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1.
Front Immunol ; 13: 884067, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35720332

RESUMO

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease caused primarily by autoantibodies (PV-IgG) against the desmosomal adhesion proteins desmoglein (Dsg)1 and Dsg3. PV patient lesions are characterized by flaccid blisters and ultrastructurally by defined hallmarks including a reduction in desmosome number and size, formation of split desmosomes, as well as uncoupling of keratin filaments from desmosomes. The pathophysiology underlying the disease is known to involve several intracellular signaling pathways downstream of PV-IgG binding. Here, we summarize our studies in which we used transmission electron microscopy to characterize the roles of signaling pathways in the pathogenic effects of PV-IgG on desmosome ultrastructure in a human ex vivo skin model. Blister scores revealed inhibition of p38MAPK, ERK and PLC/Ca2+ to be protective in human epidermis. In contrast, inhibition of Src and PKC, which were shown to be protective in cell cultures and murine models, was not effective for human skin explants. The ultrastructural analysis revealed that for preventing skin blistering at least desmosome number (as modulated by ERK) or keratin filament insertion (as modulated by PLC/Ca2+) need to be ameliorated. Other pathways such as p38MAPK regulate desmosome number, size, and keratin insertion indicating that they control desmosome assembly and disassembly on different levels. Taken together, studies in human skin delineate target mechanisms for the treatment of pemphigus patients. In addition, ultrastructural analysis supports defining the specific role of a given signaling molecule in desmosome turnover at ultrastructural level.


Assuntos
Pênfigo , Acantólise/metabolismo , Acantólise/patologia , Animais , Vesícula/metabolismo , Desmossomos/metabolismo , Humanos , Imunoglobulina G , Queratinas/metabolismo , Camundongos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
2.
Front Immunol ; 13: 884241, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35711465

RESUMO

Desmosomes are important epidermal adhesion units and signalling hubs, which play an important role in pemphigus pathogenesis. Different expression patterns of the pemphigus autoantigens desmoglein (Dsg)1 and Dsg3 across different epidermal layers have been demonstrated. However, little is known about changes in desmosome composition in different epidermal layers or in patient skin. The aim of this study was thus to characterize desmosome composition in healthy and pemphigus skin using super-resolution microscopy. An increasing Dsg1/Dsg3 ratio from lower basal (BL) to uppermost granular layer (GL) was observed. Within BL desmosomes, Dsg1 and Dsg3 were more homogeneously distributed whereas superficial desmosomes mostly comprised one of the two molecules or domains containing either one but not both. Extradesmosomal, desmoplakin (Dp)-independent, co-localization of Dsg3 with plakoglobin (Pg) was found mostly in BL and extradesmosomal Dsg1 co-localization with Pg in all layers. In contrast, in the spinous layer (SL) most Dsg1 and Dsg3 staining was confined to desmosomes, as revealed by the co-localization with Dp. In pemphigus patient skin, Dsg1 and Dsg3 immunostaining was altered especially along blister edges. The number of desmosomes in patient skin was reduced significantly in basal and spinous layer keratinocytes with only few split desmosomes found. In addition, Dsg1-Pg co-localization at the apical BL and Dsg3-Pg co-localization in SL were significantly reduced in patients, suggesting that that extradesmosomal Dsg molecules were affected. These results support the hypothesis that pemphigus is a desmosome assembly disease and may help to explain histopathologic differences between pemphigus phenotypes.


Assuntos
Pênfigo , Desmogleína 1/metabolismo , Desmogleína 3/metabolismo , Desmossomos , Epiderme , Humanos , Pele
3.
Cell Mol Life Sci ; 79(5): 223, 2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35380280

