Keratinocyte Differentiation Markers Research Articles

Enhanced Wound Healing- and Inflammasome-Associated Gene Expression in TNFAIP3-Interacting Protein 1- (TNIP1-) Deficient HaCaT Keratinocytes Parallels Reduced Reepithelialization.

TNIP1 protein is a widely expressed, cytoplasmic inhibitor of inflammatory signaling initiated by membrane receptors such as TLRs which recognize pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs). Keratinocyte TNIP1 deficiency sensitizes cells to PAMPs and DAMPs promoting hyperresponsive expression and secretion of cytokine markers (e.g., IL-8 and IL-6) relevant to cases of chronic inflammation, like psoriasis, where TNIP1 deficiency has been reported. Here, we examined the impact of TNIP1 deficiency on gene expression and cellular responses (migration and viability) relevant to acute inflammation as typically occurs in wound healing. Using siRNA-mediated TNIP1 expression knockdown in cultured HaCaT keratinocytes, we investigated TNIP1 deficiency effects on signaling downstream of TLR3 agonism with low-concentration poly (I:C), a representative PAMP/DAMP. The combination of TNIP1 knockdown and PAMP/DAMP signaling disrupted expression of specific keratinocyte differentiation markers (e.g., transglutaminase 1 and involucrin). These same conditions promoted synergistically increased expression of wound-associated markers (e.g., S100A8, TGFβ, and CCN2) suggesting potential benefit of increased inflammatory response from reduced TNIP1 protein. Unexpectedly, poly (I:C) challenge of TNIP1-deficient cells restricted reepithelialization and reduced cell viability. In these cells, there was not only increased expression for genes associated with inflammasome assembly (e.g., ASC, procaspase 1) but also for A20, a TNIP1 partner protein that represses cell-death signaling. Despite this possibly compensatory increase in A20 mRNA, there was a decrease in phospho-A20 protein, the form necessary for quenching inflammation. Hyperresponsiveness to poly (I:C) in TNIP1-deficient keratinocytes was in part mediated through p38 and JNK pathways. Taken together, we conclude that TNIP1 deficiency promotes enhanced expression of factors associated with promoting wound healing. However, the coupled, increased potential priming of the inflammasome and reduced compensatory activity of A20 has a net negative effect on overall cell recovery potential manifested by poor reepithelialization and viability. These findings suggest a previously unrecognized role for TNIP1 protein in limiting inflammation during successful progression through early wound healing stages.

MicroRNA-184 is induced by store-operated calcium entry and regulates early keratinocyte differentiation.

Extracellular calcium (Ca2+ ) and store-operated Ca2+ entry (SOCE) govern homoeostasis in the mammalian epidermis. Multiple microRNAs (miRNA) also regulate epidermal differentiation, and raised external Ca2+ modulates the expression of several such miRNAs in keratinocytes. However, little is known about the regulation of miR-184 in keratinocytes or the roles of miR-184 in keratinocyte differentiation. Here we report that exogenous Ca2+ stimulates miR-184 expression in primary epidermal keratinocytes and that this occurs in a SOCE-dependent manner. Levels of miR-184 were raised by about 30-fold after exposure to 1.5 mM Ca2+ for 5 days. In contrast, neither phorbol ester nor 1,25-dihydroxyvitamin D3 had any effect on miR-184 levels. Pharmacologic and genetic inhibitors of SOCE abrogated Ca2+ -dependent miR-184 induction by 70% or more. Ectopic miR-184 inhibited keratinocyte proliferation and led to a fourfold increase in the expression of involucrin, a marker of early keratinocyte differentiation. Exogenous miR-184 also triggered a threefold rise in levels of cyclin E and doubled the levels of γH2AX, a marker of DNA double-strand breaks. The p21 cyclin-dependent kinase inhibitor, which supports keratinocyte growth arrest, was also induced by miR-184. Together our findings point to an SOCE:miR-184 pathway that targets a cyclin E/DNA damage regulatory node to facilitate keratinocyte differentiation.

