Proteins In Keratinocytes Research Articles

Thermostable Proteins from HaCaT Keratinocytes Identify a Wide Breadth of Intrinsically Disordered Proteins and Candidates for Liquid-Liquid Phase Separation.

Intrinsically disordered proteins (IDPs) move through an ensemble of conformations which allows multitudinous roles within a cell. Keratinocytes, the predominant cell type in mammalian epidermis, have had only a few individual proteins assessed for intrinsic disorder and its possible contribution to liquid-liquid phase separation (LLPS), especially in regard to what functions or structures these proteins provide. We took a holistic approach to keratinocyte IDPs starting with enrichment via the isolation of thermostable proteins. The keratinocyte protein involucrin, known for its resistance to heat denaturation, served as a marker. It and other thermostable proteins were identified by liquid chromatography tandem mass spectrometry and subjected to extensive bioinformatic analysis covering gene ontology, intrinsic disorder, and potential for LLPS. Numerous proteins unique to keratinocytes and other proteins with shared expression in multiple cell types were identified to have IDP traits (e.g., compositional bias, nucleic acid binding, and repeat motifs). Among keratinocyte-specific proteins, many that co-assemble with involucrin into the cell-specific structure known as the cornified envelope scored highly for intrinsic disorder and potential for LLPS. This suggests intrinsic disorder and LLPS are previously unrecognized traits for assembly of the cornified envelope, echoing the contribution of intrinsic disorder and LLPS to more widely encountered features such as stress granules and PML bodies.

Anti-psoriasis activities of hydroxytyrosol on HaCaT cells under psoriatic inflammation in vitro

Background Psoriasis is a prevalent chronic inflammatory dermatosis, which can significantly impact life quality of patients. The treatment of psoriasis is no cure and novel therapeutic options are urgently needed. Hydroxytyrosol (HT) possesses multiple biological activities, such as anti-inflammatory and anti-proliferation properties, suggesting its potential to counteract hallmarks of psoriasis. However, its role in the regulation of psoriasis remains unknown. Objective In the current study, we explored the anti-proliferative activity and anti-inflammatory responses of HT in psoriatic keratinocytes in vitro. Methods We used M5 cytokines cocktail, which includes tumor necrosis factor (TNF)-α, oncostatin-M, interleukin (IL)-17A, IL-1α, and IL-22, to simulate HaCaT cells to establish the cell model of psoriasis and explore the effects of HT on psoriasis in vitro. Results This study showed that HT exerted potent anti-inflammatory effect via influencing the expression of IL-6, IL-8, and TNF-α in M5-induced cell model of psoriasis. Moreover, it suppressed the expression of antimicrobial proteins in psoriatic keratinocytes. Additionally, it inhibited cell proliferation in psoriasis cell model. Conclusions Altogether, our results suggested that HT has anti-psoriasis effects in vitro and HT may be a promising therapeutic agent in psoriasis treatment.

The Transcriptional Regulation of Genes Involved in the Immune Innate Response of Keratinocytes Co-Cultured with Trichophyton rubrum Reveals Important Roles of Cytokine GM-CSF.

Trichophyton rubrum is the most causative agent of dermatophytosis worldwide. The keratinocytes are the first line of defense during infection, triggering immunomodulatory responses. Previous dual RNA-seq data showed the upregulation of several human genes involved in immune response and epithelial barrier integrity during the co-culture of HaCat cells with T. rubrum. This work evaluates the transcriptional response of this set of genes during the co-culture of HaCat with different stages of T. rubrum conidia development and viability. Our results show that the developmental stage of fungal conidia and their viability interfere with the transcriptional regulation of innate immunity genes. The CSF2 gene encoding the cytokine GM-CSF is the most overexpressed, and we report for the first time that CSF2 expression is contact and conidial-viability-dependent during infection. In contrast, CSF2 transcripts and GM-CSF secretion levels were observed when HaCat cells were challenged with bacterial LPS. Furthermore, the secretion of proinflammatory cytokines was dependent on the conidia developmental stage. Thus, we suggest that the viability and developmental stage of fungal conidia interfere with the transcriptional patterns of genes encoding immunomodulatory proteins in human keratinocytes with regard to important roles of GM-CSF during infection.

The IL-20RB receptor and the IL-20 signaling pathway in regulating host defense in oral mucosal candidiasis.

