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Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease with a complex underlying genetic etiology. Herein, we conduct whole-exome sequencing of a multigeneration EoE pedigree (discovery set) and 61 additional multiplex families with EoE (replication set). A series of rare, heterozygous, missense variants are identified in the genes encoding the desmosome-associated proteins DSP and PPL in 21% of the multiplex families. Esophageal biopsies from patients with these variants retain dilated intercellular spaces and decrease DSP and PPL expression even during disease remission. These variants affect barrier integrity, cell motility and RhoGTPase activity in esophageal epithelial cells and have increased susceptibility to calpain-14–mediated degradation. An acquired loss of esophageal DSP and PPL is present in non-familial EoE. Taken together, herein, we uncover a pathogenic role for desmosomal dysfunction in EoE, providing a deeper mechanistic understanding of tissue-specific allergic responses. Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease with a complex underlying genetic etiology. Here, the authors identify a series of rare variants in DSP and PPL in multiplex families with EoE and uncover a pathogenic role for desmosomal dysfunction in EoE.

Epidermal stem cells interact with the extracellular matrix (ECM) to regulate their differentiation and maintain skin architecture. Here, we demonstrate a role for basal epidermal stem cells (BECs)-ECM interaction in regulating adhesion molecules expressed by the periderm—the superficial epidermal cells (SECs) of the embryonic bilayered skin. Using the developing zebrafish fin fold, we identify BECs form distinct regions of collagen- versus laminin- enriched basement membranes through integrin-mediated adhesions. Mechanistically, collagen-associated BECs form desmosomes and adherens junctions (AJs) with SECs while laminin-associated BECs display reduced desmosomes but sustain AJs and actomyosin expression with SECs. Notably, we show both in vivo and in a bilayered human keratinocyte model, that laminin, compared to collagen, is sufficient to repress desmosome formation while sustaining AJs specifically at the interlayer cell contacts. In vivo, laminin deficiency enhances desmosome expression across layers and impairs the wound-healing capacity of SECs. This defect was partially rescued by genetic reduction of the desmosome protein Desmoplakin-1a, highlighting the role of ECM-dependent junctional specialization in mediating differences in SEC injury response. Overall, our findings identify that stem cells, through their matrix, establish specialized junctions in the overlying stratified epithelium, which contribute to skin healing properties. The extracellular matrix provides a critical scaffold for the epidermal layers of the skin. Here, He and Boraas et al. discover that the foundation for resilient skin healing in embryos lies in the supportive matrix beneath stem cells.

Desmosomes are essential cell-cell adhesion organelles that enable tension-prone tissues, like the skin and heart, to withstand mechanical stress. Desmosomal anomalies are associated with numerous epidermal disorders, cardiomyopathies, and cancer. Despite their critical importance, how desmosomes sense and respond to mechanical stimuli is not understood. Here, we combine super-resolution imaging in epithelial cells and primary cardiomyocytes, FRET-based tension sensors, atomistic computer simulations, and biochemical assays to demonstrate that actomyosin forces induce a conformational change in desmoplakin, a key cytoplasmic desmosomal protein. We show that in human breast cancer MCF7 cells, keratin-19 couples F-actin filaments to desmosomes and regulates the level of actomyosin forces integrated into the desmosomal complex. We demonstrate that actomyosin contractility reorients keratin intermediate filaments and directs force to desmoplakin along the keratin network, plausibly converting the N-terminal plakin domain from a folded to an extended conformation. We also show that desmoplakin undergoes a similar actomyosin force-dependent conformational change in primary cardiomyocytes, with the extent of the change affected by myofibril orientation. Our findings establish that desmoplakin is mechanosensitive and its structural states reflect the level of forces transmitted through the actin network across cell types. Desmosomes are adhesive junctions in tension prone tissues. Here, the authors show that cytoskeletal forces alter the conformation of desmoplakin, a key desmosomal protein, and propose this may enable the desmosome to sense and respond to mechanical stimuli.

