A combination of three antioxidants decreases the impact of rural particulate pollution in Normal human keratinocytes.

OBJECTIVE: It is well established that exposure of human skin to airborne pollution, particularly in the form of particulate matter sized 2.5 µm (PM2.5 ), is associated with oxidative stress, DNA damage and inflammation, leading to premature signs of skin aging. Because much of the damage results from oxidative stress, we examined the effects of a topical composition containing three antioxidants in an in vitro model system to assess the potential for amelioration of premature aging. The use of multiple antioxidants was of interest based on the typical composition of therapeutic skincare products. It is important to determine the efficacy of multiple antioxidants together and develop a short-term assay for larger scale efficacy testing. METHODS: Normal human epidermal keratinocytes were exposed to a rural-derived source of PM2.5 in the presence and absence of an antioxidant mixture of resveratrol, niacinamide and GHK peptide. Endpoints related to inflammation, premature aging and carcinogenicity were monitored after 5 h of exposure and included IL-6, CXCL10, MMP-1 and NRF2. Differentially expressed genes were monitored by RNA-seq. RESULTS: Pre-treatment of keratinocytes with the antioxidant preparation in the absence of PM2.5 reduced baseline levels of MMP-1, IL-6 and CYP1A1 and reduced PM2.5 -induced increases in all four endpoints, MMP-1, IL-6, CXCL10 and CYP1A1. Antioxidants significantly increased NRF2 protein in the presence of PM2.5 , indicating a protective response. RNA-seq interrogation of antioxidant-treated cells further showed increased expression of NRF2 inducible genes. The expression of CYP1A1 and genes related to aryl hydrocarbon activation were induced by PM2.5 and suppressed by antioxidants. CONCLUSIONS: Specific signalling pathways known to be correlated with skin inflammation and aging were examined based on their suitability for use in efficacy testing for the prevention of skin damage due to ambient hydrocarbon pollution. Endpoints examined after only 5 h of exposure provide a useful method amenable to high through-put screening. The results obtained reinforce the concept that a multiple antioxidant preparation, topically applied, may reduce pro-inflammatory signalling and cellular damage and thereby reduce premature skin aging due to exposure to rural-derived airborne pollution.

OBJECTIF: Il est bien établi que l'exposition de la peau humaine à la pollution atmosphérique, en particulier sous forme de particules d'une taille de 2,5 µm (PM2,5 ), est associée à un stress oxydatif, à des dommages à l'ADN et à une inflammation entraînant des signes prématurés de vieillissement cutané. Étant donné que la plupart des dommages résultent du stress oxydatif, nous avons examiné les effets d'une composition topique contenant trois antioxydants dans un système de modèle in vitro afin d'évaluer le potentiel d'amélioration du vieillissement prématuré. L'utilisation de plusieurs antioxydants a été intéressante en raison de la composition typique des produits thérapeutiques de soin de la peau. Il est important de déterminer l'efficacité de plusieurs antioxydants combinés et de développer un test à court terme pour des tests d'efficacité à plus grande échelle. MÉTHODES: Des kératinocytes épidermiques humains normaux ont été exposés à une source de PM2,5 rurale en présence et en l'absence d'un mélange antioxydant de resvératrol, de niacinamide et de peptide GHK. Les critères d'évaluation liés à l'inflammation, au vieillissement prématuré et à la carcinogénicité ont été surveillés après 5 heures d'exposition et comprenaient l'IL-6, CXCL10, MMP-1 et le NRF2. Les gènes exprimés de manière différentielle ont été surveillés par séquençage de l'ARN. RÉSULTATS: Le prétraitement des kératinocytes par la préparation antioxydante en l'absence de PM2,5 a réduit les taux initiaux de MMP-1, IL-6 et de CYP1A1 et a réduit les augmentations induites par les PM2,5 dans les quatre critères d'évaluation, MMP-1, IL-6, CXCL10 et CYP1A1. Les antioxydants ont significativement augmenté la protéine NRF2 en présence de PM2,5 , ce qui indique une réponse protectrice. L'interrogation des séquences d'ARN des cellules traitées par antioxydants a également montré une expression accrue des gènes inductibles par NRF2. L'expression du CYP1A1 et des gènes liés à l'activation des hydrocarbures aryles a été induite par les PM2,5 et supprimée par les antioxydants. CONCLUSIONS: Les voies de signalisation spécifiques connues pour être corrélées à l'inflammation cutanée et au vieillissement ont été examinées en fonction de leur adéquation à l'utilisation dans les tests d'efficacité pour la prévention des lésions cutanées dues à la pollution des hydrocarbures ambiants. Les critères d'évaluation examinés après seulement 5 heures d'exposition fournissent une méthode utile pouvant être utilisée pour un dépistage à haut débit. Les résultats obtenus renforcent le principe selon lequel une préparation antioxydante multiple, appliquée par voie topique, peut réduire la signalisation pro-inflammatoire et les dommages cellulaires et ainsi réduire le vieillissement prématuré de la peau résultant de l'exposition à la pollution atmosphérique d'origine rurale.