Pseudomembranous candidiasis (thrush), erythematous candidiasis, and fungal esophagitis are infections of the barrier mucosa of the upper gastrointestinal tract. The majority of these infections are caused by Candida albicans, an opportunistic fungal pathogen that frequently exists as a harmless commensal on mucosal surfaces lining the gastrointestinal tract. Oral infections are initiated in the superficial stratified squamous epithelium, in which keratinocytes are the most abundant host cells and are the initial points of contact with C. albicans present in saliva. Intrinsic features of oral keratinocytes are likely to play important roles in host defense and tissue homeostasis in oral candidiasis. One understudied pathway that may be important for modulating oral candidiasis is the IL-20 cytokine signaling pathway that employs keratinocyte IL-20RB receptors as ligands for IL-19, IL-20, and IL-24. We report that production of human oral keratinocyte il24 mRNA and protein are stimulated during co-culture with C. albicans. To test the role of the IL-20 family signaling pathway in oral candidiasis, Il20rb-/- mice (lacking the IL-20RB receptor) were compared to wild-type mice in a murine model of oropharyngeal candidiasis. Fungal burdens and percent loss in body weight were determined. Despite comparable fungal burdens, the Il20rb-/- mice exhibited less weight loss over the course of their infection compared to the B6 mice, suggestive of reduced overall disease consequences in the mutant mice. Interference with IL-20 family cytokine signaling may be useful for augmenting the ability of the host to defend itself against pathogens.

Pyridoxine Has a Potential to Prevent the Appearance of Pigmented Spots: Effects on the Phagocytosis and Differentiation of Keratinocytes.

Pyridoxine (VB6) is a vitamin that is essential to maintain the homeostasis of the human body by contributing to various metabolic reactions. In the skin, although some studies have shown that VB6 is involved in regulating homeostasis through the attenuation of intracellular oxidative stress, there are few reports regarding the effects of VB6 on the prevention or improvement of skin aging. Thus, we conducted this study to determine the potential anti-skin pigmentation effect of VB6 focusing on the phagocytosis of melanosomes (MSs) by keratinocytes. The phagocytosis of MSs by keratinocytes is activated by oxidative stress and is an important factor of skin pigmentation and the eventual appearance of pigmented spots. First, we confirmed the antioxidant property of VB6 that enhanced the expression of several intracellular antioxidants via nuclear erythroid factor 2-related factor 2 (Nrf2). Although the incorporation of fluorescent beads (FBs), which are used as pseudo-MSs, into keratinocytes was increased under higher oxidation conditions caused by UVB and by the depletion of intracellular glutathione, treatment with VB6 suppressed the increased incorporation of FBs into those keratinocytes via Nrf2 activation. Furthermore, VB6 restored the decreased expression of differentiation marker proteins in keratinocytes caused by FB incorporation. Taken together, the results show that VB6 has the potential to prevent the appearance of pigmented spots by suppressing the activation of phagocytosis in keratinocytes caused by oxidative stress, and by restoring the differentiation of keratinocytes disrupted by FB incorporation.

050 Eotaxin-1 and matrix metalloproteinase-9 are critical in anti-BP180 IgE-induced experimental bullous pemphigoid

Bullous pemphigoid (BP) is the most common autoimmune blistering disease characterized by chronic subepidermal blistering, autoantibodies directed against hemidesmosomal components, and a predominant eosinophil infiltrate. BP autoantibodies recognize two hemidesmosomal proteins of basal keratinocytes: BP230 (also termed BPAG1) and BP180 (also termed BPAG2 or collagen XVII) with the NC16A domain of the human BP180 antigen containing the major autoantibody-reactive epitopes. Anti-BP180 autoantibodies belong to IgG and IgE isotypes.

Abstract 3186: Keratinocyte desmosomal cadherin Desmoglein 1 as a mediator and target of paracrine signaling in the melanoma niche

