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Competing Interests: CONFLICTS OF INTEREST: The authors have nothing to disclose.

Competing Interests: CONFLICTS OF INTEREST: The authors have nothing to disclose.

Background: Alopecia areata (AA) is an autoimmune skin disease difficult to manage and treat. The pathogenesis of AA features a T-cell-associated autoimmune process, and systemic immunosuppressive therapy is prescribed widely for AA., Objective: To evaluate the efficacy and tolerance of systemic low-dose methotrexate (LD-MTX) therapy in treatment of recalcitrant AA multiplex., Methods: In a retrospective, non-controlled study, we evaluated 29 patients with recalcitrant AA treated with LD-MTX and assessed the therapeutic response according to severity of disease, disease duration, cumulative dose of MTX, and drug safety., Results: MTX was administered twice weekly, and the mean maximum weekly dose was 14.48 mg. The response was A5 (regrowth=100.0%) in 14 (48.3%) patients and A4 (regrowth of 75%~90%) in 12 (41.4%) patients. Three patients had poor response to LD-MTX treatment (A2: n=2 [6.9%], A1: n=1 [3.4%]). All three of the patients showing a poor response had disease durations exceeding 24 months. Relapse was observed in 31% of patients with more than 75% regrowth. Common side-effects were elevated liver enzyme levels and gastrointestinal discomfort., Conclusion: LD-MTX appears to be an effective and well-tolerated treatment for recalcitrant AA multiplex., Competing Interests: CONFLICTS OF INTEREST: The authors have nothing to disclose.

Background: Micro-needle patches have been recently used to increase skin permeability, which improves drug delivery, and for cosmetic purposes. However, these patches may often have limited efficacy due to insufficient skin penetration and reduced compliance caused by discomfort., Objective: We evaluated the efficacy and the safety of soluble micro-spicule containing epidermal growth factor (MS-EGF) for the treatment of periocular wrinkles., Methods: Twenty healthy volunteers aged 33 to 54 years were enrolled in a randomized, controlled, split-face study. For 4 weeks, a periocular wrinkle was treated daily with either a soluble MS-EGF cream or a cream containing EGF alone. All subjects underwent 8 weeks of follow-up. Efficacy was assessed using an ultrasonic measurement of dermal depth and density, digital skin image analysis, 5-point photonumeric scale for periocular wrinkles and subjective satisfaction., Results: MS-EGF group showed statistically significant increase of dermal depth and density compared to EGF alone group after 4 and 8 weeks. In addition, there was a marked improvement shown in clinical and 3-dimensional skin image in MS-EGF group. The treatments were well-tolerated; no significant side-effect was noted., Conclusion: The MS-EGF formulation may represent an effective and biocompatible advance in the treatment of periocular wrinkles., Competing Interests: CONFLICTS OF INTEREST: The authors have nothing to disclose.

Competing Interests: CONFLICTS OF INTEREST: The authors have nothing to disclose.

Background: Interleukin-17A (IL-17A) is mainly secreted from Th17 cells that are activated by various stimuli including CpG oligodeoxynucleotide, a Toll-like receptor 9 (TLR9) ligand. Recently, it has been demonstrated that keratinocytes play an important role in the pathogenesis of psoriasis., Objective: To investigate the potential role of keratinocytes, we examined whether TLR9 ligand CpG induces IL-17A expression in keratinocytes., Methods: We used HaCaT keratinocytes as a model system, and determined CpG-induced IL-17A using enzyme-linked immunosorbent assay and Western blot., Results: When HaCaT keratinocytes were treated with CpG, the expression of several cytokines including IL-17A, tumor necrosis factor-α and CCL20 was markedly increased. Treatment with nuclear factor (NF)-κB inhibitor significantly blocked the CpG-induced IL-17A production, indicating that CpG induced IL-17A expression through the NF-κB signaling pathway. In addition, IL-17A secreted from keratinocytes stimulated the CD4 + T cells, resulting in strong induction of IL-22 production., Conclusion: Since IL-22 is an important mediator for psoriatic inflammation, our data suggest that keratinocytes can participate in the pathogenesis of psoriasis via the TLR9-dependent IL-17A production.

