Expression Of Dsg3 Research Articles

Keratin 12 mRNA expression could serve as an early corneal marker for limbal explant cultures

This investigation aimed to identify early corneal marker and conjunctival epithelial differentiation through transcriptional analysis of limbal explant cultures and study early differentiation patterns of known corneal and conjunctival differentiation markers. 2 mm punch biopsies of limbal region were obtained from 6 donors of the Lions Cornea Bank Saar-Lorloux/Trier-Westpfalz. Limbal explants were dissected into corneal and conjunctival biopsy sections. Biopsies were placed with epithelial side down into 12 Wells. As soon as the outgrowing cells had reached confluence, they were harvested. mRNA expression of corneal differentiation markers KRT12, KRT3, DSG1, PAX6, ADH7 and ALDH1A1, conjunctival markers KRT19, KRT13 and stem cell marker ABCG2 were measured via qPCR. KRT12 and PAX6 protein expressions were evaluated using Western Blot. Results suggested that KRT12 mRNA expression was significantly higher in outgrowing cells from the corneal side of the biopsies as in those from the conjunctival side (p = 0.0043). There was no significant difference in mRNA expression of other analyzed markers comparing with marker expression of outgrown cells from both limbal biopsies (p > 0.13). KRT12 and PAX6 Western Blot analysis showed no difference in cells harvested from both sides. In conclusion, KRT12 mRNA might be a marker to measure corneal origin of cells from limbal biopsies with unknown composition of corneal and conjunctival progenitor cells. KRT3, DSG1, PAX6, ADH7, ALDH1A1, KRT19, KRT13 and ABCG2 mRNA as well as KRT12 and PAX6 protein expression could not contribute to differentiate corneal from conjunctival cell identity from limbal biopsies.

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

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

Keratinocyte cadherin desmoglein 1 controls melanocyte behavior through paracrine signaling.

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.

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

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

239 Desmosomes pattern cell mechanics to govern epidermal tissue form and function

The epidermis exhibits polarization of structural and functional features across multiple cell layers, but how this polarity is created and preserved is poorly understood. Desmosomes (DSMs) are cell-cell adhesive organelles linking to intermediate filaments (IF) and are critical for signaling and tissue integrity. The DSM cadherin desmoglein 1 (Dsg1) is first expressed as cells commit to differentiate and stratify, with expression peaking in the granular layer (SG). We hypothesize the pattern of Dsg1 and associated IF is crucial for establishing proper epidermal polarity, especially mechanical polarity. To test this, we used laser ablation and whole mount imaging to assess layer-specific mechanical properties of 3D epidermal cultures. These data support an epidermal mechanical gradient: keratinocytes are under compressive forces in the basal layer and high tension in the suprabasal layers. We previously showed Dsg1 regulates basal cell forces to promote stratification. Here, a DSM/IF uncoupling mutant recapitulates Dsg1 loss, suggesting a requirement of IF attachment. High apical tension in simple epithelia helps generate barrier-critical tight junctions (TJs). TJs localize to the SG in epidermis, where tension is high and Dsg1 expression peaks. Thus, we assessed the ability of Dsg1 to polarize tension and TJs in the SG. Dsg1 loss disrupts the polarized SG distribution of the tension sensitive TJ component ZO1. Our data suggest Dsg1 modulates TJs through the mechanosensitive kinase ErbB2, which localizes to the TJ forming layer. Dsg1 loss disrupts this localization and decreases total and phosphorylated ErbB2. Uncoupling the DSM/IF linkage or inhibiting ErbB2 significantly decreases epidermal electrical barrier function. As mechanical forces regulate cell behavior, our data suggest a model in which patterning of DSM components regulates epidermal polarity by orchestrating the distribution of mechanical inputs to drive morphogenesis and development of a life-essential barrier.

