H2O2 Is an Important Mediator of UVB-Induced EGF-Receptor Phosphorylation in Cultured Keratinocytes

H2O2 Is an Important Mediator of UVB-Induced EGF-Receptor Phosphorylation in Cultured Keratinocytes Dominik Peus, Remus A. Vasa, Alexander Meves, Markus Pott, Astrid Beyerle, Karen Squillace, and Mark R. Pittelkow Departments of Dermatology, and Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota, U.S.A.

Exposure of human keratinocytes to physiologic doses of ultraviolet B (UVB) radiation induces phosphorylation of the epidermal growth factor receptor (EGFR). We demonstrate that H2O2 generated by UVB mediates EGFR phosphorylation. Using dihydrorhodamine 123 as a specific fluorescent dye probe, we show that UVB irradiation (50–800 J per m2) of keratinocytes leads within minutes to concentration-dependent intracellular production of H2O2. A corresponding concentrationdependent increase in the release of extracellular H2O2 was measured by using Amplex, a derivative of dihydrophenoxazine. The levels of intracellular H2O2 that are induced by UVB irradiation and that stimulate EGFR phosphorylation correlate strongly with the response induced by exogenously added H2O2. UVB or H2O2 demonstrated concentration- and time-dependent stimulation of EGFR phosphorylation that was initially observed within 1–5 min and exhibited a proportionate

delay for UVB-induced production of H2O2. EGFR phosphorylation induced by UVB or H2O2 declined significantly toward baseline levels by 4 h and could be restimulated after H2O2 but not after UVB exposure. Phosphorylation of EGFR was inhibited by the structurally unrelated antioxidants butylated hydroxyanisole, N-acetyl-L-cysteine, and pyrrolidine dithiocarbamate, or by the H2O2-degrading enzyme catalase. These data indicate that generation of H2O2 by UVB radiation of human keratinocytes participates in the rapid, ligandindependent phosphorylation of EGFR and implicate H2O2 as a biologic mediator in EGFR activation and regulation of the downstream signaling cascade. UVBinduced H2O2 has the potential to initiate or modulate early EGFR-mediated signaling events that could play an important role in the cellular response to oxidative stress. Key word: tyrosine kinase phosphorylation. J Invest Dermatol 110:966–971, 1998

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The complexity and interactions of cellular responses to UVB and UVR, however, have been only partially delineated. The early signaling events regulated by growth factor tyrosine kinase receptors appear to be triggered by highly sensitive intracellular mechanisms within minutes of UVR exposure (Rosette and Karin, 1996). The mediators linking photon absorption to activation of signal transduction pathways in UVR-exposed cells are particularly important but remain ill-defined. The epidermal growth factor receptor (EGFR) is a well characterized transmembrane glycoprotein of Mr 180,000 (Carpenter and Cohen, 1990; Carpenter, 1993). Upon binding EGF or related ligands, the EGFR cytoplasmic domain is autophosphorylated on tyrosine residues and initiates multiple signaling responses associated with mitogenesis and cell regulation (Yarden and Ullrich, 1988; Ullrich and Schlessinger, 1990; Hunter, 1995). Irradiation of mesenchymal cells with ultraviolet C (UVC) wavelength radiation also activates signal transduction pathways that extend from the cell membrane to the nucleus (Sachsenmaier et al, 1994a; for reviews, see Herrlich et al, 1994; Angel, 1995; Bender et al, 1997). The current investigations stem from the observations of Sachsenmaier et al (1994a), which demonstrate phosphorylation of EGFR by UVC and inhibition by pretreatment with EGF, suramin, or expression of a dominant-negative EGFR mutation. EGFR phosphorylation has been shown to be induced by UVA, UVB, or UVC in various immortalized cell lines (Zheng et al, 1993; Miller et al, 1994; Warmuth et al, 1994; Sachsenmaier et al, 1994a, b; Coffer et al, 1995; Knebel et al, 1996; Huang et al, 1996; Rosette and Karin, 1996), but the biochemical mechanisms mediating this response have not been elucidated.

he human population is intermittently but chronically exposed to ultraviolet radiation (UVR) from the sun. UVR is primarily responsible for more than 1,000,000 cutaneous malignancies each year in the U.S.A. alone, making it the most efficient environmental carcinogen known (Miller and Weinstock, 1994). Acute and chronic effects of UVR on skin include inflammation, hyperpigmentation, hyperplasia, and skin cancer (Norris et al, 1993; Young, 1993; Gilchrest et al, 1996). UVR has been identified as a complete carcinogen in the skin and, in conjunction with appropriate chemical agents, has been demonstrated to act as both a tumor initiator and a tumor promoter (Epstein, 1978; Matsui and DeLeo, 1991). In addition, skin exposure to ultraviolet B (UVB) wavelength radiation, a minor but active constituent of sunlight, induces direct and indirect biologic effects, including DNA damage and mutations (Sachsenmaier et al, 1994b), systemic immunosuppression (Kripke, 1994), activation of dormant viruses (Herrlich et al, 1992), induction of early and late gene responses (Fornace et al, 1992; Sachsenmaier et al, 1994b; Huang and Adamson, 1995), and cell cycle growth arrest (Medrano et al, 1995).

