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Cutaneous eruptions caused by the combination of Chinese and Western medicine have attracted widespread attention; however, the underlying mechanism remains unclear. This study aimed to evaluate the potential mechanism of cutaneous eruptions in vivo and in vitro using the combination of Shuanghuanglian injection powder (SHL) and aspirin (ASA) as an example. ASA and SHL co-administration induced inflammatory responses in HaCat cells, as evidenced by marked increases in the expression of IL-4 and TNF-α, and the level of apoptosis. Additionally, histopathological investigation of mice skin tissues showed local inflammatory cell infiltration. Western boltting was used to detect the effects of ASA on desmoglein-1 (DSG1) expression; we found that DSG1 expression was down-regulated in vivo and in vitro. Finally, the key components of SHL were administered to HaCat cells with down-regulated DSG1; it was seen that neochlorogenic acid and rutin have a significant effect on HaCat cell apoptosis. These results demonstrate that DSG1 deficiency is a potential cause of cutaneous eruptions caused by the combination of SHL and ASA, and neochlorogenic acid and rutin are the main allergenic components. This study provides a new research strategy for the safety evaluation of integrated traditional Chinese and Western medicine., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021. Published by Elsevier B.V.)

The epidermis is the first line of defense against ultraviolet (UV) light from the sun. Keratinocytes and melanocytes respond to UV exposure by eliciting a tanning response dependent in part on paracrine signaling, but how keratinocyte:melanocyte communication is regulated during this response remains understudied. Here, we uncover a surprising new function for the keratinocyte-specific cell-cell adhesion molecule desmoglein 1 (Dsg1) in regulating keratinocyte:melanocyte paracrine signaling to promote the tanning response in the absence of UV exposure. Melanocytes within Dsg1-silenced human skin equivalents exhibited increased pigmentation and altered dendrite morphology, phenotypes which were confirmed in 2D culture using conditioned media from Dsg1-silenced keratinocytes. Dsg1-silenced keratinocytes increased melanocyte-stimulating hormone precursor (Pomc) and cytokine mRNA. Melanocytes cultured in media conditioned by Dsg1-silenced keratinocytes increased Mitf and Tyrp1 mRNA, TYRP1 protein, and melanin production and secretion. Melanocytes in Dsg1-silenced skin equivalents mislocalized suprabasally, reminiscent of early melanoma pagetoid behavior. Together with our previous report that UV reduces Dsg1 expression, these data support a role for Dsg1 in controlling keratinocyte:melanocyte paracrine communication and raise the possibility that a Dsg1-deficient niche contributes to pagetoid behavior, such as occurs in early melanoma development., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pemphigus is an autoimmune dermatosis in which mucocutaneous blisters are induced primarily by autoantibodies against Desmoglein (Dsg) 1 and 3. Pemphigus vulgaris (PV) usually is associated with autoantibodies against Dsg3 whereas pemphigus foliaceus (PF) patients present autoantibodies against Dsg1. Several signaling pathways were proposed to cause loss of keratinocyte adhesion. However, relevance of different signaling pathways and role of Dsg1 and 3 to trigger signaling are not fully understood. Here, we show that Ca 2+ chelation reduced PV-IgG- and PF-IgG-mediated loss of HaCaT keratinocyte cohesion whereas EGFR inhibition did not inhibit effects of PF-IgG. PV-IgG activated EGFR in a Src-dependent manner whereas both PV-IgG and PF-IgG caused Ca 2+ influx independent of EGFR. ERK activation was Src-dependent in response to PV-IgG but not PF-IgG. To delineate the roles of Dsg isoforms to trigger signaling pathways, Dsg3- and Dsg2-deficient HaCaT keratinocyte cell lines were generated using CRISPR/Cas9. Dsg3- but not Dsg2-deficient cells were protected against PV-IgG-induced loss of cell adhesion. Ca 2+ influx and ERK activation in response to PF-IgG were preserved in both cell lines.

The epidermis is a multi-layered epithelium that serves as a barrier against water loss and environmental insults. Its morphogenesis occurs through a tightly regulated program of biochemical and architectural changes during which basal cells commit to differentiate and move towards the skin's surface. Here, we reveal an unexpected role for the vertebrate cadherin desmoglein 1 (Dsg1) in remodeling the actin cytoskeleton to promote the transit of basal cells into the suprabasal layer through a process of delamination, one mechanism of epidermal stratification. Actin remodeling requires the interaction of Dsg1 with the dynein light chain, Tctex-1 and the actin scaffolding protein, cortactin. We demonstrate that Tctex-1 ensures the correct membrane compartmentalization of Dsg1-containing desmosomes, allowing cortactin/Arp2/3-dependent perijunctional actin polymerization and decreasing tension at E-cadherin junctions to promote keratinocyte delamination. Moreover, Dsg1 is sufficient to enable simple epithelial cells to exit a monolayer to form a second layer, highlighting its morphogenetic potential.

