Nitric Oxide Fully Protects against UVA-induced Apoptosis in Tight Correlation with Bcl-2 Up-regulation*

Christoph V. SuschekDagger , Verena KrischelDagger , Daniela Bruch-Gerharz§, Denise BerendjiDagger , Jean Krutmann§, Klaus-Dietrich KrönckeDagger , and Victoria Kolb-BachofenDagger

From the Dagger  Research Group Immunobiology and § Department of Dermatology, MED-Heinrich-Heine-University of Düsseldorf, Postfach 10 10 07, D-40001 Düsseldorf, Germany

    ABSTRACT
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Abstract
Introduction
References

A variety of toxic and modulating events induced by UVA exposure are described to cause cell death via apoptosis. Recently, we found that UV irradiation of human skin leads to inducible nitric-oxide synthase (iNOS) expression in keratinocytes and endothelial cells (ECs). We have now searched for the role of iNOS expression and nitric oxide (NO) synthesis in UVA-induced apoptosis as detected by DNA-specific fluorochrome labeling and in DNA fragmentation visualized by in situ nick translation in ECs. Activation with proinflammatory cytokines 24 h before UVA exposure leading to iNOS expression and endogenous NO synthesis fully protects ECs from the onset of apoptosis. This protection was completely abolished in the presence of the iNOS inhibitor L-N5-(1-iminoethyl)-ornithine (0.25 mM). Additionally, preincubation of cells with the NO donor (Z)-1-[N(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate at concentrations from 10 to 1000 µM as an exogenous NO-generating source before UVA irradiation led to a dose-dependent inhibition of both DNA strand breaks and apoptosis. In search of the molecular mechanism responsible for the protective effect, we find that protection from UVA-induced apoptosis is tightly correlated with NO-mediated increases in Bcl-2 expression and a concomitant inhibition of UVA-induced overexpression of Bax protein. In conclusion, we present evidence for a protective role of iNOS-derived NO in skin biology, because NO either endogenously produced or exogenously applied fully protects against UVA-induced cell damage and death. We also show that the NO-mediated expression modulation of proteins of the Bcl-2 family, an event upstream of caspase activation, appears to be the molecular mechanism underlying this protection.

    INTRODUCTION
Top
Abstract
Introduction
References

Exposure of eukaryotic cells to UVA radiation results in cellular inactivation and death (1, 2). UVA-induced oxidative damage has been reported to occur in lipids (3), coenzymes (4), and DNA (5). On the level of organelles, nucleated mammalian cells exposed to UVA radiation were reported to bear damaged cellular structures, notably the microtubuli (6), the plasma membrane, the nuclear membranes, and the rough endoplasmic reticulum (7, 8). Besides causing membrane damage, UVA radiation is also known to result in DNA damage, mostly in the form of single-strand breaks and protein-DNA cross-links. The amount of UVA radiation-mediated damage to membranes and DNA was found to correlate with the onset of cell death via apoptosis (5, 9).

Apoptosis is a distinct form of cell death controlled by an internally encoded suicide program. The morphological changes associated with apoptosis include the condensation of nucleoplasm, blebbing of cytoplasmic membranes, and fragmentation of the cell into apoptotic bodies that are rapidly phagocytosed by neighboring cells. The biochemical markers of apoptosis include activation of a caspase cascade, DNA fragmentation into nucleosomal fragments, and cleavage of various caspase substrates (10, 11), including gelsolin, leading to rounding and detachment of cells (12), and nuclear lamin, allowing for nuclear fragmentation (13, 14).

A plethora of anti-oxidant and antiapoptotic defense mechanisms exist in mammalian cells. These can be broadly classified into the expression of enzymes, including superoxide dismutase, catalase, and several peroxidases, compounds to quench reactive oxygen intermediates like ascorbate, ureic acid, carotenoids, and glutathione (15), or apoptosis-protective proteins such as Bcl-2 as critical factors controlling the apoptosis program (14). Recently, it has also been shown that the radical nitric oxide (NO),1 which is known to exert cytotoxic effects and mediate onset of apoptosis in a variety of mammalian cells (16), may also protect against the injurious actions of superoxide, hydrogen peroxide, and alkyl peroxides (17) as putative mediators of UV radiation-induced cytotoxicity.

NO and equal amounts of citrulline are synthesized from the guanidino nitrogen of L-arginine by nitric-oxide synthases (NOSs) found in endothelial cells and neurons and upon activation in macrophages and many other cell types. This enzyme family consists of three isoenzymes: the endothelial ecNOS and the neuronal ncNOS are constitutively expressed and calcium/calmodulin-regulated and produce regulated and low amounts of NO for short pulses, whereas the cytokine-inducible and calcium-independent isoenzyme (iNOS) synthesizes large amounts of NO for long periods of time (18, 19).

During inflammatory processes cytokines are known modulators of endothelial cell functions (20). One prominent effect that cytokines can exert in endothelial cells is the induction of iNOS, followed by high output NO synthesis (21, 22). The iNOS-generated NO is thought to serve mainly as nonspecific immune protection (23), and prolonged iNOS activity was described as dangerous for the host, as evidenced by its role in the pathogenesis of septic shock and cytokine-induced hypotension (24), the suppression of various cellular functions (25-27), inflammatory tissue destruction (28), and the induction of apoptosis (18).

