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UVB Light Stimulates Production of Reactive Oxygen Species

2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês

10.1074/jbc.c300048200

1083-351X

Diane E. Heck, Anna M. Vetrano, Thomas M. Mariano, Jeffrey D. Laskin,

Skin Protection and Aging

In keratinocytes, UVB light stimulates the production of reactive oxygen species (ROS). Lysates of these cells were found to possess a non-dialyzable, trypsin- and heat-sensitive material capable of generating ROS in response to UVB light. Using ion exchange, metal affinity, and size exclusion chromatography, a 240-kDa protein was isolated with ROS generating activity. The protein exhibited strong absorption in the 320–360 nm range with additional soret peaks around 400–410 nm, suggesting the presence of heme. Sequencing using liquid chromatography-ion trap mass spectrometry identified the protein as catalase. Using purified catalases from a variety of species, the ROS generating activity was found to be temperature- and O2-dependent, stimulated by inhibitors of the catalatic activity of catalase, including 3-aminotriazole and azide, and inhibited by cyanide. A marked increase in the production of ROS was observed in UVB-treated cells overexpressing catalase and decreased generation of oxidants was found in UVB-treated keratinocytes with reduced levels of catalase. Our data indicate that catalase plays a direct role in generating oxidants in response to UVB light. The finding that catalase mediates the production of ROS following UVB treatment is both novel and highly divergent from the well known antioxidant functions of the enzyme. We hypothesize that, through the actions of catalase, high energy DNA damaging UVB light is absorbed by the enzyme and converted to reactive chemical intermediates that can be detoxified by cellular antioxidant enzymes. Accumulation of excessive ROS, generated through the action of catalase, may lead to oxidative stress, DNA damage, and the development of skin cancer. In keratinocytes, UVB light stimulates the production of reactive oxygen species (ROS). Lysates of these cells were found to possess a non-dialyzable, trypsin- and heat-sensitive material capable of generating ROS in response to UVB light. Using ion exchange, metal affinity, and size exclusion chromatography, a 240-kDa protein was isolated with ROS generating activity. The protein exhibited strong absorption in the 320–360 nm range with additional soret peaks around 400–410 nm, suggesting the presence of heme. Sequencing using liquid chromatography-ion trap mass spectrometry identified the protein as catalase. Using purified catalases from a variety of species, the ROS generating activity was found to be temperature- and O2-dependent, stimulated by inhibitors of the catalatic activity of catalase, including 3-aminotriazole and azide, and inhibited by cyanide. A marked increase in the production of ROS was observed in UVB-treated cells overexpressing catalase and decreased generation of oxidants was found in UVB-treated keratinocytes with reduced levels of catalase. Our data indicate that catalase plays a direct role in generating oxidants in response to UVB light. The finding that catalase mediates the production of ROS following UVB treatment is both novel and highly divergent from the well known antioxidant functions of the enzyme. We hypothesize that, through the actions of catalase, high energy DNA damaging UVB light is absorbed by the enzyme and converted to reactive chemical intermediates that can be detoxified by cellular antioxidant enzymes. Accumulation of excessive ROS, generated through the action of catalase, may lead to oxidative stress, DNA damage, and the development of skin cancer. Chronic exposure to sunlight is a significant causative factor in the development of skin cancer. Modifications of DNA and other critical cellular macromolecules by the higher energy shorter solar wavelengths comprising the UVB spectra (290–320 nm) are the most damaging to the skin (for reviews, see Refs. 1Matsumura Y. Ananthaswamy H.N. Front. 