Abstract Melanoma is an aggressive cancer arising from the transformation of melanocytes residing in the basal layer of the epidermis, where they are in direct contact with surrounding keratinocytes. The role of keratinocytes in shaping the melanoma tumor microenvironment, however, remains understudied. We previously showed that the keratinocyte-specific desmosomal cadherin, desmoglein 1 (Dsg1) is reduced in response to acute UV exposure. Here, we show that Dsg1 protein is reduced in keratinocytes surrounding melanoma lesions but is unchanged in keratinocytes adjacent to benign nevi. As Dsg1 reduction by UV exposure is transient, we hypothesized that the persistent loss of Dsg1 observed in the melanoma tumor niche is due to melanoma-keratinocyte paracrine crosstalk. To address this idea, keratinocytes were cultured in conditioned media from melanocytes or melanoma cells. Melanoma conditioned media reduced keratinocyte Dsg1 mRNA and protein levels, suggesting that Dsg1 downregulation in keratinocytes is maintained by melanoma cells post-transformation. Keratinocytes in melanoma conditioned media exhibited reduced Grhl1 (Grainyhead like 1), a transcriptional activator of Dsg1, and increased Snai2, a repressor of Grhl1 expression and epidermal differentiation. These data support the idea that increased Snai2 stimulated by factors secreted by melanoma cells reduces keratinocyte Dsg1 mRNA expression by repressing Grhl1. To determine the impact of keratinocyte Dsg1 loss on melanoma cell behavior, melanoma cells were grown in conditioned media from control or Dsg1-deficient keratinocytes. Melanoma cell migration increased in a trans-well assay when melanoma cells were grown in conditioned media from Dsg1-deficient keratinocytes compared to from control keratinocytes. Supporting the in vivo importance of this finding, we found a significant, negative correlation between keratinocyte Dsg1 expression and melanoma cell movement within the melanoma tumor niche in patient samples. Downregulation of keratinocyte Dsg1 by melanoma cell conditioned media increased Erk1/2 signaling upstream of pro-migratory CXCL1 production, while pharmacological inhibition of keratinocyte Erk1/2 abrogated this effect. Furthermore, inhibition of the CXCL1 receptor, CXCR2 on melanoma cells decreased migration in melanoma cells grown in Dsg1-depleted keratinocyte conditioned media. Together, these data support the idea that paracrine crosstalk between melanoma cells and keratinocytes resulting in chronic keratinocyte Dsg1 reduction is important for melanoma progression and melanoma cell movement in the early stages of metastasis. Citation Format: Hope E. Burks, Christopher Arnette, Jennifer Koetsier, Joshua Broussard, Quinn Roth-Carter, Pedram Gerami, Jodi Johnson, Kathleen Green. Keratinocyte desmosomal cadherin Desmoglein 1 as a mediator and target of paracrine signaling in the melanoma niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3186.

Effects of Autophagy Modulators and Dioxin on the Expression of Epidermal Differentiation Proteins on Psoriasis-Like Keratinocytes in vitro and ex vivo.

ObjectivePsoriasis is a chronic inflammatory skin disorder associated with impairment of epidermal differentiation. Many signaling pathways, including those involved in aryl hydrocarbon receptor (AHR) and autophagy dysfunction, are reportedly associated with the pathogenesis of psoriasis. However, the discrete effects of dioxin via AHR activation or autophagy on the epidermal barrier remain unclear. In the current study, we evaluated the effects of autophagy modulators (chloroquine [CQ] and rapamycin) and the AHR agonist TCDD on the expression of epidermal barrier proteins in psoriasis-like keratinocytes and psoriasis lesional skin tissue culture.MethodsPolycytokine-stimulated human keratinocytes and psoriasis skin biopsies were treated with TCDD, CQ, or rapamycin, and the expression of keratinocyte differentiation-related factors, such as S100A7, S100A8, HRNR, IVL, FLG, and KRT10, was examined by Western blotting or quantitative-polymerase chain reaction.ResultsTCDD upregulated S100A7 and S100A8 expression in polycytokine-stimulated HaCaT cells compared to that in unstimulated cells. CQ decreased HRNR, IVL, and KRT10 mRNA levels, while rapamycin increased HRNR, IVL, and KRT10 mRNA levels in HaCaT cells relative to that in unstimulated cells. Co-treatment with CQ reversed TCDD-induced elevation in FLG, HRNR, and IVL mRNA expression. In psoriasis skin tissue, TCDD induced the upregulation of HRNR, IVL, S100A7, and S100A8 compared with that in normal skin. In ex vivo cultures treated with CQ, IVL expression in psoriasis skin tissue was repressed compared to that in normal skin tissue.ConclusionOur data suggest that autophagy modulation or AHR activation affects processes involved in epidermal differentiation and relates to the pathogenesis of chronic inflammatory skin diseases with skin barrier abnormalities such as psoriasis.