Background: Keratinocytes are the major cells in epidermis, providing barrier components such as cornified cells through the sophisticated differentiation process. In addition, keratinocytes exerts their role as the defense cells via activation of innate immunity. It has been known that pathogen-associated molecular patterns (PAMPs) including double-strand RNA and nucleotides can provoke inflammatory reaction in keratinocytes., Objective: The aim of this study is to evaluate the effect of Ampelopsis japonica Makino extract (AE) on PAMPs-induced inflammatory reaction of keratinocytes., Methods: The effects of AE were determined using poly (I:C)-induced inflammation and imiquimod-induced psoriasiform dermatitis models., Results: In cultured keratinocytes, AE significantly inhibited poly(I:C)-induced expression of inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α. AE significantly inhibited poly(I:C)-induced release of caspase-1 active form (p20), and down-regulated nuclear factor-κB signaling pathway. In imiquimod-induced psoriasiform dermatitis model, topical application of AE resulted in significant reduction of epidermal hyperplasia., Conclusion: These results suggest that AE may be a potential candidate for the treatment of skin inflammation.

Background: S100A8 is differentially expressed in various cell types and is associated with a number of malignant disorders. S100A8 may affect tumor biology. However, its role in cutaneous squamous cell carcinoma (SCC) is not well established., Objective: This study aims to investigate the relationship between S100A8 and cutaneous SCC development., Methods: We performed immunohistochemical staining to detect S100A8 expression in facial skin specimens of premalignant actinic keratosis (AK), malignant SCC, and normal tissues. In addition, we utilized postconfluence and high calcium-induced differentiation in a culture system model. Furthermore, we constructed a recombinant adenovirus expressing GFP-tagged S100A8 to investigate the role of S100A8 in SCC cell differentiation., Results: S100A8 was significantly overexpressed in human cutaneous SCC compared to that in normal and AK tissues. S100A8 was gradually upregulated in SCC cells in a post-confluence-induced differentiation model. Overexpression of S100A8 in SCC cells induced by adenoviral transduction led to increased expression levels of differentiation markers, such as loricrin, involucrin, and filaggrin. S100A8 overexpression also increased loricrin and involucrin luciferase activity., Conclusion: S100A8 regulates cutaneous SCC differentiation and induces well-differentiated SCC formation in skin.

A20 is a negative regulator of nuclear factor κ-light‑chain-enhancer of activated B cells (NF-κB) signaling, and has been implicated in the pathogenesis of psoriasis through genome-wide association study (GWAS). In the present study, we investigated the putative role of A20 in epidermal keratinocytes. Immunohistochemical analysis showed that A20 was expressed in all layers of the epidermis, with an increasing pattern in the upper layers. In our model of calcium-induced keratinocyte differentiation, A20 expression was increased in a time-dependent manner. To investigate whether A20 affected keratinocyte differentiation, we overexpressed A20 in cultured keratinocytes. As a result, we noted that A20 overexpression did not affect keratinocyte differentiation, suggesting that A20 is not a direct modulator of keratinocyte differentiation. Interestingly, we found that A20 levels were decreased in psoriatic lesional skin compared to non-lesional areas. To investigate whether A20 played a role in the innate immune response of keratinocytes, we overexpressed A20 and then examined poly(I:C)-induced cytokine expression. We noted that A20 significantly inhibited poly(I:C)-induced cytokine production, and this effect was related to the inhibition of NF-κB signaling. These results suggest that the downregulation of A20 increased the susceptibility of keratinocytes to external stimuli, thus contributing to the development of psoriasis.

Background: Psoriasis is characterized by uncontrolled hyperproliferation, aberrant differentiation, and dermal infiltration of immune cells. Recent studies have reported that Wnt5a and Notch1 signaling are altered in psoriatic skin lesions., Objective: We aimed to investigate the interaction of Wnt5a with Notch 1 with respect to inflammation-mediated epidermal hyperproliferation in psoriasis., Methods: Expression of Wnt5a and Notch1 signaling-related proteins were examined in psoriatic skin biopsies. Wnt5a was upregulated in human keratinocytes by treating the cells with its recombinant form (rWnt5a)., Results: In psoriatic lesions, expression of Wnt5a increased while that of Notch1 decreased when compared to that in non-lesional and normal skin. Treatment with rWnt5a increased the proliferation of keratinocytes and increased their secretion of interleukin (IL)-23, IL-12, and tumor necrosis factor (TNF)-α. Further, exposure of keratinocytes to IL-1α, TNF-α, transforming growth factor-α, and interferon-γ downregulated Notch1 as well as HES 1, which is downstream to Notch1, but increased the Wnt5a levels. The upregulated Wnt5a in keratinocytes downregulated both Notch1 and HES1., Conclusion: Our data suggest that Wnt5a and Notch1 signaling exert counteracting influences on each other and are involved, in part, in the pathomechanism of psoriasis.