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Med. 2000; 28: 1456-1462Crossref PubMed Scopus (866) Google Scholar). When generated in excess, reactive oxygen species can also induce tissue injury and contribute to the development of skin cancer (16Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7532) Google Scholar, 17Kovacic P. Jacintho J.D. Curr. Med. Chem. 2001; 8: 773-796Crossref PubMed Scopus (349) Google Scholar, 18Thiele J.J. Schroeter C. Hsieh S.N. Podda M. Packer L. Curr. Probl. Dermatol. 2001; 29: 26-42Crossref PubMed Google Scholar, 19Hensley K. Robinson K.A. Gabbita S.P. Salsman S. Floyd R.A. Free Radic. Biol. Med. 2000; 28: 1456-1462Crossref PubMed Scopus (866) Google Scholar). In the present studies we report that human skin expresses an enzyme that generates reactive oxygen species in response to UVB light; unexpectedly, this enzyme was identified as catalase. The conversion of DNA damaging solar radiation into less energetic oxidant species by catalase is a novel and previously unrecognized activity of this antioxidant enzyme. Cells, UVB Light Irradiation, Detection of Reactive Oxygen Species— PAM 212 keratinocytes were kindly provided by Dr. Stuart Yuspa (National Institutes of Health, Bethesda, MD). Human keratinocytes were from Clonetics (Gaithersburg, MD). HA-1 and catalase-overexpressing OC5 Chinese hamster fibroblasts were obtained from Dr. Douglas Spitz (University of Iowa, Iowa City, IO). Bovine and mouse liver catalase, twice crystallized bovine liver catalase, and all chemicals were from Sigma unless otherwise specified. Bovine catalase was processed as indicated below to confirm that the protein was purified to homogeneity. Repurified material was used to characterize the oxidant generating activity of the enzyme. Polyclonal anti-catalase antibodies were from Abcam Ltd. (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Bio-Rad. For anion exchange and size exclusion chromatography, an AKTA fast protein liquid chromatograph (Amersham Biosciences) fitted with a Mono Q HR 5/5 column and a Superose HR 10/30 column were used. Samples were applied to the columns in buffer containing 25 mm Tris-HCl, pH 8.0 supplemented with 5% glycerol. Catalase eluted at ∼0.3 m NaCl using a linear salt gradient on the anion exchange column. Catalase eluted from the size exclusion column with a molecular mass of 240 kDa, which corresponds to the tetramer form of the enzyme. Metal affinity chromatography is a highly efficient method to purify catalase (20Yang Q. DePierre J.W. Protein Expression Purif. 1998; 12: 277-283Crossref PubMed Scopus (11) Google Scholar), and we used a 1-ml HiTrap Chelating HP column (Amersham Biosciences) charged with Ni(NO3)2 according to the manufacturer's instructions for these studies. Catalase was eluted using a 100 mm linear imidazole gradient. Purified material was analyzed on 10% SDS-polyacrylamide gels and appeared as a single 60-kDa band following silver staining. For ultraviolet light treatment, cells or reaction mixtures were irradiated in uncovered 96-well tissue culture plates (Costar, Corning, NY) with UVB light emitted from two Westinghouse FS20 light tubes as described previously (21Sur R. Heck D.E. Mariano T.M. Jin Y. Murphy W.J. Laskin J.D. Biochem. Pharmacol. 2002; 64: 1469-1481Crossref PubMed Scopus (14) Google Scholar). The UVB lights were calibrated with an IL 442A Phototherapy Radiometer (International Light, Newburyport, MA). To generate dose responses, reaction mixes were either treated with UVB for increasing periods of time at a fixed distances from the light source (25 cm) or by increasing the intensity of the light source. Results from both methods were expressed as mJ/cm2 and produced similar results. Unless otherwise indicated, intracellular reactive oxygen species were detected using 2′,7′-dichlorofluorescein diacetate (Molecular Probes, Eugene, OR) in conjunction with flow cytometry as described previously (21Sur R. Heck D.E. Mariano T.M. Jin Y. Murphy W.J. Laskin J.D. Biochem. Pharmacol. 