Biological Rhythms in the Skin.

Circadian rhythms, ≈24 h oscillations in behavior and physiology, are reflected in all cells of the body and function to optimize cellular functions and meet environmental challenges associated with the solar day. This multi-oscillatory network is entrained by the master pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which directs an organism's rhythmic expression of physiological functions and behavior via a hierarchical system. This system has been highly conserved throughout evolution and uses transcriptional-translational autoregulatory loops. This master clock, following environmental cues, regulates an organism's sleep pattern, body temperature, cardiac activity and blood pressure, hormone secretion, oxygen consumption and metabolic rate. Mammalian peripheral clocks and clock gene expression have recently been discovered and are present in all nucleated cells in our body. Like other essential organ of the body, the skin also has cycles that are informed by this master regulator. In addition, skin cells have peripheral clocks that can function autonomously. First described in 2000 for skin, this review summarizes some important aspects of a rapidly growing body of research in circadian and ultradian (an oscillation that repeats multiple times during a 24 h period) cutaneous rhythms, including clock mechanisms, functional manifestations, and stimuli that entrain or disrupt normal cycling. Some specific relationships between disrupted clock signaling and consequences to skin health are discussed in more depth in the other invited articles in this IJMS issue on Sleep, Circadian Rhythm and Skin.

Solar-simulated ultraviolet radiation induces histone 3 methylation changes in the gene promoters of matrix metalloproteinases 1 and 3 in primary human dermal fibroblasts.

Molecular signalling pathways delineating the induction of matrix metalloproteinases (MMPs) by ultraviolet radiation (UVR) are currently well-defined; however, the effects of UVR on epigenetic mechanisms of MMP induction are not as well understood. In this study, we examined solar-simulated UVR (ssUVR)-induced gene expression changes and alterations to histone methylation in the promoters of MMP1 and MMP3 in primary human dermal fibroblasts (HDF). Gene expression changes, including the increased expression of MMP1 and MMP3, were observed using Affymetrix GeneChip arrays and confirmed by qRT-PCR. Using ChIP-PCR, we showed for the first time that in HDF irradiated with 12 J/cm(2) ssUVR, the H3K4me3 transcriptional activating mark increased and the H3K9me2 transcriptional silencing mark decreased in abundance in promoters, correlating with the observed elevation of MMP1 and MMP3 mRNA levels following ssUVR exposure. Changes in mRNA levels due to a single exposure were transient and decreased 5 days after exposure.

The protein deacetylase SIRT3 prevents oxidative stress-induced keratinocyte differentiation.