In Vitro Effects of Dehydrotrametenolic Acid on Skin Barrier Function.

Dehydrotrametenolic acid (DTA) is a lanostane-type triterpene acid isolated from Poria cocos Wolf (Polyporaceae). Several studies have reported the anti-inflammatory and antidiabetic effects of DTA; however, its effects on the skin are poorly understood. In this study, we investigated the effects of DTA on skin barrier function in vitro and its regulatory mechanism in human keratinocyte cell line HaCaT cells. DTA increased the microRNA (mRNA) expression of natural moisturizing factor-related genes, such as HAS-2, HAS-3, and AQP3 in HaCaT cells. DTA also upregulated the mRNA expression of various keratinocyte differentiation markers, including TGM-1, involucrin, and caspase-14. Moreover, the protein expression of HAS-2, HAS-3, and TGM-2 were significantly increased by DTA. To examine the regulatory mechanisms of DTA, Western blotting, luciferase-reporter assays, and RT-PCR were conducted. The phosphorylation of mitogen-activated protein kinases (MAPKs) and IκBα were increased in DTA-treated HaCaT cells. In addition, AP-1 and NF-κB transcriptional factors were dose-dependently activated by DTA. Taken together, our in vitro mechanism studies indicate that the regulatory effects of DTA on skin hydration and keratinocyte differentiation are mediated by the MAPK/AP-1 and IκBα/NF-κB pathways. In addition, DTA could be a promising ingredient in cosmetics for moisturizing and increased skin barrier function.

Clinical and in vitro evaluation of new anti-redness cosmetic products in subjects with winter xerosis and sensitive skin.

ObjectiveTo demonstrate the in vitro activities of panthenol, palmitoylethanolamide (PEA), and niacinamide (NAM) and determine the biophysical properties, clinical safety, tolerability together with efficacy of two developmental anti‐redness (AR) formulations containing these ingredients, in alleviating facial redness associated with winter xerosis in healthy volunteers with sensitive skin.MethodsThe anti‐inflammatory and skin protective properties of panthenol, PEA and NAM were evaluated in vitro. The physical properties of the AR formulations were analysed using measurement of water vapour transport rate (WVTR) and infrared spectroscopy. Clinical studies were performed between the months of December and April (2014–2015) with efficacy assessed during the winter. Facial redness, irritation, sensitization potential, photo‐irritation, and photo‐sensitization were evaluated. Self‐assessed adverse reactions were reported in diaries of use.ResultsPanthenol and PEA reduced prostaglandin E2, interleukin‐6, and thymic stromal lymphopoietin levels in vitro, while NAM induced nicotinamide adenine dinucleotide (NAD) levels and the keratinocyte differentiation markers: filaggrin (2‐fold increase, P < 0.001), loricrin (2‐fold increase, P < 0.05), involucrin (2 fold increase, P < 0.001) & peroxisomal proliferator activated receptor‐alpha (1.5 fold increase, P < 0.05). The two AR products exhibited low WVTR vs. no treatment (P < 0.001) and displayed an ordered lipid structure. The day cream formulation protected against ultraviolet B radiation in vitro. A total of 382 participants were included in clinical studies which showed the AR formulations significantly improved facial redness associated with winter xerosis (Day 29 mean change from baseline: AR day cream 0.77 (P < 0.001); AR serum 0.67 (P < 0.001)). No irritation, sensitization, photo‐irritation, photo‐sensitization or product‐related adverse reactions were observed or reported in the clinical studies.ConclusionThe new products significantly improved skin redness associated with winter xerosis in participants with self‐perceived sensitive skin. Both products were well tolerated with a suitable safety profile for topical use in subjects with sensitive skin.

APOBEC3 regulates keratinocyte differentiation and expression of Notch3.

