 interaction. Inhibition of p38MAPK, a central signaling molecule in PV pathogenesis, abrogated loss of cell cohesion, but had a minor effect on loss of Dsg3 binding. Similarly, the cholesterol-depleting agent methyl-β-cyclodextrin (β-MCD) fully blocked cell dissociation, but did not restore Dsg3 interactions or prevent the activation of p38MAPK. These results demonstrate that inhibition of Dsg3 binding is not sufficient to cause loss of cell cohesion, but rather alters signaling events which, in lipid raft-dependent manner, induce cell dissociation

Desmoglein 2 is less important than desmoglein 3 for keratinocyte cohesion.

Desmosomes provide intercellular adhesive strength required for integrity of epithelial and some non-epithelial tissues. Within the epidermis, the cadherin-type adhesion molecules desmoglein (Dsg) 1-4 and desmocollin (Dsc) 1-3 build the adhesive core of desmosomes. In keratinocytes, several isoforms of these proteins are co-expressed. However, the contribution of specific isoforms to overall cell cohesion is unclear. Therefore, in this study we investigated the roles of Dsg2 and Dsg3, the latter of which is known to be essential for keratinocyte adhesion based on its autoantibody-induced loss of function in the autoimmune blistering skin disease pemphigus vulgaris (PV). The pathogenic PV antibody AK23, targeting the Dsg3 adhesive domain, led to profound loss of cell cohesion in human keratinocytes as revealed by the dispase-based dissociation assays. In contrast, an antibody against Dsg2 had no effect on cell cohesion although the Dsg2 antibody was demonstrated to interfere with Dsg2 transinteraction by single molecule atomic force microscopy and was effective to reduce cell cohesion in intestinal epithelial Caco-2 cells which express Dsg2 as the only Dsg isoform. To substantiate these findings, siRNA-mediated silencing of Dsg2 or Dsg3 was performed in keratinocytes. In contrast to Dsg3-depleted cells, Dsg2 knockdown reduced cell cohesion only under conditions of increased shear. These experiments indicate that specific desmosomal cadherins contribute differently to keratinocyte cohesion and that Dsg2 compared to Dsg3 is less important in this context

Tracheal development in the Drosophila brain is constrained by glial cells

AbstractThe Drosophila brain is tracheated by the cerebral trachea, a branch of the first segmental trachea of the embryo. During larval stages the cerebral trachea splits into several main (primary) branches that grow around the neuropile, forming a perineuropilar tracheal plexus (PNP) at the neuropile surface. Five primary tracheal branches whose spatial relationship to brain compartments is relatively invariant can be distinguished, although the exact trajectories and branching pattern of the brain tracheae are surprisingly variable. Immunohistochemical and electron microscopic studies demonstrate that all brain tracheae grow in direct contact with the glial cell processes that surround the neuropile. To investigate the effect of glia on tracheal development, embryos and larvae lacking glial cells as a result of a genetic mutation or a directed ablation were analyzed. In these animals, the tracheal branching pattern was highly abnormal. In particular, the number of secondary branches entering the central neuropile was increased. Wild-type larvae possess only two central tracheae, typically associated with the mushroom body and the antennocerebral tract. In larvae lacking glial cells, six to ten tracheal branches penetrate the neuropile in a variable pattern. This finding indicates that glia-derived signals constrained tracheal growth in the Drosophila brain and restrict the number of branches entering the neuropile

Pemphigus-A Disease of Desmosome Dysfunction caused by Multiple Mechanisms

Pemphigus is a severe autoimmune-blistering disease of the skin and mucous membranes caused by autoantibodies reducing desmosomal adhesion between epithelial cells. Autoantibodies against the desmosomal cadherins desmogleins (Dsgs) 1 and 3 as well as desmocollin 3 were shown to be pathogenic, whereas the role of other antibodies is unclear. Dsg3 interactions can be directly reduced by specific autoantibodies. Autoantibodies also alter the activity of signaling pathways, some of which regulate cell cohesion under baseline conditions and alter the turnover of desmosomal components. These pathways include Ca2+, p38MAPK, PKC, Src, EGFR/Erk, and several others. In this review, we delineate the mechanisms relevant for pemphigus pathogenesis based on the histology and the ultrastructure of patients' lesions. We then dissect the mechanisms which can explain the ultrastructural hallmarks detectable in pemphigus patient skin. Finally, we reevaluate the concept that the spectrum of mechanisms, which induce desmosome dysfunction upon binding of pemphigus autoantibodies, finally defines the clinical phenotype