RESUMO

Desmosomes are intercellular junctions which mediate cohesion and communication in tissues exposed to mechanical strain by tethering the intermediate filament cytoskeleton to the plasma membrane. While mature desmosomes are characterized by a hyperadhesive, Ca2+-independent state, they transiently loose this state during wound healing, pathogenesis and tissue regeneration. The mechanisms controlling the hyperadhesive state remain incompletely understood. Here, we show that upon Ca2+-induced keratinocyte differentiation, expression of keratin 17 (K17) prevents the formation of stable and hyperadhesive desmosomes, accompanied by a significant reduction of desmoplakin (DP), plakophilin-1 (PKP1), desmoglein-1 (Dsg1) and -3 (Dsg3) at intercellular cell borders. Atomic force microscopy revealed that both increased binding strength of desmoglein-3 molecules and amount of desmoglein-3 oligomers, known hallmarks of hyperadhesion, were reduced in K17- compared to K14-expressing cells. Importantly, overexpression of Dsg3 or DPII enhanced their localization at intercellular cell borders and increased the formation of Dsg3 oligomers, resulting in stable, hyperadhesive desmosomes despite the presence of K17. Notably, PKP1 was enriched in these desmosomes. Quantitative image analysis revealed that DPII overexpression contributed to desmosome hyperadhesion by increasing the abundance of K5/K17-positive keratin filaments in the proximity of desmosomes enriched in desmoglein-3. Thus, our data show that hyperadhesion can result from recruitment of keratin isotypes K5/K17 to desmosomes or from enhanced expression of DP and Dsg3 irrespective of keratin composition. The notion that hyperadhesive desmosomes failed to form in the absence of keratins underscores the essential role of keratins and suggest bidirectional control mechanisms at several levels.


Assuntos
Desmossomos , Queratinas , Adesão Celular , Citoesqueleto/metabolismo , Desmogleínas/metabolismo , Desmossomos/metabolismo , Queratinócitos/metabolismo , Queratinas/metabolismo
4.
Biophys J ; 121(7): 1322-1335, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35183520

RESUMO

Desmoglein (Dsg) 2 is a ubiquitously expressed desmosomal cadherin. Particularly, it is present in all cell types forming desmosomes, including epithelial cells and cardiac myocytes and is upregulated in the autoimmune skin disease pemphigus. Thus, we here characterized the binding properties of Dsg2 in more detail using atomic force microscopy (AFM). Dsg2 exhibits homophilic interactions and also heterophilic interactions with the desmosomal cadherin desmocollin (Dsc) 2, and further with the classical cadherins E-cadherin (E-Cad) and N-cadherin (N-Cad), which may be relevant for cross talk between desmosomes and adherens junctions in epithelia and cardiac myocytes. We found that all homo- and heterophilic interactions were Ca2+-dependent. All binding forces observed are in the same force range, i.e., 30 to 40 pN, except for the Dsg2/E-Cad unbinding force, which with 45 pN is significantly higher. To further characterize the nature of the interactions, we used tryptophan, a critical amino acid required for trans-interaction, and a tandem peptide (TP) designed to cross-link Dsg isoforms. TP was sufficient to prevent the tryptophan-induced loss of Dsg2 interaction with the desmosomal cadherins Dsg2 and Dsc2; however, not with the classical cadherins E-Cad and N-Cad, indicating that the interaction modes of Dsg2 with desmosomal and classical cadherins differ. TP rescued the tryptophan-induced loss of Dsg2 binding on living enterocytes, suggesting that interaction with desmosomal cadherins may be more relevant. In summary, the data suggest that the ubiquitous desmosomal cadherin Dsg2 enables the cross talk with adherens junctions by interacting with multiple binding partners with implications for proper adhesive function in healthy and diseased states.


Assuntos
Desmogleína 2 , Desmossomos , Caderinas/metabolismo , Adesão Celular , Desmogleína 2/análise , Desmogleína 2/metabolismo , Desmossomos/metabolismo , Células Epiteliais/metabolismo , Triptofano/metabolismo
5.
Ann Anat ; 241: 151904, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35131450

