Desmosome Formation Research Articles

The transmembrane domain of the desmosomal cadherin desmoglein-1 governs lipid raft association to promote desmosome adhesive strength.

Cholesterol- and sphingolipid-enriched domains called lipid rafts are hypothesized to selectively coordinate protein complex assembly within the plasma membrane to regulate cellular functions. Desmosomes are mechanically resilient adhesive junctions that associate with lipid raft membrane domains, yet the mechanisms directing raft association of the desmosomal proteins, particularly the transmembrane desmosomal cadherins, are poorly understood. We identified the desmoglein-1 (DSG1) transmembrane domain (TMD) as a key determinant of desmoglein lipid raft association and designed a panel of DSG1TMD variants to assess the contribution of TMD physicochemical properties (length, bulkiness, and palmitoylation) to DSG1 lipid raft association. Sucrose gradient fractionations revealed that TMD length and bulkiness, but not palmitoylation, govern DSG1 lipid raft association. Further, DSG1 raft association determines plakoglobin recruitment to raft domains. Super-resolution imaging and functional assays uncovered a strong relationship between the efficiency of DSG1TMD lipid raft association and the formation of morphologically and functionally robust desmosomes. Lipid raft association regulated both desmosome assembly dynamics and DSG1 cell surface stability, indicating that DSG1 lipid raft association is required for both desmosome formation and maintenance. These studies identify the biophysical properties of desmoglein transmembrane domains as key determinants of lipid raft association and desmosome adhesive function.

Enhanced desmosome assembly driven by acquired high-level desmoglein-2 promotes phenotypic plasticity and endocrine resistance in ER+ breast cancer

Acquired resistance to endocrine treatments remains a major clinical challenge. In this study, we found that desmoglein-2 (DSG2) plays a major role in acquired endocrine resistance and cellular plasticity in ER+ breast cancer (BC). By analysing the well-established fulvestrant-resistant ER+ BC model using single-cell RNA-seq, we revealed that ER inhibition leads to a specific increase in DSG2 in cancer cell populations, which in turn enhances desmosome formation in vitro and in vivo and cell phenotypic plasticity that promotes resistance to treatment. DSG2 depletion reduced tumorigenesis and metastasis in fulvestrant-resistant xenograft models and promoted fulvestrant efficiency. Mechanistically, DSG2 forms a desmosome complex with JUP and Vimentin and triggers Wnt/PCP signalling. We showed that elevated DSG2 levels, along with reduced ER levels and an activated Wnt/PCP pathway, predicted poor survival, suggesting that a DSG2high signature could be exploited for therapeutic interventions. Our analysis highlighted the critical role of DSG2-mediated desmosomal junctions following antiestrogen treatment.

Ectopic Expression of a Truncated Isoform of Hair Keratin 81 in Breast Cancer Alters Biophysical Characteristics to Promote Metastatic Propensity.

Keratins are an integral part of cell structure and function. Here, it is shown that ectopic expression of a truncated isoform of keratin 81 (tKRT81) in breast cancer is upregulated in metastatic lesions compared to primary tumors and patient-derived circulating tumor cells, and is associated with more aggressive subtypes. tKRT81 physically interacts with keratin 18 (KRT18) and leads to changes in the cytosolic keratin intermediate filament network and desmosomal plaque formation. These structural changes are associated with a softer, more elastically deformable cancer cell with enhanced adhesion and clustering ability leading to greater in vivo lung metastatic burden. This work describes a novel biomechanical mechanism by which tKRT81 promotes metastasis, highlighting the importance of the biophysical characteristics of tumor cells.

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.

