Desmosomal Plaque Proteins Research Articles

Heterozygous DSP in-frame deletion in a poodle with syndromic ichthyosis involving additional hair and tooth abnormalities.

Ichthyoses comprise a large heterogeneous group of skin disorders, characterized by generalized scaly and hyperkeratotic skin. We investigated a miniature poodle with early onset generalized scaling, dry and irregularly thickened skin, paw pad hyperkeratosis and abnormalities in hair and teeth. The clinical signs of ichthyosis were confirmed by histopathological examination, which revealed mild epidermal hyperplasia and lamellar orthokeratotic hyperkeratosis. A hereditary condition was suspected and a genetic investigation was initiated. We sequenced the whole genome of the affected dog and searched for potentially causative variants in functional candidate genes for the observed phenotype. The analysis revealed a heterozygous in-frame deletion in DSP, NC_049256.1:g.8804542_8804544del resulting from a de novo mutation event as evidenced by genotyping leukocyte DNA from both parents. The 3 bp deletion is predicted to remove one aspartic acid without disrupting the open reading frame (XM_038584124.1:c.1821_1823del, XP_038440052.1:p.(Asp608del)). The DSP gene encodes desmoplakin, a desmosomal plaque protein, responsible for cell-cell adhesion to provide resistance to mechanical stress in epidermal and cardiac tissues. We hypothesize that the deletion of one amino acid in the N-terminal globular head domain acts in a dominant negative manner and thus impairs the proper connection with other proteins. Several variants in DSP in humans and cattle have been described to result in different phenotypes associated with hair and skin abnormalities, sometimes in combination with variable cardiac and/or dental manifestations. In conclusion, we characterized a new syndromic ichthyosis phenotype in a dog and identified a de novo 3 bp deletion in the DSP gene as causal variant.

PP2A-B55alpha controls keratinocyte adhesion through dephosphorylation of the Desmoplakin C-terminus

Critical for the maintenance of epidermal integrity and function are attachments between intermediate filaments (IF) and intercellular junctions called desmosomes. The desmosomal cytoplasmic plaque protein desmoplakin (DP) is essential for anchoring IF to the junction. DP-IF interactions are regulated by a phospho-regulatory motif within the DP C-terminus controlling keratinocyte intercellular adhesion. Here we identify the protein phosphatase 2A (PP2A)-B55α holoenzyme as the major serine/threonine phosphatase regulating DP’s C-terminus and consequent intercellular adhesion. Using a combination of chemical and genetic approaches, we show that the PP2A-B55α holoenzyme interacts with DP at intercellular membranes in 2D- and 3D- epidermal models and human skin samples. Our experiments demonstrate that PP2A-B55α regulates the phosphorylation status of junctional DP and is required for maintaining strong desmosome-mediated intercellular adhesion. These data identify PP2A-B55α as part of a regulatory module capable of tuning intercellular adhesion strength and a candidate disease target in desmosome-related disorders of the skin and heart.

Plakophilin 2 regulates intestinal barrier function by modulating protein kinase C activity in vitro

ABSTRACT Previous data provided evidence for a critical role of desmosomes to stabilize intestinal epithelial barrier (IEB) function. These studies suggest that desmosomes not only contribute to intercellular adhesion but also play a role as signaling hubs. The contribution of desmosomal plaque proteins plakophilins (PKP) in the intestinal epithelium remains unexplored. The intestinal expression of PKP2 and PKP3 was verified in human gut specimens, human intestinal organoids as well as in Caco2 cells whereas PKP1 was not detected. Knock-down of PKP2 using siRNA in Caco2 cells resulted in loss of intercellular adhesion and attenuated epithelial barrier. This was paralleled by changes of the whole desmosomal complex, including loss of desmoglein2, desmocollin2, plakoglobin and desmoplakin. In addition, tight junction proteins claudin1 and claudin4 were reduced following the loss of PKP2. Interestingly, siRNA-induced loss of PKP3 did not change intercellular adhesion and barrier function in Caco2 cells, while siRNA-induced loss of both PKP2 and PKP3 augmented the changes observed for reduced PKP2 alone. Moreover, loss of PKP2 and PKP2/3, but not PKP3, resulted in reduced activity levels of protein kinase C (PKC). Restoration of PKC activity using Phorbol 12-myristate 13-acetate (PMA) rescued loss of intestinal barrier function and attenuated the reduced expression patterns of claudin1 and claudin4. Immunostaining, proximity ligation assays and co-immunoprecipitation revealed a direct interaction between PKP2 and PKC. In summary, our in vitro data suggest that PKP2 plays a critical role for intestinal barrier function by providing a signaling hub for PKC-mediated expression of tight junction proteins claudin1 and claudin4.