Recently, UV irradiation has been shown to modulate local NO production in human skin (29). Additionally, our observation of a strong epidermal as well as endothelial iNOS expression in UV-irradiated normal skin (30) led us to search for a possible role of cutaneous UV-induced NO formation. Here, we tested the role of iNOS-generated as well as exogenously added NO in the UVA-induced apoptosis of resting or cytokine-activated ECs, respectively. The experiments presented here demonstrate for the first time a NO-mediated modulation of the Bcl family of proteins as a strong protective mechanism rescuing ECs from UVA-induced cell death.

    EXPERIMENTAL PROCEDURES

Reagents and Materials-- Recombinant human interleukin 1beta (IL-1beta ), recombinant human tumor necrosis factor alpha  (TNF-alpha ), and recombinant murine IFN-gamma were obtained from HBT (Leiden, Netherlands). Endothelial cell growth supplement, Neutral Red (3% solution), type I collagen, collagenase (from Clostridium histolyticum), rabbit anti-human von Willebrand factor antiserum, and Hoechst dye H33342 were from Sigma. Monoclonal antibody Ox43 was from Serotec (Camon, Wiesbaden, Germany). The rabbit anti-rat Bcl-2 antibody and Bax antisera were from PharMingen (San Diego, CA). Peroxidase-conjugated porcine anti-rabbit IgG was from DAKO (Hamburg, Germany), and peroxidase-conjugated goat anti-mouse IgG was from Zymed Laboratories Inc. Trypsin, EDTA, fetal calf serum (FCS, endotoxin-free), RPMI 1640 medium (endotoxin-free), biotin-dUTP, Kornberg polymerase, dGTP, dATP, dCTP, dithiothreitol, oligo(dT)16 primer, reverse transcriptase, and Taq polymerase were purchased from Boehringer Mannheim or Life Technologies, Inc. 3,3'-Diaminobenzidine was from Serva GmbH (Heidelberg, Germany). The interleukin-converting enzyme inhibitor Z-VAD was obtained from Enzyme Systems (Livermore, CA). The NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) was kindly provided by Prof. H. Weber (Heinrich-Heine-University of Düsseldorf, Düsseldorf, Germany). Denitrosilated DETA/NO (DETA) was obtained by incubating stock solutions (50 mM) of DETA/NO for 10 days at 37 °C. The iNOS inhibitor L-N5-(1-iminoethyl)-ornithine (NIO) was a kind gift from Boehringer Mannheim. We used a Sellas-2000 lamp (Sellas Medizinische Geräte GmbH, Geveisberg, Germany) emitting the UVA1 spectrum (340-390 nm) as a UVA source.

Endothelial Cells-- ECs were isolated by outgrowth from male Wistar rat aortic rings exactly as described previously (31). Aortic segments were placed on top of a collagen gel (1.8 mg collagen/ml) in 24-well tissue culture plates and incubated in RPMI 1640 medium with 20% FCS and 100 µg endothelial cell growth supplement/ml in a humidified incubator at 37 °C in a 95% air/5% CO2 atmosphere for 5 days. Aortic explants were then removed, and cells were detached with 0.25% collagenase in Hanks' balanced salt solution and replated onto plastic culture dishes in RPMI 1640 medium with 20% FCS. Cells were subcultured for up to eight passages, and removal from culture dishes for each passage was performed by treatment with trypsin/EDTA.

Cellular Characterization of Cultured Endothelial Cells-- Cells were passaged from tissue culture dishes onto sterile glass coverslips and allowed to grow as subconfluent monolayers. Cells were washed with PBS and fixed with acetone at -20 °C for 10 min, followed by inhibition of endothelial peroxidase activity with 0.3% H2O2 in ethanol and three washing steps in Tris-buffered saline. After blocking unspecific binding with 0.5% bovine serum albumin in Tris-buffered saline for 30 min and rinsing, specimens were incubated with a 1:50 dilution of rabbit anti-human-von Willebrand factor antiserum, which was previously shown to cross-react with the rat antigen (32), in a moist chamber for 45 min. After an additional wash, slides were incubated in a 1:50 dilution of peroxidase-conjugated porcine anti-rabbit IgG for 45 min at room temperature. After washing, peroxidase activity was visualized with 0.05% diaminobenzidine plus 0.015% H2O2 for 10 min at room temperature. Control cultures were incubated with a nonrelevant rabbit hyperimmune serum instead of the first antiserum. Positive controls (human platelets) and negative controls (rat alveolar macrophages and the fibroblastoma cell line L929) were also tested with this anti-von Willebrand factor antiserum. The rat vascular endothelium-specific monoclonal antibody Ox43 (33) was used in a 1:50 dilution. A peroxidase-conjugated goat anti-mouse IgG was diluted 1:50 before use. Otherwise, conditions were as described above.

