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A Synthetic Superoxide Dismutase/Catalase Mimetic (EUK-134) Inhibits Membrane-Damage-Induced Activation of Mitogen-Activated Protein Kinase Pathways and Reduces p53 Accumulation in Ultraviolet B-Exposed Primary Human Keratinocytes

      Salen-manganese complexes exhibit powerful superoxide dismutase and catalase activity, with pharmacologic efficacy in several oxidative-stress-associated disease models. Ultraviolet (UV) B not only induces direct DNA damage, but also generates oxidative stress. EUK-134, a salen-manganese complex, might therefore confer a direct protection against UVB-induced oxidative stress and consequently alleviate UVB-damage-induced signal transduction. We investigated the effect of EUK-134 on the UVB-induced accumulation and stabilization of the p53 protein. p53 plays a central role in the UVB response, both as sensor of UVB damage and as a mediator of a protective response. Cells treated with EUK-134 before UVB irradiation showed a significantly lower accumulation of the p53 protein in a concentration-dependent fashion. Furthermore, EUK-134 severely reduced N-terminal phosphorylation of p53. The extracellular signal-regulated kinase ERK and the stress-activated kinases JNK and p38 have been implicated in the UVB-induced N-terminal phosphorylation and accumulation of p53. Pre-treatment with EUK-134 inhibited the UVB-induced activation of these mitogen-activated protein kinase (MAPK) pathways. We hypothesize that EUK-134, by direct protection of the membrane from UVB-induced oxidative damage, reduces oxidative stress induced MAPK signaling and consequently lowers the level of p53 induction. The protection conferred by EUK-134 resulted in a significant increase in cell survival following UVB irradiation.