Manuscript received June 9, 1997; revised February 3, 1998; accepted for publication February 10, 1998. Reprint requests to: Dr. Mark R. Pittelkow, Mayo Clinic, Dermatology Research, Gugg. 442D, 200 First Street SW, Rochester, MN 55905. Abbreviations: BHA, butylated hydroxyanisole; DHR, dihydrorhodamine 123; EGFR, epidermal growth factor receptor; NAC, N-acetyl-L-cysteine; PDTC, pyrrolidine dithiocarbamate.

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0022-202X/98/$10.50 Copyright © 1998 by The Society for Investigative Dermatology, Inc.

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Reactive oxygen species (ROS) are constitutively produced in most cell types by specific metabolic processes such as enzymatic oxidations and aerobic respiration (Cerutti, 1985; Darr and Fridovich, 1994). When cellular levels of ROS exceed the counter-regulatory antioxidant system, the redox balance within cells is perturbed, and oxidative stress results. UVR is a potent inducer of ROS, including superoxide radical (•O2–), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), which have been shown to exert potent activities in stimulating cellular damage. ROS have been implicated in cutaneous aging as well as the pathogenesis of diseases such as cancer and inflammatory disorders (Fridovich, 1978; Black, 1987; Halliwell, 1989; Floyd, 1990; Janssen et al, 1993; Tyrrell, 1994). ROS have recently been suggested to mediate induction of growth factor tyrosine kinase phosphorylation following UVC exposure (Huang et al, 1996). Interestingly, H2O2 has been found to enhance EGFR tyrosine phosphorylation in human squamous carcinoma and other cell lines, including NA, A431, HeLa, and A549 (Gamou and Shimizu et al, 1995; Huang et al, 1996; Knebel et al, 1996; Rao, 1996). These observations suggest that oxidative stress may be involved in EGFR phosphorylation. This study focuses on generation of H2O2 in normal human keratinocytes by physiologic doses of UVB radiation. We also examine the induction of EGFR phosphorylation by UVB and H2O2. MATERIALS AND METHODS Materials and chemicals The UVB source was a FS20 lamp (Westinghouse, Pittsburgh, PA) that emits an energy spectrum with high fluence in the UVB region and a peak at 313 nm (Toews et al, 1980). The emitted dose was regularly quantitated by a UVB radiometer and photodetector (IL 443 and SEE 240, International Light, Newburyport, MA). N-acetyl-L-cysteine (NAC), butylated hydroxyanisole (BHA), pyrrolidine dithiocarbamate (PDTC), aminotriazole, and bovine catalase were purchased from Sigma (St. Louis, MO). Amplex Red reagent and dihydrorhodamine 123 (DHR) were obtained from Molecular Probes (Eugene, OR). DHR detects mainly H2O2 production following conversion to the oxidized product, rhodamine 123, and very little oxidized compound leaks from cells compared with other probes such as dichlorofluorescein (Heck et al, 1992; Oyama et al, 1994; Los et al, 1995; Sundaresan et al, 1995). Culture and treatment of normal human keratinocytes Human keratinocytes were isolated from neonatal foreskin specimens, and primary cultures were initiated and maintained in a replicative state with complete, serum free MCDB 153 medium. Subconfluent keratinocytes from primary cultures were plated into secondary culture at 1–10 3 103 cells per cm2 and used at subconfluence or confluence as determined by phase-microscopic observation. Complete medium was supplemented with 0.1 mM calcium, 0.2% (vol/vol) bovine pituitary extract, EGF (10 ng per ml), insulin (5 µg per ml), hydrocortisone (5 3 10–7 M), ethanolamine (1 3 10–4 M), phosphoethanolamine (1 3 10–4 M), and supplemented amino acids (Wille et al, 1984; Pittelkow and Scott, 1986). Cells were grown to confluence, washed, and incubated in standard medium (without growth factors) for 48 h to measure EGFR phosphorylation following UVB irradiation or H2O2 treatment. No differences were observed whether cells were irradiated in phosphate buffered saline or in standard culture medium. Detection of intracellular H2O2 by flow cytometric analysis Intracellular levels of H2O2 were analyzed by flow cytometry using DHR as a specific fluorescent dye probe (Royal and Ischiropoulos, 1993; Chen et al, 1995). Keratinocytes were loaded with DHR (1 µM) for 45 min before treatment with UVB. During the intracellular release of H2O2, reduced DHR is irreversibly oxidized and converted to the red fluorescent compound rhodamine 123 (Rothe et al, 1991). Keratinocytes were detached from the culture plate by trypsin, and enzyme activity was quenched with 2% fetal calf serum in phosphate buffered saline. Keratinocytes were fixed in 1% paraformaldehyde (Sigma), placed on ice, and the intracellular rhodamine 123 fluorescence intensity of 10,000 cells was measured for each sample using a Becton-Dickenson FACS Vantage flow cytometer with the excitation source at 488 nm and emission detected with a 580 nm long pass filter. Histograms were analyzed with the software program PC-Lysis (Becton Dickenson). Extracellular hydrogen peroxide assay Measurement of extracellular H2O2 following UVB exposure was performed using Amplex, a derivative of dehydrophenoxazine, which becomes highly fluorescent upon oxidation by

