Yuri Okano

UV-induced DNA damage initiates release of MMP-1 in human skin.

Abstract:  Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase‐1 (MMP‐1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet‐B (UVB)‐irradiated and treated with liposome‐encapsulated DNA repair enzymes. The photolyase‐mediated DNA repair of epidermal UV damage was associated with a reduction of MMP‐1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP‐1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP‐1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP‐1. The MMP‐1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP‐1 release, and enhancing DNA repair can reduce MMP‐1 expression in human skin cells and tissue.

Generation of active oxygen species from advanced glycation end-products (AGEs) during ultraviolet light A (UVA) irradiation and a possible mechanism for cell damaging

Advanced glycation end-products (AGEs) have been reported to be accumulated in dermal skin. However, the role of AGEs in the photoaging of human skin remains unknown, and for this reason, we have examined the interaction between AGEs and ultraviolet A light (UVA) from both the chemical and biological aspects. Previously, we reported that exposing human dermal fibroblasts to UVA in the presence of AGEs that were prepared with bovine serum albumin (BSA) decreased the cell viability due to superoxide anion radical s (.O2(-)) and hydroxyl radicals (.OH) generated by AGEs under UVA irradiation, and active oxygen species are detected with ESR spin-trapping. To identify the active oxygen species in detail and to clarify the cell damaging mechanism, we performed several experiments and the following results were obtained. (1) In ESR spin-trapping, by addition of dimethyl sulfoxide and superoxide dismutase, ESR signals due to .O2(-) -derived DMPO-OOH and .OH-derived DMPO-OH adducts, respectively, were detectable. (2) UVA-irradiated AGEs elevated the lipid peroxide levels in both fibroblasts and liposomes. But the peroxidation in liposomes was inhibited by addition of deferoxamine. (3) Survival of fibroblasts exposed to UVA in the presence of AGEs was elevated by addition of deferoxamine. And finally, (4) survival of fibroblasts was found to be regulated by the level of H2O2. On the basis of these results, we propose a possible mechanism in which AGEs under UVA irradiation generate active oxygen species involving .O2(-), H2O2, and .OH, and the .OH species plays a harmful role in promoting cell damage.

Differential role of catalase and glutathione peroxidase in cultured human fibroblasts under exposure of H2O2 or ultraviolet B light

The purpose of this study was to elucidate the differential contribution of catalase and glutathione peroxidase (GSH-Px) to H2O2 scavenging in cultured human dermal fibroblasts. Responses of the cells in terms of both enzyme activities were examined by using two sorts of inhibitors, 3-amino-1H-1,2,4-triazole (AT) for catalase and dl-buthionine-[S, R]-sulfoximine (BSO) for GSH-Px, under exposure to H2O2 or ultraviolet (UV) B radiation. AT treatment resulted in a decrease in H2O2 scavenging activity, while BSO treatment did not affect H2O2 scavenging. When fibroblasts were exposed to a low concentration of H2O2 (100 ÌM). AT treatment resulted in a significant decrease in cell survival, but BSO treatment did not affect survival. At higher concentrations of H2O2 ranging from 500 ÌM to 1 mM, BSO-treated fibroblasts showed reduced survival. In addition, AT treatment was much more cytotoxic in the presence of UVB than BSO treatment. The intracellular levels of H2O2 in fibroblasts treated with AT or BSO were also determined. BSO-treated cells showed similar H2O2 levels to control cells, but the intracellular H2O2 levels of AT-treated fibroblasts were 1.4-fold higher than found in control cells. These results with human dermal fibroblasts indicate that catalase acts as a primary defence against oxidative stress from exogenous or endogenous H2O2 at low concentrations. In contrast, GSH-Px helps protect the cell from damage during exposure to high concentrations of H2O2.

Reactive Oxygen Species in HaCaT Keratinocytes After UVB Irradiation Are Triggered by Intracellular Ca2+ Levels

It is recognized that reactive oxygen species (ROS) are responsible for skin damage due to UVB-radiation (UVB-R). However, the triggering substance(s) for ROS generation after UVB-R is uncertain with respect to the activation of NADPH oxidase (Nox), xanthine oxidase (XOD), and respiratory chain-chain reactions in mitochondria. As a first step in identifying the trigger(s) for UVB-induced ROS generation, we examined the relationship between Ca(2+) levels and ROS generation in HaCaT keratinocytes. UVB-R exposure of HaCaT keratinocytes resulted in an immediate elevation of ROS that recurred 7 hours later. This was accompanied by immediately elevated intracellular Ca(2+) . A Ca(2+) chelating agent, BAPTA, abolished the elevation of ROS after UVB-R completely. In addition, exogenous H(2)O(2) did not increase intracellular Ca(2+) levels. This suggests that intracellular Ca(2+) is the first trigger for UVB-induced ROS generation.Journal of Investigative Dermatology Symposium Proceedings (2009) 14, 50-52; doi:10.1038/jidsymp.2009.12.

