Meeting report - Desmosome dysfunction and disease: Alpine desmosome disease meeting.

Desmosome diseases are caused by dysfunction of desmosomes, which anchor intermediate filaments (IFs) at sites of cell-cell adhesion. For many decades, the focus of attention has been on the role of actin filament-associated adherens junctions in development and disease, especially cancer. However, interference with the function of desmosomes, their molecular constituents or their attachments to IFs has now emerged as a major contributor to a variety of diseases affecting different tissues and organs including skin, heart and the digestive tract. The first Alpine desmosome disease meeting (ADDM) held in Grainau, Germany, in October 2022 brought together international researchers from the basic sciences with clinical experts from diverse fields to share and discuss their ideas and concepts on desmosome function and dysfunction in the different cell types involved in desmosome diseases. Besides the prototypic desmosomal diseases pemphigus and arrhythmogenic cardiomyopathy, the role of desmosome dysfunction in inflammatory bowel diseases and eosinophilic esophagitis was discussed.

Cytoskeletal anchorage of different Dsg3 pools revealed by combination of hybrid STED/SMFS-AFM.

Desmoglein 3 (Dsg3) is a desmosomal cadherin mediating cell adhesion within desmosomes and is the antigen of the autoimmune blistering skin disease pemphigus vulgaris. Therefore, understanding of the complex desmosome turnover process is of high biomedical relevance. Recently, super resolution microscopy was used to characterize desmosome composition and turnover. However, studies were limited because adhesion measurements on living cells were not possible in parallel. Before desmosomal cadherins are incorporated into nascent desmosomes, they are not bound to intermediate filaments but were suggested to be associated with the actin cytoskeleton. However, direct proof that adhesion of a pool of desmosomal cadherins is dependent on actin is missing. Here, we applied single-molecule force spectroscopy measurements with the novel single molecule hybrid-technique STED/SMFS-AFM to investigate the cytoskeletal anchorage of Dsg3 on living keratinocytes for the first time. By application of pharmacological agents we discriminated two different Dsg3 pools, only one of which is anchored to actin filaments. We applied the actin polymerization inhibitor Latrunculin B to modify the actin cytoskeleton and the PKCα activator PMA to modulate intermediate filament anchorage. On the cellular surface Dsg3 adhesion was actin-dependent. In contrast, at cell-cell contacts, Dsg3 adhesion was independent from actin but rather is regulated by PKC which is well established to control desmosome turn-over via intermediate filament anchorage. Taken together, using the novel STED/SMFS-AFM technique, we demonstrated the existence of two Dsg3 pools with different cytoskeletal anchorage mechanisms.

Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by Involving an Integrin-αVß6/TGF-ß Signaling Cascade.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell-cell adhesion complex, but the underlying mechanisms are not well understood. In the current study, we investigated the relevance of defective desmosomal adhesion for ACM development and progression. METHODS: We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell-cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVß6 antibody. RESULTS: The DSG2-W2A mutation impaired binding on molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVß6 and subsequent transforming growth factor-ß signaling as driver of cardiac fibrosis. Blocking integrin-αVß6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. CONCLUSIONS: We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVß6 and transforming growth factor-ß signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

Plakophilin 1 but not plakophilin 3 regulates desmoglein clustering.

Plakophilins (Pkp) are desmosomal plaque proteins crucial for desmosomal adhesion and participate in the regulation of desmosomal turnover and signaling. However, direct evidence that Pkps regulate clustering and molecular binding properties of desmosomal cadherins is missing. Here, keratinocytes lacking either Pkp1 or 3 in comparison to wild type (wt) keratinocytes were characterized with regard to their desmoglein (Dsg) 1- and 3-binding properties and their capability to induce Dsg3 clustering. As revealed by atomic force microscopy (AFM), both Pkp-deficient keratinocyte cell lines showed reduced membrane availability and binding frequency of Dsg1 and 3 at cell borders. Extracellular crosslinking and AFM cluster mapping demonstrated that Pkp1 but not Pkp3 is required for Dsg3 clustering. Accordingly, Dsg3 overexpression reconstituted cluster formation in Pkp3- but not Pkp1-deficient keratinocytes as shown by AFM and STED experiments. Taken together, these data demonstrate that both Pkp1 and 3 regulate Dsg membrane availability, whereas Pkp1 but not Pkp3 is required for Dsg3 clustering.

Adrenergic Signaling Strengthens Cardiac Myocyte Cohesion.