Experimental Design-- All measurements were performed with cells from passages two to eight. Endothelial cells (2 × 105) were cultured in 12-well tissue culture plates or on 8-well chamber Tec glass slides in a humidified incubator at 37 °C in a 95% air/5% CO2 atmosphere in RPMI 1640 medium with 20% FCS. For iNOS induction and endogenous NO production, endothelial cells were activated 24 h before UVA irradiation (2, 4, 6, 8, and 10 J/cm2) by the addition of IL-1beta (200 units/ml), IL-1beta plus TNF-alpha (500 units/ml), or IL-1beta plus TNF-alpha plus IFN-gamma (100 units/ml). Inhibition of the cytokine-induced iNOS activity was reached by adding the iNOS inhibitor NIO (0.25 or 0.5 mM) to the activated endothelial cell cultures. NIO was present in the culture medium during cytokine activation as well as during and 24 h after UVA irradiation. Additionally, 24 h before UVA irradiation, resident endothelial cells were incubated with the NO donor DETA/NO or DETA alone at the concentrations indicated. 10 min before UVA exposure, cells were washed extensively with PBS and covered with 1 ml of Hanks' balanced salt solution/HEPES or RPMI 1640 medium without phenol red; after irradiation, cells were washed in PBS, and RPMI 1640/FCS medium was added. 24 h after irradiation, the relative number of living ECs was detected by neutral red staining (34). Cells were incubated for 90 min with neutral red (1:100 dilution of the 3% solution) and then washed twice with PBS. Cells were then dried and lysed by isopropanol containing 0.5% of 1 N HCl. Extinctions of the supernatants were then measured at 530 nm. Additionally, the viability of endothelial cells was routinely controlled at the beginning and end of every experiment using the trypan blue exclusion assay.

Nitrite Determination-- After 24 h of incubation, nitrite was determined in these control culture supernatants using the diazotization reaction as modified by Wood et al. (35) and using NaNO2 as a standard.

Detection and Quantification of Nuclear DNA Fragmentation-- DNA strand breaks of cells grown on 8-well chamber Tec slides were visualized by the in situ nick translation method (36) 1-24 h after UVA irradiation (2-10 J/cm2). In acetone-fixed cells, endogenous peroxidase activity was blocked with methanol plus 0.3% H202 for 30 min. The nick translation mixture contained 3 µM biotin-dUTP, 5 units/100 µl Kornberg polymerase, 3 µM each of dGTP, dATP, and dCTP, 50 mM Tris-HCL, pH 7.5, 5 mM MgC12, and 0.1 mM dithiothreitol, and the reaction was performed at room temperature for 20 min. Slides were washed in PBS and processed for immunocytochemical detection of biotin-labeled UTP by peroxidase-labeled avidin, followed by an enzyme reaction using 3,3'-diaminobenzidine as the substrate. In each sample, a minimum of 500 cells were counted, and labeled nuclei were expressed as a percentage of the total number of nuclei.

Detection of Nuclear Chromatin Condensation and Nuclear Fragmentation-- 1-24 h after UVA irradiation (2-10 J/cm2), endothelial cells grown in 12-well culture plates were washed with PBS and stained with Hoechst dye H33342 (8 µg/ml) for 5 min, and nuclei were visualized using a Zeiss fluorescence microscope. In each sample, a minimum of 400 cells were counted, and condensed or fragmented nuclei were expressed as a percentage of the total number of nuclei.

Polymerase Chain Reaction (PCR)-- Total cellular RNA (1 µg each), prepared 16 h after irradiation, from adherent growing nonirradiated and UVA-irradiated (6 J/cm2) ECs that were resting or cytokine-activated (addition of 200 units/ml IL-1beta  + 500 units/ml TNF-alpha  + 100 units/ml IFN-gamma 24 h before UVA irradiation), or preincubated with DETA/NO or DETA (1 mM each for 24 h before UVA irradiation) (37) was used for cDNA synthesis (38). Reverse transcription was carried out at 42 °C for 60 min, using the oligo(dT)16 primer. The cDNA was then used as a template for PCR, primed by either the oligonucleotides TATGATAACCGGGAGATCGTG (sense; bases 26-45 of rat Bcl-2 cDNA) and CAGATGCCGGTTCAGGTACTC (antisense; bases 526-546 of rat Bcl-2 cDNA) for specific Bcl-2 amplification (39) or the oligonucleotides CAAGAAGCTGAGCGAGTGTCT (sense; bases 62-82 of rat Bax cDNA) and GGTTCTGATCAGCTCGGGCAC (antisense; bases 279-299 of rat Bax cDNA) for specific Bax amplification (39). For specific rat glyceraldehyde-3-phosphate dehydrogenase cDNA amplification, the oligonucleotides CAACTACATGGTTTACATGTTCC (sense; rat glyceraldehyde-3-phosphate dehydrogenase cDNA bases 153-175) and GGACTGTGGTCATGAGTCCT (antisense; rat glyceraldehyde-3-phosphate dehydro- genase cDNA bases 549-568) were used (40). Additionally, to exclude unspecific amplification by mRNA or DNA contamination, control PCR was performed with all additives but without cDNA or with the RNA probes, respectively. PCR was carried out following standard protocols (41) with the following cycle profile: 33 cycles with 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C for Bcl-2 or Bax mRNA amplification and 25 cycles with 30 s at 94 °C, 30 s at 62 °C, and 30 s at 72 °C for glyceraldehyde-3-phosphate dehydrogenase mRNA amplification. A final incubation step was performed at 72 °C for 10 min. An aliquot of each reaction was subjected to electrophoresis on 1.5% agarose gels. Bands were visualized by ethidium bromide staining.

