Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces.

Desmoglein 3 (Dsg3) plays a crucial role in cell-cell adhesion and tissue integrity. Increasing evidence suggests that Dsg3 acts as a regulator of cellular mechanotransduction, but little is known about its direct role in mechanical force transmission. The present study investigated the impact of cyclic strain and substrate stiffness on Dsg3 expression and its role in mechanotransduction in keratinocytes. A direct comparison was made with E-cadherin, a well-characterized mechanosensor. Exposure of oral and skin keratinocytes to equiaxial cyclic strain promoted changes in the expression and localization of junction assembly proteins. The knockdown of Dsg3 by siRNA blocked strain-induced junctional remodeling of E-cadherin and Myosin IIa. Importantly, the study demonstrated that Dsg3 regulates the expression and localization of yes-associated protein (YAP), a mechanosensory, and an effector of the Hippo pathway. Furthermore, we showed that Dsg3 formed a complex with phospho-YAP and sequestered it to the plasma membrane, while Dsg3 depletion had an impact on both YAP and phospho-YAP in their response to mechanical forces, increasing the sensitivity of keratinocytes to the strain or substrate rigidity-induced nuclear relocation of YAP and phospho-YAP. Plakophilin 1 (PKP1) seemed to be crucial in recruiting the complex containing Dsg3/phospho-YAP to the cell surface since its silencing affected Dsg3 junctional assembly with concomitant loss of phospho-YAP at the cell periphery. Finally, we demonstrated that this Dsg3/YAP pathway has an influence on the expression of YAP1 target genes and cell proliferation. Together, these findings provide evidence of a novel role for Dsg3 in keratinocyte mechanotransduction.

The Regulation of the Hippo Pathway by Intercellular Junction Proteins.

The Hippo pathway is an evolutionarily conserved pathway that serves to promote cell death and differentiation while inhibiting cellular proliferation across species. The downstream effectors of this pathway, yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), are considered vital in promoting the output of the Hippo pathway, with activation of upstream kinases negatively regulating YAP/TAZ activity. The upstream regulation of the Hippo pathway is not entirely understood on a molecular level. However, several studies have shown that numerous cellular and non-cellular mechanisms such as cell polarity, contact inhibition, soluble factors, mechanical forces, and metabolism can convey external stimuli to the intracellular kinase cascade, promoting the activation of key components of the Hippo pathway and therefore regulating the subcellular localisation and protein activity of YAP/TAZ. This review will summarise what we have learnt about the role of intercellular junction-associated proteins in the activation of this pathway, including adherens junctions and tight junctions, and in particular our latest findings about the desmosomal components, including desmoglein-3 (DSG3), in the regulation of YAP signalling, phosphorylation, and subcellular translocation.

YAP Inhibition by Verteporfin Causes Downregulation of Desmosomal Genes and Proteins Leading to the Disintegration of Intercellular Junctions.

The Hippo-YAP pathway serves as a central signalling hub in epithelial tissue generation and homeostasis. Yes-associated protein (YAP) is an essential downstream transcription cofactor of this pathway, with its activity being negatively regulated by Hippo kinase-mediated phosphorylation, leading to its cytoplasmic translocation or degradation. Our recent study showed phospho-YAP complexes with Desmoglein-3 (Dsg3), the desmosomal cadherin known to be required for junction assembly and cell-cell adhesion. In this study, we show that YAP inhibition by Verteporfin (VP) caused a significant downregulation of desmosomal genes and a remarkable reduction in desmosomal proteins, including the Dsg3/phospho-YAP complex, resulting in attenuation of cell cohesion. We also found the desmosomal genes, along with E-cadherin, were the YAP-TEAD transcriptional targets and Dsg3 regulated key Hippo components, including WWTR1/TAZ, LATS2 and the key desmosomal molecules. Furthermore, Dsg3 and phospho-YAP exhibited coordinated regulation in response to varied cell densities and culture durations. Overexpression of Dsg3 could compensate for VP mediated loss of adhesion components and proper architecture of cell junctions. Thus, our findings suggest that Dsg3 plays a crucial role in the Hippo network and regulates junction configuration via complexing with phospho-YAP.

Oxidative Stress-Mediated YAP Dysregulation Contributes to the Pathogenesis of Pemphigus Vulgaris.

Pemphigus Vulgaris (PV) is a life-threatening autoimmune disease manifested with blisters in the skin and mucosa and caused by autoantibodies against adhesion protein desmoglein-3 (Dsg3) expressed in epithelial membrane linings of these tissues. Despite many studies, the pathogenesis of PV remains incompletely understood. Recently we have shown Dsg3 plays a role in regulating the yes-associated protein (YAP), a co-transcription factor and mechanical sensor, and constraining reactive oxygen species (ROS). This study investigated the effect of PV sera as well as the anti-Dsg3 antibody AK23 on these molecules. We detected elevated YAP steady-state protein levels in PV cells surrounding blisters and perilesional regions and in keratinocytes treated with PV sera and AK23 with concomitant transient ROS overproduction. Cells treated with hydrogen peroxide also exhibited augmented nuclear YAP accompanied by reduction of Dsg3 and α-catenin, a negative regulator of YAP. As expected, transfection of α-catenin-GFP plasmid rendered YAP export from the nucleus evoked by hydrogen peroxide. In addition, suppression of total YAP was observed in hydrogen peroxide treated cells exposed to antioxidants with enhanced cell-cell adhesion being confirmed by decreased fragmentation in the dispase assay compared to hydrogen peroxide treatment alone. On the other hand, the expression of exogenous YAP disrupted intercellular junction assembly. In contrast, YAP depletion resulted in an inverse effect with augmented expression of junction assembly proteins, including Dsg3 and α-catenin capable of abolishing the effect of AK23 on Dsg3 expression. Finally, inhibition of other kinase pathways, including p38MAPK, also demonstrated suppression of YAP induced by hydrogen peroxide. Furthermore, antioxidant treatment of keratinocytes suppressed PV sera-induced total YAP accumulation. In conclusion, this study suggests that oxidative stress coupled with YAP dysregulation attributes to PV blistering, implying antioxidants may be beneficial in the treatment of PV.

