a-Tocopherol transfer protein: Regulatory mechanism for skin vitamin E?
M.G. Traber, J.J. Thiele, Y. Terasawa, R.V. Farese, Jr., W.L. Epstein, S. Weber, M. Podda, and L. Packer
Department of Molecular & Cell Biology, UC Berkeley, and UC San Francisco
Destruction of epidermal vitamin E by UV-light and the mechanism for its subsequent recovery were investigated in humans and in hairless mice. Solar simulated UV-light (0.5 minimal erythemal dose, M2 cm control sites were not irradiated. 0 and 24 h later, 1 non- irradiated and 2 irradiated sites were anesthetized and the epidermis was removed by curettage. Epidermal antioxidants (a- and g-tocopherols, ubiquinol, and ubiquinone) were extracted and measured. Only a-to copherol levels changed; they were 23 ± 3 nmol/g (mean ± SEM) and were significantly depleted by 0.5 MED UV-light exposure (15 ± 3 nmol/g; p < 0.001). Only 60% ± 8% remained after UV-irradiation, but 24 h later epidermal a-tocopherol was nearly ED) was used to irradiate four-1 cm sites of non-sun exposed skin on the buttocks of humans (5 males and 5 females); two- replete (92% ± 9%). In hairless mice 24 h after UV-irradiation (0.5, 1.5 or 3 MED), the epidermis contained nearly double the a-tocopherol concentrations. Murine epidermal cholesterol concentrations were unchanged by UV-irradia tion, while ubiquinol was depleted and remained depleted 24 h after 3 MED UV-irradiation. Since the a-tocopherol transfer protein (a-TTP ) is involved in a-tocopherol secretion from the liver, its role in skin a-tocopherol secretion was investigated. By RT-PCR, both mouse and rat skin contained a-TTP mRNA. Six hs after removal of murine stratum corneum by tape stripping, a process that causes skin barrier disruption and increases epidermal lamellar lipid secretion, epidermal a-tocopherol increased (p < 0.03), suggesting a relationship between lamellar lipid and a-tocopherol secretion and a-TTP. Hence, UV- irradiation, insufficient to cause reddening of the skin, was sufficient to significantly deplete human epidermal a-tocopherol, decreasing antioxidant protection. Furthermore, by 24 h, a-tocopherol contents of the irradiated skin increased to almost baseline levels in humans and double baseline levels in mice, suggesting that the skin has the ability to replace lost a-tocopherol in response to oxidative stress. Finally, studies in mice suggest that regulation of skin vitamin E occurs in conjunction with lamellar lipid secretion via a a-TTP-mediat ed process.
Skin barrier a-tocopherol: A highly sensitive biomarker of environmental oxidative stress
Jens J. Thiele and Lester Packer
Department of Molecular & Cell Biology, UC Berkeley, USA
The stratum corneum (SC) is the outermost skin layer and penetration barrier of the body. Thus, it is frequently and directly exposed to a pro-oxidative environment, including ultraviolet solar radiation (UVB and UVA), air pollutants (such as ozone), and chemicals. We sought to investigate the physiological antioxidant gradients in SC, their response to various types of oxidant exposures (ozone, UVB/UVA, and benzoyl peroxide), and possible regulatory mechanisms for epidermal a-toco-pherol.
Methods were developed to measure antioxidants a-tocopherol, as-corbic acid, and urate in vivo in murine and human SC. Murine skin was exposed to defined levels of ozone and solar simulated UVA and UVB radiation; furthermore, baseline levels of SC antioxidants and their responses to topical benzoyl peroxide were evaluated in 12 human volunteers.
Both in mice and humans, the SC contained unique antioxidant distribution gradients. a-Tocopherol appeared to be the predominating SC antioxidant with respect to both its concentration and its remarkable susceptibility to acute exposures of UV-irradiation, ozone, and benzoyl peroxide. Intriguingly, we demonstrated repletion of a-tocopherol in the SC of chronically UV-exposed murine SC, indicating the existence of mechanisms to control a-tocopherol levels in skin; these may involve a cutaneous form of the a-tocopherol transfer protein, and/or secretion by sebaceous glands. Depletion of barrier a-tocopherol may represent an early pathophysiological event leading to the initiation of barrier disruption and inflammation in environmentally damaged skin.
