Journal of Dermatological Science 74 (2014) 9–17
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Peroxiredoxin I plays a protective role against UVA irradiation through reduction of oxidative stress Takaaki Ito a, Shintaro Kimura b, Kahori Seto a, Eiji Warabi c, Yasuhiro Kawachi d, Junichi Shoda e, Katsuhiko Tabuchi f, Kenji Yamagata g, Shogo Hasegawa g, Hiroki Bukawa g, Tetsuro Ishii c, Toru Yanagawa g,* a
Oral and Maxillofacial Surgery, Clinical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan Environmental Molecular Biology, Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan c Department of Environmental Molecular Biology, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan d Department of Dermatology, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan e Department of Medical Science, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan f Department of Neurophysiology, Faculty of Medicine, Shinshu University, Matsumoto, Nagano 390-8621, Japan g Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 September 2013 Received in revised form 3 December 2013 Accepted 4 December 2013
Background: Exposure of skin to long-wave UV radiation (UVA) increases the cellular levels of reactive oxygen species (ROS), which have been linked to apoptosis induction through the damage of lipids, proteins, and nucleic acids. Peroxiredoxin I (Prx I) is one of a family of antioxidant proteins that plays a protective role against oxidative damage; however the role of Prx I in UVA-induced damage remains to be clarified. Objective: Here we investigated the protective role of Prx I against UVA-induced changes using mouse embryonic fibroblasts (MEFs) derived from Prx I homozygous knockout (Prx I (/)) mice. Methods: Prx I (/) and wild-type (Prx I (+/+)) MEFs were subjected to UVA irradiation, and the resulting apoptosis was analyzed using flow cytometry, quantitative real-time PCR, and western blotting. Results: Prx I (/) MEFs showed enhanced sensitivity to UVA treatment, exhibiting increased apoptosis and ROS production compared to Prx I (+/+) MEFs. Consistent with the increase in apoptosis, p53 expression was significantly higher, while Bcl-2, Bcl-xL, and Nrf2 expressions were all lower in Prx I (/ ) versus (+/+) MEFs. The UVA-induced inflammatory response was upregulated in Prx I (/) MEFs, as indicated by increased expressions of IkB, TNFa, and IL-6. Evidence was presented indicating that Prx I impacts these pathways by modifying critical signaling intermediates including p53, IkB, and Nrf2. Conclusion: Our results indicate that Prx I plays a protective role against UVA-induced oxidative damage by controlling ROS accumulation. Both the UVA-induced apoptotic and inflammatory signals were found to be modulated by Prx I. ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Peroxiredoxin I UVA Apoptosis p53 ROS
1. Introduction The major environmental cause of skin damage is excessive ultraviolet (UV) radiation, which leads to acute reactions such as
erythema and sunburn, and over the long-term can result in premature skin aging and carcinoma. UV radiation in sunlight is composed of long-wave (UVA: 320–400 nm) and shorter wavelength (UVB: 280–320 nm) UV radiation. UVA is approximately 20-fold
Abbreviations: ROS, reactive oxygen species; UV, ultraviolet; PCR, polymerase chain reaction; Cys, cysteine; GCLM, glutamate-cysteine ligase modifier subunit; MEF, mouse embryo fibroblast; cisplatin, cis-diamminedichloroplatinum(II); Prx, Peroxiredoxin; Prx I, Peroxiredoxin I; MSP23, macrophage stress protein 23 kDa; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nrf2, NF-E2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; HO-1, hemeoxygenase-1; DCFH-DA, 20 ,70 dichlorofluoresceindiacetate; RIPA buffer, Radioimmunoprecipitation assay buffer; PVDF, polyvinylidene difluoride; PI, propidium iodide; NFkB, nuclear factor-kappa B; Bcl2, B-cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra large; Bax, Bcl-2-associated X protein; PUMA, p53 up-regulated modulator of apoptosis; TNFa, tumor necrosis factor alpha; IL-6, Interleukin 6. * Corresponding author at: Oral and Maxillofacial Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. Tel.: +81 29 853 3052; fax: +81 29 853 3052. E-mail address: [email protected] (T. Yanagawa). 0923-1811/$36.00 ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2013.12.002
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more abundant than UVB and penetrates the basal germinative layers [1]. Exposure of skin to UVA increases the cellular levels of reactive oxygen species (ROS), which have been linked to apoptosis through the damage of lipids, proteins, and nucleic acids [2]. A recent study indicated that UVA irradiation generates ROS, resulting in an altered intracellular redox balance and the translocation of p53 to the mitochondria [3]. Murine Prx I, first termed MSP23 (macrophage stress protein 23 kDa) was cloned by our group from peritoneal macrophages as an oxidative stress-inducible protein [4]. Previous studies established that Peroxiredoxins (Prxs) are an important family of antioxidants, protecting cells from oxidative damage by reducing peroxides such as H2O2, and scavenging radicals [5–7]. There are currently six Prx family members, which share a common reactive Cys residue in the N-terminal region. Prx I is the major cytosolic Prx and is widely expressed in various tissues [8]. Since H2O2 is an intracellular signaling molecule produced in response to cell surface receptor activation, the Prx peroxidase activity is reversibly and effectively inactivated to allow for transient H2O2 induction and the transmission of intracellular signals [9,10]. Recently, various signals associated with ROS have been reported, and the signaling modulatory roles of the Prx enzymes have been elucidated. A major cytotoxic effect of the cancer chemotherapeutic agent cis-diamminedichloroplatinum(II) (cisplatin) is cellular oxidative stress that results in acute renal failure. Studies using Prx I-deficient mice demonstrated that Prx I plays a protective role against cisplatin-induced apoptosis [11,12]. However, there have been no reports investigating the role of Prx I in UVA-induced damage. In the present study, we used mouse embryonic fibroblasts (MEFs) from Prx I-deficient mice to explore the role of Prx I in protecting cells from UVA-induced damage.
in 6-well plates at 1 105 cells per well and incubated overnight at 37 8C. Following treatment with various doses of UVA irradiation, MTT was added, and the cells were further incubated for 5 h at 37 8C. The absorbance at 540 nm was determined using a Varioskan plate reader (Thermo Fisher Scientific. Co., Yokohama, Japan). Cytotoxicity was expressed as the ratio of the UVAirradiated cell absorbance to that of untreated control cells. 2.4. Apoptosis analysis using flow cytometry UVA-induced apoptosis of Prx I (+/+) and Prx I (/) MEFs was assessed using an Annexin V-FLUOS Staining Kit (Roche Diagnostic. Co., Tokyo) followed by flow cytometry analysis. Cells were seeded at 1 105 cells per 35-mm dish, cultured overnight in 10% FBS/ DMEM, and then UVA irradiated at 15 J/cm2 and further cultured for 5 h. The washed and resuspended cells were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V antibody and propidium iodide (PI), according to the manufacturer’s instructions. The cells (2 104 per sample) were then analyzed using a flow cytometer (FACSCalibur; Becton Dickinson Co., Tokyo) equipped with a 488-nm argon laser and the CellQuest Pro software. 2.5. Detection of ROS generation and mitochondrial damage Prx I (/) and (+/+) MEFs were subjected to UVA radiation. Then, 20 mM 20 ,70 -dichlorofluoresceindiacetate (DCFH-DA) and 150 mM MitoTracker Deep Red were added to the culture medium and the cells were incubated for 30 min. The cells were then washed five times with culture medium, and fluorescent images were acquired with a confocal laser scanning microscope (TSC SP2, Leica Microsystems, Tokyo, Japan).
