Keratinocyte Adhesion Research Articles

Integrin-mediated regulation of epidermal wound functions.

During cutaneous wound healing, keratinocyte proliferation and migration are critical for re-epithelialization. In addition the epidermis secretes growth factors, cytokines, proteases, and matricellular proteins into the wound microenvironment that modify the extracellular matrix and stimulate other wound cells that control the inflammatory response, promote angiogenesis and facilitate tissue contraction and remodeling. Wound keratinocytes express at least seven different integrins-the major cell adhesion receptors for the extracellular matrix-that collectively control essential cell-autonomous functions to ensure proper re-epithelialization, including migration, proliferation, survival and basement membrane assembly. Moreover, it has become evident in recent years that some integrins can regulate paracrine signals from wound epidermis that stimulate other wound cells involved in angiogenesis, contraction and inflammation. Importantly, it is likely that abnormal integrin expression or function in the epidermis contributes to wound pathologies such as over-exuberant healing (e.g., hypertrophic scar formation) or diminished healing (e.g., chronic wounds). In this review, we discuss current knowledge of integrin function in the epidermis, which implicates them as attractive therapeutic targets to promote wound healing or treat wound pathologies. We also discuss challenges that arise from the complex roles that multiple integrins play in wound epidermis, which may be regulated through extracellular matrix remodeling that determines ligand availability. Indeed, understanding how different integrin functions are temporally coordinated in wound epidermis and which integrin functions go awry in pathological wounds, will be important to determine how best to target them clinically to achieve maximum therapeutic benefit. Graphical abstract In addition to their well-characterized roles in keratinocyte adhesion, migration and wound re-epithelialization, epidermal integrins play important roles in modifying the wound microenvironment by regulating the expression and secretion of growth factors, extracellular proteases, and matricellular proteins that stimulate other wound cells, including vascular endothelial cells and fibroblasts/myofibroblasts.

Nanofibrous asymmetric membranes self-organized from chemically heterogeneous electrospun mats for skin tissue engineering

Asymmetric membranes, which mimic the structure and functions of human skin, have been extensively pursued as ideal skin tissue engineering constructs. In this study, we demonstrated that nanostructured asymmetric membranes can be prepared by the self-organization of chemically heterogeneous bilayer electrospun membranes in aqueous solutions. Structurally, the skin layer consists of hydrophobic β-glucan butyrate nanofibers and its inner layer consists of hydrophilic β-glucan acetate nanofibers. After the electrospinning process, both of the layers are in a dense state. When placed in water, the skin layer absorbs little water and still remains dense, but the fibers in the inner layer become extensively hydrated and spontaneously reorganize into a fully stretched structure, resulting in a significant volume increase and a density decrease of the inner layer. SEM imaging showed that β-glucan ester nanofibers exhibited a bead-free and uniform structure. Contact angle measurement and swelling tests showed that the inner layer was highly hydrophilic with extensive swelling, but the skin layer was highly hydrophobic with little swelling. Mechanical tests indicated that the nanofibrous asymmetric membranes had good mechanical properties in both the dry and wet states. In vitro cytocompatibility tests showed that nanofibrous asymmetric membranes could promote the adhesion and proliferation of fibroblasts and keratinocytes. A preliminary in vivo study performed on a full thickness mouse skin wound model demonstrated that the nanofibrous asymmetric membranes significantly accelerated the wound healing process by promoting re-epithelialization, tissue remodeling and collagen deposition. Taken together, our study provides a novel model for the design and fabrication of nanostructured asymmetric membranes, and our β-glucan based nanofibrous asymmetric membranes could be used as an advanced platform for skin tissue engineering.