Keratinocyte differentiation is a key process in the formation and maintenance of the protective skin barrier. Dysregulation in the balance of reactive oxygen species homeostasis may play a role in keratinocyte differentiation. We have identified the mitochondrial deacetylase SIRT3 as a key regulator of mitochondrial reactive oxygen species in keratinocytes. Our studies demonstrate that SIRT3 expression is down-regulated during keratinocyte differentiation, consistent with an increase in mitochondrial superoxide levels. Importantly, loss of SIRT3 expression in keratinocytes increased superoxide levels and promoted the expression of differentiation markers, whereas overexpression decreased superoxide levels and reduced the expression of differentiation markers. These findings identify a new role for SIRT3 in the suppression of epidermal differentiation via lowering oxidative stress.

Mitochondrial common deletion mutation and extrinsic skin ageing in German and Japanese women.

The mitochondrial common deletion (CD) mutation is induced by oxidative stress. One main source of oxidative stress is the error-prone process of the respiratory chain located in the mitochondria. Another important source is the exposure to environmental factors, which further induces oxidative stress in the cells. For human skin, the primary damaging environmental factor is ultraviolet (UV) radiation, which is able to induce CD mutations and the characteristic extrinsic skin ageing signs. Traditionally, levels of UV exposure differ between German and Japanese populations, as tanned skin represents beauty and health in Western cultures, whereas photo-protected skin is considered ideal in Asia. We hypothesize that (i) this cultural-related UV exposure pattern might be reflected by CD concentrations in environmentally exposed skin and (ii) CD concentrations in environmentally exposed areas might be associated with the manifestation of extrinsic skin ageing. In this study, we determined the concentration of CD in skin from the neck (environmentally exposed area) and the buttock (environmentally protected area) of 22 German and 46 Japanese women between 30 and 70 years of age. We evaluated skin ageing signs by a validated clinical score, and exposure to environmental factors, such as UV exposure and smoking, was assessed using a questionnaire-based interview. Higher levels of CD were detected in neck skin than in buttock skin in both German and Japanese women. CD also increased with age in the neck skin. German women had higher CD concentrations in the neck skin than Japanese women. The CD concentrations in the buttock skin samples were similar in both populations. These findings suggest higher environmental UV exposure resulted in higher levels of CD in the skin of German women compared with Japanese women. However, only in Japanese women were the signs of extrinsic skin ageing associated with higher CD concentrations in the neck skin, in agreement with the hypothesis (ii). In German women, we did not find this latter association, which might be due to reaching a maximum level of CD, beyond which cells undergo negative selection and are lost to the population samples. In conclusion, under some conditions, there seems to be an association between the CD mutation concentration and extrinsic skin ageing, but this may be modified by cellular and tissue processes which affect the sampling rate for CD mutation concentrations and prevent a statistical association with extrinsic skin ageing.

Sunburn protection as a function of sunscreen application thickness differs between high and low SPFs.

BACKGROUND: Sunscreens are an important component of healthy sun-protection behavior. To achieve satisfactory protection, sunscreens must be applied consistently, evenly and correctly. Consumers do not apply sunscreen properly and, therefore, do not achieve the protection indicated by the label 'sun protection factor' (SPF). The objective of the present study was to determine the actual sun(burn) protection given by a range of sunscreen application thickness levels for both low and high SPF formulas. SUBJECTS AND METHODS: Forty study subjects were recruited from each of three geographical regions in China. Sunscreens with label SPFs of 4, 15, 30, and 55 were tested at application levels of 0.5, 1.0, 1.5, and 2.0 mg/cm(2) in three laboratories using a standard SPF protocol. RESULTS: Sunscreens with lower SPFs (4 and 15) showed a linear dose-response relationship with application level, but higher SPF (30 and 55) product protection was exponentially related to application thickness. CONCLUSION: Sunscreen protection is not related in one uniform way to the amount of product applied to human skin. Consumers may achieve an even lower than expected sunburn protection from high SPF products than from low SPF sunscreens.

Quasi-drugs developed in Japan for the prevention or treatment of hyperpigmentary disorders.