2. Materials and methods 2.6. Western blot analysis 2.1. Cell culture Prx I homozygous knockout (Prx I (/)) (OmniBank, Lexicon Pharmaceuticals, Inc.) and wild-type (Prx I (+/+)) mice were treated as described previously [8]. Primary MEFs were generated from 13.5-day Prx I (/) and Prx I (+/+) embryos as described previously [11]. Briefly, the brain and dark-red (internal) organs were dissected away from the embryos, and the remaining tissue was finely minced. The cells were dissociated using 0.25% trypsin, which was removed by centrifugation. The University of Tsukuba Animal Research Committee approved all the animal experiments (No. 11-171). The resulting MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing streptomycin (100 U/ml), penicillin (0.1 mg/ml), and heat-inactivated 10% FBS. The cells were cultured in a humidified atmosphere with 5% CO2 at 37 8C. All experiments with subcultures of MEFs were measured from three individual culture plates (n = 3). 2.2. UVA irradiation MEFs were washed with PBS, and the medium was replaced with phenol-red-free DMEM containing 5% FBS and 30 mM HEPES. Next, the cells were irradiated with UVA using an FL20SBLB lamp (Toshiba, Tokyo, Japan) with a peak emission frequency of 352 nm. Wavelengths below 320 nm were blocked by an ATG filter UV-35 (Asahi Technoglass, Japan). The dose of UVA irradiation was measured using a radiometer (UVR-305/365; Toshiba, Tokyo, Japan).
Whole-cell extracts were generated by lysing cells in RIPA buffer, followed by centrifugation at 12,000 g for 10 min, and collection of the supernatants. The samples were separated on a 12.5% polyacrylamide gel with SDS and then transferred onto PVDF membranes for 2 h at 80 V. The membranes were incubated overnight at 4 8C in blocking buffer (Tris buffered saline-Tween20 (TBS-T) containing 5% fat-free dried milk powder (Yukijirushi, Tokyo, Japan)). The membranes were then incubated with primary antibody diluted 1:1000 in fresh blocking buffer for 1 h at room temperature. p53, Bcl-2, and Bcl-xL antibodies were purchased from Cell Signaling Technology (Cell Signaling Technology Japan Co., Tokyo, Japan). The Prx I and Nrf2 polyclonal antibodies were described previously [8,13]. After treatment with peroxidaseconjugated anti-mouse IgG antibody (Sigma) or anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, USA) for 1 h at room temperature, the labeled bands in washed blots were detected using the ECL Plus kit (GE Healthcare Bio-Sciences Co., Tokyo, Japan). The membranes were exposed to Fuji Medical X-ray Film (Fuji Photo Film Co., Ltd., Tokyo, Japan) at room temperature. 2.7. Quantitative real-time PCR analysis Quantitative real-time PCR was carried out as described previously [2,14]. The mRNA expression levels were normalized to that of GAPDH. 2.8. Statistical analysis
2.3. MTT assay for cell viability Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded
All data are presented as mean SD. Differences among data were determined using Student’s t-test (StatView, version 5.0, Abacus Concepts, Berkeley, USA).
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3. Results 3.1. Enhanced UVA sensitivity in Prx I-deficient MEFs Prx I (+/+) and Prx I (/) MEFs were irradiated using various doses of UVA, and cell survival was evaluated using the MTT assay. The resulting cell viabilities are shown in Fig. 1A. Prx I (/) MEFs were significantly more sensitive to UVA treatment at all doses tested (10 J/cm2 (P < 0.05), 12.5 J/cm2 (P < 0.01), and 15 J/cm2 (P < 0.001)). Next, we analyzed the UVA-treated MEFs by staining with an Annexin V antibody and PI (propidium iodide) followed by flow cytometry to determine whether the increased UVA sensitivity of the Prx I-deficient MEFs was a result of apoptosis. A representative scatterplot of Prx I (/) and (+/+) MEFs is shown in Fig. 1B. The percentage of cells in the upper- and lower-right quadrants (late and early apoptosis) was defined as the apoptotic index. The analysis indicated that UVA-irradiated Prx I (/) MEFs contained an increased percentage of Annexin V positive cells compared to identically treated Prx I (+/+) MEFs. Fig. 1C shows the apoptotic indexes of the two cell types. After treatment with 15 J/cm2 UVA there was a significant difference between the apoptotic index of Prx I (/) cells (85.6%) and Prx I (+/+) cells (16.7%) (P < 0.001), suggesting that Prx I (/) cells are more susceptible to UVAinduced apoptosis. 3.2. UVA-induced apoptosis in Prx I-deficient MEFs is mediated by p53 To explore the mechanistic basis for the increased apoptosis seen in Prx I (/) MEFs, the cells were further analyzed by treating with UVA followed by immunoblotting with antibodies to p53, Bcl-2, and Bcl-xL (Fig. 2A and B). The expression of p53 was induced in both Prx I (/) and (+/+) MEFs exposed to UVA irradiation, and the basal levels of p53 were significantly higher in Prx I (/) versus (+/+) MEFs (Fig. 2A and B). In contrast, the Bcl-2 and Bcl-xL expressions were significantly reduced in the UVAtreated Prx I (/) MEFs in comparison with UVA-treated Prx I (+/ +) MEFs. Next, we examined the expression of p53-regulated genes. Quantitative real-time PCR revealed that p21, Noxa, PUMA, and Bax mRNA expression levels were all increased in the Prx I (/) versus (+/+) MEFs (Fig. 2C). These results indicate that the p53dependent apoptotic signaling pathway was altered in Prx I (/) MEFs. 3.3. Regulation of ROS accumulation in Prx I-deficient MEFs To compare the accumulation of ROS in Prx I (+/+) and Prx I (/ ) MEFs, cells were treated with DCFH-DA to visualize the ROS production following UVA irradiation. As shown in Fig. 3A, strong fluorescent signals derived from activated DCFH-DA were detected in the Prx I (/) MEFs exposed to 5 J/cm2 UVA. In contrast, DCFHDA signals were barely detectable in the UVA-treated Prx I (+/+) MEFs or in untreated cells. To quantify the signals, the intracellular ROS generation was measured by flow cytometry. This analysis showed that the ROS production in Prx I (/) MEFs was significantly elevated when compared to Prx I (+/+) MEFS (P < 0.01) (Fig. 3B). 3.4. Altered UVA-induced Nrf2 regulation in PrxI-deficient MEFs UVA irradiation induces the nuclear accumulation of the transcription factor Nrf2, which plays a critical role in protecting dermal cells from UVA-induced apoptosis [15]. Furthermore, Nrf2 regulates the gene expression of various electrophile- and oxidative stress-inducible proteins, including Prx I [13]. Thus,
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we investigated the effects of UVA irradiation on the expression of Nrf2 and Nrf2-regulated oxidative stress-inducible proteins in Prx I (/) and (+/+) MEFs. The expression levels of Nrf2, Prx I, and hemeoxygenase 1 (HO-1), a representative downstream gene product of Nrf2, are shown in Fig. 4A and B. In the Prx I (+/+) MEFs all three proteins, Nrf2, Prx I, and HO-1 were significantly upregulated by UVA irradiation. However, in Prx I (/) MEFs, the UVA-inducible expression of both Nrf2 and HO-1 was significantly lower. As expected, Prx I protein was not detected in the Prx I (/) cells. These results showed UVA-inducible expression of oxidative stress-inducible proteins was reduced in Prx I (/) MEFs. Next, we used quantitative real-time PCR to analyze the mRNA expression levels of both HO-1 and glutamate-cysteine ligase modifier subunit (GCLM), another Nrf2-regulated gene. HO-1 and GCLM genes are located downstream of Nrf2 binding element, and their up-regulation means that transcriptional activity of Nrf2 was enhanced. UVA irradiation significantly induced both HO-1 and GCLM mRNA expression in Prx I (+/+) and (/) MEFs; however, the degree of GCLM up-regulation was significantly smaller in the Prx I (/) than the Prx I (+/+) MEFs (Fig. 4C). These findings suggest that Nrf2-regulated gene expression was reduced in the Prx I-deficient MEFs. 3.5. Elevated UVA-induced inflammatory response in Prx I-deficient MEFs Since it was previously reported that Nrf2 signaling can be transcriptionally repressed by NFkB [16], we next focused on the involvement of NFkB. To examine NFkB signaling following exposure to UVA irradiation, we first examined the protein levels of NFkB’s associated inhibitor protein IkB, by western blotting. As shown in Fig. 5A and B, there was a strong basal expression of IkB in Prx I (+/+) MEFs that was significantly down-regulated following UVA treatment. In contrast, in the Prx I (/) MEFs, IkB protein expression was barely detectable in the control or UV-treated cells. Quantitative real time PCR analysis revealed that UVA irradiation induced TNFa and IL-6 mRNA expression in both Prx I (/) and (+/+) MEFs; however, the induced levels of both cytokines were significantly elevated in the Prx-I (/) cells (Fig. 5C). These findings indicate that Prx I deficiency resulted in reduced IkB expression, which led to an increased inflammatory response to UVA treatment. 4. Discussion In the current study, we found that Prx I (/) MEFs were significantly more sensitive to UVA than Prx I (+/+) MEFs, resulting in increased induction of apoptosis. Since previous reports showed that UVA-induced apoptosis was caused by extensive oxidative damage leading to the p53-regulated mitochondrial release of cytochrome c and caspase activation [3], we investigated the role of p53 in our system. We examined the expression of p53 and p53regulated pro-apoptotic genes including Bax, Noxa, PUMA, and p21 (a cell cycle regulator of G1), as well as the anti-apoptotic proteins Bcl-2 and Bcl-xL. We observed enhanced expressions of p53 and the four p53-regulated genes following the UVA treatment of Prx I (/) MEFs. In contrast the expression of Bcl-2 and Bcl-xL was found to be suppressed in UVA-treated Prx I (/) MEFs. Taken together, these data suggest that the increased apoptosis observed in Prx I (/) MEFs was mediated by p53 signaling. We hypothesized that the UVA-induced apoptosis in Prx I (/) MEFs was caused by excessive oxidative stress. To test, this we examined the ROS levels in Prx I (/) and (+/+) MEFs using DCFHDA. Following irradiation with 5 J/cm2 UVA, ROS were abundantly detected in the Prx I (/) MEFs; in contrast, ROS were not detected in Prx I (+/+) MEFs treated with the same dose of
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Fig. 1. Enhanced UVA sensitivity in Prx I-deficient MEFs. (A) MTT cell viability analysis of Prx I homozygous knockout (Prx I (/)) and wild-type (Prx I (+/+)) MEFs irradiated with various doses of UVA. Prx I (/) MEFs were more sensitive to UVA irradiation at all doses tested (10, 12.5, and 15 J/cm2) (*P < 0.05, **P < 0.01, ***P < 0.001) (n = 3). (B) Flow cytometric analysis of Prx I (+/+) and Prx I (/) MEFs stained with propidium iodide and fluorescently labeled Annexin V antibodies. Apoptotic cells were defined as the Annexin V-positive cells in the upper- and lower-right quadrants. Vertical axis: propidium iodide (PI); horizontal axis: Annexin V. (C) Quantification of flow cytometric analysis. The percentage of cells in the upper- and lower-right quadrants was defined as the apoptotic index. After 15 J/cm2 UVA irradiation the apoptotic index of Prx I (/) MEFs was significantly increased in comparison to the apoptotic index of Prx I (+/+) cells. Values are the means SD from three independent experiments (***P < 0.001) (n = 3).
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Fig. 2. Apoptotic signaling in Prx (+/+) and Prx (/) MEFs. (A) Western blot analysis of p53, Bcl-2, and Bcl-xL in Prx I (+/+) and (/) MEFs before and after 5 J/cm2 UVA irradiation. (B) Quantification of p53, Bcl-2, and Bcl-xL western blots. p53 expression was increased, while Bcl-2 and Bcl-xL expression levels were significantly reduced in the Prx I (/) MEFs (*P < 0.05, **P < 0.01, ***P < 0.001) (n = 3). (C) Quantitative real time PCR analysis of p21, Noxa, PUMA, and Bax mRNAs. The expression levels were normalized to that of GAPDH. All fourp53-regulated mRNAs were significantly upregulated in the Prx I (/) MEFs following UVA irradiation (*P < 0.05, **P < 0.01, ***P < 0.001) (n = 3).
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Fig. 3. ROS generation induced by UVA in Prx I (/) and Prx I (+/+) MEFs. (A) Analysis of ROS production in MEFs. Cells were exposed to 5 J/cm2 UVA irradiation and then stained with DCFH-DA. Fluorescently labeled ROS signals were visualized with confocal laser scanning microscopy. (B) Quantification of ROS signals by flow cytometric analysis. UVA-induced production of ROS was significantly greater in Prx I (/) MEFs compared to Prx I (+/+) MEFs (***P < 0.001) (n = 3).
radiation. This result indicated that the antioxidant capacity was reduced in the Prx (/) MEFs, resulting in the ineffective elimination of ROS and the accumulation of excessive oxidative stress, leading to the induction of apoptosis. These findings are consistent with previous reports that Prx I is a member of a ubiquitous family of antioxidant enzymes that regulate the levels of cytokine-induced peroxides [17]. The role of ROS in UVA-induced apoptosis was also observed in several studies using various antioxidants [18–20], and those reports also support our hypothesis. Since the Nrf2-Keap1 system is a central regulator of the cellular antioxidant response [21], we examined the UVA induction of Nrf2 and Nrf2-regulated genes. The expressions of Nrf2 and its downstream genes encoding HO-1 and GCLM were found to be reduced in the Prx I (/) versus (+/+) MEFs. This was
an unexpected finding, given a previous report showing that ROS counteract the degradation of Nrf2 and provoke its activation, resulting in the expression of a battery of genes encoding antioxidative stress enzymes and proteins [21]. However, another report showed that p53 and Nrf2 interact, and that p53 suppresses the transcription of Nrf2 target genes [22]. Thus, in Prx I (/) cells the upregulated p53 may interact with Nrf2 leading to the suppression of Nrf2’s target genes. In contrast, a recent report showed an interaction between the p53-regulated p21 and Nrf2. p21 was shown to interact with the DLG and ETGE motifs within Nrf2, thereby attenuating the Keap1-based ubiquitination and subsequent proteosomal degradation of Nrf2. [23]. Thus, there may be some mechanism like a cytoprotective feedback loop among p53, p21, and Nrf2 in response to oxidative stress. Our results show
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Fig. 4. UVA induction of Nrf2 and Nrf2-regulated genes. (A) Western blot analysis of Nrf2, Prx I, and HO-1 in Prx I (+/+) and (/) MEFs before and after UVA irradiation. (B) Quantification of Nrf2, HO-1, and Prx I western blots, indicating the UVA-induced upregulation of Nrf-2 and HO-1 in Prx (/) and (+/+) MEFs. Prx I was upregulated in UVAtreated Prx I (+/+) MEFs and as expected, was not detectable in Prx I (/) MEFs. (C) Quantitative real time PCR of Nrf2-regulated genes, HO-1 and GCLM. The mRNA expression levels were normalized to that of GAPDH (*P < 0.05, **P < 0.01, **P < 0.001) (n = 3).
that in the absence of Prx I, the p21 levels were upregulated, while Nrf2 and the Nrf2 signaling were suppressed. Thus, the absence of Prx I may alter the cytoprotective feedback loop, resulting in the induction of apoptosis following UVA exposure as one hypothesis.