Cortical tension regulates desmosomal morphogenesis.

Mechanical stability is a fundamental and essential property of epithelial cell sheets. It is in large part determined by cell-cell adhesion sites that are tightly integrated by the cortical cytoskeleton. An intimate crosstalk between the adherens junction-associated contractile actomyosin system and the desmosome-anchored keratin intermediate filament system is decisive for dynamic regulation of epithelial mechanics. A major question in the field is whether and in which way mechanical stress affects junctional plasticity. This is especially true for the desmosome-keratin scaffold whose role in force-sensing is virtually unknown. To examine this question, we inactivated the actomyosin system in human keratinocytes (HaCaT) and canine kidney cells (MDCK) and monitored changes in desmosomal protein turnover.Partial inhibition of myosin II by para-nitro-blebbistatin led to a decrease of the cells' elastic modulus and to reduced desmosomal protein turnover in regions where nascent desmosomes are formed and, to a lower degree, in regions where larger, more mature desmosomes are present. Interestingly, desmosomal proteins are affected differently: a significant decrease in turnover was observed for the desmosomal plaque protein desmoplakin I (DspI), which links keratin filaments to the desmosomal core, and the transmembrane cadherin desmoglein 2 (Dsg2). On the other hand, the turnover of another type of desmosomal cadherin, desmocollin 2 (Dsc2), was not significantly altered under the tested conditions. Similarly, the turnover of the adherens junction-associated E-cadherin was not affected by the low doses of para-nitro-blebbistatin. Inhibition of actin polymerization by low dose latrunculin B treatment and of ROCK-driven actomyosin contractility by Y-27632 treatment also induced a significant decrease in desmosomal DspI turnover. Taken together, we conclude that changes in the cortical force balance affect desmosome formation and growth. Furthermore, they differentially modulate desmosomal protein turnover resulting in changes of desmosome composition. We take the observations as evidence for a hitherto unknown desmosomal mechanosensing and mechanoresponse pathway responding to an altered force balance.

401 PP2A-B55α dephosphorylates desmoplakin's C-terminus to regulate cell adhesion

Critical for the maintenance of epidermal stability and function are attachments between intermediate filaments (IF) and intercellular junctions called desmosomes. The desmosomal cytoplasmic plaque protein desmoplakin (DP) is essential for anchoring IF to the junction. DP-IF interactions are regulated by a phospho-regulatory motif within the DP C-terminus. Previously we showed that hypo-phosphorylated DP increased DP-IF interaction strength, impairing desmosome formation due to DP retention on IF, but generated stronger, more stable desmosomes in mature cell sheets.

Plakophilin3 loss leads to an increase in autophagy and radio-resistance

Loss of the desmosomal plaque protein plakophilin3 (PKP3) leads to increased tumor progression and metastasis. As metastatic tumors are often resistant to therapy, we wished to determine whether PKP3 loss led to increased radioresistance. PKP3 knockdown cells showed increased resistance to radiation in vitro and in vivo. The increase in resistance was accompanied by an increased ability to clear reactive oxygen species (ROS) and increased autophagy. The increase in autophagy was required for radioresistance and ROS clearance as inhibiting autophagy using either chloroquine or knocking down ATG3 re-sensitized the PKP3 knockdown clones to radiotherapy. These experiments suggest that autophagy inhibitors could target therapy-resistant PKP3 deficient tumors.