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H2O2 in the presence of horseradish peroxidase. Fluorescence was detected using a Cytofluor II microplate fluorometer (Perspectives Biosystems, Framingham, MA). The excitation/emission wavelength filter was set at 590/ 615. Fluorescence intensity of Amplex was measured within the linear region of the H2O2 concentration–response curve. Wells without cells containing the same substrates and exposed to the same treatment represented controls. The control fluorescent values and background fluorescence of untreated cells were monitored over time and subtracted from the relative fluorescence intensity of experimental samples. These values were converted into absolute pmoles using an H2O2 standard curve. All H2O2 measurements were performed in 24 well plates containing confluent keratinocytes, 100 µM Amplex, and 1 U horseradish peroxidase per ml (Mohanty et al, 1997). Immunoprecipitation, immunoblot, and tyrosine phosphorylation assays Cell lysates were obtained by scraping cultures into 1 ml of Frackelton buffer, centrifugation for 5 min, and adding to the supernatant monoclonal antibody LA1 to EGFR (Upstate Biotechnology, Lake Placid, NY) coupled to Protein G-Plus Agarose beads (Oncogene, Cambridge, MA). Beads were rocked for several hours, repeatedly washed, released after adding 20 µl of sample buffer by incubation at 100°C for 5 min, and separated by centrifugation. The samples were then separated electrophoretically by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were electro-transferred to Immobilon-P (Millipore, Bedford, MA), and the membrane was blocked with 2% bovine serum albumin before addition of antiphosphotyrosine antibody (PY20) coupled to peroxidase (Transduction Laboratories, Lexington, KY). Total EGFR was quantitated by immunoblotting each immunoprecipitation membrane with EGFR monoclonal antibody 15E11 specific for peptide epitopes of EGFR extracellular domain (Baron et al, 1997). The bands were visualized using the ECL detection system (Amersham, Arlington Heights, IL).

RESULTS H2O2 is dose-dependently induced by UVB H2O2 production in keratinocytes was analyzed by DHR loading followed by irradiation with UVB over a range of doses and incubation for 20 min. Compared with unirradiated control cells, 100–800 J UVB per m2 induced a dose-dependent increase in relative fluorescent intensity (Fig 1A) that demonstrated intracellular H2O2 generation. These results correlated closely with extracellular release of H2O2 30 min after UVB exposure as measured by the in situ assay using Amplex (Fig 1B). Correlation of DHR fluorescence after UVB or H2O2 treatment and inhibition of UVB-induced H2O2 by antioxidants PDTC or BHA Using DHR as an indicator of intracellular H2O2 levels, we found that treatment of keratinocytes with 60–80 µM H2O2 resulted in DHR fluorescence similar to 200 J UVB per m2, and exposure to 100 µM H2O2 induced fluorescence equivalent in intensity to 400 J per m2 (Fig 2A). DHR fluorescent intensity of cells exposed to 200 J UVB per m2 was specifically inhibited by 50 µM PDTC (Fig 2B) or 50 µM BHA (Fig 2C). Baseline levels of fluorescence in untreated cells were also decreased by these antioxidants. These findings provide evidence that H2O2- and UVB-induced DHR fluorescence are strongly correlated, and structurally different antioxidants modulate H2O2 levels in UVB-stimulated cells. H2O2 and UVB phosphorylate EGFR in a time- and dosedependent fashion The biologic activity of UVB or H2O2 was measured by phosphorylation of EGFR in keratinocytes. Cells were exposed to 200 µM H2O2 for 1–20 min or irradiated with 400 J UVB per m2 and incubated for 5–20 min (Fig 3). Within 1 min of exposure, the level of EGFR phosphorylation was markedly enhanced by H2O2, and maximum EGFR phosphorylation was observed by 5 min. UVB irradiation induced a modest increase in EGFR phosphorylation at 5 min, and the level of EGFR phosphorylation was further enhanced by 10–20 min. Immunoblotting with EGFR monoclonal antibody 15E11 revealed equal loading of protein (Fig 3). A dose-dependent increase in EGFR phosphorylation was observed over the range of 3– 300 µm H2O2 (Fig 4A). Increasing doses of UVB (50–800 J per m2) proportionately enhanced levels of phosphorylated EGFR (Fig 4B). The rapid onset of UVB- or H2O2-induced EGFR phosphorylation implicates a ligand-independent mechanism of activity.