However, there are many other target genes that have interaction with Nrf2, then further investigation is necessary. Another hypothesis that may explain this paradox involves the NFkB signal. In a recent report, NFkB was shown to directly repress
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Fig. 5. Enhanced UVA-induced inflammatory responses in Prx (/) MEFs. A. Western blot analysis of IkB in Prx I (/) and (+/+) MEFs before and after treatment with 5 J/cm2 UVA irradiation. B. Quantification of IkB western blot. IkB was strongly expressed in Prx I (+/+) MEFs and was suppressed by UVA irradiation. IkB expression was significantly lower in Prx I (/) MEFs and remained low following UVA treatment (*P < 0.05) (n = 3). (C) Quantitative real time PCR analysis of TNFa and IL-6 mRNAs. UVA-inducible TNFa and IL-6 mRNA levels were significantly higher in the Prx I (/) versus Prx I (+/+) MEFs (*P < 0.05, **P < 0.01, **P < 0.001) (n = 3).
Nrf2 signaling at the transcriptional level. NFkB was found to compete with Nrf2 for the co-activator CREB binding protein (CBP) and to recruit histone deacetylase 3 (HDAC3), resulting in local hypoacetylation and the repression of Nrf2 signaling [24]. The NFkB family includes the Rel family proteins p65, c-Rel, Rel B, p50, and p52. Various stimuli such as pro-inflammatory signals, and genotoxic or oxidative stress lead to activation of the IkB kinase signalosome, resulting in IkB degradation and movement of the NFkB dimer to the nucleus, where it induces the transcription of target genes. In the current study, IkB protein levels were significantly reduced following UVA irradiation in Prx I (+/+) MEFs. However, in the Prx I (/) MEFs, IkB protein was barely detectable both before and after UV irradiation. Thus, some
stimulus, most likely oxidative stress, evoked NFkB signaling in the basal state of Prx I (/) MEFs. Following UVA irradiation, the expression of NFkB-regulated genes, namely TNFa and IL-6, was enhanced in the Prx I (/) MEFs. Thus, the Prx I (/) MEFs may be predisposed to developing an elevated inflammatory response, or affected by other mechanisms associated with inflammation caused by Prx I deficiency. In this report, we demonstrated that Prx I plays a protective role in UVA-induced apoptosis through controlling the ROS accumulation. However, the mechanistic basis of these observations is not fully understood. Prx I protects cells and tissues from oxidative damage through a number of mechanisms besides its enzymatic peroxidase activity. Prx I can also suppress oxidative
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stress-induced cell death through direct and indirect interactions with different types of molecules that play key roles in regulating cell death and/or apoptosis, depending on the cell type and stimulus. For instance, Prx I has been shown to suppress ASK1-JNK signaling, and to inhibit both p66shc and c-Abl activation. Furthermore, Prx I associates with some transcription factors, such as c-Myc, NFkB, and the androgen receptor, thereby modulating gene expression [25]. Thus, the specific mechanism by which Prx I functions in suppressing the apoptotic signaling caused by UVA irradiation is unclear. Further studies are necessary to define the exact role of Prx I in UVA signaling pathways. Acknowledgements The first three authors (T.I., S.K. and K.S.) contributed to this work equally. This work was supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research (25670843). References [1] Bruls WA, Slaper H, van der Leun JC, Berrens L. Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem Photobiol 1984;40:485–94. [2] Kimura S, Warabi E, Yanagawa T, Ma D, Itoh K, Ishii Y, et al. Essential role of Nrf2 in keratinocyte protection from UVA by quercetin. Biochem Biophys Res Commun 2009;387:109–14. [3] Waster PK, Ollinger KM. Redox-dependent translocation of p53 to mitochondria or nucleus in human melanocytes after UVA- and UVB-induced apoptosis. J Invest Dermatol 2009;129:1769–81. [4] Ishii T, Yamada M, Sato H, Matsue M, Taketani S, Nakayama K, et al. Cloning and characterization of a 23-kDa stress-induced mouse peritoneal macrophage protein. J Biol Chem 1993;268:18633–36. [5] Rhee SG, Chae HZ, Kim K. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic Biol Med 2005;38:1543–52. [6] Flohe L, Harris JR. Introduction. History of the peroxiredoxins and topical perspectives. Subcell Biochem 2007;44:1–25. [7] Fujii J, Ikeda Y. Advances in our understanding of peroxiredoxin, a multifunctional, mammalian redox protein. Redox Rep 2002;7:123–30. [8] Uwayama J, Hirayama A, Yanagawa T, Warabi E, Sugimoto R, Itoh K, et al. Tissue Prx I in the protection against Fe-NTA and the reduction of nitroxyl radicals. Biochem Biophys Res Commun 2006;339:226–31.
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[9] Rhee SG. Cell signaling, H2O2, a necessary evil for cell signaling. Science 2006;312:1882–3. [10] Woo HA, Yim SH, Shin DH, Kang D, Yu DY, Rhee SG. Inactivation of peroxiredoxin I by phosphorylation allows localized H(2)O(2) accumulation for cell signaling. Cell 2010;140:517–28. [11] Ma D, Warabi E, Yanagawa T, Kimura S, Harada H, Yamagata K, et al. Peroxiredoxin I plays a protective role against cisplatin cytotoxicity through mitogen activated kinase signals. Oral Oncol 2009;45:1037–43. [12] Okada K, Ma D, Warabi E, Morito N, Akiyama K, Murata Y, et al. Amelioration of cisplatin-induced nephrotoxicity in peroxiredoxin I-deficient mice. Cancer Chemother Pharmacol 2013;71:503–9. [13] Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y, et al. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem 2000;275:16023–29. [14] Warabi E, Takabe W, Minami T, Inoue K, Itoh K, Yamamoto M, et al. Shear stress stabilizes NF-E2-related factor 2 and induces antioxidant genes in endothelial cells: role of reactive oxygen/nitrogen species. Free Radic Biol Med 2007;42:260–9. [15] Hirota A, Kawachi Y, Itoh K, Nakamura Y, Xu X, Banno T, et al. Ultraviolet A irradiation induces NF-E2-related factor 2 activation in dermal fibroblasts: protective role in UVA-induced apoptosis. J Invest Dermatol 2005;124: 825–32. [16] Li W, Khor TO, Xu C, Shen G, Jeong WS, Yu S, et al. Activation of Nrf2antioxidant signaling attenuates NFkappaB-inflammatory response and elicits apoptosis. Biochem Pharmacol 2008;76:1485–9. [17] Wood ZA, Schroder E, Robin Harris J, Poole LB. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 2003;28:32–40. [18] Chung JH, Han JH, Hwang EJ, Seo JY, Cho KH, Kim KH, et al. Dual mechanisms of green tea extract (EGCG)-induced cell survival in human epidermal keratinocytes. FASEB J 2003;17:1913–5. [19] Dou M, Han Y, Han Z, Chen X, Wang Y, Tan J, et al. Inhibitory effect of polypeptide from Chlamys farreri on UVA-induced apoptosis in human keratinocytes. Invest New Drugs 2004;22:391–8. [20] Schneider LA, Dissemond J, Brenneisen P, Hainzl A, Briviba K, Wlaschek M, et al. Adaptive cellular protection against UVA-1-induced lipid peroxidation in human dermal fibroblasts shows donor-to-donor variability and is glutathione dependent. Arch Dermatol Res 2006;297:324–8. [21] Itoh K, Tong KI, Yamamoto M. Molecular mechanism activating Nrf2-Keap1 pathway in regulation of adaptive response to electrophiles. Free Radic Biol Med 2004;36:1208–13. [22] Faraonio R, Vergara P, Di Marzo D, Pierantoni MG, Napolitano M, Russo T, et al. p53 suppresses the Nrf2-dependent transcription of antioxidant response genes. J Biol Chem 2006;281:39776–84. [23] Chen W, Sun Z, Wang XJ, Jiang T, Huang Z, Fang D, et al. Direct interaction between Nrf2 and p21(Cip1/WAF1) upregulates the Nrf2-mediated antioxidant response. Mol Cell 2009;34:663–73. [24] Liu GH, Qu J, Shen X. NF-kappaB/p65 antagonizes Nrf2-ARE pathway by depriving CBP from Nrf2 and facilitating recruitment of HDAC3 to MafK. Biochim Biophys Acta 2008;1783:713–27. [25] Ishii T, Warabi E, Yanagawa T. Novel roles of peroxiredoxins in inflammation, cancer and innate immunity. J Clin Biochem Nutr 2012;50:91–105.
