Meeting report - Desmosome dysfunction and disease: Alpine desmosome disease meeting.

Desmosome diseases are caused by dysfunction of desmosomes, which anchor intermediate filaments (IFs) at sites of cell-cell adhesion. For many decades, the focus of attention has been on the role of actin filament-associated adherens junctions in development and disease, especially cancer. However, interference with the function of desmosomes, their molecular constituents or their attachments to IFs has now emerged as a major contributor to a variety of diseases affecting different tissues and organs including skin, heart and the digestive tract. The first Alpine desmosome disease meeting (ADDM) held in Grainau, Germany, in October 2022 brought together international researchers from the basic sciences with clinical experts from diverse fields to share and discuss their ideas and concepts on desmosome function and dysfunction in the different cell types involved in desmosome diseases. Besides the prototypic desmosomal diseases pemphigus and arrhythmogenic cardiomyopathy, the role of desmosome dysfunction in inflammatory bowel diseases and eosinophilic esophagitis was discussed.

Abnormal keratinocyte differentiation in the nasal planum of Labrador Retrievers with hereditary nasal parakeratosis (HNPK).

Hereditary nasal parakeratosis (HNPK) is an inherited disorder described in Labrador Retrievers and Greyhounds. It has been associated with breed-specific variants in the SUV39H2 gene encoding a histone 3 methyltransferase involved in epigenetic silencing. Formalin-fixed biopsies of the nasal planum of Labrador Retrievers were screened by immunofluorescence microscopy for the presence and distribution of epidermal proliferation and differentiation markers. Gene expression of these markers was further analysed using RNA sequencing (RNA-seq) and ultrastructural epidermal differences were investigated by electron microscopy. Differentiation of the nasal planum in the basal and suprabasal epidermal layers of HNPK-affected dogs (n = 6) was similar compared to control dogs (n = 6). In the upper epidermal layers, clear modifications were noticed. Loricrin protein was absent in HNPK-affected nasal planum sections in contrast to sections of the same location of control dogs. However, loricrin was present in the epidermis of paw pads and abdominal skin from HNPK dogs and healthy control dogs. The patterns of keratins K1, K10 and K14, were not markedly altered in the nasal planum of HNPK-affected dogs while the expression of the terminal differentiation marker involucrin appeared less regular. Based on RNA-seq, LOR and IVL expression levels were significantly decreased, while KRT1, KRT10 and KRT14 levels were up-regulated (log2fold-changes of 2.67, 3.19 and 1.71, respectively) in HNPK-affected nasal planum (n = 3) compared to control dogs (n = 3). Electron microscopical analysis revealed structural alterations in keratinocytes and stratum corneum, and disrupted keratinocyte adhesions and distended intercellular spaces in lesional samples (n = 3) compared to a sample of a healthy control dog (n = 1). Our findings demonstrate aberrant keratinocyte terminal differentiation of the nasal planum of HNPK-affected Labrador Retrievers and provide insights into biological consequences of this inactive SUV39H2 gene variant.

Toll-like receptor-ligand induced thymic stromal lymphopoietin expression in primary equine keratinocytes.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) plays a key role in the development of allergic inflammation. Little is known about possible triggers of equine TSLP expression. HYPOTHESIS/OBJECTIVES: To investigate TSLP expression in equine insect bite hypersensitivity (IBH) skin lesions. The capacity of TLR 1-8 ligands (L) and of atopic cytokine milieu as potential triggers of TSLP and of interleukin (IL)-6 as a downstream effector molecule of TLR signalling, were examined in primary equine keratinocyte cultures. ANIMALS: Lesional skin from IBH-affected and healthy skin from control-horses (n = 9 each group) was sampled. METHODS AND MATERIALS: Keratinocyte cultures were established from six healthy horses and stimulated with TLR 1-8-L, and with IL-4 and tumor necrosis factor-α, to mimic an atopic inflammation cytokine milieu. TSLP and IL-6 gene expression was assessed by quantitative real-time PCR. RESULTS: Expression of TSLP was significantly greater in IBH lesions compared to healthy skin. TLR 1-8-L significantly upregulated TSLP expression in keratinocytes. The strongest upregulation was induced by TLR 1/2-L and TLR 3-L. Combination of atopic cytokine milieu and TLR 1/2-L or TLR 3-L further increased TSLP expression. TLR-L 1-5 stimulation significantly upregulated IL-6 expression. CONCLUSIONS AND CLINICAL IMPORTANCE: The data herein suggest that the upregulation of TSLP expression in lesional skin of IBH-affected horses might play a role in IBH development. Moreover, TSLP expression is induced by TLR-L, in particular by TLR 1/2-L and TLR 3-L, and is further increased by atopic cytokine milieu, indicating a mechanism for TSLP-mediated exacerbation of IBH.

