Epidermal Junction Research Articles

LB965 Esrp regulates an alternative splicing program essential for epidermal tight junction

LB965 Esrp regulates an alternative splicing program essential for epidermal tight junction

Cdc42 regulates junctional actin but not cell polarization in the Caenorhabditis elegans epidermis.

During morphogenesis, adherens junctions (AJs) remodel to allow changes in cell shape and position while preserving adhesion. Here, we examine the function of Rho guanosine triphosphatase CDC-42 in AJ formation and regulation during Caenorhabditis elegans embryo elongation, a process driven by asymmetric epidermal cell shape changes. cdc-42 mutant embryos arrest during elongation with epidermal ruptures. Unexpectedly, we find using time-lapse fluorescence imaging that cdc-42 is not required for epidermal cell polarization or junction assembly, but rather is needed for proper junctional actin regulation during elongation. We show that the RhoGAP PAC-1/ARHGAP21 inhibits CDC-42 activity at AJs, and loss of PAC-1 or the interacting linker protein PICC-1/CCDC85A-C blocks elongation in embryos with compromised AJ function. pac-1 embryos exhibit dynamic accumulations of junctional F-actin and an increase in AJ protein levels. Our findings identify a previously unrecognized molecular mechanism for inhibiting junctional CDC-42 to control actin organization and AJ protein levels during epithelial morphogenesis.

Biphasic influence of Staphylococcus aureus on human epidermal tight junctions.

Bacterial infections (e.g., with Staphylococcus aureus) are serious problems in skin with a compromised barrier, such as in patients with atopic dermatitis. Previously, it was shown that tight junction (TJ) proteins are influenced by staphylococcal infection, and TJ function is impaired after infection of the keratinocyte cell line HaCaT. However, functional studies in cells or models more similar to human skin are missing. Therefore, we investigated bacterial colonialization and infection with live S. aureus in primary human keratinocytes and reconstructed human epidermis (RHE). We show that short-term inoculation results in increased TJ barrier function-which could not be seen in HaCaT cells-hinting at an early protective effect. This is accompanied by occludin phosphorylation and sustained localization of occludin and claudin-4 at cell membranes. Long-term incubation resulted in decreased presence of claudin-1 and claudin-4 at cell membranes and decreased TJ barrier function. The agr regulon of S. aureus plays a role in the increasing but not in the decreasing effect. Proinflammatory cytokines, which are produced as a result of S. aureus inoculation, influence both phases. In summary, we show here that S. aureus can have short-term promoting effects on the TJ barrier, while in the long term it results in disturbance of TJs.

119 Breaking down barriers: Defining the role of EphA2 in building epidermal tight junctions in cancer

119 Breaking down barriers: Defining the role of EphA2 in building epidermal tight junctions in cancer

442 ER stress regulates epidermal tight junction and PPARα activation attenuates ER stress-induced unfolded protein response and tight junction damage in cultured keratinocytes

442 ER stress regulates epidermal tight junction and PPARα activation attenuates ER stress-induced unfolded protein response and tight junction damage in cultured keratinocytes

470 Epidermal tight junctions are strengthened by short-term staphylococcal challenge in an agr dependent manner

470 Epidermal tight junctions are strengthened by short-term staphylococcal challenge in an agr dependent manner

750 Improvement of the dermal epidermal junction to prevent senile lentigines formation

750 Improvement of the dermal epidermal junction to prevent senile lentigines formation

Semi-automated localization of dermal epidermal junction in optical coherence tomography images of skin.

Identifying the location of the dermal epidermal junction (DEJ) in skin images is essential in several clinical applications of dermatology such as epidermal thickness determination in healthy versus unhealthy skins, such as basal cell carcinoma. Optical coherence tomography (OCT) facilitates the visual detection of DEJ in vivo. However, due to the granular texture of speckle and a low contrast between dermis and epidermis, a skin border detection method is required for DEJ localization. Current DEJ algorithms work well for skins with a visible differentiable epidermal layer but not for the skins of different body sites. In this paper, we present a semi-automated DEJ localization algorithm based on graph theory for OCT images of skin. The proposed algorithm is performed in an interactive framework by a graphical representation of an attenuation coefficient map through a uniform-cost search method. For border thinning, a fuzzy-based nonlinear smoothing technique is used. For evaluation, the DEJ detection method is used by several experts, and the results are compared with manual segmentation. The mean thickness error between the proposed algorithm and the experts' opinion in the Bland-Altman plot is computed as 14 μm; this is comparable to the resolution of the OCT. The results suggest that the proposed image processing method successfully detects DEJ.