RESUMO

Pemphigus vulgaris (PV) is a severe autoimmune blistering skin disease caused primarily by autoantibodies (PV-IgG) against the desmosomal cadherins desmoglein (Dsg) 1 and Dsg 3. Pemphigus is a model disease to study desmosome regulation because patient lesions are characterized by ultrastructural hallmarks including loss, shrinkage and splitting of desmosomes as well as by retraction of keratin filaments. The mechanisms underlying the disease are not completely understood but involve several intracellular signaling pathways triggered by autoantibody binding. Recently, we demonstrated that Phosphoinositid-Phospholipase C (PLC) and Ca2+ signaling are required for acantholysis in human epidermis. Here, we used transmission electron microscopy to characterize the role of PLC and Ca2+ signaling with regard to the pathogenic effects of PV-IgG on desmosome ultrastructure in human ex vivo skin model. First, we observed that the PV-IgG used in this study significantly reduced desmosome length and caused uncoupling of desmosomes from keratin filaments. Moreover, PV-IgG enhanced the number of split desmosomes but did not cause a significant loss of desmosomes. We found that inhibition of PLC and Ca2+ signaling significantly blocked keratin filament uncoupling but not shrinkage of desmosomes. Blocking Ca2+ flux prevented desmosome splitting. The ultrastructural analysis revealed that for preventing skin blistering it is sufficient to enhance keratin filament insertion, which is regulated by PLC/ Ca2+. Here, we underscore the unique role of electron microscopy to investigate the underlying mechanisms by which a signaling pathway regulates desmosome ultrastructure in pemphigus.


Assuntos
Pênfigo , Desmossomos , Humanos , Imunoglobulina G , Queratinócitos/metabolismo , Queratinas/metabolismo , Microscopia Eletrônica , Pênfigo/metabolismo , Pênfigo/patologia , Transdução de Sinais , Fosfolipases Tipo C/análise
7.
J Cell Biol ; 221(3)2022 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-35139142

RESUMO

The coordinated interplay of cytoskeletal networks critically determines tissue biomechanics and structural integrity. Here, we show that plectin, a major intermediate filament-based cytolinker protein, orchestrates cortical cytoskeletal networks in epithelial sheets to support intercellular junctions. By combining CRISPR/Cas9-based gene editing and pharmacological inhibition, we demonstrate that in an F-actin-dependent context, plectin is essential for the formation of the circumferential keratin rim, organization of radial keratin spokes, and desmosomal patterning. In the absence of plectin-mediated cytoskeletal cross-linking, the aberrant keratin-desmosome (DSM)-network feeds back to the actin cytoskeleton, which results in elevated actomyosin contractility. Also, by complementing a predictive mechanical model with Förster resonance energy transfer-based tension sensors, we provide evidence that in the absence of cytoskeletal cross-linking, major intercellular junctions (adherens junctions and DSMs) are under intrinsically generated tensile stress. Defective cytoarchitecture and tensional disequilibrium result in reduced intercellular cohesion, associated with general destabilization of plectin-deficient sheets upon mechanical stress.


Assuntos
Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Plectina/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Citoesqueleto/ultraestrutura , Desmossomos/metabolismo , Desmossomos/ultraestrutura , Cães , Células Epiteliais/ultraestrutura , Técnicas de Inativação de Genes , Humanos , Queratinas/metabolismo , Células MCF-7 , Células Madin Darby de Rim Canino , Camundongos , Isoformas de Proteínas/metabolismo , Resistência à Tração
8.
Stem Cell Reports ; 17(2): 337-351, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35063130

RESUMO

Loss-of-function mutations in PKP2, which encodes plakophilin-2, cause arrhythmogenic cardiomyopathy (AC). Restoration of deficient molecules can serve as upstream therapy, thereby requiring a human model that recapitulates disease pathology and provides distinct readouts in phenotypic analysis for proof of concept for gene replacement therapy. Here, we generated isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of plakophilin-2 from a patient with AC carrying a heterozygous frameshift PKP2 mutation. After monolayer differentiation, plakophilin-2 deficiency led to reduced contractility, disrupted intercalated disc structures, and impaired desmosome assembly in iPSC-CMs. Allele-specific fluorescent labeling of endogenous DSG2 encoding desmoglein-2 in the generated isogenic lines enabled real-time desmosome-imaging under an adjusted dose of plakophilin-2. Adeno-associated virus-mediated gene replacement of PKP2 recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome-imaging in plakophilin-2-deficient iPSC-CMs. Our isogenic set of iPSC-CMs recapitulates AC pathology and provides a rapid and convenient cellular platform for therapeutic development.