      Keywords

      Abbreviations

      AAPH
      2,2′-azobis(2-amidinopropane)dihydrochloride
      ERK
      extracellular signal-regulated kinase
      JNK/SAPK
      c-Jun N-terminal kinase/stress-activated protein kinase
      MAPK
      mitogen-activated protein kinase
      ROS
      reactive oxygen species
      SOD
      superoxide dismutase; UV, ultraviolet
      Exposure of human skin to ultraviolet (UV) radiation has well-known acute and chronic effects. The acute effects include erythema, tanning, and immune suppression. Long-term adverse effects of UV exposure are photoaging and photocarcinogenesis (
      • Gilchrest B.A.
      • Park H.Y.
      • Eller M.S.
      • Yaar M.
      Mechanisms of ultraviolet light-induced pigmentation.
      ;
      • Taylor C.R.
      • Sober A.J.
      Sun exposure and skin disease.
      ;
      • Clydesdale G.J.
      • Dandie G.W.
      • Muller H.K.
      Ultraviolet light induced injury: Immunological and inflammatory effects.
      ). Not only UV-induced direct DNA damage, but also UV-induced reactive oxygen species (ROS) are involved in these processes (
      • de Gruijl F.R.
      • van Kranen H.J.
      • Mullenders L.H.
      UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer.
      ;
      • Scharffetter-Kochanek K.
      • Wlaschek M.
      • Brenneisen P.
      • Schauen M.
      • Blaudschun R.
      • Wenk J.
      UV-induced reactive oxygen species in photocarcinogenesis and photoaging.
      ). ROS can cause genetic damage, both to mitochondrial and to nuclear DNA. ROS are also involved in photodamage of the dermal connective tissue, characteristic for photoaging, both through direct damage to connective tissue cells and proteins and through the induction of proteolytic pathways (
      • Ma W.
      • Wlaschek M.
      • Tantcheva-Poor I.
      • et al.
      Chronological ageing and photoageing of the fibroblasts and the dermal connective tissue.
      ).
      In a healthy epidermis, a keratinocyte protects itself from the deleterious effects of UVB damage by undergoing a typical UVB response. The cell undergoes a cell-cycle arrest and repairs its DNA at low UVB doses, whereas high UVB doses bring about a last escape mechanism: the induction of programmed cell death or apoptosis. p53 plays a central role in this response both as a sensor of UVB damage and as an initiator of a program of expression of genes involved in growth arrest, repair, or apoptosis. DNA damage elicited by UVB is thought to an important trigger for p53 accumulation and transcriptional activation (
      • Decraene D.
      • Agostinis P.
      • Pupe A.
      • de Haes P.
      • Garmyn M.
      Acute response of human skin to solar radiation: Regulation and function of the p53 protein.
      ).
      Nevertheless, a growing amount of data suggest that the complex cellular response induced by UVB (290–320 nm) not only involves events triggered by DNA damage, but also depends on cytosolic signaling pathways originating at the plasma membrane (
      • Assefa Z.
      • Garmyn M.
      • Bouillon R.
      • Merlevede W.
      • Vandenheede J.R.
      • Agostinis P.
      Differential stimulation of ERK and JNK activities by ultraviolet B irradiation and epidermal growth factor in human keratinocytes.
      ;
      • Kulms D.
      • Zeise E.
      • Poppelmann B.
      • Schwarz T.
      DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation-induced apoptosis in an essential and independent way.
      ). Although UVA is generally regarded as the prime source for ROS in sunlight-induced damage, UVB also produces a substantial amount of ROS through interaction with endogenous photosensitizers (
      • Peus D.
      • Meves A.
      • Vasa R.A.
      • Beyerle A.
      • O'Brien T.
      • Pittelkow M.R.
      H2O2 is required for UVB-induced EGF receptor and downstream signaling pathway activation.
      ), leading to the depletion of enzymatic and nonenzymatic antioxidant systems (
      • Sander C.S.
      • Chang H.
      • Salzmann S.
      • Muller C.S.
      • Ekanayake-Mudiyanselage S.
      • Elsner P.
      • Thiele J.J.
      Photoaging is associated with protein oxidation in human skin in vivo.
      ). UVB-generated ROS damage the plasma membrane, resulting in the ligand-independent clustering of membrane receptors. The consequent activation of receptors in the absence of ligand binding initiates specific signaling cascades, particularly the stress-induced mitogen-activated protein kinase (MAPK), within minutes of UVB exposure (
      • Rosette C.
      • Karin M.
      Ultraviolet light and osmotic stress: Activation of the JNK cascade through multiple growth factor and cytokine receptors.
      ;
      • Peus D.
      • Vasa R.A.
      • Beyerle A.
      • Meves A.
      • Krautmacher C.
      • Pittelkow M.R.
      UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.
      ).
      A reduction in the level of UVB-generated oxidative damage is therefore bound to have an impact on the major UVB-responsive pathways. We therefore investigated the impact of a salen-manganese complex, EUK-134, on the UVB response of primary human keratinocytes. The synthetic salen-manganese complexes, a class of stable low-molecular-weight compounds that contain tightly bound manganese, exhibit powerful superoxide dismutase (SOD) and catalase activity, targeting and destroying both superoxide and hydrogen peroxide, respectively (
      • Baker K.
      • Marcus C.B.
      • Huffman K.
      • Kruk H.
      • Malfroy B.
      • Doctrow S.R.
      Synthetic combined superoxide dismutase/catalase mimetics are protective as a delayed treatment in a rat stroke model: A key role for reactive oxygen species in ischemic brain injury.
      ;
      • Baudry M.
      • Etienne S.
      • Bruce A.
      • Palucki M.
      • Jacobsen E.
      • Malfroy B.
      Salen-manganese complexes are superoxide dismutase-mimics.
      ;
      • Doctrow S.R.
      • Huffman K.
      • Marcus C.B.
      • Musleh W.
      • Bruce A.
      • Baudry M.
      • Malfroy B.
      Salen-manganese complexes: Combined superoxide dismutase/catalase mimics with broad pharmacological efficacy.
      ). These SOD/catalase mimetics have shown efficacy in a variety of oxidative stress paradigms, including in vivo models for stroke (
      • Baker K.
      • Marcus C.B.
      • Huffman K.
      • Kruk H.
      • Malfroy B.
      • Doctrow S.R.
      Synthetic combined superoxide dismutase/catalase mimetics are protective as a delayed treatment in a rat stroke model: A key role for reactive oxygen species in ischemic brain injury.
      ), Parkinson's disease (
      • Melov S.
      • Doctrow S.R.
      • Schneider J.A.
      • et al.
      Lifespan extension and rescue of spontiform encephalopathy in superoxide dismutase 2 nullizygous mice treated with superoxide dismutase-catalase mimetics.
      ), autoimmune disease (
      • Malfroy B.
      • Doctrow S.R.
      • Orr P.L.
      • Tocco G.
      • Fedoseyeva E.V.
      • Benichou G.
      Prevention and suppression of autoimmune encephalomyelitis by EUK-8, a synthetic catalytic scavenger of oxygen-reactive metabolites.
      ), and excitotoxic neuronal death (
      • Rong Y.
      • Doctrow S.R.
      • Tocco G.
      • Baudry M.
      EUK-134, a synthetic superoxide dismutase and catalase mimetic, prevents oxidative stress and attenuates kainate-induced neuropathology.
      ). Furthermore, an augmentation of the natural antioxidant system with synthetic salen manganese complexes was shown to increase life span in certain animal models (
      • Melov S.
      • Ravenscroft J.
      • Malik S.
      • et al.
      Extension of life-span with superoxide dismutase/catalase mimetics.
      ) and to reverse age-related learning deficits in mice (
      • Melov S.
      • Doctrow S.R.
      • Schneider J.A.
      • et al.
      Lifespan extension and rescue of spontiform encephalopathy in superoxide dismutase 2 nullizygous mice treated with superoxide dismutase-catalase mimetics.
      ;
      • Liu R.
      • Liu I.Y.
      • Bi X.
      • Thompson R.F.
      • Doctrow S.R.
      • Malfroy B.
      • Baudry M.
      Reversal of age-related learning deficits and brain oxidative stress in mice with superoxide dismutase/catalase mimetics.
      ). Studies have shown that activation of MAPK signal transduction pathways in response to UVB is presumably triggered by ROS-mediated membrane damage (
      • Peus D.
      • Meves A.
      • Vasa R.A.
      • Beyerle A.
      • O'Brien T.
      • Pittelkow M.R.
      H2O2 is required for UVB-induced EGF receptor and downstream signaling pathway activation.
      ;
      • Rosette C.
      • Karin M.
      Ultraviolet light and osmotic stress: Activation of the JNK cascade through multiple growth factor and cytokine receptors.
      ).
      We show that a pre-treatment with EUK-134 inhibits the UVB-induced activation of MAPK signal transduction pathways in human keratinocytes. This results in a lower accumulation of p53, a protein central in the response to UVB damage. In turn, this leads to a significant increase in cell survival. Our results indicate that low concentrations of EUK-134 can effectively protect cells against oxidative damage by UVB. Our results also show that in addition to direct damage, ROS-dependent events are involved in the accumulation of p53 upon UVB exposure. These data shed new light on the importance of ROS-mediated signal transduction in the UVB-response of human skin. They point out a potential use for salen-manganese complexes as protective agents against the ROS-dependent deleterious effects of chronic UV exposure: photoaging, and photocarcinogenesis.