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Peroxiredoxin I plays a protective role against UVA irradiation through reduction of oxidative stress Takaaki Ito a, Shintaro Kimura b, Kahori Seto a, Eiji Warabi c, Yasuhiro Kawachi d, Junichi Shoda e, Katsuhiko Tabuchi f, Kenji Yamagata g, Shogo Hasegawa g, Hiroki Bukawa g, Tetsuro Ishii c, Toru Yanagawa g,* a
Oral and Maxillofacial Surgery, Clinical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan Environmental Molecular Biology, Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan c Department of Environmental Molecular Biology, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan d Department of Dermatology, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan e Department of Medical Science, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan f Department of Neurophysiology, Faculty of Medicine, Shinshu University, Matsumoto, Nagano 390-8621, Japan g Department of Oral and Maxillofacial Surgery, Faculty of Medicine, University of Tsukuba, Ibaraki 305-8575, Japan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 September 2013 Received in revised form 3 December 2013 Accepted 4 December 2013
Background: Exposure of skin to long-wave UV radiation (UVA) increases the cellular levels of reactive oxygen species (ROS), which have been linked to apoptosis induction through the damage of lipids, proteins, and nucleic acids. Peroxiredoxin I (Prx I) is one of a family of antioxidant proteins that plays a protective role against oxidative damage; however the role of Prx I in UVA-induced damage remains to be clarified. Objective: Here we investigated the protective role of Prx I against UVA-induced changes using mouse embryonic fibroblasts (MEFs) derived from Prx I homozygous knockout (Prx I (/)) mice. Methods: Prx I (/) and wild-type (Prx I (+/+)) MEFs were subjected to UVA irradiation, and the resulting apoptosis was analyzed using flow cytometry, quantitative real-time PCR, and western blotting. Results: Prx I (/) MEFs showed enhanced sensitivity to UVA treatment, exhibiting increased apoptosis and ROS production compared to Prx I (+/+) MEFs. Consistent with the increase in apoptosis, p53 expression was significantly higher, while Bcl-2, Bcl-xL, and Nrf2 expressions were all lower in Prx I (/ ) versus (+/+) MEFs. The UVA-induced inflammatory response was upregulated in Prx I (/) MEFs, as indicated by increased expressions of IkB, TNFa, and IL-6. Evidence was presented indicating that Prx I impacts these pathways by modifying critical signaling intermediates including p53, IkB, and Nrf2. Conclusion: Our results indicate that Prx I plays a protective role against UVA-induced oxidative damage by controlling ROS accumulation. Both the UVA-induced apoptotic and inflammatory signals were found to be modulated by Prx I. ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Peroxiredoxin I UVA Apoptosis p53 ROS
1. Introduction The major environmental cause of skin damage is excessive ultraviolet (UV) radiation, which leads to acute reactions such as
erythema and sunburn, and over the long-term can result in premature skin aging and carcinoma. UV radiation in sunlight is composed of long-wave (UVA: 320–400 nm) and shorter wavelength (UVB: 280–320 nm) UV radiation. UVA is approximately 20-fold
Abbreviations: ROS, reactive oxygen species; UV, ultraviolet; PCR, polymerase chain reaction; Cys, cysteine; GCLM, glutamate-cysteine ligase modifier subunit; MEF, mouse embryo fibroblast; cisplatin, cis-diamminedichloroplatinum(II); Prx, Peroxiredoxin; Prx I, Peroxiredoxin I; MSP23, macrophage stress protein 23 kDa; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nrf2, NF-E2-related factor 2; Keap1, Kelch-like ECH-associated protein 1; HO-1, hemeoxygenase-1; DCFH-DA, 20 ,70 dichlorofluoresceindiacetate; RIPA buffer, Radioimmunoprecipitation assay buffer; PVDF, polyvinylidene difluoride; PI, propidium iodide; NFkB, nuclear factor-kappa B; Bcl2, B-cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra large; Bax, Bcl-2-associated X protein; PUMA, p53 up-regulated modulator of apoptosis; TNFa, tumor necrosis factor alpha; IL-6, Interleukin 6. * Corresponding author at: Oral and Maxillofacial Surgery, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan. Tel.: +81 29 853 3052; fax: +81 29 853 3052. E-mail address: [email protected] (T. Yanagawa). 0923-1811/$36.00 ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2013.12.002
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more abundant than UVB and penetrates the basal germinative layers [1]. Exposure of skin to UVA increases the cellular levels of reactive oxygen species (ROS), which have been linked to apoptosis through the damage of lipids, proteins, and nucleic acids [2]. A recent study indicated that UVA irradiation generates ROS, resulting in an altered intracellular redox balance and the translocation of p53 to the mitochondria [3]. Murine Prx I, first termed MSP23 (macrophage stress protein 23 kDa) was cloned by our group from peritoneal macrophages as an oxidative stress-inducible protein [4]. Previous studies established that Peroxiredoxins (Prxs) are an important family of antioxidants, protecting cells from oxidative damage by reducing peroxides such as H2O2, and scavenging radicals [5–7]. There are currently six Prx family members, which share a common reactive Cys residue in the N-terminal region. Prx I is the major cytosolic Prx and is widely expressed in various tissues [8]. Since H2O2 is an intracellular signaling molecule produced in response to cell surface receptor activation, the Prx peroxidase activity is reversibly and effectively inactivated to allow for transient H2O2 induction and the transmission of intracellular signals [9,10]. Recently, various signals associated with ROS have been reported, and the signaling modulatory roles of the Prx enzymes have been elucidated. A major cytotoxic effect of the cancer chemotherapeutic agent cis-diamminedichloroplatinum(II) (cisplatin) is cellular oxidative stress that results in acute renal failure. Studies using Prx I-deficient mice demonstrated that Prx I plays a protective role against cisplatin-induced apoptosis [11,12]. However, there have been no reports investigating the role of Prx I in UVA-induced damage. In the present study, we used mouse embryonic fibroblasts (MEFs) from Prx I-deficient mice to explore the role of Prx I in protecting cells from UVA-induced damage.