Accumulation of C-reactive protein in basal keratinocytes of normal skins

BackgroundC-reactive protein (CRP) is a prototypic acute phase protein which increases dramatically in the blood during the first 48h of tissue inflammation and has been recognized as a risk factor for atherosclerosis. CRP interacts with a variety of proteins. ObjectiveTo know the role of accumulated CRP in the skin. MethodsInteraction of CRP with basal keratinocytes was studied using immunohistochemical method and keratinocyte culture system. ResultsWe found an immunohistochemical deposition of CRP on the basal keratinocyte membrane in some normal human skins (23 out of 46 skins). When added to cultured keratinocytes, heat-denatured but not native CRP was found to adhere to keratinocyte cell membrane after 1h, then internalized into cytoplasm after 24h. The heat-denatured CRP recognized at least four keratinocyte polypeptides with the molecular weights of 56, 42, 32 and 24kDa. Ligand binding assays suggested that multiple populations of receptor-ligand interactions were involved in the binding between CRP and keratinocyte. Cultured dermal microvascular endothelial cells were found to express CRP of which expression was greatly induced by interleukin-1β (IL-1β) treatment, suggesting that the deposited CRP in the basal keratinocytes can be derived from local dermal microvasculatures as well as from systemic circulation (serum). Treatment of cultured keratinocytes with heat-denatured CRP induced interleukin-8 (IL-8) expression, a potent leukocyte chemotactic cytokine. CRP in the medium (liquid phase) and CRP-coated dishes (solid phase) both inhibited the adhesion of keratinocytes in culture. ConclusionAccumulation of CRP may regulate the skin inflammation and keratinocyte proliferation by modulating keratinocyte cytokine expression and adhesion to substrate.

Increased Frequency of T Follicular Helper Cells and Elevated Interleukin-27 Plasma Levels in Patients with Pemphigus.

Pemphigus is an autoimmune disease in which IgG auto-antibodies (auto-ab) against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 cause loss of epidermal keratinocyte adhesion. Aim of this study was to investigate cytokines derived from antigen-presenting cells (APC) and their relation to CD4+ T cell subpopulations and to the auto-ab response in pemphigus. In this regard, patients with pemphigus were compared to patients with myasthenia gravis (MG), an unrelated auto-ab–mediated autoimmune disease, and healthy controls. In pemphigus and MG, the plasma concentrations of the APC-derived immunomodulatory cytokine IL-27 were highly increased. Strikingly, IL-27 strongly correlated with Dsg-specific IgG auto-ab titers. T helper (Th) 17 cells were augmented in both pemphigus and MG patients while T follicular helper (Tfh) cells, which are essential in providing B cell help, were increased only in pemphigus along with increasing plasma concentrations of IL-21, a cytokine produced by Th17 and Tfh cells. Moreover, we could detect Dsg3-specific autoreactive T cells producing IL-21 upon ex vivo stimulation with Dsg3. These findings suggest that IL-27 and IL-21-producing T cells, are involved in the pathogenesis of pemphigus. The further characterization of IL-21-producing T cells and of the role of IL-27 will lead to a more defined understanding of the auto-ab response in pemphigus.

Biomimetic interconnected porous keratin–fibrin–gelatin 3D sponge for tissue engineering application

The medicated wound dressing material with highly interconnected pores, mimicking the function of the extracellular matrix was fabricated for the promotion of cell growth. In this study, keratin (K), fibrin (F) and gelatin (G) composite scaffold (KFG-SPG) was fabricated by freeze drying technique and the mupirocin (D) drug was successfully incorporated with KFG-SPG (KFG-SPG-D) intended for tissue engineering applications. The fabrication of scaffold was performed without the use of any strong chemical solvents, and the solid sponge scaffold was obtained with well interconnected pores. The porous morphology of the scaffold was confirmed by SEM analysis and exhibited competent mechanical properties. KFG-SPG and KFG-SPG-D possess high level of biocompatibility, cell proliferation and cell adhesion of NIH 3T3 fibroblast and human keratinocytes (HaCaT) cell lines thereby indicating the scaffolds potential as a suitable medicated dressing for wound healing.

Comparative transcriptomic profiling of hydrogen peroxide signaling networks in zebrafish and human keratinocytes: Implications toward conservation, migration and wound healing.