2002; 64: 1469-1481Crossref PubMed Scopus (14) Google Scholar, 22Heck D.E. Laskin D.L. Gardner C.R. Laskin J.D. J. Biol. Chem. 1992; 267: 21277-21280Abstract Full Text PDF PubMed Google Scholar). For standard in vitro assays, reaction mixes contained 50 mm phosphate buffer, pH 7.4, and 4.3 μm catalase in a reaction volume of 100 μl. The reaction was initiated by the addition of the fluorescent probe (5 μm, final concentration). Fluorescence was quantified using an HTS 7000 plus bio-assay reader (PerkinElmer Life Sciences, Beaconsfield, Buckinghamshire, UK) with 495 nm excitation and 520 nm emission filters. In some assays, peroxides were detected using 1,2,3-dihydrorhodamine (1,2,3-DHR, Molecular Probes). To determine the effects of pH on UVB light-stimulated hydrogen peroxide production, the reaction was performed in buffers ranging in pH from 3.5 to 10.0 (sodium acetate, pH 3.5 and 4.5; sodium citrate, pH 5.5 and 6.0; potassium phosphate, pH 7.0 and 7.4; sodium borate, pH 8.0, 8.5, 9.0, and 10.0). Cytochrome c assay and nitro blue tetrazolium assays were used to measure superoxide anion production in reaction mixes (23Bors W. Saran M. Michel C. Oberly L. Superoxide Dismutase. II. CRC Press, Boca Raton, FL1982: 31-62Google Scholar, 24Weinberger B. Fakhrzadeh L. Heck D.E. Laskin J.D. Gardner C.R. Laskin D.L. Am. J. Respir. Crit. Care Med. 1998; 158: 931-938Crossref PubMed Scopus (50) Google Scholar). Catalase Variants, Transfections, Protein Sequencing, and Western Blotting—Protein was quantified using either the BCA protein reagent kit (Pierce) or the detergent-compatible (Dc) protein assay (Bio-Rad) with bovine serum albumin (BSA) 1The abbreviations used are: BSA, bovine serum albumin; DCFH-DA, 2′,7′-dichlorofluorescin diacetate; 1,2,3-DHR, 1,2,3-dihydrorhodamine; 3-AT, 3-amino-1,2,4-triazole; BSO, buthionine sulfoximine. as the standard. Western blots were run as previously described (21Sur R. Heck D.E. Mariano T.M. Jin Y. Murphy W.J. Laskin J.D. Biochem. Pharmacol. 2002; 64: 1469-1481Crossref PubMed Scopus (14) Google Scholar, 22Heck D.E. Laskin D.L. Gardner C.R. Laskin J.D. J. Biol. Chem. 1992; 267: 21277-21280Abstract Full Text PDF PubMed Google Scholar). Briefly, lysates containing catalase were separated on 10% SDS-polyacrylamide gels and then transferred onto nitrocellulose membranes. After blocking with 5% BSA in tTBS buffer (Tris-buffered saline with 0.1% Tween 20) for 1 h, membranes were incubated with anti-catalase antibodies overnight at 4 °C followed by horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Catalase-antibody complexes were visualized using enhanced chemiluminescence (ECL) reagents (PerkinElmer Life Sciences, Boston, MA). Protein fragments derived from tryptic digests of purified protein were analyzed using a Finigan LCQ DECA XP mass spectrometer interfaced with a Thermo separation system. Fragments were aligned and sequences compared using NCBI Blast algorithms (25Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59933) Google Scholar). Catalase morpholino antisense oligonucleotides were synthesized that contained nucleotide bases linked to a six-membered morpholino ring (Gene-Tools, Philomath, OR). Morpholino oligonucleotides were delivered to the keratinocytes using an ethoxylated polyethylenimine delivery system according to the manufacturer's directions. In some experiments oligos were biotinylated and transfection efficiency evaluated using fluorescent labeled streptavidin. In these experiments, ∼80–85% of the cells contained morpholino oligos. The 25-base morpholino probes, designed in conjunction with Dr. Shannon Knuth (Gene-Tools) were targeted to positions 57–81 of the cDNA sequence encoding murine catalase 1. In our initial studies, we analyzed the effects of UVB light on the generation of intracellular oxidants in human and mouse keratinocytes. We found that UVB light, in the range of 1–100 mJ/cm2, caused a marked increase in the formation of reactive oxygen species in both of these cell types (Fig. 1 and data not shown). This response was dependent on the dose of UVB light. To defend against damaging free radical-mediated reactions, cells possess enzymes and small molecular weight molecules with antioxidant activity (16Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7532) Google Scholar, 19Hensley K. Robinson K.A. Gabbita S.P. Salsman S. Floyd R.A. Free Radic. Biol. Med. 2000; 28: 1456-1462Crossref PubMed Scopus (866) Google Scholar, 26Engelhardt J.F. Antioxid. Redox Signal. 1999; 1: 5-27Crossref PubMed Scopus (59) Google Scholar, 27Bauer V. Bauer F. Gen. Physiol. Biophys. 