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Background: Sensitive skin represents hyperactive sensory symptoms showing exaggerated reactions in response to internal stimulants or external irritants. Although sensitive skin is a very common condition affecting an estimated 50% of the population, its pathophysiology remains largely elusive, particularly with regard to its metabolic aspects., Objective: The objective of our study was to investigate the pathogenesis of sensitive skin., Methods: We recruited healthy participants with 'sensitive' or 'non-sensitive' skin based on standardized questionnaires and 10% lactic acid stinging test, and obtained skin samples for microarray analysis and subsequent experiments., Results: Microarray transcriptome profiling revealed that genes involved in muscle contraction, carbohydrate and lipid metabolism, and ion transport and balance were significantly decreased in sensitive skin. These altered genes could account for the abnormal muscle contraction, decreased ATP amount in sensitive skin. In addition, pain-related transcripts such as TRPV1, ASIC3 and CGRP were significantly up-regulated in sensitive skin, compared with non-sensitive skin., Conclusions: Our findings suggest that sensitive skin is closely associated with the dysfunction of muscle contraction and metabolic homeostasis., (Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: β-Catenin, the transducer of Wnt signaling, is critical for the development and growth of hair follicles. In the absence of Wnt signals, cytoplasmic β-catenin is phosphorylated by glycogen synthase kinase (GSK)-3 and then degraded. Therefore, inhibition of GSK-3 may enhance hair growth via β-catenin stabilization. Valproic acid is an anticonvulsant and a mood-stabilizing drug that has been used for decades. Recently, valproic acid was reported to inhibit GSK-3β in neuronal cells, but its effect on human hair follicles remains unknown., Objectives: To determine the effect of VPA on human hair growth., Methods: We investigated the effect of VPA on cultured human dermal papilla cells and outer root sheath cells and on an in vitro culture of human hair follicles, which were obtained from scalp skin samples of healthy volunteers. Anagen induction by valproic acid was evaluated using C57BL/6 mice model., Results: Valproic acid not only enhanced the viability of human dermal papilla cells and outer root sheath cells but also promoted elongation of the hair shaft and reduced catagen transition of human hair follicles in organ culture model. Valproic acid treatment of human dermal papilla cells led to increased β-catenin levels and nuclear accumulation and inhibition of GSK-3β by phosphorylation. In addition, valproic acid treatment accelerated the induction of anagen hair in 7-week-old female C57BL/6 mice., Conclusions: Valproic acid enhanced human hair growth by increasing β-catenin and therefore may serve as an alternative therapeutic option for alopecia., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Chemicals with a molecular weight <500 and adequate lipid solubility can penetrate the intact human skin. As many lipid ingredients in moisturizers have molecular weights <500, the lipid ingredients may penetrate into the skin and affect skin responses to UV; however, little is known about this phenomenon., Objective: To evaluate the effects of major lipid ingredients in moisturizers on skin responses to UV in tape-stripped human skin in vivo., Methods: We evaluated the effects of three major lipid ingredients in moisturizers (cholesterol, linoleic acid, and a synthetic ceramide, N-oleoyl-phytosphingosine) on skin responses to UV in the tape-stripped skin of healthy volunteers. After 2 days of lipid-application, the areas were irradiated with UV, and skin samples were obtained 24h after irradiation. Histologic features and the expression of the markers of collagen metabolism and inflammatory mediators were evaluated., Results: Compared to vehicle, topical cholesterol significantly decreased the degree of dermal inflammatory infiltrates and exocytosis, and also decreased the expression of MMP-1, IL-6, and IL-1ß mRNA. In contrast, topical linoleic acid increased the induction of apoptotic cells, and the expression of MMP-1 and IL-6 mRNA. N-oleoyl-phytosphingosine increased the expression of MMP-1 and IL-6 mRNA, while decreasing the expression of COX-2 mRNA., Conclusions: Topical cholesterol can protect the barrier-disrupted skin against UV-induced damage, while linoleic acid or N-oleoyl-phytosphingosine alone has the potential to aggravate the damage., (Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Glycosaminoglycans (GAGs) have various structural and physiological regulatory functions in skin, including tissue water maintenance, due to their high water-holding capacity., Objective: To investigate changes of GAGs during intrinsic aging and photoaging of human skin and their correlations with water content., Methods: Samples of sun-protected buttock and sun-exposed forearm skin were obtained from young male (21-30 years, n=8) and female (20-33 years, n=8) subjects, as well as old male (70-78 years, n=8) and female (70-80 years, n=8) subjects, and their epidermal and dermal contents of hyaluronic acid (HA), total sulfated GAG (tsGAG), total uronic acid (tUA), and tissue water were measured. HA content was determined by enzyme-linked immunosorbent assay using HA-binding protein, tsGAG by the sulfated GAG assay kit using 1,9-dimethylmethylene blue, tUA by carbazole reaction, and tissue water by subtraction of tissue dry weight from wet weight., Results: In the buttock, HA was higher in dermis than in epidermis, while tsGAG and tUA were higher in epidermis. In intrinsically aged buttock, epidermal HA and dermal tsGAG and tUA decreased. However, when analyzed for each gender, epidermal tsGAG, tUA, and tissue water decreased only in females. Forearm/buttock ratios of each molecule were compared for determination of photoaging-dependent changes. Forearm/buttock ratios of HA, tsGAG, tUA, and tissue water increased in aged dermis, but showed no change in aged epidermis. When analyzed for each gender, ratios of epidermal HA and tissue water increased only in aged females, while ratios of epidermal tsGAG, tUA, and tissue water decreased only in aged males. Correlations of water content with HA, tsGAG, and tUA were found in epidermis, but not with tsGAG in dermis., Conclusion: These intrinsic aging- and photoaging-dependent GAG changes and their correlations with water content provide new insights into the pathophysiology of dry skin in the elderly., (Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Thrombospondin-1 (TSP-1) is a matricellular glycoprotein and recognized as an inhibitor of angiogenesis and an activator of transforming growth factor-beta (TGF-beta). Although TSP-1 expression has been shown to be regulated by various stimuli including UV in some types of cell, more work need to be done to understand the regulation of TSP-1 expression and its functional significances in many other types of cell., Objective: In this study, we investigated the effect of UV on TSP-1 expression in human skin dermis and dermal fibroblasts and the role of TSP-1 on the type I procollagen expression after UV exposure., Methods: Human buttock skin and human dermal fibroblasts were irradiated with UV. The mRNA and the protein levels of TSP-1 or type I procollagen were measured by real-time polymerase chain reaction and Western blotting, respectively., Results: We found that UV irradiation increased TSP-1 expression at the mRNA and protein levels in human skin dermis and dermal fibroblasts. UV-induced TSP-1 expression was greatly suppressed by inhibition of the PI3K, Akt, or mTOR pathways. The inhibition of TSP-1 activity by a blocking peptide or suppression of TSP-1 expression by specific TSP-1 small interfering RNA augmented the UV-induced decrease of type I procollagen expression., Conclusion: Our results suggest that UV-induced TSP-1 plays an important role on alleviating and/or recovering the type I procollagen expression downregulated by UV in human dermal fibroblasts., (Copyright (c) 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