FAM83G/Fam83g genetic variants affect canine and murine hair formation.

FAM83G/Fam83g genetic variants have been described in dogs, mice and recently also in humans. They are associated with palmoplantar keratoderma and altered hair or coat phenotype, reported as wooly phenotype in mice. FAM83G/Fam83g is an unexplored effector of temporally and spatially coordinated Wnt and BMP signalling which are key pathways in pre- and postnatal hair follicle morphogenesis and differentiation. The aim of this study was to unravel phenotypic consequences of FAM83G/Fam83g variants on hair coat formation in dogs and mice. Our results show differences in hair types and hair shaft structures in both species. Additionally, mice exhibit deregulated hair cycle progression which timely correlates with defective Wnt signalling (Axin2) and Bmp2/4 expression. These results affirm the involvement of FAM83G in hair morphogenesis, hair follicle differentiation and cycling.

A Splice Defect in the EDA Gene in Dogs with an X-Linked Hypohidrotic Ectodermal Dysplasia (XLHED) Phenotype.

X-linked hypohidrotic ectodermal dysplasia (XLHED) caused by variants in the EDA gene represents the most common ectodermal dysplasia in humans. We investigated three male mixed-breed dogs with an ectodermal dysplasia phenotype characterized by marked hypotrichosis and multifocal complete alopecia, almost complete absence of sweat and sebaceous glands, and altered dentition with missing and abnormally shaped teeth. Analysis of SNP chip genotypes and whole genome sequence data from the three affected dogs revealed that the affected dogs shared the same haplotype on a large segment of the X-chromosome, including the EDA gene. Unexpectedly, the whole genome sequence data did not reveal any nonsynonymous EDA variant in the affected dogs. We therefore performed an RNA-seq experiment on skin biopsies to search for changes in the transcriptome. This analysis revealed that the EDA transcript in the affected dogs lacked 103 nucleotides encoded by exon 2. We speculate that this exon skipping is caused by a genetic variant located in one of the large introns flanking this exon, which was missed by whole genome sequencing with the illumina short read technology. The altered EDA transcript splicing most likely causes the observed ectodermal dysplasia in the affected dogs. These dogs thus offer an excellent opportunity to gain insights into the complex splicing processes required for expression of the EDA gene, and other genes with large introns.

Stem Cell-Associated Marker Expression in Canine Hair Follicles.

Functional hair follicle (HF) stem cells (SCs) are crucial to maintain the constant recurring growth of hair. In mice and humans, SC subpopulations with different biomarker expression profiles have been identified in discrete anatomic compartments of the HF. The rare studies investigating canine HF SCs have shown similarities in biomarker expression profiles to that of mouse and human SCs. The aim of our study was to broaden the current repertoire of SC-associated markers and their expression patterns in the dog. We combined analyses on the expression levels of CD34, K15, Sox9, CD200, Nestin, LGR5 and LGR6 in canine skin using RT-qPCR, the corresponding proteins in dog skin lysates, and their expression patterns in canine HFs using immunohistochemistry. Using validated antibodies, we were able to define the location of CD34, Sox9, Keratin15, LGR5 and Nestin in canine HFs and confirm that all tested biomarkers are expressed in canine skin. Our results show similarities between the expression profile of canine, human and mouse HF SC markers. This repertoire of biomarkers will allow us to conduct functional studies and investigate alterations in the canine SC compartment of different diseases, like alopecia or skin cancer with the possibility to extend relevant findings to human patients.