Effects of the epidermal growth factor receptor inhibitor, gefitinib, on lipid and hyaluronic acid synthesis in cultured HaCaT keratinocytes.

Epidermal growth factor receptor inhibitors (EGFRIs) are widely used for treating various cancers, including lung, colon, head and neck cancers. However, EGFRIs have unique dermatological side effects, including acneiform eruption, dry skin, paronychia and pruritus. In this study, we investigated the molecular changes induced by an EGFRI, gefitinib, in the expression of lipogenic enzymes and hyaluronic acid (HA) regulatory proteins in HaCaT keratinocytes, and whether EGF restored these changes. HaCaT cells were treated with gefitinib, with or without EGF, and treated with tumor necrosis factor α (TNFα) for inducing an inflammatory response. The mRNA and protein expression was analyzed by real-time RT-PCR, enzyme-linked immunosorbent assay (ELISA) and western blotting. Gefitinib enhanced the TNFα-induced expression of C-C motif chemokine ligand 2 (CCL2), CCL5 and C-X-C motif chemokine ligand 10 (CXCL10), and the expression of TNFα in HaCaT cells, while EGF restored these changes. At a similar concentration range, gefitinib reduced the mRNA and/or protein expression of various lipogenic enzymes for fatty acid, cholesterol and ceramide synthesis, except acidic sphingomyelinase. Gefitinib suppressed the mRNA and protein expression of HA synthase 2 (HAS2), HAS3, cluster of differentiation 44 (CD44), hyaluronidase 1 (HYAL1) and HYAL2, except the mRNA expression of HYAL1. EGF restored the changes induced by gefitinib, except for the mRNA expression of fatty acid synthase (FASN) and elongation of very long-chain fatty acid protein (ELOVL) 6. In conclusion, EGFRIs suppress lipogenesis and HA metabolism, which may contribute to adverse dermatological effects, including barrier function impairment in cancer patients treated with EGFRIs.

The DPA-derivative 11S, 17S-dihydroxy 7,9,13,15,19 (Z,E,Z,E,Z)-docosapentaenoic acid inhibits IL-6 production by inhibiting ROS production and ERK/NF-κB pathway in keratinocytes HaCaT stimulated with a fine dust PM10

11 S, 17S-dihydroxy 7,9,13,15,19 (Z,E,Z,E,Z)-docosapentaenoic acid (DoPE) is a derivative of docosapentaenoic acid, a specialized pro-resolving mediator of inflammation such as lipoxins, resolvins, maresins, and protectins. PM10 is a fine dust particle that induces oxidative stress, DNA damage, inflammation, aging, and cancer. The anti-inflammatory mechanism of DoPE, however, has not yet been elucidated. In these studies, we investigated whether DoPE has anti-inflammatory effects in human keratinocyte HaCaT cells. We demonstrated that DoPE suppressed PM10-induced expressions of IL-6 mRNA and protein in human HaCaT keratinocytes. We also investigated the modulating effects of DoPE on reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK). ROS production, extracellular signal regulated kinase (ERK) phosphorylation, and translocation of nuclear factor-kappa B (NF-kB) p65 and NF-kB activity were suppressed by DoPE in PM10-stimulated HaCaT cells. Collectively, our results demonstrated that DoPE inhibited IL-6 expression by reducing ROS generation, suppressing ERK phosphorylation, and inhibiting translocation of NF-kB p65 and NF-kB activity in PM10-stimulated HaCaT cells, suggesting that DoPE can be useful for the resolution of the inflammation caused by IL-6.

124 Investigation of the circadian system in psoriasis

Circadian rhythm in psoriatic skin has been studied in vitro and in vivo animal experiments. However, its human cellular implications are less well known. Epidemiological data have shown that psoriasis is more common among shift workers. We investigated the expression and daily rhythm of clock genes and proteins in psoriatic skin biopsies and HaCaT keratinocytes after stimulation with cytokines (TNFα, IL-23, IL-22, IL-1β, IL-17A). Clock, bmal1, cry1, per1, per2 and rev-erbα circadian genes and cop1 and mdm2 cell cycle regulators were examined in mRNA expression experiments.

Seeing Keratinocyte Proteins through the Looking Glass of Intrinsic Disorder

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid–liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology.