PKA Compartmentalization via AKAP220 and AKAP12 Contributes to Endothelial Barrier Regulation

cAMP-mediated PKA signaling is the main known pathway involved in maintenance of the endothelial barrier. Tight regulation of PKA function can be achieved by discrete compartmentalization of the enzyme via physical interaction with A-kinase anchoring proteins (AKAPs). Here, we investigated the role of AKAPs 220 and 12 in endothelial barrier regulation. Analysis of human and mouse microvascular endothelial cells as well as isolated rat mesenteric microvessels was performed using TAT-Ahx-AKAPis peptide, designed to competitively inhibit PKA-AKAP interaction. In vivo microvessel hydraulic conductivity and in vitro transendothelial electrical resistance measurements showed that this peptide destabilized endothelial barrier properties, and dampened the cAMP-mediated endothelial barrier stabilization induced by forskolin and rolipram. Immunofluorescence analysis revealed that TAT-Ahx-AKAPis led to both adherens junctions and actin cytoskeleton reorganization. Those effects were paralleled by redistribution of PKA and Rac1 from endothelial junctions and by Rac1 inactivation. Similarly, membrane localization of AKAP220 was also reduced. In addition, depletion of either AKAP12 or AKAP220 significantly impaired endothelial barrier function and AKAP12 was also shown to interfere with cAMP-mediated barrier enhancement. Furthermore, immunoprecipitation analysis demonstrated that AKAP220 interacts not only with PKA but also with VE-cadherin and beta-catenin. Taken together, these results indicate that AKAP-mediated PKA subcellular compartmentalization is involved in endothelial barrier regulation. More specifically, AKAP220 and AKAP12 contribute to endothelial barrier function and AKAP12 is required for cAMP-mediated barrier stabilization

Keratin Retraction and Desmoglein3 Internalization Independently Contribute to Autoantibody-Induced Cell Dissociation in Pemphigus Vulgaris

Pemphigus vulgaris (PV) is a potentially lethal autoimmune disease characterized by blister formation of the skin and mucous membranes and is caused by autoantibodies against desmoglein (Dsg) 1 and Dsg3. Dsg1 and Dsg3 are linked to keratin filaments in desmosomes, adhering junctions abundant in tissues exposed to high levels of mechanical stress. The binding of the autoantibodies leads to internalization of Dsg3 and a collapse of the keratin cytoskeleton-yet, the relevance and interdependence of these changes for loss of cell-cell adhesion and blistering is poorly understood. In live-cell imaging studies, loss of the keratin network at the cell periphery was detectable starting after 60 min of incubation with immunoglobulin G fractions of PV patients (PV-IgG). These rapid changes correlated with loss of cell-cell adhesion detected by dispase-based dissociation assays and were followed by a condensation of keratin filaments into thick bundles after several hours. Dsg3 internalization started at 90 min of PV-IgG treatment, thus following the early keratin changes. By inhibiting casein kinase 1 (CK-1), we provoked keratin alterations resembling the effects of PV-IgG. Although CK-1-induced loss of peripheral keratin network correlated with loss of cell cohesion and Dsg3 clustering in the membrane, it was not sufficient to trigger the internalization of Dsg3. However, additional incubation with PV-IgG was effective to promote Dsg3 loss at the membrane, indicating that Dsg3 internalization is independent from keratin alterations. Vice versa, inhibiting Dsg3 internalization did not prevent PV-IgG-induced keratin retraction and only partially rescued cell cohesion. Together, keratin changes appear very early after autoantibody binding and temporally overlap with loss of cell cohesion. These early alterations appear to be distinct from Dsg3 internalization, suggesting a crucial role for initial loss of cell cohesion in PV

Atomic Force Microscopy Provides New Mechanistic Insights into the Pathogenesis of Pemphigus

Autoantibodies binding to the extracellular domains of desmoglein (Dsg) 3 and 1 are critical in the pathogenesis of pemphigus by mechanisms leading to impaired function of desmosomes and blister formation in the epidermis and mucous membranes. Desmosomes are highly organized protein complexes which provide strong intercellular adhesion. Desmosomal cadherins such as Dsgs, proteins of the cadherin superfamily which interact via their extracellular domains in Ca2+ -dependent manner, are the transmembrane adhesion molecules clustered within desmosomes. Investigations on pemphigus cover a wide range of experimental approaches including biophysical methods. Especially atomic force microscopy (AFM) has recently been applied increasingly because it allows the analysis of native materials such as cultured cells and tissues under near-physiological conditions. AFM provides information about the mechanical properties of the sample together with detailed interaction analyses of adhesion molecules. With AFM, it was recently demonstrated that autoantibodies directly inhibit Dsg interactions on the surface of living keratinocytes, a phenomenon which has long been considered the main mechanism causing loss of cell cohesion in pemphigus. In addition, AFM allows to study how signaling pathways altered in pemphigus control binding properties of Dsgs. More general, AFM and other biophysical studies recently revealed the importance of keratin filaments for regulation of Dsg binding and keratinocyte mechanical properties. In this mini-review, we reevaluate AFM studies in pemphigus and keratinocyte research, recapitulate what is known about the interaction mechanisms of desmosomal cadherins and discuss the advantages and limitations of AFM in these regards