Preclinical studies identify non-apoptotic low-level caspase-3 as therapeutic target in pemphigus vulgaris.

The majority of pemphigus vulgaris (PV) patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis). The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg) 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG), PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice) as well as PV patients' biopsies (n=6). A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP) and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the in vitro and in vivo PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other diseases including cancer.

EGFR inhibitors erlotinib and lapatinib ameliorate epidermal blistering in pemphigus vulgaris in a non-linear, V-shaped relationship.

Novel insights into intra-cellular signalling involved in pemphigus vulgaris (PV), an autoimmune blistering disease of skin and mucous membranes, are now revealing new therapeutic approaches such as the chemical inhibition of PV-associated signals in conjunction with standard immunosuppressive therapy. However, extensive inhibition of signalling molecules that are required for normal tissue function and integrity may hamper this approach. Using a neonatal PV mouse model, we demonstrate that epidermal blistering can be prevented in a dose-dependent manner by clinically approved EGFR inhibitors erlotinib and lapatinib, but only up to approximately 50% of normal EGFR activity. At lower EGFR activity, blisters again aggravated and were highly exacerbated in mice with a conditional deletion of EGFR. Statistical analysis of the relation between EGFR activity and the extent of skin blistering revealed the best fit with a non-linear, V-shaped curve with a median break point at 52% EGFR activity (P = 0.0005). Moreover, lapatinib (a dual EGFR/ErbB2 inhibitor) but not erlotinib significantly reduced blistering in the oral cavity, suggesting that signalling mechanisms differ between PV predilection sites. Our results demonstrate that future clinical trials evaluating EGFR/ErbB2 inhibitors in PV patients must select treatment doses that retain a specific level of signal molecule activity. These findings may also be of relevance for cancer patients treated with EGFR inhibitors, for whom skin lesions due to extensive EGFR inhibition represent a major threat.

Canine keratinocytes upregulate type I interferons and proinflammatory cytokines in response to poly(dA:dT) but not to canine papillomavirus.

Papillomaviruses (PV) are double stranded (ds) DNA viruses that infect epithelial cells within the skin or mucosa, most often causing benign neoplasms that spontaneously regress. The immune system plays a key role in the defense against PVs. Since these viruses infect keratinocytes, we wanted to investigate the role of the keratinocyte in initiating an immune response to canine papillomavirus-2 (CPV-2) in the dog. Keratinocytes express a variety of pattern recognition receptors (PRR) to distinguish different cutaneous pathogens and initiate an immune response. We examined the mRNA expression patterns for several recently described cytosolic nucleic acid sensing PRRs in canine monolayer keratinocyte cultures using quantitative reverse transcription-polymerase chain reaction. Unstimulated normal cells were found to express mRNA for melanoma differentiation associated gene 5 (MDA5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activation of interferon regulatory factors, leucine rich repeat flightless interacting protein 1, and interferon inducible gene 16 (IFI16), as well as their adaptor molecules myeloid differentiation primary response gene 88, interferon-ß promoter stimulator 1, and endoplasmic reticulum-resident transmembrane protein stimulator of interferon genes. When stimulated with synthetic dsDNA [poly(dA:dT)] or dsRNA [poly(I:C)], keratinocytes responded with increased mRNA expression levels for interleukin-6, tumor necrosis factor-α, interferon-ß, RIG-I, IFI16, and MDA5. There was no detectable increase in mRNA expression, however, in keratinocytes infected with CPV-2. Furthermore, CPV-2-infected keratinocytes stimulated with poly(dA:dT) and poly(I:C) showed similar mRNA expression levels for these gene products when compared with expression levels in uninfected cells. These results suggest that although canine keratinocytes contain functional PRRs that can recognize and respond to dsDNA and dsRNA ligands, they do not appear to recognize or initiate a similar response to CPV-2.