Background: Protease-activated receptor 2 (PAR-2) participates in various biological activities, including the regulation of epidermal barrier homeostasis, inflammation, pain perception, and melanosome transfer in the skin., Objective: To evaluate the basic physiological role of PAR-2 in skin., Methods: We investigated PAR-2 expression in human epidermis, skin tumors, and cultured epidermal cells using western blot and immunohistochemical analysis. Additionally, we examined the effect of the PAR-2 agonist, SLIGRL-NH2, on cultured keratinocytes., Results: Strong PAR-2 immunoreactivity was observed in the granular layer of normal human skin and the acrosyringium of the eccrine sweat glands. In contrast, weak PAR-2 immunoreactivity was seen in the granular layer of callused skin and in the duct and gland cells of the eccrine sweat glands. Interestingly, PAR-2 immunoreactivity was very weak or absent in the tumor cells of squamous cell carcinoma (SCC) and syringoma. PAR-2 was detected in primary keratinocytes and SV-40T-transformed human epidermal keratinocytes (SV-HEKs), an immortalized keratinocyte cell line, but not in SCC12 cells. SV-HEKs that were fully differentiated following calcium treatment displayed higher PAR-2 expression than undifferentiated SV-HEKs. Treatment of cultured SV-HEKs with PAR-2 agonist increased loricrin and filaggrin expression, a terminal differentiation marker., Conclusion: Our data suggest that PAR-2 is associated with terminal differentiation of epidermis and eccrine sweat glands.

Background: Adenosine is a nucleoside, in which an adenine molecule is attached to a ribofuranose sugar moiety. It can be released into the microenvironment by metabolically active cells, and then fulfills a multitude of functions in regulation of cell proliferation, by activating four subtypes of G protein-coupled adenosine receptors., Objective: In this study, we investigated the effect of adenosine on melanogenesis, using B16 melanoma cells., Methods: The toxic effects of adenosine on B16 melanoma cells were assessed. To understand the mechanism of the effect of adenosine on melanogenesis in B16 cells, melanin content and tyrosinase activity were measured. Tyrosinase, tyrosinase-related protein-1, and dopachrome tautomerase were monitored by Western blotting. Finally, adenosine was applied to zebrafish embryos, and its in vivo effect on pigmentation investigated., Results: At a low concentration, adenosine increased melanin content and tyrosinase activity, while a high dose of adenosine resulted in inhibition of tyrosinase activity. Western blotting showed that adenosine increased tyrosinase protein levels slightly, while high-dose adenosine decreased the expression of tyrosinase. In zebrafish tests, adenosine slightly inhibited body pigmentation., Conclusion: In this study, we investigated the effect of adenosine on melanogenesis, using the well-established B16 melanoma cell and zebrafish models. The results suggest that adenosine may inhibit pigmentation, through negative regulation of tyrosinase.

Background: The fibrous proteins of extracellular matrix (ECM) produced by dermal fibroblast contributes to the maintenance of connective tissue integrity., Objective: This study is carried out to identify the bioactive ingredient from natural products that enhances ECM production in dermal fibroblasts., Methods: Bioassay-directed fractionation was used to isolate the active ingredient from natural extracts. The effects of rasatiol (isolated from Raphanus sativus) on ECM production in primary cultured human dermal fibroblasts was investigated by enzyme linked immunosorbent assay and western blot analysis., Results: Rasatiol accelerated fibroblast growth in a dose-dependent manner and increased the production of type 1 collagen, fibronectin and elastin. Phosphorylation of p42/44 extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and Akt was remarkably increased by rasatiol, indicating that enhanced ECM production is linked to the activation of intracellular signaling cascades., Conclusion: These results indicate that rasatiol stimulates the fibrous components of ECM production, and may be applied to the maintenance of skin texture.