Western Blot Analysis of Bcl-2 and Bax Protein Expression-- After a 16-h incubation after UVA irradiation (6 J/cm2), resting, cytokine-activated (200 units/ml IL-1beta  + 500 units/ml TNF-alpha  + 100 units/ml IFN-gamma ), EC cultures preincubated with DETA/NO (1 mM for 24 h before UVA irradiation) or DETA (1 mM for 24 h before UVA irradiation) were washed, and adherent growing cells were lysed, scraped from the dishes, transferred to a microcentrifuge tube, and boiled for 5 min exactly as described previously (42). Proteins (50 µg/lane) were separated by electrophoresis in a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. To control equal loading of total protein in all lanes, blots were stained with Ponceau S solution immediately after transfer. Blots were incubated for 2 h with blocking buffer (2% bovine serum albumin, 5% non-fat milk powder, and 0.1% Tween 20 in PBS buffer), incubated for 1 h with a 1:1000 dilution of the anti-Bcl-2 or anti-Bax antiserum, washed, incubated for 1 h with a 1:1000 dilution of the secondary horseradish peroxidase-conjugated porcine anti-rabbit IgG antibody, incubated for 5 min in ECL reagent (Pierce, Rockford, IL), placed into a plastic bag, and exposed to an enhanced autoradiographic film.

Statistical Analysis-- Data are given as arithmetical means ± S.D. Values were calculated using Student's t test (two-tailed for independent samples).

    RESULTS

UVA irradiation of endothelial cells in a dose-dependent manner leads to cell death via apoptosis. Resting ECs were exposed to UVA radiation with increasing intensities as indicated. The relative number of viable or dead cells was determined using neutral red staining or trypan blue exclusion, respectively. Nuclear fragmentation or DNA strand breaks were visualized using Hoechst DNA dye H33342 or the in situ nick translation method, respectively.

Irradiation of ECs with UVA in a dose-dependent manner led to endothelial cell death (Fig. 1). We find that 24 h after maximal UVA irradiation with 10 J/cm2, 37 ± 7% of the initial cell number are still adherent; of these, about half were trypan blue-positive (Fig. 1A). Thus, maximal UVA irradiation leads to the cell death of 80% of cells within 24 h. About 60% of cells detach from the dish as a first indication for ongoing apoptosis, in which cellular detachment and cytoskeletal disruption occur. Half-maximal cytotoxicity was found at a UVA dose of 6 ± 1 J/cm2 (Fig. 1A). Indeed, at and above 6 J/cm2, staining with Hoechst DNA dye or in situ nick translation revealed nuclear alterations such as chromatin condensation and fragmentation, as well as DNA strand breaks in 35-80% of adherent cells (Figs. 1B and 2, C and D).


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Fig. 1.   UVA irradiation of endothelial cells leads to cell death. Resting endothelial cells were exposed to UVA radiation with increasing intensities, as indicated. The relative number of viable (open circle ) or dead adherent cells (black-square) was determined by neutral red staining or trypan blue exclusion, respectively. The relative number of all adherent cells (viable plus dead) is also indicated (). Furthermore, apoptotic nuclei and nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 () or the in situ nick translation method () and calculated as a percentage of all adherent cells, respectively. A, irradiation of ECs with UVA in a dose-dependent manner led to endothelial cell death as detected 24 h after irradiation. B, increasing UVA intensities led to an increasing number of cells with pygnotic nuclei, nuclear chromatin condensation, and nuclear fragmentation as well as DNA strand breaks as detected 24 h after irradiation. C-F, time course of UVA-induced cell death and apoptotic events; 4 or 8 h after irradiation with 6 (D) or 10 J/cm2 (F), a low but significant increase in the number of fragmented nuclei and nuclei with DNA strand breaks was found. At >= 16 h after UVA irradiation, the number of apoptotic nuclei is significantly increased. Values are the mean ± S.D. of 3-12 individual experiments.


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Fig. 2.   UVA irradiation of endothelial cells leads to apoptosis and DNA strand breaks. Resident or Z-VAD-treated (30 µM) ECs were exposed to UVA radiation (6 J/cm2), and after 16 h, apoptotic nuclei or nuclear DNA strand breaks were visualized using Hoechst DNA dye H33342 (A, C, and E) or the in situ nick translation method (B, D, and F), respectively. UVA irradiation leads to nuclear chromatin condensation and nuclear fragmentation (C) as well as DNA strand breaks in most of the nuclei (D) as indicators of ongoing apoptosis, whereas Z-VAD (30 µM) inhibits the development of these apoptosis symptoms (E and F). A and B, nonirradiated control cultures. Magnification: ×650 in A and E, ×700 in C, ×300 in B, D, and F.