In this study, a vacuum distillation of a mixture of LiCl-KCl eutectic salt and rare-earth oxidative precipitates was performed to separate a pure LiCl-KCl eutectic salt from the mixture. Also, a dechlorination and oxidation of the rare-earth oxychlorides was carried out to stabilize a final waste form. The mixture was distilled under a range of 710-759.5Torr of a reduced pressure at a fixed heating rate of 4 degrees C/min and the LiCl-KCl eutectic salt was completely separated from the mixture. The required time for the salt distillation and the starting temperature for the salt vaporization were lowered with a reduction in the pressure. Dechlorination and oxidation of the rare-earth oxychlorides was completed at a temperature below 1300 degrees C and this was dependent on the partial pressure of O2. The rare-earth oxychlorides (NdOCl/PrOCl) were transformed to oxides (Nd2O3/PrO2) during the dechlorination and oxidation process. These results will be utilized to design a concept for a process for recycling the waste salt from an electrorefining process.

Eicosapentaenoic acid (EPA) is an omega-3 (omega-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-alpha-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-alpha induced MMP-9 expression by NF-kappaB-dependent pathway. Pretreatment of EPA inhibited TNF-alpha-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IkappaB-alpha phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-kappaB. EPA inhibited TNF-alpha-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKalpha-dependent event. Taken together, we demonstrate that EPA inhibits TNF-alpha-induced MMP-9 expression through inhibition of p38 and Akt activation.