in 6-well plates at 1 105 cells per well and incubated overnight at 37 8C. Following treatment with various doses of UVA irradiation, MTT was added, and the cells were further incubated for 5 h at 37 8C. The absorbance at 540 nm was determined using a Varioskan plate reader (Thermo Fisher Scientific. Co., Yokohama, Japan). Cytotoxicity was expressed as the ratio of the UVAirradiated cell absorbance to that of untreated control cells. 2.4. Apoptosis analysis using flow cytometry UVA-induced apoptosis of Prx I (+/+) and Prx I (/) MEFs was assessed using an Annexin V-FLUOS Staining Kit (Roche Diagnostic. Co., Tokyo) followed by flow cytometry analysis. Cells were seeded at 1 105 cells per 35-mm dish, cultured overnight in 10% FBS/ DMEM, and then UVA irradiated at 15 J/cm2 and further cultured for 5 h. The washed and resuspended cells were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V antibody and propidium iodide (PI), according to the manufacturer’s instructions. The cells (2 104 per sample) were then analyzed using a flow cytometer (FACSCalibur; Becton Dickinson Co., Tokyo) equipped with a 488-nm argon laser and the CellQuest Pro software. 2.5. Detection of ROS generation and mitochondrial damage Prx I (/) and (+/+) MEFs were subjected to UVA radiation. Then, 20 mM 20 ,70 -dichlorofluoresceindiacetate (DCFH-DA) and 150 mM MitoTracker Deep Red were added to the culture medium and the cells were incubated for 30 min. The cells were then washed five times with culture medium, and fluorescent images were acquired with a confocal laser scanning microscope (TSC SP2, Leica Microsystems, Tokyo, Japan).
2. Materials and methods 2.6. Western blot analysis 2.1. Cell culture Prx I homozygous knockout (Prx I (/)) (OmniBank, Lexicon Pharmaceuticals, Inc.) and wild-type (Prx I (+/+)) mice were treated as described previously [8]. Primary MEFs were generated from 13.5-day Prx I (/) and Prx I (+/+) embryos as described previously [11]. Briefly, the brain and dark-red (internal) organs were dissected away from the embryos, and the remaining tissue was finely minced. The cells were dissociated using 0.25% trypsin, which was removed by centrifugation. The University of Tsukuba Animal Research Committee approved all the animal experiments (No. 11-171). The resulting MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing streptomycin (100 U/ml), penicillin (0.1 mg/ml), and heat-inactivated 10% FBS. The cells were cultured in a humidified atmosphere with 5% CO2 at 37 8C. All experiments with subcultures of MEFs were measured from three individual culture plates (n = 3). 2.2. UVA irradiation MEFs were washed with PBS, and the medium was replaced with phenol-red-free DMEM containing 5% FBS and 30 mM HEPES. Next, the cells were irradiated with UVA using an FL20SBLB lamp (Toshiba, Tokyo, Japan) with a peak emission frequency of 352 nm. Wavelengths below 320 nm were blocked by an ATG filter UV-35 (Asahi Technoglass, Japan). The dose of UVA irradiation was measured using a radiometer (UVR-305/365; Toshiba, Tokyo, Japan).
Whole-cell extracts were generated by lysing cells in RIPA buffer, followed by centrifugation at 12,000 g for 10 min, and collection of the supernatants. The samples were separated on a 12.5% polyacrylamide gel with SDS and then transferred onto PVDF membranes for 2 h at 80 V. The membranes were incubated overnight at 4 8C in blocking buffer (Tris buffered saline-Tween20 (TBS-T) containing 5% fat-free dried milk powder (Yukijirushi, Tokyo, Japan)). The membranes were then incubated with primary antibody diluted 1:1000 in fresh blocking buffer for 1 h at room temperature. p53, Bcl-2, and Bcl-xL antibodies were purchased from Cell Signaling Technology (Cell Signaling Technology Japan Co., Tokyo, Japan). The Prx I and Nrf2 polyclonal antibodies were described previously [8,13]. After treatment with peroxidaseconjugated anti-mouse IgG antibody (Sigma) or anti-rabbit IgG antibody (Jackson ImmunoResearch, West Grove, USA) for 1 h at room temperature, the labeled bands in washed blots were detected using the ECL Plus kit (GE Healthcare Bio-Sciences Co., Tokyo, Japan). The membranes were exposed to Fuji Medical X-ray Film (Fuji Photo Film Co., Ltd., Tokyo, Japan) at room temperature. 2.7. Quantitative real-time PCR analysis Quantitative real-time PCR was carried out as described previously [2,14]. The mRNA expression levels were normalized to that of GAPDH. 2.8. Statistical analysis
2.3. MTT assay for cell viability Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded
All data are presented as mean SD. Differences among data were determined using Student’s t-test (StatView, version 5.0, Abacus Concepts, Berkeley, USA).
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3. Results 3.1. Enhanced UVA sensitivity in Prx I-deficient MEFs Prx I (+/+) and Prx I (/) MEFs were irradiated using various doses of UVA, and cell survival was evaluated using the MTT assay. The resulting cell viabilities are shown in Fig. 1A. Prx I (/) MEFs were significantly more sensitive to UVA treatment at all doses tested (10 J/cm2 (P < 0.05), 12.5 J/cm2 (P < 0.01), and 15 J/cm2 (P < 0.001)). Next, we analyzed the UVA-treated MEFs by staining with an Annexin V antibody and PI (propidium iodide) followed by flow cytometry to determine whether the increased UVA sensitivity of the Prx I-deficient MEFs was a result of apoptosis. A representative scatterplot of Prx I (/) and (+/+) MEFs is shown in Fig. 1B. The percentage of cells in the upper- and lower-right quadrants (late and early apoptosis) was defined as the apoptotic index. The analysis indicated that UVA-irradiated Prx I (/) MEFs contained an increased percentage of Annexin V positive cells compared to identically treated Prx I (+/+) MEFs. Fig. 1C shows the apoptotic indexes of the two cell types. After treatment with 15 J/cm2 UVA there was a significant difference between the apoptotic index of Prx I (/) cells (85.6%) and Prx I (+/+) cells (16.7%) (P < 0.001), suggesting that Prx I (/) cells are more susceptible to UVAinduced apoptosis. 