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Figure 1. Dose-dependent intracellular H2O2 production and extracellular release of H2O2 after UVB exposure. DHR-loaded, subconfluent keratinocytes were irradiated with UVB, released by trypsinization, fixed in 0.5% formaldehyde, and analyzed by flow cytometry as described in the Materials and Methods. (a) Dose-dependent increase in relative fluorescence following increasing UVB doses and incubation for 30 min. (b) Amplex (100 µM), a dihydrophenoxazine derivative, and horseradish peroxidase (1 U per ml) were added to confluent keratinocytes in 24 well plates. Thirty minutes after exposure to different doses of UVB, fluorescence intensity was measured by Cytofluor II and converted into absolute picomoles of H2O2 using an H2O2 standard curve.

Time-dependent decrease in H2O2- and UVB-induced EGFR phosphorylation and differences in restimulation responses EGFR phosphorylation decreased markedly by 4 h after treatment with H2O2 or UVB (Fig 5A, B). 200 µM H2O2 induced the maximum increase in EGFR phosphorylation by 10 min, and a steady decline to baseline levels was observed over 4 h (Fig 5A). Following 400 J UVB per m2 exposure, the maximum level of EGFR phosphorylation was reached after µ1 h and declined progressively thereafter (Fig 5B). Baseline levels of phosphorylated EGFR varied among experiments due to cell density and autocrine activity (Peus et al, 1997). H2O2 restimulation strongly enhanced EGFR phosphorylation in keratinocytes (Fig 5A); however, UVB-exposed keratinocytes exhibited a higher baseline level of EGFR phosphorylation that persisted at 4 h and showed no significant enhancement in response to UVB restimulation (Fig 5C). Catalase decreases and aminotriazole increases EGFR phosphorylation after UVB Exposure of keratinocytes to 400 J UVB per m2 following 1 h pretreatment with 300 U catalase, a H2O2

Figure 2. Correlation between H2O2- and UVB-induced DHR fluorescence intensity and inhibition by antioxidants PDTC and BHA. DHR-loaded confluent keratinocytes were treated with different doses of H2O2 or UVB (a). Pretreatment of cells with (b) PDTC (50 µM) or (c) BHA (50 µM) for 30 min and exposure to UVB (200 J per m2) resulted in decreased relative fluorescence intensity 30 min after UVB irradiation. Error bars, 6 SD.

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Figure 3. H2O2 and UVB phosphorylate EGFR in a time-dependent manner. Confluent, resting human keratinocytes were exposed to H2O2 (200 µM) or UVB (400 J per m2) and incubated for designated times. Total cell protein was extracted, immunoprecipitated with monoclonal EGFR antibody, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunoblotted with horseradish peroxidase coupled anti-phosphotyrosine antibody, PY20. Autophosphorylation of EGFR was stimulated by EGF (10 ng per ml) for 20 min. Equal loading was demonstrated by immunoblotting with EGFR monoclonal antibody 15E11 after stripping of PY20 from membrane.

Figure 5. Time-dependent decrease and restimulation of H2O2- and UVB-induced EGFR phosphorylation. Resting, confluent keratinocytes were exposed to: (a) H2O2 (200 µM) for designated times or EGF (10 ng per ml) for 20 min, 4 h later H2O2-stimulated cells were restimulated for 10 min with the same dose of H2O2; or (b) UVB (400 J per m2) exposure and incubation for designated times or EGF (10 ng per ml). (c) Four hours after UVB (400 J per m2) exposure, cells were restimulated for 20 min with the same dose of UVB. EGFR phosphorylation analysis was performed as described in Fig 3.

DISCUSSION Figure 4. Dose-dependent EGFR phosphorylation by H2O2 and UVB. Confluent, resting keratinocyte cultures were exposed to H2O2 or UVB, and EGFR phosphorylation was quantitated as described in Fig 3. (a) H2O2 doses, 3–300 µM for 10 min treatment; (b) UVB radiation, 50–800 J per m2 and 20 min incubation or EGF (10 ng per ml) for 20 min.