Steroid hormones and human choriogonadotropin influence the distribution of alpha6-integrin and desmoplakin 1 in gland-like endometrial epithelial spheroids

In human glandular endometrial epithelial cells, desmosomal and adherens junction proteins have been shown to extend from a subapically restricted lateral position to the entire lateral membrane during the implantation window of the menstrual cycle. Similarly, a menstrual cycle stage-dependent redistribution of the extracellular matrix adhesion protein α6-integrin has been reported. These changes are believed to be important for endometrial receptiveness and successful embryo implantation. To prove the hypothesis that steroid hormones and human choriogonadotropin can induce the redistribution of these adhesion molecules, we used the human endometrial cell line Ishikawa in a 3D culture system. Gland-like spheroids were grown in reconstituted basement membrane (Matrigel™). The lumen-bearing spheroids were treated for 2 or 4 days with ovarian steroids or human choriogonadotropin and then assessed by immunofluorescence microscopy. In addition, human endometrial biopsies were obtained from patients, who were in therapy for assisted reproductive technology, and were examined in parallel. Lateral redistribution of the desmosomal plaque protein desmoplakin 1 was observed in the spheroids treated either with progesterone, medroxyprogesterone acetate or human choriogonadotropin. Furthermore, the extracellular matrix adhesion protein α6-integrin showed an increased lateral membrane localization upon gestagen stimulation in the 3D culture system. The results of this study demonstrate that the 3D endometrial Ishikawa cell culture might be suited as an experimental model system to prove the effect of hormonal changes like those occurring during the window of implantation.

Plakophilin 1 but not plakophilin 3 regulates desmoglein clustering.

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

Plakophilin‐dependent Dsg3 oligomerization and binding properties are involved in desmosomal hyper‐adhesion

Under mechanical stress, intercellular adhesion of keratinocytes is critically dependent on desmosomes which, during maturation, acquire a hyper‐adhesive state – at which desmosomal cadherins become independent from Calcium (Ca2+). The desmosomal plaque proteins plakophilins (Pkps) are known to be involved in desmosomal turnover and hyper‐adhesion. However, the molecular mechanism underlying is not elucidated yet. Thus, we here used atomic force microscopy (AFM) in combination with biochemical approaches in murine keratinocytes lacking either Pkp1 or Pkp3 to dissect the underlying mechanism by which Pkps regulate desmosomal cadherin binding properties and desmosomal hyper‐adhesion. Intercellular adhesion was impaired in Pkp3‐ and more severe in Pkp1‐deficient keratinocytes as revealed by keratinocytes dissociation assay. In accordance, Pkps regulated the single molecule binding properties of desmoglein 3 in a manner that Pkp loss led to decreased binding frequency and strength. This was further accompanied by compromised oligomerization of Dsg3. Furthermore, loss of Pkp1 but not Pkp3 causes increased lateral mobility and impaired cluster size of Dsg3. Thus, we questioned whether Pkp‐dependent changes in Dsg clustering and mobility participate in development of a hyper‐adhesive state in those keratinocytes. Interestingly, in keratinocyte dissociation assays wt keratinocytes become partly hyper‐adhesive after 72h whereas Pkp‐loss abolished the development of Ca2+‐independency. Further, Pkp1‐deficiency reduced development of Ca2+‐independent Dsg3 oligomers on the contrary to wt and Pkp3 k.o. keratinocytes. Furthermore, in wt but not in Pkp‐deficient keratinocytes, Dsg3 single molecule binding strength was increased in wt keratinocytes when cells where hyper‐adhesive. Whereas this increase was missing in Pkp‐deficient keratinocytes, which could serve as a molecular explanation for their failure to reach a proper hyper‐adhesive state. In summary, the data provided show that Pkp‐dependent changes in Dsg3 oligomerization as well as binding properties provide a new concept of the molecular mechanism of hyper‐adhesion.Support or Funding InformationElse‐Kröner‐Fresenius‐Stiftung 2016_AW157DFG SPP1782This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inotropic Agent Digitoxin Strengthens Desmosomal Adhesion in Cardiac Myocytes in an ERK1/2‐dependent Manner