3.2. UVA-induced apoptosis in Prx I-deficient MEFs is mediated by p53 To explore the mechanistic basis for the increased apoptosis seen in Prx I (/) MEFs, the cells were further analyzed by treating with UVA followed by immunoblotting with antibodies to p53, Bcl-2, and Bcl-xL (Fig. 2A and B). The expression of p53 was induced in both Prx I (/) and (+/+) MEFs exposed to UVA irradiation, and the basal levels of p53 were significantly higher in Prx I (/) versus (+/+) MEFs (Fig. 2A and B). In contrast, the Bcl-2 and Bcl-xL expressions were significantly reduced in the UVAtreated Prx I (/) MEFs in comparison with UVA-treated Prx I (+/ +) MEFs. Next, we examined the expression of p53-regulated genes. Quantitative real-time PCR revealed that p21, Noxa, PUMA, and Bax mRNA expression levels were all increased in the Prx I (/) versus (+/+) MEFs (Fig. 2C). These results indicate that the p53dependent apoptotic signaling pathway was altered in Prx I (/) MEFs. 3.3. Regulation of ROS accumulation in Prx I-deficient MEFs To compare the accumulation of ROS in Prx I (+/+) and Prx I (/ ) MEFs, cells were treated with DCFH-DA to visualize the ROS production following UVA irradiation. As shown in Fig. 3A, strong fluorescent signals derived from activated DCFH-DA were detected in the Prx I (/) MEFs exposed to 5 J/cm2 UVA. In contrast, DCFHDA signals were barely detectable in the UVA-treated Prx I (+/+) MEFs or in untreated cells. To quantify the signals, the intracellular ROS generation was measured by flow cytometry. This analysis showed that the ROS production in Prx I (/) MEFs was significantly elevated when compared to Prx I (+/+) MEFS (P < 0.01) (Fig. 3B). 3.4. Altered UVA-induced Nrf2 regulation in PrxI-deficient MEFs UVA irradiation induces the nuclear accumulation of the transcription factor Nrf2, which plays a critical role in protecting dermal cells from UVA-induced apoptosis [15]. Furthermore, Nrf2 regulates the gene expression of various electrophile- and oxidative stress-inducible proteins, including Prx I [13]. Thus,
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we investigated the effects of UVA irradiation on the expression of Nrf2 and Nrf2-regulated oxidative stress-inducible proteins in Prx I (/) and (+/+) MEFs. The expression levels of Nrf2, Prx I, and hemeoxygenase 1 (HO-1), a representative downstream gene product of Nrf2, are shown in Fig. 4A and B. In the Prx I (+/+) MEFs all three proteins, Nrf2, Prx I, and HO-1 were significantly upregulated by UVA irradiation. However, in Prx I (/) MEFs, the UVA-inducible expression of both Nrf2 and HO-1 was significantly lower. As expected, Prx I protein was not detected in the Prx I (/) cells. These results showed UVA-inducible expression of oxidative stress-inducible proteins was reduced in Prx I (/) MEFs. Next, we used quantitative real-time PCR to analyze the mRNA expression levels of both HO-1 and glutamate-cysteine ligase modifier subunit (GCLM), another Nrf2-regulated gene. HO-1 and GCLM genes are located downstream of Nrf2 binding element, and their up-regulation means that transcriptional activity of Nrf2 was enhanced. UVA irradiation significantly induced both HO-1 and GCLM mRNA expression in Prx I (+/+) and (/) MEFs; however, the degree of GCLM up-regulation was significantly smaller in the Prx I (/) than the Prx I (+/+) MEFs (Fig. 4C). These findings suggest that Nrf2-regulated gene expression was reduced in the Prx I-deficient MEFs. 3.5. Elevated UVA-induced inflammatory response in Prx I-deficient MEFs Since it was previously reported that Nrf2 signaling can be transcriptionally repressed by NFkB [16], we next focused on the involvement of NFkB. To examine NFkB signaling following exposure to UVA irradiation, we first examined the protein levels of NFkB’s associated inhibitor protein IkB, by western blotting. As shown in Fig. 5A and B, there was a strong basal expression of IkB in Prx I (+/+) MEFs that was significantly down-regulated following UVA treatment. In contrast, in the Prx I (/) MEFs, IkB protein expression was barely detectable in the control or UV-treated cells. Quantitative real time PCR analysis revealed that UVA irradiation induced TNFa and IL-6 mRNA expression in both Prx I (/) and (+/+) MEFs; however, the induced levels of both cytokines were significantly elevated in the Prx-I (/) cells (Fig. 5C). These findings indicate that Prx I deficiency resulted in reduced IkB expression, which led to an increased inflammatory response to UVA treatment. 4. Discussion In the current study, we found that Prx I (/) MEFs were significantly more sensitive to UVA than Prx I (+/+) MEFs, resulting in increased induction of apoptosis. Since previous reports showed that UVA-induced apoptosis was caused by extensive oxidative damage leading to the p53-regulated mitochondrial release of cytochrome c and caspase activation [3], we investigated the role of p53 in our system. We examined the expression of p53 and p53regulated pro-apoptotic genes including Bax, Noxa, PUMA, and p21 (a cell cycle regulator of G1), as well as the anti-apoptotic proteins Bcl-2 and Bcl-xL. We observed enhanced expressions of p53 and the four p53-regulated genes following the UVA treatment of Prx I (/) MEFs. In contrast the expression of Bcl-2 and Bcl-xL was found to be suppressed in UVA-treated Prx I (/) MEFs. Taken together, these data suggest that the increased apoptosis observed in Prx I (/) MEFs was mediated by p53 signaling. We hypothesized that the UVA-induced apoptosis in Prx I (/) MEFs was caused by excessive oxidative stress. To test, this we examined the ROS levels in Prx I (/) and (+/+) MEFs using DCFHDA. Following irradiation with 5 J/cm2 UVA, ROS were abundantly detected in the Prx I (/) MEFs; in contrast, ROS were not detected in Prx I (+/+) MEFs treated with the same dose of
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Fig. 1. Enhanced UVA sensitivity in Prx I-deficient MEFs. (A) MTT cell viability analysis of Prx I homozygous knockout (Prx I (/)) and wild-type (Prx I (+/+)) MEFs irradiated with various doses of UVA. Prx I (/) MEFs were more sensitive to UVA irradiation at all doses tested (10, 12.5, and 15 J/cm2) (*P < 0.05, **P < 0.01, ***P < 0.001) (n = 3). (B) Flow cytometric analysis of Prx I (+/+) and Prx I (/) MEFs stained with propidium iodide and fluorescently labeled Annexin V antibodies. Apoptotic cells were defined as the Annexin V-positive cells in the upper- and lower-right quadrants. Vertical axis: propidium iodide (PI); horizontal axis: Annexin V. (C) Quantification of flow cytometric analysis. The percentage of cells in the upper- and lower-right quadrants was defined as the apoptotic index. After 15 J/cm2 UVA irradiation the apoptotic index of Prx I (/) MEFs was significantly increased in comparison to the apoptotic index of Prx I (+/+) cells. Values are the means SD from three independent experiments (***P < 0.001) (n = 3).
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Fig. 2. Apoptotic signaling in Prx (+/+) and Prx (/) MEFs. (A) Western blot analysis of p53, Bcl-2, and Bcl-xL in Prx I (+/+) and (/) MEFs before and after 5 J/cm2 UVA irradiation. (B) Quantification of p53, Bcl-2, and Bcl-xL western blots. p53 expression was increased, while Bcl-2 and Bcl-xL expression levels were significantly reduced in the Prx I (/) MEFs (*P < 0.05, **P < 0.01, ***P < 0.001) (n = 3). (C) Quantitative real time PCR analysis of p21, Noxa, PUMA, and Bax mRNAs. The expression levels were normalized to that of GAPDH. All fourp53-regulated mRNAs were significantly upregulated in the Prx I (/) MEFs following UVA irradiation (*P < 0.05, **P < 0.01, ***P < 0.001) (n = 3).