1999; 18: 7-14PubMed Google Scholar). One intracellular mediator recognized as critical for cellular defense against reactive oxygen intermediates is glutathione. In addition to its role as a substrate for glutathione-dependent antioxidant enzymes, this thiol tripeptide participates in the regeneration of ascorbate and α-tocopherol and functions to directly detoxify reactive species via its ability to conjugate pro-oxidants (16Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7532) Google Scholar, 28Sheehan D. Meade G. Foley V.M. Dowd C.A. Biochem. J. 2001; 360: 1-16Crossref PubMed Scopus (1421) Google Scholar, 29Monks T.J. Anders M.W. Dekant W. Stevens J.L. Lau S.S. van Bladeren P.J. Toxicol. Appl. Pharmacol. 1990; 106: 1-19Crossref PubMed Scopus (203) Google Scholar, 30Strange R.C. Spiteri M.A. Ramachandran S. Fryer A.A. Mutat. Res. 2001; 482: 21-26Crossref PubMed Scopus (682) Google Scholar). We found that depleting keratinocytes of glutathione using buthionine sulfoximine, an inhibitor of glutathione synthesis, increased intracellular levels of reactive oxygen species (Fig. 1, center panel). Moreover, glutathione-depleted cells were significantly more sensitive to the oxidant-generating effects of UVB light (Fig. 1, center panel). These results demonstrate that glutathione is critical for limiting the accumulation of potentially damaging reactive oxygen species in UVB-treated keratinocytes. To analyze mechanisms by which UVB light induces the formation of reactive oxygen species in keratinocytes, we determined whether intact cells were required for this process. Lysates, prepared by successive freeze thawing of mouse keratinocytes, were found to retain UVB light-induced oxidant generating activity. Following low speed centrifugation (1000 × g, 10 min), ∼80% of the activity remained in cell supernatants. This was reduced to 30% by subsequent higher speed centrifugation (12,000 × g, 10 min). Thus, ∼70% of the oxidant generating activity was found in the resulting cell pellet. This fraction was largely comprised of actin and other insoluble polymers, intracellular organelles including peroxisomes, mitochondria, and the endoplasmic reticulum. We found that the reactive oxygen species generating activity was non-dialyzable, denatured by heating, and trypsin-sensitive, suggesting that the material was a protein (Fig. 2 and data not shown). Material generating reactive oxygen intermediates in response to UVB light was purified using ion exchange, metal affinity, and size exclusion chromatography. An example of the purification using an ion exchange column and a representative Western blot are shown in Fig. 2A. On the size exclusion column, this activity eluted with a molecular mass of 240 kDa and exhibited strong absorption in the range of 320–360 nm with additional soret peaks at 400–410 nm indicating the potential presence of iron-bound heme (not shown). Trypsin digests of the material were analyzed by liquid chromatography-ion trap mass spectrometry and found to contain peptide fragments that were highly homologous with the amino acid sequence predicted for catalase (residues Gly48–Arg68 and Gly78–Arg93 of mouse catalase 1, Cancer Genomics Office, NCI, National Institutes of Health). Interestingly, these peptide sequences were 99.6% homologous with catalase sequences from Rattus norvegicus, 97.2% homologous with human erythrocyte catalase, 95.6% homologous with bovine liver catalase, and 89.9% homologous with the sequence of Danio rerio catalase 1, reflecting the significant similarities found in vertebrate catalases and consistent with a protein containing an iron-ligated heme. These data suggested that the oxidant generating protein was catalase. This was confirmed by Western blot analysis of the column fractions using catalase-specific antibodies (Fig. 2, A and B, not shown). Moreover, commercially available catalase proteins, including twice crystallized bovine liver catalase and purified bovine and mouse liver catalase, produced reactive oxidant species when stimulated by UVB light (Fig. 2B). Taken together these data indicate that catalase is responsible for the UVB light-induced reactive oxygen species generating activity in keratinocytes. In further studies, we investigated this activity using highly purified bovine liver catalase. We found that the ability of the enzyme to generate reactive oxidants was