Background: The skin has many important functions such as protection, preservation, temperature regulation, and vitamin D synthesis. It is composed of a variety of cell types including keratinocytes, melanocytes and fibroblasts., Objective: We attempted to compare the gene expression profiles between keratinocytes, melanocytes and fibroblast, using cDNA microarray., Methods: Keratinocytes, melanocytes and fibroblasts were primary cultured from five foreskin specimens. Total RNAs were extracted and pooled to reduce the individual variations, and then used for cDNA microarray., Results: Total 12,028 genes were selected as the reliable genes whose expression was detected in at least one of the three cell types. By comparing the relative expression levels with cutoff limitation as a fourfold change, we obtained 126 fibroblast-specific, 179 keratinocyte-specific and 173 melanocyte-specific genes, many of which are known to be characteristically expressed in each cell type. In addition, we identified many genes whose skin-specific functions have not yet been determined., Conclusion: Our data provide important information on which to base further investigation into the specification of skin cell types.

In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation.

Background: Epigenetic modulation of gene expression occurs by various methods, including DNA methylation and histone modification. DNA methylation of specific genes may affect the chromatin structure, preventing access by the transcriptional machinery. Although gene expression is dramatically changed during keratinocyte differentiation, there is no evidence of epigenetic modulation during the process of epidermal stratification., Objective: We investigated whether epigenetic modulation is involved in keratinocyte differentiation-specific gene regulation., Methods: We used trypsin to produce epidermal fragmentation (named T1-T4) and performed a morphological analysis using hematoxylin-eosin stain and cytokeratin expression based on reverse transcription polymerase chain reaction. We then constructed a DNA methylation microarray., Results: Each epidermal fragment showed morphological features of the epithelial layer. T1 represented the basal layer, T2 was the spinous layer, T3 was the granular layer, and T4 was the cornified layer. The level of the K14 proliferation marker was increased in the T1 fraction, and the level of K10 differentiation marker was increased in the T2-T4 fractions. Using a methylation microarray with the T1 and T4 fractions, we obtained many hypermethylated and hypomethylated genes from differentiated keratinocytes., Conclusion: The importance of epigenetic modulation in target gene expression during keratinocyte differentiation is identified.

Hair regression and balding are distressing concerns for an increasing number of people due to changes in lifestyle and serious nutritional imbalances. Therapies for treatment of hair loss are needed. Among potential therapeutics, adenosine has been suggested as a potent regulator of hair growth. In this study, we investigated the effects of adenosine on hair follicles and dermal papilla (DP) cells, and the mechanism underlying the action of adenosine. Hair follicles are organs, including DP cells, that are responsible for the production of hair fibers by inducing and maintaining the hair growth phase (anagen). In a culture of DP cells in vitro, adenosine stimulated proliferation of DP cells by increasing thymidine uptake. Subsequently, adenosine activated and elongated the anagen phase by increasing the uptake of radiolabeled cysteine in an organ culture of mouse vibrissae hair follicles. We also confirmed that adenosine promoted the expression of several growth factors that are responsible for hair growth, including fibroblast growth factors (FGF)-7, FGF-2, insulin-like growth factor (IGF)-1, and vascular endothelial growth factor (VEGF) in a cDNA microarray with semi-quantitative RT-PCR. Transcriptional activation of β-catenin in DP cells was increased by adenosine in a luciferase assay. β-catenin is a co-activator of Wnt/β-catenin signaling that induces morphogenesis and differentiation of hair follicles and also acts to transactivate downstream signaling pathways, including the ERK pathway. Using Western blotting, we found that adenosine stimulated phosphorylation of ERK, CREB and AKT. These results suggest that adenosine stimulates growth of hair follicles by triggering the expression of growth factors and β-catenin, and by inducing their downstream target signaling pathways.