Aberrant differentiation of keratinocytes has been demonstrated to be associated with a number of skin diseases. A growing number of studies have showed that long noncoding RNAs (lncRNAs) have an important part in gene regulation, however, the role of lncRNAs in keratinocyte differentiation remains to be largely unknown. In the present study, we demonstrated that lncRNA-H19 act as an endogenous 'sponge', which binds directly to miR-130b-3p and therefore inhibits its activity on Dsg1. MiR-130b-3p was illustrated to inhibit keratinocyte differentiation by targeting Dsg1. H19 regulates Dsg1 expression and the consequent keratinocyte differentiation through miR-130b-3p. Our study casts light on a novel regulatory model of keratinocyte differentiation, which may provide new therapeutic targets of skin diseases.

Background: Upregulation of kallikreins (KLKs) including KLK5 has been reported in atopic dermatitis (AD). KLK5 has biological functions that include degrading desmosomal proteins and inducing proinflammatory cytokine secretion through protease-activated receptor 2 (PAR2). However, due to the complex interactions between various cells in AD inflamed skin, it is difficult to dissect the precise and multiple roles of upregulated KLK5 in AD skin., Objective: We investigated the effect of upregulated KLK5 on the expression of epidermal-related proteins and cytokines in keratinocytes and on skin architecture., Methods: Lesional and nonlesional AD skin biopsies were collected for analysis of morphology and protein expression. The relationship between KLK5 and barrier-related molecules was investigated using an ex vivo dermatitis skin model with transient KLK5 expression and a cell model with persistent KLK5 expression. The influence of upregulated KLK5 on epidermal morphology was investigated using an in vivo skin graft model., Results: Upregulation of KLK5 and abnormal expression of desmoglein 1 (DSG1) and filaggrin, but not PAR2 were identified in AD skin. PAR2 was increased in response to transient upregulation of KLK5, whereas persistently upregulated KLK5 did not show this effect. Persistently upregulated KLK5 degraded DSG1 and stimulated secretion of IL-8, IL-10, and thymic stromal lymphopoietin independent of PAR2 activity. With control of higher KLK5 activity by the inhibitor sunflower trypsin inhibitor G, restoration of DSG1 expression and a reduction in AD-related cytokine IL-8, thymic stromal lymphopoietin, and IL-10 secretion were observed. Furthermore, persistently elevated KLK5 could induce AD-like skin architecture in an in vivo skin graft model., Conclusions: Persistently upregulated KLK5 resulted in AD-like skin architecture and secretion of AD-related cytokines from keratinocytes in a PAR2 independent manner. Inhibition of KLK5-mediated effects may offer potential as a therapeutic approach in AD., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Cell junctions are scaffolds that integrate mechanical and chemical signaling. We previously showed that a desmosomal cadherin promotes keratinocyte differentiation in an adhesion-independent manner by dampening Epidermal Growth Factor Receptor (EGFR) activity. Here we identify a potential mechanism by which desmosomes assist the de-neddylating COP9 signalosome (CSN) in attenuating EGFR through an association between the Cops3 subunit of the CSN and desmosomal components, Desmoglein1 (Dsg1) and Desmoplakin (Dp), to promote epidermal differentiation. Silencing CSN or desmosome components shifts the balance of EGFR modifications from ubiquitination to neddylation, inhibiting EGFR dynamics in response to an acute ligand stimulus. A reciprocal relationship between loss of Dsg1 and neddylated EGFR was observed in a carcinoma model, consistent with a role in sustaining EGFR activity during tumor progression. Identification of this previously unrecognized function of the CSN in regulating EGFR neddylation has broad-reaching implications for understanding how homeostasis is achieved in regenerating epithelia.

Pemphigus is an autoimmune blistering skin disease caused primarily by autoantibodies against desmoglein (Dsg)1 and 3. Here, we characterized the mechanisms engaged by pemphigus IgG from patients with different clinical phenotypes and autoantibody profiles. All pemphigus vulgaris (PV) and pemphigus foliaceus (PF) IgG and AK23, a monoclonal mouse antibody against Dsg3, caused loss of cell cohesion, cytokeratin retraction and p38MAPK activation. Strong alterations in Dsg3 distribution were caused by mucosal (aDsg3 antibodies), mucocutaneous (aDsg1 + aDsg3) as well as atypical (aDsg3) PV-IgG. All PV-IgG fractions and AK23 compromised Dsg3 but not Dsg1 binding and enhanced Src activity. In contrast, rapid Ca 2+ influx and Erk activation were induced by mucocutaneous PV-IgG and pemphigus foliaceus (PF) IgG (aDsg1) whereas cAMP was increased by mucosal and mucocutaneous PV-IgG only. Selective inhibition of p38MAPK, Src or PKC blocked loss of keratinocyte cohesion in response to all autoantibody fractions whereas Erk inhibition was protective against mucocutaneous PV-IgG and PF-IgG only. These results demonstrate that signaling patterns parallel the clinical phenotype as some mechanisms involved in loss of cell cohesion are caused by antibodies targeting Dsg3 whereas others correlate with autoantibodies against Dsg1. The concept of key desmosome regulators may explain observations from several experimental models of pemphigus.