Next we examined the time course of UVA-induced cell death, nuclear damage, and DNA damage; between 8 and 16 h after irradiation with 6 (Fig. 1C) or 10 J/cm2 (Fig. 1F), we found a highly significant increase in the number of nuclei with DNA strand breaks, indicating that DNA damage is not an immediate consequence of UVA irradiation. At >= 16 h after UVA irradiation, the number of altered nuclei increased to 35-65% of adherent cells at 6 J/cm2 or 40-90% of adherent cells at 10 J/cm2 .

Three lines of evidence argue in favor of apoptosis as the main course of cell death. As shown in Fig. 2, Hoechst staining of UVA-irradiated cells led to the formation of shrunken pygnotic nuclei and nuclear fragmentation (Fig. 2C). Furthermore, DNA strand breaks occur (Fig. 2D) in focal areas of unbroken nuclei 16 h after half-maximal UVA irradiation (6 J/cm2). Finally, incubation of ECs with the caspase inhibitor Z-VAD (30 µM) for 6 h before and for 24 h after UVA irradiation completely inhibits all morphological alterations of nuclei (Fig. 2, E and F).

Cytokine-induced Activation and Concomitant Endothelial iNOS Expression Fully Protects against Cell Damage-- ECs were activated by proinflammatory cytokines (200 units/ml IL-1beta , 500 units/ml TNF-alpha , and 100 units/ml IFN-gamma ). Activation led to the production of NO as evidenced by increased nitrite concentrations in culture supernatants. Incubation of endothelial cell cultures with different combinations of cytokines (200 units/ml IL-1beta , IL-1beta plus 500 units/ml TNF-alpha , and IL-1beta plus TNF-alpha plus 100 units/ml IFN-gamma ) shows a specific pattern of iNOS activity (Fig. 3A), exactly as described previously (32, 43). The cultures of resident or activated cells were exposed to UVA radiation, and the relative number of living cells was determined after an additional 24 h.


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Fig. 3.   iNOS expression and activity protects endothelial cells from injurious effects of UVA irradiation. ECs were activated by cytokines for high output NO production in the presence or absence of the iNOS inhibitor NIO. a, resident cells; b, IL-1beta (200 units/ml); c, IL-1beta  + TNF-alpha (500 units/ml); d, IL-1beta  + TNF-alpha  + IFN-gamma (100 units/ml); e, IL-1beta  + TNF-alpha  + IFN-gamma (100 units/ml) + NIO (0.25 mM); f, IL-1beta  + TNF-alpha  + IFN-gamma (100 units/ml) + NIO (0.5 mM). Nitrite concentrations in culture supernatants were determined 24 h after cytokine challenge. Cells were then exposed to UVA radiation, and after an additional incubation of 24 h, the relative number of living cells was determined by neutral red staining. A and B, EC cultures incubated with IL-1beta (b; black-square) exhibit a half-maximal iNOS activity and a half-maximal protection. Maximal endothelial iNOS activity after incubation with combinations of the cytokines (c and d;  and ) results in a highly significant protection from UVA-induced cell death. C, activation of ECs in the presence of the NOS inhibitor NIO (0.25 mM and 0.5 mM; e and f; triangle  and black-triangle) abrogates the protective effects. D, protection against UVA-induced (10 J/cm2) cell death is in linear correlation (R2 = 0.9763) with the amount of NO produced. Values are the mean ± S.D. of four to eight individual experiments. *, p < 0.001; **, p < 0.0001.

A highly significant protection against UVA-induced cell death in close correlation to the amount of NO produced was observed (Fig. 3, B-D). EC cultures incubated with the single cytokine IL-1beta show a half-maximal iNOS activity, leading to 3.5 ± 0.6 nmol of nitrite in culture supernatants. This leads to significantly protective effects against UVA-induced cell death (40 ± 5% surviving cells at 8 J/cm2; 33 ± 5% surviving cells at 10 J/cm2). Maximal endothelial iNOS activity was achieved after incubation with the cytokine combinations IL-1beta plus TNF-alpha (10.7 ± 1.0 nmol of nitrite) or Il-1beta plus TNF-alpha plus IFN-gamma (11.8 ± 1.7 nmol of nitrite) and resulted in a highly significant protection (90 ± 6% of surviving cells at 6 J/cm2; 82 ± 7% of surviving cells at 10 J/cm2). When activation of ECs was performed in the presence of the NOS inhibitor NIO (0.25 mM), complete abrogation of the protective effects further indicated that iNOS activity does indeed mediate the protective mechanism (Fig. 3C). Cell survival after UVA irradiation shows a linear correlation with iNOS activity as measured by nitrite concentrations in culture supernatants (Fig. 3D).

Additionally, parallel to these experiments, apoptotic nuclei and DNA strand breaks were visualized 16 h after half-maximal UVA irradiation (6 J/cm2). In cytokine-activated cultures (IL-1beta plus TNF-alpha plus IFN-gamma ), cells are fully protected from UVA-induced apoptosis (Fig. 4, A and B) as seen in comparison to resident and UVA-irradiated cells (Fig. 2, C and D) where apoptosis is frequent. Inhibition of endothelial NO production by the addition of NIO (0.25 mM) abrogates this protection (Fig. 4, C and D).


