Combination of FLNC and JUP variants causing arrhythmogenic cardiomyopathy in an Iranian family with different clinical features.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) characterized by progressive myocardial loss and replacement with fibro-fatty tissue is a major cause of sudden cardiac death (SCD). In particular, ACM with predominantly left ventricular involvement, known as arrhythmogenic left ventricular cardiomyopathy (ALVC), has a poor prognosis. METHODS: The proband underwent whole-exome sequencing (WES) to determine the etiology of ALVC. Family members were then analyzed using PCR and Sanger sequencing. Clinical evaluations including 12-lead ECG, transthoracic echocardiography, and cardiac MRI were performed for all available first-degree relatives. RESULTS: WES identified two variants in the FLNC (c.G3694A) and JUP (c.G1372A) genes, the combination of which results in ALVC and SCD. CONCLUSION: The present study comprehensively investigates the involvement of two discovered variants of FLNC and JUP in the pathogenesis of ALVC. More study is necessary to elucidate the genetic factors involved in the etiology of ALVC.

Mycn ameliorates cardiac hypertrophy-induced heart failure in mice by mediating the USP2/JUP/Akt/ß-catenin cascade.

BACKGROUND: Pathological cardiac hypertrophy is associated with cardiac dysfunction and is a key risk factor for heart failure and even sudden death. This study investigates the function of Mycn in cardiac hypertrophy and explores the interacting molecules. METHODS: A mouse model of cardiac hypertrophy was induced by isoproterenol (ISO). The cardiac dysfunction was assessed by the heart weight-to-body weight ratio (HW/BW), echocardiography assessment, pathological staining, biomarker detection, and cell apoptosis. Transcriptome alteration in cardiac hypertrophy was analyzed by bioinformatics analysis. Gain- or loss-of-function studies of MYCN proto-oncogene (Mycn), ubiquitin specific peptidase 2 (USP2), and junction plakoglobin (JUP) were performed. The biological functions of Mycn were further examined in ISO-treated cardiomyocytes. The molecular interactions were verified by luciferase assay or immunoprecipitation assays. RESULTS: Mycn was poorly expressed in ISO-treated mice, and its upregulation reduced HW/BW, cell surface area, oxidative stress, and inflammation while improving cardiac function of mice. It also reduced apoptosis of cardiomyocytes in mice and those in vitro induced by ISO. Mycn bound to the USP2 promoter to activate its transcription. USP2 overexpression exerted similar myocardial protective functions. It stabilized JUP protein by deubiquitination modification, which blocked the Akt/ß-catenin pathway. Knockdown of JUP restored phosphorylation of Akt and ß-catenin protein level, which negated the protective effects of USP2. CONCLUSION: This study demonstrates that Mycn activates USP2 transcription, which mediates ubiquitination and protein stabilization of JUP, thus inactivating the Akt/ß-catenin axis and alleviating cardiac hypertrophy-induced heart failure.

Isolated JUP plakoglobin gene mutation with left ventricular fibrosis in familial arrhythmogenic right ventricular cardiomyopathy.

