Eliane J. Müller

Pemphigus vulgaris identifies plakoglobin as key suppressor of c-Myc in the skin

The autoimmune disease pemphigus vulgaris (PV) manifests as loss of keratinocyte cohesion triggered by autoantibody binding to desmoglein (Dsg)3, an intercellular adhesion molecule of mucous membranes, epidermis, and epidermal stem cells. Here we describe a so far unknown signaling cascade activated by PV antibodies. It extends from a transient enhanced turn over of cell surface‐exposed, nonkeratin‐anchored Dsg3 and associated plakoglobin (PG), through to depletion of nuclear PG, and as one of the consequences, abrogation of PG‐mediated c‐Myc suppression. In PV patients (6/6), this results in pathogenic c‐Myc overexpression in all targeted tissues, including the stem cell compartments. In summary, these results show that PV antibodies act via PG to abolish the c‐Myc suppression required for both maintenance of epidermal stem cells in their niche and controlled differentiation along the epidermal lineage. Besides a completely novel insight into PV pathogenesis, these data identify PG as a potent modulator of epithelial homeostasis via its role as a key suppressor of c‐Myc.

Are desmoglein autoantibodies essential for the immunopathogenesis of pemphigus vulgaris, or just ‘witnesses of disease'?

Abstract:  Pemphigus vulgaris (PV) is fascinating to dermatologists, epithelial biologists and immunologists alike, as its pathogenesis has been clarified to a much greater extent than that of most other organ‐specific autoimmune diseases, and as it has provided abundant novel insights into desmoglein biology and pathology along the way. Historically, the most influential PV pathogenesis concept is that of Stanley and Amagai. This concept holds that autoantibodies against desmogleins are both essential and sufficient for epidermal blister formation (acantholysis) by impeding the normal functioning of these major adhesion proteins. However, as with most good theories, this landmark concept has left a number of intriguing and important questions open (or at least has not managed to answer these to everyones satisfaction). Moreover, selected dissenting voices in the literature have increasingly called attention to what may or may not be construed as inconsistencies in this dominant PV pathogenesis paradigm of the recent past. The present debate feature therefore bravely rises to the challenge of re‐examining the entire currently available evidence, as rationally and as undogmatically as possible, by provocatively asking a carefully selected congregation of experts (who have never before jointly published on this controversial topic!) to discuss how essential anti‐desmoglein autoantibodies really are in the immunopathogenesis of PV. Not surprisingly, some of our expert ‘witnesses’ in this animated debate propose diametrically opposed answers to this question. While doing so, incisive additional questions are raised that relate to the central one posed, and our attention is called to facts that may deserve more careful consideration than they have received so far. Together with the intriguing (often still very speculative) complementary or alternative pathogenesis scenarios proposed in the following pages, this offers welcome ‘food for thought’ as well as very specific suggestions for important future research directions – within and beyond the camp of PV aficionados. The editors trust that this attempt at a rational public debate of the full evidence that is currently at hand will constructively contribute to further dissecting the exciting – and clinically very relevant! – immunopathogenesis of PV in all its complexity.

Mechanisms of Plakoglobin-dependent Adhesion DESMOSOME-SPECIFIC FUNCTIONS IN ASSEMBLY AND REGULATION BY EPIDERMAL GROWTH FACTOR RECEPTOR

Plakoglobin (PG) is a member of the Armadillo family of adhesion/signaling proteins that can be incorporated into both adherens junctions and desmosomes. Loss of PG results in defects in the mechanical integrity of heart and skin and decreased adhesive strength in keratinocyte cultures established from the skin of PG knock-out (PG-/-) mice, the latter of which cannot be compensated for by overexpressing the closely related β-catenin. In this study, we examined the mechanisms of PG-regulated adhesion in murine keratinocytes. Biochemical and morphological analyses indicated that junctional incorporation of desmosomal, but not adherens junction, components was impaired in PG-/- cells compared with PG+/- controls. Re-expression of PG, but not β-catenin, in PG-/- cells largely reversed these effects, indicating a key role for PG in desmosome assembly. Epidermal growth factor (EGF) receptor activation resulted in Tyr phosphorylation of PG, which was accompanied by a loss of desmoplakin from desmosomes and decreased adhesive strength following 18-h EGF treatment. Importantly, introduction of a phosphorylation-deficient PG mutant into PG null cells prevented the EGF receptor-dependent loss of desmoplakin from junctions, attenuating the effects of long term EGF treatment on cell adhesion. Therefore, PG is essential for maintaining and regulating adhesive strength in keratinocytes largely through its contributions to desmosome assembly and structure. As a target for modulation by EGF, regulation of PG-dependent adhesion may play an important role during wound healing and tumor metastasis.

Cardiac tissue-restricted deletion of plakoglobin results in progressive cardiomyopathy and activation of {beta}-catenin signaling.

ABSTRACT Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein via its ability to modulate the Wnt/β-catenin signaling pathway. To investigate the role of plakoglobin in ARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice. Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients. Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to induced arrhythmias. Ablation of plakoglobin caused increase β-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3β. Finally, β-catenin/TCF transcriptional activity may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of ARVC demonstrates for the first time how plakoglobin affects β-catenin activity in the heart and its implications for disease pathogenesis.Mutations in the plakoglobin (JUP) gene have been identified in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients. However, the mechanisms underlying plakoglobin dysfunction involved in the pathogenesis of ARVC remain poorly understood. Plakoglobin is a component of both desmosomes and adherens junctions located at the intercalated disc (ICD) of cardiomyocytes, where it functions to link cadherins to the cytoskeleton. In addition, plakoglobin functions as a signaling protein via its ability to modulate the Wnt/β-catenin signaling pathway. To investigate the role of plakoglobin in ARVC, we generated an inducible cardiorestricted knockout (CKO) of the plakoglobin gene in mice. Plakoglobin CKO mice exhibited progressive loss of cardiac myocytes, extensive inflammatory infiltration, fibrous tissue replacement, and cardiac dysfunction similar to those of ARVC patients. Desmosomal proteins from the ICD were decreased, consistent with altered desmosome ultrastructure in plakoglobin CKO hearts. Despite gap junction remodeling, plakoglobin CKO hearts were refractory to induced arrhythmias. Ablation of plakoglobin caused increase β-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3β. Finally, β-catenin/TCF transcriptional activity may contribute to the cardiac hypertrophy response in plakoglobin CKO mice. This novel model of ARVC demonstrates for the first time how plakoglobin affects β-catenin activity in the heart and its implications for disease pathogenesis.

Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures.

Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro.

Proprotein cleavage of E-cadherin by furin in baculovirus over-expression system: potential role of other convertases in mammalian cells.

Sequence analysis of the adhesion molecule E‐cadherin had revealed a multibasic motif [4PArg‐Gln‐Lys‐Arg1P], reminiscent of the minimal cleavage signal for furin, the prototype of the proprotein convertase family, and/or other members sharing similar sequence specificity. Mutation of this site was sufficient to abolish processing of E‐cadherin in fibroblasts reinforcing the possibility that proprotein convertases are involved in the maturation of this adhesion molecule. Here we demonstrate that even though furin can efficiently and specifically cleave proE‐cadherin in a baculovirus‐based co‐expression system, the furin‐deficient LoVo cells were found to process endogenous E‐cadherin as efficiently as normal cell lines. This suggests, for the first time, that E‐cadherin is not only a substrate for furin but for other mammalian convertases sharing similar sequence specificity.

The Metalloprotease Meprinβ Processes E-Cadherin and Weakens Intercellular Adhesion

Background Meprin (EC 3.4.24.18), an astacin-like metalloprotease, is expressed in the epithelium of the intestine and kidney tubules and has been related to cancer, but the mechanistic links are unknown. Methodology/Principal Findings We used MDCK and Caco-2 cells stably transfected with meprinα and or meprinβ to establish models of renal and intestinal epithelial cells expressing this protease at physiological levels. In both models E-cadherin was cleaved, producing a cell-associated 97-kDa E-cadherin fragment, which was enhanced upon activation of the meprin zymogen and reduced in the presence of a meprin inhibitor. The cleavage site was localized in the extracellular domain adjacent to the plasma membrane. In vitro assays with purified components showed that the 97-kDa fragment was specifically generated by meprinβ, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins β-catenin and plakoglobin were processed by an intracellular protease, whereas α-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprinβ and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprinβ-expressing cells displayed a reduced strength of cell-cell contacts and a significantly lower tendency to form multicellular aggregates. Conclusions/Significance By identifying E-cadherin as a substrate for meprinβ in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprinβ in the control of adhesiveness via cleavage of E-cadherin with potential implications in a wide range of biological processes including epithelial barrier function and cancer progression.

Missing C‐terminal filaggrin expression, NFkappaB activation and hyperproliferation identify the dog as a putative model to study epidermal dysfunction in atopic dermatitis

Please cite this paper as: Missing C‐terminal filaggrin expression, NFkappaB activation and hyperproliferation identify the dog as a putative model to study epidermal dysfunction in atopic dermatitis. Experimental Dermatology 2010; 19: e343–e346.

β-Catenin is not required for proliferation and differentiation of epidermal mouse keratinocytes

Despite the pivotal role of β-catenin in a variety of biological processes, conditional β-catenin gene ablation in the skin of transgenic mice failed to affect interfollicular epidermal morphogenesis. We elucidated the molecular mechanisms underlying this phenomenon. Long-term cultures of homozygous, heterozygous and β-catenin-null mutant keratinocytes were established to demonstrate that epidermal keratinocyte proliferation, cell cycle progression and cyclin D1 expression occur independently of β-catenin and correlate with repression of transcription from Tcf/Lef-responsive promoters. Moreover, during differentiation,β -catenin-null cells assemble normal intercellular adhesion junctions owing to the substitution of β-catenin with plakoglobin, whereas the expression of the other adhesion components remains unaffected. Taken together, our results demonstrate that epidermal proliferation and adhesion are independent of β-catenin.

Plakoglobin Is Required for Effective Intermediate Filament Anchorage to Desmosomes

Desmosomes are adhesive junctions that provide mechanical coupling between cells. Plakoglobin (PG) is a major component of the intracellular plaque that serves to connect transmembrane elements to the cytoskeleton. We have used electron tomography and immunolabeling to investigate the consequences of PG knockout on the molecular architecture of the intracellular plaque in cultured keratinocytes. Although knockout keratinocytes form substantial numbers of desmosome-like junctions and have a relatively normal intercellular distribution of desmosomal cadherins, their cytoplasmic plaques are sparse and anchoring of intermediate filaments is defective. In the knockout, beta-catenin appears to substitute for PG in the clustering of cadherins, but is unable to recruit normal levels of plakophilin-1 and desmoplakin to the plaque. By comparing tomograms of wild type and knockout desmosomes, we have assigned particular densities to desmoplakin and described their interaction with intermediate filaments. Desmoplakin molecules are more extended in wild type than knockout desmosomes, as if intermediate filament connections produced tension within the plaque. On the basis of our observations, we propose a particular assembly sequence, beginning with cadherin clustering within the plasma membrane, followed by recruitment of plakophilin and desmoplakin to the plaque, and ending with anchoring of intermediate filaments, which represents the key to adhesive strength.