Background: The extracellular matrix (ECM) produced by dermal fibroblasts supports skin structure, and degradation and/or reduced production of ECM are the main causes of wrinkle formation., Objective: The aim of this study was to identify the active ingredient that enhances ECM production in dermal fibroblasts., Methods: Polarity-based fractionation was used to isolate the active ingredient from natural extracts, and the effects of cedrol (isolated from Pterocarpus indicusirginia) on ECM production in cultured human dermal fibroblasts was investigated by reverse transcription-polymerase chain reaction, enzyme linked immunosorbent assay, and Western blot analysis., Results: Cedrol accelerated fibroblast growth in a dose-dependent manner and increased the production of type 1 collagen and elastin. Phosphorylation of p42/44 extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and Akt was markedly increased by cedrol, indicating that enhanced ECM production is linked to activation of intracellular signaling cascades., Conclusion: These results indicate that cedrol stimulates ECM production, with possible applications to the maintenance of skin texture.

Background: Autologous platelet-rich plasma has attracted attention in various medical fields recently, including orthopedic, plastic, and dental surgeries and dermatology for its wound healing ability. Further, it has been used clinically in mesotherapy for skin rejuvenation., Objective: In this study, the effects of activated platelet-rich plasma (aPRP) and activated platelet-poor plasma (aPPP) have been investigated on the remodelling of the extracellular matrix, a process that requires activation of dermal fibroblasts, which is essential for rejuvenation of aged skin., Methods: Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared using a double-spin method and then activated with thrombin and calcium chloride. The proliferative effects of aPRP and aPPP were measured by [(3)H]thymidine incorporation assay, and their effects on matrix protein synthesis were assessed by quantifying levels of procollagen type I carboxy-terminal peptide (PIP) by enzyme-linked immunosorbent assay (ELISA). The production of collagen and matrix metalloproteinases (MMP) was studied by Western blotting and reverse transcriptase-polymerase chain reaction., Results: Platelet numbers in PRP increased to 9.4-fold over baseline values. aPRP and aPPP both stimulated cell proliferation, with peak proliferation occurring in cells grown in 5% aPRP. Levels of PIP were highest in cells grown in the presence of 5% aPRP. Additionally, aPRP and aPPP increased the expression of type I collagen, MMP-1 protein, and mRNA in human dermal fibroblasts., Conclusion: aPRP and aPPP promote tissue remodelling in aged skin and may be used as adjuvant treatment to lasers for skin rejuvenation in cosmetic dermatology.

Background: β-catenin plays a pivotal role in hair follicle development and hair growth cycle., Objective: The aim of this study was to identify β-catenin-regulated genes in cultured human hair outer root sheath (ORS) cells., Methods: Primary cultured ORS cells were transduced with recombinant adenovirus expressing N-terminal truncated β-catenin (constitutive active form), and β-catenin-regulated genes were identified., Results: Overexpression of the constitutively active form of β-catenin led to induction of Sox9 expression at both mRNA and protein levels. To investigate the potential role of Sox9, we made the recombinant adenovirus expressing green fluorescent protein-tagged Sox9, and then transduced into cultured ORS cells. Interestingly, Sox9 induced the expression of keratin 15, increased the proliferation of ORS cells in vitro, and enhanced colony-forming activity., Conclusion: Our results suggest that Sox9 is a β-catenin-regulated gene in ORS cells, and has potential importance in the regulation of hair follicle homeostasis.

Nuclear factor of activated T-cells (NFAT) proteins are, calcium-regulated transcription factors, key regulator of stimulation-dependent gene activation. In our microarray analysis for the genes expressed in human black and white hairs, NFAT2 was significantly upregulated in the white hair, compared to the black hair. The aim of this study was to investigate functional role of NFAT2 in melanogenesis. Western blot analysis was performed to investigate the expression of NFAT2 protein in B16 melanoma cells. Our data showed that NFAT2 expression was increased in the hypopigmented B16 cells, while tyrosinase and MITF expression was decreased. To investigate the potential role of NFAT2, the recombinant adenovirus expressing microRNA specific for NFAT2 was transduced into the cultured B16 melanoma cells. Consistently, inhibition of NFAT2 enhanced tyrosinase activity and melanin content. Moreover, cyclosporine A, which is known as a calcineurin inhibitor blocking NFAT activation, enhanced tyrosinase activity and melanin content. These data suggest that NFAT2 may play an important role in regulation of melanogenesis in melanocyte.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. In this study, we investigated the role of the POU domain-containing transcription factor Brn2 in keratinocyte differentiation. Immunohistochemical analysis showed that Brn2 is expressed primarily in the upper granular layer. Consistent with its epidermal localization, Brn2 expression was highly induced at 14 days after calcium treatment of cultured normal human epidermal keratinocytes. When Brn2 was overexpressed by adenoviral transduction, Brn2 led to increased expression of the differentiation-related genes involucrin, filaggrin, and loricrin in addition to inhibition of their proliferation. Chromatin immunoprecipitation demonstrated that Brn2 bound to the promoter regions of these differentiation-related genes. We injected the purified Brn2 adenovirus into rat skin, which led to a thickened epidermis with increased amounts of differentiation related markers. The histopathologic features of adenovirus-Brn2 injected skin tissues looked similar to the features of lichen planus, a human skin disease showing chronic inflammation and well-differentiated epidermal changes. Moreover, Brn2 is shown to be expressed in almost all cell nuclei of the thickened epidermis of lichen planus, and Brn2 also attracts T lymphocytes. Our results demonstrate that Brn2 is probably a transcriptional factor playing an important role in keratinocyte differentiation and probably also in the pathogenesis of lichen planus lesions.