Excess production of melanin or its abnormal distribution, or both, can cause irregular hyperpigmentation of the skin, leading to melasma and age spots. To date, various quasi-drugs that prevent or improve hyperpigmentary disorders have been developed and officially approved by the Ministry of Health, Labor and Welfare of Japan. Many of these inhibit the activity of tyrosinase, an enzyme required for melanin synthesis, for example, by competitive or non-competitive inhibition of its catalytic activity, by inhibiting its maturation, or by accelerating its degradation. In this review, we categorize the quasi-drugs developed in Japan to prevent or treat hyperpigmentary disorders, or both, and discuss perspectives for future development.

Vitamin D Update.

Purpose: The goal of this review is to provide an update in the field of vitamin D, in particular, the role of vitamin D in non-skeletal health, the complexity of providing patient guidance regarding obtaining sufficient vitamin D, and the possible involvement of vitamin D in morbidity and mortality due to SARS-CoV-2 (COVID-19). Recent Findings: In addition to bone health, vitamin D may play a role in innate immunity, cardiovascular disease, and asthma. Although rickets is often regarded as an historical disease of the early twentieth century, it appears to be making a comeback worldwide, including "first-world" countries. Broad-spectrum sunscreens (with high UVA filters) that prevent erythema are unlikely to compromise vitamin D status in healthy populations. Summary: New attention is now focused on the role of vitamin D in a variety of diseases, and more individualized patient recommendation schemes are being considered that take into account more realistic and achievable goals for achieving sufficient vitamin D through diet, supplements, and sun behavior.

Trichloroacetic acid model to accurately capture the efficacy of treatments for postinflammatory hyperpigmentation.

Postinflammatory hyperpigmentation (PIH) occurs following cutaneous injury and is common following resolution of acne especially in patients with skin of color. The objective of this study was to further validate a trichloroacetic acid (TCA)-induced PIH model and compare it to acne-induced PIH using topical bakuchiol, a botanical extract that has been shown to have antimicrobial, anti-inflammatory, antioxidant, and antiacne properties. A prospective, non-randomized clinical trial was conducted on subjects with skin phototypes IV-VI with a history of acne-induced PIH. Subjects applied bakuchiol or vehicle cream twice daily to 2 acne-induced and 2 TCA-induced PIH lesions for 28 days with a third lesion serving as a control in each group. Degree of improvement was defined as the change in the Investigator Global Assessment (IGA) score over 28 days of treatment. Twenty subjects (6 males, 14 females) completed the study. For TCA-induced PIH sites, there was a statistically significant (p < 0.05) degree of improvement with bakuchiol treatment (- 0.50 ± 0.18) compared to vehicle (0.05 ± 0.15) and control (- 0.06 ± 0.17). For acne-induced PIH, there was a greater degree of improvement for bakuchiol (- 1.06 ± 0.23) when compared to vehicle (- 0.56 ± 0.16) and control (- 0.69 ± 0.18); however, statistical significance was not reached (p > 0.05). TCA-induced PIH sites were uniform in size and pigment intensity thereby allowing better comparison among sites. This emphasizes the relevance of using this model for PIH which may help reduce the barriers in clinical trials and help improve access to treatments for patients who suffer from PIH. The results suggest that topical bakuchiol may decrease the severity of PIH.

Topical application of green and white tea extracts provides protection from solar-simulated ultraviolet light in human skin.

BACKGROUND: Tea polyphenols have been found to exert beneficial effects on the skin via their antioxidant properties. AIMS: We sought to determine whether topical application of green tea or white tea extracts would prevent simulated solar radiation-induced oxidative damages to DNA and Langerhans cells that may lead to immune suppression and carcinogenesis. METHODS: Skin samples were analysed from volunteers or skin explants treated with white tea or green tea after UV irradiation. In another group of patients, the in vivo immune protective effects of green and white tea were evaluated using contact hypersensitivity to dinitrochlorobenzene. RESULTS: Topical application of green and white tea offered protection against detrimental effects of UV on cutaneous immunity. Such protection is not because of direct UV absorption or sunscreen effects as both products showed a sun protection factor of 1. There was no significant difference in the levels of protection afforded by the two agents. Hence, both green tea and white tea are potential photoprotective agents that may be used in conjunction with established methods of sun protection.