      Results

      EUK-134 protects primary human keratinocytes against oxidative stress

      The structure of the salen-manganese complex EUK-134 is shown in Figure 1. EUK-134 is an analog of EUK-8 with increased catalase activity and equivalent SOD activity (
      • Doctrow S.R.
      • Huffman K.
      • Marcus C.B.
      • et al.
      Salen-manganese complexes as catalytic scavengers of hydrogen peroxide and cytoprotective agents: Structure–activity relationship studies.
      ).
      Figure thumbnail gr1
      Figure 1Structures of the salen-manganese complexes EUK-8 and EUK-134. The ring substituents (R) of EUK-8 and EUK-134 differ as shown (with permission from Eukarion, Inc.).
      We first investigated whether EUK-134 retains its catalase- and SOD-like activity under UVB irradiation. EUK-134 solutions were irradiated with doses up to 300 mJ per cm2 UVB Figure 2a and subsequently subjected to analysis. The catalase-like activity, the ability to neutralize H2O2, was assayed by monitoring the rate of conversion of hydrogen peroxide to oxygen as a decrease in absorbance at 240 nm, as originally described by
      • Beers R.F.
      • Sober A.J.
      A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase.
      . Catalase activity of sham-irradiated EUK-134, extrapolated from a standard curve of bovine liver catalase, was calculated to be 26 U per mg. The SOD-like activity of EUK-134, the ability to neutralize O2, was determined by its ability to compete with luminol for superoxide anion, leading to a decrease in the chemiluminescent signal, as modified from
      • Kimura H.
      • Nakano M.
      Highly sensitive and reliable chemiluminescence method for the assay of superoxide dismutase in human erythrocytes.
      . Superoxide was generated in situ through the hypoxanthine/xanthine oxidase system. SOD-like activity of sham-irradiated EUK-134, extrapolated from a standard curve of bovine erythrocyte SOD, was estimated to be 0.33 U per mg. Both the SOD-like and the catalase-like activity of EUK-134 proved to be stable under increasing UVB doses Figure 2a.
      Figure thumbnail gr2
      Figure 2EUK-134 pre-treatment protects keratinocytes against oxidative stress. (A) EUK-134 solutions were either sham irradiated or exposed to increasing UVB doses. Following UVB-irradiation, the catalase-like (U/mg) and the SOD-like activity (U/mg) of the solution was calculated as described under Materials and Methods. (B) The ability of EUK-134 to prevent lipid peroxidation in vitro was tested using AAPH as a radical initiator and linoleic acid as a substrate, as described under Materials and Methods. The concentration of EUK-134 varied from 0.25 to 5 μM as indicated. Ethanol represents the control without EUK-134. (C) Cells were pre-treated for 24 h with increasing EUK-134 doses (12.5–50 μM as indicated) and subsequently exposed to increasing concentrations of either hydrogen peroxide (100–400 μM) or xanthine oxidase (0.125–2 U/mL). The uptake of neutral red dye was monitored 18 h after H2O2 or xanthine oxidase exposure, as described under Materials and Methods (mean±SD). *p<0.05 (Student's t test). The above data are representative of three independent experiments.
      Oxidative damage to membranes is thought to be the main cause for the activation of several UVB-induced signaling cascades (
      • Rosette C.
      • Karin M.
      Ultraviolet light and osmotic stress: Activation of the JNK cascade through multiple growth factor and cytokine receptors.
      ;
      • Peus D.
      • Vasa R.A.
      • Beyerle A.
      • Meves A.
      • Krautmacher C.
      • Pittelkow M.R.
      UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.
      ). We therefore sought whether the antioxidative properties of EUK-134 could confer protection against the oxidation of lipids. The ability of EUK-134 to protect linoleic acid against ROS-induced lipid peroxidation was tested using the radical initiator AAPH Figure 2b. Thermal decomposition (37°C) of the azo compound AAPH generates free radicals that rapidly react with oxygen to give peroxyl radicals. The peroxyl radicals abstract hydrogen atoms from the linoleic acid to give lipid radicals. These lipid radicals then induce a sequence of propagation reactions to give lipid hydroperoxides. The progress of lipid peroxidation was followed by the rate of appearance of conjugated dienes at 234 nm as a function of time. EUK-134 strongly reduced the propagation rate of the peroxidation reaction, resulting in a concentration-dependent decrease of the slope Figure 2b. The concentration causing 50% inhibition (IC50) was estimated to be approximately 0.8 μM EUK-134 under the test conditions described.
      We next investigated whether EUK-134 could effectively protect cultured human keratinocytes against oxidative stress. To demonstrate a protective effect of EUK-134 against oxidative-stress-induced cytotoxicity, we performed a neutral red viability assay (
      • Fautz R.
      • Husein B.
      • Hechenberger C.
      Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: Rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS).
      ). Neutral red is readily incorporated into the lysosomes of living cells. The uptake of the neutral red dye, monitored by an increase in absorbance at 540 nm, is therefore a direct indication for the amount of viable cells. A 24-h pre-treatment with EUK-134 (12.5–50 μM) significantly reduced hydrogen peroxide induced cytotoxicity (150–300 μM H2O2; Figure 2c). Likewise, a 24-h pre-treatment with 25 to 50 μM EUK-134 significantly reduced xanthine oxidase/xanthine-induced cytotoxicity (0.5–2 U/mL xanthine oxidase, Figure 2c). Xanthine oxidase catalyzes the oxidation of xanthine, present in cell culture medium, leading to the production of superoxide (
      • Kimura H.
      • Nakano M.
      Highly sensitive and reliable chemiluminescence method for the assay of superoxide dismutase in human erythrocytes.
      ).