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Fig. 4.   Endogenous NO production protects ECs from UVA-induced apoptosis and DNA strand breaks. Cytokine-activated (200 units/ml IL-1beta plus 500 units/ml TNF-alpha plus 100 units/ml IFN-gamma ) cells incubated in the presence or absence of NOS inhibitor NIO (0.25 mM) were exposed to UVA radiation as described in the Fig. 2 legend (A and C, Hoechst dye; B and D, in situ nick translation). In cytokine-activated EC cultures, cells are protected from UVA-induced apoptosis (A) and DNA strand breaks (B), whereas inhibition of NO production by the addition of NIO abrogates this protection (C and D). Magnification: ×650 in A and C; ×300 in B and D.

Exogenous NO Is Sufficient to Protect against UVA Irradiation-induced Cell Damage-- To test whether NO alone is sufficient to mediate the above described protection, resident ECs were incubated 24 h before UVA irradiation with various concentrations of the chemical NO donor DETA/NO.

Neutral red staining of living cells performed 24 h after UVA irradiation revealed that at and above concentrations of 50 µM DETA/NO, protection was always significant (Fig. 5A). With 1 mM DETA/NO, full protection was achieved (86 ± 9% of surviving cells at 6 J/cm2 UVA; 78 ± 7% of surviving cells at 10 J/cm2 UVA). In contrast, DETA without NO had no protective effect at all (Fig. 5B), proving the NO specificity. A linear correlation between protection and the concentration of the exogenous NO donor was found (Fig. 5C). Neither DETA/NO nor any of the other compounds used showed any cytotoxic effects toward the ECs within the incubation times.


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Fig. 5.   Incubation of ECs with DETA/NO before UVA irradiation protects cells from UVA-induced cell damage. Resident ECs were incubated for 24 h before UVA irradiation with the NO donor DETA/NO (open circle , control cells; black-diamond , 10 µM; diamond , 50 µM; black-square, 0.1 mM; , 0.5 mM; , 1 mM) or control additives, respectively. Neutral red staining of living cells was performed 24 h after UVA irradiation. A, incubation of cells with DETA/NO before UVA irradiation leads to a concentration-dependent protection from UVA-induced cell damage. At >= 50 µM DETA/NO, the protection against UVA-induced toxicity at 4-10 J/cm2 was highly significant (dotted cage) as compared with the controls (open circle ). Values are the mean of 12 individual experiments. B, as a control additive, denitrosilated DETA/NO (black-triangle, 1 mM) has no protective effect, providing evidence for NO specificity. C, a linear correlation of protection against UVA-induced cell death (10 J/cm2) with the concentration of NO donor present was observed (R2 = 0.9404). Values are the mean ± S.D. of 6-12 individual experiments. **, p < 0.0001.

Again, visualization of apoptotic nuclei and DNA strand breaks (Fig. 6) exactly as described above demonstrated that EC cultures preincubated with the NO donor before irradiation were exactly like the untreated and nonirradiated controls (Fig. 2), whereas DETA alone had no protective action against UVA-induced nucleus aberrations.


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Fig. 6.   Exogenous NO protects ECs from UVA-induced apoptosis and DNA strand breaks. Resident ECs were incubated 24 h before UVA irradiation (6 J/cm2) with the NO donor DETA/NO (1 mM) or DETA (1 mM) alone, respectively. After 16 h, apoptotic nuclei or DNA strand breaks were detected using Hoechst dye (A and C) or in situ nick translation (B and D), exactly as described in Figs. 1 and 3. Incubation of EC cultures with DETA/NO before UVA irradiation protects cells from UVA-induced apoptosis (A) and DNA strand breaks (B), whereas DETA alone (C and D) has no protective action. Magnification: ×650 in A and C, ×300 in B and D.

NO or UVA Modulates the Expression of Endothelial Bcl-2 or Bax mRNA as well as Bcl-2 or Bax Protein-- In search of the molecular mechanism(s) responsible for the protective effects, we examined the mRNA expression and the relative amounts of the two proteins Bax and Bcl-2 after the various treatments.

As described above, 16 h after UVA irradiation (6 J/cm2), 72 ± 7% of ECs were still adhering to the plate, and only these could be used for PCR or Western blot analysis. Of the adherent population, 79% were live cells positively stained by neutral red and excluding trypan blue.

Both endogenously produced NO after cytokine challenge (Fig. 7A) and exogenously applied NO (Fig. 7C) result in a 2.5-3-fold increase in Bcl-2 mRNA expression that persists for 24 h, whereas activation of cells in the presence of the NOS inhibitor NIO (0.25 mM) or incubation with DETA alone had no effects on Bcl-2 mRNA expression (Fig. 7, B and D), indicating the NO specificity of these effects. Moreover, the time course also reveals that UVA will not counteract the up-regulated Bcl-2 gene expression.