Clinical and histological characterization of hair coat and glandular tissue of Chinese crested dogs.

BACKGROUND: Two varieties exist in the Chinese crested dog breed, namely hairless Chinese crested dogs presenting with hypotrichosis and dentition abnormalities, and the coated powderpuffs. Hairless Chinese crested dogs are obligate heterozygotes for a FOXI3 mutation, and this phenotype is classified as a form of canine ectodermal dysplasia. OBJECTIVES: We provide a detailed histological description of hair follicles and their density for the three subphenotypes (true hairless, semi-coated and powderpuffs) of Chinese crested dogs. Apocrine and exocrine glands of the skin and other tissues were compared with findings reported from dogs with X-linked ectodermal dysplasia. ANIMALS: Skin biopsies were collected from 22 Chinese crested dogs. Additionally, the glands of the skin and other tissues were examined from another two dogs available for postmortem examination. METHODS: Skin biopsies and tissues were processed, stained and evaluated in a blinded fashion. RESULTS: Hair follicular anomalies decreased with increasing number of hairs in the different phenotypes. The FOXI3 mutants had only simple primary hair follicles, whereas the nonmutant powderpuffs had compound follicles identical to other dog breeds. All Chinese crested dogs had an anagen-dominated hair cycle. Furthermore, apocrine glands in the skin and respiratory mucous glands of the mutant Chinese crested dogs were present and normal. CONCLUSIONS AND CLINICAL IMPORTANCE: We have identified striking histopathological differences between the three subphenotypes of Chinese crested dogs. We clearly demonstrated distinct differences between the canine ectodermal dysplasia in Chinese crested dogs and dogs with X-linked ectodermal dysplasia.

Expression of thymic stromal lymphopoietin in canine atopic dermatitis.

BACKGROUND: In humans, thymic stromal lymphopoietin (TSLP) plays a central role in the development of allergic inflammation, such as atopic dermatitis (AD), but it is unknown whether it is involved in the pathogenesis of canine AD (CAD). HYPOTHESIS/OBJECTIVES: Our aim was to characterize canine TSLP and to assess its expression in CAD. METHODS: Canine TSLP was identified based on sequence homology with human TSLP and the complementary DNA (cDNA) cloned by RT-PCR. Real-time quantitative RT-PCR was established to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsy specimens from lesional and nonlesional skin of 12 dogs with CAD and eight healthy control dogs. RESULTS: Partial canine TSLP cDNA was cloned and characterized. It contained four exons that shared 70 and 73% nucleotide identity with human and equine TSLP, respectively, encoding the signal peptide and full-length secreted protein. We found significantly increased TSLP expression in lesional and nonlesional skin of dogs with CAD compared with healthy control dogs (P < 0.05), whereas no difference was measured between lesional and nonlesional samples. In cultured primary canine keratinocytes, we found increased TSLP expression after stimulation with house dust mite allergen extract or Toll-like receptor ligands lipopolysaccharide and poly I:C. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased TSLP expression in the skin of dogs with CAD supports an involvement of TSLP in the pathogenesis of CAD similar to that in humans. Further studies should elucidate the function and therapeutic potential of TSLP in CAD.

Analysis of a Jup hypomorphic allele reveals a critical threshold for postnatal viability.

