PKP3 interactions with MAPK-JNK-ERK1/2-mTOR pathway regulates autophagy and invasion in ovarian cancer.

Armadillo-related proteins function in both signal transduction and cell adhesion, it also plays a central role in tumorigenesis. Plakophilin 3 (PKP3) is a member of the armadillo protein family. PKP3 has demonstrated a role in melanoma, breast cancer, gastric cancer, and other kind of cancers; however its role in ovarian cancer was not fully understood. In this study we explored the function and mechanisms of PKP3 in ovarian cancer. An elevated level of PKP3 was found in ovarian cancer tissues compared with normal tissues. PKP3 also modulate cellular proliferation and invasion in ovarian cancer. The ability of cellular proliferation, formation, and invasion was significantly decreased after the silencing of PKP3 in SKOV3 cells. While an over-expression of PKP3 in A2780 cells up-regulates the ability of cellular proliferation, formation, and invasion. As for the mechanism of PKP3, mTOR pathway was activated to regulate autophagy according to the interaction of PKP3 with the upstream of MAPK pathway. The result of this study support PKP3 as the oncogene candidate and a potential target for the treatment of ovarian cancer.

Comparison of cellular location and expression of Plakophilin-2 in epidermal cells from nonlesional atopic skin and healthy skin in German shepherd dogs.

BACKGROUND: Canine atopic dermatitis (CAD) is an inflammatory and pruritic allergic skin disease caused by interactions between genetic and environmental factors. Previously, a genome-wide significant risk locus on canine chromosome 27 for CAD was identified in German shepherd dogs (GSDs) and Plakophilin-2 (PKP2) was defined as the top candidate gene. PKP2 constitutes a crucial component of desmosomes and also is important in signalling, metabolic and transcriptional activities. OBJECTIVES: The main objective was to evaluate the role of PKP2 in CAD by investigating PKP2 expression and desmosome structure in nonlesional skin from CAD-affected (carrying the top GWAS SNP risk allele) and healthy GSDs. We also aimed at defining the cell types in the skin that express PKP2 and its intracellular location. ANIMALS/METHODS: Skin biopsies were collected from nine CAD-affected and five control GSDs. The biopsies were frozen for immunofluorescence and fixed for electron microscopy immunolabelling and morphology. RESULTS: We observed the novel finding of PKP2 expression in dendritic cells and T cells in dog skin. Moreover, we detected that PKP2 was more evenly expressed within keratinocytes compared to its desmosomal binding-partner plakoglobin. PKP2 protein was located in the nucleus and on keratin filaments attached to desmosomes. No difference in PKP2 abundance between CAD cases and controls was observed. CONCLUSION: Plakophilin-2 protein in dog skin is expressed in both epithelial and immune cells; based on its subcellular location its functional role is implicated in both nuclear and structural processes.

TGF-beta1 pathway activation and adherens junction molecular pattern in nonsyndromic mitral valve prolapse.

AIMS: Dysregulation of the transforming growth factor beta (TGF-ß) 1 pathway has been associated with either syndromic or isolated mitral valve (MV) prolapse due to myxoid degeneration (floppy MV). The activation of Smad receptor-mediated intracellular TGF-ß pathway and its effect on adherens junction (AJ) molecular pattern of activated valvular interstitial cells (VICs) in MV prolapse are herein investigated. METHODS: Floppy MV leaflets were obtained from 30 patients (24 males, mean age 55.5±12.7 years) who underwent surgical repair, and 10 age- and sex-matched Homograft Tissue Bank samples served as controls. MV leaflet cellular and extracellular matrix composition, including collagen I and III, was evaluated by histology and transmission electron microscopy. Smad2 active phosphorylated form (p-Smad2), α-smooth muscle actin (α-SMA), and junctional proteins (N-cadherin, cadherin-11, ß-catenin, plakoglobin, plakophilin-2) in VICs were assessed by immunohistochemistry and immunofluorescence and confirmed by immunoblotting. Quantitative real-time polymerase chain reaction was carried out for components of TGF-ß pathway cascade and filamin A (FLN-A). RESULTS: Floppy MV leaflets were thicker (P<.001) and had higher α-SMA+ cell density (P=.002) and collagen III expression (P<.001) than controls. Enhanced p-Smad2 nuclear immunoreactivity (P<.001) and TGF-ß1 gene (P=.045), TIMP1 (P=.020), and CTGF (P=.047) expression but no differences in FLN-A and total Smad2 gene expression levels were found between floppy MV and controls. Higher expression of cadherin-11, either exclusively or in colocalization with N-cadherin, and aberrant presence of plakophilin-2 at the AJ were found in floppy MV vs. CONCLUSIONS: TGF-ß1 pathway activation in nonsyndromic MV prolapse induces VICs differentiation into contractile myofibroblasts and is associated with changes in the molecular pattern of the AJ, with increased cadherin-11 and aberrant plakophilin-2 expression. AJ reinforcement might promote latent TGF-ß1 activation leading to extracellular matrix remodeling in floppy MV.