Desmosomal proteins including desmoglein‐2 (DSG2), plakoglobin (PG), and desmoplakin (DP) are components of the intercalated disc and mediate cardiac myocyte cell‐cell adhesion. Enhancement of cardiac myocyte cohesion, referred to as “positive adhesiotropy”, was demonstrated to be a function of sympathetic signaling in the heart and to be relevant for a sufficient positive inotropic response to adrenergic stimulation. Here we used the positive inotropic agent digitoxin to further investigate the link between inotropy and adhesiotropy in cardiac myocytes. Digitoxin failed to enhance cardiac contractility in Langendorff‐perfused mouse hearts lacking the desmosomal plaque protein PG indicating that a positive inotropic response requires intact desmosomal adhesion. To investigate a potential effect of digitoxin on desmosomal adhesion, atomic force microscopy was applied and revealed that digitoxin increased the binding force of the adhesion molecule DSG2 at cell‐cell contact areas. This was paralleled by enhanced cardiac myocyte cohesion in both HL‐1 cardiac myocytes and murine cardiac slices as determined by dissociation assays as well as by accumulation of DSG2, DP and PG at cell‐cell contact areas. However, total protein levels or cytoskeletal anchorage of desmosomal proteins was not affected. siRNA‐mediated depletion of DSG2, PG or DP abrogated increase of cell cohesion demonstrating that intact desmosomal adhesion is required for positive adhesiotropy. Mechanistically, digitoxin caused activation of ERK1/2 but not of p38MAPK or Src. In line with this, inhibition of ERK1/2 signaling using the inhibitor UO 126 abrogated the effects of digitoxin on cell‐cell adhesion and desmosomal reorganization. These results show that the positive inotropic agent digitoxin enhances cardiac myocyte cohesion with reorganization of desmosomal proteins in an ERK1/2‐dependent manner. Desmosomal adhesion seems to be important for a sufficient positive inotropic response of digitoxin treatment which can be medical relevant for the treatment of heart failure.Support or Funding InformationThis work was supported by the Ludwig‐Maximilians‐Universität Munich with the Wifomed program and the Deutsche Forschungsgemeinschaft DFG [grant number WA2474/11‐1 to J.W.]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calcium Independence of Epithelial Junctions is Regulated by Protein Mobility

Desmosomes are macromolecular cell adhesion complexes that provide mechanical integrity to epithelial tissues. Unlike other cell junctions, desmosomes can adopt a unique calcium-independent adhesive state where adhesion is maintained in the absence of calcium, also known as hyperadhesion. Regulation of adhesion between the calcium-dependent and calcium-independent states is critical for balancing desmosome strength with plasticity in processes including epithelial tissue homeostasis, embryogenesis, and wound healing. Though changes in signaling coordinated with desmosome adhesion are well understood, adhesion is ultimately determined by binding of cadherins at the desmosome core, and the structural differences between states remain unknown. Here we investigate the mechanism of hyperadhesion by exploring the physical properties of desmosomes, including protein order, desmosome architecture, and protein mobility. We used a human keratinocyte cell line (HaCaT) and confirmed treatment with the PKCa inhibitor Gö6976 induced hyperadhesion. We then used fluorescence polarization microscopy to measure cadherin order. Previously we showed that the cadherin Desmoglein 3 (Dsg3) was ordered in calcium-dependent desmosomes and became disordered after calcium depletion. Here we show when calcium was depleted from hyperadhesive desmosomes, Dsg3 became disordered following the same kinetics, even though desmosomes remained adhesive. This demonstrates that cadherin order is not what drives hyperadhesion. Next, we investigated desmosome plaque architecture by super-resolution STORM and found no changes between calcium-dependent or -independent states. Finally, we investigated protein mobility using FRAP. In calcium-dependent desmosomes cadherins exhibited an average 20% mobile fraction, while we found that there was little to no exchange of cadherins or desmosomal plaque proteins in hyperadhesive desmosomes. This loss of mobility was confirmed using the DP point mutant S2849G, that has been shown to induce hyperadhesion. We conclude that changes in protein mobility represent the structural mechanism by which desmosome hyperadhesion is established.