Protective effects of cannabidiol on the membrane proteins of skin keratinocytes exposed to hydrogen peroxide via participation in the proteostasis network

Hydrogen peroxide (H2O2) is widely used in clinical practice due to its antiseptic properties and its ability to heal wounds. However, due to its involvement in the formation of ROS, H2O2 causes several side effects, including disorders of the metabolism of skin cells and the development of chronic inflammation mediated by oxidative stress. Therefore, this study evaluated the effects of cannabidiol (CBD), a phytocannabinoid known for its antioxidant and anti-inflammatory properties, on the proteome of keratinocyte membranes exposed to H2O2. Overall, the hydrogen peroxide caused the levels of several proteins to increase, while the treatment with CBD prevented these changes. Analysis of the protein-protein interaction network showed that the significant changes mainly involved proteins with important roles in the proteasomal activity, protein folding processes (regulatory subunit of the proteasome 26S 6A, beta proteasome subunit type 1, chaperonin 60 kDa), protein biosynthesis (40S ribosomal proteins S16, S2 and ubiquitin-S27a), regulation of the redox balance (carbonyl reductase [NADPH] 1 and NAD(P)H [quinone] 1 dehydrogenase) and cell survival (14-3-3 theta protein). Additionally, CBD reduced the total amount of MDA, 4-HNE and 4-ONE-protein adducts. Therefore, we conclude that CBD partially prevents the changes induced by hydrogen peroxide by reducing oxidative stress and maintaining proteostasis networks. Moreover, our results indicate that combination therapy with CBD may bring a promising approach in the clinical use of hydrogen peroxide by preventing its pro-oxidative and pro-inflammatory effect through potential participation of CBD in membrane mediated molecular signaling.

Peroxisomal Fatty Acid Oxidation and Glycolysis Are Triggered in Mouse Models of Lesional Atopic Dermatitis

Alterations of the lipid profile of the stratum corneum have an important role in the pathogenesis of atopic dermatitis (AD) because they contribute to epidermal barrier impairment. However, they have not previously been envisioned as a cellular response to altered metabolic requirements in AD epidermis. In this study, we report that the lipid composition in the epidermis of flaky tail, that is, ft/ft mice mimics that of human lesional AD (ADL) epidermis, both showing a shift toward shorter lipid species. The amounts of C24 and C26 free fatty acids and C24 and C26 ceramides—oxidized exclusively in peroxisomes—were reduced in the epidermis of ft/ft mice despite increased lipid synthesis, similar to that seen in human ADL edpidermis. Increased ACOX1 protein and activity in granular keratinocytes of ft/ft epidermis, altered lipid profile in human epidermal equivalents overexpressing ACOX1, and increased ACOX1 immunostaining in skin biopsies from patients with ADL suggest that peroxisomal β-oxidation significantly contributes to lipid signature in ADL epidermis. Moreover, we show that increased anaerobic glycolysis in ft/ft mouse epidermis is essential for keratinocyte proliferation and adenosine triphosphate synthesis but does not contribute to local inflammation. Thus, this work evidenced a metabolic shift toward enhanced peroxisomal β-oxidation and anaerobic glycolysis in ADL epidermis.

Effect of scratching and friction on human skin in vivo.

To investigate effect of scratching and friction on human skin function and functional differences between scratching and friction. Forty healthy volunteers were enrolled. Scratching and friction behavior was modeled by scalpel and sandpaper simulation to forearm for 80 times, respectively. Noninvasive bioengineering devices were used to measure basic skin physiological parameters and exfoliated stratum corneum collected and protein quantified. Parameters were recorded at baseline (BL) and after every 20 times interventions (20, 40, 60, and 80 times). Compared to BL, transepidermal water loss (TEWL) value increased significantly at both scratched and friction sites (P<.001) with a significant higher value for friction (P<.001). There was no significant difference in stratum corneum hydration (SCH) value postscratching (P>.05), while it decreased first and then increased significantly at friction site (P<.001). Roughness values (contract (CONT), variety (VAR), and scaliness (SEsc)) were raised significantly at both sites (P<.001). Net change in CONT and SEsc values of friction was higher than scratched sites (P>.05). There was no significant difference in blood flow after both scratching and friction (P>.05). Quantity of keratinocyte protein from friction sites was statistically higher than scratching after 80 times interventions (P<.05). Both noninvasive detections and protein quantification indicated more damage from friction, which may have significance for behavior guidance of patients with pruritus and implication for further investigation.