Skin wounds need to be repaired rapidly after injury to restore proper skin barrier function. Hydrogen peroxide (H2O2) is a conserved signaling factor that has been shown to promote a variety of skin wound repair processes, including immune cell migration, angiogenesis and sensory axon repair. Despite growing research on H2O2 functions in wound repair, the downstream signaling pathways activated by this reactive oxygen species in the context of injury remain largely unknown. The goal of this study was to provide a comprehensive analysis of gene expression changes in the epidermis upon exposure to H2O2 concentrations known to promote wound repair. Comparative transcriptome analysis using RNA-seq data from larval zebrafish and previously reported microarray data from a human epidermal keratinocyte line shows that H2O2 activates conserved cell migration, adhesion, cytoprotective and anti-apoptotic programs in both zebrafish and human keratinocytes. Further assessment of expression characteristics and signaling pathways revealed the activation of three major H2O2–dependent pathways, EGF, FOXO1, and IKKα. This study expands on our current understanding of the clinical potential of low-level H2O2 for the promotion of epidermal wound repair and provides potential candidates in the treatment of wound healing deficits.

Aquaporin 3 and E-Cadherin Expression in Perilesional Vitiligo Skin.

Vitiligo is a common dermatologic disorder with debated aetiology. Most studies focused on role of melanocytes and few investigated the role of keratinocytes in pathogenesis of the disease. To investigate the keratinocyte adhesion in perilesional vitiligo skin through the immunolocalization of Aquaporin-3 (AQP3) and E-cadherin. Sixty five subjects were selected. These included 40 cases with vitiligo and 25 age and gender-matched healthy subjects as a control group. Skin biopsies were taken from perilesional skin of cases and from site-matched areas of control subjects. The expression of AQP3 and E-cadherin was evaluated by immunohistochemical techniques. Results were statistically analysed using IBM personal computer and the statistical package SPSS version 11. Fisher-exact and Chi-square tests were used to study the association between two qualitative variables. Mann-Whitney test was used for comparison between quantitative variables not normally distributed. Spearman's correlation coefficient was used to assess correlation between two quantitative variables. The p≤0.05 was considered significant. Regarding AQP3 expression, strong intensity, diffuse distribution, higher percent of expression and higher H-score (p<0.001 for all) were significantly associated with control skin compared with perilesional skin in follicular and inter-follicular epidermis. Regarding E-cadherin expression, moderate intensity, higher percent of expression and higher H- score (p<0.001 for all) were significantly associated with control skin compared with perilesional skin in follicular and inter-follicular epidermis. No significant association was found between E-cadherin and AQP3 H-scores or percent of expression and clinical data of selected cases. No significant correlation was detected between E-cadherin and AQP3 H-scores or percent of expression and age of cases, disease duration or Vitiligo Disease Activity (VIDA) score. The following sequence of events can be suggested for vitiligo pathogenesis, based on findings in perilesional skin: AQP3 is downregulated by a primary unknown factor and this will lead to down regulation of its downstream molecules, mainly phosphatidylinositol 3-kinase, E-cadherin and catenins, which is followed by defective keratinocyte adhesion and decreased release of keratinocyte-derived growth factors. Subsequently a secondary event, physical trauma, oxidative stress or autoantibodies, may lead to exfoliation of keratinocytes and pigmented cells.

Homozygous Deletion Mutation of the FERMT1 Gene in a Chinese Patient with Kindler Syndrome.