metabolizing enzyme, per ml, showed a decrease in the level of phosphorylated EGFR (Fig 6A). By contrast, inhibition of endogenous catalase by 2 or 20 mM aminotriazole increased EGFR phosphorylation after UVB exposure (Fig 6B). Treatment of cells with catalase alone had no effect and 20 mM aminotriazole alone slightly increased the baseline level of phosphorylated EGFR (Fig 6B). These findings provide further evidence that modulating the levels of H2O2 produced in keratinocytes by UVB exposure exerts an effect on EGFR phosphorylation. Inhibition of UVB-induced EGFR phosphorylation by antioxidants Keratinocyte cultures were pretreated with the structurally unrelated antioxidants NAC (30 mM or 3 mM) or PDTC (10 µM) prior to 200 J UVB per m2 exposure. These agents partially inhibited UVB-induced EGFR phosphorylation (Fig 7A). Another antioxidant, BHA (50 µM), also inhibited UVB-induced EGFR phosphorylation (Fig 7B). Addition of both NAC and PDTC or BHA resulted in additive inhibition (Fig 7C). Non-irradiated cells treated with NAC, PDTC, or BHA in combination or alone, showed no significant difference in the levels of phosphorylated EGF compared with control. The inhibition of EGFR phosphorylation by antioxidants indicates that this process is dependent on ROS generation and the cellular redox state.

The chemical mediators of various cellular responses to UVR are only beginning to be identified and characterized. Our findings provide several lines of evidence to indicate that H2O2 functions as a prominent mediator of UVB-induced EGFR phosphorylation. Firstly, EGFR phosphorylation is inhibited by structurally unrelated antioxidant reagents, including PDTC, BHA, and NAC. Secondly, keratinocytes treated with catalase show a substantial reduction of EGFR phosphorylation, whereas aminotriazole, a catalase inhibitor, enhances phosphorylated levels of EGFR. Thirdly, concentrations of exogenous H2O2 correlate closely with the intensity of EGFR phosphorylation and concentrations of intracellular H2O2 that are generated. Fourthly, the time-course of UVB-induced generation of H2O2 as measured by specific fluorescence of oxidized DHR correlates closely with the kinetics of increase in EGFR phosphorylation; and lastly, H2O2 directly and rapidly induces phosphorylation of EGFR, mimicking selected effects of UVB. UVB doses of 50–800 J per m2 stimulate the keratinocyte to phosphorylate EGFR, and these levels of irradiation represent exposures that typically induce the physiologic response of solar erythema in skin types I–III (Norris et al, 1993). This range of UVB irradiation also routinely produces a minimal erythema dose (MED) under standard skin testing conditions. Therefore, the dose range of UVB that induces EGFR phosphorylation in cultured keratinocytes has physiologic relevance for epidermis and skin. Based on the low doses of UVB (50–100 J per m2) required to stimulate ligand-independent EGFR phosphorylation, we hypothesize that this keratinocyte response is a cellular defence mechanism rather than a toxic insult and serves a protective role in epidermis. Generation of ROS has been more extensively studied after UVA radiation than after UVB irradiation. In fibroblasts, production of ROS

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Figure 6. Effects of catalase and aminotriazole on EGFR phosphorylation after UVB. Confluent, resting keratinocytes were pretreated with: (a) catalase (cat, 300 U per ml) for 1 h (lanes 3, 4) before UVB (400 J per m2) exposure for 20 min (lanes 2, 3); or (b) the catalase inhibitor, aminotriazole (20 or 2 mM), for 20 min before UVB exposure (200 J per m2) for 20 min. EGFR phosphorylation analysis was performed as described in Fig 3.

such as singlet oxygen and H2O2 has been reported (Tyrrell and Pidoux, 1989; Vile and Tyrrell, 1995); however, little is known about the generation of ROS in keratinocytes. These studies begin to address this gap by demonstrating H2O2 generation after UVB irradiation in keratinocytes. Intracellular production of H2O2, as a cell-generated mediator of EGFR phosphorylation in response to UVB irradiation, was identified using flow cytometry and cell loading of DHR, a highly sensitive fluorescent dye probe. Within 20 min of UVB exposure, a significant increase in H2O2 generation was measured in DHR-loaded keratinocytes that was inhibited by PDTC or BHA. These antioxidants are also able to inhibit phosphorylation of EGFR that demonstrates that this process is dependent on ROS and indicates that H2O2 may be involved as a mediator. In addition, for the induction of DHR fluorescence, similar doses of UVB and H2O2 are needed that are necessary to induce EGFR phosphorylation. In parallel with the generation of intracellular H2O2, we also found a concentrationdependent increase of the release of H2O2 as measured by Amplex. Even though H2O2 freely crosses cell membranes, only a fraction of intracellularly generated H2O2 is likely to be released. Therefore, intracellular H2O2 may be significantly higher and/or more effective because the site of generation may be in close proximity to the site of action. Several recent investigations have examined UVR-induced receptor phosphorylation events and have speculated on the cellular mechanisms that mediate these responses. Rosette and Karin (1996) showed that UVB exposure induced receptor clustering followed by internalization. The response was assumed to result from physical perturbation of the plasma membrane or conformational changes in membrane proteins caused by UVR energy absorption, but a potential role for ROS generated by UVB-treated cells was not addressed. In this regard, the early responses of ligand-induced receptor clustering and aggregation that is believed to be crucial for transducing a signal (Metzger, 1992), have not been previously elucidated as UVR-mediated mechanisms (Anderson, 1993). UVR-induced EGFR phosphorylation has been shown to be predominantly a cell membrane-associated event, rather than arising from the nucleus as had previously been suggested (Sachsenmaier et al, 1994b; Herrlich et al, 1994; Huang et al, 1996). UVC as well as UVB stimulated EGFR phosphorylation of isolated cell membranes (Knebel