Psoriasin, which is highly expressed in psoriasis, is encoded by a gene located within the epidermal differentiation complex. The aim of this study was to investigate the effect of endogenous psoriasin on disturbed keratinocyte differentiation in psoriasis. Immunohistochemical staining revealed a gradient of psoriasin expression in the psoriatic epidermis with highest expression in the suprabasal, differentiated layers. Induction of keratinocyte differentiation caused concurrent expression of psoriasin and the differentiation marker involucrin. The differentiation-induced psoriasin expression was found to be mediated by the protein kinase C pathway. The downregulation of psoriasin expression by small interfering RNA revealed that psoriasin mediates the expression of involucrin, desmoglein 1, transglutaminase 1 and CD24 in normal differentiation. The lentivirus-mediated overexpression of psoriasin, mimicking the psoriatic milieu, gave rise to an altered regulation of differentiation genes and an expression pattern reminiscent of that in psoriatic epidermis. These findings suggest that psoriasin contributes to the dysregulated differentiation process in the psoriasis epidermis.

Background: Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies (Abs) against desmoglein 1 (Dsg1). PF sera contain polyclonal Abs which are heterogeneous mixture of both pathogenic and non-pathogenic Abs, as shown by isolation of monoclonal Abs (mAbs)., Objective: To investigate how pathogenic and non-pathogenic anti-Dsg1 Abs contribute to blister formation in PF., Methods: Using organ-cultured human skin, we compared the effect of a single pathogenic anti-Dsg1 IgG mAb, a single non-pathogenic anti-Dsg1 IgG mAb, and their mixture on blister formation as analyzed by histology, subcellular localization of IgG deposits and desmosomal proteins by confocal microscopy, and desmosomal structure by electron microscopy. In addition, we measured keratinocyte adhesion by an in vitro dissociation assay., Results: 24h after injection, a single pathogenic anti-Dsg1 IgG caused a subcorneal blister with IgG and Dsg1 localized linearly on the cell surface of keratinocytes. A single non-pathogenic anti-Dsg1 IgG bound linearly on the keratinocytes but did not induce blisters. A pathogenic and a non-pathogenic IgG mAb injected together caused an aberrant granular pattern of IgG and Dsg1 in the lower epidermis with blister formation in the superficial epidermis. Electron microscopy demonstrated that the mixture of mAbs shortened desmosomal lengths more than a single mAb in the basal and spinous layers. Furthermore, although Dsg1 clustering required both cross-linking of Dsg1 molecules by the non-pathogenic IgG plus a pathogenic antibody, the latter could be in the form of a monovalent single chain variable fragment, suggesting that loss of trans-interaction of Dsg1 is required for clustering. Finally, a p38MAPK inhibitor blocked Dsg1 clustering. When pathogenic strength was measured by the dissociation assay, a mixture of pathogenic and non-pathogenic IgG mAbs disrupted keratinocyte adhesion more than a single pathogenic mAb. This pathogenic effect was only partially suppressed by the p38MAPK inhibitor., Conclusion: These findings indicate that a polyclonal mixture of anti-Dsg1 IgG antibodies enhances pathogenic activity for blister formation associated with p38MAPK-dependent Dsg1 clustering and that not only pathogenic antibodies but also non-pathogenic antibodies coordinately contribute to blister formation in PF., (Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Bacteria that reside on the skin can influence the behavior of the cutaneous immune system, but the mechanisms responsible for these effects are incompletely understood. Colonization of the skin by Staphylococcus aureus (S. aureus) is increased in atopic dermatitis and can result in increased severity of the disease. In this study, we show that S. aureus stimulates human keratinocytes to increase their endogenous protease activity, including specific increases in trypsin activity. This increased protease activity coincided with increased expression of mRNA for kallikreins (KLKs), with KLK6, 13, and 14 showing the greatest induction after exposure to S. aureus. Suppression of mRNA for these KLKs in keratinocytes by targeted small interfering RNA silencing before S. aureus exposure blocked the increase in protease activity. Keratinocytes exposed to S. aureus showed enhanced degradation of desmoglein-1 and filaggrin, whereas small interfering RNA for KLK6, KLK13, and KLK14 partially blocked this degradation. These data illustrate how S. aureus directly influences the skin barrier integrity by stimulating endogenous proteolytic activity and defines a previously unknown mechanism by which S. aureus may influence skin diseases., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