Dear Editor: Kindler syndrome (KS, OMIM173650) is a rare autosomal recessive genodermatosis characterized by trauma-induced skin blistering, cutaneous atrophy, progressive poikiloderma and photosensitivity1. KS is caused by mutation in the FERMT1 gene1. This gene encodes kindlin-1, which regulates not only keratinocyte adhesion through β1-class integrins but also proliferation and differentiation of cutaneous stem cells by promoting αvβ6 integrin–mediated transforming growth factor-β activation and inhibiting Wnt ligand expression2. A 12-year-old Chinese boy presented with recurrent blistering after mild trauma from the age of 1 year. There is no family history. Physical examination revealed generalized skin atrophy with some scattered resolving blisters on the extremities and poikiloderma on the face and neck (Fig. 1). He has also suffered from pruritus and photosensitivity. A skin biopsy from non-blistered area on his leg revealed hyperkeratosis and epidermal atrophy (Fig. 2A). Immunofluorescence (IF) mapping of a blister showed that keratins 5 and 14 were localized at the roof of the blister while laminin 5 and type IV collagen were localized at the base (Fig. 2B, C). Electron microscopic findings of non-blistered skin revealed widening of the lamina lucida and reduplication of the lamina densa (Fig. 2D). DNA extracted from the patient's blood was processed for direct nucleotide sequencing and we detected a homozygous dinucleotide deletion mutation at c.994_995delCA in exon 8. The mutation causes frameshift and a premature stop codon (Fig. 2E). Based on these results, we diagnosed the patient with KS. Fig. 1 Clinical features of the patient. (A, B) Poikiloderma on the face and neck. (C) Atrophic change on the dorsal aspect of hand. (D) Skin atrophy with resolving blisters on the leg. Fig. 2 (A) Histological examination. Hyperkeratosis and epidermal atrophy (H&E, ×400). (B, C) Immunofluorescence mapping shows separation between keratin 5 (KRT5) and type IV collagen (COL4). KRT5 is localized at the roof of the blister (B) while ... KS usually manifests at birth with trauma-induced skin blistering that is more prominent on the extremities and tends to regress with age3. However, as patients age, skin atrophy localized to the dorsal aspects of the hands and feet become generalized and progressive skin poikiloderma manifests. Most patients also show photosensitivity3. Although our patient had no history of mucosal lesions, mucosal involvement is common1. Histopathology and IF mapping of a blister are not informative for diagnosis because the plane of cleavage can be variable at the level of the basement membrane zone1. Transmission electron microscopy of the non-blistered skin shows extensive reduplication of the lamina densa which is a characteristic diagnostic feature of KS4. The diagnostic golden standard is sequencing of the FERMT1 gene1. To date, more than 60 FERMT1 gene mutations and 150 KS patients have been reported and one Chinese and 4 Japanese patients have been reported in Eastern Asia5 (https://grenada.lumc.nl/LOVD2/mendelian_genes/home.php?select_db=FERMT1). Most mutations cause premature termination of translation, therefore loss of kindlin-1 function and most patients are related with consanguineous marriages3. Recently, Youssefian et al.4 first reported the c.994_995delCA mutation in a 19-year-old Iranian female patient. Interestingly, that patient had no mucosal involvement except dental plaques. This may imply an association between the mutation and mucosal-sparing features. Has et al.3 suggested genotype-phenotype correlations in KS because some patients with a missense mutation or in-frame deletion demonstrate a mild phenotype. However, only two cases including ours have been reported to date and our patient might be too young to have developed mucosal lesions. Further studies are needed to clarify this association.

Is Endoplasmic Reticulum Stresslinked to the Pathogenesis of Pemphigus Vulgaris?