Ultrastructural changes in endometrial desmosomes of desmoglein 2 mutant mice.

The intercellular binding of desmosomal junctions is mediated by cadherins of the desmoglein (Dsg) and desmocollin (Dsc) type. Dsg2 mutant mice with deletion of a substantial segment of the extracellular EC1-EC2 domain, which is believed to participate in homo- and heterophilic desmosomal cadherin interactions, develop cardiac fibrosis and ventricular dilation. Widening of the intercellular cleft and complete intercalated disc ruptures can be observed in the hearts of these mice. Since a reduced litter size of homozygous Dsg2 mutant mice was noted and a functional correlation between desmosomes and embryo implantation has been deduced from animal studies, we looked for an alteration of desmosomes in uterine endometrial epithelium. Shape and number of desmosomes as well as the expression of Dsg2 and the desmosomal plaque protein desmoplakin (Dsp) were investigated by electron microscopy and immunohistochemistry in 12 oestrous-dated mice (7 wildtype and 5 homozygous Dsg2 mutant mice) at the age of 9-17weeks. The immunohistochemical detection of Dsg2 was diminished in the mutants and the number of desmosomes was significantly reduced as revealed by electron microscopy. In addition, the intercellular desmosomal space measured in electron micrographs was considerably widened in the Dsg2 mutants. The increased intercellular spacing can be explained by the partial deletion of the extracellular EC1-EC2 domain of Dsg2. Whether these changes explain the reduced number of offspring of homozygous Dsg2 mutant mice remains to be further investigated.

14-3-3 proteins regulate desmosomal adhesion via plakophilins.

Desmosomes are essential for strong intercellular adhesion and are abundant in tissues exposed to mechanical strain. At the same time, desmosomes need to be dynamic to allow for remodeling of epithelia during differentiation or wound healing. Phosphorylation of desmosomal plaque proteins appears to be essential for desmosome dynamics. However, the mechanisms of how context-dependent post-translational modifications regulate desmosome formation, dynamics or stability are incompletely understood. Here, we show that growth factor signaling regulates the phosphorylation-dependent association of plakophilins 1 and 3 (PKP1 and PKP3) with 14-3-3 protein isoforms, and uncover unique and partially antagonistic functions of members of the 14-3-3 family in the regulation of desmosomes. 14-3-3γ associated primarily with cytoplasmic PKP1 phosphorylated at S155 and destabilized intercellular cohesion of keratinocytes by reducing its incorporation into desmosomes. In contrast, 14-3-3σ (also known as stratifin, encoded by SFN) interacted preferentially with S285-phosphorylated PKP3 to promote its accumulation at tricellular contact sites, leading to stable desmosomes. Taken together, our study identifies a new layer of regulation of intercellular adhesion by 14-3-3 proteins.

Plakophilin3 increases desmosome assembly, size and stability by increasing expression of desmocollin2

Previous reports show that the desmosomal plaque protein plakophilin3 (PKP3) is essential for desmosome formation. Here, we report that PKP3 over-expression decreases calcium dependency for de novo desmosome formation and makes existing cell-cell adhesion junctions more resilient in low calcium medium due to an increase in desmocollin2 expression. PKP3 overexpression increases the stability of other desmosomal proteins independently of the increase in DSC2 levels and regulates desmosome formation and stability by a multimodal mechanism affecting transcription, protein stability and cell border localization of desmosomal proteins.

Up-regulation of plakophilin-2 is correlated with the progression of glioma.