RATIONALE: The sympathetic nervous system is a major mediator of heart function. Intercalated discs composed of desmosomes, adherens junctions, and gap junctions provide the structural backbone for coordinated contraction of cardiac myocytes. OBJECTIVE: Gap junctions dynamically remodel to adapt to sympathetic signaling. However, it is unknown whether such rapid adaption also occurs for the adhesive function provided by desmosomes and adherens junctions. METHODS AND RESULTS: Atomic force microscopy revealed that ß-adrenergic signaling enhances both the number of desmoglein 2-specific interactions along cell junctions and the mean desmoglein 2-mediated binding forces, whereas N-cadherin-mediated interactions were not affected. This was accompanied by increased cell cohesion in cardiac myocyte cultures and murine heart slices. Enhanced desmoglein 2-positive contacts and increased junction length as revealed by immunofluorescence and electron microscopy reflected cAMP-induced reorganization of intercellular contacts. The mechanism underlying cAMP-mediated strengthening of desmoglein 2 binding was dependent on expression of the intercalated disc plaque protein plakoglobin (Pg) and direct phosphorylation at S665 by protein kinase A: Pg deficiency as well as overexpression of the phospho-deficient Pg-mutant S665A abrogated both cAMP-mediated junctional remodeling and increase of cohesion. Moreover, Pg knockout hearts failed to functionally adapt to adrenergic stimulation. CONCLUSIONS: Taken together, we provide first evidence for positive adhesiotropy as a new cardiac function of sympathetic signaling. Positive adhesiotropy is dependent on Pg phosphorylation at S665 by protein kinase A. This mechanism may be of high medical relevance because loss of junctional Pg is a hallmark of arrhythmogenic cardiomyopathy.

Premacular membranes in tissue culture.

PURPOSE: To investigate integrity and characteristics of human premacular membranes (PMM) with and without standard tissue culturing using mechanical traction. METHODS: Premacular membranes were harvested from 32 eyes of 32 patients with idiopathic macular pucker during standard vitrectomy. By flat-mount preparation with phase contrast and interference microscopy, specimens were prepared for time-lapse microscopy, immunocytochemistry, and transmission electron microscopy. Sixteen of 32 specimens were held in tissue culture with tangential traction by using entomological pins. Of these, specimens of 7 eyes were analyzed with and without tissue culturing for comparison. Primary antibodies were used for myofibroblasts, hyalocytes, macro-/microglial cells, and retinal pigment epithelial and immune cells. RESULTS: Hyalocytes, macroglia, and microglia composed the main cell composition of surgically removed PMM. Correlation of time-lapse microscopy with immunofluorescence microscopy identified fast and unidirectional moving small round cells as microglia. Slowly moving elongated large cells were characterized as alpha-smooth muscle actin (α-SMA)-positive myofibroblasts. Following tissue culturing with tangential stretch, enhanced positive immunolabelling for α-SMA and integrins-αv was seen. All other labelling results were demonstrated to be similar with pre-culture conditions. Ultrastructural analysis revealed fibroblasts, myofibroblasts, and proliferation of glial cells following tissue culture. CONCLUSION: This study demonstrates abundance of fibroblasts, myofibroblasts, and glial cells in PMM from idiopathic macular pucker following tissue culture with tangential stretch application. We found enhanced contractive properties of the cultured PPM that appear to indicate transdifferentiation of the cell composition. This in vitro model may improve understanding of pathogenesis in traction maculopathies and help to establish further anti-fibrosis treatment strategies.

Loss of desmoglein 2 promotes tumorigenic behavior in pancreatic cancer cells.

The ability to maintain cell-cell adhesion is crucial for tissue integrity and organization. Accordingly, loss of cohesiveness plays a critical role in cancer invasion and metastasis. Desmosomes are cell junctions providing strong intercellular adhesive strength and dysregulation of desmosomal constituents contributes to cancer progression through altered cell signaling pathways. Here, we focused on the desmosomal adhesion molecules Desmoglein 2 (Dsg2) and Desmocollin 2 (Dsc2), and their contribution to migration and invasion in pancreatic cancer cells. Silencing of Dsg2 but not Dsc2 resulted in loss of cell cohesion and enhanced migration, and invasion of pancreatic adenocarcinoma cells. To identify potential pathways regulated by Dsg2, we performed kinase arrays and detected the activity of ERK and growth factor receptors to be significantly enhanced in Dsg2-deficient cells. Consequently, inhibition of ERK phosphorylation in Dsg2 knockdown cells normalized migration. Loss of Dsg2 resulted in reduced levels of the desmosomal adapter protein and transcriptional regulator Plakoglobin (PG) in an ERK-dependent manner, whereas other desmosomal molecules were not altered. Overexpression of PG rescued enhanced migration induced by silencing of Dsg2. These results identify a novel pro-migratory pathway of pancreatic cancer cells in which loss of Dsg2 reduces the levels of PG via deregulated MAPK signaling.