Background: Through differentiation processes, keratinocytes provide a physical barrier to our bodies and control skin features such as moisturization, wrinkles and pigmentation. Keratinocyte differentiation is disturbed in several skin diseases such as psoriasis and atopic dermatitis., Objective: The aim of this study is to evaluate the keratinocyte differentiation-enhancing effect of rose absolute oil (RAO)., Methods: Primary cultured human normal keratinocytes were treated with RAO, and differentiation then checked by the expression of marker genes., Results: RAO did not induce cytotoxicity on cultured keratinocytes at a dose of 10microM. The level of involucrin, an early marker for keratinocyte differentiation, was significantly increased by RAO. Concomitantly, RAO increased involucrin promoter activity, indicating that RAO increased involucrin gene expression at the mRNA level. Furthermore, RAO increased the level of filaggrin in cultured keratinocytes, and in the granular layer of mouse skin. In line with these results, RAO decreased the proliferation of keratinocytes cultured in vitro. When RAO was applied topically on the tape-stripped mouse skins, it accelerated the recovery of disturbed barrier function., Conclusion: These results suggest that RAO may be applicable for the control of skin texture and keratinocyte differentiation-related skin diseases.

Background: Fibroblasts produce many components of the extracellular matrix (ECM) and so they contribute to the maintenance of connective tissue integrity., Objective: The aim of this study is to evaluate the effect of velvet antler extract (VAE) on the ECM production of dermal fibroblasts cultured in vitro., Methods: Primary cultured human dermal fibroblasts were treated with VAE, and then the ECM production was determined by RT-PCR, ELISA and Western blot analysis. Furthermore, the change of gene expression according to VAE treatment was evaluated by cDNA microarray., Results: VAE accelerated the growth of fibroblasts in a dose-dependent manner. VAE increased the production of several ECM components, including type 1 collagen, fibronectin and elastin. In line with these results, the phosphorylations of p42/44 ERK and p38 mitogen-activated protein kinase were markedly increased by VAE, suggesting that the enhancement of ECM production may be linked to the activation of intracellular signaling cascades. VAE also significantly increased cell migration on an in vitro scratch wound test. In cDNA microarray, many genes related with connective tissue integrity were identified to be up-regulated by VAE., Conclusion: These results suggest that VAE has a potential to stimulate ECM production, and VAE may be applicable for maintaining the skin's texture.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. To examine the gene expression profile in calcium-induced keratinocyte differentiation, we constructed a normalized cDNA library using mRNA isolated from these calcium-treated keratinocytes. After sequencing about 10,000 clones, we were able to obtain 4,104 independent genes. They consisted of 3,699 annotated genes and 405 expressed sequence tags (ESTs). Some were the genes involved in constituting epidermal structures and others were unknown genes that are probably associated with keratinocytes. In particular, we were able to identify genes located at the chromosome 1q21, the locus for the epidermal differentiation complex, and 19q13.1, another probable locus for epidermal differentiation-related gene clusters. One EST located at the chromosome 19q13.1 showed increased expression by calcium treatment, suggesting a novel candidate gene relevant to keratinocyte differentiation. These results demonstrate the complexity of the transcriptional profile of keratinocytes, providing important clues on which to base further investigations of the molecular events underlying keratinocyte differentiation.