The Role of Desmoglein 1 in Gap Junction Turnover Revealed through the Study of SAM Syndrome

An effective epidermal barrier requires structural and functional integration of adherens junctions, tight junctions, gap junctions (GJ), and desmosomes. Desmosomes govern epidermal integrity while GJs facilitate small molecule transfer across cell membranes. Some patients with severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome, caused by biallelic desmoglein 1 (DSG1) mutations, exhibit skin lesions reminiscent of erythrokeratodermia variabilis, caused by mutations in connexin (Cx) genes. We, therefore, examined whether SAM syndrome-causing DSG1 mutations interfere with Cx expression and GJ function. Lesional skin biopsies from SAM syndrome patients (n= 7) revealed decreased Dsg1 and Cx43 plasma membrane localization compared with control and nonlesional skin. Cultured keratinocytes and organotypic skin equivalents depleted of Dsg1 exhibited reduced Cx43 expression, rescued upon re-introduction of wild-type Dsg1, but not Dsg1 constructs modeling SAM syndrome-causing mutations. Ectopic Dsg1 expression increased cell-cell dye transfer, which Cx43 silencing inhibited, suggesting that Dsg1 promotes GJ function through Cx43. As GJA1 gene expression was not decreased upon Dsg1 loss, we hypothesized that Cx43 reduction was due to enhanced protein degradation. Supporting this, PKC-dependent Cx43 S368 phosphorylation, which signals Cx43 turnover, increased after Dsg1 depletion, while lysosomal inhibition restored Cx43 levels. These data reveal a role for Dsg1 in regulating epidermal Cx43 turnover.

The secreted Ly-6/uPAR related protein-1 (SLURP1) stabilizes epithelial cell junctions and suppresses TNF-α-induced cytokine production

The secreted Ly-6/uPAR related protein-1 (SLURP1) is an anti-angiogenic and anti-inflammatory peptide highly expressed by the mucosal epithelial cells. SLURP1 is abundantly expressed by the corneal epithelial cells and is significantly downregulated when these cells are transformed and adapted for culture in vitro. Here we studied the effect of overexpressing SLURP1 in Human Corneal Limbal Epithelial (HCLE) cells cultured in vitro. The expression of DSP1, DSG1, TJP1 and E-Cadherin was significantly upregulated in two different SLURP1-overexpressing HCLE cell (HCLE-SLURP1) clones. HCLE-SLURP1 cells also displayed a significant decrease in tumor necrosis factor-α (TNF-α)-induced upregulation of (i) IL-8 from 7.4- to 2.9- and 2.1-fold, (ii) IL-1β from 4.9- to 3.9- and 2.9-fold, (iii) CXCL1 from 9- to 3.3- and 5.5-fold, and (iv) CXCL2 from 4.8- to 2.1- and 2.8-fold. ELISAs revealed a concomitant decrease in IL-8 levels in cell culture supernatants from 789 pg/ml in the control, to 503 and 352 pg/ml in HCLE-SLURP1 cells. Consistently, cytosolic IκB expression was elevated in HCLE-SLURP1 cells with a concurrent suppression of TNF-α-activated nuclear translocation of NF-κB. Collectively, these results elucidate the beneficial effects of SLURP1 in stabilizing the HCLE intercellular junctions and suppressing the TNF-α-induced upregulation of inflammatory cytokines by suppressing NF-κB nuclear translocation.

A potential link between desmoglein 3 and epidermal growth factor receptor in oral squamous cell carcinoma and its effect on cetuximab treatment efficacy.