Anomaly of cornea and ocular adnexa in spinster homolog 2 (Spns2) knockout mice

Spinster 2 (Spns2) is a transporter that pumps sphingosine-1-phosphate (S1P), a bioactive lipid mediator synthesized in the cytoplasm, out of cells into the inter cellular space. S1P is a signal that modulates cellular behavior during embryonic development, inflammation and tissue repair, etc. A Spns2-null (KO) mouse is born with failure of eyelid closure (eyelid-open-at birth; EOB) and develop corneal fibrosis in adulthood. It remains elusive whether corneal lesion is caused by exposure to keratitis (lagophthalmos) of EOB phenotype or the loss of Spns2 directly perturbs the corneal tissue morphogenesis and intra-eyelid structures. Therefore, we investigated differences between the cornea and ocular adnexa morphogenesis in KO and wild-type (WT) embryos and adults as well.The loss of Spns2 perturbs cornea morphogenesis during embryonic development as early as E16.5 besides EOB phenotype. Histology showed that the corneal stroma was thinner with less extracellular matrix accumulation, e.g., collagen and keratocan in the KO mouse. Epithelial stratification, expression of keratin 12 and formation of desmosomes and hemidesmosomes were also perturbed in these KO corneas. Lacking Spns2 impaired morphogenesis of the Meibomian glands and of orbicularis oculi muscles. KO glands were labeled for ELOVL4 and PPARγ and were Oil-Red O-positive, suggesting KO acinar cells possessed functionality as the glands.This is the first report on the roles of Spns2 in corneal and Meibomian gland morphogenesis. Corneal tissue destruction in an adult KO mouse might be due to not only lagophthalmos but also to an impaired morphogenesis of cornea, Meibomian glands, and orbicularis oculi muscle.

143 Structure of a novel endoplasmic reticulum-desmosome complex and its role in skin disease

Desmosomes are adhesive intercellular junctions that provide strength and integrity to the epidermis and other epithelia. Dysfunction of the endoplasmic reticulum (ER) resident calcium pump, SERCA2, causes Darier’s disease (DD), an epidermal disorder characterized by compromised desmosomal adhesion and abnormal keratinocyte differentiation. However, the mechanism by which ER dysfunction impairs desmosome formation is unclear. Using electron microscopy and live-cell fluorescence imaging, we found that peripheral ER tubules are in close proximity to desmosomes and form mirror image arrangements at desmosomes.

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.

Mechanisms Causing Acantholysis in Pemphigus-Lessons from Human Skin.

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease caused primarily by autoantibodies (PV-IgG) against the desmosomal adhesion proteins desmoglein (Dsg)1 and Dsg3. PV patient lesions are characterized by flaccid blisters and ultrastructurally by defined hallmarks including a reduction in desmosome number and size, formation of split desmosomes, as well as uncoupling of keratin filaments from desmosomes. The pathophysiology underlying the disease is known to involve several intracellular signaling pathways downstream of PV-IgG binding. Here, we summarize our studies in which we used transmission electron microscopy to characterize the roles of signaling pathways in the pathogenic effects of PV-IgG on desmosome ultrastructure in a human ex vivo skin model. Blister scores revealed inhibition of p38MAPK, ERK and PLC/Ca2+ to be protective in human epidermis. In contrast, inhibition of Src and PKC, which were shown to be protective in cell cultures and murine models, was not effective for human skin explants. The ultrastructural analysis revealed that for preventing skin blistering at least desmosome number (as modulated by ERK) or keratin filament insertion (as modulated by PLC/Ca2+) need to be ameliorated. Other pathways such as p38MAPK regulate desmosome number, size, and keratin insertion indicating that they control desmosome assembly and disassembly on different levels. Taken together, studies in human skin delineate target mechanisms for the treatment of pemphigus patients. In addition, ultrastructural analysis supports defining the specific role of a given signaling molecule in desmosome turnover at ultrastructural level.

Bidirectional regulation of desmosome hyperadhesion by keratin isotypes and desmosomal components

Desmosomes are intercellular junctions which mediate cohesion and communication in tissues exposed to mechanical strain by tethering the intermediate filament cytoskeleton to the plasma membrane. While mature desmosomes are characterized by a hyperadhesive, Ca2+-independent state, they transiently loose this state during wound healing, pathogenesis and tissue regeneration. The mechanisms controlling the hyperadhesive state remain incompletely understood. Here, we show that upon Ca2+-induced keratinocyte differentiation, expression of keratin 17 (K17) prevents the formation of stable and hyperadhesive desmosomes, accompanied by a significant reduction of desmoplakin (DP), plakophilin-1 (PKP1), desmoglein-1 (Dsg1) and -3 (Dsg3) at intercellular cell borders. Atomic force microscopy revealed that both increased binding strength of desmoglein-3 molecules and amount of desmoglein-3 oligomers, known hallmarks of hyperadhesion, were reduced in K17- compared to K14-expressing cells. Importantly, overexpression of Dsg3 or DPII enhanced their localization at intercellular cell borders and increased the formation of Dsg3 oligomers, resulting in stable, hyperadhesive desmosomes despite the presence of K17. Notably, PKP1 was enriched in these desmosomes. Quantitative image analysis revealed that DPII overexpression contributed to desmosome hyperadhesion by increasing the abundance of K5/K17-positive keratin filaments in the proximity of desmosomes enriched in desmoglein-3. Thus, our data show that hyperadhesion can result from recruitment of keratin isotypes K5/K17 to desmosomes or from enhanced expression of DP and Dsg3 irrespective of keratin composition. The notion that hyperadhesive desmosomes failed to form in the absence of keratins underscores the essential role of keratins and suggest bidirectional control mechanisms at several levels.