We obtained metastasized melanoma tissue from a primary acral lentiginous melanoma (ALM) patient and established a melanoma cell line named primary culture of melanoma cell derived from lymph node (PML)-1. PML-1 cells had a light brown color and decreased the expression of melanogenesis markers, including tyrosinase (TYR), microphthalmia-associated transcription factor, and tyrosinase-related protein-1. To identify genes differentially regulated in PML-1 melanoma cells, we performed DNA microarray and two-dimensional matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. Among the candidate genes identified, we chose NAD(P)H:quinone oxidoreductase-1 (NQO1) for further study. Reverse transcription-PCR and western blot analyses showed that NQO1 was markedly decreased in PML-1 cells and in several amelanotic melanoma cell lines. To investigate whether NQO1 affects the melanogenesis, we treated the cultured normal human melanocytes (NHMC) and zebrafish with NQO1 inhibitors, ES936 and dicoumarol. Interestingly, melanogenesis was significantly decreased by the addition of NQO1 inhibitors in both NHMC and zebrafish models. In contrast, overexpression of NQO1 using a recombinant adenovirus clearly induced melanogenesis, concomitantly with an increase of TYR protein level. These results suggest that NQO1 is a positive regulator of the pigmentation process.

Background: Nkx2.5 is a homeodomain-containing nuclear transcription protein that has been associated with acute T-lymphoblastic leukemia. In addition, Nkx2.5 has an essential role in cardiomyogenesis. However, the expression of Nkx2.5 in the skin has not been investigated., Objective: In an attempt to screen the differentially regulated genes involved in keratinocyte differentiation, using a cDNA microarray, we identified Nkx2.5 as one of the transcription factors controlling the expression of proteins associated with keratinocyte differentiation., Methods: To investigate the expression of Nkx2.5 during keratinocyte differentiation, we used a calcium-induced keratinocyte differentiation model., Results: RT-PCR and Western blot analysis revealed that the expression of Nkx2.5, in cultured human epidermal keratinocytes, increased with calcium treatment in a time-dependent manner. In normal skin tissue, the expression of Nkx2.5 was detected in the nuclei of the keratinocytes in all layers of the epidermis except the basal layer by immunohistochemistry. In addition, the expression of Nkx2.5 was significantly increased in psoriasis and squamous cell carcinoma, but was barely detected in atopic dermatitis and basal cell carcinoma., Conclusion: These results suggest that Nkx2.5 may play a role in the change from proliferation to differentiation of keratinocytes and in the pathogenesis of skin disease with aberrant keratinocyte differentiation.

Background: Psoriasis is a chronic inflammatory skin disease that affects approximately 1~3% of the general population., Objective: We performed cDNA microarray analysis with using the dendrimer labelling method to investigate the gene expression profile in the peripheral blood mononuclear cells (PBMCs) of psoriatic patients., Methods: The peripheral blood mononuclear cells of 5 patients with psoriasis and 8 control subjects were used in the gene expression analyses of psoriasis., Results: We identified 212 differentially expressed genes that showed at least a two-fold induction and/or reduction in psoriatic patients. Among those, 63 genes, including CD44, CD56 and IL7R, were induced, while 139 genes, including the sphingosine kinase 1 and p16-INK genes, were reduced in the psoriatic patients., Conclusion: We can speculate that these genes may have a role for the pathogenesis of psoriasis via their affecting different cellular functions. Our results suggest a possible mechanism by which activated immune cells migrate from the blood to the skin in psoriatic patients, and we provide novel putative targets for developing drugs to treat psoriasis.

Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern-recognition receptors expressed in keratinocytes. We found that keratinocyte Toll-like receptors (TLRs) 2 and 4 and Dectin-1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL-37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin-1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL-37. In addition, TLR2, Dectin-1 and, to an extent, TLR4 are essential for the MU-mediated expression of CXCL8, CCL2 and LL-37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin-1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL-37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin-1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.