Desmoglein (DSG) 3 is overexpressed in oral squamous cell carcinoma (OSCC). Epidermal growth factor receptor (EGFR) inhibitor cetuximab is widely used for OSCC treatment. Several evidences suggest a correlation between DSG3 and EGFR in epidermal keratinocytes. EGFR inhibition has been shown to enhance cell-cell adhesion and induce terminal differentiation in epidermal cells. Thus, here we investigated the DSG3-EGFR interaction in OSCC and its effect on cetuximab treatment. Cell lines established from the primary tumor and metastatic lymph nodes of four OSCC patients and three commercial OSCC cell lines were used for the experiments. Cells from metastatic lymph nodes of each patient expressed increased DSG3 and EGFR than cells from the primary tumor in the same patient. Cetuximab treatment increased DSG3 expression by up to 3.5-fold in seven of the 11 cell lines. A high calcium concentration increased the expression of DSG3 and EGFR in a dose-dependent manner. Strikingly, a high calcium-associated DSG3 induction enhanced cetuximab efficacy by up to 23% increase in cetuximab-low-sensitive cell lines. Our findings also suggest a correlation between DSG3 and EGFR in OSCC, and this affects cetuximab treatment efficacy.

Desmoglein 1 Regulates Invadopodia by Suppressing EGFR/Erk Signaling in an Erbin-Dependent Manner.

Loss of the desmosomal cell-cell adhesion molecule, Desmoglein 1 (Dsg1), has been reported as an indicator of poor prognosis in head and neck squamous cell carcinomas (HNSCC) overexpressing epidermal growth factor receptor (EGFR). It has been well established that EGFR signaling promotes the formation of invadopodia, actin-based protrusions formed by cancer cells to facilitate invasion and metastasis, by activating pathways leading to actin polymerization and ultimately matrix degradation. We previously showed that Dsg1 downregulates EGFR/Erk signaling by interacting with the ErbB2-binding protein Erbin (ErbB2 Interacting Protein) to promote keratinocyte differentiation. Here, we provide evidence that restoring Dsg1 expression in cells derived from HNSCC suppresses invasion by decreasing the number of invadopodia and matrix degradation. Moreover, Dsg1 requires Erbin to downregulate EGFR/Erk signaling and to fully suppress invadopodia formation. Our findings indicate a novel role for Dsg1 in the regulation of invadopodia signaling and provide potential new targets for development of therapies to prevent invadopodia formation and therefore cancer invasion and metastasis. IMPLICATIONS: Our work exposes a new pathway by which a desmosomal cadherin called Dsg1, which is lost early in head and neck cancer progression, suppresses cancer cell invadopodia formation by scaffolding ErbB2 Interacting Protein and consequent attenuation of EGF/Erk signaling.

Characteristics of multicellular tumor spheroids formed by pancreatic cells expressing different adhesion molecules

AimsMulticellular tumor spheroids (MCTS) produced by different methods vary in forms, sizes, and properties. The aim of this work was to characterize MCTS formed by six pancreatic cell lines on a non-adherent surface. Materials and methodsHuman pancreatic cells were grown in 2D and 3D conditions and compared for the expression of E- and desmosomal cadherins (PCR, confocal microscopy), growth, cell cycling, apoptosis (flow cytometry), and a response to antitumor drugs doxorubicin and gemcitabine (MTT-assay). Key findingsThree types of MCTS were identified: BxPC-3, T3M4 formed small number of large and dense spheroids representing type I MCTS; COLO-357 and AsPC-1 generated type II multiple and loose MCTS of different sizes while MiaPaCa-2 and PANC-1 represented type III cultures which grew almost as floating monolayer films. Formation of type I MCTS depended on the simultaneous expression of DSG3 and several DSC proteins; II MCTS expressed solely DSG2-DSC2 but not DSG3, while type III cells either did not express E-cadherin or a pair of DSG and DSC proteins. Cells in type I MCTS but not in types II and III ones quickly became quiescent which correlated with a decrease in the proliferation, increased apoptosis, and a higher resistance to antitumor drugs doxorubicin and gemcitabine. SignificanceTaken collectively, pancreatic cells significantly vary in the expression of desmosomal cadherins, resulting in the formation of MCTS with different characteristics. The sensitivity of MCTS to various drugs depends on the type of cells and the method of spheroid preparation used.

Desmoglein-2 overexpression predicts poor prognosis in hepatocellular carcinoma patients.

