Department of Dermatology, University Medical Center Benjamin Franklin, Free University of Berlin, Berlin, Germany

Correspondence: Dr Beate Tebbe, Department of Dermatology, University Medical Center Benjamin Franklin, Free University of Berlin, Fabeckstr. 60–62, 14195 Berlin, Germany. Email: teb@zedat.fu-berlin.de

Received 22 August 2000; Revised 8 February 2001; Accepted 23 February 2001.

To the Editor:

The biologic effects of UV on skin may occur either as an acute alteration shortly after UV exposure (inflammation, sunburn cells) or as a chronic damage (photoaging, photocarcinogenesis) (^{Beissert and Granstein, 1996}). Reactive oxygen species (ROS) are involved in the pathophysiologic mechanisms leading to photooxidative damage on nucleic acids, lipids, and proteins. ROS are also responsible for the induction of proinflammatory cytokines and adhesion molecules (^{Punnonen et al, 1991;Krutmann and Grewe, 1995}). ROS are constantly generated in epidermal keratinocytes and are rapidly removed by nonenzymic and enzymic antioxidant substances that prevent harmful effects of free radicals and maintain a prooxidant/antioxidant balance, thus resulting in cell and tissue stabilization (^{Fuchs et al, 1989;Yohn et al, 1991}).

The intracellular pathway of ROS signal transduction is not fully understood. The nuclear transcription factor kappa B (NFB) may be one candidate for the intracellular response of UVA-induced ROS in keratinocytes. NFB is an inducible transcription factor involved in the regulation of genes participating in either inflammatory or immune responses. In most cell types NFB resides in the cytoplasm in a latent form composed of several subunits. NFB activation occurs by dissociation from its inhibitor protein (IB) and its translocation into the nucleus (^{Baldwin, 1996}). NFB can be activated by various agents including UV. The fact that NFB activation may be inhibited by antioxidants, suggests that ROS are involved in activating NFB (^{Devary et al, 1993;Bender et al, 1998}).

Activation of NFB can be induced in human skin by UVB (^{Fisher et al, 1996}). In cultured human keratinocytes NFB activation occurs after combined UVA/UVB irradiation and also in cultured fibroblasts UVA irradiation activates NFB; however, high UVA doses lead to decreased NFB (^{Vile et al, 1995;Djavaheri-Mergny et al, 1999;Saliou et al, 1999}). UVA-induced upregulation of interleukin-1 (IL-1) can be inhibited by ^L-ascorbic acid (^{Tebbe et al, 1997}). In this investigation, we tested the influence of ^L-ascorbic acid on NFB binding activity, in order to obtain a linkage between UVA-induced ROS and immune response in keratinocytes.

HaCaT cells, an immortalized aneuploid human keratinocyte cell line (^{Boukamp et al, 1988}) shows a higher constitutive NFB binding activity than normal keratinocytes. The response to some cell cycle regulating factors in normal keratinocytes and HaCaT cells has been compared (^{Chaturvedi et al, 1999;Qin et al, 1999}). A strong activation of NFB by factors inducing cell cycle arrest is only observed in normal keratinocytes. The absence on responsiveness to cell cycle regulatory factors correlates well with the immortalized state of HaCaT cells. In spite of the constitutive level of binding activity present in HaCaT, these cells provide a homogenous system that allow to compare other NFB inducing agents.^{Saliou et al (1999)} showed in HaCaT cells that NFB activation after UV radiation can be modulated by different agents. These studies are not possible to do by using a highly heterogeneous system like human normal keratinocytes coming from different individuals.

UVA irradiation increases TBARS formation in HaCaT keratinocytes. This effect can be partially inhibited by ^L-ascorbic acid indicating that ROS are involved (data not shown). NFB binding acitivity, measured in the nuclear protein extract of HaCaT keratinocytes, is only increased within a short time period after UVA irradiation Figure 1. ^L-ascorbic acid supplementation and UVA irradiation (1 J per cm²⁾ showed a synergistic effect on NFB binding activity in HaCaT keratinocytes. NFB binding activity was found to be increased to 156% (SEM 19%) in UVA-irradiated cells. ^L-ascorbic acid alone did not change NFB binding activity; however, UVA irradiation-increased NFB binding activity in ^L-ascorbic acid supplemented cells showed remarkable increase Figure 2a, b. Both NFB subunits p50 and p65 were detectable in nuclear protein extracts of HaCaT keratinocytes Figure 2c.

Figure 1.