T helper type 2 (Th2) cytokines, IL-4 and IL-13, attenuate the expression of genes that regulate epidermal cellular structures and the barrier function at the terminal stage of keratinocyte differentiation. However, whether these Th2 cytokines act at earlier stages remains unknown. We investigated the roles of cytokines in expression levels of mRNAs and/or proteins in primary mouse keratinocytes and human keratinocyte HaCaT cells at earlier stages. We showed that IL-4 downregulated the expression levels of Krt1, Krt10, Dsg1, and Dsc1 via IL-4Rα- and signal transducer and activator of transcription factor 6 (STAT6)-dependent mechanisms in differentiating mouse keratinocytes at early stages. As the expression levels of keratin-1 and -10 in the keratinocytes transiently expressing an active form of STAT6 were not downregulated, STAT6 and other IL-4-induced molecules may synergistically regulate this expression. The restoration of the downregulated expression levels of Krt1 and Krt10 induced by IL-4 with the MEK (mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase) inhibitor U0126 indicated the involvement of the p44/42 MAPK signaling pathway in the attenuated expression. IL-13 also downregulated the expression of the four genes. Furthermore, IL-4 or IL-13 caused the downregulation of these genes in HaCaT cells and promoted the fragmentation of cell sheets with mechanical stress. Our results showed that IL-4 or IL-13 acted on differentiating keratinocytes in vitro at early stages to attenuate the gene expression.

Many skin infections are caused by Staphylococcus aureus, a bacterial pathogen that produces virulence factors associated with these conditions such as exfoliative toxins A and B (ETA, ETB) and the leukotoxin Panton-Valentine leukocidin (PVL). Herein, we examine the potential of skin-infecting S. aureus to produce virulence factors and their impact on the local immune response. Toxin gene profiles were generated from 188 S. aureus isolated as single infecting organisms from skin lesions and demonstrated a higher potential to express ETA, ETB, and PVL than community isolates (p < 0.001). Within the study isolate group, the prevalence of genes encoding PVL was higher among methicillin-resistant S. aureus (MRSA; n = 49), while genes encoding ETs were more prevalent in methicillin-susceptible S. aureus (MSSA; n = 139). When lesion-associated white blood cell (WBC) counts were dichotomized into high- or low-WBC-count-associated bacteria, the gene for ETA was found to be associated with a low WBC count among MSSA (p = 0.001). The ETA-induced mouse model of staphylococcal scalded skin syndrome was used to investigate the link between ETA and cytokine production. Elevated IL-6 levels in the serum and increased expression of IL-6 mRNA in the skin were detected in response to ETA exposure. These findings were recapitulated in vitro using primary human keratinocytes. Thus, S. aureus may influence the local immune response via ETA cleavage of desmoglein 1 and the induction of cutaneous IL-6 expression.

Although much is known about signaling factors downstream of Rho GTPases that contribute to epidermal differentiation, little is known about which upstream regulatory proteins (guanine nucleotide exchange factors [GEFs] or GTPase-activating proteins [GAPs]) are involved in coordinating Rho signaling in keratinocytes. Here we identify the GEF breakpoint cluster region (Bcr) as a major upstream regulator of RhoA activity, stress fibers, and focal adhesion formation in keratinocytes. Loss of Bcr reduced expression of multiple markers of differentiation (such as desmoglein-1 [Dsg1], keratin-1, and loricrin) and abrogated MAL/SRF signaling in differentiating keratinocytes. We further demonstrated that loss of Bcr or MAL reduced levels of Dsg1 mRNA in keratinocytes, and ectopic expression of Dsg1 rescued defects in differentiation seen upon loss of Bcr or MAL signaling. Taken together, these data identify the GEF Bcr as a regulator of RhoA/MAL signaling in keratinocytes, which in turn promotes differentiation through the desmosomal cadherin Dsg1.

Desmoglein-1 (DSG1), a desmosomal protein, maintains the structure of epidermis through its adhesive function. However, heterozygous mutations in DSG1 in humans result in abnormal differentiation, as does downregulation of DSG1 in human skin organ culture, suggesting that it may have important signaling functions. In this issue of the JCI, Harmon et al. elucidate how the binding of the DSG1 cytoplasmic tail to the scaffolding protein Erbin decreases signaling through the Ras-Raf pathway to promote stratification and differentiation of keratinocytes in the epidermis.