Non-sunscreen photoprotection: antioxidants add value to a sunscreen.

The association between ultraviolet radiation (UVR) exposure and both skin cancer and photo-aging is well documented. In addition to the conventional organic-chemical and physical-mineral type sunscreens, other non-sunscreen protective strategies have been developed. These include topically applied botanical extracts and other antioxidants as well as topical DNA repair enzymes. Standard terms of photoprotection such as sun protection factor (SPF) do not accurately reflect the photoprotection benefits of these materials. For example, in spite of minimal SPF, tea extract containing polyphenols such as (-)-epigallocatechin-3-gallate (EGCG) has been shown to protect against UV-induced DNA damage and immune suppression, in part through its ability to reduce oxidative stress and inhibit NF-kB. The addition of botanical antioxidants and vitamins C and E to a broad-spectrum sunscreen may further decrease UV-induced damage compared with sunscreen alone. These agents have been shown to enhance protection against UV-induced epidermal thickening, overexpression of MMP-1and MMP-9, and depletion of CD1a(+) Langerhans cells. Non-sunscreen materials such as botanical extracts, antioxidants, and DNA repair enzymes can contribute value when applied topically to human skin in vivo.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 56-59; doi:10.1038/jidsymp.2009.14.

Norepinephrine modulates human dendritic cell activation by altering cytokine release.

Norepinephrine (NE) can modulate dendritic cell (DC) activation in animal models, but the response of human DC to NE and other response modifiers is as yet not completely understood. Here we report the effect of NE on the cytokine response of a mixed population of human DC cells to extracellular stimuli. These cells were obtained by differentiating human cord blood CD34+ precursor cells. NE inhibited the lipopolysaccharide (LPS)-stimulated production of interleukin (IL)-23, IL-12 p40, tumor necrosis factor (TNF)-alpha and IL-6 whereas the expression of IL-10 was not significantly affected. Thus, human cord blood-derived DC respond to NE in a manner similar to mouse Langerhans cells (LC). Furthermore, forskolin also inhibited the LPS-induced levels of TNF-alpha, IL-12 p40, IL-23 p19 and IL-6, supporting the hypothesis that the effects of NE are mediated by cAMP. Data from experiments using inhibitors of adrenergic receptors suggest that NE acts through beta-adrenergic receptors. As IL-23 promotes the differentiation of CD4+ T cells required for T(H)1-mediated immunity, we suggest that NE decreases the differentiation of CD4+ T cells needed for T(H)1-mediated contact hypersensitivity and that NE is a candidate regulator of human DC functions in the skin.

Green tea phenol extracts reduce UVB-induced DNA damage in human cells via interleukin-12.

Green tea chemoprevention has been a focus of recent research, as a polyphenolic fraction from green tea (GTP) has been suggested to prevent UV radiation-induced skin cancer. Recently, it was demonstrated that GTP reduced the risk for skin cancer in a murine photocarcinogenesis model. This was accompanied by a reduction in UV-induced DNA damage. These effects appeared to be mediated via interleukin (IL)-12, which was previously shown to induce DNA repair. Therefore, we studied whether GTP induction of IL-12 and DNA repair could also be observed in human cells. KB cells and normal human keratinocytes were exposed to GTP 5 h before and after UVB. UVB-induced apoptosis was reduced in UVB-exposed cells treated with GTP. GTP induced the secretion of IL-12 in keratinocytes. The reduction in UV-induced cell death by GTP was almost completely reversed upon addition of an anti-IL-12-antibody, indicating that the reduction of UV-induced cell death by GTP is mediated via IL-12. The ability of IL-12 to reduce DNA damage and sunburn cells was confirmed in "human living skin equivalent" models. Hence the previously reported UV-protective effects of GTP appear to be mediated in human cells via IL-12, most likely through induction of DNA repair.