Restoration of cutaneous pigmentation by transplantation to mice of isogeneic human melanocytes in dermal-epidermal engineered skin substitutes.

Autologous engineered skin substitutes (ESS) containing melanocytes (hM) may restore pigmentation and photoprotection after grafting to full-thickness skin wounds. In this study, normal hM were isolated from discard skin, propagated with or without tyrosinase inhibitors, cryopreserved, recovered into culture, and added to ESS (ESS-P) before transplantation. ESS-P were incubated in either UCMC160/161 or UCDM1 medium, scored for hM densities, and grafted to mice. The results showed that sufficient hM can be propagated to expand donor tissue by 100-fold; incubation of hM in tyrosinase inhibitors reduced pigment levels but did not change hM recovery after cryopreservation; hM densities in ESS-P were greater after incubation in UCDM1 than UCMC160 medium; hM were localized to the dermal-epidermal junction of ESS-P; and UCDM1 medium promoted earlier pigment distribution and density. These results indicate that hM can be incorporated into ESS-P efficiently to restore cutaneous pigmentation and UV photoprotection after full-thickness skin loss conditions.

427 Epidermal Tight Junctions are strengthened by short-term Staphylococcal challenge in an agr dependent manner

427 Epidermal Tight Junctions are strengthened by short-term Staphylococcal challenge in an agr dependent manner

448 Sub-toxic hydrogen peroxide exposure significantly reduces expression of extracellular matrix and dermal epidermal junction components in 3-D skin tissue, mimicking key features of aging skin

448 Sub-toxic hydrogen peroxide exposure significantly reduces expression of extracellular matrix and dermal epidermal junction components in 3-D skin tissue, mimicking key features of aging skin

393 Molecular efficacy of cosmetic formulations by a large-scale approach based on gene expression and ex vivo skin model

The skin aging represents a multifactorial and complex condition involving an association of genetic background and environmental influences, resulting in a multitude of skin phenotypes as a consequence of changes in gene expression. With the purpose of promote anti-aging benefits to skin, a number of 12 different cosmetic formulations and 24 different combinations of active ingredient were evaluated comparatively with its respective placebos formulations and non-treated controls after treatment of ex vivo skin fragments during 72 hours. Subsequently, the gene expression screening of 180 genes were performed. A bioinformatic analysis in ExpressionSuite and Ingenuity Pathway Analysis (IPA) softwares retrieved the identification of genes modulated and the main biological function and relevant pathway altered by each treatment such as those related to cell proliferation and differentiation, oxidative stress, inflammation, integrin-mediated adhesion, stimulation of collagen and elastic fibers, dermal epidermal junction, among others. After that, the proteins expression related to the main molecular mechanism identified were assessed by a complementary immunofluorescence assay to confirm those results. In conclusion, this methodology proved to be a useful tool to identify the molecular efficacy of anti-aging cosmetic formulations and combinations of actives ingredients in a wider and more complete strategy enabling targeting female skin ages ranges.

EpCAM Expressed by Murine Epidermal Langerhans Cells Modulates Immunization to an Epicutaneously Applied Protein Antigen

Langerhans cells (LCs) induce type 2 antibodies reactive with protein antigens that are applied to murine skin in the absence of adjuvant after extending their dendrites through tight junctions to acquire antigens and migrating to regional lymph nodes. In response to contact sensitizers, epithelial cell adhesion molecule (EpCAM) on LCs promotes LC dendrite mobility and LC migration. In epithelial cells, EpCAM regulates expression and distribution of selected tight junctions-associated claudins. To determine if EpCAM regulates claudins in LC and immune responses to externally applied proteins, we studied conditional knockout mice with EpCAM-deficient LCs. Although LC claudin-1 levels were dramatically reduced in the absence of EpCAM, conditional knockout mice with EpCAM-deficient LCs and control LC dendrites docked with epidermal tight junctions with equal efficiencies and ingested surface proteins. Topical immunization of conditional knockout mice with EpCAM-deficient LCs with ovalbumin led to increased induction of type 2 Ova-specific antibodies and enhanced proliferation of ovalbumin-reactive T cells associated with increased accumulation of LCs in lymph nodes. These results suggest that, in the absence of strong adjuvants, EpCAM-deficient LCs exhibit increased migration to regional lymph nodes. EpCAM appears to differentially regulate LC mobility/migration inthe setting of limited inflammation as compared with the intense inflammation triggered by contact sensitizers.