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family consists of deaminases. Some isozymes of APOBEC3 are induced upon human papillomavirus infection or development of psoriasis skin lesions. However, the involvement of APOBEC3 in keratinocyte differentiation has not been addressed. We herein sought to evaluate the roles of APOBECs in mouse primary keratinocyte differentiation. We found that expression levels of APOBEC1 and APOBEC3 were increased during calcium-induced keratinocyte differentiation. Unexpectedly, however, the expression levels of keratinocyte differentiation markers keratin 1/10, involucrin, loricrin and filaggrin were higher in keratinocytes treated with APOBEC3 siRNAs than in those treated with control RNAs. In addition, the treatment of keratinocytes with APOBEC3 siRNAs increased the gene expression levels of Notch3, a master regulator of keratinocyte differentiation. Moreover, calcium-induced increase in Notch3 expression and keratinocyte differentiation were impaired by transfection with an APOBEC3 expression plasmid. Furthermore, co-treatment with Notch3 siRNAs reduced the APOBEC3 siRNA-mediated upregulation of Notch3 expression and in part attenuated the increased expression levels of keratinocyte differentiation markers. These results suggest that APOBEC3 is induced upon keratinocyte differentiation and negatively regulates the keratinocyte differentiation in part by its inhibitory role for Notch3 expression.

Comparative genomics suggests loss of keratin K24 in three evolutionary lineages of mammals

Keratins are the main cytoskeletal proteins of epithelial cells and changes in the expression of keratins have contributed to the evolutionary adaptation of epithelia to different environments. Keratin K24 was proposed to be a differentiation marker of epidermal keratinocytes but the significance of K24 expression in the epidermis versus other tissues has remained elusive. Here, we show by RT-PCR, western blot, and immunofluorescence analyses that K24 is highly expressed in the epithelium of the cornea whereas its expression levels are significantly lower in other stratified epithelia including in the epidermis. To investigate the evolutionary history of K24, we screened the genome sequences of vertebrates for orthologs of the human KRT24 gene. The results of this comparative genomics study suggested that KRT24 originated in a common ancestor of amniotes and that it was lost independently in three clades of mammals, i.e. camels, cetaceans, and a subclade of pinnipeds comprising eared seals and the walrus. Together, the results of this study identify K24 as component of the cytoskeleton in the human corneal epithelium and reveal previously unknown differences of keratin gene content among mammalian species.

Eucalyptus citriodora extract regulates cutaneous homeostasis including immune dysregulation and skin barrier dysfunction via the modulation of peroxisome proliferator-activated receptor-delta (PPAR-delata) pathway

Perturbation of cutaneous homeostasis including immune dysregulation and skin barrier dysfunction evokes skin disorders. In this study, we examined the effect of Eucalyptus citriodora (Euc-c) extract on cytokine production, cell proliferation and cell differentiation in HaCaT cells to elucidate its influence on cutaneous homeostasis. Euc-c suppressed significantly LPS-induced IL-6 and TNF-a-induced IL-8 production from HaCaT cells. Conversely IL-1ra production was significantly enhanced by Euc-c. The expressions of IVL, CERS3 and CERS4, keratinocyte differentiation markers, were upregulated to 3.1, 2.8 and 2.7-fold respectively by Euc-c treatment, compared to the control, while the proliferation was downregulated. The lipid contents in Euc-c-treated cells tended to increase, compared with non-treated cells. To explore the underlying mechanism of these effect, we next performed siRNA experiments against PPAR-b/d. Euc-c enhanced PPAR-b/d mRNA expression to 3.25-fold, while PPAR-b/d mRNA expression in transfected cells was suppressed. The expressions of IVL, CERS3 and CERS4 in transfected cells were suppressed to 1.48, 0.82 and 0.72-fold respectively, concomitant with suppression of PPAR-b/d mRNA expression. These results indicated that Euc-c exerts anti-inflammatory effects and regulates keratinocyte differentiation via the modulation of PPAR-b/d pathway. Therefore, the application of Euc-c is expected to exert beneficial effect on skin disorders evoked by perturbation of skin homeostasis.Key words: Eucalyptus citriodora, PPAR-b/d, inflammation, barrier function, cutaneous homeostasis