An alternative promoter of the human plakophilin-3 gene controls the expression of the new isoform PKP3b.

The plakophilin family (PKP1 to PKP3) is an essential component of the desmosomal adhesion complex with differentiation-dependent and partially overlapping expression and possible participation of the corresponding genes in malignant transformation. Here, we describe a new protein variant of the human PKP3 gene, namely PKP3b, which differs from the published PKP3a only at the amino-terminus by the splicing in of the newly identified exon 1b. Specific antibodies have demonstrated differential expression patterns of the two variants. Whereas PKP3a is broadly expressed among epithelial cells, PKP3b is abundant in the desmosomes of stratified epithelial cells, such as HaCaT but absent or heterogeneous in simple epithelial cells such as CaCo2 or MCF7. The differential expression of the PKP3 variants has been observed in a similar manner in selected normal human tissues and carcinomas derived thereof. Both variants are localized to the desmosomes of all cells of stratified tissues, whereas the new PKP3b is heterogeneously expressed in the colon and its tumors. Therefore, we assume that both variants are controlled by alternative promoters. Reporter gene assays have confirmed that a fragment upstream of exon 1b exhibits transcriptional activity only in HaCaT cells but not in CaCo2 cells and thus has been identified as an alternative promoter driving the expression of PKP3b. Finally, by using electromobility shift assays, we found a potential binding site in the PKP3b promoter for transcription factor C/EBP regulating keratinocyte differentiation and probably also PKP3b expression. We discuss the properties of the new variant PKP3b as a possible marker protein for the analyses of differentiation and malignant transformation.

Super-resolution fluorescence microscopy of the cardiac connexome reveals plakophilin-2 inside the connexin43 plaque.

AIMS: Cell function requires formation of molecular clusters localized to discrete subdomains. The composition of these interactomes, and their spatial organization, cannot be discerned by conventional microscopy given the resolution constraints imposed by the diffraction limit of light (∼200-300 nm). Our aims were (i) Implement single-molecule imaging and analysis tools to resolve the nano-scale architecture of cardiac myocytes. (ii) Using these tools, to map two molecules classically defined as components 'of the desmosome' and 'of the gap junction', and defined their spatial organization. METHODS AND RESULTS: We built a set-up on a conventional inverted microscope using commercially available optics. Laser illumination, reducing, and oxygen scavenging conditions were used to manipulate the blinking behaviour of individual fluorescent reporters. Movies of blinking fluorophores were reconstructed to generate subdiffraction images at ∼20 nm resolution. With this method, we characterized clusters of connexin43 (Cx43) and of 'the desmosomal protein' plakophilin-2 (PKP2). In about half of Cx43 clusters, we observed overlay of Cx43 and PKP2 at the Cx43 plaque edge. SiRNA-mediated loss of Ankyrin-G expression yielded larger Cx43 clusters, of less regular shape, and larger Cx43-PKP2 subdomains. The Cx43-PKP2 subdomain was validated by a proximity ligation assay (PLA) and by Monte-Carlo simulations indicating an attraction between PKP2 and Cx43. CONCLUSIONS: (i) Super-resolution fluorescence microscopy, complemented with Monte-Carlo simulations and PLAs, allows the study of the nanoscale organization of an interactome in cardiomyocytes. (ii) PKP2 and Cx43 share a common hub that permits direct physical interaction. Its relevance to excitability, electrical coupling, and arrhythmogenic right ventricular cardiomyopathy, is discussed.