Current treatments and developments in pemphigoid diseases as paradigm diseases for autoantibody-driven, organ-specific autoimmune diseases

Pemphigoid diseases constitute a group of difficult-to-treat, remitting-relapsing autoimmune skin diseases. They are autoantibody-driven diseases with well-defined autoantigens in the hemidesmosomal complex at the dermal–epidermal junction (DEJ). Autoantibody deposition initiates the recruitment of granulocytes to the DEJ, where these cells degrade the hemidesmosomal complex, disrupting dermal-epidermal adherence, which clinically manifests as blisters and erosions. Due to their well-defined autoantigens and the location at the body surface, allowing the direct observation of inflammation throughout its course, pemphigoid diseases are excellent paradigm diseases to study the pathomechanisms of autoantibody-driven diseases. Current treatments of pemphigoid diseases largely rely on systemic immunosuppression despite the restriction of inflammation to the skin. The iatrogenic immunosuppression may contribute to the high 1-year mortality rate in pemphigoid diseases. Thus, the overall survival does not depend on the extent of skin lesions before treatment, but depends on whether topical or systemic glucocorticoid application is initially used. Systemic glucocorticoid application is linked to higher mortality than topical application, illustrating that systemic immunosuppression can harm the overall prognosis and that immunomodulatory treatment strategies are required. New mouse models of pemphigoid diseases have been instrumental in approaching the introduction of novel immunomodulatory therapies in the treatment of autoimmune diseases.

Perlecan expression influences the keratin 15-positive cell population fate in the epidermis of aging skin.

The epidermis is continuously renewed by stem cell proliferation and differentiation. Basal keratinocytes append the dermal‐epidermal junction, a cell surface‐associated, extracellular matrix that provides structural support and influences their behaviour. It consists of laminins, type IV collagen, nidogens, and perlecan, which are necessary for tissue organization and structural integrity. Perlecan is a heparan sulfate proteoglycan known to be involved in keratinocyte survival and differentiation. Aging affects the dermal epidermal junction resulting in decreased contact with keratinocytes, thus impacting epidermal renewal and homeostasis. We found that perlecan expression decreased during chronological skin aging. Our in vitro studies revealed reduced perlecan transcript levels in aged keratinocytes. The production of in vitro skin models revealed that aged keratinocytes formed a thin and poorly organized epidermis. Supplementing these models with purified perlecan reversed the phenomenon allowing restoration of a well‐differentiated multi‐layered epithelium. Perlecan down‐regulation in cultured keratinocytes caused depletion of the cell population that expressed keratin 15. This phenomenon depended on the perlecan heparan sulphate moieties, which suggested the involvement of a growth factor. Finally, we found defects in keratin 15 expression in the epidermis of aging skin. This study highlighted a new role for perlecan in maintaining the self‐renewal capacity of basal keratinocytes.

Infection of Maize by Clavibacter michiganensis subsp. nebraskensis Does Not Require Severe Wounding.