Increase of cystathionine-γ-lyase (CSE) during late wound repair: Hydrogen sulfide triggers cytokeratin 10 expression in keratinocytes

The gaseous mediators nitric oxide (NO), carbon monoxide (CO) and lately also hydrogen sulfide (H2S) have been described to contribute to the interplay of protein type- and lipid mediators in the regulation of wound healing. In particular, the recently reported role of H2S in skin repair remains largely unresolved. Therefore we assessed the expressional kinetics of potential H2S-producing enzymes during undisturbed skin repair: the cystathionine-γ-lyase (CSE), the cystathionine-β-synthase (CBS) and the 3-mercaptopyruvate sulfurtransferase (MPST). All three enzymes were not transcriptionally induced upon wounding and remained silent through the acute inflammatory and proliferative phase of skin repair. By contrast, CSE expression started to increase significantly at the later stages of healing, when cellular proliferation ceases within the granulation tissue and neoepidermis. The importance of H2S production in late healing phases was supported by a strong induction of otherwise not-induced CBS to complement the loss of CSE function in CSE-deficient mice. Immunohistochemistry revealed hair follicle keratinocytes and basal keratinocytes of the neo-epidermis covering the wound area as sources of CSE expression. Subsequent in vitro studies implicated a role of CSE-derived H2S for keratinocyte differentiation: the H2S-donor GYY4137 markedly increased the Ca2+-triggered expression of the early keratinocyte differentiation markers cytokeratin 10 (CK10) and involucrin (IVN) in cultured human keratinocytes. Here, GYY4137-derived H2S strongly enhanced CK10 expression by increasing the binding of RNA polymerase II to the CK10 promoter.

Three Constituents of Moringa oleifera Seeds Regulate Expression of Th17-Relevant Cytokines and Ameliorate TPA-Induced Psoriasis-Like Skin Lesions in Mice.

As a folk medicine, Moringa oleifera L. is used effectively to treat inflammatory conditions and skin diseases. However, its mechanism of action is not well understood, limiting its medical use. We isolated and identified three compounds, namely niazirin, marumoside A and sitosterol-3-O-β-d-glucoside, from the seeds of Moringa oleifera, and studied their effects on the expression of Th17-relevant cytokines (IL-12/IL-23 p40, IL-17A, IL-22 and IL-23 p19) using lipopolysaccharide-stimulated THP-1 cells. Additionally, as Th17 plays a critical role in the pathogenesis of psoriasis, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced psoriasis-like skin lesion mouse model to study their potential therapeutic application in vivo. The compounds suppressed the expression of IL-12/IL-23 p40, IL-17A, IL-22 and IL-23 p19 in vitro, and in vivo they ameliorated psoriasis-like skin lesions, decreased IL-17A mRNA expression, and increased the expression of keratinocyte differentiation markers. To our knowledge, this is the first report regarding the mechanism and therapeutic application of Moringa oleifera seeds to treat psoriasis-like lesions in vivo.

Adiponectin Enhances Human Keratinocyte Lipid Synthesis via SIRT1 and Nuclear Hormone Receptor Signaling