Desmoglein-3 acts as a pro-survival protein by suppressing reactive oxygen species and doming whilst augmenting the tight junctions in MDCK cells.

Kidney disease prevalence increases with age, with a common feature of the disease being defects in the epithelial tight junctions. Emerging evidence suggests that the desmosomal adhesion protein Desmoglein-3 (Dsg3) functions beyond the desmosomal adhesion and plays a role in regulating the fundamental pathways that govern cell fate decisions in response to environmental chemical and mechanical stresses. In this study, we explored the role of Dsg3 on dome formation, reactive oxygen species (ROS) production and transepithelial electrical resistance (TER) in MDCK cells, a kidney epithelial cell model widely used to study cell differentiation and tight junction formation and integrity. We show that overexpression of Dsg3 constrained nuclear ROS production and cellular doming in confluent cell cultures and these features coincided with augmented TER and enhanced tight junction integrity. Conversely, cells expressing dominant-negative Dsg3ΔC mutants exhibited heightened ROS production and accelerated doming, accompanied by increased apoptosis, as well as cell proliferation, with massive disruption in F-actin organization and accumulation, and alterations in tight junctions. Inhibition of actin polymerization and protein synthesis was able to sufficiently block dome formation in mutant populations. Taken together, these findings underscore that Dsg3 has a role in controlling cellular viability and differentiation as well as the functional integrity of tight junctions in MDCK cells.

The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53.

Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell-cell adhesion and regulates various signaling pathways involved in the progression and metastasis of cancer where it is upregulated. We show here that expression of Dsg3 impacts on the expression and function of p53, a key transcription factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect enhanced by treating cells with UVB, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 was Dsg3 specific since neither E-cadherin nor desmoplakin knockdown caused similar effects. Analysis of Dsg3-/- mouse skin also indicated an increase of p53/p21WAF1/CIP1 and cleaved caspase-3 relative to Dsg3+/- controls. Finally, we evaluated whether this pathway was operational in the autoimmune disease PV in which Dsg3 serves as a major antigen involved in blistering pathogenesis. We uncovered increased p53 with diffuse cytoplasmic and/or nuclear staining in the oral mucosa of patients, including cells surrounding blisters and the pre-lesional regions. This finding was verified by in vitro studies where treatment of keratinocytes with PV sera, as well as a characterized pathogenic antibody specifically targeting Dsg3, evoked pronounced p53 expression and activity accompanied by disruption of cell-cell adhesion. Collectively, our findings suggest a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, and this pathway is disrupted in PV.

Desmoglein 3 regulates membrane trafficking of cadherins, an implication in cell-cell adhesion.

E-cadherin mediated cell-cell adhesion plays a critical role in epithelial cell polarization and morphogenesis. Our recent studies suggest that the desmosomal cadherin, desmoglein 3 (Dsg3) cross talks with E-cadherin and regulates its adhesive function in differentiating keratinocytes. However, the underlying mechanism remains not fully elucidated. Since E-cadherin trafficking has been recognized to be a central determinant in cell-cell adhesion and homeostasis we hypothesize that Dsg3 may play a role in regulating E-cadherin trafficking and hence the cell-cell adhesion. Here we investigated this hypothesis in cells with loss of Dsg3 function through RNAi mediated Dsg3 knockdown or the stable expression of the truncated mutant Dsg3ΔC. Our results showed that loss of Dsg3 resulted in compromised cell-cell adhesion and reduction of adherens junction and desmosome protein expression as well as the cortical F-actin formation. As a consequence, cells failed to polarize but instead displayed aberrant cell flattening. Furthermore, retardation of E-cadherin internalization and recycling was consistently observed in these cells during the process of calcium induced junction assembling. In contrast, enhanced cadherin endocytosis was detected in cells with overexpression of Dsg3 compared to control cells. Importantly, this altered cadherin trafficking was found to be coincided with the reduced expression and activity of Rab proteins, including Rab5, Rab7 and Rab11 which are known to be involved in E-cadherin trafficking. Taken together, our findings suggest that Dsg3 functions as a key in cell-cell adhesion through at least a mechanism of regulating E-cadherin membrane trafficking.

Evidence for Dsg3 in regulating Src signaling by competing with it for binding to caveolin-1.

This data article contains extended, complementary analysis related to the research articles entitled "Desmoglein 3, via an interaction with E-cadherin, is associated with activation of Src" (Tsang et al., 2010) [1] and figures related to the review article entitled "Desmoglein 3: a help or a hindrance in cancer progression?" (Brown et al., 2014) [2]. We show here that both Src and caveolin-1 (Cav-1) associate with Dsg3 in a non-ionic detergent soluble pool and that modulation of Dsg3 levels inversely alters the expression of Src in the Cav-1 complex. Furthermore, immunofluorescence analysis revealed a reduced colocalization of Cav-1/total Src in cells with overexpression of Dsg3 compared to control cells. In support, the sequence analysis has identified a region within the carboxyl-terminus of human Dsg3 for a likelihood of binding to the scaffolding domain of Cav-1, the known Src binding site in Cav-1, and this region is highly conserved across most of 18 species as well as within desmoglein family members. Based on these findings, we propose a working model that Dsg3 activates Src through competing with its inactive form for binding to Cav-1, thus leading to release of Src followed by its auto-activation.