Pemphigus vulgaris (PV) is anorgan-specific autoimmune blistering disease, affecting the skin and the mucous membranes. Despite the breakthroughs in therapy for advanced disease in the recent years, the management of PV remains challenging with poor prognosis and limited therapeutic options.Cloning of cDNAs encoding pemphigus antigens has provided ample evidence that IgG autoantibodies recognize the desmogleins (DSGs) that are found in the adhering junctions, the desmosomes. Binding of such IgGs in the DSGs results in the weakeningof intercellular adhesion of keratinocytes in the upper part of the epidermis and eventually to acantholysis. Desmosomes have instrumental roleas a protective barrier in maintaining the integrity and function of the epidermis and the mucous epithelia.Mechanistically, the production of IgG autoantibodies against DSG1 and DSG3 is linked to disease pathogenesis. Recently,endoplasmic reticulum (ER) stress,a highly conserved cellular stress response,has been proposed to play a role in the development and progression of PV. ER stress triggers the activation ofseveral intracellular signaling pathwaysthat collectively constitute the unfolded protein response (UPR) thatinitially aims to restore homeostaticbalance while subsequently becomes proapoptotic. The discovery that ER stress occurs during PV development implies that deregulated UPR may contribute to the pathogenesis of the disease. Future studies should be directed toward understanding how modulation of the ER can provide new therapeutic possibilities for the treatment of PV patients.Key Words:Pemphigus Vulgaris (PV), desmogleins (DSGs), Endoplasmic Reticulum (ER) stress ER-stress, Unfolded protein response (UPR), C/EBP-homologous protein (CHOP), autoimmune disease

ADF and Cofilin1 Control Actin Stress Fibers, Nuclear Integrity, and Cell Survival.

SummaryGenetic co-depletion of the actin-severing proteins ADF and CFL1 triggers catastrophic loss of adult homeostasis in multiple tissues. There is impaired cell-cell adhesion in skin keratinocytes with dysregulation of E-cadherin, hyperproliferation of differentiated cells, and ultimately apoptosis. Mechanistically, the primary consequence of depleting both ADF and CFL1 is uncontrolled accumulation of contractile actin stress fibers associated with enlarged focal adhesions at the plasma membrane, as well as reduced rates of membrane protrusions. This generates increased intracellular acto-myosin tension that promotes nuclear deformation and physical disruption of the nuclear lamina via the LINC complex that normally connects regulated actin filaments to the nuclear envelope. We therefore describe a pathway involving the actin-severing proteins ADF and CFL1 in regulating the dynamic turnover of contractile actin stress fibers, and this is vital to prevent the nucleus from being damaged by actin contractility, in turn preserving cell survival and tissue homeostasis.

Distinct Impact of Two Keratin Mutations Causing Epidermolysis Bullosa Simplex on Keratinocyte Adhesion and Stiffness

Keratin filaments constitute the major component of the epidermal cytoskeleton from heterodimers of type I and type II keratin subunits. Missense mutations in keratin 5 or keratin 14, highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS) in humans by rendering the keratin cytoskeleton sensitive to mechanical stress; yet, the mechanisms by which individual mutations cause cell fragility are incompletely understood. Here, we compared the K14p.Arg125Pro with the K5p.Glu477Asp mutation, both giving rise to severe generalized EBS, by stable expression in keratin-free keratinocytes. This revealed distinctly different effects on keratin cytoskeletal organization, in agreement with in vivo observations, thus validating the cell system. Although the K14p.Arg125Pro mutation led to impaired desmosomes, downregulation of desmosomal proteins, and weakened epithelial sheet integrity upon shear stress, the K5p.Glu477Asp mutation did not impair these functions, although causing EBS with squamous cell carcinoma in vivo. Atomic force microscopy demonstrated that K14 mutant cells were even less resistant against deformation compared with keratin-free keratinocytes. Thus, a keratin mutation causing EBS compromises cell stiffness to a greater extent than the lack of keratins. Finally, re-expression of K14 in K14 mutant cells did not rescue the above defects. Collectively, our findings have implications for EBS therapy approaches.