Assuntos
Arritmias Cardíacas/patologia , Desmossomos/fisiologia , Contração Miocárdica/fisiologia , Placofilinas/metabolismo , Arritmias Cardíacas/genética , Sistemas CRISPR-Cas/genética , Diferenciação Celular , Feminino , Edição de Genes , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Modelos Biológicos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Linhagem , Placofilinas/genética
10.
J Invest Dermatol ; 142(2): 272-274, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34799122

RESUMO

Single gene disorders are ideally suited to establish robust genotype‒phenotype correlations and provide excellent opportunities to understand molecular pathomechanisms with relevance to complex disorders. The observation that patients diagnosed with the same causative mutation can present with phenotypic disease variability illustrates the significant role of disease modifiers and warns against oversimplification. In a new article in the Journal of Investigative Dermatology, Zimmer et al. (2021) analyze two mutations located in the desmoglein (DSG) 1 transmembrane domain (TMD) and find that both mutants fail to assemble into desmosomes owing to reduced membrane trafficking and lipid raft targeting. One mutation maintained normal protein expression levels and turnover relative to those of wild-type (WT) DSG1, and behaved as a dominant negative. The second mutant showed reduced stability and increased turnover compared with WT DSG1 as well as reduced desmosome size and abundance. A full understanding of the TMD of DSG1 requires cell biological approaches, underscoring the value of cell biology in biomedical research in general.


Assuntos
Desmogleína 1 , Desmossomos , Desmogleína 1/genética , Desmossomos/genética , Humanos , Microdomínios da Membrana , Mutação
11.
J Invest Dermatol ; 142(2): 323-332.e8, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34352264

RESUMO

Dominant and recessive mutations in the desmosomal cadherin, desmoglein (DSG) 1, cause the skin diseases palmoplantar keratoderma (PPK) and severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome, respectively. In this study, we compare two dominant missense mutations in the DSG1 transmembrane domain (TMD), G557R and G562R, causing PPK (DSG1PPK-TMD) and SAM syndrome (DSG1SAM-TMD), respectively, to determine the differing pathomechanisms of these mutants. Expressing the DSG1TMD mutants in a DSG-null background, we use cellular and biochemical assays to reveal the differences in the mechanistic behavior of each mutant. Super-resolution microscopy and functional assays showed a failure by both mutants to assemble desmosomes due to reduced membrane trafficking and lipid raft targeting. DSG1SAM-TMD maintained normal expression levels and turnover relative to wildtype DSG1, but DSG1PPK-TMD lacked stability, leading to increased turnover through lysosomal and proteasomal pathways and reduced expression levels. These results differentiate the underlying pathomechanisms of these disorders, suggesting that DSG1SAM-TMD acts dominant negatively, whereas DSG1PPK-TMD is a loss-of-function mutation causing the milder PPK disease phenotype. These mutants portray the importance of the DSG TMD in desmosome function and suggest that a greater understanding of the desmosomal cadherin TMDs will further our understanding of the role that desmosomes play in epidermal pathophysiology.


Assuntos
Desmogleína 1/genética , Desmossomos/patologia , Epiderme/patologia , Ceratodermia Palmar e Plantar/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Desmogleína 1/metabolismo , Caderinas de Desmossomos/metabolismo , Desmossomos/metabolismo , Epiderme/metabolismo , Humanos , Ceratodermia Palmar e Plantar/patologia , Mutação com Perda de Função , Microdomínios da Membrana/metabolismo , Mutação de Sentido Incorreto , Domínios Proteicos/genética , Estabilidade Proteica
12.
Can J Cardiol ; 38(1): 41-48, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34500006