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Fig. 7.   Effects of NO and UVA irradiation on endothelial Bcl-2 mRNA expression. Using specific reverse transcription-PCR, we examined the effects of nitric oxide on the Bcl-2 mRNA expression of cytokine-activated and DETA/NO (1 mM)-preincubated ECs, exactly as described under "Experimental Procedures." Endogenously produced NO in cytokine-activated ECs (A) and exogenously applied NO (C) led to a 2.5-3-fold increase in Bcl-2 mRNA expression that persisted for 48 h, irrespective of UVA irradiation (6 J/cm2). Activation of cells in the presence of the NOS inhibitor NIO (B; 0.25 mM) or incubation of cells with DETA alone (D) had no effects on Bcl-2 mRNA expression, indicating the NO specificity of these effects.

Furthermore, 1 h after UVA irradiation (6 J/cm2) of resting cells, we found an increased Bax mRNA expression that was augmented after 24 h by a factor of 2.5-3.5 compared with the controls (Fig. 8, A and B), whereas in cytokine-preactivated ECs (Fig. 8A) or cells pretreated for 24 h with DETA/NO (Fig. 8B), UVA had no influence on endothelial Bax mRNA expression.


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Fig. 8.   Effects of NO and UVA irradiation on endothelial Bax mRNA expression. Using specific reverse transcription-PCR, we examined the effects of UVA (6 J/cm2) and nitric oxide on Bax mRNA expression in cytokine-activated as well as DETA/NO (1 mM)-preincubated ECs, exactly as described under "Experimental Procedures." A and B, as compared with the controls, UVA irradiation of resting cells led to a 2.5-3.5-fold increase in Bax mRNA expression, whereas in cytokine-activated ECs (A) with high output NO production or in DETA/NO-preincubated cells (B), UVA had no influence on endothelial Bax mRNA expression. Inhibition of iNOS activity (A) by NIO (0.25 mM) or incubation of ECs with DETA alone (B) led to a pattern of Bax mRNA expression after UVA irradiation similar to that seen with resting cells.

Protein expression as studied by Western blots showed analogous effects of NO or UVA (Fig. 9). Untreated resident ECs express both proteins (lanes 1 and 4). Cytokine activation and concomitant high output NO production (as seen in Fig. 3A) resulted in a significant increase in Bcl-2 levels (Fig. 9B, lanes 2 and 5), and this Bcl-2 overexpression was absent when cells were activated in the presence of NIO (0.25 mM). UVA irradiation of resting cells significantly increased endothelial Bax levels (Fig. 9A). This UVA-induced Bax overexpression was blocked by the preactivation of cells; instead, a simultaneous overexpression of Bcl-2 was found. Again, both effects were absent when activation was performed in the presence of the NOS inhibitor. Controls with resident cells and NIO addition showed no differences from untreated controls (data not shown). Parallel experiments with or without exogenous NO (24 h preincubation with 1 mM DETA/NO) and with or without UVA irradiation resulted in identical patterns of NO-mediated Bcl-2 increase and abrogation of UVA-induced Bax overexpression (Fig. 9B).


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Fig. 9.   Effects of NO and UVA irradiation on endothelial Bcl-2 and Bax protein expression. 16 h after UVA irradiation (6 J/cm2) of resting, cytokine-activated (11 nmol nitrite/1 × 105 cells × 24 h after incubation with 200 units/ml IL-1beta  + 500 units/ml TNF-alpha  + 100 units/ml IFN-gamma ) or DETA/NO (1 mM for 24 h before UVA irradiation)-preincubated EC cultures, the expression of Bax and Bcl-2 protein was examined by Western blots (50 µg total protein/lane) exactly as described under "Experimental Procedures." A, UVA irradiation of resting EC cultures led to a strongly increased expression of Bax as compared with nonirradiated ECs. Endogenous NO production by iNOS inhibited the UVA-induced increase in Bax expression, whereas after inhibition of the endogenous NO formation with NIO, Bax expression was similar to that seen with irradiated controls. In sham-treated EC cultures, endogenous NO in both nonirradiated and UVA-irradiated EC cultures increased the expression of the antiapoptotic protein Bcl-2, but UVA or incubation with NIO did not affect Bcl-2 expression. Equal loading of protein lysates was confirmed by Ponceau S staining of loaded membranes (Ponceau S; figures show the dominant protein bands at 35-40 kDa). B, parallel experiments with exogenous NO (DETA/NO) gave similar results.


    DISCUSSION

A consequence of excessive UV exposure of human skin is the occurrence of programmed cell death or sunburn cells, which appears to contribute to aging and tumor development (1). We recently observed that UV challenge of normal human skin leads to the induction of iNOS expression in keratinocytes and capillary endothelial cells.

Higher amounts of NO as produced by iNOS are known inducers of cell death that often follows the route of apoptosis (44), and it was generally accepted that apoptosis induction represents one mode of action for the cytotoxic effects of NO (16). Thus, it was tempting to speculate that iNOS expression after UV challenge may contribute to cell death in skin.