Propiedades biomecánicas de la membrana limitante interna tras recibir tratamiento intravítreo con ocriplasmina.

Objetivo: Evaluar la rigidez de la membrana limitante interna (MLI) humana y evaluar los posibles cambios de las propiedades mecánicas tras administrar una inyección intravítrea de ocriplasmina para tratar la tracción vitreomacular. Métodos: Este estudio se compone de una serie de casos intervencionales y comparativos de 12 muestras de MLI extraídas mediante cirugía y obtenidas de forma consecutiva de 9 ojos de 9 pacientes después de someterse sin éxito a vitreólisis farmacológica con ocriplasmina. Durante el mismo periodo de tiempo, 16 muestras de otros 13 ojos sin tratamiento con ocriplasmina se obtuvieron mediante vitrectomía y sirvieron como controles. Todos los pacientes presentaron agujeros maculares o tracción vitreomacular y se sometieron a vitrectomía con disección de la MLI tanto con tinción con azul brillante (AB) como sin ella. Todas las muestras se analizaron con un microscopio de fuerza atómica con imágenes de las regiones de 25 × 25 µm. En todas las muestras, se analizaron tanto la parte de la retina como la del vítreo de la MLI. Resultados: La microscopia de fuerza atómica no reveló diferencias significativas en cuanto a elasticidad de las muestras de MLI extraídas de ojos con o sin tratamiento con ocriplasmina. Las áreas onduladas de la parte de la retina presentaron una mayor rigidez que la parte del vítreo de la MLI. La cartografía topográfica tanto de la parte del vítreo como de la retina de la MLI no mostró ninguna alteración aparente de la morfología en ojos tratados con ocriplasmina en comparación con los ojos no tratados. La tinción con azul brillante conllevó un aumento de la rigidez tisular. Conclusiones: Las inyecciones intravítreas de ocriplasmina no varían las propiedades biomecánicas de la MLI humana. No existen pruebas de un posible efecto enzimático que interfiera con la rigidez de esta membrana basal.

Cleavage and cell adhesion properties of human epithelial cell adhesion molecule (HEPCAM).

Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one ß-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aß-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and ß-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable.

E-cadherin and Src associate with extradesmosomal Dsg3 and modulate desmosome assembly and adhesion.

Desmosomes provide strong intercellular cohesion essential for the integrity of cells and tissues exposed to continuous mechanical stress. For desmosome assembly, constitutively synthesized desmosomal cadherins translocate to the cell-cell border, cluster and mature in the presence of Ca(2+) to stable cell contacts. As adherens junctions precede the formation of desmosomes, we investigated in this study the relationship between the classical cadherin E-cadherin and the desmosomal cadherin Desmoglein 3 (Dsg3), the latter of which is indispensable for cell-cell adhesion in keratinocytes. By using autoantibodies from patients with the blistering skin disease pemphigus vulgaris (PV), we showed in loss of function studies that E-cadherin compensates for effects of desmosomal disassembly. Overexpression of E-cadherin reduced the loss of cell cohesion induced by PV autoantibodies and attenuated activation of p38 MAPK. Silencing of E-cadherin abolished the localization of Dsg3 at the membrane and resulted in a shift of Dsg3 from the cytoskeletal to the non-cytoskeletal protein pool which conforms to the notion that E-cadherin regulates desmosome assembly. Mechanistically, we identified a complex consisting of extradesmosomal Dsg3, E-cadherin, ß-catenin and Src and that the stability of this complex is regulated by Src. Moreover, Dsg3 and E-cadherin are phosphorylated on tyrosine residues in a Src-dependent manner and Src activity is required for recruiting Dsg3 to the cytoskeletal pool as well as for desmosome maturation towards a Ca(2+)-insensitive state. Our data provide new insights into the role of E-cadherin and the contribution of Src signaling for formation and maintenance of desmosomal junctions.

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 × 25 µm. 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.

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.

Desmoglein 2 compensates for desmoglein 3 but does not control cell adhesion via regulation of p38 mitogen-activated protein kinase in keratinocytes.