dependent on the dose of UV light utilized and the concentration of catalase (Fig. 2C). It was also temperature- and oxygen-dependent and inhibited by heat denaturation (Fig. 2C and not shown). These experiments were performed using DCFH-DA as the probe for peroxide. Similar results were also found using the peroxide sensitive probe 1,2,3-DHR (Fig. 2C). In further experiments, we found that Met (30 mm) did not inhibit peroxide production by UVB-treated catalase when either DCFH-DA or 1,2,3-DHR was used as a probe suggesting that singlet oxygen was not formed (data not shown). At the doses of UVB used (<1–100 mJ/cm2), the superoxide anion could not be detected by cytochrome c and nitro blue tetrazolium assays, suggesting that it may not be an intermediate in the reaction under these conditions. However, we were able to detect superoxide anion at doses of UVB greater that 200 mJ/cm2 (data not shown). At the present time, we cannot rule out the possibility that the superoxide anion is formed in the reactions at low concentrations and/or that it is rapidly degraded. As observed in intact keratinocytes, the production of reactive oxygen species by purified catalase in response to UVB light was diminished in the presence of reduced glutathione (Figs. 3, lower panel, and not shown). The optimum pH for oxidant generation was 7.1 (Fig. 2C). Although the reaction was carried out under aqueous conditions, no additional substrates were required. The temperature dependence and pH sensitivity of the activity indicate that UV-mediated reactive oxygen species production is limited by the intracellular environment and the conformation of the protein. It is well established that catalase possesses an activity responsible for degrading hydrogen peroxide via the reaction: 2H2O2 → 2H2O + O2, a process also referred to as catalatic activity (31Chance B. Oshino N. Biochem. J. 1971; 122: 225-233Crossref PubMed Scopus (84) Google Scholar, 32Oshino N. Oshino R. Chance B. Biochem. J. 1973; 131: 555-563Crossref PubMed Scopus (145) Google Scholar, 33Deisseroth A. Dounce A.L. Physiol. Rev. 1970; 50: 319-375Crossref PubMed Scopus (533) Google Scholar, 34Handler J.A. Thurman R.G. Sipes I. McQueen C.A. Gandolfi A.J. Comprehensive Toxicology. 3. Elsevier Science Publishing Co., Inc., New York1997: 217-227Google Scholar). In addition, in the presence of low concentrations of hydrogen peroxide (<10–6m), catalase exhibits peroxidatic activity. In this reaction, oxygen containing electron donors such as ethanol and low molecular weight phenolic compounds are oxidized (for reviews, see Refs. 32Oshino N. Oshino R. Chance B. Biochem. 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Recent studies indicate that catalatic and peroxidatic enzymatic activity may also require a charge-relay network that stabilizes reaction intermediates and facilitates the heterolytic cleavage of peroxide. This network has been hypothesized to regulate catalase activity at the metal site by minimizing the charge of a porphyrin π-cation radical and an electron-deficient oxyferryl moiety at Tyr358 (42Putnam C.D. Arvai A.S. Bourne Y. Tainer J.A. J. Mol. Biol. 2000; 296: 295-309Crossref PubMed Scopus (349) Google Scholar). Although increased electron density formed by the presence of cyanide at the heme iron may serve to alter this charge relay network, interactions between amino acid residues forming this network, and azide moieties that disrupt channel activity, are likely to account for the competitive inhibition of the enzyme by sodium azide (33Deisseroth A. Dounce A.L. Physiol. Rev. 1970; 50: 319-375Crossref PubMed Scopus (533) Google Scholar, 42Putnam C.D. Arvai A.S. Bourne Y. Tainer J.A. J. Mol. Biol. 2000; 296: 295-309Crossref PubMed Scopus (349) Google Scholar). Our observations that the effects of UVB light on catalase were highly pH-sensitive and oxygen-dependent, but did not require additional substrates, suggest that reactive oxygen species may be formed by the transfer of water-derived protons and subsequent interactions with molecular oxygen. To investigate this possibility, inhibitors of the catalatic and peroxidatic activity of catalase were used. We found that sodium cyanide induced a biphasic response, enhancing catalase activity at low concentrations