Skin aging can be attributed to photoaging (extrinsic) and chronological (intrinsic) aging. Photoaging and intrinsic aging are induced by damage to human skin attributable to repeated exposure to ultraviolet (UV) irradiation and to the passage of time, respectively. In our previous report, eicosapentaenoic acid (EPA) was found to inhibit UV-induced matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Therefore, we investigated the effects of EPA on UV-induced skin damage and intrinsic aging by applying EPA topically to young and aged human skin, respectively. By immunohistochemical analysis and Western blotting, we found that topical application of EPA reduced UV-induced epidermal thickening and inhibited collagen decrease induced by UV light. It was also found that EPA attenuated UV-induced MMP-1 and MMP-9 expression by inhibiting UV-induced c-Jun phosphorylation, which is closely related to UV-induced activator protein-1 activation, and by inhibiting JNK and p38 activation. EPA also inhibited UV-induced cyclooxygenase-2 (COX-2) expression without altering COX-1 expression. Moreover, it was found that EPA increased collagen and elastic fibers (tropoelastin and fibrillin-1) expression by increasing transformin growth factor-beta expression in aged human skin. Together, these results demonstrate that topical EPA has potential as an anti-skin-aging agent.

To understand the molecular alterations occurring during the aging process, we compared mitogen-activated protein (MAP) kinase activities in the intrinsically aged and photoaged skins in the same individuals. Furthermore, we investigated the molecular events related to MAP kinase changes in intrinsically aged and photoaged skins. We found that extracellular-signal-regulated kinase (ERK) activity in photoaged skin was reduced, and that the activities of c-Jun N-terminal kinase (JNK) and p38 kinase were increased compared with intrinsically aged skin in the same individuals. Phospho-c-Jun levels and activator protein 1 activities in photoaged skin were also higher than in intrinsically aged skin. Moreover, catalase activity was found to be much reduced in primary dermal fibroblasts from photoaged skin, and as a result, H2O2 accumulated more in primary dermal fibroblasts in photoaged skin. In addition, treating primary dermal fibroblasts from photoaged skin with catalase reduced H2O2 levels, reversed aging-dependent MAP kinase changes, and inhibited matrix metalloproteinase (MMP)-1 expression. Our results indicate that the accumulation of reactive oxygen species due to catalase attenuation may be a critical aspect of the MAP kinase signaling changes that may lead to skin aging and photoaging in human skin in vivo. Thus, the induction and regulation of endogenous antioxidant enzymes including catalase may offer a strategy for preventing and treating skin aging.

Ultraviolet (UV) irradiation regulates UV-responsive genes, including matrix metalloproteinases (MMPs). Moreover, UV-induced MMPs cause connective tissue damage and the skin to become wrinkled and aged. Here, we investigated the effect of eicosapentaenoic acid (EPA), a dietary omega-3 fatty acid, on UV-induced MMP-1 expression in human dermal fibroblasts (HDFs). We found that UV radiation increases MMP-1 expression and that this is mediated by p44 and p42 MAP kinase (ERK) and Jun-N-terminal kinase (JNK) activation but not by p38 activation. Pretreatment of HDFs with EPA inhibited UV-induced MMP-1 expression in a dose-dependent manner and also inhibited the UV-induced activation of ERK and JNK by inhibiting ERK kinase (MEK1) and SAPK/ERK kinase 1 (SEK1) activation, respectively. Moreover, inhibition of ERK and JNK by EPA resulted in the decrease of c-Fos expression and c-Jun phosphorylation/expression induced by UV, respectively, which led to the inhibition of UV-induced activator protein-1 DNA binding activity. This inhibitory effect of EPA on MMP-1 was not mediated by an antioxidant effect. We also found that EPA inhibited 12-O-tetradecanoylphorbol-13-acetate- or tumor necrosis factor-alpha-induced MMP-1 expression in HDFs and UV-induced MMP-1 expression in HaCaT cells. In conclusion, our results demonstrate that EPA can inhibit UV-induced MMP-1 expression by inhibiting the MEK1/ERK/c-Fos and SEK1/JNK/c-Jun pathways. Therefore, EPA is a potential agent for the prevention and treatment of skin aging.