Critical Role of the Neonatal Fc Receptor (FcRn) in the Pathogenic Action of Antimitochondrial Autoantibodies Synergizing with Anti-desmoglein Autoantibodies in Pemphigus Vulgaris

Pemphigus vulgaris (PV) is a life-long, potentially fatal IgG autoantibody-mediated blistering disease targeting mucocutaneous keratinocytes (KCs). PV patients develop pathogenic anti-desmoglein (Dsg) 3 ± 1 and antimitochondrial antibodies (AMA), but it remained unknown whether and how AMA enter KCs and why other cell types are not affected in PV. Therefore, we sought to elucidate mechanisms of cell entry, trafficking, and pathogenic action of AMA in PV. We found that PVIgGs associated with neonatal Fc receptor (FcRn) on the cell membrane, and the PVIgG-FcRn complexes entered KCs and reached mitochondria where they dissociated. The liberated AMA altered mitochondrial membrane potential, respiration, and ATP production and induced cytochrome c release, although the lack or inactivation of FcRn abolished the ability of PVIgG to reach and damage mitochondria and to cause detachment of KCs. The assays of mitochondrial functions and keratinocyte adhesion demonstrated that although the pathobiological effects of AMA on KCs are reversible, they become irreversible, leading to epidermal blistering (acantholysis), when AMA synergize with anti-Dsg antibodies. Thus, it appears that AMA enter a keratinocyte in a complex with FcRn, become liberated from the endosome in the cytosol, and are trafficked to the mitochondria, wherein they trigger pro-apoptotic events leading to shrinkage of basal KCs uniquely expressing FcRn in epidermis. During recovery, KCs extend their cytoplasmic aprons toward neighboring cells, but anti-Dsg antibodies prevent assembly of nascent desmosomes due to steric hindrance, thus rendering acantholysis irreversible. In conclusion, FcRn is a common acceptor protein for internalization of AMA and, perhaps, for PV autoantibodies to other intracellular antigens, and PV is a novel disease paradigm for investigating and elucidating the role of FcRn in this autoimmune disease and possibly other autoimmune diseases.

Abstract 2380: Integrin αv is necessary for skin tissue generation and SCC tumor invasion, but dispensable for tissue maintenance

Abstract Integrin heterodimers play crucial roles in epithelial adhesion, proliferation, differentiation, wound healing and tumorigenesis. However, the effects of acute loss of many of the 26 integrin subunits in epidermis have not been determined. This information is critical since many inhibitors for specific integrin heterodimers are entering clinical trials for various cancers. We performed an shRNA-based multiplexed screen to define roles for each integrin subunit in a 3-dimensional in vitro organotypic skin model. In this screen, we transduced individual populations of human keratinocytes with shRNAs against each integrin subunit. We then quantitatively determined the fitness of each cell population in a competition assay in reconstituted human skin. This screen identified the integrin αv class of heterodimers as essential for generation of human skin tissue: the population of keratinocytes lacking αv was selected against 125-fold compared to non-silencing control cells (p&amp;lt;0.001). An identical screen in human melanocytes showed no change in fitness upon loss of integrin αv, indicating cell-intrinsic functions for this subunit. Loss of integrin αv alone in keratinocytes leads to cell cycle arrest with a 4-5 fold increase in the G1/S ratio. Surprisingly, we found that integrin αv is not localized within focal adhesions in human keratinocytes and does not control focal adhesion dynamics in this cell type. Instead, it forms an independent signaling complex at the cellular membrane that controls keratinocyte growth through a FAK c-myc signaling axis. Both FAK and c-myc total protein levels are lost upon αv knock down, and re-expression of c-myc in αv-null keratinocytes is sufficient to completely rescue the cell cycle defects and skin tissue phenotypes. The effects of integrin αv loss are dependent on αv's binding partners β5 and β6, but not β1 or β8. Using a doxycycline-inducible system, we show that αv loss plays a crucial role in tissue generation and tumor invasion, but is not necessary for epidermal tissue maintenance. Furthermore, both β5 and β6 binding partners are required for tumor invasion, thus identifying αvβ5 and αvβ6 as therapeutic targets for metastatic squamous cell carcinoma. Citation Format: Elizabeth K. Duperret, Ankit Dahal, Todd W. Ridky. Integrin αv is necessary for skin tissue generation and SCC tumor invasion, but dispensable for tissue maintenance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2380. doi:10.1158/1538-7445.AM2015-2380

Fibroblast-specific upregulation of Flightless I impairs wound healing.

The cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell-specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue-specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast-specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue-specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non-inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non-tissue-specific upregulation of Flii suggesting that fibroblast-derived Flii may have an important role in the wound healing response.

E-cadherin and Src associate with extradesmosomal Dsg3 and modulate desmosome assembly and adhesion.

Desmosomes provide strong intercellular cohesion essential for the integrity of cells and tissues exposed to continuous mechanical stress. For desmosome assembly, constitutively synthesized desmosomal cadherins translocate to the cell-cell border, cluster and mature in the presence of Ca(2+) to stable cell contacts. As adherens junctions precede the formation of desmosomes, we investigated in this study the relationship between the classical cadherin E-cadherin and the desmosomal cadherin Desmoglein 3 (Dsg3), the latter of which is indispensable for cell-cell adhesion in keratinocytes. By using autoantibodies from patients with the blistering skin disease pemphigus vulgaris (PV), we showed in loss of function studies that E-cadherin compensates for effects of desmosomal disassembly. Overexpression of E-cadherin reduced the loss of cell cohesion induced by PV autoantibodies and attenuated activation of p38 MAPK. Silencing of E-cadherin abolished the localization of Dsg3 at the membrane and resulted in a shift of Dsg3 from the cytoskeletal to the non-cytoskeletal protein pool which conforms to the notion that E-cadherin regulates desmosome assembly. Mechanistically, we identified a complex consisting of extradesmosomal Dsg3, E-cadherin, β-catenin and Src and that the stability of this complex is regulated by Src. Moreover, Dsg3 and E-cadherin are phosphorylated on tyrosine residues in a Src-dependent manner and Src activity is required for recruiting Dsg3 to the cytoskeletal pool as well as for desmosome maturation towards a Ca(2+)-insensitive state. Our data provide new insights into the role of E-cadherin and the contribution of Src signaling for formation and maintenance of desmosomal junctions.

Hailey-Hailey disease: A fold (intertriginous) dermatosis

Hailey-Hailey disease, also called benign familial pemphigus, is a late-onset blistering disorder that affects the flexures. There are typically painful erosions and cracks in affected areas. Lesions generally begin between 20 and 40 years of age. In two third of all cases, positive family history is detected. In pathogenesis, there is a defect in keratinocyte adhesion due to ATP2 C1 gene mutation. The result of the desmosomal decomposition is acantholysis. Menstruation, pregnancy, skin infections, physical trauma, excessive sweating and exposure to ultraviolet radiation are important triggering factors. Histopathologic changes are suprabasal acantholysis and formation of intraepidermal bullae. In the epidermis, a partial acantholysis that looks like broken bricks is observed.

Alpha Actinin-1 Regulates Cell-Matrix Adhesion Organization in Keratinocytes: Consequences for Skin Cell Motility

The migration of keratinocytes in wound healing requires coordinated activities of the motility machinery of a cell, the cytoskeleton and matrix adhesions. In this study we assessed the role of alpha actinin-1 (ACTN1), one of the two alpha actinin isoforms expressed in keratinocytes, in skin cell migration via an shRNA-mediated knockdown approach. Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity and impaired lamellipodial dynamics. They also display aberrant directed motility and move slower than their wild-type counterparts. Moreover, they have abnormally arranged matrix adhesion sites. Specifically, the focal adhesions in ACTN1 knockdown keratinocytes are not organized as distinct entities. Rather, focal adhesion proteins are arranged in a circle subjacent to cortical fibers of actin. In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells and β4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes. In summary, our data suggest a mechanism by which ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion and hemidesmosome proteins complexes, thereby modulating cell speed, lamellipodial dynamics and directed migration.

Nuclear receptor peroxisome proliferator activated receptor (PPAR) β/δ in skin wound healing and cancer.