UV protective effects of DNA repair enzymes and RNA lotion.

Solar UV radiation is known to cause immune suppression, believed to be a critical factor in cutaneous carcinogenesis. Although the mechanism is not entirely understood, DNA damage is clearly involved. Sunscreens function by attenuating the UV radiation that reaches the epidermis. However, once DNA damage ensues, repair mechanisms become essential for prevention of malignant transformation. DNA repair enzymes have shown efficacy in reducing cutaneous neoplasms among xeroderma pigmentosum patients. In vitro studies suggest that RNA fragments increase the resistance of human keratinocytes to UVB damage and enhance DNA repair but in vivo data are lacking. This study aimed to determine the effect of topical formulations containing either DNA repair enzymes (Micrococcus luteus) or RNA fragments (UVC-irradiated rabbit globin mRNA) on UV-induced local contact hypersensitivity (CHS) suppression in humans as measured in vivo using the contact allergen dinitrochlorobenzene. Immunohistochemistry was also employed in skin biopsies to evaluate the level of thymine dimers after UV. Eighty volunteers completed the CHS portion. A single 0.75 minimum erythema dose (MED) simulated solar radiation exposure resulted in 64% CHS suppression in unprotected subjects compared with unirradiated sensitized controls. In contrast, UV-induced CHS suppression was reduced to 19% with DNA repair enzymes, and 7% with RNA fragments. Sun protection factor (SPF) testing revealed an SPF of 1 for both formulations, indicating that the observed immune protection cannot be attributed to sunscreen effects. Biopsies from an additional nine volunteers showed an 18% decrease in thymine dimers by both DNA repair enzymes and RNA fragments, relative to unprotected UV-irradiated skin. These results suggest that RNA fragments may be useful as a photoprotective agent with in vivo effects comparable to DNA repair enzymes.

Genetic variants associated with skin aging in the Chinese Han population.

BACKGROUND: The progression and manifestation of human skin aging has a strong genetic basis; however, most of the supporting evidence has been gathered in Caucasian populations. The genetic contribution to the variation in skin aging in non-Caucasian populations is poorly understood. OBJECTIVE: To investigate the genetic risk factors of relevance for skin aging in East Asians, we conducted the first candidate gene study for signs of skin aging in Han Chinese. METHODS: We collected skin aging and genotype data in 502 female Han Chinese from the Taizhou cohort. We evaluated skin aging by the validated skin aging score SCINEXA™. Confounding factors were assessed through a questionnaire. We obtained the genotype data for 21 candidate SNPs and for a further 509 SNPs from 16 related candidate genes. Associations were tested by linear and logistic regression analyses and adjusted for potential confounders. RESULTS: Our candidate study found a significant association between SNP rs2066853 in exon 10 of the aryl hydrocarbon receptor gene AHR and crow's feet. In addition, we found a significant association between SNP rs10733310 in intron 5 of BNC2 and pigment spots on the arms, and between SNP rs11979919, 3kb downstream of COL1A2, and laxity of eyelids. CONCLUSIONS: Our results identified genetic risk factors for signs of skin aging (pigmentation, wrinkles or laxity) in Han Chinese. We also found that the manifestation of skin aging is further modified by anatomical site. Together with previous work, our results also suggest that different genetic variants could be responsible for distinct skin aging signs characteristic of Caucasians compared to East Asians.

Transcription factors and stress response gene alterations in human keratinocytes following Solar Simulated Ultra Violet Radiation.