116 Weak Links in the Permeability Barrier of the Skin

The microbiota composition and chemical milieu have been shown to prominently differ in topographically distinct healthy skin areas, and recently, our workgroup identified differences in the immune activity of distinct regions as well. As a continuation of our work, we aimed to study the barrier function and compare the expression levels of major permeability barrier components (stratum corneum and tight junction [TJ]) at the molecular level in healthy sebaceous gland-rich (SGR), apocrine gland-rich (AGR), and gland-poor (GP) skin regions. Molecules involved in cornified envelope (CE) formation, desquamation and desmosome formation were investigated in parallel with TJ molecules at the mRNA and protein levels by qRT-PCR and immunohistochemistry. Desmosome and TJ structures were also visualized and quantified by confocal microscopy. While CE structure components were similarly expressed, kallikrein proteases showed significantly higher mRNA levels in SGR and AGR regions compared to GP, and immunohistochemistry indicated their prominent presence in sebaceous and apocrine glands. Desmosome and TJ components’ mRNA levels were similar in the three regions, in contrast, significantly lower protein levels of DSG1, CDSN (desmosome), and OCLN (TJ) molecule were detected in the SGR region, while in AGR skin, the presence of DSG1 (desmosome), OCLN and CLDN1 (TJ) was significantly decreased compared to GP areas. We propose that the increased levels of KLK enzymes and their degradation processes are responsible for the weakened cell junctions in SGR and AGR skin which may explain why certain acantholytic skin disorders (e.g. Darier’s disease, Hailey-Hailey disease) are localized to these areas.

Large lamellar bodies and their role in the growing oocytes of the armadillo Chaetophractus villosus.

Oogenesis in the armadillo Chaetophractus villosus, a representative species of a mammalian basal clade, was investigated by light microscopy, transmission electron microscopy, and immunohistochemical localization of keratin. At the beginning of the growth phase, oocyte follicles showed one, and sometimes several, large bodies composed of lamellae (multilamellar bodies [MLBs]), which entrap other cytoplasmic organelles at more advanced stages. Lamellae diameter is described in cross-section (37 nm) and tangential sections (50 nm). The MLB of early oocytes is most frequently located close to the nucleus. In large oocytes, both, this body and the free organelles are relocated at the oocyte periphery. The MLB grows from the primary follicle up to its full development at the follicular phase characterized by tall granulosa cells. Mitochondria, smooth small vesicles, and lipofuscin granules are trapped between lamellae. MLBs engage in the formation of different sets of organelles, both trapped and free ones. When oocytes are well developed and the zona pellucida is formed, the MLB is reduced to small remnants detected only by transmission electron microscopy. The MLB disintegrates when an antrum develops. Immunohistochemical localization techniques showed the presence of cytokeratin in the MLBs. This cytokeratin pool may be involved in the filament and desmosome formation found in the periphery of late oocytes.

Rab13 and Desmosome Redistribution in Uterine Epithelial Cells During Early Pregnancy.

The luminal uterine epithelial cells are the first point of contact with the implanting blastocyst. Dramatic changes occur in the structure and function of these cells at the time of receptivity including changes in the lateral junctional complex. While these morphological changes are important for uterine receptivity, currently there is no known mechanism of regulation of the lateral junctional complexes. Rab13, a member of the Rab (Ras-related in the brain) family of GTPases has a critical role in endosomal trafficking to the lateral plasma membrane and is involved in modulation of the tight junction in several cell types. The aim of this study is to investigate the role of Rab13 in changes to the lateral junctional complex at the time of receptivity. Immunofluorescence microscopy demonstrated no association between Rab13 and ZO-1 (a tight junction protein) or Rab13 and E-cadherin (an integral component of adherens junctions). Co-localisation was demonstrated between Rab 13 and desmoglein-2 at the time of fertilization and also at receptivity suggesting involvement of Rab13 in relocalisation of desmoglein-2 and formation of giant desmosomes in the apical part of the lateral plasma membrane at the time of uterine receptivity. We suggest that despite the loss of the adherens junction at the time of receptivity, the presently reported redistribution of desmosomes regulated by Rab13 allows the uterine epithelium to maintain structural integrity.