Genetic disorders of the Ras/MAPK pathway, termed RASopathies, produce numerous abnormalities, including cutaneous keratodermas. The desmosomal cadherin, desmoglein-1 (DSG1), promotes keratinocyte differentiation by attenuating MAPK/ERK signaling and is linked to striate palmoplantar keratoderma (SPPK). This raises the possibility that cutaneous defects associated with SPPK and RASopathies share certain molecular faults. To identify intermediates responsible for executing the inhibition of ERK by DSG1, we conducted a yeast 2-hybrid screen. The screen revealed that Erbin (also known as ERBB2IP), a known ERK regulator, binds DSG1. Erbin silencing disrupted keratinocyte differentiation in culture, mimicking aspects of DSG1 deficiency. Furthermore, ERK inhibition and the induction of differentiation markers by DSG1 required both Erbin and DSG1 domains that participate in binding Erbin. Erbin blocks ERK signaling by interacting with and disrupting Ras-Raf scaffolds mediated by SHOC2, a protein genetically linked to the RASopathy, Noonan-like syndrome with loose anagen hair (NS/LAH). DSG1 overexpression enhanced this inhibitory function, increasing Erbin-SHOC2 interactions and decreasing Ras-SHOC2 interactions. Conversely, analysis of epidermis from DSG1-deficient patients with SPPK demonstrated increased Ras-SHOC2 colocalization and decreased Erbin-SHOC2 colocalization, offering a possible explanation for the observed epidermal defects. These findings suggest a mechanism by which DSG1 and Erbin cooperate to repress MAPK signaling and promote keratinocyte differentiation.

There is mounting evidence that Th2 cytokines adversely affect skin barrier functions and contribute to the pathogenesis of atopic dermatitis (AD). AD is also characterized by abnormal cohesion in the stratum corneum (SC). However, the contribution of Th2 cytokines to this abnormality remains unknown. This study examined the effects of IL-4, a prototypic Th2 cytokine, on the cohesion of the SC. Structural and physiological assessments revealed that repeated intradermal injections of IL-4 compromised the cohesion of the SC of normal hairless mice. Two potential mechanisms were explored to account for the altered cohesion. First, IL-4 decreased the amount of corneodesmosomes and down-regulated the expression of desmoglein 1, but not of corneodesmosin (CDSN) or loricrin expression, in murine skin and in cultured human keratinocytes (KC). IL-4 did not affect the skin surface pH, and in situ zymography revealed no net change in total serine protease activity in the IL-4-treated SC. Yet, IL-4 enhanced expression of kallikrein (KLK)7, while simultaneously down-regulating KLK5 and KLK14. Finally, IL-4 did not alter the expression of the lympho-epithelial Kazal-type inhibitor (LEKTI) in KC. This study suggests that IL-4 abrogates the cohesion of SC primarily by reducing epidermal differentiation., (© 2012 John Wiley & Sons A/S.)

EphA2 is a receptor tyrosine kinase that is engaged and activated by membrane-linked ephrin-A ligands residing on adjacent cell surfaces. Ligand targeting of EphA2 has been implicated in epithelial growth regulation by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2)-mitogen activated protein kinase (MAPK) pathway. Although contact-dependent EphA2 activation was required for dampening Erk1/2-MAPK signaling after a calcium switch in primary human epidermal keratinocytes, the loss of this receptor did not prevent exit from the cell cycle. Incubating keratinocytes with a soluble ephrin-A1-Fc peptide mimetic to target EphA2 further increased receptor activation leading to its down-regulation. Moreover, soluble ligand targeting of EphA2 restricted the lateral expansion of epidermal cell colonies without limiting proliferation in these primary cultures. Rather, ephrin-A1-Fc peptide treatment promoted epidermal cell colony compaction and stratification in a manner that was associated with increased keratinocyte differentiation. The ligand-dependent increase in keratinocyte adhesion and differentiation relied largely upon the up-regulation of desmoglein 1, a desmosomal cadherin that maintains the integrity and differentiated state of suprabasal keratinocytes in the epidermis. These data suggest that keratinocytes expressing EphA2 in the basal layer may respond to ephrin-A1-based cues from their neighbors to facilitate entry into a terminal differentiation pathway.