Ultraviolet radiation (UVR) from sunlight is the major effector for skin aging and carcinogenesis. However, genes and pathways altered by solar-simulated UVR (ssUVR), a mixture of UVA and UVB, are not well characterized. Here we report global changes in gene expression as well as associated pathways and upstream transcription factors in human keratinocytes exposed to ssUVR. Human HaCaT keratinocytes were exposed to either a single dose or 5 repetitive doses of ssUVR. Comprehensive analyses of gene expression profiles as well as functional annotation were performed at 24 hours post irradiation. Our results revealed that ssUVR modulated genes with diverse cellular functions changed in a dose-dependent manner. Gene expression in cells exposed to a single dose of ssUVR differed significantly from those that underwent repetitive exposures. While single ssUVR caused a significant inhibition in genes involved in cell cycle progression, especially G2/M checkpoint and mitotic regulation, repetitive ssUVR led to extensive changes in genes related to cell signaling and metabolism. We have also identified a panel of ssUVR target genes that exhibited persistent changes in gene expression even at 1 week after irradiation. These results revealed a complex network of transcriptional regulators and pathways that orchestrate the cellular response to ssUVR.

Indoor PM2.5 exposure affects skin aging manifestation in a Chinese population.

Traffic-related air pollution is known to be associated with skin aging manifestations. We previously found that the use of fossil fuels was associated with skin aging, but no direct link between indoor air pollutants and skin aging manifestations has ever been shown. Here we directly measured the indoor PM2.5 exposure in 30 households in Taizhou, China. Based on the directly measured PM2.5 exposure and questionnaire data of indoor pollution sources, we built a regression model to predict the PM2.5 exposure in larger datasets including an initial examination group (N = 874) and a second examination group (N = 1003). We then estimated the association between the PM2.5 exposure and skin aging manifestations by linear regression. In the initial examination group, we showed that the indoor PM2.5 exposure levels were positively associated with skin aging manifestation, including score of pigment spots on forehead (12.5% more spots per increase of IQR, P-value 0.0371), and wrinkle on upper lip (7.7% more wrinkle on upper lip per increase of IQR, P-value 0.0218). The results were replicated in the second examination group as well as in the pooled dataset. Our study provided evidence that the indoor PM2.5 exposure is associated with skin aging manifestation in a Chinese population.

Neuropeptide (calcitonin gene-related peptide) induction of nitric oxide in human keratinocytes in vitro.

Nitric oxide (NO) is an important signaling molecule in both the central nervous system and the periphery, where it is involved in neurotransmission, vascular and bronchial tone, inflammation, and cutaneous immune function. More recently, NO has been implicated in intracellular signaling and may have a role in cellular differentiation, cytokine expression, and apoptosis. The experiments described herein examined the effect of calcitonin gene-related protein (CGRP), a cutaneous nerve neuropeptide, on NO production in human keratinocytes in vitro. CGRP stimulated two distinct increases in NO production: one within 30 minutes and a second at 24 hours. CGRP stimulated a modest increase in inducible nitric oxide synthase (iNOS) at 3-6 hours. Experimental evidence suggested that CGRP stimulated both constitutive NOS activity and generation of NO via nitrosothiol degradation within the first hour. Production of NO was paralleled by a decrease in nitrosothiol levels for 2 hour, suggesting that immediate NO release may originate from pre-existing stores. Nitrosothiols are ubiquitous molecules that comprise an important NO pool and have intracellular regulatory roles, particularly linked to oxidative stress. The present data indicate that, in addition to its known cAMP signaling pathway, CGRP may act to regulate keratinocyte biology through intracellular NO by modulation of S-nitrosothiol stores and stimulation of NOS activity.

Induction of the 72-kilodalton heat shock protein and protection from ultraviolet B-induced cell death in human keratinocytes by repetitive exposure to heat shock or 15-deoxy-delta(12,14)-prostaglandin J2.

It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42 degrees C or to 15dPGJ2 for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ2 treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ2 are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ2 is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ2 up to a concentration of 1 microg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.

Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.

Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.