RESUMO

BACKGROUND: Deleterious rare variants in genes encoding desmosome proteins have been identified as the essential basis of arrhythmogenic cardiomyopathy (ACM) and detected in dilated cardiomyopathy, but the relationship between deleterious rare desmosomal variants and hypertrophic cardiomyopathy (HCM) remains unknown. METHODS: Whole exome sequencing was performed in 1000 patients with HCM and 761 non-HCM controls to search for deleterious rare variants in genes encoding desmosomal proteins including PKP2, JUP, DSC2, DSG2, and DSP. Clinical phenotypes were assessed in patients with HCM, and patients with deleterious rare desmosomal variants underwent evaluation of ACM revised Task Force Criteria. RESULTS: A total of 27 deleterious rare desmosomal variants were present in 24 (2.4%) patients with HCM and 5 (0.66%) controls. The variants were more prevalent in the patients with HCM than in the controls (P = 0.004). The majority of patients possessing deleterious rare desmosomal variants could not be diagnosed as ACM. Moreover, the patients with deleterious rare desmosomal variants possessed several distinctive clinical features compared with patients without such variants, including a higher incidence of nonsustained ventricular tachycardia (29.2% vs 4.5%, P < 0.001), left bundle branch block (33.3% vs 1.6%, P < 0.001), and right ventricular involvement for an HCM phenotype (29.2% vs 0.30%, P < 0.001). CONCLUSIONS: We screened deleterious rare desmosomal variants in a large HCM case-control cohort and found deleterious rare desmosomal variants can be relevant to HCM. Moreover, our data indicated deleterious rare desmosomal variants were associated with distinctive clinical features of HCM. These findings require validation in other HCM cohorts.


Assuntos
Cardiomiopatia Hipertrófica/genética , DNA/genética , Desmossomos/genética , Mutação , Função Ventricular Direita/fisiologia , Adulto , Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/fisiopatologia , Análise Mutacional de DNA , Desmossomos/metabolismo , Eletrocardiografia Ambulatorial/métodos , Feminino , Seguimentos , Predisposição Genética para Doença , Humanos , Imagem Cinética por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Estudos Retrospectivos , Sequenciamento Completo do Genoma/métodos
13.
Annu Rev Pathol ; 17: 47-72, 2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-34425055

RESUMO

Desmosomal cadherins are a recent evolutionary innovation that make up the adhesive core of highly specialized intercellular junctions called desmosomes. Desmosomal cadherins, which are grouped into desmogleins and desmocollins, are related to the classical cadherins, but their cytoplasmic domains are tailored for anchoring intermediate filaments instead of actin to sites of cell-cell adhesion. The resulting junctions are critical for resisting mechanical stress in tissues such as the skin and heart. Desmosomal cadherins also act as signaling hubs that promote differentiation and facilitate morphogenesis, creating more complex and effective tissue barriers in vertebrate tissues. Interference with desmosomal cadherin adhesive and supra-adhesive functions leads to a variety of autoimmune, hereditary, toxin-mediated, and malignant diseases. We review our current understanding of how desmosomal cadherins contribute to human health and disease, highlight gaps in our knowledge about their regulation and function, and introduce promising new directions toward combatting desmosome-related diseases.


Assuntos
Desmocolinas , Desmossomos , Caderinas/fisiologia , Adesão Celular/fisiologia , Desmossomos/fisiologia , Humanos , Transdução de Sinais
15.
Cell Mol Gastroenterol Hepatol ; 13(4): 1181-1200, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34929421