The corneodesmosomes in the stratum corneum are critical for the maintenance of stratum corneum integrity. To evaluate the normal and diseased keratinization states in the epidermis, we studied the distribution of desmoglein 1 (DSG1), a major component of corneodesmosomes, in samples of the stratum corneum obtained by tape stripping, a non-invasive method. Samples were collected from lesional skin of four patients with psoriasis and three with lichen planus, and from non-lesional skin of three volunteers. Upper stratum corneum cells were obtained by tape stripping and skin biopsies were obtained from adjacent sites. Tape-stripped samples were examined by immunofluorescence microscopy using anti-DSG1 monoclonal antibody, in combination with histopathology of skin biopsies. In normal human stratum corneum, which shows basket-woven orthokeratosis, DSG1-containing fluorescent dots were distributed on the lateral cell-cell contact areas of plasma membrane, but not on the dorsal/ventral plasma membrane, and formed a well-ordered hexagonal network structure. In psoriatic stratum corneum, fluorescent dots were distributed throughout the cell membrane at ventral aspects of corneocytes as well as at the lateral cell-cell contacts. In lichen planus, fluorescent dots were distributed homogeneously and/or heterogeneously on the ventral surface in some cells. Adjacent cells lacked DSG1 at the lateral cell-cell contacts, but were instead separated by distinctive black-gap lines. These results suggest that the intercellular adhesion by DSG1 may depend on the lateral plasma membrane in normal human stratum corneum, on the dorsal/ventral plasma membrane in lichen planus, and on both lateral and dorsal/ventral plasma membranes in psoriatic stratum corneum. Tape stripping and DSG1 immunofluorescence visualizes adhesion features of corneocytes and has considerable potential for evaluation of abnormal keratinization and the process of healing in response to treatment., (© 2010 Japanese Dermatological Association.)

In confluent keratinocyte monolayers, desmosomal adhesion gradually becomes calcium-independent and this is associated with an increase in the strength of intercellular adhesion (hyper-adhesion). In this study, we investigated the functional and molecular significance of hyper-adhesion in a system challenged by autoimmune sera from patients with Pemphigus Vulgaris (PV), a disease primarily targeting desmosomal adhesion. The results show that keratinocytes with calcium-independent desmosomes are resistant to disruption of intercellular contacts (acantholysis) in experimental PV. Furthermore, both the desmosomal cadherins desmoglein (Dsg) 1 and Dsg3 and the adherens junction protein E-cadherin were decreased in confluent keratinocytes at Day 1, but not in hyper-adhesive cells (Day 6) after incubation with PV serum. Pharmacological induction of the hyper-adhesive state with the PKC inhibitor Go6976 reduced both the acantholysis rate and the processing of cell adhesion molecules induced by PV serum. When the establishment of the hyper-adhesive state was prevented by cell adhesion recognition (CAR) peptides that perturbed desmosomal interactions, Go6976 could still partially attenuate PV acantholysis. Taken together, these data demonstrate that keratinocyte hyper-adhesion decreases the morphological, functional and biochemical dys-cohesive effects of PV serum via mechanisms that involve, at least in part, the function of PKC. This suggests that reinforcing keratinocyte adhesion may be a promising way to inhibit the effects of this most debilitating disorder., (Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.)

Desmocollin (Dsc) 1-3 and desmoglein (Dsg) 1-4, transmembrane proteins of the cadherin family, form the adhesive core of desmosomes. Here we provide evidence that Dsc3 homo- and heterophilic trans-interaction is crucial for epidermal integrity. Single molecule atomic force microscopy (AFM) revealed homophilic trans-interaction of Dsc3. Dsc3 displayed heterophilic interaction with Dsg1 but not with Dsg3. A monoclonal antibody targeted against the extracellular domain reduced homophilic and heterophilic binding as measured by AFM, caused intraepidermal blistering in a model of human skin, and a loss of intercellular adhesion in cultured keratinocytes. Because autoantibodies against Dsg1 are associated with skin blistering in pemphigus, we characterized the role of Dsc3 binding for pemphigus pathogenesis. In contrast to AFM experiments, laser tweezer trapping revealed that pemphigus autoantibodies reduced binding of Dsc3-coated beads to the keratinocyte cell surface. These data indicate that loss of heterophilic Dsc3/Dsg1 binding may contribute to pemphigus skin blistering.

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca(2+)-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor-Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.

Epidermal expression of adhesion molecules such as desmogleins (Dsg) and cadherins is strongly affected by the differentiation status of keratinocytes. We have previously shown that certain protein kinase C (PKC) isoforms differentially alter the growth and differentiation of human epidermal HaCaT keratinocytes. In this paper, using recombinant overexpression and RNA interference, we define the specific roles of the different PKC isoenzymes in modulation of expression of adhesion molecules in HaCaT keratinocytes. The level of Dsg1, a marker of differentiating keratinocytes, was antagonistically regulated by two Ca-independent 'novel' nPKC isoforms; i.e. it increased by the differentiation-promoting nPKCdelta and decreased by the growth-promoting nPKCepsilon. The expression of Dsg3 (highly expressed in proliferating epidermal layers) was conversely regulated by these isoenzymes, and was also inhibited by the differentiation inducer Ca-dependent 'conventional' cPKCalpha. Finally, the expression of P-cadherin (a marker of proliferating keratinocytes) was regulated by all of the examined PKCs, also in an antagonistic manner (inhibited by cPKCalpha/nPKCdelta and stimulated by cPKCbeta/nPKCepsilon). Collectively, the presented results strongly argue for the marked, differential, and in some instances antagonistic roles of individual Ca-dependent and Ca-independent PKC isoforms in the regulation of expression of adhesion molecules of desmosomes and adherent junctions in human epidermal keratinocytes.