INTRODUCTION: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare inherited disorder usually affecting the right ventricle (RV), characterized by fibro-fatty tissue replacement of the healthy ventricular myocardium. It often predisposes young patients to ventricular tachycardia, heart failure, and/or sudden cardiac death. However, recent studies have suggested predominantly left ventricle (LV) involvement with variable and/or atypical manifestations. Cardiac magnetic resonance (CMR) imaging has emerged as the noninvasive gold standard for the diagnosis of ARVC. CASE SUMMARY: A 21-year-old athletic male with a family history of unknown ventricular arrhythmias, presented with near syncope, chest pain, and exertional palpitations. He had an initial work-up that was grossly unremarkable including an electrocardiogram (ECG), echocardiogram and a CMR study. Six months later, he presented again with recurrent symptoms of presyncope during exercise and his ECG demonstrated new findings of a terminal activation delay in his precordial leads. He had markedly elevated cardiac biomarkers, (troponin I > 100 ng/dl, normal value < 0.04 ng/dl) and demonstrated ventricular tachycardia with a right bundle branch morphology. An endomyocardial biopsy did not reveal any pathology. A follow-up CMR demonstrated the new development and prominent left ventricular epicardial scar in the lateral wall. The patient underwent familial genetic testing, which confirmed the presence of an isolated junction plakoglobin (JUP) gene mutation and showed multiple genes consistent with ARVC in his mother. Thus, he manifested a partial transmission of only one abnormal gene for ARVC and exhibited a markedly different expression in his disease without evidence of typical right-sided heart pathology. A third CMR study was performed, which showed partial improvement in myocardial fibrosis after exercise cessation. CONCLUSION: We present a case of a young athletic male with a newly diagnosed isolated JUP gene mutation and a genetically diagnosed family history of ARVC. During his course, he demonstrated the progression of new, atypical, left ventricular fibrosis. This case demonstrates a complex interplay between genetic penetrance, phenotypical heterogeneity, and lifestyle factors such as exercise in disease progression and provides insight into the natural course of an isolated JUP mutation. Although rare, clinicians should have a high threshold for the clinical suspicion of ARVC or variants of this disorder even in the absence of classic right-sided pathologies.

miR-195-5p as Regulator of γ-Catenin and Desmosome Junctions in Colorectal Cancer.

Desmosomes play a key role in the regulation of cell adhesion and signaling. Dysregulation of the desmosome complex is associated with the loss of epithelial cell polarity and disorganized tissue architecture typical of colorectal cancer (CRC). The aim of this study was to investigate and characterize the effect of miR-195-5p on desmosomal junction regulation in CRC. In detail, we proposed to investigate the deregulation of miR-195-5p and JUP, a gene target that encodes a desmosome component in CRC patients. JUP closely interacts with desmosomal cadherins, and downstream, it regulates several intracellular transduction factors. We restored the miR-195-5p levels by transient transfection in colonic epithelial cells to examine the effects of miR-195-5p on JUP mRNA and protein expression. The JUP regulation by miR-195-5p, in turn, determined a modulation of desmosome cadherins (Desmoglein 2 and Desmocollin 2). Furthermore, we focused on whether the miR-195-5p gain of function was also able to modulate the expression of key components of Wnt signaling, such as NLK, LEF1 and Cyclin D1. In conclusion, we have identified a novel mechanism controlled by miR-195-5p in the regulation of adhesive junctions, suggesting its potential clinical relevance for future miRNA-based therapy in CRC.

Downregulation of γ-Catenin by miR-195-5p Inhibits Colon Cancer Progression, Regulating Desmosome Function.

Desmosomes are essential structures for ensuring tissue functions, and their deregulation is involved in the development of colorectal cancer (CRC). JUP (γ-catenin) is a desmosome adhesion component that also acts as a signaling hub, suggesting its potential involvement in CRC progression. In this context, we recently demonstrated that miR-195-5p regulated JUP and desmosome cadherins expression. In addition, miR-195-5p gain of function indirectly modulated the expression of key effectors of the Wnt pathway involved in JUP-dependent signaling. Here, our purpose was to demonstrate the aberrant expression of miR-195-5p and JUP in CRC patients and to functionally characterize the role of miR-195-5p in the regulation of desmosome function. First, we showed that miR-195-5p was downregulated in CRC tumors compared to adjacent normal tissue. Then, we demonstrated that JUP expression was significantly increased in CRC tissues compared to adjacent normal tissues. The effects of miR-195-5p on CRC progression were assessed using in vitro transient transfection experiments and in vivo miRNA administration. Increased miR-195-5p in colonic epithelial cells strongly inhibits cell proliferation, viability, and invasion via JUP. In vivo gain of function of miR-195-5p reduced the numbers and sizes of tumors and significantly ameliorated the histopathological changes typical of CRC. In conclusion, our findings indicate a potential pharmacological target based on miR-195-5p replacement as a new therapeutic approach in CRC.

ICAT promotes colorectal cancer metastasis via binding to JUP and activating the NF-κB signaling pathway.