      EUK-134 inhibits p53 N-terminal phosphorylation and p53 accumulation in UVB-irradiated primary human keratinocytes

      UVB causes both direct DNA damage and oxidative stress. Both induce phosphorylation, stabilization, and consequent accumulation of the p53 protein. To further demonstrate the antioxidative capacity of EUK-134, we exposed cells for 10 min to increasing concentrations of hydrogen peroxide (0.01–0.2 mM). Hydrogen peroxide induced a concentration-dependent accumulation of the p53 protein 6 h after treatment Figure 3a. A 24-h pre-treatment with 50 μM EUK-134 led to a near complete inhibition of this oxidative stress-induced p53 accumulation.
      Figure thumbnail gr3
      Figure 3EUK-134 pre-treatment reduces p53 N-terminal phosphorylation and p53 accumulation following UVB irradiation. p53 protein levels and the p53 phosphorylation status following oxidative stress or UVB damage were determined by western blotting as described under Materials and Methods. (A) Where indicated, primary human keratinocytes were pre-treated for 24 h with 50 μM EUK-134. Cells were then exposed for 10 min to increasing concentrations of H2O2 (0.01–0.2 mM). The cell lysate was collected 6 h later and analyzed for total p53 level. (B) Primary human keratinocytes were pre-treated for 24 h with increasing concentrations of EUK-134, as indicated. Cells were scraped 6 h after sham irradiation or irradiation with 12 mJ per cm2 UVB and analyzed for total p53 levels. (C) Dose response of p53 accumulation, 6 h after UVB irradiation. Where indicated, cells were pre-treated for 24 h with 50 μM EUK-134. Cells were irradiated with increasing UVB doses, as indicated, and evaluated for total p53 levels. (D) Time course analysis of the phosphorylation status of the p53 protein following irradiation with 32 mJ per cm2 UVB. Cells, pre-treated with 50 μM EUK-134 where indicated, were scraped at the appropriate time point and analyzed for total p53 levels, p53 phosphorylation at Ser46, and p53 phosphorylation at Ser15.
      Next, we investigated whether a pre-treatment with EUK-134 was also able to prevent or reduce p53 accumulation following UVB irradiation. Indeed, a 24-h pre-treatment with increasing doses (0.5–50 μM) of EUK-134 led to a concentration-dependent decrease of p53 accumulation 6 h after irradiation with 12 mJ per cm2 UVB Figure 3b. This dramatic decline of UVB-induced p53 accumulation in EUK-134-pre-treated cells (50 μM, 24-h pre-incubation) was consistent across a wide range of UVB doses Figure 3c. Next, the phosphorylation of two serine sites was monitored by western blotting. A 24-h pre-incubation with 50 μM EUK-134 completely inhibited the UVB-induced (32 mJ/cm2) phosphorylation of serine 46 Figure 3d. In EUK-134-treated cells a diminished accumulation of the p53 protein following UVB-damage also strongly correlated with a decreased phosphorylation of the serine 15 phosphorylation site Figure 3d.

      EUK-134 pre-treatment attenuates UVB-induced MAPK activation in primary human keratinocytes

      One of the predominant UVB-responsive signaling pathways is the MAPK pathway, which has three distinct components: the ERK pathway, the stress-activated JNK pathway, and the stress-activated p38 pathway. MAPK play an important role in p53 stabilization and accumulation by phosphorylating the N-terminus of the p53 protein at several serine and threonine sites (Ser15, Ser20, Ser33, Ser46, Thr81) in response to oxidative membrane damage. The phospho-p44/42 MAPK antibody recognizes dually phosphorylated ERK1 (p44) as well as dually phosphorylated ERK2 (p42) MAPK. The UVB-dependent increase in both ERK1 and ERK2 activation was completely inhibited by a 24-h pre-treatment with 50 μM EUK-134 Figure 4a. Basal activation of the ERK pathway, owing to the presence of epidermal growth factor, was unaffected by EUK-134 pre-treatment.
      Figure thumbnail gr4
      Figure 4EUK-134 pre-treatment prevents the UVB-induced activation of three major MAPK pathways. Where indicated, cells were treated with 50 μM EUK-134 24 h before UVB irradiation. Cell lysates were collected 15 min after irradiation with the indicated UVB dose. Proteins were separated by Western blotting as described under Materials and Methods. Membranes were probed with phosphospecific antibodies. (A) ERK1 and ERK2 phosphorylation status (ERK-MAPK pathway). (B) MKK4, JNK1, and JNK2 phosphorylation status (JNK/SAPK pathway). (C) MKK3, MKK6, and p38 MAPK phosphorylation status (p38 MAPK pathway).
      The UVB-induced activation of JNK protein kinases was largely inhibited in cells pre-treated with 50 μM EUK-134 Figure 4b. The antibody recognizes two alternative splicing variants (a 46- and a 54-kDa variant) of both the JNK1 and the JNK2 protein kinase (
      • Gupta S.
      • Barrett T.
      • Whitmarsh A.J.
      • Cavanagh J.
      • Sluss H.K.
      • Derijard B.
      • Davis R.J.
      Selective interaction of JNK protein kinase isoforms with transcription factors.
      ). The UVB-induced activation of SEK1/MKK4, a MAPK that phosphorylates and activates JNK1 and JNK2, was also severely inhibited by a 24-h pre-treatment with 50 μM EUK-134 Figure 4b.
      The activation of the p38 MAPK pathway could only be observed when cells were irradiated with higher UVB doses (32–64 mJ/cm2; Figure 4c). EUK-134 pre-treatment led to a near complete inhibition of p38 MAPK phosphorylation and activation. Correspondingly, the activation of both MKK3 and MKK6, two closely related protein kinases that phosphorylate p38 MAP kinase following UVB damage (32–64 mJ/cm2), was almost completely inhibited Figure 4c.