Desmoglein-2 (Dsg2) plays a crucial role in the assembly and adhesion of desmosomes. The absent or aberrant expression of Dsg2 was reported to be associated with the progression of varies human cancers. However, the expression of Dsg2 in hepatocellular carcinoma (HCC) and its association with tumor prognosis is still unknown. The aim of this study was to evaluate the expression level of Dsg2 in HCC and of the correlation between Dsg2 expression and clinicopathological variables. A total of 104 patients diagnosed with HCC were enrolled in this study. Real time-quantitative PCR (RT-qPCR) and Western blot were performed to determine the expression level of Dsg2 in HCC tumor tissues and matched noncancerous tissues. Cell proliferation and cell cycle were measured by cell counting kit-8 (CCK-8) assay and flow-cytometry assay, respectively. Our results revealed that Dsg2 expression was significantly higher in HCC tumor tissues than in matched noncancerous tissues (p < 0.01), positively correlated with tumor size (p = 0.035) and tumor stage (p = 0.021). Univariate and multivariate analyses demonstrated Dsg2 expression was an independent prognostic factor for overall survival. Meanwhile, we found knockdown the expression of Dsg2 using small interfering RNA (siRNA) could efficiently impaired HCC cell proliferation rate and cell cycle progression (p < 0.05). Taken together, our results suggest that increased Dsg2 expression was associated with tumor progression in HCC and may function as a promising biomarker for unfavorable prognosis of HCC.

Abstract 4176: Abrogation of desmoglein 2-mediated c-met activation as a novel therapeutic strategy for reducing anaplastic thyroid cancer metastasis

Abstract Anaplastic thyroid cancer (ATC) is a fast growing and highly metastatic cancer with limited therapeutic options. Thus, targeted therapies need to be developed. This study aims to determine desmoglein 2 (Dsg2) expression in ATC and to elucidate its biological role and potential as a therapeutic target in ATC. Dsg2 expression was low or abnormal in ATC tissues. The ATC patients with reduced Dsg2 expression also had distant metastasis. Dsg2 depletion significantly increased cell migration and invasion, with relatively little effect on ATC cell proliferation in vitro, and increased distant metastasis in vivo. Dsg2 knockdown induced cell motility through the hepatocyte growth factor receptor (HGFR, c-Met)/Src/Rac1 signaling axis, with no alterations in the expression of EMT-related molecules. Further, specific targeting of c-Met significantly inhibited the motility of shDsg2-depleted ATC cells. Decreased membrane expression of Dsg2 increases the metastatic potential of ATC cells. Dsg2 plays an important role in migration and invasiveness in cell lines derived from ATC. Therapies targeting c-Met might be effective for ATC patients with decreased membrane expression of Dsg2. Thus, analysis of Dsg2 expression may provide a novel therapeutic strategy for ATC treatment. Keywords Anaplastic Thyroid Cancer, Desmoglein 2, Metastasis, c-Met, Tivantinib Citation Format: Dong G. Lee, Jeong-Ki Min. Abrogation of desmoglein 2-mediated c-met activation as a novel therapeutic strategy for reducing anaplastic thyroid cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4176.

Abstract LB-355: Loss of keratinocyte desmoglein 1 occurs during melanoma development and alters crosstalk between keratinocytes and melanocytes