Adiponectin is known to have beneficial effects on lipid and insulin metabolism, wound healing, and cellular senescence, but its effect on skin barrier formation remains unknown. We investigated the effects of adiponectin on keratinocyte lipid synthesis with respect to skin barrier function. Lipid staining revealed an adiponectin-mediated increase in keratinocyte intracellular and reconstructed epidermal lipid content. Moreover, significant increases in the levels of ceramide and its downstream metabolites (sphingosine and sphingosine-1-phosphate) following adiponectin stimulation were detected using liquid chromatography-mass spectrometry. Expression levels of keratinocyte differentiation markers were also increased. Adiponectin also increased expression of lipid biosynthesis enzymes (fatty acid synthase, HMG-CoA reductase, and serine palmitoyltransferase), nuclear hormone receptors (peroxisome proliferator-activated receptors and liver X receptors), and the adiponectin signal molecule SIRT1. Suppression of SIRT1, liver X receptor-α, or peroxisome proliferator-activated receptor-α downregulated the expression of lipid synthetic enzymes, decreasing lipid content. Inhibition of adiponectin receptors decreased expression of nuclear hormone receptors, SIRT1, lipid-synthesizing enzymes, and sphingolipids. Thus, activation of adiponectin signaling increases the expression of transcription factors, including SIRT1, liver X receptor-α, and peroxisome proliferator-activated receptor-α, enhancing lipid synthesis and keratinocyte cell differentiation and possibly aiding in the maintenance of skin barrier homeostasis.

Cutaneous immunohistochemical staining pattern of p53β isoforms

p53 is considered the guardian of the genome and as such has numerous functions. The TP53 gene is the most commonly mutated gene in cancer, and yet the exact biological...

LGI3 promotes human keratinocyte differentiation via the Akt pathway.

Leucine-rich repeat LGI family member 3 (LGI3), a member of the LGI family, is a secreted protein that is expressed not only in the brain and adipose tissues, but also in various skin cells. We previously reported that LGI3 was secreted after exposure to ultraviolet B and promoted the migration of HaCaT human keratinocytes. In the present study, we investigated whether LGI3 influences the differentiation of keratinocytes. The results show that the expression of involucrin, a keratinocyte differentiation marker, was reduced in tissue from LGI3-knockout mice. Those results indicate that LGI3 plays an important role in keratinocyte differentiation. Therefore, we treated HaCaT cells with LGI3 to examine its effect on keratinocyte differentiation. Protein levels of various differentiation markers were enhanced by treatment with LGI3. Furthermore, expression of differentiation markers was inhibited when keratinocytes were transfected with an siRNA for LGI3. LGI3 strongly activated Akt, whereas it had no apparent effect on extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, or the c-Jun N-terminal kinase. A specific inhibitor of phosphoinositide 3-kinase, LY294002, reduced LGI3-induced expression of differentiation markers in HaCaT cells. Taken together, these results suggest that LGI3 promotes keratinocyte differentiation and could be used as a therapeutic agent to recover skin barrier function in epidermal barrier disruption.

673 11β-hydroxysteroid dehydrogenase type 1 activation induced by UVB might be a possible mechanism of skin barrier dysfunction due to UV exposure

Ultraviolet light (UV) radiation to the skin causes dose- and wavelength- dependent reactions, including skin barrier dysfunction. Long-term exposure to even low dose UV induces the alteration of skin barrier presenting prominent ceramide decrease in the stratum corneum (SC) intercellular lipids. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts inactive cortisone to active cortisol in peripheral tissues and inhibits keratinocytes and fibroblasts proliferation, is known to be induced by UVB. We performed in vitro, in vivo, ex-vivo studies to elucidate whether skin barrier dysfunction induced by UV could be resulted from 11β-HSD1 activation after UV irradiation. We observed that 11β-HSD1 expression was increased by cortisol treatment itself and UV irradiation, thereby rapidly converting cortisone into cortisol. UVB irradiation increases 11β-HSD1 expression as well as inflammatory cytokines using HSD11B1 siRNA transfected normal human epidermal keratinocytes. 11β-HSD1 mRNA was prominently higher in UVB irradiated primary human keratinocytes compared with control. Keratinocytes transfected with HSD11B1 siRNA decreased the expression of 11β-HSD1 mRNA and did not increase cortisol even when the keratinocytes were irradiated by UVB. In vitro, increased cortisol converted by 11β-HSD1 inhibits keratinocyte differentiation. Cortisone decreased the mRNA and protein expression of keratinocyte differentiation markers in UVB-exposed keratinocytes. 11β-HSD1 expression remarkably increased in UV exposed skin and intrinsic aged skin. Skin barrier function presented by transpidermal water loss, SC hydration and SC integrity was impaired in long term UV exposed skin and intrinsic aged skin. Therefore, 11β-HSD1 activation by UVB might suggest another possible mechanism on skin barrier dysfunction due to UV exposure.