Desmosomal cadherins are transmembrane adhesion molecules that provide cell adhesion by interacting in the intercellular space of adjacent cells. In keratinocytes, several desmoglein (Dsg1-4) and desmocollin (Dsc1-3) isoforms are coexpressed. We have shown previously that Dsg2 is less important for keratinocyte cohesion compared with Dsg3 and that the latter forms a complex with p38 MAPK. In this study, we compared the involvement of Dsg2 and Dsg3 in the p38 MAPK-dependent regulation of keratinocyte cohesion. We show that loss of cell adhesion and keratin filament retraction induced by Dsg3 depletion is ameliorated by specific p38 MAPK inhibition. Furthermore, in contrast to depletion of Dsg2, siRNA-mediated silencing of Dsg3 induced p38 MAPK activation, which is in line with immunoprecipitation experiments demonstrating the interaction of activated p38 MAPK with Dsg3 but not with Dsg2. Cell fractionation into a cytoskeleton-unbound and a cytoskeleton-anchored desmosome-containing pool revealed that Dsg3, in contrast to Dsg2, is present in relevant amounts in the unbound pool in which activated p38 MAPK is predominantly detectable. Moreover, because loss of cell adhesion by Dsg3 depletion was partially rescued by p38 MAPK inhibition, we conclude that, besides its function as an adhesion molecule, Dsg3 is strengthening cell cohesion via modulation of p38 MAPK-dependent keratin filament reorganization. Nevertheless, because subsequent targeting of Dsg3 in Dsg2-depleted cells led to drastically enhanced keratinocyte dissociation and Dsg2 was enhanced at the membrane in Dsg3 knockout cells, we conclude that Dsg2 compensates for Dsg3 loss of function.

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.

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 Ca(2+) 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 editor: Using 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.

Desmosomes and extradesmosomal adhesive signaling contacts in pemphigus.

Desmosomes are adhering junctions present in all cells of simple and complex epithelia. They are most abundant in cells of tissues subjected to extensive mechanical stress such as keratinocytes and cardiomyocytes. The core of desmosomes is built up of desmosomal cadherins, cadherin-type adhesion molecules that are tethered to intermediate filaments via adaptor proteins of the armadillo and the plakin family. In addition, desmosomal cadherins are present outside of desmosomes. Recent investigations indicate that these molecules are involved in adhesion-dependent and adhesion-independent signaling and thus have functions different from the adhesive properties of their counterparts within desmosomes. Impaired adhesion of desmosomal cadherins both within and outside of desmosomes is the cause of the blistering skin disease pemphigus. Autoantibodies interfere with the binding of desmosomal cadherins and alter intracellular signaling pathways, the latter of which is necessary for loss of cell adhesion. Among the plethora of signaling molecules reported, altered activities of p38MAPK, protein kinase C, and epidermal growth factor receptor (EGFR) are most relevant. This review highlights the recent data on signaling by desmosomal cadherins and the mechanisms involved in pemphigus skin blistering.

CD109 drives pro-tumorigenic cell properties in human non-small cell lung cancer through interaction with desmoglein-2.

Cluster of differentiation 109 (CD109) is a glycosylphosphatidylinositol (GPI) anchored cell surface protein, expressed on epithelial and endothelial cells, CD4+ and CD8+ T-cells, and premature lymphocytes. CD109 interacts with different cell surface receptors and thereby modulates intracellular signaling pathways, which ultimately changes cellular functions. One well-studied example is the interaction of CD109 with the TGFß/TGFß-receptor complex at the cell surface. CD109 silences intracellular SMAD2/3 signaling and targets TGFß/TGFß-receptor to the endosomal/lysosomal compartment. In recent years, CD109 emerged as a tumor marker for different tumor entities and expression of CD109 could be linked to adverse outcome in patients. In this study, we show that silencing of CD109 in human non-small cell lung cancer (NSCLC) cells, returns these cells to an epithelial like growth phenotype. On the transcriptional level, we describe changes in cell-cell contact and epithelial-mesenchymal transition associated gene clusters. At the cell surface, we identify desmoglein-2 (DSG2) as a new interaction partner of CD109 and demonstrate CD109 dependent targeting of DSG2 to the apical cell surface, where it forms desmosomes between apical and basal cell poles. Both, CD109 and DSG2 are genetic risk factors, linked to reduced overall survival in lung adenocarcinoma patients (subtype of NSCLC). In this study, we show the expression of both proteins in the same tumor and suggest a new CD109-DSG2 axis in NSCLC patients that could present a targetable therapeutic option in the future.