The target antigen of autoantibodies in human pemphigus foliaceus (PF) is a desmosomal cadherin, desmoglein 1 (Dsg1). It was demonstrated by immunoelectron microscopy (IEM) that the location of the binding sites of PF autoantibodies in the human epidermis was the extracellular regions of the desmosomes. Only a limited number of canine PF sera were shown to react with canine Dsg1, and the target proteins have not yet been identified. The purpose of this study was to demonstrate the ultrastructural binding site of canine PF autoantibodies to the canine skin by two kinds of IEM methods. Three canine PF sera, which were shown to react with the keratinocyte cell surface by immunofluorescence, were tested in this study. Using a technique of immunoprecipitation-immunoblotting, one out of the three canine PF sera were shown to react with recombinant canine Dsg1. By post-embedding IEM using cryofixation technique, one serum, which did not react with canine Dsg1 by immunoprecipitation-immunoblotting, bound broadly to the extra- and intracellular regions of the desmosomes of normal canine skin. By pre-embedding IEM using canine cultured keratinocytes (MCA-B1 cells), the autoantibodies of all three canine PF sera were identified to be bound to the cell-cell contact area of the adjacent cytoplasmic projections. When double stained with human PF serum and canine PF sera, the binding sites of both human and canine autoantibodies were co-localized on the MCA-B1 cells where the cytoplasmic projections contacted each other. Therefore, it may be concluded that the serum antibodies of canine PF targeted desmosomal proteins, regardless of whether or not they react with canine Dsg1 by immunoprecipitation-immunoblotting method.

We have previously demonstrated that serum of patients with pemphigus vulgaris induces reduction of desmoglein 3 (Dsg3) half-life in keratinocytes (FEBS Lett 2006: 580: 3276). This phenomenon seems to occur as a consequence of the progressive depletion of Dsg3 from desmosomes. Here we reported that reduction of full-length Dsg3 may be due to its progressive cleavage, leading to the formation of two fragmentation products with apparent molecular masses of about 60 kDa (fragment 1) and 70 kDa (fragment 2), as revealed by Western blotting. Unexpectedly, analysis of fragmentation pattern suggested cleavage to occur intracellularly. Consistently, fragment 1 was shed and localized within the cytosol, as shown by living cell immunofluorescence microscopy. Total amounts of full-length plakoglobin and Dsg1 were apparently unchanged. Taken together, our findings provide evidence that proteolytic processing of Dsg3 can lead to depletion of Dsg3 from the cell.

We recently showed that p120 catenin (p120ctn), which is an armadillo family protein member that binds to E-cadherin (E-cad), is also localized to desmosomes by directly or indirectly binding to desmogleins (Dsg). We examined whether p120ctn is associated with Dsg1 and Dsg3, as compared with E-cad and plakoglobin (PG), in keratinocytes grown in high or low Ca2+, using a human squamous cell carcinoma cell line, DJM-1 cells. The cell lysate of DJM-1 cells grown in high- or low-Ca2+ media was immunoprecipitated with anti-Dsg1/2 and Dsg3 antibodies, and we examined whether p120ctn is associated with Dsg1 and Dsg3. Then, we observed the co-localization between Dsg3 and p120ctn in cells grown in high- or low-Ca2+ medium on double-staining immunofluorescence microscopy using anti-p120ctn and anti-Dsg3 antibodies. Immunoprecipitates with anti-Dsg1/2 and Dsg3 antibodies in cells grown in high-Ca2+ medium contained p120ctn. In contrast, in low-Ca2+ medium, p120ctn was co-immunoprecipitated with neither Dsg1 nor Dsg3, but was co-immunoprecipitated with E-cad in cells grown in both high- and low-Ca2+ media. Dsg3 was associated with PG in cells grown in both low- and high-Ca2+ media. On immunofluorescence microscopy, p120ctn and Dsg3 were independently observed in cells grown in low-Ca2+ medium; p120ctn, but not Dsg3, was observed in a linear pattern at the cell-cell boundary. However, they were co-localized at cell-cell contacts in cells grown in high-Ca2+ medium. Thus, these proteins are not co-localized in low Ca2+ medium. These results suggest that p120ctn plays an important role in Ca2+-induced desmosome formation.