Glioma is the most common type of primary brain tumor in the CNS. Due to its poor prognosis and high mortality rates, it is urgent to find out more effective therapies. Plakophilin-2 (PKP2) is a widespread desmosomal plaque protein. Recently, the important roles of PKP2 in the proliferation and migration of cancer cells and tumor progression has been shown. However, the expression and potential function of PKP2 in glioma was still unclear. In this study, we demonstrated that PKP2 protein expression level was increased in glioma tissues compared with normal brain tissues, and its level was significantly associated with the Ki-67 expression and WHO grade by Western blot analysis and immunohistochemistry. Clinically, high PKP2 expression was tightly related to poor prognosis of glioma patients. Interestingly, we found that down-regulated PKP2 expression was shown to inhibit the migration of cells in glioma. Moreover, cell counting kit (CCK)-8 and colony formation analyses proved that reduced expression of PKP2 could weaken glioma cell proliferation. Taken together, these data uncover a potential role for PKP2 in the pathogenic process of glioma, suggesting that PKP2 may be a promising therapeutic target of glioma.

Abstract 5052: Pro-oncogenic role of desmosomal plaque-related proteins in non-small cell lung cancer (NSCLC)

Abstract Squamous cell carcinomas and adenocarcinomas are the most frequent forms of lung carcinoma, and have different prognoses and therapeutic approaches. During recent years, accumulating evidence has supported an important role of several junctional proteins in carcinogenesis, tumor invasion and metastasis. Contact between epithelial cells is mediated by several types of cell-cell junction, which are composed by complex arrays of transmembrane and plaque proteins and are connected typically to cytoskeletal components. Desmosomes are cell-cell complexes found primarily in epithelial tissues. In addition to the well-known structural role of desmosome genes, evidences such as their dual subcellular location, the interaction with single-stranded DNA and translation initiation factors, etc. suggest there is a poorly understood role of these proteins in signalling pathways. Desmosomal components have been traditionally considered as tumoral suppressors although current evidences suggest potential pro-oncogenic functions of desmosomal-plaque proteins, probably due to an unknown regulatory role in intracellular signaling -such as in Wnt and Akt pathways. Previously, our group showed differentially expressed gene sequences corresponding to several desmosomal plaque-related proteins as a function of tumor type, stage and differentiation grade in NSCLC. The staining pattern of some junctional proteins also differed between squamous-cell carcinomas and adenocarcinomas. Expression of these proteins marked intercellular junctions that are characteristic of the squamous stratum of stratified flattened epithelium and of neoplasias with this type of differentiation. We observed more intense staining of these proteins in better-differentiated areas. In order to gain greater insight into the specific function of some desmosomal components in NSCLC tumors, we have performed several functional assays, as cell cycle and proliferation among others, in selected SCC lung cancer cell lines. Furthermore, we are developing stable cell lines with over-expression and knockdown of some desmosomal proteins by lentiviral vectors and inducible clonal cell lines; and also carrying out knockout cell lines using CRISPR-Cas9 system. Presumably, they will allow us to perform long-term functional assays and expression arrays in order to identify subcellular location and potential pro-oncogenic roles of desmosomal proteins in NSCLC carcinogenesis which may lead to improvements in the treatment and prevention of this disease. Our preliminary results indicate some desmosomal proteins as potential targets for NSCLC diagnosis and treatment. In a future perspective, xenografts will lead to understand the molecular origins and progression of lung cancer in regard to structural components of the desmosome. Corresponding authors: pedromedina@ugr.es, efarez@ugr.es Citation Format: Pedro P. Medina, Laura Boyero, Joel Martin-Padrón, Esther Farez. Pro-oncogenic role of desmosomal plaque-related proteins in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5052.