Hyaluronic acid (HA) stimulates the in vitro expression of CD44 proteins but not HAS1 proteins in normal human epidermal keratinocytes (NHEKs) and is HA molecular weight dependent.

In the skin, hyaluronic acid is broken down to smaller fragments by hyaluronidase enzymes, particularly when skin is wounded. The impact of various molecular weight fragments of HA on normal human epidermal keratinocytes (NHEK) with regard to expression of important cellular proteins has not been deeply explored. Examination of three molecular weight (Mw) fractions of hyaluronic acid: 1) average Mw of the high fraction: 1.5-2MDa, 2) average Mw of the medium fraction: 200-500kDa, and 3) average Mw of the low fraction: 5-10kDa and a unique 1:1:1 composite complex of the three HA fragments (Triluronic® Acid) was done to examine the influence of the HA on two critical skin cell protein targets: hyaluronan synthase-1 (HAS-1) and the HA binding protein cluster of differentiation 44 (CD44). NHEKs were treated in vitro with a 1.0% stock solution of each HA Mw fraction at 1.0, 0.5, and 0.1% concentrations of the 1.0% solution and the polysaccharide composite at the same concentrations for 48 Hrs. The cells were than analyzed by ELISA protein assays for HAS-1 and CD44 protein content. Examination of HAS-1 protein expression indicates that none of the HA test materials influenced the expression of HAS-1 at any concentration. Examination of the CD44 protein expression indicated that the low Mw fraction and the commercial complex of the three Mw fractions upregulated CD44 protein expression in NHEKs, but the medium Mw and high Mw HA fractions did not. In this work, it was demonstrated that HA can influence the expression of CD44 protein, a critical HA transmembrane HA binding protein, and the influence appears to be molecular weight dependent.

Epsilon toxin from Clostridium perfringens induces toxic effects on skin tissues and HaCaT and human epidermal keratinocytes

Epsilon toxin (ETX) is a key pathogenic factor of C. perfringens type B and D, causing fatal enterotoxemia in sheep and goats. Excessive production of ETX increases intestinal permeability; its entrance into the bloodstream leads to severe edema in organs such as the brain and kidneys. At present, very few cell lines are known to be sensitive to ETX, with the most sensitive cell model for in vitro research being the MDCK cell line. Recently, more tissue-derived cell lines have been shown to be sensitive to ETX, but the mechanism of cytotoxicity remains unknown. Herein, for the first time, we aimed to evaluate the effects of ETX on HaCaT keratinocytes and human epidermal keratinocytes (HEKa). In addition, the median lethal dose of subcutaneous injection of ETX in mice was 109 ng/kg. At this dose, ETX rapidly entered the blood circulation, causing hemorrhage and edema in the brain and kidneys. ETX also increased the expression of aquaporin 3 in the muscle layer and hair follicles of the skin. We further showed the presence of the MAL protein in HaCaT keratinocytes and HEKa and skin tissues, supporting the hypothesis that it is a key element in the mechanism of cytotoxicity of ETX. In conclusion, skin cell lines were used for the first time as a model for studying the toxic effects of ETX, which will help elucidate the cytotoxicity induced by ETX and the related molecular mechanisms.

Expression of epidermal antimicrobial peptides is increased in tinea pedis.

Tinea pedis is often chronic or recurrent, but not all individuals are equally susceptible to this infection. Dermatophytes are able to induce the expression of antimicrobial peptides and proteins (AMPs) in human keratinocytes and certain AMPs can inhibit the growth of dermatophytes. The focus of this study was to analyse the secretion of relevant AMPs, especially RNase 7, human beta-defensin-2 (hBD-2) and the S-100 protein psoriasin (S100A7), in patients with confirmed tinea pedis. To verify the diagnosis, skin scales were obtained from all patients (n=13) and the dermatophytes were identified by potassium hydroxide mount, culture and molecular analysis. To determine the AMP concentrations, the lesional skin area of the foot was rinsed with a buffer that was subsequently analysed by ELISA. The corresponding area of the other unaffected foot as well as defined healthy skin areas of the forearm and forehead and samples from age and gender-matched healthy volunteers served as controls. In tinea pedis patients the AMP concentrations were higher in lesional skin than in non-lesional skin and in healthy skin of controls. In particular, concentrations of hBD-2 and psoriasin were significantly elevated. The induction of AMPs in tinea pedis might be triggered directly by the dermatophytes; furthermore, attendant inflammation and/or differentiation processes may play a role. Our results indicate that there is no defect in the constitutive expression and induction of the analysed AMPs by dermatophytes in the epidermis of affected patients. However, it is not known why the elevated AMP concentrations fail to efficiently combat dermatophyte growth.