Resident T Cells in Resolved Psoriasis Steer Tissue Responses that Stratify Clinical Outcome

Psoriasis is driven by focal disruptions of the immune-homeostasis in human skin. Local relapse following cessation of therapy is common and unpredictable, which complicates clinical management of psoriasis. We have previously shown that pathogenic resident T cells accumulate in active and resolved psoriasis, but whether these cells drive psoriasiform tissue reactions is less clear. Here, we activated T cells within skin explants using the pan-T cell activating antibody OKT-3. To explore if T cells induced different tissue response patterns in healthy and psoriasis afflicted skin, transcriptomic analyses were performed with RNA-sequencing and Nanostring. Core tissue responses dominated by IFN-induced pathways were triggered regardless of the inflammatory status of the skin. In contrast, pathways induced by IL-17A, including Defensin beta 2 and keratinocyte differentiation markers, were activated in psoriasis samples. An integrated analysis of IL-17A and IFN-related responses revealed that IL-17 dominated tissue response correlated with early relapse following UVB treatment. Stratification of tissue responses to T cell activation in resolved lesions could potentially offer individualized prediction of disease relapse during long-term immunomodulatory treatment.

Sophora japonica extracts accelerates keratinocyte differentiation through miR-181a

BackgroundThe Sophora japonica extracts contain flavonol triglycoside, isoflavonol, coumarone chromone, saponin, triterpene glucoside, phospholipids, alkaloids, amino acids, polysaccharides, and fatty acids. These components have physiological effects such as anti-infertility and anti-cancer activities. This study investigated the regulation of keratinocyte differentiation upon treatment with the S. japonica extracts in keratinocyte and the molecular cell biological mechanism involved.MethodsTo determine whether the S. japonica extracts or troxerutin, which is its main component, regulates keratinocyte differentiation, quantitative real-time polymerase chain reaction (qRT-PCR) was performed on keratinocyte differentiation markers such as keratin 1 (K1), keratin 10 (K10), involucrin, and filaggrin after treatment with the S. japonica extracts. miR-181a knockdown confirmed that keratinocyte differentiation was regulated by increased miR-181a expression upon treatment with the S. japonica extracts or troxerutin.ResultsThe expression of keratinocyte differentiation markers such as K1, K10, involucrin, and filaggrin increased upon treatment with the S. japonica extracts and troxerutin. Furthermore, miR-181a expression, which is known to increase during keratinocyte differentiation, increased upon treatment with the S. japonica extracts and troxerutin. When miR-181a was knocked down, the increased expression of keratinocyte differentiation markers upon treatment with the S. japonica extracts and troxerutin decreased again. Finally, it was confirmed that miR-181a directly regulated and reduced the expression of Notch2, which reduces keratinocyte differentiation, and that the decrease in Notch2 expression by miR-181a regulated keratinocyte differentiation.ConclusionsThese results suggest that the S. japonica extracts or troxerutin accelerates keratinocyte differentiation through miR-181a. This accelerated keratinocyte differentiation was confirmed to have resulted from the regulation of Notch2 expression by miR-181a. The results of this study provide an opportunity to confirm the molecular cell biological mechanism of S. japonica extracts or troxerutin on skin keratinization, and we expected that this study contribute to develop a moisturizing cosmetic material that can strengthen the skin barrier through regulating keratinocyte differentiation.

Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.