Modulation of UV-induced signal transduction in human keratinocytes by antioxidants
Claude Saliou, Laura McLaughlin, and Lester Packer
Department of Molecular and Cell Biology, University of California, Berkeley, California, USA
Sunburn, a common reaction occurring to the skin following exposure to solar ultraviolet radiation, is the manifestation of an inflammatory process indirectly triggered by generation of Reactive Oxygen Intermediates, activation of transcription factors (e.g. NF-kB and AP-1), and subsequent induction of cytokines, growth factors and matrix metallo-proteinases gene expression. Antioxidants protect against UV damages and can also reduce the extent of chronic deleterious responses.
Using a solar simulator, keratinocytes (HaCaT) were irradiated with UV. Activation of NF-kB and AP-1 and expression of genes implicated in early inflammatory stages were examined. NF-kB and AP-1 were activated in a dose (75-150 mJ/cm2) and time (2-8 hours) dependent manner. Significantly, a-lipoic acid (1 mM), N-acetyl-L- cysteine (10 mM) and the plant extract silymarin (25µg/ml) inhibited NF-kB activation. In addition, combinations including a-tocopherol (10-25µM) and either a-lipoic acid, N-acetyl-L-cysteine or silymarin synergistically inhibited this activation.
Induction of the proinflammatory chemokine interleukin-8, involved in psoriasis, and the metalloproteinase type-I collagenase, responsible for the cell remodeling in response to skin injury, were also determined. Their induction was observed in a dose (35-150 mJ/cm2) dependent manner within 24 hours after UV exposure.
These observations suggest that keratinocytes, direct target of UV, contribute by their gene expression to the early stages of the skin inflammation. Moreover, antioxidants can efficiently modulate the extent of these responses.
Signaling events by UVA radiation and singlet oxygen
Karlis Briviba, Lars-Oliver Klotz, Karin Scharffetter-Kochanek, Meinhard Wlaschek, Susanne Grether-Beck, Jean Krutmann and Helmut Sies
Institut für Physiologische Chemie I and Hautklinik, Heinrich-Heine-Universität Düsseldorf, and Hautklinik, Universität zu Köln, Germany
Singlet oxygen has damaging effects on biomolecules and exerts genotoxic, virucidal, and cytotoxic effects. This is of relevance for biological systems because singlet oxygen can be produced photochemically as a result of the irradiation of endogenous or exogenously applied photosensitizers with visible or ultraviolet light, or in dark reactions, e.g. by stimulated phagocytes during the oxidative burst. In addition, there is increasing evidence that singlet oxygen has pronounced effects on cellular signaling events leading to the induced expression of a variety of proteins.
Singlet oxygen mimics and mediates effects of UVA radiation on gene expression. Induction of gene expression of heme oxygenase-1, interstitial collagenase (matrix metalloproteinase-1, MMP-1), the interleukins IL-1a,b/ and IL-6, and intercellular adhesion molecule-1 (ICAM-1) and FAS ligand was reported. NF-kB is induced upon treatment with singlet oxygen generated photochemically by Rose Bengal or methylene blue plus light. Transcription factor AP-2 is responsive to singlet oxygen released from NDPO2 as was shown using ICAM-1 promoter constructs. Singlet oxygen generated photochemically by Rose Bengal plus light as well as by UVA irradiation activates c-Jun N-terminal kinase (JNK) activity in human dermal fibroblasts. Furthermore, UVA irradiation of human skin cells leads to the activation of AP-1, NF-kB, and AP-2. When singlet oxygen was generated outside the cells it was not able to induce NF-kB or JNKs.