Clustering of desmosomal cadherins by desmoplakin is essential for cell-cell adhesion.

Desmoplakin (Dp) is a crucial component of the desmosome, a supramolecular cell junction complex anchoring intermediate filaments. The mechanisms how Dp modulates cell-cell adhesion are only partially understood. Here, we studied the impact of Dp on the function of desmosomal adhesion molecules, desmosome turnover and intercellular adhesion. CRISPR/Cas9 was used for gene editing of human keratinocytes which were characterized by Western blot and immunostaining. Desmosomal ultrastructure and function were assessed by electron microscopy and cell adhesion assays. Single molecule binding properties and localization of desmosomal cadherins were studied by atomic force microscopy and super-resolution imaging. Knockout (ko) of Dp impaired cell cohesion to drastically higher extents as ko of another desmosomal protein, plakoglobin (Pg). In contrast to Pg ko, desmosomes were completely absent in Dp ko. Binding properties of the desmosomal adhesion molecules desmocollin (Dsc) 3 and desmoglein (Dsg) 3 remained unaltered under loss of Dp. Dp was required for assembling desmosomal cadherins into large clusters, as Dsg2 and Dsc3, adhesion molecules primarily localized within desmosomes, were redistributed into small puncta in the cell membrane of Dp ko cells. Additional silencing of desmosomal cadherins in Dp ko did not further increase loss of intercellular adhesion. Our data demonstrate that Dp is essential for desmosome formation but does not influence intercellular adhesion on the level of individual cadherin binding properties. Rather, macro-clustering of desmosomal adhesion molecules through Dp is crucial. These results may help to better understand severe diseases which are caused by Dp dysfunction.

The Actin-Binding Protein α-Adducin Modulates Desmosomal Turnover and Plasticity

Intercellular adhesion is essential for tissue integrity and homeostasis. Desmosomes are abundant in the epidermis and the myocardium-tissues, which are under constantly changing mechanical stresses. Yet, it is largely unclear whether desmosomal adhesion can be rapidly adapted to changing demands, and the mechanisms underlying desmosome turnover are only partially understood. In this study we show that the loss of the actin-binding protein α-adducin resulted in reduced desmosome numbers and prevented the ability of cultured keratinocytes or murine epidermis to withstand mechanical stress. This effect was not primarily caused by decreased levels or impaired adhesive properties of desmosomal molecules but rather by altered desmosome turnover. Mechanistically, reduced cortical actin density in α-adducin knockout keratinocytes resulted in increased mobility of the desmosomal adhesion molecule desmoglein 3 and impaired interactions with E-cadherin, a crucial step in desmosome formation. Accordingly, the loss of α-adducin prevented increased membrane localization of desmoglein 3 in response to cyclic stretch or shear stress. Our data demonstrate the plasticity of desmosomal molecules in response to mechanical stimuli and unravel a mechanism of how the actin cytoskeleton indirectly shapes intercellular adhesion by restricting the membrane mobility of desmosomal molecules.

Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.

SummaryThe glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.

Plakophilin 3 phosphorylation by ribosomal S6 kinases supports desmosome assembly.

Desmosome remodeling is crucial for epidermal regeneration, differentiation and wound healing. It is mediated by adapting the composition, and by post-translational modifications, of constituent proteins. We have previously demonstrated in mouse suprabasal keratinocytes that plakophilin (PKP) 1 mediates strong adhesion, which is negatively regulated by insulin-like growth factor 1 (IGF1) signaling. The importance of PKP3 for epidermal adhesion is incompletely understood. Here, we identify a major role of epidermal growth factor (EGF), but not IGF1, signaling in PKP3 recruitment to the plasma membrane to facilitate desmosome assembly. We find that ribosomal S6 kinases (RSKs) associate with and phosphorylate PKP3, which promotes PKP3 association with desmosomes downstream of the EGF receptor. Knockdown of RSKs as well as mutation of an RSK phosphorylation site in PKP3 interfered with desmosome formation, maturation and adhesion. Our findings implicate a coordinate action of distinct growth factors in the control of adhesive properties of desmosomes through modulation of PKPs in a context-dependent manner.