BACKGROUND: The inhibitor of ß-catenin and T-cell factor (ICAT) is a direct negative regulator of the canonical Wnt signaling pathway, which is an attractive therapeutic target for colorectal cancer (CRC). Accumulating evidence suggests that ICAT interacts with other proteins to exert additional functions, which are not yet fully elucidated. METHODS: The overexpression of ICAT of CRC cells was conducted by lentivirus infection and plasmids transfection and verified by quantitative real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) and Western blotting. The effect of ICAT on the mobility of CRC cells was assessed by wound healing assay and transwell assay in vitro and lung metastasis in vivo. New candidate ICAT-interacting proteins were explored and verified using the STRING database, silver staining, co-immunoprecipitation mass spectrometry analysis (Co-IP/MS), and immunofluorescence (IF) staining analysis. RESULT: Inhibitor of ß-catenin and T-cell factor overexpression promoted in vitro cell migration and invasion and tumor metastasis in vivo. Co-IP/MS analysis and STRING database analyses revealed that junction plakoglobin (JUP), a homolog of ß-catenin, was involved in a novel protein interaction with ICAT. Furthermore, JUP downregulation impaired ICAT-induced migration and invasion of CRC cells. In addition, ICAT overexpression activated the NF-κB signaling pathway, which led to enhanced CRC cell migration and invasion. CONCLUSION: Inhibitor of ß-catenin and T-cell factor promoted CRC cell migration and invasion by interacting with JUP and the NF-κB signaling pathway. Thus, ICAT could be considered a protein diagnostic biomarker for predicting the metastatic ability of CRC.

Junction plakoglobin, a potential prognostic marker of oral squamous cell carcinoma, promotes proliferation, migration and invasion.

BACKGROUND: Junction plakoglobin (JUP) is an important cell-cell junction protein. Recently, its deregulation has been correlated with the initiation and progression of various malignancies. Our aim was to investigate the expression of JUP in oral squamous cell carcinoma (OSCC) and its correlation with prognosis and to further study the effects of JUP on the proliferation, apoptosis, migration and invasion of OSCC cells. METHODS: We detected JUP expression in 273 OSCC specimens using immunohistochemistry. We assessed the correlation of JUP expression with clinicopathologic parameters and patient survival by Cox regression. Then, expression levels of JUP in normal oral keratinocytes (NOKs) and OSCC cell lines were detected by Western blotting and quantitative real-time PCR (qPCR). Next, we used HSC3 cells to study the effect of JUP on tumor cell proliferation, apoptosis, migration, and invasion by using cell counting kit-8, flow cytometry, and transwell assays, respectively. RESULTS: Cox regression showed that high expression of JUP was related to the poor prognosis of OSCC patients. Western blotting and qPCR assays showed that the expression level of JUP in OSCC cell lines was higher than that in NOKs. Overexpression of JUP promoted the proliferation, metastasis, and invasion of HSC3 cells and inhibited apoptosis, while the opposite was observed after JUP knockdown. CONCLUSION: This study initially revealed that JUP was overexpressed in OSCC, and that JUP promoted the proliferation, migration, and invasion of OSCC cells and inhibited apoptosis. Moreover, high expression of JUP could be used as a potential prognostic marker of OSCC.

ß-catenin coordinates with Jup and the TCF1/GATA6 axis to regulate human embryonic stem cell fate.

ß-catenin-mediated signaling has been extensively studied in regard to its role in the regulation of human embryonic stem cells (hESCs). However, the results are controversial and the mechanism by which ß-catenin regulates the hESC fate remains unclear. Here, we report that ß-catenin and γ-catenin are functionally redundant in mediating hESC adhesion and are required for embryoid body formation, but both genes are dispensable for hESC maintenance, as the undifferentiated state of ß-catenin and γ-catenin double deficient hESCs can be maintained. Overexpression of ß-catenin induces rapid hESC differentiation. Functional assays revealed that TCF1 plays a crucial role in hESC differentiation mediated by ß-catenin. Forced expression of TCF1, but not other LEF1/TCF family members, resulted in hESC differentiation towards the definitive endoderm. Conversely, knockdown of TCF1 or inhibition of the interaction between TCF1 and ß-catenin delayed hESC exit from pluripotency. Furthermore, we demonstrated that GATA6 plays a predominant role in TCF1-mediated hESC differentiation. Knockdown of GATA6 completely eliminated the effect of TCF1, while forced expression of GATA6 induced hESC differentiation. Our data thus reveal more detailed mechanisms for ß-catenin in regulating hESC fate decisions and will expand our understanding of the self-renewal and differentiation circuitry in hESCs.