      Inhibition of the p38 MAPK pathway decreases p53 N-terminal phosphorylation and accumulation following UVB-damage

      By use of a pharmacologic inhibitor, we wanted to investigate whether an inhibition of the p38 MAPK pathway had similar effects on the UVB-induced N-terminal phosphorylation and accumulation of the p53 protein. By binding to the ATP pocket of p38 MAPK, the pyridinyl imidazole inhibitor PD169316 potently inhibits p38 MAPK activation (
      • Young P.R.
      • McLaughlin M.M.
      • Kumar S.
      • et al.
      Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site.
      ). Inhibition of the p38 MAPK pathway by a 30-min pre-treatment with increasing concentrations of PD169316 (1–10 μM) resulted in a strong inhibition of the UVB-induced p53 phosphorylation at serine 15 and serine 46 Figure 5. Consequently, the inhibition of the p38 MAPK pathway by a 30-min PD169316 pre-treatment (1–10 μM) resulted in a corresponding decrease of p53 accumulation following irradiation with 32 mJ per cm2 UVB, similar to a EUK-134 pre-treatment.
      Figure thumbnail gr5
      Figure 5Inhibition of the p38 MAPK pathway reduces p53 N-terminal phosphorylation and p53 accumulation following UVB irradiation. Half an hour before UVB irradiation, cells were pre-treated with increasing concentrations of the p38 MAPK inhibitor PD169316 (1–10 μM). Cells were then irradiated with 32 mJ per cm2 UVB and scraped at the indicated time point. p53 phosphorylation at serine 46, p53 phosphorylation at serine 15, and total p53 level was determined by western blotting as described under Materials and Methods.

      EUK-134 pre-treatment increases cell survival after UVB irradiation

      Next, we wanted to investigate how pre-treatment with EUK-134 influenced the fate of a cell following UVB irradiation. The amount of viable cells was determined by a trypan blue exclusion assay. Cells were treated with 5 or 50 μM EUK-134 24 h before UVB irradiation. UVB induced a concentration-dependent loss of cell viability in keratinocytes Figure 6. EUK-134 treatments were found to increase cell viability following UVB irradiation in primary human keratinocytes. A 24-h pre-treatment with either 5 or 50 μM EUK-134 significantly reduced cell death with 16 and 25%, respectively (p<0.01) following irradiation with 24 mJ per cm2 UVB. A pre-treatment with 50 μM EUK-134 also significantly reduced cell death following UVB irradiation with 16 mJ per cm2 with 7% (p<0.01).
      Figure thumbnail gr6
      Figure 6Protection against oxidative damage with EUK-134 increases cell survival after UVB irradiation. Cells either were left untreated or were treated with 5 or 50 μM EUK-134 24 h before UVB irradiation. Twenty-four hours after UVB irradiation the percentage of UVB-induced cell death was evaluated by a trypan blue exclusion assay as described under Materials and Methods. Data are expressed as percentages trypan blue exclusion (%TBE). One representative of two independent experiments is shown (mean±SEM., n=3). *p<0.01 (Student's t test).