Abstract Ultraviolet (UV)-induced mutations in melanocytes are a well-established cause of melanoma. However, histologically normal “fields” of altered keratinocytes present in sun-exposed epidermis could also drive initiation and progression of melanoma. We previously demonstrated that UV radiation reduced expression of the keratinocyte desmosomal cadherin, desmoglein 1 (Dsg1), and that loss-of-function mutations in Dsg1 increased keratinocyte cytokine production in Severe dermatitis, multiple Allergies, and Metabolic wasting (SAM) syndrome. Based on these observations we hypothesized that loss of Dsg1 alters the microenvironment through increased cytokine signaling. Here, we show that Dsg1 expression is progressively reduced in regions surrounding human dysplastic nevi, melanoma in situ, and stage I/II melanomas. In contrast, keratinocyte E-cadherin was not significantly altered in early tumors. Dsg1 knockdown in keratinocytes in vitro increased phospho-EGFR, phospho-Stat3, and nuclear NFκB, while increasing keratinocyte mRNAs encoding IL-1α, IL-1β, IL-6 and IL-8 pro-inflammatory cytokines. Re-expressing silencing-resistant Dsg1 returned cytokine levels to control levels. To determine whether Dsg1 loss in keratinocytes elicits paracrine-mediated changes in melanocyte behavior, we treated normal primary melanocytes with conditioned media from Dsg1-silenced keratinocytes. Short-term (overnight) exposure to Dsg1-deficient conditioned media resulted in significantly increased melanocyte dendrite length, similar to morphology changes induced by UV exposure; however, long-term (7 day) exposure resulted in significantly shorter dendrites. Melanocytes exposed to Dsg1-deficient media increased melanin secretion and decreased melanin retention, a phenomenon associated with the process of transformation. Melanocytes incorporated into Dsg1-deficient 3D human skin equivalents exhibited aberrant behavior, including increased numbers and mis-localization to suprabasal layers. Based on these observations, we propose that under homeostatic conditions Dsg1 functions to maintain baseline levels of cytokine signaling, but that Dsg1 loss due to environmental stress disrupts keratinocyte:melanocyte communication through altered paracrine signaling to promote melanoma initiation. Citation Format: Christopher Arnette, Jodi L. Johnson, Hope E. Burks, Jennifer L. Koetsier, Kathleen J. Green. Loss of keratinocyte desmoglein 1 occurs during melanoma development and alters crosstalk between keratinocytes and melanocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-355.

Abstract 1082: Loss of desmoglein 2 promotes tumor growth and progression through the activation of Src and facilitates the internalization of EGFR in biliary tract carcinoma cells

Abstract Understanding the molecular carcinogenesis pathways is critical to establish novel individualized molecular targeted therapies and finally to improve overall clinical outcomes of patients with a highly heterogeneous group of malignancies such as biliary tract cancers (BTCs). In this context, we investigated the patho-physiological roles of a desmosomal component, desmoglein 2 (Dsg2) and found that Dsg2 functions as a tumor suppressor protein in BTC, as Dsg2 loss induced a number of cellular biological activities including proliferation, motility, invasion, transendothelial migration in vitro and tumor growth and metastasis in vivo. Clinico-pathological analyses of BTC patients indicated that lower expression of Dsg2 is associated with a poor prognosis of patients as perineural and lymphatic invasion were highly increased in those patients. In addition, loss of Dsg2 showed a significant increase of EGFR clearance from the cell membrane in BTC cells and patients' tumor tissues. Those cells were significantly decreased in the EGF-driven activation of EGFR and highly resistant to the anti-EGFR antibody treatment. Src appears to be involved in the Dsg2-mediated clearance of EGFR, as this significantly blocked by the suppression of cSrc expression in Dsg2-depleted cells, possibly through the direct binding of Dsg2 preferentially with an inactive form of Src through its SH2 domain. Suppression of cSrc by Src inhibitors or siRNA dramatically reduced BTC tumor growth in vitro and in vivo. In comparison with the anti-EGFR antibody therapy, Src-targeted therapies produced consistent anti-tumor activities independent with the expression status of EGFR in the BTC tumors. In conclusion, targeting Src would be a promising therapeutic approach to patients with advanced BTC and potentially useful in overcoming resistance to current anti-EGFR therapies, although further clinical studies are essentially required to translate the current preclinical knowledge into clinical practice. Citation Format: Sang-Hyun Lee, Jeong-Ki Min. Loss of desmoglein 2 promotes tumor growth and progression through the activation of Src and facilitates the internalization of EGFR in biliary tract carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1082.

Ultrastructural changes in endometrial desmosomes of desmoglein 2 mutant mice.