α-Tocopherol Increases the Intracellular Glutathione Level in HaCaT Keratinocytes

f -Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of f -tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with f -tocopherol for 24 h, the intracellular GSH was increased at every concentration of f -tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with f -tocopherol at 50 w M for 24 h exhibited resistance against H 2 O 2 . However, a short exposure of HaCaT keratinocytes to f -tocopherol for 1 h did not influence either the GSH level or the resistance to H 2 O 2 . These findings suggest that GSH, which is inductively synthesized by f -tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of f -tocopherol on the GSH level, BSO, which is a typical inhibitor of n -glutamylcysteine synthetase ( n -GCS), was used. When BSO was added to HaCaT keratinocytes, no action of f -tocopherol on the GSH level was observed. On the other hand, f -tocopherol resulted in the up-regulation of n -GCS-HS (heavy subunit) mRNA. In addition, water soluble f -tocopherol derivatives ( f -tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that f -tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of n -GCS-HS mRNA.

Effects of UV irradiation on the sebaceous gland and sebum secretion in hamsters

BACKGROUND Although an understanding of the photobiology of the skin has been extensively advanced recently, the effect of ultraviolet (UV) radiation on sebaceous glands is not well known. OBJECTIVE In this study, we examined the direct effect of UV radiation on cultured sebocytes from hamsters in vitro experimental system. Moreover, we examined whether UV-induced peroxidation of skin surface lipids may affect barrier function of horney layer. METHODS We irradiated cultured sebocytes from hamsters, which have similar biological characteristics to the human sebocytes, with UV radiation. Moreover, transepidermal water loss (TEWL) was examined after topical application of cholesterol or triglyceride (TG) and UV exposures on the back of hamsters. RESULTS The number of sebocytes were increased significantly (120-140%) after 4 days as compared with the non-irradiated controls. Lipid production in sebocytes was also increased on day 7 in an irradiation-dependent manner up to 4.1 times of the pre-irradiated level. When UVB was irradiated to TG- or cholesterol-applied skin at the minimum ear-swelling dose, TEWL increased twice or more as compared with UVB irradiation to unapplied sites. When in vitro-irradiated TG, in vitro-irradiated cholesterol, TG-peroxide (TG-OOH), and cholesterol-peroxide (CHO-OOH) were applied to the skin, TEWL increased significantly. CONCLUSION These results suggest that UVB may directly activate the functions of the sebaceous gland in vivo to produce increased amounts of sebum, which may undergo peroxidation by UV light and damage the barrier functions of the skin.

Pentosidine in advanced glycation end-products (AGEs) during UVA irradiation generates active oxygen species and impairs human dermal fibroblasts

Our previous study reported that advanced glycation end-products (AGE)-modified BSA produced active oxygen species, *O2-, H2O2, and *OH under UVA irradiation and enhanced the cytotoxicity of UVA light. We examined whether pentosidine in AGE-modified BSA was involved in one of the mechanisms generating the active oxygen species. In biological investigations, fibroblasts exposed to UVA (20 J/cm2) in the presence of pentosidine-rich compounds (PRCs), which were prepared with L-arginine, L-lysine and glucose, showed a time-dependent leakage of the cytosolic enzyme LDH. In addition, release of LDH was suppressed by addition of DMSO and deferoxamine under UVA irradiation. From these results, it was determined that PRCs exposed to UVA damaged the plasma membrane of human dermal fibroblasts due to the conversion of *OH from H2O2 via a Fenton-like reaction. These features of PRCs exposed to UVA were consistent with those of AGE-modified BSA. In an ESR study, PRCs under UVA irradiation yielded DMPO-OH (DMPO-OH adduct) using DMPO as a spin-trapping reagent. *O2- generation from UVA-irradiated PRCs was also indicated by the combination of NBT reduction and SOD. When PRCs were exposed to UVA light controlled with a long-pass filter, WG-360, it was found that their production of *O2- was prohibited less than 50% in the NBT reduction assay. The *O2- production profile of PRCs depending on the wavelength of UVA light was similar to that of AGE-modified BSA. Furthermore, it was found that the H2O2 level was increased by PRCs exposed to UVA. These results indicated that pentosidine is an important factor of AGE-modified BSA in active oxygen generation under UVA irradiation.