Mutations in the human Jup gene cause arrhythmogenic right ventricular cardiomyopathy (ARVC), a heart muscle disease that often leads to sudden cardiac death. Inactivation of the murine Jup gene (also known as plakoglobin) results in embryonic lethality due to cardiac rupture. In an effort to generate a conditional knockout allele, a neomycin cassette was introduced into the murine plakoglobin (PG) gene. This allele (PG F(N)) functions as a hypomorph when combined with a null allele (PG Δ). About half of the PG F(N)/Δ animals were smaller than their littermates and died before weaning age, whereas the remaining PG F(N)/Δ animals survived. Despite the reduced levels of PG in the heart, there were no signs of cardiomyopathy or cardiac dysfunction as determined by echocardiography. Importantly, the PG homolog, ß-catenin (CTNNB1), was increased in the PG F(N)/Δ hearts. In addition to its structural role as part of the N-cadherin/catenin adhesion complex, ß-catenin is a downstream effector of Wnt signaling. However, no change in ß-catenin/TCF reporter activity was observed in PG F(N)/Δ embryos suggesting that excess ß-catenin was not likely causing increased transcription of Wnt/ß-catenin target genes. These data suggest novel function(s) for PG beyond the heart and define a critical threshold of PG expression that is necessary for postnatal survival.

An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris.

Evidence has accumulated that changes in intracellular signaling downstream of desmoglein 3 (Dsg3) may have a significant role in epithelial blistering in the autoimmune disease pemphigus vulgaris (PV). Currently, most studies on PV involve passive transfer of pathogenic antibodies into neonatal mice that have not finalized epidermal morphogenesis, and do not permit analysis of mature hair follicles (HFs) and stem cell niches. To investigate Dsg3 antibody-induced signaling in the adult epidermis at defined stages of the HF cycle, we developed a model with passive transfer of AK23 (a mouse monoclonal pathogenic anti-Dsg3 antibody) into adult 8-week-old C57Bl/6J mice. Validated using histopathological and molecular methods, we found that this model faithfully recapitulates major features described in PV patients and PV models. Two hours after AK23 transfer, we observed widening of intercellular spaces between desmosomes and EGFR activation, followed by increased Myc expression and epidermal hyperproliferation, desmosomal Dsg3 depletion, and predominant blistering in HFs and oral mucosa. These data confirm that the adult passive transfer mouse model is ideally suited for detailed studies of Dsg3 antibody-mediated signaling in adult skin, providing the basis for investigations on novel keratinocyte-specific therapeutic strategies.

Survivin in skin pathologies.

Survivin is a member of the inhibitor of apoptosis (IAP) protein family acting at the intersection between proliferation and cell survival. This protein exhibits low or undetectable expression in most adult tissues but is increased in the majority of cancers. Suggested to be one of the most cancer-specific proteins identified to date, survivin acts as a signalling node in tumour maintenance and, after first promising results, is now attracting increasing attention as a target in anti-cancer therapy. In the skin, survivin has been implicated in a number of pathological conditions such as psoriasis and tumours of melanocytic and epithelial origin. Its expression can correlate with tumour severity, metastasis and decreased patient survival and has been inversely correlated with the sensitivity to cytotoxic agents used in anti-cancer therapy. Survivin may also be of importance for normal epidermal homeostasis possibly supporting self-renewal of epidermal stem cells. In this review, the authors summarize and discuss current data of survivin in skin biology and provide a comprehensive compilation of survivin expression in skin pathologies with focus on future therapeutical use.

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

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/ß-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 ß-catenin stabilization associated with activated AKT and inhibition of glycogen synthase kinase 3ß. Finally, ß-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 ß-catenin activity in the heart and its implications for disease pathogenesis.

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

Filaggrin loss-of-function mutations resulting in C-terminal protein truncations are strong predisposing factors in human atopic dermatitis (AD). To assess the possibility of similar truncations in canine AD, an exclusion strategy was designed on 16 control and 18 AD dogs of various breeds. Comparative immunofluorescence microscopy was performed with an antibody raised against the canine filaggrin C-terminus and a commercial N-terminal antibody. Concurrent with human AD-like features such as generalized NFKB activation and hyperproliferation, four distinctive filaggrin expression patterns were identified in non-lesional skin. It was found that 10/18 AD dogs exhibited an identical pattern for both antibodies with comparable (category I, 3/18) or reduced (category II, 7/18) expression to that of controls. In contrast, 4/18 dogs displayed aberrant large vesicles revealed by the C-terminal but not the N-terminal antibody (category III), while 4/18 showed a control-like N-terminal expression but lacked the C-terminal protein (category IV). The missing C-terminal filaggrin in category IV strongly points towards loss-of function mutations in 4/18 (22%) of all AD dogs analysed.