Dysregulation of epidermal barrier in mouse skin wounds induced by sulfur mustard

Sulfur mustard (2,2′-dichlorodiethyl sulfide, SM), is a potent chemical vesicant. Skin exposure to SM results in blistering, persistent epidermal hyperplasia, severe tissue damage, and prolonged wound repairs. SM, a bifunctional alkylating agent, causes DNA damage in mouse skin cells; however, the mechanistic action of SM in tissue injuries is still unclear. Studies showed mouse skin following SM exposure activates several stress signaling pathways including oxidative stress, and induces strong inflammatory responses. Skin structured with multilayers of keratinocytes (KCs), serves as a protection barrier against toxic chemicals. Keratins (Ks), the major structural intermediate filaments proteins in KCs, may regulate skin wound healing. In normal skin epidermis, KCs express K5 in basal cells and K10 in the differentiated suprabasal layers. Upon skin injury, KCs, activated by inflammation, express K6 and K17 to promote KCs migration for wound repair. Dysregulation of activated epidermal KCs may result in barrier breach and lead to uncontrolled inflammation and skin diseases. In addition, the cornified envelope (CE) precursor molecules, the small proline-rich (Sprr) proteins, present to protect skin from reactive oxidative species (ROS) damage. The present study was focused on SM induced alteration of skin barrier alarmin molecules in mouse ear skin. Time course studies (1-7 days) of mouse ear skin exposing to a single dose (0.08 mg) of SM showed characteristic histopathology changes over time. Significant changes of skin barrier alamin molecules (K10, K6, K17, and Sprr1B) gene expression were observed in SM injured skin post exposure. Immunohistochemistry results showed the K6 and K17 staining was restricted to the skin appendages in the naïve control, whereas samples exposed to SM had increased expression over time in the suprabasal epithelium. The activated KCs highly expressed K6, K17, and Sprr1B at the wound edge. In contrast, reduced expression of the differentiation marker, K10, indicated incompletely differentiated epithelium, suggesting weakened barrier protection in skin damaged by SM. Multiplex immunofluorescent studies showed heterogeneous coexpression of K5/ K6/K10 and K5/K10/Sprr1B in the hyperplasia behind the leading edge, suggesting hyperactivated KCs in the SM skin wounds. Overall, K10, K6, K17, and Sprr1B have value as skin wound and repair markers of SM induced injury. Understanding the molecular mechanism of action of vesicant-induced skin injury and repair involved in the dysregulation of epidermal barrier may help to identify new targets of medical countermeasures to chemical vesicant skin injury.

Inhibitory effect of strawberry geranium (Saxifraga stolonifera) on Toll-like receptor 2-mediated inflammatory response in human skin keratinocytes

Ethnopharmacological relevanceStrawberry geranium (Saxifraga stolonifera [L.] Meeb) has traditionally been used as a drug to treat skin disorders in Japan. However, little is known about its physiological effects on skin keratinocytes.Aim of the study: We investigated the anti-inflammatory effects of a strawberry geranium extract (SGE) on human skin keratinocytes. Materials and methodsThe human keratinocyte cell line, HaCaT, was treated with SGE, and then stimulated with tumor necrosis factor (TNF)-α. The expression of 207 genes related to the innate immune system was analyzed using DNA microarrays. The effect of SGE on the target proteins in primary human epidermal keratinocytes was confirmed by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The mechanisms of action and active components involved in the suppressive effect of SGE were evaluated by fractionation and a transcription assay. ResultsThe microarray analysis revealed that SGE primarily suppressed Toll-like receptor (TLR)2 expression through procyanidin B2 3,3′-di-O-gallate, without TLR2 downregulation, in TNF-α-stimulated HaCaT cells. SGE suppressed TLR2 expression and interleukin (IL)-8 production induced by TLR2 ligands in primary human epidermal keratinocytes and HaCaT cells. Multiple components downregulating TLR2 expression suppressed the Sp1 activity. ConclusionsWe identified a novel physiological function of SGE, which suppresses TLR2 expression and TLR2-mediated inflammation in human skin keratinocytes. This study provides significant insights into the anti-inflammatory effect of SGE in human skin.