We review the functions of peroxisome proliferator activated receptor (PPAR) β/δ in skin wound healing and cancer. In particular, we highlight the roles of PPARβ/δ in inhibiting keratinocyte apoptosis at wound edges via activation of the PI3K/PKBα/Akt1 pathway and its role during re-epithelialization in regulating keratinocyte adhesion and migration. In fibroblasts, PPARβ/δ controls IL-1 signalling and thereby contributes to the homeostatic control of keratinocyte proliferation. We discuss its therapeutic potential for treating diabetic wounds and inflammatory skin diseases such as psoriasis and acne vulgaris. PPARβ/δ is classified as a tumour growth modifier; it is activated by chronic low-grade inflammation, which promotes the production of lipids that, in turn, enhance PPARβ/δ transcription activity. Our earlier work unveiled a cascade of events triggered by PPARβ/δ that involve the oncogene Src, which promotes ultraviolet-induced skin cancer in mice via enhanced EGFR/Erk1/2 signalling and the expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, PPARβ/δ expression is correlated with the expression of SRC and EMT markers in human skin squamous cell carcinoma. Furthermore, there is a positive interaction between PPARβ/δ, SRC, and TGFβ1 at the transcriptional level in various human epithelial cancers. Taken together, these observations suggest the need for evaluating PPARβ/δ modulators that attenuate or increase its activity, depending on the therapeutic target.

Manganese Redistribution by Calcium-stimulated Vesicle Trafficking Bypasses the Need for P-type ATPase Function

Regulation of intracellular ion homeostasis is essential for eukaryotic cell physiology. An example is provided by loss of ATP2C1 function, which leads to skin ulceration, improper keratinocyte adhesion, and cancer formation in Hailey-Hailey patients. The yeast ATP2C1 orthologue PMR1 codes for a Mn(2+)/Ca(2+) transporter that is crucial for cis-Golgi manganese supply. Here, we present evidence that calcium overcomes the lack of Pmr1 through vesicle trafficking-stimulated manganese delivery and requires the endoplasmic reticulum Mn(2+) transporter Spf1 and the late endosome/trans-Golgi Nramp metal transporter Smf2. Smf2 co-localizes with the putative Mn(2+) transporter Atx2, and ATX2 overexpression counteracts the beneficial impact of calcium treatment. Our findings suggest that vesicle trafficking promotes organelle-specific ion interchange and cytoplasmic metal detoxification independent of calcineurin signaling or metal transporter re-localization. Our study identifies an alternative mode for cis-Golgi manganese supply in yeast and provides new perspectives for Hailey-Hailey disease treatment.

Loss-of-Function Mutations in CAST Cause Peeling Skin, Leukonychia, Acral Punctate Keratoses, Cheilitis, and Knuckle Pads

Calpastatin is an endogenous specific inhibitor of calpain, a calcium-dependent cysteine protease. Here we show that loss-of-function mutations in calpastatin (CAST) are the genetic causes of an autosomal-recessive condition characterized by generalized peeling skin, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads, which we propose to be given the acronym PLACK syndrome. In affected individuals with PLACK syndrome from three families of different ethnicities, we identified homozygous mutations (c.607dup, c.424A>T, and c.1750delG) in CAST, all of which were predicted to encode truncated proteins (p.Ile203Asnfs∗8, p.Lys142∗, and p.Val584Trpfs∗37). Immunohistochemistry shows that staining of calpastatin is reduced in skin from affected individuals. Transmission electron microscopy revealed widening of intercellular spaces with chromatin condensation and margination in the upper stratum spinosum in lesional skin, suggesting impaired intercellular adhesion as well as keratinocyte apoptosis. A significant increase of apoptotic keratinocytes was also observed in TUNEL assays. In vitro studies utilizing siRNA-mediated CAST knockdown revealed a role for calpastatin in keratinocyte adhesion. In summary, we describe PLACK syndrome, as a clinical entity of defective epidermal adhesion, caused by loss-of-function mutations in CAST.