Three miR-34 family members (miR-34a, miR-34b, and miR-34c) are clustered on two different chromosomal loci, Mir34a and Mir34b/c . These miRNAs have identical seed sequences, which are predicted to target the same set of genes. However, miR-34a and miR-34c have different sets of negatively correlated genes in lung adenocarcinoma data from The Cancer Genome Atlas. Therefore, we hypothesized that the individual miR-34 family members, which are tumor suppressive miRNAs, would have varying effects on lung tumorigenesis. To show this, we overexpressed each miR-34 cluster in murine lung cancer cells. MiR-34b/c enhanced cancer cell attachment and suppressed cell growth and invasion compared with miR-34a. In a syngeneic mouse model, both miR-34a and miR-34b/c blocked lung metastasis. However, miR-34b/c suppressed tumor growth more than miR-34a. MiR-34b/c also decreased the expression of mesenchymal markers ( Cdh2 and Fn1 ) and increased the expression of epithelial markers ( Cldn3 , Dsp , and miR-200 ) to a greater degree than miR-34a. These results imply that miR-34b and miR-34c inhibit epithelial-to-mesenchymal transition. Furthermore, knockout of all three miR-34 members promoted mutant Kras -driven lung tumor progression in mice. Similarly, lung adenocarcinoma patients with higher miR-34a/b/c levels had better survival rates than did those with lower levels. In summary, we suggest that miR-34b and miR-34c are more effective tumor suppressors than miR-34a. Cancer: Regulatory micro-RNAs could combine for treatment Exploring the effects of three similar small RNA molecules called micro-RNAs (miRNAs) that can restrict the activity of specific genes reveals how they might be used in cancer treatment. RNA is best known as messenger RNA, which carries a copy of a gene’s information into the cell cytoplasm to direct protein manufacture. Many small RNAs play less well-known but crucial roles by binding to messenger RNA molecules to regulate their activity. Researchers in South Korea and USA, led by Young-Ho Ahn at Ewha Womans University in Seoul, investigated how these miRNAs can suppress lung cancer in mice. Their results reveal details of how the miRNAs inhibit the expression of specific tumor-supporting genes. They suggest that three of the RNAs administered together might treat cancer more effectively than using only one as in previous trials.

Arrhythmogenic cardiomyopathy (AC) is a common cause of sudden cardiac arrest and death in young adults. It can be induced by different types of mutations throughout the desmoplakin gene including the R2834H mutation in the extreme carboxyterminus tail of desmoplakin (DP CT) which remains structurally uncharacterized and poorly understood. Here, we have created 3D models of DP CT which show the structural effects of AC-inducing mutations as well as the implications of post-translational modifications (PTMs). Our results suggest that, in absence of PTMs, positively charged wildtype DP CT likely folds back onto negatively-charged plectin repeat 14 of nearby plakin repeat domain C (PRD C) contributing to the recruitment of intermediate filaments (IFs). When phosphorylated and methylated, negatively-charged wildtype DP CT would then fold back onto positively-charged plectin repeat 17 of PRD C, promoting the repulsion of intermediate filaments. However, by preventing PTMs, the R2834H mutation would lead to the formation of a cytoplasmic mutant desmoplakin with a constitutively positive DP CT tail that would be aberrantly recruited by cytoplasmic IFs instead of desmosomes, potentially weakening cell-cell contacts and promoting AC. Virtual screening of FDA-approved drug libraries identified several promising drug candidates for the treatment of cardiocutaneous diseases through drug repurposing.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is a genetic disease caused by mutations in desmosomal proteins. The phenotypic hallmark of ARVC is fibroadipocytic replacement of cardiac myocytes, which is a unique phenotype with a yet-to-be-defined molecular mechanism. We established atrial myocyte cell lines expressing siRNA against desmoplakin (DP), responsible for human ARVC. We show suppression of DP expression leads to nuclear localization of the desmosomal protein plakoglobin and a 2-fold reduction in canonical Wnt/beta-catenin signaling through Tcf/Lef1 transcription factors. The ensuing phenotype is increased expression of adipogenic and fibrogenic genes and accumulation of fat droplets. We further show that cardiac-restricted deletion of Dsp, encoding DP, impairs cardiac morphogenesis and leads to high embryonic lethality in the homozygous state. Heterozygous DP-deficient mice exhibited excess adipocytes and fibrosis in the myocardium, increased myocyte apoptosis, cardiac dysfunction, and ventricular arrhythmias, thus recapitulating the phenotype of human ARVC. We believe our results provide for a novel molecular mechanism for the pathogenesis of ARVC and establish cardiac-restricted DP-deficient mice as a model for human ARVC. These findings could provide for the opportunity to identify new diagnostic markers and therapeutic targets in patients with ARVC.

Arrhythmogenic cardiomyopathy (ACM) is a rare genetic cardiac disease characterized by the progressive substitution of myocardium with fibro-fatty tissue. Clinically, ACM shows wide variability among patients; symptoms can include syncope and ventricular tachycardia but also sudden death, with the latter often being its sole manifestation. Approximately half of ACM patients have been found with variations in one or more genes encoding cardiac intercalated discs proteins; the most involved genes are plakophilin 2 (PKP2), desmoglein 2 (DSG2), and desmoplakin (DSP). Cardiac intercalated discs provide mechanical and electro-metabolic coupling among cardiomyocytes. Mechanical communication is guaranteed by the interaction of proteins of desmosomes and adheren junctions in the so-called area composita, whereas electro-metabolic coupling between adjacent cardiac cells depends on gap junctions. Although ACM has been first described almost thirty years ago, the pathogenic mechanism(s) leading to its development are still only partially known. Several studies with different animal models point to the involvement of the Wnt/β-catenin signaling in combination with the Hippo pathway. Here, we present an overview about the existing murine models of ACM harboring variants in intercalated disc components with a particular focus on the underlying pathogenic mechanisms. Prospectively, mechanistic insights into the disease pathogenesis will lead to the development of effective targeted therapies for ACM.