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Fig. 3. ROS generation induced by UVA in Prx I (/) and Prx I (+/+) MEFs. (A) Analysis of ROS production in MEFs. Cells were exposed to 5 J/cm2 UVA irradiation and then stained with DCFH-DA. Fluorescently labeled ROS signals were visualized with confocal laser scanning microscopy. (B) Quantification of ROS signals by flow cytometric analysis. UVA-induced production of ROS was significantly greater in Prx I (/) MEFs compared to Prx I (+/+) MEFs (***P < 0.001) (n = 3).
radiation. This result indicated that the antioxidant capacity was reduced in the Prx (/) MEFs, resulting in the ineffective elimination of ROS and the accumulation of excessive oxidative stress, leading to the induction of apoptosis. These findings are consistent with previous reports that Prx I is a member of a ubiquitous family of antioxidant enzymes that regulate the levels of cytokine-induced peroxides [17]. The role of ROS in UVA-induced apoptosis was also observed in several studies using various antioxidants [18–20], and those reports also support our hypothesis. Since the Nrf2-Keap1 system is a central regulator of the cellular antioxidant response [21], we examined the UVA induction of Nrf2 and Nrf2-regulated genes. The expressions of Nrf2 and its downstream genes encoding HO-1 and GCLM were found to be reduced in the Prx I (/) versus (+/+) MEFs. This was
an unexpected finding, given a previous report showing that ROS counteract the degradation of Nrf2 and provoke its activation, resulting in the expression of a battery of genes encoding antioxidative stress enzymes and proteins [21]. However, another report showed that p53 and Nrf2 interact, and that p53 suppresses the transcription of Nrf2 target genes [22]. Thus, in Prx I (/) cells the upregulated p53 may interact with Nrf2 leading to the suppression of Nrf2’s target genes. In contrast, a recent report showed an interaction between the p53-regulated p21 and Nrf2. p21 was shown to interact with the DLG and ETGE motifs within Nrf2, thereby attenuating the Keap1-based ubiquitination and subsequent proteosomal degradation of Nrf2. [23]. Thus, there may be some mechanism like a cytoprotective feedback loop among p53, p21, and Nrf2 in response to oxidative stress. Our results show
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Fig. 4. UVA induction of Nrf2 and Nrf2-regulated genes. (A) Western blot analysis of Nrf2, Prx I, and HO-1 in Prx I (+/+) and (/) MEFs before and after UVA irradiation. (B) Quantification of Nrf2, HO-1, and Prx I western blots, indicating the UVA-induced upregulation of Nrf-2 and HO-1 in Prx (/) and (+/+) MEFs. Prx I was upregulated in UVAtreated Prx I (+/+) MEFs and as expected, was not detectable in Prx I (/) MEFs. (C) Quantitative real time PCR of Nrf2-regulated genes, HO-1 and GCLM. The mRNA expression levels were normalized to that of GAPDH (*P < 0.05, **P < 0.01, **P < 0.001) (n = 3).
that in the absence of Prx I, the p21 levels were upregulated, while Nrf2 and the Nrf2 signaling were suppressed. Thus, the absence of Prx I may alter the cytoprotective feedback loop, resulting in the induction of apoptosis following UVA exposure as one hypothesis.
However, there are many other target genes that have interaction with Nrf2, then further investigation is necessary. Another hypothesis that may explain this paradox involves the NFkB signal. In a recent report, NFkB was shown to directly repress
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Fig. 5. Enhanced UVA-induced inflammatory responses in Prx (/) MEFs. A. Western blot analysis of IkB in Prx I (/) and (+/+) MEFs before and after treatment with 5 J/cm2 UVA irradiation. B. Quantification of IkB western blot. IkB was strongly expressed in Prx I (+/+) MEFs and was suppressed by UVA irradiation. IkB expression was significantly lower in Prx I (/) MEFs and remained low following UVA treatment (*P < 0.05) (n = 3). (C) Quantitative real time PCR analysis of TNFa and IL-6 mRNAs. UVA-inducible TNFa and IL-6 mRNA levels were significantly higher in the Prx I (/) versus Prx I (+/+) MEFs (*P < 0.05, **P < 0.01, **P < 0.001) (n = 3).
Nrf2 signaling at the transcriptional level. NFkB was found to compete with Nrf2 for the co-activator CREB binding protein (CBP) and to recruit histone deacetylase 3 (HDAC3), resulting in local hypoacetylation and the repression of Nrf2 signaling [24]. The NFkB family includes the Rel family proteins p65, c-Rel, Rel B, p50, and p52. Various stimuli such as pro-inflammatory signals, and genotoxic or oxidative stress lead to activation of the IkB kinase signalosome, resulting in IkB degradation and movement of the NFkB dimer to the nucleus, where it induces the transcription of target genes. In the current study, IkB protein levels were significantly reduced following UVA irradiation in Prx I (+/+) MEFs. However, in the Prx I (/) MEFs, IkB protein was barely detectable both before and after UV irradiation. Thus, some
stimulus, most likely oxidative stress, evoked NFkB signaling in the basal state of Prx I (/) MEFs. Following UVA irradiation, the expression of NFkB-regulated genes, namely TNFa and IL-6, was enhanced in the Prx I (/) MEFs. Thus, the Prx I (/) MEFs may be predisposed to developing an elevated inflammatory response, or affected by other mechanisms associated with inflammation caused by Prx I deficiency. In this report, we demonstrated that Prx I plays a protective role in UVA-induced apoptosis through controlling the ROS accumulation. However, the mechanistic basis of these observations is not fully understood. Prx I protects cells and tissues from oxidative damage through a number of mechanisms besides its enzymatic peroxidase activity. Prx I can also suppress oxidative
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stress-induced cell death through direct and indirect interactions with different types of molecules that play key roles in regulating cell death and/or apoptosis, depending on the cell type and stimulus. For instance, Prx I has been shown to suppress ASK1-JNK signaling, and to inhibit both p66shc and c-Abl activation. Furthermore, Prx I associates with some transcription factors, such as c-Myc, NFkB, and the androgen receptor, thereby modulating gene expression [25]. Thus, the specific mechanism by which Prx I functions in suppressing the apoptotic signaling caused by UVA irradiation is unclear. Further studies are necessary to define the exact role of Prx I in UVA signaling pathways. Acknowledgements The first three authors (T.I., S.K. and K.S.) contributed to this work equally. This work was supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research (25670843). References [1] Bruls WA, Slaper H, van der Leun JC, Berrens L. Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem Photobiol 1984;40:485–94. [2] Kimura S, Warabi E, Yanagawa T, Ma D, Itoh K, Ishii Y, et al. Essential role of Nrf2 in keratinocyte protection from UVA by quercetin. Biochem Biophys Res Commun 2009;387:109–14. [3] Waster PK, Ollinger KM. Redox-dependent translocation of p53 to mitochondria or nucleus in human melanocytes after UVA- and UVB-induced apoptosis. J Invest Dermatol 2009;129:1769–81. [4] Ishii T, Yamada M, Sato H, Matsue M, Taketani S, Nakayama K, et al. Cloning and characterization of a 23-kDa stress-induced mouse peritoneal macrophage protein. J Biol Chem 1993;268:18633–36. [5] Rhee SG, Chae HZ, Kim K. Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic Biol Med 2005;38:1543–52. [6] Flohe L, Harris JR. Introduction. History of the peroxiredoxins and topical perspectives. Subcell Biochem 2007;44:1–25. [7] Fujii J, Ikeda Y. Advances in our understanding of peroxiredoxin, a multifunctional, mammalian redox protein. Redox Rep 2002;7:123–30. [8] Uwayama J, Hirayama A, Yanagawa T, Warabi E, Sugimoto R, Itoh K, et al. Tissue Prx I in the protection against Fe-NTA and the reduction of nitroxyl radicals. Biochem Biophys Res Commun 2006;339:226–31.
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