Ultrastructural localization of desmoglein and plakophilin in the human hair suggests that the cell membrane complex is a long desmosomal remnant.

Unlike the superficial part of the corneous layer of the epidermis (Stratum corneum) where desmosomes are degraded and corneocytes flake away, the trichocytes in the hair remain attached to each other after cornification. The permanence and fine localization of cell junctions, in particular of desmosomal proteins in the cornifying and mature human hair, is not known. The present electron microscope immunolocalization study indicates that two protein markers for desmosomes such as desmoglein 4 and plakophilins 1 and 3 are still present in mature cortical and cuticle cells. These proteins remain mainly localized in the cornified cytoplasmic side of desmosomal remnants of cortical cells, but also in the delta layer of the extracellular region of the membrane complex. This suggests that the delta layer represents an extensive desmosomal remnant formed between mature cortical cells and in cuticle cells. The endocuticle appears to be the site of accumulation of desmosomal proteins and degraded nuclear material. The cornification of desmosomal junctions in both cortical and cuticle cells likely contributes to stabilize the integrity of the hair shaft.

p0071/PKP4, a multifunctional protein coordinating cell adhesion with cytoskeletal organization.

P0071 is a member of a subfamily of armadillo proteins that also comprises p120-catenin (p120ctn), δ-catenin/NPRAP, ARVCF and the more distantly related plakophilins 1-3. These proteins share a conserved central domain consisting of a series of repeated motifs, the armadillo repeats, which is flanked by more diverse amino- and carboxy-terminal domains. P0071 and the related proteins were first described as components of adherens junctions with a function in clustering and stabilizing cadherins, thereby controlling intercellular adhesion. In addition, these proteins show a cytoplasmic and a nuclear localization. Major progress in understanding their cytoplasmic role has been made in recent years. One common theme appears to be the spatiotemporal control of the small GTPases of the Rho family in various cellular contexts, such as cell adhesion and motility, cell division or neurite outgrowth. In this review article, we focus on the functions of the p0071 protein and its closest relatives in regulating cell adhesion and cytoskeletal organization, which are critically involved in the control of cell polarity. Understanding p0071's multiple functions requires assigning specific functions to particular binding partners and subcellular compartments. The identification of several new p0071 interacting proteins has promoted our understanding of the complex functions of this protein. Moreover, an initial analysis of its regulation begins to shed light on how these functions are coordinated in a cellular context.

A novel kind of tumor type-characteristic junction: plakophilin-2 as a major protein of adherens junctions in cardiac myxomata.

Using novel antibodies of high avidity to--and specificity for--the constitutive desmosomal plaque protein, plakophilin-2 (Pkp2), in a systematic study of the molecular composition of junctions connecting the cells of soft tissue tumors, we have discovered with immunocytochemical, biochemical and electron microscopical methods, a novel type of adherens junctions in all 32 cardiac myxomata examined. These junctions contain cadherin-11 as their major transmembrane glycoprotein, which we could repeatedly show in colocalization with N-cadherin, anchored in a cytoplasmic plaque formed by α- and ß-catenin, together with the further armadillo-type proteins plakoglobin, p120, p0071 and ARVCF. Surprisingly, all adherens junctions of these tumors contained, in addition, another major armadillo protein Pkp2, hitherto known as an obligatory and characteristic constituent of desmosomes in epithelium-derived tumors. We have not detected Pkp2 in a series of noncardiac myxomata studied in parallel. Therefore, we conclude that this acquisition of Pkp2, which we have recently also observed in some mesenchymally derived cells growing in culture, can also occur in tumorigenic transformations in situ. We propose to examine the marker value of Pkp2 in clinical diagnoses of cardiac myxomata and to develop Pkp2-targeted therapeutic reagents.