Goss's bacterial wilt and leaf blight of maize is caused by Clavibacter michiganensis subsp. nebraskensis. Infested residue is the primary source of inoculum and infection occurs via wounds caused by sand blasting, hail, or wind damage. The pathogen survives as an epiphyte on maize leaves and, because the disease has been observed on plants in the field with no obvious wounding, we wondered whether infection by epiphytic C. michiganensis subsp. nebraskensis and disease development could occur in the absence of severe wounding. Consequently, greenhouse experiments were done to evaluate disease development in the absence of wounding in ambient and increased humidity conditions. Maize plants at the V4 to V5 crop development stage were spray inoculated with a suspension of C. michiganensis subsp. nebraskensis (108 cells ml-1). Leaf blight incidence was assessed on whole plants and individual leaves; epiphytic populations of C. michiganensis subsp. nebraskensis were monitored by dilution plating of leaf washes; and epiphytic C. michiganensis subsp. nebraskensis colonization was visualized using scanning electron microscopy (SEM). Goss's leaf blight symptoms were observed on nonwounded plants in ambient (37.0% plant incidence) and increased humidity conditions (60.0% plant incidence). Populations of epiphytic C. michiganensis subsp. nebraskensis survived and increased on maize leaves, particularly at increased humidity. We observed C. michiganensis subsp. nebraskensis colonizing maize leaves in localized sites that included epidermal junctions, cuticle depressions, in and around stomata, and at the base of trichomes. Single cells and aggregates of C. michiganensis subsp. nebraskensis were observed within substomatal chambers using SEM. These data indicate that severe wounding is not necessary for C. michiganensis subsp. nebraskensis infection of maize, and stomata or trichomes may serve as entry points for the bacterium.

Non-centrosomal epidermal microtubules act in parallel to LET-502/ROCK to promote C. elegans elongation

C. elegans embryonic elongation is a morphogenetic event driven by actomyosin contractility and muscle-induced tension transmitted through hemidesmosomes. A role for the microtubule cytoskeleton has also been proposed, but its contribution remains poorly characterized. Here, we investigate the organization of the non-centrosomal microtubule arrays present in the epidermis and assess their function in elongation. We show that the microtubule regulators γ-tubulin and NOCA-1 are recruited to hemidesmosomes and adherens junctions early in elongation. Several parallel approaches suggest that microtubule nucleation occurs from these sites. Disrupting the epidermal microtubule array by overexpressing the microtubule-severing protein Spastin or by inhibiting the C. elegans ninein homolog NOCA-1 in the epidermis mildly affected elongation. However, microtubules were essential for elongation when hemidesmosomes or the activity of the Rho kinase LET-502/ROCK were partially compromised. Imaging of junctional components and genetic analyses suggest that epidermal microtubules function together with Rho kinase to promote the transport of E-cadherin to adherens junctions and myotactin to hemidesmosomes. Our results indicate that the role of LET-502 in junctional remodeling is likely to be independent of its established function as a myosin II activator, but requires a microtubule-dependent pathway involving the syntaxin SYX-5. Hence, we propose that non-centrosomal microtubules organized by epidermal junctions contribute to elongation by transporting junction remodeling factors, rather than having a mechanical role.

EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions.

EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions.

Ptbp1 and Exosc9 knockdowns trigger skin stability defects through different pathways

In humans, genetic diseases affecting skin integrity (genodermatoses) are generally caused by mutations in a small number of genes that encode structural components of the dermal–epidermal junctions. In this article, we first show that inactivation of both exosc9, which encodes a component of the RNA exosome, and ptbp1, which encodes an RNA-binding protein abundant in Xenopus embryonic skin, impairs embryonic Xenopus skin development, with the appearance of dorsal blisters along the anterior part of the fin. However, histological and electron microscopy analyses revealed that the two phenotypes are distinct. Exosc9 morphants are characterized by an increase in the apical surface of the goblet cells, loss of adhesion between the sensorial and peridermal layers, and a decrease in the number of ciliated cells within the blisters. Ptbp1 morphants are characterized by an altered goblet cell morphology. Gene expression profiling by deep RNA sequencing showed that the expression of epidermal and genodermatosis-related genes is also differentially affected in the two morphants, indicating that alterations in post-transcriptional regulations can lead to skin developmental defects through different routes. Therefore, the developing larval epidermis of Xenopus will prove to be a useful model for dissecting the post-transcriptional regulatory network involved in skin development and stability with significant implications for human diseases.

Unusual presentation of a distal cholangiocarcinoma

We report a case of a 51-year-old male presented with abdominal pain and itching, whereas jaundice occurred afterwards. Diagnosis was made by means of CT and better assessed by MRI and endoscopic ultrasounds. Patient was treated with an open Whipple's procedure, and after an 18-month follow-up he was doing well.