Plakophilins 2 and 3 are involved in desmosome formation in the intestine

The integrity of desmosomes is crucial to maintain intestinal epithelial barrier (IEB) function in health and disease. In addition to adherens junctions, desmosome‐dependent intercellular adhesion between cells is provided by the cadherins, Desmoglein2 (DSG2) and Desmocollin2 (DSC2) in the intestine. Both are tethered to the intermediate filament system via different adaptor proteins, such as the Plakophilins (PKPs). The specific role of the different isoforms of PKP 1–3 has been recognized in different organs. However, there is a wide lack of literature regarding the existence of the PKPs and their critical role in the intestine. Accordingly, it remains unclear whether PKPs play a specific role for IEB stabilization. Therefore, the aim of this study was to perform a basal characterization of intestinal PKPs.To analyze the expression pattern of the different PKP isoforms, immunostaining, qRT‐PCR and Western blotting were used. In healthy human mucosa that derived from surgical specimens, PKP2 and PKP3, but not PKP1, were present at the cell borders of tissue sections. In co‐staining experiments with DSG2, both PKP2 and PKP3 were found to be present at desmosomal junctions. The expression of PKP2 and PKP3 but not PKP1 was confirmed on the RNA level by qRT‐PCR and on the protein level by Western Blot analysis. Similar results were obtained in enteroids generated from human mucosa and in Caco2 cells which are commonly used as models to investigate the mechanisms underlying changes of IEB showed a similar expression pattern of PKPs. Interestingly, in Caco2 cells that are deficient for DSG2, a compensatory upregulation of PKP2 RNA and total protein levels was detected when compared to Caco2 WT cells whereas no changes were evident for PKP3 in DSG2‐deficient cells. Stimulation of Caco2 WT cells with Tumor necrosis factor α (TNFα) resulted in a loss of IEB as revealed by measurements of transepithelial resistance. This was associated with a loss of DSG2, PKP2 and PKP3 at the cell borders. However, while total protein levels of PKP2 and DSG2 were unaltered under these conditions, PKP3 protein levels were upregulated.In summary, for the first time, we provide evidence that PKP2 and 3 but not PKP1 are expressed in human intestinal epithelial cells as well as in human enteroids and Caco2 cells. Compensatory upregulation of PKP2 following loss of DSG2 suggests a functional role for PKP2 to stabilize desmosomal integrity. Furthermore, since TNFα treatment was accompanied with an upregulation of PKP3, a potential role of PKP3 during inflammatory processes can be hypothesized.

The Desmoglein‐1 Transmembrane Domain Drives Lipid Raft Association and Desmosome Assembly

The epidermis is a barrier to the outside world, but mutations in genes encoding desmosomal proteins can inhibit this function, causing human diseases of skin fragility. Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a severe homozygous recessive disorder of skin fragility caused by mutations in the gene encoding desmoglein 1 (DSG1), a component of desmosomes. Desmosomes are junctional complexes that function in mechanical stress resistance across tissues. In these junctions, desmosomal cadherins such as DSG1 interact across extracellular spaces and with intracellular adaptor proteins to link cytoskeletal elements of adjacent cells. Both assembly and disassembly of desmosomes have been shown to rely on lipid raft membrane microdomains, highly ordered cholesterol and sphingolipid‐enriched regions of the plasma membrane important in signaling and trafficking. Our current model predicts that raft association promotes clustering of desmosomal proteins to drive assembly and mediate adhesion. However, the mechanisms of raft association and desmosome assembly are not fully understood. We created a DSG‐null A431 cell line to examine the relationship between DSG raft association and desmosomal adhesion. A431s are an immortal human epidermal carcinoma cell line commonly used in desmosomal studies that express a single DSG species, DSG2. The absence of DSG2 in these cells was confirmed by western blot and immunofluorescence. Further characterization of these DSG‐null A431 cells revealed mislocalized desmoplakin, a marker of mature desmosomes, and a lack of strong cell‐cell adhesion. Exogenous stable expression of GFP‐tagged wildtype DSG1 (DSG1WT‐GFP) rescued both desmosome formation and function, suggesting that these DSG‐null cells would be suitable for probing the relationship between DSG raft association and desmosome assembly and function. Recently, we discovered a novel, heterozygous mutation in the transmembrane domain (TMD)‐encoding region of DSG1 that causes a glycine to arginine substitution and SAM syndrome. DSG1SAM is a lipid raft targeting mutant suggesting an important role for the TMD in raft association. Stable expression of DSG1SAM‐GFP could not rescue desmosome formation or function in the DSG‐null cells, suggesting that a loss of raft association also impacts desmosome assembly and adhesive function. Intriguingly, a chimeric DSG1 protein containing the TMD from E‐cadherin (DSG1Ecad‐GFP), a non‐raft associating adhesive protein, only partly rescued desmosome formation and function. Furthermore, these results correlated with the predicted lipid raft affinity of each TMD, suggesting that desmosome assembly and function is dependent on raft association. We are currently analyzing a panel of GFP‐tagged DSG1TMD variants with differential raft targeting affinities and assessing their ability to support desmosome assembly and function. In conclusion, the TMD targets desmogleins to lipid rafts and modulates desmoglein adhesive function.Support or Funding InformationNIH R01AR050501, NIH R01AR048266