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Figure 7. Antioxidants NAC, PDTC, and BHA inhibit UVB-induced EGFR phosphorylation. Keratinocytes were irradiated with UVB (200 J per m2) after pretreatment with (a) NAC (30 mM, lane 3, or 3 mM, lane 4) or PDTC (10 µM) for 30 min or (b) BHA (50 µM) for 30 min before UVB exposure and incubation for 20 min. (c) Pretreatment with a combination of NAC (10 mM) and PDTC (10 µM) or BHA (50 µM) for 30 min before UVB (200 J per m2) exposure and incubation for 30 min exerted additive inhibition of UVB-induced EGFR phosphorylation. NAC was added 8 h before UVB exposure. EGFR phosphorylation analysis was performed as described in Fig 3.

et al, 1996). The response is independent of ligand binding as shown by EGFR containing a truncated extracellular domain that still undergoes phosphorylation following UVR (Knebel et al, 1996). Knebel et al also demonstrated that the inhibition of membrane-bound protein tyrosine phosphatases by UVR and H2O2 may also be a target to modulate EGFR phosphorylation. UVB-induced phosphorylation of EGFR is similar to ligand-induced autophosphorylation of EGFR (Peus et al, 1997); however, a recent report demonstrates distinct differences in specific sites of phosphorylation of EGFR induced by H2O2 versus the peptide ligand, EGF (Gamou and Shimizu et al, 1995). The relative kinase activity and biologic potency of phosphorylated EGFR induced by EGF versus H2O2 remain to be determined. Furthermore, it is not yet known whether specific EGFR phosphorylation sites induced by UVB are identical or significantly overlap with those sites phosphorylated by H2O2. In this regard, the role of other ROS, such as •OH and •O2– in phosphorylating EGFR and other receptor tyrosine kinases, also require further investigation. These findings imply that UVR- and H2O2-mediated phosphorylation of EGFR are, in large part, rapid, membrane-associated responses induced by oxidative stress. Additional transmembrane receptor targets such as FGFR and interleukin-1 and tumor necrosis factor-α receptors of HeLa cells, have also been shown to be rapidly phosphorylated by UVB, UVC, and osmotic stress (Sachsenmaier et al, 1994a; Rosette and Karin, 1996). Growth factor receptors other than EGFR, such as platelet-derived growth factor receptor, may also activate signalling responses via H2O2 or other ROS (Sundaresan et al, 1995). The activation or modulation of multiple receptor families, including T cell receptor-coupled tyrosine kinases (Schieven et al, 1994) in the absence of ligand binding, have the potential to be mediated by oxidative molecules and likely contribute to the initiation of specific signaling cascades and stress-

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related responses within cells (Bender et al, 1997). Osmotic stress as well as UVR are known inducers of ROS. We suggest that ligandindependent H2O2-induced receptor phosphorylation and ‘‘activation’’ or modulation of receptor activity may involve multiple cell surface receptors in keratinocytes and play roles in coordinating crucial physiologic responses, such as triggering cellular defence mechanisms or mediating injurious or toxic events. Based on these findings, we conjecture that the balance between pro- and anti-oxidative mechanisms of the cell will determine the activities of downstream signaling cascades initiated by UVB stimulation of EGFR and other receptor phosphorylation; however, the specific downstream elements that are activated by UVB- and H2O2-induced EGFR phosphorylation remain to be identified. Collectively, these results provide clearer evidence that H2O2 generated by keratinocytes acts as a proximate second messenger to upregulate the phosphorylation of EGFR in response to UVB. These observations are instrumental in focusing future investigations on specific UVR-related intracellular signalling pathways of keratinocytes and epidermis. Cellular and molecular mechanisms that underlie acute and chronic skin responses to UVR, including solar erythema, skin aging, and carcinogenesis, may be mediated, at least in part, by H2O2 and other ROS produced by epidermal keratinocytes.

The excellent technical assistance provided by N.N. Swanson is gratefully acknowledged. We thank Dr. N. Maihle for the gift of EGFR monoclonal antibody 15E11. R. Vasa was funded by a grant from the Heinrich-Hertz Stiftung, Duesseldorf, Germany. D. Peus is supported by a grant (PE 635/1–2) of the Deutsche Forschungsgemeinschaft (DFG). Additional support was provided by the Mayo Foundation.