Plakin repeat domains (PRDs) are globular modules that mediate the interaction of plakin proteins with the intermediate filament (IF) cytoskeleton. These associations are vital for maintaining tissue integrity in cardiac muscle and epithelial tissues. PRDs are subject to mutations that give rise to cardiomyopathies such as arrhythmogenic right ventricular cardiomyopathy, characterised by ventricular arrhythmias and associated with an increased risk of sudden heart failure, and skin blistering diseases. Herein, we have examined the functional and structural effects of 12 disease-linked missense mutations, identified from the human gene mutation database, on the PRDs of the desmosomal protein desmoplakin. Five mutations (G2056R and E2193K in PRD-A, G2338R and G2375R in PRD-B and G2647D in PRD-C) rendered their respective PRD proteins either fully or partially insoluble following expression in bacterial cells. Each of the residues affected are conserved across plakin family members, inferring a crucial role in maintaining the structural integrity of the PRD. In transfected HeLa cells, the mutation G2375R adversely affected the targeting of a desmoplakin C-terminal construct containing all three PRDs to vimentin IFs. The deletion of PRD-B and PRD-C from the construct compromised its targeting to vimentin. Bioinformatic and structural modelling approaches provided multiple mechanisms by which the disease-causing mutations could potentially destabilise PRD structure and compromise cytoskeletal linkages. Overall, our data highlight potential molecular mechanisms underlying pathogenic missense mutations and could pave the way for informing novel curative interventions targeting cardiomyopathies and skin blistering disorders.

Background Arrhythmogenic cardiomyopathy (ACM) is a genetically inherited desmosome heart disease leading to life-threatening arrhythmias and sudden cardiac death. Currently, ACM treatment paradigms are merely symptom targeting. Recently, apremilast was shown to stabilize keratinocyte adhesion in the desmosomal disease pemphigus vulgaris. Therefore, this study investigated whether apremilast can be a therapeutic option for ACM. Methods Human induced pluripotent stem cells from a healthy control (hiPSC) and an ACM index patient (ACM-hiPSC) carrying a heterozygous desmoplakin ( DSP ) gene mutation (c.2854G > T, p.Glu952Ter), confirmed by whole exome sequencing (WES), were established. Cyclic-AMP ELISA, dissociation assay, immunostaining, and Western blotting analyses were performed in human iPSC-derived cardiomyocytes (hiPSC-CMs), murine HL-1 cardiomyocytes, and cardiac slices derived from wild-type (WT) mice, plakoglobin (PG, Jup ) knockout ( Jup −/− ) (murine ACM model) or PG Serine 665 phosphodeficient (JUP-S665A) mice. Microelectrode array (MEA) analyses in ventricular cardiac slices and Langendorff heart perfusion were performed to analyze heart rate variability and arrhythmia. Results ACM-hiPSC derived cardiomyocytes (ACM-hiPSC-CMs) revealed a significant loss of cohesion, which was rescued by apremilast. Further, treatment with apremilast strengthened basal cardiomyocyte cohesion in HL-1 cells and WT murine cardiac slices, paralleled by phosphorylation of PG at Serine 665 in human and murine models. In HL-1 cells, apremilast in addition activated ERK1/2, inhibition of which abolished apremilast-enhanced cardiomyocyte cohesion. Further, dissociation assays in slice cultures from JUP-S665A and Jup −/− mice revealed that PG is crucial for apremilast-enhanced cardiomyocyte cohesion. In parallel to enhanced cell adhesion, MEA and Langendorff measurements from WT and Jup −/− mice demonstrated decreased heart rate variability and arrhythmia after apremilast treatment. Conclusions Apremilast improves loss of cardiomyocyte cohesion, enhances localization of DSG2, and reduces arrhythmia in human and/or murine models of ACM ex vivo and in vitro, providing a novel treatment strategy for ACM by preserving desmosome function.

The cytoplasmic surface of intercellular junctions is a complex network of molecular interactions that link the extracellular region of the desmosomal cadherins with the cytoskeletal intermediate filaments. Although 3D structures of the major plaque components are known, the overall architecture remains unknown. We used cryoelectron tomography of vitreous sections from human epidermis to record 3D images of desmosomes in vivo and in situ at molecular resolution. Our results show that the architecture of the cytoplasmic surface of the desmosome is a 2D interconnected quasiperiodic lattice, with a similar spatial organization to the extracellular side. Subtomogram averaging of the plaque region reveals two distinct layers of the desmosomal plaque: a low-density layer closer to the membrane and a high-density layer further away from the membrane. When combined with a heuristic, allowing simultaneous constrained fitting of the high-resolution structures of the major plaque proteins (desmoplakin, plakophilin, and plakoglobin), it reveals their mutual molecular interactions and explains their stoichiometry. The arrangement suggests that alternate plakoglobin-desmoplakin complexes create a template on which desmosomal cadherins cluster before they stabilize extracellularly by binding at their N-terminal tips. Plakophilins are added as a molecular reinforcement to fill the gap between the formed plaque complexes and the plasma membrane.