      Discussion

      The SOD and catalase mimic EUK-134, stable under UVB irradiation, was found to potently protect cultured human keratinocytes against oxidative-stress-induced damage. Because UVB-generated oxidative stress is known to be an important trigger for UVB-induced cytosolic signaling (
      • Peus D.
      • Meves A.
      • Vasa R.A.
      • Beyerle A.
      • O'Brien T.
      • Pittelkow M.R.
      H2O2 is required for UVB-induced EGF receptor and downstream signaling pathway activation.
      ;
      • Peus D.
      • Vasa R.A.
      • Beyerle A.
      • Meves A.
      • Krautmacher C.
      • Pittelkow M.R.
      UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.
      ), we wanted to investigate whether a supplementation of primary human keratinocytes with the antioxidant EUK-134 could consequently affect the UVB response of these keratinocytes.
      Our results show that EUK-134 inhibits the UVB-induced activation of three members of the MAPK signal transduction pathway: ERK, JNK, and p38. Inhibition of these UVB-activated pathways by EUK-134 was accompanied by a prevention of UVB-induced p53 N-terminal phosphorylation at Ser15 and Ser46 and subsequently p53 accumulation. In turn this led to a small but significant increase in cell survival upon UVB irradiation. These results underline the importance of oxidative membrane damage as an initiator of MAPK signal transduction and p53 accumulation following UVB irradiation. Furthermore, EUK-134 strongly reduced oxidative-stress-elicited damage response pathways following UVB-irradiation. The antioxidative properties of this molecule could thus prove useful against both short- and long-term deleterious effects of UVB irradiation.
      The salen-manganese complexes have proven their efficacy as oxidative stress scavengers in a number of experimental models (
      • Malfroy B.
      • Doctrow S.R.
      • Orr P.L.
      • Tocco G.
      • Fedoseyeva E.V.
      • Benichou G.
      Prevention and suppression of autoimmune encephalomyelitis by EUK-8, a synthetic catalytic scavenger of oxygen-reactive metabolites.
      ;
      • Baker K.
      • Marcus C.B.
      • Huffman K.
      • Kruk H.
      • Malfroy B.
      • Doctrow S.R.
      Synthetic combined superoxide dismutase/catalase mimetics are protective as a delayed treatment in a rat stroke model: A key role for reactive oxygen species in ischemic brain injury.
      ;
      • Rong Y.
      • Doctrow S.R.
      • Tocco G.
      • Baudry M.
      EUK-134, a synthetic superoxide dismutase and catalase mimetic, prevents oxidative stress and attenuates kainate-induced neuropathology.
      ;
      • Melov S.
      • Ravenscroft J.
      • Malik S.
      • et al.
      Extension of life-span with superoxide dismutase/catalase mimetics.
      ). We have shown that the antioxidative properties of EUK-134, member of the salen-manganese complex family, are preserved under UVB irradiation and potently protect cultured keratinocytes against oxidative damage. Our data further suggest that EUK-134 effectively protects against oxidative membrane damage. Indeed EUK-134 was found to prevent lipid peroxidation elicited by a radical initiator and linoleic acid as a substrate. Oxidative membrane damage is thought to be the initiator of several UVB-responsive signaling pathways that originate at the plasma membrane (
      • Peus D.
      • Vasa R.A.
      • Beyerle A.
      • Meves A.
      • Krautmacher C.
      • Pittelkow M.R.
      UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.
      ;
      • Rosette C.
      • Karin M.
      Ultraviolet light and osmotic stress: Activation of the JNK cascade through multiple growth factor and cytokine receptors.
      ). A supplementation with EUK-134 before UVB irradiation might replenish the cell's capacity to neutralize oxidative stress, because UVB irradiation depletes the endogenous antioxidant levels in human skin (
      • Sander C.S.
      • Chang H.
      • Salzmann S.
      • Muller C.S.
      • Ekanayake-Mudiyanselage S.
      • Elsner P.
      • Thiele J.J.
      Photoaging is associated with protein oxidation in human skin in vivo.
      ).
      A major UVB-responsive pathway is the MAPK pathway. The MAPK pathway consists of three components in mammals, the ERK pathway, the JNK/SAPK pathway, and the p38 kinase pathway. Stress-induced MAPK activation is the result of ROS-mediated, ligand-independent clustering of membrane receptors (
      • Rosette C.
      • Karin M.
      Ultraviolet light and osmotic stress: Activation of the JNK cascade through multiple growth factor and cytokine receptors.
      ). If a pre-treatment with EUK-134 is able to protect the cell membrane against UVB damage in vivo, this would consequently result in a severely decreased activation of the MAPK pathways. Indeed, a pre-treatment with EUK-134 strongly downregulated the UVB-induced activation of all three MAPK pathways Figure 4. Furthermore, the ERK pathway is primarily activated in response to growth factors and is only modestly activated by UV irradiation (
      • Assefa Z.
      • Garmyn M.
      • Bouillon R.
      • Merlevede W.
      • Vandenheede J.R.
      • Agostinis P.
      Differential stimulation of ERK and JNK activities by ultraviolet B irradiation and epidermal growth factor in human keratinocytes.
      ). The presence of epidermal growth factor in the keratinocyte medium leads to a sustained basal activation of the ERK pathway Figure 4. Although EUK-134 pre-treatment did not affect the basal growth factor-dependent activation of this pathway, it completely inhibited UVB-damage-induced activation of this pathway. EUK-134 thus only inhibits UVB-damage-induced MAPK signaling, not mitogen-induced MAPK signaling. These data reinforce our hypothesis and suggest that EUK-134 might also effectively protect the cellular membrane against UVB-induced oxidative stress in vivo.
      The p53 protein quickly accumulates following various forms of environmental stresses, including DNA-damaging agents, decreased oxygen, heat shock, and redox stress (
      • Giaccia A.J.
      • Kastan M.B.
      The complexity of p53 modulation: Emerging patterns from divergent signals.
      ). UVB induces both direct DNA damage and oxidative stress (
      • Assefa Z.
      • Garmyn M.
      • Bouillon R.
      • Merlevede W.
      • Vandenheede J.R.
      • Agostinis P.
      Differential stimulation of ERK and JNK activities by ultraviolet B irradiation and epidermal growth factor in human keratinocytes.
      ;
      • Kulms D.
      • Zeise E.
      • Poppelmann B.
      • Schwarz T.
      DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation-induced apoptosis in an essential and independent way.
      ). In our experimental setup p53 accumulation following oxidative stress (in the form of exogenously applied H2O2) was shown to be nearly completely prevented by a pre-treatment with the antioxidant EUK-134. In agreement with this observation, p53 accumulation upon UVB exposure was severely reduced by an EUK-134 pre-treatment. This again indicates an important role for UVB-elicited oxidative stress in the stress signaling to and accumulation of p53 following UVB damage.
      We further investigated the mechanisms involved in this reduction of p53 accumulation. A severe reduction in the amount of oxidative stress and thus membrane damage following UVB irradiation can be expected to lower p53 accumulation through the suppression of MAPK activation. Indeed, MAPK are activated by ROS induced ligand-independent clustering of membrane receptors and these MAPK can phosphorylate several N-terminal phosphorylation sites on the p53 protein. Phosphorylation of these sites plays an important role in p53 stabilization, subsequent accumulation, and transcriptional activation (
      • Oda K.
      • Arakawa H.
      • Tanaka T.
      • et al.
      p53AIP1, a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53.
      ;
      • Buschmann T.
      • Potapova O.
      • Bar-Shira A.
      • et al.
      Jun NH2-terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptional activities in response to stress.
      ). Among the N-terminal phosphorylation sites of p53 inducible by MAPK are serine 15 and serine 46. Serine 15 has been shown to be a targeted by ERK and the p38 MAPK. Phosphorylation of serine 15 results in a disruption of the p53-MDM2 complex, a key element in the stabilization and transcriptional activation following UV-damage (
      • She Q.B.
      • Chen N.
      • Dong Z.
      ERKs and p38 kinase phosphorylate p53 protein at serine 15 in response to UV radiation.
      ). Likewise, serine 46 phosphorylation is p38 MAPK dependent and also results in increased stabilization and transcriptional activation following UV damage (
      • Bulavin D.V.
      • Saito S.
      • Hollander M.C.
      • Sakaguchi K.
      • Anderson C.W.
      • Appella E.
      • Fornace Jr., A.J.
      Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation.
      ). p53 accumulation following UVB damage was strongly reduced in EUK-134-pre-treated cells over a wide range of UVB doses (8–64 mJ/cm2). This reduction in p53 accumulation was associated with a strong reduction of serine 15 phosphorylation and an almost complete inhibition of serine 46 phosphorylation. To investigate the importance of p38 MAPK activation in the stabilization and accumulation of p53 following UVB damage, we treated cells with an inhibitor of the p38 MAPK before UVB irradiation. A pre-treatment with 10 μM PD169316 strongly reduced the accumulation of p53 following UVB irradiation. Similarly to the effect of EUK-134, the reduction in p53 accumulation was associated with a strong reduction of both serine 15 phosphorylation and serine 46 phosphorylation.
      These data again confirm the hypothesis that the cellular response to UVB irradiation is not only governed by nuclear events triggered by direct DNA damage. On the contrary, cytosolic signaling pathways induced by membrane damage also play an important role in the UVB response of human keratinocytes. This is further demonstrated by the fact that a pre-treatment with EUK-134 slightly but significantly reduced cell death following irradiation with higher UVB doses.
      The downstream targets of p53 determine the fate of the cell, either cell-cycle arrest or apoptosis, depending on the amount of damage induced (
      • Decraene D.
      • Agostinis P.
      • Pupe A.
      • de Haes P.
      • Garmyn M.
      Acute response of human skin to solar radiation: Regulation and function of the p53 protein.
      ). This response effectively prevents the proliferation of cells containing damaged DNA and is vital for cellular homeostasis. p53 is therefore often called “the guardian of the genome.” Nevertheless, p53 induction seems to act as a double-edged sword, because p53 plays a role in several theories of human aging: involving DNA damage, telomere shortening, and also oxidative stress (
      • Sharpless N.E.
      • DePinho R.A.
      p53: Good cop/bad cop.
      ). Pharmacologic targetting of p53 itself is unwanted, because the resulting inhibition of p53 function might be complicated by increased tumorigenesis. Clearly, the ultimate beneficial action of a product on cells is the reduction of stress itself (be it DNA damage, direct or indirect, or oxidative damage). EUK-134 appears to be an efficient product in this respect, because it addresses at least one part of this spectrum, the inhibition of oxidative stress.
      UVB-generated oxidative stress contributes to its chronic deleterious effects, for example, photoaging and photocarcinogenesis. Increased oxidative stress may lead to increased mitochondrial and nuclear DNA damage, subsequent mutation, and in the long run cancer. UVB-induced oxidative stress is also involved in connective tissue degradation, characteristic of dermal photodamage and signature of photoaging.
      • Sander C.S.
      • Chang H.
      • Salzmann S.
      • Muller C.S.
      • Ekanayake-Mudiyanselage S.
      • Elsner P.
      • Thiele J.J.
      Photoaging is associated with protein oxidation in human skin in vivo.
      have recently shown that the endogenous SOD and catalase enzyme levels in the epidermis are depleted both by chronic and by acute UV exposures and that photoaging is associated with protein oxidation in human skin in vivo. By replenishing the cell's antioxidant defense, EUK-134 may prove to be very efficient in the fight against both the short and the long-term (photoaging and photocarcinogenesis) deleterious effects of UVB irradiation on skin.