REFERENCES Anderson RG: Caveolae: where incoming and outgoing messengers meet. Proc Natl Acad Sci USA 90:10909–10913, 1993 Angel P: The role and regulation of the jun proteins in response to phorbol ester and UV light. In: Baeuerle PA (ed.). Inducible Gene Expression, Vol. 1. Birkhauser, Boston, 1995, pp. 62–92 Baron AT, Huntley BK, Lafky JM, et al: Monoclonal antibodies specific for peptide epitopes of the epidermal growth factor receptor’s extracellular domain. Hybridoma 16:259– 271, 1997 Bender K, Blattner C, Knebel A, Iordanov M, Herrlich P, Rahmsdorf HJ: UV-induced signal transduction. J Photochem Photobiol 37:1–17, 1997 Black HS: Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage. Photochem Photobiol 46:213–221, 1987 Carpenter G: EGF: new tricks for an old growth factor. Curr Opin Cell Biol 5:261–264, 1993 Carpenter G, Cohen S: Epidermal growth factor. J Biol Chem 265:7709–7712, 1990 Cerutti PA: Prooxidant states and tumor promotion. Science 227:375–381, 1985 Chen Q, Olashaw N, Wu J: Participation of reactive oxygen species in the lysophosphatidic acid-stimulated mitogen-activated protein kinase kinase activation pathway. J Biol Chem 270:28499–28502, 1995 Coffer PJ, Burgering BM, Peppelenbosch MP, Bos JL, Kruijer W: UV activation of receptor tyrosine kinase activity. Oncogene 11:561–569, 1995 Darr D, Fridovich I: Free radicals in cutaneous biology. J Invest Dermatol 102:671–675, 1994 Epstein JH: Photocarcinogenesis: a review. Natl Cancer Inst Monographs 50:13–25, 1978 Floyd RA: Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J 4:2587–2597, 1990 Fornace AJ Jr, Jackman J, Hollander MC, Hoffman-Liebermann B, Liebermann DA: Genotoxic-stress response genes and growth-arrest genes, gadd, MyD, and other genes induced by treatments eliciting growth arrest. Ann NY Acad Sci 663:139–153, 1992 Fridovich I: The biology of oxygen radicals. Science 201:875–880, 1978 Gamou S, Shimizu N: Hydrogen peroxide preferentially enhances the tyrosine phosphorylation of epidermal growth factor receptor. FEBS Lett 357:161–164, 1995 Gilchrest BA, Park HY, Eller MS, Yaar M: Mechanisms of ultraviovlet light-induced pigmentation. Photochem Photobiol 63:1–10, 1996 Halliwell B: Free radicals, reactive oxygen species and human disease: a critical evaluation with special reference to atherosclerosis. Br J Exp Pathol 70:737–757, 1989 Heck DE, Laskin DL, Gardner CR, Laskin JD: Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing. J Biol Chem 267:21277–21280, 1992 Herrlich P, Ponta H, Rahmsdorf HJ: DNA damage-induced gene expression: signal transduction and relation to growth factor signaling. Rev Physiol Biochem Pharmacol 119:187–223, 1992 Herrlich P, Sachsenmaier C, Radler-Pohl A, Gebel S, Blattner C, Rahmsdorf HJ: The mammalian UV response: Mechanism of DNA damage induced gene expression. Adv Enzyme Regul 34:381–395, 1994 Huang R-P, Adamson ED: A biological role for Egr-1 in cell survival following ultra-violet irradiation. Oncogene 10:467–475, 1995 Huang R-P, Wu JX, Fan Y, Adamson ED: UV activates growth factor receptors via reactive oxygen intermediates. J Cell Biol 133:211–220, 1996