Highest NFB binding activity in HaCaT cells was seen 1 h after UVA irradiation (1 J per cm²). HaCaT cells were irradiated with 1, 5, or 10 J per cm². UVA irradiation was performed from the bottom of the cell culture dishes using a lamp with a range of 320–400 nm (Waldmann, Villingen, Germany). The output measured by a radiometer through culture dish was 6.7 mW per cm². Nuclear proteins were extracted from the cultured cells according to^{Corsini et al (1997)}. Electrophoretic mobility shift assays (EMSA) were performed according to^{Hirano et al (1998)}. A double-stranded oligonucleotide containing the NFB binding site (sense 5'-AGCTTCAGAGGGGACTTTCCGAG AGG-3', antisense 5'-TCGACCTCTCGGAAAGTCCCCTCTGA-3') was labeled with [-³²P]dCTP (NEN Life Science Products, Boston, MA) using Klenow Fragment (Gibco BRL, Gaithersburg, MD). Data for NFB binding activity were quantified using computer-based densitometry (TINA).

Full figure and legend (16K)

Figure 2.

(a, b) ^L-ascorbic acid increases UVA-induced NFB binding activity in HaCaT keratinocytes. NFB binding activity was measured by EMSA in UVA-irradiated (1 J per cm²) HaCaT keratinocytes supplemented with ^L-ascorbic acid (10^-4M). NFB binding activity was quantified using computer-based densitometry and related to nonirradiated HaCaT keratinocytes without ^L-ascorbic acid supplementation. UVA-induced NFB binding acitivity was markedly increased by ^L-ascorbic acid (mean SEM). (c) NFB subunits p50 and p65 are both expressed in HaCaT cells after UVA irradiation. NFB supershift assay for detection of p50, p65, and c-rel subunits was used in HaCaT keratinocytes irradiated with UVA (1 J per cm²). Both p50 and p65 specific antibodies were found positive, whereas c-rel was not detectable. Specific antibodies directed against NFB subunits p50, p65, c-rel (Santa Cruz Biotechnology, Santa Cruz) were used for supershift assay.

Full figure and legend (45K)

The regulation of NFB by ROS is cell type specific. In certain cell types, such as Wurzburg subclone of T cells, L6 skeletal muscle myotubes, human breast CF-7, and 70Z/3 pre-B cells, H₂O₂ was shown to be an effective inducer of NFB-binding activity (^{Sen and Packer, 1996;Manna et al, 1998}). It has been reported that H₂O₂ can also induce NFB binding activity in HeLa cells and Jurkat cells (^{Schreck et al, 1991;Brennan and O'Neill, 1995}); however, some other groups could not detect NFB binding activity in these cell lines after H₂O₂ stimulation (^{Anderson et al, 1994;Li and Karin, 1999}).

The effect of ascorbic acid on NFB activation varies in different cell types. Ascorbate increases the binding of NFB to DNA in TNF- stimulated Jurkat cells. The ability of ascorbate to enhance cytoplasmic inhibitory IB protein degradation correlates completely with its capacity to induce NFB binding to DNA that potentiate NFB-mediated transactivation of the HIV-1 long-terminal repeat (^{Muñoz et al, 1997}).

Beside its antioxidative properties, ascorbic acid can also interact with metal ions that contribute to oxidative damage through the production of hydroxyl and lipid alkoxyl radicals (^{Carr and Frei, 1999}). Ascorbate increases NO production by increasing the amount of iNOS in activated macrophages (^{Mizutani and Tsukagoshi, 1999}). This is caused by a delay of IB recovery and prolonged NFB DNA binding in lipopolysaccharide and interferon- activated macrophages leading to an increase in the iNOS protein level (^{Mizutani and Tsukagoshi, 1999}).

HaCaT keratinocytes possess very efficient systems to maintain high levels of both intracellular and extracellular ascorbic acid (^{Savini et al, 1999}). The regeneration and uptake of ascorbic acid from extracellular medium contributes to the intracellular antioxidant capacity, and consequently cells become more resistant to free radical generation and cell death induced by UVB irradiation (^{Savini et al, 1999}).

Our results indicate that UVA induces NFB binding activity in HaCaT keratinocytes, and this effect can be increased by ^L-ascorbic acid indicating that the latter has either an effect on IB degradation or prooxidative properties. Redox regulation of cell nutrients may induce changes in cell signalling or protein conformation that represent an important mechanism in the regulation of transcription factors and gene expression.