A Zn(II)-glycine complex suppresses UVB-induced melanin production by stimulating metallothionein expression

Oxidative stress caused by ultraviolet (UV) radiation generates reactive oxygen species (ROS) in the skin, induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyper‐pigmentation. Thus, increasing the anti‐oxidative ability of skin cells is expected to be a good strategy for skin‐lightening cosmetics. Metallothionein (MT) is one of the stress‐induced proteins and is known to exhibit a strong anti‐oxidative property. We previously reported that a zinc(II) complex with glycine (Zn(II)(Gly)2) effectively induces MT expression in cultured human keratinocytes. To determine its potential as a new skin lightening active, we examined whether Zn(II)(Gly)2 regulates the release of melanocyte‐activating factors from UVB‐irradiated keratinocytes and affects melanin production in a reconstructed human epidermal equivalent. Conditioned medium from UVB‐irradiated keratinocytes accelerated melanocyte proliferation to 110%, and that increase could be prevented by pre‐treatment with Zn(II)(Gly)2. In addition, Zn(II)(Gly)2 significantly reduced both the production of prostaglandin E2 and proopiomelanocortin expression in UVB‐irradiated keratinocytes. Zn(II)(Gly)2 also decreased melanin production in a reconstructed human epidermal equivalent. These results indicate that MT‐induction in the epidermis effectively up‐regulates tolerance against oxidative stress and inhibits the secretion of melanocyte growth and activating factors from keratinocytes. Thus, Zn(II)(Gly)2 is a good candidate as a new skin‐lightening active.

Lysophospholipids improve skin moisturization by modulating of calcium-dependent cell differentiation pathway.

Recent studies have demonstrated that lysophospholipids (LPL) play critical roles in several biological signal transduction pathways to maintain the homoeostasis of cells, tissues and organs. Among them, lysophosphatidic acid (LPA) has been identified as a lipid mediator that induces morphological improvement in the epidermis in mice. In this study, we examined the effects of LPL (soybean‐derived phospholipids modified with phospholipase A2 and C) compared with LPA. We initially examined the effects of LPA on normal human epidermal keratinocytes (NHEK) focusing on the expression of profilaggrin and serine palmitoyltransferase (SPT) mRNAs. LPA enhanced the expression of profilaggrin and SPT mRNAs via the modulation of Ca2+ influx. Based on those results, the influence of LPL on NHEK was examined and was expanded to analyse the expression of two tight junction–related proteins, occludin and claudin‐1. LPL had similar effects to increase profilaggrin and SPT mRNA expression and also stimulated the expression of occludin and claudin‐1 at the mRNA and protein levels. In accordance with these results, LPL elicited significant improvements in surface water content in human skin. These findings indicate that LPL has the potential to strengthen the skin moisturizing capability by up‐regulating the expression of mRNAs encoding components important to skin barrier function and skin hydration.

Resistance of Cultured Human Skin Fibroblasts from Old and Young Donors to Oxidative Stress and Their Glutathione Peroxidase Activity

Background: It has been suggested that oxidative stress is involved in the aging process and that the resistance of animals to oxidative stress may decrease with advancing aging. However, there are only a limited number of reports of studies on the relationship between aging and resistance to oxidative stress. Objective: The aim of this work is to examine the relationship between the resistance of human skin fibroblasts to oxidative stress and donor age, and the relevance of antioxidant enzyme activities to this resistance. Methods: Percent cell survival was determined by the trypan blue exclusion test and the neutral red method. Superoxide dismutase activity was assayed by the method of Oyanagi, catalase activity by the method of Aebi, and glutathione peroxidase activity by the method of Flohé and Günzler. Reduced glutathione concentration was measured by the method of Griffith. Antioxidant enzyme mRNA levels were estimated by reverse transcription polymerase chain reactions (RT-PCR). Results: The percent survivals of cultured human skin fibroblasts, derived from young and old donors (referred to as young and old cells, respectively), under oxidative stress from hydrogen peroxide, linoleic acid hydroperoxide, or ultraviolet light B were examined. Old cells were more resistant to such oxidative stress than young cells. The activity of glutathione peroxidase was higher by 46.1% in old cells than in young cells, although there was no difference between their relative glutathione peroxidase mRNA levels. Further, there was no difference between their activities of copper/zinc superoxide dismutase, manganese superoxide dismutase, or catalase. However, the relative mRNA levels of copper/zinc superoxide dismutase and manganese superoxide dismutase were lower by 13.9 and 20.9% in old cells than in young cells, respectively, while there was no difference between the levels of catalase. Conclusions: These results suggest that old cells are more resistant to oxidative stress than young cells, presumably because of an increase in cellular glutathione peroxidase activity.