A Novel Mutation in Junctional Plakoglobin Causing Lethal Congenital Epidermolysis Bullosa.

We report a case of neonatal generalized erythema and epidermolysis resulting from a novel mutation in the junctional plakoglobin gene causing truncation of the plakoglobin protein. Expedited genetic testing enabled diagnosis while the patient was in the neonatal intensive care unit, providing valuable information for the clinicians and family.

Plakoglobin and High-Mobility Group Box 1 Mediate Intestinal Epithelial Cell Apoptosis Induced by Clostridioides difficile TcdB.

Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated intestinal disease, resulting in severe diarrhea and fatal pseudomembranous colitis. TcdB, one of the essential virulence factors secreted by this bacterium, induces host cell apoptosis through a poorly understood mechanism. Here, we performed an RNA interference (RNAi) screen customized to Caco-2 cells, a cell line model of the intestinal epithelium, to discover host factors involved in TcdB-induced apoptosis. We identified plakoglobin, also known as junction plakoglobin (JUP) or γ-catenin, a member of the catenin family, as a novel host factor and a previously known cell death-related chromatin factor, high-mobility group box 1 (HMGB1). Disruption of those host factors by RNAi and CRISPR resulted in resistance of cells to TcdB-mediated and mitochondrion-dependent apoptosis. JUP was redistributed from adherens junctions to the mitochondria and colocalized with the antiapoptotic factor Bcl-XL. JUP proteins could permeabilize the mitochondrial membrane, resulting in the release of cytochrome c. Our results reveal a novel role of JUP in targeting the mitochondria to promote the mitochondrial apoptotic pathway. Treatment with glycyrrhizin, an HMGB1 inhibitor, resulted in significantly increased resistance to TcdB-induced epithelial damage in cultured cells and a mouse ligated colon loop model. These findings demonstrate the critical roles of JUP and HMGB1 in TcdB-induced epithelial cell apoptosis. IMPORTANCE Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea. Toxins, especially TcdB, cause epithelial cell apoptosis, but the underlying cell death mechanism is less clear. Through an apoptosis-focused RNAi screen using a bacterium-made small interfering (siRNA) library customized to a human colonic epithelial cell model, we found a novel host factor, plakoglobin (γ-catenin), as a key factor required for cell apoptosis induced by TcdB. Plakoglobin targets and permeabilizes mitochondria after stimulation by TcdB, demonstrating a hitherto underappreciated role of this catenin family member in the apoptosis of intestinal epithelial cells. We also found a previously known cell death-related chromatin factor, HMGB1, and explored the inhibition of HMGB1 for CDI therapy in vivo.

Extracellular vesicles of human diabetic retinopathy retinal tissue and urine of diabetic retinopathy patients are enriched for the junction plakoglo bin protein.