Actinic lentigos (AL) are benign hyperpigmented skin lesions associated with photoageing. Despite their high prevalence, biological mechanisms driving their formation remain unclear. To provide new insights about the physiopathology of AL through a comprehensive description of their histological and molecular features. Quantitative analysis of dermoscopic images was used to select AL containing elongated patterns, predicted to display a highly deformed dermal-epidermal junction (DEJ), on the back of the hands of 15 Caucasian women. Biopsies from lesional and adjacent nonlesional (NL) areas were processed for histological analysis or gene expression profiling. Histological staining confirmed a drastic deformation of the DEJ in AL, with deep epidermal invaginations into the dermis. Although the melanin content was significantly higher in AL compared with NL epidermis, the distribution of melanocytes along the DEJ was unchanged. Transcriptomic analysis revealed a signature of 529 genes differently expressed in AL vs. NL skin. Alteration of epidermal homeostasis was confirmed by the dysregulation of keratinocyte proliferation and differentiation markers. Surprisingly, canonical genes involved in melanogenesis were not significantly modulated in AL. A striking finding was the overexpression of a large group of genes involved in dermal extracellular matrix organization and remodelling. Dermal alterations were confirmed by immunolabellings on AL and NL sections. Drastic disorganization of the cutaneous structure in AL is accompanied by a specific molecular signature revealing alterations in both epidermal and dermal compartments. In particular, our results suggest that local modifications of the dermal extracellular matrix might contribute to hyperpigmentation in AL.

The expression pattern of keratin 24 in tissue-engineered dermo-epidermal human skin substitutes in an in vivo model.

The use of autologous tissue-engineered skin substitutes is a promisingapproach to cover large skin defects in patients. Preclinical investigation is pivotal to test and improve the quality of these bio-engineered substitutes. In the skin, the epidermis, formed mainly by keratinocytes, provides the first physical barrier protecting from the environment. Proper keratinocyte differentiation and, thus, formation of astratified epidermis is essential for this function. Keratins, the main structural support of keratinocytes, play a vital role regarding differentiation of keratinocytes. Here, we examined the expression pattern of a recently described keratinocyte differentiation marker, namely Keratin 24, in our skin substitutes. Human epidermal keratinocytes, melanocytes, dermal fibroblasts, palmar fibroblasts or sweat gland cells were used to prepare skin substitutes. Fibroblast-containing collagen hydrogels were prepared, and keratinocytes or sweat gland cells and melanocytes were seeded onto the hydrogels. The generated tissue-engineered dermo-epidermal skin analogs were transplanted onto full-thickness skin wounds created on the back of immuno-incompetent rats. The skin substitutes were excised at different time points and histologically examined with regard to Keratin 24 expression. We observed the expression of Keratin 24 in keratinocytes of the upper stratum spinosum of the epidermis. In particular, we observed an intensified expression of Keratin 24 13weeks after transplantation compared to 4weeks after transplantation. Importantly, we noticed a markedly higher presence of Keratin 24 in more spinous layers if we used palmar fibroblasts or sweat gland cells in our skin substitutes compared non-palmar fibroblasts or epidermal keratinocytes. Our observations prove that the keratinocyte differentiation marker Keratin 24 is expressed in our dermo-epidermal skin substitutes in a normal pattern. This highlights that our bio-engineered skinanalogs mature and reach homeostasis in an in vivo assay. These findings harbor favorable implications regarding future clinical application.

Physiological Doses of Red Light Induce IL-4 Release in Cocultures between Human Keratinocytes and Immune Cells.