Mice with Hepatic Loss of the Desmosomal Protein γ-Catenin Are Prone to Cholestatic Injury and Chemical Carcinogenesis

γ-Catenin, an important component of desmosomes, may also participate in Wnt signaling. Herein, we dissect the role of γ-catenin in liver by generating conditional γ-catenin knockout (KO) mice and assessing their phenotype after bile duct ligation (BDL) and diethylnitrosamine-induced chemical carcinogenesis. At baseline, KO and wild-type littermates showed comparable serum biochemistry, liver histology, and global gene expression. β-Catenin protein was modestly increased without any change in Wnt signaling. Desmosomes were maintained in KO, and despite no noticeable changes in gene expression, differential detergent fractionation revealed quantitative and qualitative changes in desmosomal cadherins, plaque proteins, and β-catenin. Enhanced association of β-catenin to desmoglein-2 and plakophilin-3 was observed in KO. When subjected to BDL, wild-type littermates showed specific changes in desmosomal protein expression. In KO, BDL deteriorated baseline compensatory changes, which manifested as enhanced injury and fibrosis. KO also showed enhanced tumorigenesis to diethylnitrosamine treatment because of Wnt activation, as also verified in vitro. γ-Catenin overexpression in hepatoma cells increased its binding to T-cell factor 4 at the expense of β-catenin-T-cell factor 4 association, induced unique target genes, affected Wnt targets, and reduced cell proliferation and viability. Thus, γ-catenin loss in liver is basally well tolerated. However, after insults like BDL, these compensations at desmosomes fail, and KO show enhanced injury. Also, γ-catenin negatively regulates tumor growth by affecting Wnt signaling.

Two Novel Homozygous Desmoplakin Mutations in Carvajal Syndrome.

Mutations in various desmosomal proteins were shown to cause inherited forms of cardiomyopathy. Carvajal syndrome (Online Mendelian Inheritance in Man [OMIM] 605676) is characterized by the association of dilated cardiomyopathy, striate palmoplantar keratoderma, and woolly hair. It is caused by homozygous as well as heterozygous mutations in DSP, which encodes the desmosomal plaque protein desmoplakin. An overlapping cardiocutaneous phenotype was also described with homozygous mutations in genes encoding two other desmosomal proteins; plakoglobin (Naxos disease; OMIM 601214) and desmocollin-2 (OMIM 610476). We performed clinical and molecular workups in two consanguineous Arab Palestinian families manifesting an autosomal recessive pattern of inheritance of the above mentioned clinical findings. Whole exome sequencing was employed in the search for the causing mutation. Affected family members suffered from biventricular involvement and arrhythmogenic right ventricular dysplasia based on echocardiography and magnetic resonance imaging. One patient who underwent implantation of an implantable cardioverter-defibrillator (ICD) is still alive at the age of 59 years. Whole exome sequencing revealed two novel homozygous mutations in DSP, each affecting one family. The association of woolly hair with palmoplantar keratoderma in a child should lead to a cardiac workup in the search for those at increased risk for sudden cardiac death. Early diagnosis and ICD implantation may be lifesaving. Whole exome sequencing should be utilized for rapid genetic analysis since the cardiocutaneous phenotype may result from mutations in one of several genes.

Expression of desmosomal proteins in acantholytic squamous cell carcinoma of the skin.

Acantholytic (adenoid) squamous cell carcinoma (ASCC) is a subtype of squamous cell carcinoma (SCC) in which neoplastic tumour cells form gland-like structures. Little is known about the pathogenetic mechanisms of ASCC. We hypothesised that they may be related to the compositon of desmosomes. We analysed the immunohistochemical expression of desmosomal proteins in 5 cases of ASCC of the skin, in comparison to 5 cases of conventional SCC of the skin. The most consistent findings were loss of desmoglein 1 (DSG 1), desmoglein 3 (DSG3), desmocollin 1 (DSC1), desmocollin 2 (DSC2), desmocollin 3 (DSC 3), and plakophilin 1 (PKP 1), and decreased expression of desmoplakin 1 (DP 1) and plakoglobin (PG). In conventional well to moderately differentiated SCC, the expression of desmosomal proteins was decreased, but membranous staining was mostly preserved with patterns similar to normal epidermis. Our results suggest that loss of desmosomal cadherins and decreased expression of desmosomal plaque proteins might be responsible for the formation of gland-like structures in ASCC. It seems that desmosomal cadherins, which correspond to the transmembrane core of desmosomes, are predominantly affected in ASCC, while DP 1 and PG, which correspond to cytoplasmic plaque of desmosomes, probably play a lesser role in maintenance of tumour cell cohesion. Our results also indicate that, in addition to previously described verrucous and spindle cell carcinoma, ASCC is another subtype of SCC with a characteristic expression pattern of desmosomal proteins.