Introduction: Diabetic Retinopathy (DR) is a potentially blinding retinal disorder that develops through the pathogenesis of diabetes. The lack of disease predictors implies a poor prognosis with frequent irreversible retinal damage and vision loss. Extracellular Vesicles (EVs) present a novel opportunity for pre-symptomatic disease diagnosis and prognosis, both severely limited in DR. All biological fluids contain EVs, which are currently being studied as disease biomarkers. EV proteins derived from urine have emerged as potential noninvasive biomarkers. Methods: In this study, we isolated EVs from DR retinal tissue explants and from DR patients' urine, and characterized the vesicles, finding differences in particle number and size. Next, we performed proteomic analysis on human explanted DR retinal tissue conditioned media, DR retinal EVs and DR urinary EVs and compared to normal human retinal tissue, retinal EVs, and urinary EVs, respectively. Results: Our system biology analysis of DR tissue and EV expression profiles revealed biological pathways related to cell-to-cell junctions, vesicle biology, and degranulation processes. Junction Plakoglobin (JUP), detected in DR tissue-derived EVs and DR urinary EVs, but not in controls, was revealed to be a central node in many identified pathogenic pathways. Proteomic results were validated by western blot. Urinary EVs obtained from healthy donors and diabetic patient without DR did not contain JUP. Conclusion: The absence of JUP in healthy urinary EVs provide the basis for development of a novel Diabetic Retinopathy biomarker, potentially facilitating diagnosis.

Effects of differential distributed-JUP on the malignancy of gastric cancer.

JUP, a homologue of ß-catenin, is a cell-cell junction protein involved in adhesion junction and desmosome composition. JUP may have a controversial role in different malignancies dependence of its competence with or collaboration with ß-catenin as a transcription factor. In this study, we reveal that the function of JUP is related to its cellular location in GC development process from epithelium-like, low malignant GC to advanced EMT-phenotypic GC. Gradual loss of membrane and/or cytoplasm JUP is closely correlated with GC malignancy and poor prognostics. Knockdown of JUP in epithelium-like GC cells causes EMT and promotes GC cell migration and invasion. Ectopic expression of wild JUP in malignant GC cells leads to an attenuated malignant phenotype such as reduced cell invasive potential. In mechanism, loss of membrane and/or cytoplasm JUP abolishes the restrain of JUP to EGFR at cell membrane and results in increased p-AKT levels and AKT/GSK3ß/ß-catenin signaling activity. In addition, nuclear JUP interacts with nuclear ß-catenin and TCF4 and plays a synergistic role with ß-catenin in promoting TCF4 transcription and its downstream target MMP7 expression to fuel GC cell invasion.

[Genetics of complex and syndromic palmoplantar keratoderma]. / Génétique des kératodermies palmoplantaires complexes et syndromiques.

Palmoplantar keratodermas (PPK) comprise a heterogenous group of acquired and hereditary disorders marked by excessive thickening of the epidermis of palms and soles. Hereditary PPKs can be classified into 3 groups: 1) isolated non-syndromic PPKs; 2) complex non-syndromic PPKs associated with other ectodermal defects; and 3) syndromic PPKs associated with extracutaneous manifestations. All types of inheritance have been observed: autosomal dominant, autosomal recessive, X-linked recessive, and mitochondrial. Some of these disorders are restricted to geographic isolates. This review describes the different genetic causes of hereditary syndromic and complex PPKs for which the genes have been identified. The identification of pathogenic variants has consequences in terms of genetic counseling, appropriate medical care and follow-up, especially for PPKs predisposing to hearing loss, cardiomyopathies, benign tumors or carcinomas. In addition, the development of targeted therapies based on pathophysiology of disorders should allow a more effective treatment of these conditions in the near future.

JUP/plakoglobin is regulated by salt-inducible kinase 2, and is required for insulin-induced signalling and glucose uptake in adipocytes.