During a search for keratinocyte differentiation-related genes, we obtained a cDNA fragment from the 5'-untranslated region of a previously identified splicing variant of desmoglein 3 (Dg3). This transcript encodes a protein of 282 amino acids, which corresponds to the N-terminal truncated intracellular domain of Dg3 (deltaNDg3). Northern blot analysis detected a 4.6-kb transcript matching the predicted size of deltaNDg3 mRNA, and Western blot analysis with an antibody raised against the Dg3 C-terminus (H-145) detected a 31-kDa protein. Increased deltaNDg3 expression was observed in differentiating keratinocytes by RT-PCR and Western blot analysis, suggesting that deltaNDg3 is indeed a differentiation-related gene product. In immunohistochemical studies of normal and pathologic tissues, H-145 antibody detected the protein in the cytoplasm of suprabasal layer cells, whereas an antibody directed against the N-terminal region of Dg3 (AF1720) reacted with a membrane protein in the basal layer. In addition, deltaNDg3 transcript and protein were upregulated in psoriatic epidermis, and protein expression appeared to increase in epidermal tumors including Bowen's disease and squamous cell carcinoma. Moreover, overexpression of deltaNDg3 led to increased migration and weakening of cell adhesion. These results suggest that deltaNDg3 have a role in keratinocyte differentiation, and that may be related with tumorigenesis of epithelial origin.

In a previous search for the differentially expressed genes in keratinocyte differentiation, we identified neutrophil gelatinase-associated lipocalin (NGAL) as a calcium-induced gene. In this study, we further verified the expression of NGAL in cultured keratinocytes as well as in several skin diseases. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and ELISA clearly showed that NGAL expression was markedly increased in calcium-induced keratinocyte differentiation in vitro. However, in our previous report, NGAL expression was not detected in normal skin tissue except for hair follicle by in situ hybridization and immunohistochemistry, indicating the difference of cell status between in vitro and in vitro conditions. Interestingly, NGAL expression was highly increased in psoriasis-like inflammatory disorders (lichen planus and pityriasis rubura pilaris) and skin cancers (keratoacanthoma and squamous cell carcinoma), implying that NGAL may be related with the epidermal hyperplasia. Collectively, these results reveal the potential importance of NGAL in the maintenance of skin homeostasis.

Ceramide is an important constituent of stratum corneum lipids, which act as both physical barriers and signal modulators. We synthesized several ceramide derivatives and investigated their effects on keratinocyte differentiation. RT-PCR and Western blotting showed that the novel synthetic ceramide derivatives K6PC-4 [N-(2,3-dihydroxypropyl)-2-hexyl-3-oxo-decanamide], K6PC-5, [N-(1,3-dihydroxypropyl-2-hexyl-3-oxo-decanamide] and K6PC-9 (N-ethanol-2-hexyl-3-oxo-decanamide) [corrected] These ceramide derivatives elicited a rapid transient increase in intracellular calcium levels, which were measured using laser scanning confocal microscopy. In addition, K6PC-4, K6PC-5, and K6PC-9 stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In a reconstituted epidermis model, K6PC-4, K6PC-5, and K6PC-9 significantly increased keratin 1 expression in the suprabasal layer. These results indicate that these novel synthetic ceramide derivatives have the potential to promote keratinocyte differentiation, suggesting that the lipid molecules are applicable for treating skin diseases involving abnormal keratinocyte differentiation.

The diverse functions of microtubules (MT) in different cells and tissues may be facilitated by compositional changes in tubulin isotypes. We obtained partial cDNA clones of class II beta-tubulin from a library of differentiating normal human epidermal keratinocytes (NHEK) cells, whereas screening via subtractive hybridization for genes involved in calcium-induced keratinocyte differentiation. Analysis of the isotypic composition of beta-tubulin from NHEK cells revealed elevations in class II beta-tubulin concentrations at both protein and message levels during cell differentiation, resulting in increased rates of incorporation of class II beta-tubulin into MT. Immunohistochemistry of normal and pathologic skin tissues showed that class II beta-tubulin occurred in the granular layer of the epidermis and in differentiated areas of carcinomas. Class II beta-tubulin was, however, not observed in the uppermost granular and cornified layers of normal epidermis. Further experiments showed that MT were likely to decay in the final stage of terminal differentiation during formation of the cornified envelope. Our results suggest that there is differential modulation of MT composition and stability during keratinocyte differentiation.