      Materials and methods

      Cell culture

      Primary human keratinocytes were isolated and pooled from foreskins of five different donors (less than 6 y) as described (
      • Gilchrest B.A.
      In vitro assessment of keratinocyte aging.
      ). The procedure has been approved by the ethical committee of the University of Leuven. Keratinocytes were grown in serum-free medium (Keratinocyte-SFM, Invitrogen, Merelbeke, Belgium) supplemented with bovine pituitary extract (50 μg/mL) and human recombinant epidermal growth factor (5 ng/mL). Third- to fifth-passage cells were used in experiments.

      Antibodies and reagents

      Phospho-p44/42 MAP kinase (Thr202/Tyr204, recognizes dually phosphorylated extracellular signal-regulated kinase (ERK) 1 and ERK2), phospho-stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) (Thr183/Tyr185, recognizes dually phosphorylated JNK1 and JNK2 isoforms), phospho-MKK3/6 (Ser189/207), phospho-SEK1/MKK4 (Thr261), phospho-p53 (Ser46), phospho-p38 MAP kinase (Thr180/Tyr182), and p38 MAP kinase antibodies were purchased from Cell Signaling (Beverly, MA). Anti-human p53 (clone DO-1) antibody was purchased from PharMingen (BD Biosciences, Erembodegem, Belgium). EUK-134 was purchased from Eukarion Inc. (Bedford, MA). Hydrogen peroxide and xanthine oxidase were purchased from Sigma-Aldrich (Bornem, Belgium).

      UVB irradiation

      Before UVB irradiation cells were washed twice with phosphate-buffered saline, irradiated through a thin film of phosphate-buffered saline, and then refed with their own medium. Cells were exposed through the cover of the dish, which filters out residual UVC (
      • Brown D.B.
      • Peritz A.E.
      • Uitto J.
      • Gasparro F.P.
      Ultraviolet-filtering properties of commonly used tissue cell culture plasticware.
      ). The UVB source was a parallel bank of three Philips TL 20W12 tubes with a peak output of 310 nm. Output was measured through the cover of a tissue culture dish with an IL700 radiometer (International light, Newburyport, MA).