Inhibition of Rho A activity causes pemphigus skin blistering

The autoimmune blistering skin diseases pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are mainly caused by autoantibodies against desmosomal cadherins. In this study, we provide evidence that PV–immunoglobulin G (IgG) and PF-IgG induce skin blistering by interference with Rho A signaling. In vitro, pemphigus IgG caused typical hallmarks of pemphigus pathogenesis such as epidermal blistering in human skin, cell dissociation, and loss of desmoglein 1 (Dsg 1)–mediated binding probed by laser tweezers. These changes were accompanied by interference with Rho A activation and reduction of Rho A activity. Pemphigus IgG–triggered keratinocyte dissociation and Rho A inactivation were p38 mitogen-activated protein kinase dependent. Specific activation of Rho A by cytotoxic necrotizing factor-y abolished all pemphigus-triggered effects, including keratin retraction and release of Dsg 3 from the cytoskeleton. These data demonstrate that Rho A is involved in the regulation of desmosomal adhesion, at least in part by maintaining the cytoskeletal anchorage of desmosomal proteins. This may open the possibility of pemphigus treatment with the epidermal application of Rho A agonists

Biomechanical Properties of the Internal Limiting Membrane after Intravitreal Ocriplasmin Treatment

Purpose: To assess the stiffness of the human internal limiting membrane (ILM) and evaluate potential changes of mechanical properties following intravitreal ocriplasmin injection for vitreomacular traction. Methods: This is an interventional comparative case series of 12 surgically excised ILM specimens consecutively obtained from 9 eyes of 9 patients after unsuccessful pharmacologic vitreolysis with ocriplasmin. During the same time period, 16 specimens from 13 other eyes without ocriplasmin treatment were harvested during vitrectomy and served as controls. All patients presented with macular holes or vitreomacular traction and underwent vitrectomy with ILM peeling either with or without brilliant blue (BB) staining. All specimens were analyzed using atomic force microscopy with scan regions of 25 x 25 mu m. In all specimens, both the retinal side and vitreal side of the ILM were analyzed. Results: Atomic force microscopy revealed no significant differences in elasticity of ILM specimens removed from eyes with or without ocriplasmin treatment. Undulated areas of the retinal side presented stiffer than the vitreal side of the ILM. Topographical mapping of both the vitreal and retinal side of the ILM showed no apparent alteration of the morphology in ocriplasmin-treated eyes compared to untreated eyes. Staining with BB resulted in an increase of tissue stiffness. Conclusions: Intravitreal injection of ocriplasmin does not change biomechanical properties of the human ILM. There is no evidence of a potential enzymatic effect of ocriplasmin interfering with the stiffness of this basement membrane. (C) 2016 S. Karger AG, Base

Protective Endogenous Cyclic Adenosine 5'-Monophosphate Signaling Triggered by Pemphigus Autoantibodies

Pemphigus vulgaris (PV) is an autoimmune skin disease mediated by autoantibodies directed against the cadherin-type cell adhesion molecules desmoglein (Dsg) 3 and Dsg1 and is characterized by loss of keratinocyte cohesion and epidermal blistering. Several intracellular signaling pathways, such as p38MAPK activation and RhoA inhibition, have been demonstrated to be altered following autoantibody binding and to be causally involved in loss of keratinocyte cohesion. In this paper, we demonstrate that cAMP-mediated signaling completely prevented blister formation in a neonatal pemphigus mouse model. Furthermore, elevation of cellular cAMP levels by forskolin/rolipram or β receptor agonist isoproterenol blocked loss of intercellular adhesion, depletion of cellular Dsg3, and morphologic changes induced by Ab fractions of PV patients (PV-IgG) in cultured keratinocytes. Incubation with PV-IgG alone increased cAMP levels, indicating that cAMP elevation may be a cellular response pathway to strengthen intercellular adhesion. Our data furthermore demonstrate that this protective pathway may involve protein kinase A signaling because protein kinase A inhibition attenuated recovery from PV-IgG–induced cell dissociation. Finally, cAMP increase interfered with PV-IgG–induced signaling by preventing p38MAPK activation both in vitro and in vivo. Taken together, our data provide insights into the cellular response mechanisms following pemphigus autoantibody binding and point to a possible novel and more specific therapeutic approach in pemphigus