DPM1 modulates desmosomal adhesion and epidermal differentiation through SERPINB5.

Glycosylation is essential to facilitate cell-cell adhesion and differentiation. We determined the role of the dolichol phosphate mannosyltransferase (DPM) complex, a central regulator for glycosylation, for desmosomal adhesive function and epidermal differentiation. Deletion of the key molecule of the DPM complex, DPM1, in human keratinocytes resulted in weakened cell-cell adhesion, impaired localization of the desmosomal components desmoplakin and desmoglein-2, and led to cytoskeletal organization defects in human keratinocytes. In a 3D organotypic human epidermis model, loss of DPM1 caused impaired differentiation with abnormally increased cornification, reduced thickness of non-corneal layers, and formation of intercellular gaps in the epidermis. Using proteomic approaches, SERPINB5 was identified as a DPM1-dependent interaction partner of desmoplakin. Mechanistically, SERPINB5 reduced desmoplakin phosphorylation at serine 176, which was required for strong intercellular adhesion. These results uncover a novel role of the DPM complex in connecting desmosomal adhesion with epidermal differentiation.

EGFR Inhibition by Erlotinib Rescues Desmosome Ultrastructure and Keratin Anchorage and Protects Against Pemphigus Vulgaris IgG-Induced Acantholysis in Human Epidermis.

Pemphigus is a severe blistering disease caused by autoantibodies primarily against the desmosomal cadherins desmoglein (DSG)1 and DSG3 which impair desmosome integrity. Especially for the acute phase, additional treatment options allowing to reduce corticosteroids would fulfill an unmet medical need. Here, we provide evidence that epidermal growth factor receptor (EGFR) inhibition by erlotinib ameliorates pemphigus vulgaris immunoglobulin G (PV-IgG) -induced acantholysis in intact human epidermis. PV-IgG caused phosphorylation of EGFR (Y845) and SRC in human epidermis. In line with that, a phosphotyrosine kinome analysis revealed a robust response associated with EGFR and SRC family kinase signaling in response to PV-IgG but not pemphigus foliaceus autoantibodies. Erlotinib inhibited PV-IgG-induced epidermal blistering and EGFR phosphorylation, loss of desmosomes as well as ultrastructural alterations of desmosome size, plaque symmetry, keratin filament insertion and restored the desmosome midline considered as hallmark of mature desmosomes. Erlotinib enhanced both single molecule DSG3 binding frequency and strength and delayed DSG3 fluorescence recovery supporting that EGFR inhibition increases DSG3 availability and cytoskeletal anchorage. Our data indicate that EGFR is a promising target for pemphigus therapy due to its link to several signaling pathways known to be involved in pemphigus pathogenesis.

IgG against the Membrane-Proximal Portion of the Desmoglein 3 Ectodomain Induces Loss of Keratinocyte Adhesion, a Hallmark in Pemphigus Vulgaris.

Pemphigus vulgaris is a severe autoimmune blistering disease characterized by IgG autoantibodies (auto-abs) against the desmosomal adhesion molecules desmoglein (DSG) 3 and DSG1. Underlying mechanisms leading to blister formation upon binding of DSG-specific IgG auto-abs are not fully understood. Numerous studies showed the pathogenicity of IgG auto-ab binding to the aminoterminal region 1 (EC1) of the DSG3 ectodomain. However, auto-abs in pemphigus vulgaris are polyclonal, including IgG against both aminoterminal- and membrane-proximal epitopes of the DSG3 ectodomain. In this study, the pathogenicity of a previously uncharacterized murine monoclonal IgG antibody, 2G4, directed against the membrane-proximal region (EC5) of the DSG3 ectodomain was characterized and tested in various specificity and functionality assays. The results clearly show that 2G4 is capable of inhibiting intercellular keratinocyte adhesion and of inducing cellular DSG3 redistribution by activation of the p38MAPK signal transduction pathway. In this study, we provide evidence that an IgG auto-abs directed against the membrane-proximal region EC5 of DSG3 induces acantholysis, the hallmark in pemphigus vulgaris. These findings challenge the current concept that IgG auto-abs targeting the NH2-terminal portion of the DSG3 ectodomain are pathogenic only. Our study provides further aspects for a deeper understanding of desmosomal keratinocyte adhesion and improves our insight into the complex auto-ab‒induced blister formation in pemphigus vulgaris.