Upregulation of plakophilin-2 and its acquisition to adherens junctions identifies a novel molecular ensemble of cell-cell-attachment characteristic for transformed mesenchymal cells.

In contrast to the desmosome-containing epithelial and carcinoma cells, normal and malignantly transformed cells derived from mesenchymal tissues and tumors are connected only by adherens junctions (AJs) containing N-cadherins and/or cadherin-11, anchored in a cytoplasmic plaque assembled by alpha- and beta-catenin, plakoglobin, proteins p120 and p0071. Here, we report that the AJs of many malignantly transformed cell lines are characterized by the additional presence of plakophilin-2 (Pkp2), a protein hitherto known only as a major component of desmosomal plaques, i.e., AJs of epithelia and carcinomatous cells. This massive acquisition of Pkp2 and its integration into AJ plaques of a large number of transformed cell lines is demonstrated with biochemical and immunolocalization techniques. Upregulation of Pkp2 and its integration into AJs has also been noted in some soft tissue tumors insitu and some highly proliferative colonies of cultured mesenchymal stem cells. As Pkp2 has recently been identified as a functionally important major regulatory organizer in AJs and related junctions in epithelial cells and cardiomyocytes, we hypothesize that the integration of Pkp2 into AJs of "soft tissue tumor" cells also can serve functions in the upregulation of proliferation, the promotion of malignant growth in general as well as the close-packing of diverse kinds of cells and the metastatic behavior of such tumors. We propose to examine its presence in transformed mesenchymal cells and related tumors and to use it as an additional diagnostic criterion.

Cardiomyopathies: New test for arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is challenging to diagnose because of nonspecific findings, particularly in the early phases of the disease. Clinical diagnosis is made on the basis of several criteria, but these lack sensitivity. Asimaki et al. suggest that immunohistochemical analysis of myocardial desmosomal proteins is a highly sensitive and specific diagnostic test for ARVD/C.

A new diagnostic test for arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: The diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) can be challenging because the clinical presentation is highly variable and genetic penetrance is often low. METHODS: To determine whether a change in the distribution of desmosomal proteins can be used as a sensitive and specific diagnostic test for ARVC, we performed immunohistochemical analysis of human myocardial samples. RESULTS: We first tested myocardium from 11 subjects with ARVC; of these samples, 8 had desmosomal gene mutations. We also tested myocardium obtained at autopsy from 10 subjects with no clinical or pathological evidence of heart disease as control samples. All ARVC samples but no control samples showed a marked reduction in immunoreactive signal levels for plakoglobin (also known as gamma-catenin), a protein that links adhesion molecules at the intercalated disk to the cytoskeleton. Other desmosomal proteins showed variable changes, but signal levels for the nondesmosomal adhesion molecule N-cadherin were normal in all subjects with ARVC. To determine whether a diminished plakoglobin signal level was specific for ARVC, we analyzed myocardium from 15 subjects with hypertrophic, dilated, or ischemic cardiomyopathies. In every sample, levels of N-cadherin and plakoglobin signals at junctions were indistinguishable from those in control samples. Finally, we performed blinded immunohistochemical analysis of heart-biopsy samples from the Johns Hopkins ARVC registry. We made the correct diagnosis in 10 of 11 subjects with definite ARVC on the basis of clinical criteria and correctly ruled out ARVC in 10 of 11 subjects without ARVC, for a sensitivity of 91%, a specificity of 82%, a positive predictive value of 83%, and a negative predictive value of 90%. The plakoglobin signal level was reduced diffusely in ARVC samples, including those obtained in the left ventricle and the interventricular septum. CONCLUSIONS: Routine immunohistochemical analysis of a conventional endomyocardial-biopsy sample appears to be a highly sensitive and specific diagnostic test for ARVC.