RESUMO

BACKGROUND & AIMS: Desmosomes are intercellular junctions connecting keratin intermediate filaments of neighboring cells. The cadherins desmoglein 2 (Dsg2) and desmocollin 2 mediate cell-cell adhesion, whereas desmoplakin (Dsp) provides the attachment of desmosomes to keratins. Although the importance of the desmosome-keratin network is well established in mechanically challenged tissues, we aimed to assess the currently understudied function of desmosomal proteins in intestinal epithelia. METHODS: We analyzed the intestine-specific villin-Cre DSP (DSPΔIEC) and the combined intestine-specific DSG2/DSPΔIEC (ΔDsg2/Dsp) knockout mice. Cross-breeding with keratin 8-yellow fluorescent protein knock-in mice and generation of organoids was performed to visualize the keratin network. A Dsp-deficient colorectal carcinoma HT29-derived cell line was generated and the role of Dsp in adhesion and mechanical stress was studied in dispase assays, after exposure to uniaxial cell stretching and during scratch assay. RESULTS: The intestine of DSPΔIEC mice was histopathologically inconspicuous. Intestinal epithelial cells, however, showed an accelerated migration along the crypt and an enhanced shedding into the lumen. Increased intestinal permeability and altered levels of desmosomal proteins were detected. An inconspicuous phenotype also was seen in ΔDsg2/Dsp mice. After dextran sodium sulfate treatment, DSPΔIEC mice developed more pronounced colitis. A retracted keratin network was seen in the intestinal epithelium of DSPΔIEC/keratin 8-yellow fluorescent protein mice and organoids derived from these mice presented a collapsed keratin network. The level, phosphorylation status, and solubility of keratins were not affected. Dsp-deficient HT29 cells had an impaired cell adhesion and suffered from increased cellular damage after stretch. CONCLUSIONS: Our results show that Dsp is required for proper keratin network architecture in intestinal epithelia, mechanical resilience, and adhesion, thereby protecting from injury.


Assuntos
Desmossomos , Queratinas , Animais , Adesão Celular , Desmoplaquinas/metabolismo , Desmossomos/metabolismo , Queratina-8/metabolismo , Queratinas/metabolismo , Camundongos
16.
J Clin Invest ; 132(3)2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34905516

RESUMO

Desmoglein 1 (Dsg1) is a cadherin restricted to stratified tissues of terrestrial vertebrates, which serve as essential physical and immune barriers. Dsg1 loss-of-function mutations in humans result in skin lesions and multiple allergies, and isolated patient keratinocytes exhibit increased proallergic cytokine expression. However, the mechanism by which genetic deficiency of Dsg1 causes chronic inflammation is unknown. To determine the systemic response to Dsg1 loss, we deleted the 3 tandem Dsg1 genes in mice. Whole transcriptome analysis of embryonic Dsg1-/- skin showed a delay in expression of adhesion/differentiation/keratinization genes at E17.5, a subset of which recovered or increased by E18.5. Comparing epidermal transcriptomes from Dsg1-deficient mice and humans revealed a shared IL-17-skewed inflammatory signature. Although the impaired intercellular adhesion observed in Dsg1-/- mice resembles that resulting from anti-Dsg1 pemphigus foliaceus antibodies, pemphigus skin lesions exhibit a weaker IL-17 signature. Consistent with the clinical importance of these findings, treatment of 2 Dsg1-deficient patients with an IL-12/IL-23 antagonist originally developed for psoriasis resulted in improvement of skin lesions. Thus, beyond impairing the physical barrier, loss of Dsg1 function through gene mutation results in a psoriatic-like inflammatory signature before birth, and treatment with a targeted therapy significantly improved skin lesions in patients.


Assuntos
Desmogleína 1/imunologia , Desmossomos/imunologia , Queratinócitos/imunologia , Pênfigo/imunologia , Células Th17/imunologia , Animais , Desmogleína 1/genética , Desmossomos/genética , Camundongos , Pênfigo/genética
17.
Nat Commun ; 12(1): 6795, 2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34815391

RESUMO

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.


Assuntos
Desmoplaquinas/genética , Esofagite Eosinofílica/genética , Mucosa Esofágica/patologia , Plaquinas/genética , Adolescente , Biópsia , Calpaína/metabolismo , Estudos de Casos e Controles , Criança , Análise Mutacional de DNA , Desmoplaquinas/metabolismo , Desmossomos/patologia , Esofagite Eosinofílica/patologia , Mucosa Esofágica/citologia , Feminino , Células HEK293 , Células HaCaT , Heterozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Plaquinas/metabolismo , Proteólise , RNA-Seq , Análise de Célula Única , Sequenciamento Completo do Exoma
18.
J Cell Sci ; 134(21)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34633031