UVB INDUCES EGF-RECEPTOR PHOSPHORYLATION VIA H2O2

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Hunter T: Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80:225–236, 1995 Janssen YM, Van Houten B, Borm PJ, Mossman BT: Cell and tissue responses to oxidative damage. Lab Invest 69:261–274, 1993 Knebel A, Rahmsdorf HJ, Ullrich A, Herrlich P: Dephosphorylation of receptor tyrosine kinases as target of regulation by radiation, oxidants or alkylating agents. EMBO J 15:5314–5325, 1996 Kripke ML: Ultraviolet radiation and immunology: Something new under the sun. Cancer Res 54:6102–6105, 1994 Los M, Schenk H, Hexel K, Baeuerle PA, Droge W, Schulze-Osthoff K: IL-2 gene expression and NF-kB activation through CD28 requires reactive oxygen production by. 5lipoxygenase. EMBO J 14:3731–3740, 1995 Matsui MS, DeLeo VA: Longwave ultraviolet radiation and promotion of skin cancer. Cancer Cells 3:8–12, 1991 Medrano EE, Im S, Yang F, Abdel-Malek ZA: Ultraviolet B light induces G1 arrest in human melanocytes by prolonged inhibition of retinoblastoma protein phosphorylation associated with long-term expression of the p21Waf-1/SDI-1/Cip-1 protein. Cancer Res 55:4047–4052, 1995 Metzger H: Transmembrane signaling: The joy of aggregation. J Immunol 149:1477, 1992 Miller CC, Hale P, Pentland AP: Ultraviolet B injury increases prostaglandin synthesis through a tyrosine kinase-dependent pathway. Evidence for UVB-induced epidermal growth factor receptor activation. J Biol Chem 269:3529–3533, 1994 Miller DL, Weinstock MA: Nonmelanoma skin cancer in the United States: incidence. J Am Acad Dermatol 30:774–778, 1994 Mohanty JG, Jaffe JS, Schulman ES, Raible DG: A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative. J Immunol Meth 202:133–141, 1997 Norris PG, Gange RW, Hawk JLM: Acute effects of ultraviolet radiation on the skin. In: Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM, Austen KF (eds). Dermatology in General Medicine, 4th edn. McGraw Hill, New York, 1993, pp. 1651–1658 Oyama Y, Hayashi A, Ueha T, Maekawa K: Characterization of 29,79-dichlorofluorescin fluorescence in dissociated mammalian brain neurons: Estimation on intracellular content of hydrogen peroxide. Brain Res 635:113–117, 1994 Peus P, Hamacher L, Pittelkow MR: EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation. J Invest Dermatol 109:751– 756, 1997 Pittelkow MR, Scott RE: New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns. Mayo Clin Proc 61:771–777, 1986 Rao GN: Hydrogen peroxide induces complex formation of SHC-Grb2-SOS with receptor tyrosine kinase and activates Ras and extracellular signal-regulated protein kinases group of mitogen-activated protein kinases. Oncogene 13:713–719, 1996 Rosette C, Karin M: Ultraviolet light and osmotic stress: Activation of the JNK cascade through multiple growth factor and cytokine receptors. Science 173:1194–1197, 1996 Rothe G, Emmendorffer A, Oser A, Roesler J, Valet G: Flow cytometric measurement of the respiratory burst activity of phagocytes using dihydrorhodamine 123 [letter; comment]. J Immunol Meth 138:133–135, 1991 Royal JA, Ischiropoulos H: Evaluation of 29,79-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys 302:348–355, 1993 Sachsenmaier C, Radler-Pohl A, Zinck R, Nordheim A, Herrlich P, Rahmsdorf HJ: Involvement of growth factor receptors in the mammalian UVC response. Cell 78:963– 972, 1994a Sachsenmaier C, Radler-Pohl A, Muller A, Herrlich P, Rahmsdorf HJ: Damage to DNA by UV light and activation of transcription factors. Biochem Pharmacol 47:129–136, 1994b Schieven GL, Mittler RS, Nadler SG, Kirihara JM, Bolen JB, Kanner SB, Ledtetter JA: ZAP70 tyrosine kinase, CD45 and T-cell receptor involvement in UV and H2O2 induced signal transduction. J Biol Chem 269:20718–20726, 1994 Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T: Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270:296–299, 1995 Toews GB, Bergstresser PR, Streilein JW: Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J Immunol 134:445–453, 1980 Tyrrell RM: The molecular and cellular pathology of solar ultraviolet radiation. Mol Aspects Med 15:1–77, 1994 Tyrrell RM, Pidoux M: Singlet oxygen involvement in the inactivation of cultured human fibroblasts by UVA (334 nm, 365 nm) and near-visible (405 nm) radiations. Photochem Photobiol 49:407–412, 1989 Ullrich A, Schlessinger J: Signal transduction by receptors with tyrosine kinase activity. Cell 61:203–212, 1990 Vile GF, Tyrrell RM: UVA radiation-induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen. Free Radic Biol Med 18:721–730, 1995 Warmuth I, Harth Y, Matsui MS, Wang N, DeLeo VA: Ultraviolet radiation induces phosphorylation of the epidermal growth factor receptor. Cancer Res 54:374–376, 1994 Wille JJ Jr, Pittelkow MR, Shipley GR, Scott RE: Integrated control of growth and differentiation of normal human prokeratinocytes cultures in serum-free medium: Clonal analyses, growth kinetics and cell cycle studies. J Cell Physiol 121:31–44, 1984 Yarden Y, Ullrich A: Growth factor receptor tyrosine kinases. Ann Rev Biochem 57:443– 478, 1988 Young AR: Chronic effects of ultraviolet radiation on the skin: experimental aspects. In: Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg IM, Austen KF, (eds). Dermatology in General Medicine, 4th edn. 1993, pp. 1658–1660 Zheng ZS, Chen RZ, Prystowsky JH: UVB radiation induces phosphorylation of the epidermal growth factor receptor, decreases EGF binding and blocks EGF induction of ornithine decarboxylase gene expression in SV40-transformed human keratinocytes. Exp Dermatol 2:257–265, 1993