BACKGROUND: Salt-inducible kinase 2 (SIK2) is abundant in adipocytes, but downregulated in adipose tissue from individuals with obesity and insulin resistance. Moreover, SIK isoforms are required for normal insulin signalling and glucose uptake in adipocytes, but the underlying molecular mechanisms are currently not known. The adherens junction protein JUP, also termed plakoglobin or γ-catenin, has recently been reported to promote insulin signalling in muscle cells. OBJECTIVE: The objective of this study was to analyse if JUP is required for insulin signalling in adipocytes and the underlying molecular mechanisms of this regulation. METHODS: Co-expression of SIK2 and JUP mRNA levels in adipose tissue from a human cohort was analysed. siRNA silencing and/or pharmacological inhibition of SIK2, JUP, class IIa HDACs and CRTC2 was employed in 3T3-L1- and primary rat adipocytes. JUP protein expression was analysed by western blot and mRNA levels by qPCR. Insulin signalling was evaluated by western blot as levels of phosphorylated PKB/Akt and AS160, and by monitoring the uptake of 3H-2-deoxyglucose. RESULTS: mRNA expression of SIK2 correlated with that of JUP in human adipose tissue. SIK2 inhibition or silencing resulted in downregulation of JUP mRNA and protein expression in 3T3-L1- and in primary rat adipocytes. Moreover, JUP silencing reduced the expression of PKB and the downstream substrate AS160, and consequently attenuated activity in the insulin signalling pathway, including insulin-induced glucose uptake. The known SIK2 substrates CRTC2 and class IIa HDACs were found to play a role in the SIK-mediated regulation of JUP expression. CONCLUSIONS: These findings identify JUP as a novel player in the regulation of insulin sensitivity in adipocytes, and suggest that changes in JUP expression could contribute to the effect of SIK2 on insulin signalling in these cells.

Increased expression of plakoglobin is associated with upregulated MAPK and PI3K/AKT signalling pathways in early resectable pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer types, and it is associated with a 5-year survival rate of <10% due to limited early detection methods and ineffective therapeutic options. Thus, an improved understanding of the mechanisms involved in the early stages of PDAC tumorigenesis is crucial in order to identify potential novel diagnostic and therapeutic targets. The most common signalling aberrations in PDAC occur in the Wnt/Notch signalling pathway, as well as within the epidermal growth factor receptor (EGFR) pathway and its associated ligands, EGF and transforming growth factor-ß. In addition, the RAS family of oncogenes, which act downstream of EGFR, are found mutated in most pancreatic cancer samples. Plakoglobin, a component of the EGFR signalling pathway, serves an important role in normal cell adhesion; however, its role in PDAC is largely unknown. The present study used transcriptome sequencing and focussed proteome microarrays to identify dysregulated genes and proteins in PDAC. The presence of upregulated plakoglobin expression levels was identified as a distinguishing feature between the PDAC microenvironment and normal pancreatic tissue. Furthermore, plakoglobin was demonstrated to be associated with the differential upregulation of the PI3K/AKT and MAPK signalling pathways in the tumour microenvironment, which suggested that it may serve an important role in PDAC tumourigenesis.

Identification of testicular cancer driver genes by a cross-species comparative oncology approach.

BACKGROUND: Germ cell tumors arise in the testis, ovary, or extragonadal locations and have a wide range of histopathological and clinical presentations. The relative lack of animal models of germ cell tumors has impeded functional assessment of candidate driver genes. Previously, we described the development of testicular germ cell tumors in zebrafish carrying a mutation in bmpr1bb, a BMP family receptor, and demonstrated that human germ cell tumors have defects in BMP signaling. OBJECTIVE: To further credential the zebrafish model for studies of human germ cell tumor, and to elucidate conserved genetic programs underlying the development of germ cell tumor. MATERIALS AND METHODS: We used genetic techniques to ablate the germ cell lineage in developing fish and tested tumors for loss-of-heterozygosity of the wild-type allele of bmpr1bb. We performed comparative gene expression profiling of zebrafish and human germ cell tumors and carried out functional studies of selected genes. RESULTS: Ablation of germ cells completely prevents testis tumor formation in the fish, definitively establishing the germ cell origin of the tumors. Germ cell tumors in bmpr1bb heterozygous mutants retain the wild-type allele, indicating haploinsufficiency of bmpr1bb as the mechanism of tumor formation. Comparison of RNA-Seq and microarray data from human and zebrafish germ cell tumors revealed a unique overlapping signature shared by the zebrafish tumors with human seminomas, yolk sac tumors, and embryonal carcinomas. The most highly conserved gene set in this cross-species analysis included potential driver genes such as JUP, which we show to be essential for germ cell tumor cell growth. CONCLUSION: Our findings highlight the value of cross-species comparative oncology for the identification of candidate human cancer genes.