Dehydroepiandrosterone (DHEA) and its sulfate conjugate (DHEA-S) are the most abundantly produced human adrenal steroids to be reduced with age. DHEA may be related to the process of skin aging through the regulation and degradation of extracelluar matrix protein. In this study, we demonstrate that DHEA can increase procollagen synthesis and inhibit collagen degradation by decreasing matrix metalloproteinases (MMP)-1 synthesis and increasing tisuue inhibitor of matrix metalloprotease (TIMP-1) production in cultured dermal fibroblasts. DHEA was found to inhibit ultraviolet (UV)-induced MMP-1 production and the UV-induced decrease of procollagen synthesis, probably due to the inhibition of UV-induced AP-1 activity. DHEA (5%) in ethanol:olive oil (1:2) was topically applied to buttock skin of volunteers 12 times over 4 weeks, and was found to significantly increase the expression of procollagen alpha1(I) mRNA and protein in both aged and young skin. On the other hand, topical DHEA significantly decreased the basal expression of MMP-1 mRNA and protein, but increased the expression of TIMP-1 protein in aged skin. We also found that DHEA induced the expressions of transforming growth factor-beta1 and connective tissue growth factor mRNA in cultured fibroblasts and aged skin, which may play a role in the DHEA-induced changes of procollagen and MMP-1 expression. Our results suggest the possibility of using DHEA as an anti-skin aging agent.

Photoaged skin contains elastotic materials in the upper reticular dermis. This phenomenon is commonly known as solar elastosis. In this study, we investigated the effects of heat on the expression of tropoelastin and fibrillin-1, two main components of elastic fibers, and on matrix metalloproteinase (MMP)-12, the most active MMP against elastin, in human skin in vivo. Heat was found to increase tropoelastin mRNA and protein expression in the epidermis and in the dermis. Fibrillin-1 mRNA and protein expression were increased by heat in the epidermis, but were decreased in the dermis. We found that pre-treatment of skin with N-acetyl cysteine or genistein for 24 h prior to heat treatment inhibited the heat-induced expression of tropoelastin, but not of fibrillin-1. These data indicate that reactive oxygen species may play a role in tropoelastin expression by heat, but not in fibrillin-1 expression. We also found that heat treatment increases MMP-12 mRNA and protein expression in human skin. Our results suggest that the abnormal production of tropoelastin and fibrillin by heat in human skin and that their degradation by various MMP, such as MMP-12, may contribute to the accumulation of elastotic material in photoaged skin.

Although many studies have been performed to elucidate the molecular consequences of ultraviolet irradiation, little is known about the effect of infrared radiation on skin aging. In addition to photons, heat is likely to be generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stress. Here we investigated the effect of heat shock on the expressions of matrix metalloproteinase (MMP)-1, MMP-2, and MMP-3 in cultured human skin fibroblasts. Heat shock induced the expression of MMP-1 and MMP-3, but not MMP-2, at the mRNA and protein levels in a temperature-dependent manner, and caused the rapid activation of three distinct mitogen-activated protein kinases (MAPK), extracelluar signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. The heat shock-induced MMP-1 and MMP-3 expression was suppressed by the inhibition of ERK and JNK but not by p38 MAPK inhibition. Furthermore, heat shock increased the synthesis and release of interleukin-6 (IL-6) into culture media. The specific inhibition of IL-6 using a monoclonal antibody against IL-6 greatly reduced the expression of MMP-1 and MMP-3 induced by heat shock. Taken together, our results suggest that ERK and JNK play an important role in the induction of MMP-1 and MMP-3 by heat shock and that the heat shock-induced expression of MMP-1 and MMP-3 is mediated via an IL-6-dependent autocrine mechanism.

Background: Minoxidil has been widely used to treat androgenetic alopecia, but little is known about its pharmacological activity or about the identity of its target cells in hair follicles. We hypothesized that minoxidil has direct effects on the proliferation and apoptosis of dermal papilla cells (DPCs) of human hair follicle., Objective: To elucidate the mechanism of topical minoxidil action in terms of stimulating hair growth., Methods: We evaluated cell proliferations in cultured DPCs by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and measured the expressions of extracellular signal-regulated kinase (ERK), Akt, Bcl-2, and Bax by Western blot. We also measured elongation of hair follicles in organ culture., Results: Minoxidil significantly increased the proliferation of DPCs. The levels of ERK phosphorylation and of phosphorylated Akt increased significantly 1 h post-treatment; percentage increase of ERK phosphorylation was 287% at 0.1 microM and 351% at 1.0 microM of minoxidil, and that of Akt phosphorylation was 168% at 0.1 microM and 257% at 1.0 microM of minoxidil. 1.0 microM of minoxidil increased Bcl-2 expression over 150%, while 1.0 microM of minoxidil decreased Bax expression by more than 50%. Moreover, a significant elongation of individual hair follicles in organ culture was observed after adding minoxidil., Conclusion: Minoxidil promotes the survival of human DPCs by activating both ERK and Akt and by preventing cell death by increasing the ratio of Bcl-2/Bax. We suggest that minoxidil stimulates the growth of human hairs by prolonging anagen through these proliferative and anti-apoptotic effects on DPCs.