The intercellular binding of desmosomal junctions is mediated by cadherins of the desmoglein (Dsg) and desmocollin (Dsc) type. Dsg2 mutant mice with deletion of a substantial segment of the extracellular EC1-EC2 domain, which is believed to participate in homo- and heterophilic desmosomal cadherin interactions, develop cardiac fibrosis and ventricular dilation. Widening of the intercellular cleft and complete intercalated disc ruptures can be observed in the hearts of these mice. Since a reduced litter size of homozygous Dsg2 mutant mice was noted and a functional correlation between desmosomes and embryo implantation has been deduced from animal studies, we looked for an alteration of desmosomes in uterine endometrial epithelium. Shape and number of desmosomes as well as the expression of Dsg2 and the desmosomal plaque protein desmoplakin (Dsp) were investigated by electron microscopy and immunohistochemistry in 12 oestrous-dated mice (7 wildtype and 5 homozygous Dsg2 mutant mice) at the age of 9-17weeks. The immunohistochemical detection of Dsg2 was diminished in the mutants and the number of desmosomes was significantly reduced as revealed by electron microscopy. In addition, the intercellular desmosomal space measured in electron micrographs was considerably widened in the Dsg2 mutants. The increased intercellular spacing can be explained by the partial deletion of the extracellular EC1-EC2 domain of Dsg2. Whether these changes explain the reduced number of offspring of homozygous Dsg2 mutant mice remains to be further investigated.

Enhancement of Cutaneous Wound Healing by Dsg2 Augmentation of uPAR Secretion.

In addition to playing a role in adhesion, desmoglein 2 (Dsg2) is an important regulator of growth and survival signaling pathways, cell proliferation, migration and invasion, and oncogenesis. Although low-level Dsg2 expression is observed in basal keratinocytes and is downregulated in nonhealing venous ulcers, overexpression has been observed in both melanomas and nonmelanoma malignancies. Here, we show that transgenic mice overexpressing Dsg2 in basal keratinocytes primed the activation of mitogenic pathways, but did not induce dramatic epidermal changes or susceptibility to chemical-induced tumor development. Interestingly, acceleration of full-thickness wound closure and increased wound-adjacent keratinocyte proliferation was observed in these mice. As epidermal cytokines and their receptors play critical roles in wound healing, Dsg2-induced secretome alterations were assessed with an antibody profiler array and revealed increased release and proteolytic processing of the urokinase-type plasminogen activator receptor. Dsg2 induced urokinase-type plasminogen activator receptor expression in the skin of transgenic compared with wild-type mice. Wounding further enhanced urokinase-type plasminogen activator receptor in both epidermis and dermis with a concomitant increase in the prohealing laminin-332, a major component of the basement membrane zone, in transgenic mice. This study demonstrates that Dsg2 induces epidermal activation of various signaling cascades and accelerates cutaneous wound healing, in part, through urokinase-type plasminogen activator receptor-related signaling cascades.

Keratinocyte Binding Assay Identifies Anti-Desmosomal Pemphigus Antibodies Where Other Tests Are Negative.

The serological diagnosis of pemphigus relies on the detection of IgG autoantibodies directed against the epithelial cell surface by indirect immunofluorescence (IIF) on monkey esophagus and against desmoglein 1 (Dsg1) and Dsg3 by ELISA. Although being highly sensitive and specific tools, discrepancies can occur. It is not uncommon that sera testing positive by ELISA give a negative result by IIF and vice versa. This brings diagnostic challenges wherein pemphigus has to be ascertained or ruled out, especially when no biopsy is available. We utilized the ability of anti-Dsg3 and anti-Dsg1 IgG to bind in specific desmosomal patterns to living cells to investigate these discrepancies between IIF and ELISA. Living cultured primary normal human keratinocytes were grown under differentiating conditions to induce adequate expression of Dsg1 and Dsg3, incubated with patient serum for 1 h, and then stained to visualize bound IgG. We investigated two different groups; sera from patients with a positive direct immunofluorescence (DIF) and inconsistent serological findings (n = 43) and sera with positive ELISA or IIF but with negative DIF (n = 60). As positive controls we used 50 sera from patients who fulfilled all diagnostics criteria, and 10 sera from normal human subjects served as negative controls. In the DIF positive group, IgG from 39 of the 43 sera bound to the cells in a desmosomal pattern while in the DIF negative group none of the 60 sera bound to the cells. This shows that for pemphigus patients, ELISA and IIF can be negative while anti-desmosomal antibodies are present and vice versa that ELISA and IIF can be positive in non-pemphigus cases. In absence of a biopsy for DIF, such findings may lead to misdiagnosis.