RESUMO

The vast majority of breast cancer-associated deaths are due to metastatic spread of cancer cells, a process aided by epithelial-to-mesenchymal transition (EMT). Mounting evidence has indicated that long non-coding RNAs (lncRNAs) also contribute to tumor progression. We report the identification of 114 novel lncRNAs that change their expression during TGFß-induced EMT in murine breast cancer cells (referred to as EMT-associated transcripts; ETs). Of these, the ET-20 gene localizes in antisense orientation within the tenascin C (Tnc) gene locus. TNC is an extracellular matrix protein that is critical for EMT and metastasis formation. Both ET-20 and Tnc are regulated by the EMT master transcription factor Sox4. Notably, ablation of ET-20 lncRNA effectively blocks Tnc expression and with it EMT. Mechanistically, ET-20 interacts with desmosomal proteins, thereby impairing epithelial desmosomes and promoting EMT. A short transcript variant of ET-20 is shown to be upregulated in invasive human breast cancer cell lines, where it also promotes EMT. Targeting ET-20 appears to be a therapeutically attractive lead to restrain EMT and breast cancer metastasis in addition to its potential utility as a biomarker for invasive breast cancer.


Assuntos
Neoplasias da Mama , RNA Longo não Codificante , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Desmossomos/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Invasividade Neoplásica/genética , RNA Longo não Codificante/genética , Fatores de Transcrição SOXC
19.
J Cell Sci ; 134(21)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635908

RESUMO

Desmosomes, strong cell-cell junctions of epithelia and cardiac muscle, link intermediate filaments to cell membranes and mechanically integrate cells across tissues, dissipating mechanical stress. They comprise five major protein classes - desmocollins and desmogleins (the desmosomal cadherins), plakoglobin, plakophilins and desmoplakin - whose individual contribution to the structure and turnover of desmosomes is poorly understood. Using live-cell imaging together with fluorescence recovery after photobleaching (FRAP) and fluorescence loss and localisation after photobleaching (FLAP), we show that desmosomes consist of two contrasting protein moieties or modules: a very stable moiety of desmosomal cadherins, desmoplakin and plakoglobin, and a highly mobile plakophilin (Pkp2a). As desmosomes mature from Ca2+ dependence to Ca2+-independent hyper-adhesion, their stability increases, but Pkp2a remains highly mobile. We show that desmosome downregulation during growth-factor-induced cell scattering proceeds by internalisation of whole desmosomes, which still retain a stable moiety and highly mobile Pkp2a. This molecular mobility of Pkp2a suggests a transient and probably regulatory role for Pkp2a in desmosomes. This article has an associated First Person interview with the first author of the paper.


Assuntos
Desmossomos , Placofilinas , Caderinas , Membrana Celular , Desmogleínas , Desmoplaquinas/genética , Humanos , Placofilinas/genética , gama Catenina
20.
FEBS Lett ; 595(21): 2675-2690, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34626438

RESUMO

14-3-3 proteins are conserved, dimeric, acidic proteins that regulate multiple cellular pathways. Loss of either 14-3-3ε or 14-3-3γ leads to centrosome amplification. However, we find that while the knockout of 14-3-3ε leads to multipolar mitoses, the knockout of 14-3-3γ results in centrosome clustering and pseudo-bipolar mitoses. 14-3-3γ knockouts demonstrate compromised desmosome function and a decrease in keratin levels, leading to decreased cell stiffness and an increase in centrosome clustering. Restoration of desmosome function increased multipolar mitoses, whereas knockdown of either plakoglobin or keratin 5 led to decreased cell stiffness and increased pseudo-bipolar mitoses. These results suggest that the ability of the desmosome to anchor keratin filaments maintains cell stiffness, thus inhibiting centrosome clustering, and that phenotypes observed upon 14-3-3 loss reflect the dysregulation of multiple pathways.


Assuntos
Proteínas 14-3-3 , Centrossomo , Desmossomos , Mitose , Células HCT116 , Humanos , Fuso Acromático
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