Quercetin Induces the Expression of Peroxiredoxins 3 and 5 Via the Nrf2/NRF1 Transcription Pathway

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Glaucoma Quercetin Induces the Expression of Peroxiredoxins 3 and 5 via the Nrf2/NRF1 Transcription Pathway

Naoya Miyamoto,1,2 Hiroto Izumi,1 Rie Miyamoto,2 Hiroyuki Kondo,2 Akihiko Tawara,2 Yasuyuki Sasaguri,3 and Kimitoshi Kohno1

PURPOSE. The flavonoids have potent antioxidant and free-rad- lavonoids such as quercetin (3,5,7,3Ј,4Ј-pentahydroxy fla- ical scavenging properties and are beneficial in the prevention Fvone) can protect cells from oxidative stress.1–4 Querce- and treatment of ocular diseases including glaucoma. The au- tin—present in fruit, vegetables, and many other dietary sourc- thors have previously reported that antiglaucoma agents could es—is one of the most widely distributed flavonoids.5 It has transcriptionally activate the antioxidant protein peroxire- been shown that certain flavonoids can induce antioxidant doxin (PRDX)2. The purpose of this study was to investigate responsive element-dependent gene expression through the 6 whether quercetin can activate transcription factors and in- activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). duce the expression of the PRDX family. Oxidative stress plays an important role in the pathogenesis of multiple ocular diseases, including glaucoma.7 METHODS. To demonstrate whether quercetin can transcrip- Glaucoma is a major cause of irreversible blindness world- tionally induce the expression of the PRDX family, trabecular wide and is characterized by cupping of the optic nerve head meshwork cells were treated with quercetin, and PRDX ex- and irreversible loss of retinal ganglion cells.8 Elevated intraoc- pression and transcription factors were both investigated by ular pressure (IOP) caused by a reduction in aqueous outflow Western blot analysis, reporter assays, and siRNA strategies. is a major risk factor in the development of glaucoma9 and the Subsequently, cellular sensitivity to oxidative stress was deter- progression of glaucomatous damage to the optic nerve.10–12 mined. The trabecular meshwork (TM) is a reticulated tissue at the RESULTS. Expression of the PRDX3 and PRDX5 genes was iridocorneal junction that makes intimate contact in the juxta- induced by quercetin in a time- and dose-dependent manner. canalicular region with the canal of Schlemm for aqueous 13 NRF1 transactivates the promoter activity of both PRDX3 humor filtration. Oxidative stress is reported to trigger de- and PRDX5 but not PRDX2 and PRDX4. Quercetin can also generation in the human TM and its endothelial cell compo- induce the expression of Nrf2 and NRF1 but not of Ets1, nents, subsequently leading to an increase in IOP and glau- Ets2, or Foxo3a. Knockdown of NRF1 expression signifi- coma. Increasing evidence indicates that reactive oxygen species (ROS) play a key role in the pathogenesis of glau- cantly reduced the expression of both PRDX3 and PRDX5. 14–17 Reporter assays showed that NRF1 transactivated the pro- coma. moter activity of both PRDX3 and PRDX5 and that the Peroxiredoxins (PRDXs) are a family of enzymes that cata- lyze the reduction of hydrogen peroxide.18–22 There are five downregulation of NRF1 with siRNA repressed the pro- 2-Cys types that contain two conserved cysteine residues. moter activity of both PRDX3 and PRDX5. Furthermore, the These PRDXs are expressed in a wide variety of cell types. downregulation of NRF1, PRDX3, and PRDX5 renders tra- However, the precise mechanisms controlling PRDX expres- becular meshwork cells sensitive to hydrogen peroxide. sion are not well understood. We have previously shown that Finally, NRF1 activation by quercetin was completely abol- oxidative stress can induce PRDX1 and PRDX5 through acti- ished by the knockdown of Nrf2. vation of the Ets1 transcription factor.23 Furthermore, we have CONCLUSIONS. Quercetin upregulates the antioxidant peroxire- reported that antiglaucoma agents transcriptionally upregulate doxins through the activation of the Nrf2/NRF1 transcription the PRDX2 gene through the activation of Foxo3a.24 Thus, pathway and protects against oxidative stress-induced ocular several transcription factors can regulate each PRDX gene. disease. (Invest Ophthalmol Vis Sci. 2011;52:1055–1063) DOI: Here, we investigated whether quercetin induces gene expres- 10.1167/iovs.10-5777 sion of PRDX3 and PRDX5 through the Nrf2/NRF1 transcription pathway.

From the Departments of 1Molecular Biology, 2Ophthalmology, EXPERIMENTAL PROCEDURES and 3Pathology and Cell Biology, School of Medicine, University of Occupational and Environmental Health, Fukuoka, Japan. Cell Culture Supported in part by Grants-in-Aid for Scientiﬁc Research from the The immortalized TM cell line, NTM5, derived from a normal trabec- Ministry for Education, Culture, Sports, Science and Technology of ular meshwork, was kindly provided by Abott F. Clark (Glaucoma Japan (17016075), UOEH Grant for Advanced Research, and The Ve- hicle Racing Commemorative Foundation. Research, Alcon Research, Ltd., Fort Worth, TX) and was cultured in Submitted for publication April 26, 2010; revised September 7, Dulbecco’s modiﬁed Eagle’s medium (Nissui Seiyaku Co., Tokyo, Ja- 2010; accepted September 28, 2010. pan).24,25 The primary TM cell (HTMC) was purchased from Sciencell Disclosure: N. Miyamoto, None; H. Izumi, None; R. Miyamoto, Research Laboratories (San Diego, CA) None; H. Kondo, None; A. Tawara, None; Y. Sasaguri, None; K. Kohno, None Antibodies and Drugs Corresponding author: Kimitoshi Kohno, Department of Molecu- lar Biology, School of Medicine, University of Occupational and Envi- Antibodies against FKHRL1 (Foxo3a) (sc-9812), Ets1 (sc-111), Ets2 ronmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, (sc-351), Nrf2 (sc-30915), PCNA (sc-56), PRDX2 (sc-23967), and Japan; [email protected]. PRDX4 (sc-23974) were purchased from Santa Cruz Biotechnology