858 Desmoglein 3 acts as a mechanosensor in keratinocytes

The desmosomal cadherin, Desmoglein 3 (Dsg3), plays a crucial role in cell-cell cohesion and tissue integrity that is highlighted in the autoimmune blistering disease pemphigus vulgaris where Dsg3 serves as a major autoantigen (PVA). Increasing evidence suggests that Dsg3 acts as a surface regulator in cell mechano-transduction. However, little is known about its direct response to mechanical forces. The aim of this study was to investigate the impact of cyclic strain and substrate stiffness on Dsg3 expression, compared with E-cadherin a well-characterized mechanosensor. Both oral and skin keratinocytes were tested using the Flexcell system and were subjected to mechanical stretching for various time periods. Although no apparent change was found at the mRNA level in response to strain for up to 24 hours, increased protein expression was detectable for Dsg3 and associated junctional proteins (to a lesser extent for E-cadherin). We found that oral keratinocytes appeared to be more mechano-sensitive than skin cells which exhibited a more delayed response. Marked increases of cell-surface Dsg3 and E-cadherin, along with actin-myosin cytoskeleton changes, was detected in strained cells. Conversely Dsg3 depletion had a negative impact on these processes. It was revealed that cyclic strain did not necessarily enhance cell-cell adhesion strength or levels of calcium-independent desmosomes but, instead, exacerbated junction remodeling with accelerated desmoplakin turnover and increased PKCa expression. Significantly, we showed that Dsg3 regulated the expression of YAP, a mechanosensor and an effecter of the Hippo pathway. Dsg3 knockdown resulted in pronounced attenuation of YAP/pYAP in both strained and non-strained cells. Colocalisation of Dsg3 and pYAP was observed at the plasma membrane of cells with their physical interaction being demonstrable by co-IP. Together, our findings strongly suggest a novel role for Dsg3 in keratinocyte mechano-transduction.

Conditional reprogrammed human limbal epithelial cells represent a novel in vitro cell model for drug responses

In this study, we established human limbal epithelial cells (LECs) from normal limbal tissues by using Conditional Reprogramming (CR) technology (refer to CR-LEC cells in this study). We have successfully established CR-LEC cell strains from three human donors (3 out of 3), and normal rabbits (2 out of 2) and pig (1 out of 1) as well. CR-LEC cells sustained a continuous and stable proliferation status with a normal karyotype, normal response to DNA damage, well-defined structured spheres in matrigel 3D culture. Responses of CR-LEC cells to IFN α2b, Ganciclovir and 5-Fluorouracil were different, suggesting that these drugs had different toxicities to these cells as expected. More important, there was no significant difference of responses to drugs between early and late passages of CR-LEC cells (p>0.05), indicating CR-LEC cells can serve a stable normal human cell model for toxicity assessment. Toxicity tests with monolayer cultures of CR-LEC cells were measured by staining the F-actin and Dsg-1 expression. Toxicity of three drugs at LD50 concentration resulted in a gradually increased destruction of monolayer, which is, in accordance with the irritation grade of three drugs on human cornea epithelium. Therefore, CR-LEC cells provide a novel and reliable in vitro physiological cell model for corneal toxicity assessment.

H19 lncRNA regulates keratinocyte differentiation by targeting miR-130b-3p

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.