Epidermal Langerhans cells are potent antigen-presenting cells in the epidermis. The establishment of a cryopreservation method for human Langerhans cells would greatly contribute to our ability to successfully conduct various experiments dealing with Langerhans cells. Since Langerhans cells are known to be sensitive to cold injury, there have been no reports concerning the cryopreservation of Langerhans cells. We have investigated the effect of cryopreservation on the function and phenotype of human Langerhans cells. Langerhans cells from human foreskins were isolated with the immunomagnetic microbead method using monoclonal antibodies for CD1a. Langerhans cells were cryopreserved in the presence of dimethylsulfoxide (DMSO) 10% and fetal calf serum 90%. Cryopreserved Langerhans cells were phenotypically assessed by flowcytometry using monoclonal antibodies to HLA-DR and CD1a. The ultrastructures of the Langerhans cells were compared using electron microscopy. An autologous T cell stimulation test was performed to compare the functions of cryopreserved Langerhans cells and fresh Langerhans cells. The viability of the cryopreserved Langerhans cells was able to be maintained at more than 90%. Cryopreserved Langerhans cells expressed high levels of HLA-DR and CD1a antigens and stimulated autologous T cells to an extent almost identical to that obtained from fresh Langerhans cells. These findings indicate that the cryopreservation of human Langerhans cells could lead to a breakthrough in various experiments dealing with human Langerhans cells.

Human macrophage metalloelastase is a member of the matrix metalloproteinase family and is involved in degradation of elastin. We investigated the ultraviolet modulation of human macrophage metalloelastase in human skin in vivo. Ultraviolet induced human macrophage metalloelastase mRNA maximally (11.9-fold) within 16 h post-ultraviolet in human skin. This induction of human macrophage metalloelastase by ultraviolet was inhibited by pretreatment with the antioxidant N-acetyl cystein (20%) and vitamin E (5%) by an average of 54% and 47%, respectively, in human skin in vivo. Ultraviolet (30 mJ per cm2) and phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, 50 nM) treatment increased expression of human macrophage metalloelastase mRNA and protein in the cultured human dermal fibroblasts, but not in the keratinocytes. Chronically sun-exposed human skin expressed significant amounts of human macrophage metalloelastase protein, which colocalized with the material of solar elastosis, whereas there was little expression in sun-protected skin of the same individuals. This study demonstrates that ultraviolet irradiation increases human macrophage metalloelastase expression in human skin in vivo, possibly in macrophages and fibroblasts, and ultraviolet-induced expression of human macrophage metalloelastase can be inhibited by antioxidant (N-acetyl cystein and vitamin E) pretreatment. Association of human macrophage metalloelastase with elastotic material suggests that it may play an important role in the development of solar elastosis, the hallmark of sun-induced damage in human skin in vivo.

UV light is absorbed in the epidermis and induces sunburn cell formation. It has been reported that HSP70 increases the UVB resistance of cell lines by in vitro experiments using various cell lines. In this study, hsp70.1(-/-) KO mouse was used in order to study the role of HSP70 after UVB irradiation. Western blotting showed a decreased level of HSP70 in hsp70.1(-/-) KO mouse compared with wild type FVB mouse. Six h after UVB irradiation, there were no significant histologic differences between the hsp70.1(-/-) KO mouse and the wild type FVB mouse. A similar degree of nuclear swelling was observed. However, there were significant differences at 12 and 24 h after UVB irradiation. After 12 h, a few apoptotic cells were observed in the wild type mouse, but a large number of cells were apoptotic in the hsp70.1(-/-) KO mouse. After 24 h, the epidermis of the wild type FVB mouse was relatively intact, but almost the entire epidermis was necrotic in the hsp70.1(-/-) KO mouse. These results showed that epidermal injury of hsp70.1(-/-) KO mouse was much more severe than that of wild type mouse although initial changes are similar in both species of mice. These results suggest that susceptibility of hsp70.1(-/-) KO mouse to UVB irradiation may originate from a defect in the repair mechanism. This HSP deficient model may be useful in studies of the effects of tissue injury that relate to the impaired tissue repair mechanisms.