The metalloprotease meprinbeta 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 meprin alpha and or meprin beta 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 meprin beta, but not by ADAM-10 or MMP-7. Concomitantly with E-cadherin cleavage and degradation of the E-cadherin cytoplasmic tail, the plaque proteins beta-catenin and plakoglobin were processed by an intracellular protease, whereas alpha-catenin, which does not bind directly to E-cadherin, remained intact. Using confocal microscopy, we observed a partial colocalization of meprin beta and E-cadherin at lateral membranes of incompletely polarized cells at preconfluent or early confluent stages. Meprin beta-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 meprin beta in a cellular context, this study reveals a novel biological role of this protease in epithelial cells. Our results suggest a crucial role for meprin beta 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.

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.

Outside-in signaling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation?

The process of epidermal renewal persists throughout the entire life of an organism. It begins when a keratinocyte progenitor leaves the stem cell compartment, undergoes a limited number of mitotic divisions, exits the cell cycle, and commits to terminal differentiation. At the end of this phase, the postmitotic keratinocytes detach from the basement membrane to build up the overlaying stratified epithelium. Although highly coordinated, this sequence of events is endowed with a remarkable versatility, which enables the quiescent keratinocyte to reintegrate into the cell cycle and become migratory when necessary, for example after wounding. It is this versatility that represents the Achilles heel of epithelial cells allowing for the development of severe pathologies. Over the past decade, compelling evidence has been provided that epithelial cancer cells achieve uncontrolled proliferation following hijacking of a "survival program" with PI3K/Akt and a "proliferation program" with growth factor receptor signaling at its core. Recent insights into adhesion receptor signaling now propose that integrins, but also cadherins, can centrally control these programs. It is suggested that the two types of adhesion receptors act as sensors to transmit extracellular stimuli in an outside-in mode, to inversely modulate epidermal growth factor receptor signaling and ensure cell survival. Hence, cell-matrix and cell-cell adhesion receptors likely play a more powerful and wide-ranging role than initially anticipated. This Perspective article discusses the relevance of this emerging field for epidermal growth and differentiation, which can be of importance for severe pathologies such as tumorigenesis and invasive metastasis, as well as psoriasis and Pemphigus vulgaris.

Keratinocyte transcriptional regulation of the human c-Myc promoter occurs via a novel Lef/Tcf binding element distinct from neoplastic cells.

The proto-oncogene c-Myc is involved in early neoplastic transformations. Two consensus Lef/Tcf binding elements (TBE) were found to be prerequisite for transcriptional transactivation by the armadillo proteins beta-catenin and plakoglobin (PG) together with Tcf4 in human neoplastic cells. In epidermal keratinocytes, c-Myc was reported to be repressed by Lef-1 and PG. Using reporter gene assays, here we demonstrate that deletion of the two consensus TBE fails to abrogate transcriptional regulation by Lef-1/PG in wildtype and beta-catenin-/- keratinocytes, while it reduces transcription in pre-neoplastic PG-/- keratinocytes. We identified a TBE sequence variant downstream of the major transcriptional initiation site that binds Lef-1 in vitro and in vivo, and its mutation compromised transcriptional regulation by Lef-1/PG. Collectively, this study demonstrates that the two consensus TBE's reported in neoplastic cells are dispensable for c-Myc regulation in normal keratinocytes, which instead use a novel TBE sequence variant. This unprecedented finding may have important implications for armadillo target genes involved in carcinogenesis.