but inhibiting the generation of reactive oxygen species at higher concentrations (Fig. 3). In contrast, both sodium azide and 3-AT enhanced UVB light-mediated reactive oxygen species production (Fig. 3). The distinct actions of these inhibitors on the different activities of catalase suggest that the mechanism mediating UVB light-induced responses is unique. The increases in production of reactive oxygen species at low concentrations of sodium cyanide, or in the presence of 3-AT or sodium azide, may result from inhibition of a competing hydrogen peroxide degrading activity of catalase, potentially through disruption of peroxide binding. However, in the presence of higher concentrations of cyanide, interaction of the anion with catalase heme is likely to block access to heme iron and to limit the activity of the charge relay network, rendering both structures unable to participate in the UVB-induced production of reactive oxygen species. The increased generation of reactive oxygen species that is observed in the presence of 3-AT and sodium azide suggests that the presence of reactants at the peroxide binding site, and the activity of the charge relay network, are both important in UVB light-mediated oxidant production. Analysis of our findings prompted us to hypothesize that, similar to 3AT, UVB light-induced effects alter the peroxide binding site allowing water molecules to access the heme iron. In our scheme, proton extraction from water molecules is also facilitated by azide-induced disruption of the charge relay network. Therefore, acting as substrates, water molecules provide a source for the generation of protons, which subsequently interact with O2 to generate reactive oxygen species. A question arises as to the significance of catalase as a mediator of oxidant production in keratinocytes exposed to UVB light. We reasoned that if catalase is important in this activity, then modulating expression of the enzyme should alter quantities of reactive oxidants produced by the cells. To test this we initially reduced levels of catalase protein in cells by transfecting morpholino-stabilized antisense oligonucleotides specific for murine catalase into PAM 212 keratinocytes. We found that antisense oligonucleotides effectively reduced both intracellular levels of catalase and the production of reactive oxygen species in response to UVB light (Fig. 4, lower panel). This was not observed in cells that were mock-transfected or in cells transfected with oligonucleotides specific for the inverted sequence or with random oligonucleotides. We also examined oxidant production in response to UVB light using a catalase expressing fibroblast cell line (HA-1 cells) and a variant clone adapted to hydrogen peroxide (OC5) that overexpresses catalase ∼30-fold (Fig. 4, upper panel) (47Spitz D.R. Li G.C. McCormick M.L. Sun Y. Oberley L.W. Radiat. Res. 1998; 114: 114-124Crossref Scopus (61) Google Scholar). Both the parental and OC5 cells produced peroxides in response to UVB light treatment. However, significantly greater amounts of hydroperoxides were produced by OC5 cells in response to UVB light. Taken together these data indicate that intracellular catalase mediates oxidant formation in cells exposed to UVB light. Our observation that, in response to UVB light, catalase generates reactive oxygen intermediates is both novel and highly divergent from the well known antioxidant functions of this enzyme. We hypothesize that, depending on the intracellular oxidant status, catalase activity in response to UVB light can be either protective or cytotoxic. Thus, through the actions of catalase, high energy DNA-damaging short wave ultraviolet light is absorbed by the enzyme and converted to reactive chemical intermediates that can be further metabolized and detoxified by cellular antioxidant enzymes. However, when antioxidant moieties are limited, UVB light-induced oxidants formed through the actions of catalase can cause cellular damage and tissue injury. We speculate that reactive peroxides produced in excessive or inappropriate quantities through the actions of catalase have the capacity to induce oxidative stress in keratinocytes, damage critical cellular molecules including DNA, and contribute to the development of skin cancer.