Interaction of human trophoblast cells with gland-like endometrial spheroids: a model system for trophoblast invasion.

Do maternal endometrial epithelial cell (EEC) differentiation and polarity impact the invasive capacity of extravillous trophoblast (EVT) cells during early human implantation? In a three dimensional (3D) confrontation co-culture the invasiveness of the human trophoblast cell line AC-1M88 was inversely correlated with the degree of differentiation and polarization of human endometrial adenocarcinoma cell spheroids. In a previous study desmosomal and adherens junction proteins were shown to spread from a subapically restricted lateral position to the entire lateral membrane in human glandular EECs during the implantation window of the menstrual cycle. Whether this change in EEC junction localization has an impact on the interaction of EVT cells with glandular EECs during early human implantation is not known. A new 3D cell culture system was developed in order to mimic early implantation events in humans. As a model for the invasion of endometrial glands by EVT cells, spheroids of three differently differentiated and polarized endometrial adenocarcinoma cell lines were confronted with an EVT cell line in co-culture experiments. Three human adenocarcinoma EEC lines were chosen for this study because of their differences in differentiation and polarization: HEC-1-A, which is well differentiated and highly polarized, Ishikawa, which is well differentiated and moderately polarized, and RL95-2, which is moderately differentiated and poorly polarized. When the cell lines were grown in reconstituted basement membrane, they formed gland-like, multicellular spheroids. The degree of polarization within the different EEC spheroids was assessed by 3D confocal immunofluorescence microscopy detecting the basal membrane protein integrin α6, the apical tight junction-associated protein ZO-1 and the desmosomal plaque protein desmoplakin 1/2 (Dsp). Cells of the human EVT cell line AC-1M88, which is a fusion cell line of primary EVT cells and choriocarcinoma-derived JEG-3 cells, were added to the different EEC spheroids to examine their interaction. For the analyses of trophoblast-endometrial confrontation sites, HLA-G was used as a specific EVT cell marker. The endometrial HEC-1-A and Ishikawa cells formed gland-like structures in reconstituted basement membrane with apicobasal polarization towards their well-developed internal lumina, while most of the RL95-2 spheroids showed no lumen formation at all. The three EEC lines strongly differed in their apicobasal distribution pattern of Dsp. Ishikawa and HEC-1-A spheroids showed a subapical concentration of Dsp. In contrast, an equal distribution of Dsp was discerned along the entire lateral membranes in RL95-2 spheroids. In 3D confrontation co-cultures the highest invasiveness of AC-1M88 was observed in the poorly polarized RL95-2 spheroids. Human endometrial and trophoblast cell lines were used for this study because of ethical and legal restrictions for implantation studies with human blastocysts and because of limited access to primary human endometrial cells. The presented 3D cell culture system can be used to investigate the contribution of epithelial junctions to trophoblast-endometrial interactions. The identified impact of endometrial differentiation and polarity on the invasiveness of EVT cells improves our understanding of the relevance of endometrial receptivity for early implantation and may contribute to higher success rates in assisted reproductive technology. This work was supported by Grant 146/14, 'START-Program', Medical Faculty, RWTH Aachen University, to V.U.B., by Grant Lec_16_12, 'RWTH Lecturer Award', RWTH Aachen University to I.C.-L. and by the German Research Council (Grant LE 566-20-1). The authors declare no conflict of interest.