Collagenase (MMP-1) induction by singlet oxygen and UVA seems to be an indirect effect mediated by interleukins IL-1a,b/ and IL-6. Singlet oxygen generated inside cells by Rose Bengal plus light or outside by Rose Bengal-agarose plus light or by NDPO2 induced expression of IL 1a,b/ and IL-6. Singlet oxygen is made responsible for a leakage of pre-formed cytosolic IL-1 which then binds to its receptor, thereby stimulating gene transcription of collagenase, of its own gene and of IL-6. IL-6, in turn, once synthesised and secreted, binds to the IL-6 receptor and stimulates collagenase synthesis. IL-1 is known to exert its effects on gene expression via activation of JNK1 and JNK2 eventually leading to an activation of transcription factor AP-1 and by activating the NF-kB system. The promoter region of the MMP-1 gene contains functional binding sites for AP-1. Transcription factor AP-2 but neither AP-1 nor NF-kB is responsible for singlet oxygen-mediated induction of ICAM-1 in human keratinocytes.
Thus, the involvement of AP-1, AP-2 and NF-kB in the singlet oxygen response is a recurring motif, and the question about how these transcription factors can be activated by singlet oxygen is now becoming of interest. JNK activation by UVA in human skin fibroblasts is mimicked by incubating the cells with the tyrosine phosphatase inhibitor orthovanadate; no further activation is found with UVA, pointing to the inactivation of a phosphatase by UVA treatment eventually leading to JNK activation.
Supported by the Deutsche Forschungsgemeinschaft, SFB 503, and the National Foundation for Cancer Research, Bethesda.
Possible roles for iron, heme oxygenase and ferritin in UVA-mediated oxidative stress and cell death
R.M. Tyrrell, C. Eden, V. Hejmadi, E. Kvam, C, Pourzand, S. Ryter, and A, Stacey
Department of Pharmacy and Pharmacology, University of Bath, BA2 7AY, Bath, United Kingdom
UVA (320-380nm) radiation generates an oxidative stress in cultured cells that can lead to the induction of pre-mutagenic and cytotoxic damage, membrane damage and induction of gene expression. In certain cell types, the cell death pathway is clearly via an apoptotic process that proceeds within hours of irradiation. Many of the effects of UVA radiation can be modulated by chemical mediators that include agents that modify singlet oxygen lifetime as well as several classes of antioxidants and iron chelators. Glutathione appears to be the most critical endogenous antioxidant. Fairly low doses of UVA radiation lead to both immediate and delayed increases in the pool of labile iron in cells and a dramatic drop in levels of intact microsomal heme proteins. UVA radiation also strongly induces transcription of the heme oxygenase 1 (HO-1) gene in many cell types and this in turn will lead to the appearance of increased levels of the protein. The changes in labile iron and heme-containing protein are clearly related to the activation of HO-1 and the eventual increases in ferritin synthesis. Transient increases in free iron (that arise, at least partly, as a result of increased HO activity) would be expected to make cells more sensitive to oxidising treatments. We are now testing this using heme oxygenase 2 over-ex pressing cell lines and find that these cells are especially sensitive to both UVA and hydrogen peroxide treatment. However, our current data remains consistent with the model that the temporary enhancement in susceptibility provoked by transient increases in labile iron will eventually be counterbalanced by the longer-term protection produced by increases in ferritin levels.
Maurizio Podda, Sandra Diehl, Roland Kaufmann, Rainer Milbradt, Thomas M. Zollner
Department of Dermatology, University of Frankfurt Medical School, Germany
Cutaneous lymphocyte-associated antigen (CLA) is the most relevant receptor for the recruitment of T-cells into the skin. There is an increasing body of evidence that T-cell CLA expression is strongly involved in the pathogenesis of T-cell mediated dermatoses, such as psoriasis, cutaneous T-cell lymphoma or allergic contact dermatitis. Several cytokines (IL-6, TGFß) and superantigens (TSST-1, SPEC) are well known inducers of CLA expression in T-cells, however, no mechanisms for the downregula-tion of CLA have been described so far. We were able to show that non-toxic concentrations of different, structurally unrelated antioxidants inhibit the expression of CLA in resting and activated T-cells. Resting PBMC, highly purified T-cells, the CLA expressing HL-60 cell line, superantigen (TSST-1) stimulated PBMC and cytokine (TGFß+IL-2+PHA) stimulated T-cells were cultured with antioxidants and T-cell CLA, sialyl Lewis X (sLex), and P-selectin glycoprotein ligand-1 (PSGL-1) expression were analyzed by flow cytometry at various time points (day 1-10). We were able to show that N-acetyl-L-cysteine (NAC, >1mM), a-lipoic acid (LA, > 300µM), a-tocopherol (TOC, > 500µM) and ascorbate (ASC, >500µM) inhibited the expression of CLA in resting T-cells starting at day 2 (up to 80% at day 5 with NAC being the most effective antioxidant) and in HL-60 cells (up to 40% with LA being the most effective antioxidant). In superantigen- and in cytokine-activated T-cell cultures, CLA expression was almost completely blocked (up to 95% inhibition with NAC being most effective). sLex expression was also affected by antioxidants, whereas the expression of the CLA protein backbone PSGL-1 remained untouched. Furthermore, in HL-60 cells treatment with antioxidants selectively affected the expression of fucosyltrans ferase VII regulated glycoproteins as CLA, whereas the fucosyltrans ferase IV regulated glycoproteins CD15 and CDw65 remained unaffected. The antioxidant inhibition of CLA expression seems specific for skin homing, since no changes were detectable on the gut homing receptor CD103. The antioxidant effect on CLA expression is of functional relevance since a dose dependent up to 80% reduction (with NAC) of T-cell binding to a recombinant E-selectin-human-IgG chimera was found. We conclude that T-cell CLA expression can be abrogated by antioxidants suggesting a redox-regulation of CLA expression. These results may form the basis of new therapeutic strategies in T-cell mediated skin disorders.
Cancer chemoprevention by polyphenolic antioxidants in green tea
Hasan Mukhtar, Sanjay Gupta, and Nihal Ahmad
Department of Dermatology, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, Ohio 44106, USA
Chemoprevention is a means of cancer prevention and control where through the use of naturally occurring and/or synthetic compounds the occurrence of the disease is completely prevented, blocked or reversed. In this regard, the naturally occurring antioxidants present in the diet and beverages consumed by humans are receiving increasing attention. Green tea extract and the polyphenolic compounds present therein (GTP) have been shown to possess cancer chemopreventive effects in a variety of animal tumor models and human epidemiological studies. The biological mechanism(s) of the cancer chemopreventive response of GTP and its polyphenolic constituents are not well defined. The major polyphenolic antioxidant present in green tea is epigallocatechin-3-gallate (EGCG). We found that EGCG treatment (80 mg/ml, 48 hrs) results in DNA fragmentation in various human and mouse carcinoma cells that included HaCaT, L5178Y and DU145. Interestingly no such effect was seen in normal human epidermal keratinocytes (NHEK). The confocal microscopy provided additional support for the induction of apoptosis by EGCG. The quantification of apoptosis by flow cytometry showed a dose dependent apoptosis in A431 cells, 48 hrs following EGCG (40, 80 and 160 mg/ml) treatment. The DNA cell cycle analysis showed that EGCG (40 and 80 mg/ml for 24 hrs) treatment also resulted in an appreciable arrest in G0-G1 phase of the cell cycle in A431 cells. The same treatment, however, did not affect the NHEKs.
We hypothesized the involvement of NFkB in cell cycle deregu latory response of EGCG. NFkB, a nuclear transcription factor that controls a variety of genes, is known to be involved in cellular proliferation and cancer. We investigated the effect of EGCG on the expression and activation of NFkB protein in A431 cells. Western blot analysis showed that EGCG treatment (40 mg/ml for 24 hours), to the growing A431 cells results in a significant decrease (~42% as compared to control) in the constitutive expression of nuclear NFkB/p65 protein without any change in the constitutive expression of its cytosolic precursor protein IkB-a. EGCG treatment also resulted in an inhibition of lipopolysaccharide-mediated activation of NFkB in the nucleus. These responses of EGCG were dose dependent with a significant effect even at 10 mg/ml dose. These results demonstrate that EGCG inhibits both the constitutive expression as well as the activation of NFkB. We suggest that EGCG-mediated inhibition in the nuclear translocation of NFkB is responsible for triggering the growth-arrest signals by imposing commands on cell cycle machinery. This may be involved in the anti-proliferative response of this antioxidant. These results indicate that antioxidants in green tea in general may have wider implications in prevention and control of cancer. We suggest that clinical trials with green tea antioxidants be undertaken.