      Western blotting

      At the indicated time points, cells were scraped in lysis buffer (25 mM HEPES, 0.3 mM NaCl, 1.5 mM MgCl2, 20 mM β-glycerolphosphate, 2 mM ethylenediaminetetraacetic acid, 2 mM ethylene glycol-bis(β-amino ethyl ether), pH 7.5) containing 1% Triton, 10% glycerol, 1 mM Na3VO4, 0.5 mM dithiothreitol, 10 μg per mL leupeptin, 10 μg per mL aprotinin, and 10 μg per mL antipain. Extracts were incubated on ice and spun down at 21,000g for 20 min. Protein concentration was determined using the bicinchoninic acid protein assay reagent (Pierce Chemical Company, Rockford, IL). A quantity of 50 to 80 μg of total protein extract was separated by a 10% SDS-PAGE, followed by wet electrotransfer onto Hybond-C Super membrane (Amersham Pharmacia, Roosendaal, Netherlands). Equal loading of proteins was verified using Ponceau-S in coloring (
      • Klein D.
      • Kern R.M.
      • Sokol R.Z.
      A method for quantification and correction of proteins after transfer to immobilization membranes.
      ). Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween and 5% nonfat dry milk. The membrane was incubated overnight at 4°C with the primary antibody, washed, and incubated for 1 h at room temperature with the peroxidase-conjugated secondary antibody. Protein bands were visualized using enhanced chemiluminescence as described by the supplier (Amersham Pharmacia, Roosendaal, Netherlands).

      Quantification of the Catalase-like and SOD-like activity of EUK-134

      The catalase-like activity of EUK-134 was assayed by monitoring the rate of conversion of hydrogen peroxide to oxygen as a decrease in absorbance at 240 nm, as originally described by
      • Beers R.F.
      • Sober A.J.
      A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase.
      . EUK-134 solutions (6 μg/mL in 50 mM phosphate buffer, pH 7.0) were incubated on ice and exposed to UVB irradiation. Following UVB irradiation, an equal volume of H2O2 (Acros, Geel, Belgium; 34 mM in phosphate buffer) was added. The decrease in UV absorbance at 240 nm was monitored over a period of 2 min. Catalase-like activity (U/mg) was extrapolated from a standard curve of different concentrations of catalase (from bovine liver).
      SOD-like activity of EUK-134 was determined as modified from
      • Kimura H.
      • Nakano M.
      Highly sensitive and reliable chemiluminescence method for the assay of superoxide dismutase in human erythrocytes.
      . The protocol is based on the ability of EUK-134 to compete with luminol for the superoxide anion, produced in situ using a hypoxanthine/xanthine oxidase system and resulting in a decrease in chemiluminescent signal. Briefly, EUK-134 solutions (120 μg/mL in 50 mM phosphate buffer, pH 7.0) were incubated on ice and exposed to UVB. Following UVB irradiation, 30 μL of EUK-134 solution was transferred to an equal volume of xanthine oxidase (0.1 U/mL). Fifteen microliters of luminol (0.5 mM) and 75 μL of hypoxanthine (0.735 mM) were added and the chemiluminescence intensity was measured for 30 s. SOD-like activity (U/mg) was extrapolated from a standard curve with different concentrations of SOD (from bovine erythrocytes). All reagents were purchased from Sigma, unless stated otherwise.

      Linoleic acid peroxidation assay

      The antioxidant activity of EUK-134 was expressed as the ability to inhibit linoleic acid peroxidation in vitro according to
      • Liegeois C.
      • Lermusieau G.
      • Collin S.
      Measuring antioxidant efficiency of wort, malt, and hops against the 2,2-azobis(2-amidinopropane) dihydrochloride-induced oxidation of an aqueous dispersion of linoleic acid.
      . Briefly, oxidation of 16 mM linoleic acid in aqueous dispersions (in borate buffer, pH 9, 0.5% Tween 20, 25 mM KOH, with or without EUK-134) was initiated by addition of 4 mM of the radical initiator 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) (Aldrich Chemical Company, Milwaukee, WI). The progress of lipid peroxidation at 37°C in the presence of EUK-134 was continuously monitored by spectrophotometric reading of the rate of appearance of conjugated dienes at 234 nm as a function of time.

      Neutral red viability assay

      Primary human keratinocytes were grown in 96-well plates to approximately 75% confluency. A pre-treatment of EUK-134 at 12.5, 25, and 50 μM was carried out. Cells were then exposed to increasing concentrations of H2O2 (100–400 μM) or xanthine oxidase (0.5–2 U/mL). Following an 18-h incubation, 50 μg per mL neutral red dye (Aldrich Chemical Company) was added to the medium. Three hours later, cells were fixed in 1% formaldehyde-1% calcium chloride, rinsed three times with phosphate-buffered saline, and lyzed with 1% acetic acid-50% ethanol for 30 min. Absorbance at 540 nm was measured by spectrophotometry.

      Trypan blue exclusion assay

      Primary human keratinocytes were plated in 60-mm cell culture dishes and grown to 75% confluency. The keratinocytes were pre-incubated with EUK-134 at 0, 5, and 50 μM for a 24-h period before UVB irradiation at 8, 16, and 24 mJ per cm2. Cells were harvested 24 h after UVB irradiation by trypsinization and centrifugation. Cell viability was determined by the trypan blue exclusion assay (
      • Kaltenbach J.P.
      • Kaltenbach M.H.
      • Lyons W.B.
      Nigrosin as a dye for differentiating live and dead ascites cells.
      ). Briefly, pelleted cells where stained with 0.4% trypan blue (Aldrich Chemical Company, Milwaukee, WI), followed by examination with a hemacytometer under an inverted microscope. Cells that excluded the dye were considered viable and the data are expressed as the percentage of trypan blue exclusion.

      ACKNOWLEDGMENTS

      This work was supported in part by Grant OT/00/33 from the University of Leuven and Grant 0211.99 from the “Fonds voor Wetenschappelijk Onderzoek-Vlaanderen,” Belgium. M.G. is clincal research associate (FWO).

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