Connexin43 remodeling caused by inhibition of plakophilin-2 expression in cardiac cells.

Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts. We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins. The results are discussed in the context of arrhythmogenic right ventricular cardiomyopathy, an inherited disease involving mutations in desmosomal proteins, including PKP2.

The transcription factor ZEB1 (deltaEF1) represses Plakophilin 3 during human cancer progression.

Plakophilin 3 (PKP3) belongs to the p120ctn family of armadillo-related proteins predominantly functioning in desmosome formation. Here we report that PKP3 is transcriptionally repressed by the E-cadherin repressor ZEB1 in metastatic cancer cells. ZEB1 physically associates with two conserved E-box elements in the PKP3 promoter and partially represses the activity of corresponding human and mouse PKP3 promoter fragments in reporter gene assays. In human tumours ZEB1 is upregulated in invasive cancer cells at the tumour-host interface, which is accompanied by downregulation of PKP3 expression levels. Hence, the transcriptional repression of PKP3 by ZEB1 contributes to ZEB1-mediated disintegration of intercellular adhesion and epithelial to mesenchymal transition.

The area composita of adhering junctions connecting heart muscle cells of vertebrates. I. Molecular definition in intercalated disks of cardiomyocytes by immunoelectron microscopy of desmosomal proteins.

Among sarcomeric muscles the cardiac muscle cells are unique by, inter alia, a systemic and extended cell-cell contact structure, the intercalated disk (ID), comprising frequent and closely spaced arrays of plaque-coated cell-cell adhering junctions (AJs). As some of these junctions may look somewhat like desmosomes and others like fasciae adhaerentes, the dogma has emerged in the literature that IDs contain - like epithelial cells - both kinds of AJs formed by - for the most - mutually exclusive molecular ensembles. This, however, is not the case. In comprehensive immunoelectron microscopic studies of mammalian (human, bovine, rat, mouse) and non-mammalian (chicken, amphibia, fishes) heart muscle tissues, we have localized major constituents of the desmosomal plaques of polar epithelia, desmoplakin, plakophilin-2 and plakoglobin, as well as the desmosomal cadherins, desmoglein Dsg2 and desmocollin Dsc2, in both kinds of ID AJs, independent of the specific morphological appearance. The desmosomal molecules are not restricted to the desmosome-like-looking junctions but can also be detected in junctions appearing similar to the zonula or fascia adhaerens structures. These AJs of cardiac ID are therefore subsumed under the collective term area composita. We discuss our results with respect to the importance of ID junction molecules for the formation, maintenance and function of the heart, particularly in relation to recent findings that deletions of - or mutations in - genes encoding such proteins can cause severe, sometimes lethal damages.

Microarray analysis of differentially expressed genes in vaginal tissues from women with stress urinary incontinence compared with asymptomatic women.

BACKGROUND: The pathophysiology of pelvic floor dysfunction resulting in stress urinary incontinence (SUI) in women is complex. Evidence suggests that there is also a genetic predisposition towards SUI. We sought to identify differentially expressed genes involved in extracellular matrix (ECM) metabolism in vaginal tissues from women with SUI in the secretory phase of menses compared with asymptomatic women. METHODS: Tissue samples were taken from the periurethral vaginal wall of five pairs of premenopausal, age-matched SUI and continent women and subjected to microarray analysis using the GeneChip Human Genome U133 oligonucleotide chip set. RESULTS: Extensive statistical analyses generated a list of 79 differentially expressed genes. Elafin, keratin 16, collagen type XVII and plakophilin 1 were consistently identified as up-regulated ECM genes. Elafin, a serine protease inhibitor involved in the elastin degradation pathway and wound healing, was expressed in pelvic fibroblasts and confirmed by Western blot, quantitative competitive PCR and immunofluorescence cell staining. CONCLUSIONS: Genes involved in elastin metabolism were differentially expressed in vaginal tissue from women with SUI, suggesting that elastin remodelling may be important in the molecular aetiology of SUI.