Pemphigus is a group of autoimmune blistering diseases of the skin and mucous membranes. In human patients with pemphigus vulgaris (PV) and paraneoplastic pemphigus (PNP), IgG autoantibodies against desmoglein (Dsg) 3 and Dsg1 play pathogenic roles in blister formation. In contrast, the target for IgG autoantibodies that induce keratinocyte dissociation has not been elucidated in canine pemphigus. The aim of the present study was to determine whether anti-Dsg IgG autoantibodies are present and disrupt the cell-cell adhesion of keratinocytes in canine PV and PNP. The extracellular domains of canine Dsg3 were recognized by IgG in 3/5 (60%) canine PV sera tested. IgG against the extracellular domains of canine Dsg1 was detected exclusively in two dogs that had PV with the mucocutaneous phenotype. In addition, anti-Dsg3 IgG was identified in canine PNP serum. Furthermore, incubation of normal human keratinocytes (NHK) with mucocutaneous canine PV serum and canine PNP serum resulted in dissociation of the NHK sheets, whereas the removal of anti-Dsg3 IgG from these canine sera blocked this dissociation. The present study indicates for the first time that circulating anti-Dsg3 IgG antibodies capable of dissociating keratinocytes are present in dogs with PV and PNP.

Kallikrein type serine proteases, KLK8/neuropsin, KLK6, and KLK7, have been implicated in the proliferation and differentiation of epidermal keratinocytes and in the pathogenesis of psoriasis. However, their mechanistic roles in these processes remain largely unknown. We applied 12-O-tetradecanoylphorbol-13-acetate on the wild type (WT) and the Klk8 gene-disrupted (Klk8(-/-)) mouse skin, inducing keratinocyte proliferation similar to the human psoriatic lesion. Klk8 mRNA as well as Klk6 and Klk7 mRNA were up-regulated after 12-O-tetradecanoylphorbol-13-acetate application in the WT mice. In contrast, Klk8(-/-) mice showed minimum increases of Klk6 and Klk7 transcripts, the proteins, and enzymatic activities. Relative to the WT, the Klk8(-/-) skin showed less proliferation and an increase in the number of cell layers in the stratum corneum. However, overexpression of Klk8 by adenovirus vector in knock-out keratinocytes did not result in an increase in Klk6 or Klk7 mRNA. The inefficient cleavage of adhesion molecules DSG1 and CDSN in Klk8(-/-) skin contributes to a delay in corneocyte shedding, resulting in the hyperkeratosis phenotype. We propose that in psoriatic lesion, KLK8 modulates hyperproliferation and prevents excessive hyperkeratosis by shedding the corneocytes.

The complexity of pemphigus acantholysis together with the weak expression of desmoglein 1 (Dsg1) in cultured keratinocytes have made the study on the pathogenic action of anti-Dsg1 antibodies quite difficult. The pathophysiology of the acantholytic phenomenon could depend on the reduction of Dsg1 adhesion function occurring after its massive internalization or decrease of its synthesis. Here, we have investigated this hypothesis by using sera of patients having antibodies against Dsg1 or monoclonal anti-Dsg1 antibodies to simulate pemphigus autoimmunity in Dsg1-rich keratinocytes. Similar to pemphigus foliaceus (PF) and vulgaris (PV) sera, monoclonal anti-Dsg1 antibodies induced transient internalization of Dsg1 and reduced the adhesion strength among keratinocytes. However, binding of IgG to Dsg1 did not determine its early depletion from the adhesion complexes but reduced the amount of Dsg1 found in the Triton X-100 soluble pool of proteins. Taken together, our results represent the first demonstration that anti-Dsg1 antibodies induce similar alterations on the subcellular distribution of Dsg1 irrespective of the disease where they come from. Furthermore, the present study provides insight into the mechanisms underlying epithelial blistering observed in the skin type of pemphigus.

Although a number of cell adhesion proteins have been identified as caspase substrates, the potential role of differentiation-specific desmosomal cadherins during apoptosis has not been examined. Here, we demonstrate that UV-induced caspase cleavage of the human desmoglein 1 cytoplasmic tail results in distinct 17- and 140- kDa products, whereas metalloproteinase-dependent shedding of the extracellular adhesion domain generates a 75-kDa product. In vitro studies identify caspase-3 as the preferred enzyme that cleaves desmoglein 1 within its unique repeating unit domain at aspartic acid 888, part of a consensus sequence not conserved among the other desmosomal cadherins. Apoptotic processing leads to decreased cell surface expression of desmoglein 1 and re-localization of its C terminus diffusely throughout the cytoplasm over a time course comparable with the processing of other desmosomal proteins and cytoplasmic keratins. Importantly, whereas classic cadherins have been reported to promote cell survival, short hairpin RNA-mediated suppression of desmoglein 1 in differentiated keratinocytes protected cells from UV-induced apoptosis. Collectively, our results identify desmoglein 1 as a novel caspase and metalloproteinase substrate whose cleavage likely contributes to the dismantling of desmosomes during keratinocyte apoptosis and also reveal desmoglein 1 as a previously unrecognized regulator of apoptosis in keratinocytes.