Differentiation of human colon tissue in culture: Effects of calcium on trans-epithelial electrical resistance and tissue cohesive properties.

Background and aimsHuman colonoid cultures maintained under low-calcium (0.25 mM) conditions undergo differentiation spontaneously and, concomitantly, express a high level of tight junction proteins, but not desmosomal proteins. When calcium is included to a final concentration of 1.5–3.0 mM (provided either as a single agent or as a combination of calcium and additional minerals), there is little change in tight junction protein expression but a strong up-regulation of desmosomal proteins and an increase in desmosome formation. The aim of this study was to assess the functional consequences of calcium-mediated differences in barrier protein expression.MethodsHuman colonoid-derived epithelial cells were interrogated in transwell culture under low- or high-calcium conditions for monolayer integrity and ion permeability by measuring trans-epithelial electrical resistance (TEER) across the confluent monolayer. Colonoid cohesiveness was assessed in parallel.ResultsTEER values were high in the low-calcium environment but increased in response to calcium. In addition, colonoid cohesiveness increased substantially with calcium supplementation. In both assays, the response to multi-mineral intervention was greater than the response to calcium alone. Consistent with these findings, several components of tight junctions were expressed at 0.25 mM calcium but these did not increase substantially with supplementation. Cadherin-17 and desmoglein-2, in contrast, were weakly-expressed under low calcium conditions but increased with intervention.ConclusionsThese findings indicate that low ambient calcium levels are sufficient to support the formation of a permeability barrier in the colonic epithelium. Higher calcium levels promote tissue cohesion and enhance barrier function. These findings may help explain how an adequate calcium intake contributes to colonic health by improving barrier function, even though there is little change in colonic histological features over a wide range of calcium intake levels.

Role of PKC and ERK Signaling in Epidermal Blistering and Desmosome Regulation in Pemphigus.

Desmosomes reinforce cohesion of epithelial cells at the interface between adjacent cells. They include the cadherin-type adhesion molecules desmoglein 1 (Dsg1) and Dsg3. Pemphigus vulgaris (PV) is an autoimmune disease in which circulating autoantibodies (PV-IgG) targeting Dsg1 and 3 cause characteristic epidermal blister formation. It has been shown that PV-IgG binding induced activation of kinases such as ERK and PKC, and inhibition of these signaling pathways prevented loss of cell cohesion in cell cultures. However, the role of Erk and PKC in blister formation and regulation of desmosome ultrastructure in human skin are unknown. Accordingly, we assessed the role of PKC and ERK signaling pathways in blister formation and regulation of desmosome ultrastructure in human epidermis. Here we performed electron microscopy analyses using human skin explants injected with PV-IgG together with inhibitors for PKC or ERK signaling. Inhibition of PKC was not effective to prevent suprabasal blister formation or ultrastructural alterations of desmosomes. In contrast, inhibition of ERK signaling significantly ameliorated blister formation and decrease in the number of desmosomes whereas shortening and splitting of desmosomes and keratin filament insertion were not different from samples treated with PV-IgG alone. However, apical desmosomes between basal and suprabasal cells remained unaltered when ERK signaling was inhibited. Therefore, our results show that inhibition of ERK but not PKC signaling appears to be effective to ameliorate blistering and alterations of desmosome ultrastructure triggered by PV-IgG in human skin.