However, very recent reports indicate that NO can also exert antiapoptotic activity (45, 46) by inhibiting caspase 3 via S-nitrosation (47, 48) or by modulating the expression of the antiapoptotic protein Bcl-2 (49), respectively. Indeed, with the results of our experiments, we provide evidence that NO, either endogenously produced or exogenously generated, is a powerful tool to prevent UVA-induced apoptosis.

In search of the intracellular events that help to explain this protective effect, we find that events upstream of caspase 3 activation appear to be responsible, namely, the modulation of proteins from the Bcl family at their relative expression levels by both UVA and NO, albeit in opposing patterns. Proteins of the Bcl family regulate apoptosis either positively (as, for instance, Bax) or negatively (as, for instance, Bcl-2 (11, 50)). Both proteins are found as intracellular membrane-associated proteins of the outer mitochondrial membrane, endoplasmatic reticulum, and nuclear envelope (51). Bax was shown to form channels in lipid membranes, and the proapoptotic effect of Bax appears to be elicited through an intrinsic pore-forming activity (52), thus leading to leakage of cytochrome c from the mitochondrial intermembrane compartment into the cytosol. Cytosolic cytochrome c initiates the apoptotic program by activating specific caspases, leading to the cleavage of fodrin and lamin (53) and apoptotic nuclear morphology (54). In contrast, Bcl-2 overexpression prevents the release of cytochrome c and ensuing events and thus mediates antiapoptotic effects (42, 55), and an inhibitory role of Bcl-2 on Bax channel-forming activity appears likely (52). Thus, onset of apoptosis is controlled by the ratio of death agonists (like Bax) to antagonist (like Bcl-2). This death-life rheostat is mediated, at least in part, by competitive dimerization: with excess Bax, Bax homodimer formation will dominate, and apoptosis occurs; with excess Bcl-2, Bax/Bcl-2 heterodimers will dominate, and cells are protected (56, 57).

As mentioned above, the antiapoptotic activity of NO has been mainly attributed to NO-mediated S-nitrosation and the partial inhibition of caspase 3 or activation of Bcl-2. With the results presented here, we now show that NO can also act upstream of caspase 3, mainly by regulating the expression levels of Bcl-2. The proapoptotic effects of UVA lead to significant increases in relative Bax expression. In contrast, cytokine-activated cells exhibit a significant increase in Bcl-2 levels and also an apparent suppression of UVA-induced Bax overexpression. Endogenous NO synthesis and exogenously applied NO thus very effectively counteract the proapoptotic UVA activity. Because NO-mediated protection tightly correlates with Bcl-2 overexpression at the time of UVA challenge, data presented are in full agreement with several publications demonstrating that Bcl-2 protects against diverse cytotoxic insults including UV irradiation (for a review, see Ref. 58).

The concentration of exogenously added NO necessary to achieve full protection lies well within physiological levels as deduced from published data. It has been shown (59, 60) that DETA/NO spontaneously releases NO with a predictable first-order kinetic and a half-life of 7.7 h, thus providing a constant NO supply over a period of hours. According to the first-order kinetic laws (t1/2 = ln2/k and c = c0 × e-kt), 1 mM DETA/NO generates 2.7 µM NO/min. Recently, a steady-state concentration of about 4-5 µM NO has been calculated to be present in the immediate vicinity of a cell monolayer that enzymatically generates NO (61). In consideration of these calculations and measured data, the amount of NO needed for full protection is readily available under physiological conditions.

In summary, we show that NO protects against UVA-induced cell death and that endogenously produced or exogenously generated NO is sufficient to achieve full protection. Thus, iNOS expression in human skin after UV exposure appears to represent a beneficial response helping to minimize sunburn-associated skin damage. This concept is further backed by our recent finding in cutaneous Lupus erythematosus patients (30). These patients are known to exhibit photosensitivity leading to skin lesion formation after UV exposure. We could demonstrate that in these patients, a significantly delayed UV-induced iNOS expression correlates with lesion formation as further evidence for the beneficial activity of iNOS expression in UV exposure.

    ACKNOWLEDGEMENTS

We thank Prof. H. Weber for kindly providing DETA/NO, Christa-Maria Wilkens, Marija Lenzen, and Ulla Lammersen for technical assistance, and Martha Turken for photographic assistance.

    FOOTNOTES

* This study was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 503, A3, to V. K.-B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Institute of Immunobiology Geb. 14.80, MED-Heinrich-Heine-University, P. O. Box 10 10 07, D-40001 Düsseldorf, Germany. Tel.: 49-211-81-19184; Fax: 49-211-81-19532; E-mail: Bachofen{at}uni-duesseldorf.de.

    ABBREVIATIONS

The abbreviations used are: EC, endothelial cell; NO, nitric oxide; NOS, nitric-oxide synthase; iNOS, inducible NOS; IL-1beta , interleukin 1beta ; TNF-alpha , tumor necrosis factor alpha ; DETA/NO, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; DETA, denitrosilated DETA/NO; NIO, L-N5-(1-iminoethyl)-ornithine; PBS, phosphate-buffered saline; FCS, fetal calf serum; PCR, polymerase chain reaction.

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