Phototherapy is routinely used for the treatment of various skin conditions and targeted therapy of superficial cancers. However, the molecular mechanisms behind their biological effects and the need for efficacy enhancing photosensitizers are not well addressed. Particularly, not much is known about the inherent effect of light from the visible spectrum on cytokine release and its downstream effects in keratinocytes and immune cells located in skin and therefore exposed to light. To address this, we delivered calibrated doses of well-defined light qualities (380 to 660nm) to cocultures of human keratinocytes and macrophage/dendritic cells in the absence or presence of the commonly used photosensitizer 8-methoxypsoralen (8-MOP). The experiments identified IL-4 as a key effector cytokine released by this coculture model with need for 8-MOP in the UVA1 /blue (380nm) and no requirement for photosensitizer in the red light spectrum (627nm). 3D organotypic skin cultures treated with IL-4 showed thickening of the epidermal layer and delayed differentiation. However unlike IL-4 and UVA1 /blue light treatment, red light did not reduce the expression of keratinocyte differentiation markers or increase signs of photo-oxidative damage. This supports the application of isolated red light as a possible alternative for photo-immunotherapy without need for additional photosensitizers.

Achillea asiatica extract and its active compounds induce cutaneous wound healing

Ethnopharmacological relevanceAchillea asiatica Serg. is a perennial herb belonging to the Asteraceae family that has long been traditionally used to treat acute intestinal and stomach inflammation, persistent fever, ulcers, wounds, and rheumatism. Aim of the studyWe investigated the effect of A. asiatica extract (AAE) on cutaneous wound healing. Materials and methodsTo assess the effect of AAE on wounds, an incisional Sprague-Dawley (SD) rat model was topically treated with AAE for 2 weeks. HaCaT keratinocytes, Hs68 dermal fibroblasts, and RAW 264.7 macrophages were used for in vitro experiments. After treatment with AAE, cell viability, cell migration, and production of nitric oxide (NO) and prostaglandin E2 (PGE2) were investigated. mRNA expression of collagen type I and III and inflammatory cytokines was measured by RT-PCR. The effect of AAE on activation of β-catenin and other markers was determined by Western blot analysis. ResultsAAE treatment significantly increased epithelialization and accelerated wound healing in SD rats. Meanwhile, AAE and its active compounds reduced NO and PGE2 release and mRNA expression of inflammatory cytokines in RAW 264.7 macrophages, reflecting anti-inflammatory activity. Furthermore, AAE and its constituents stimulated collagen expression in Hs68 fibroblasts by activating transforming growth factor-β and stimulated keratinocyte differentiation and motility by inducing β-catenin, Akt, and keratinocyte differentiation markers. ConclusionsAAE improves skin wounds in SD rats and supports keratinocyte development.

Molecular and cellular impact of Psoriasin (S100A7) on the healing of human wounds.

Psoriasin, which is also known as S100A7, is a member of the S100 protein family, a group of calcium-responsive signalling proteins. Psoriasin expression remains high in patients with psoriasis, whereas it is downregulated in patients with invasive breast carcinoma. This observation suggests that this protein may be a notable marker of keratinocyte function and differentiation during wound healing. The aim of the present study was to determine the cellular impact of Psoriasin in keratinocytes, which are the primary cell type associated with wound healing. Psoriasin expression in wound tissues was examined using reverse transcription-quantitative polymerase chain reaction and immunochemical staining. Knockdown of Psoriasin in HaCaT cells was performed using anti-Psoriasin ribozyme transgenes and the effect on growth, adhesion and migration of keratinocytes was subsequently determined using in vitro cellular functional assays. Psoriasin expression is upregulated in wounds, particularly at the wound edges. The present study demonstrated that Psoriasin is expressed in keratinocytes and is a fundamental regulator of keratinocyte migration. Significant increases in the rate of keratinocyte adhesion, migration and growth were observed in Psoriasin-deficient cells (P<0.01 vs. control). Application of small inhibitors identified the potential association of neural Wiskott-Aldrich syndrome protein, focal adhesion primase and rho-associated protein kinase signalling pathways with Psoriasin-regulated cell adhesion and motility. In conclusion, Psoriasin serves an important role in the wound healing process, suggesting that it may be utilized as a potential wound healing biomarker.