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(Santa Cruz, CA). Anti–␤-actin antibody (AC-15) was purchased from TGGCAAAGGCTAGACGCACGG-3Ј for PRDX2-Luc; 5Ј-AGATCTTAGCT- Sigma. Generation of antibodies against PRDX1 and PRDX5,23 NRF1,26 TATTAACGGACTAAAAC-3Ј and 5Ј-AAGCTTCAGTGCACTCGGGCGC- and mitochondrial transcription factor A (mtTFA)27 has been described CACGG-3Ј for PRDX3-Luc; 5Ј-AGATCTGTGAGGGGCTTGTGTGCAG-3Ј previously. The anti–PRDX3 antibody was a kind gift from Hiroki Nanri and 5Ј-AAGCTTCACGCGAGCGCAGAAACACG-3Ј for PRDX4-Luc; and (Seinan Jogakuin University, Kyushu, Japan).28 Quercetin dihydrate 5Ј-AGATCTAAGATGCAAATCATATGC-3Ј and 5Ј-AAGCTTCCCACGGC- was purchased from Sigma-Aldrich Co. (St. Louis, MO). Drug concen- CACTTCCACTCC-3Ј for PRDX5-Luc. trations in this study corresponded with those used in clinical practice. Plasmid Construction Knockdown Analysis Using Small Interfering RNAs (siRNAs) To obtain full-length cDNAs for human NRF1, PCR was carried out on a cDNA library (SuperScript; Invitrogen Life Technologies, Carlsbad, The following double-stranded RNA 25-bp oligonucleotides were CA) using the following primer pairs (underlining indicates the start commercially generated (Invitrogen): PRDX3 small interfering RNA codon and stop codon): 5Ј-ATGGAGGAACACGGAGTGACCC-3Ј and (siRNA), 5Ј-UUUACCUUCUGAAAGUACUCUUUGG-3Ј (sense) and 5Ј- 5Ј-TCACTGTTCCAATGTCACCACCTCC-3Ј. The resultant PCR product CCAAAGAGUACUUUCAGAAGGUAAA-3Ј (antisense); PRDX5 was cloned (pGEM-T Easy Vector; Promega, Madison, WI). To con- siRNA, 5Ј-AGAACCUCUUGAGACGUCGAUUCCC-3Ј (sense) and 5Ј- struct a plasmid expressing Flag-tagged NRF1, N-terminal Flag-tagged GGGAAUCGACGUCUCAAGAGGUUCU-3Ј (antisense); NRF1#1 NRF1 cDNA was ligated into the pcDNA3 vector (Invitrogen). The siRNA, 5Ј-AUUAGACUCAAAUACAUGAGGCCGU-3Ј (sense) and 5Ј- luciferase (Luc) constructs PRDX2-Luc (Ϫ402 to ϩ68), PRDX3-Luc ACGGCCUCAUGUAUUUGAGUCUAAU-3Ј (antisense); NRF1#2 (Ϫ357 to ϩ42), PRDX4-Luc (Ϫ306 to ϩ36), and PRDX5-Luc (Ϫ314 to siRNA, 5Ј-AUCUGAGUCAUCGUAAGAGGUGUCC-3Ј (sense) and 5Ј- ϩ113) have been described previously.23 The following primer pairs GGACACCUCUUACGAUGACUCAGAU-3Ј (antisense); Nrf2 siRNA, were used: 5Ј-AGATCTTAGATGCTGCAGCCTCAGC-3Јand 5Ј-AAGCT- 5Ј-AAUCACUGAGGCCAAGUAGUGUGUC-3Ј (sense) and 5Ј-GACA-

FIGURE 1. (A) Effect of quercetin on PRDX expression. NTM5 cells were incubated with 1 ␮M quercetin for the times indicated. Whole cell lysates (50 ␮g) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of ␤-actin is shown as a loading control. Rela- tive intensity is shown under each blot. (B) NTM5 cells were cultured for 12 hours in the control medium or medium containing the indicated concentrations of quercetin. Whole cell lysates (50 ␮g) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of ␤-actin is shown as a loading control. Rela- tive intensity is shown under each blot. (C) Primary HTMCs were incubated with 1 ␮M quercetin for the times indicated. Whole cell lysates (50 ␮g) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of ␤-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.

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CACUACUUGGCCUCAGUGAUU-3Ј (antisense). siRNA transfections dilution of anti-NRF1, 1:5000 dilution of anti-mtTFA, 1:5000 dilution of were performed as described previously.29,30 Briefly, 250 pmol of anti-PCNA, and 1:20,000 dilution of anti–␤-actin. Membranes were the indicated siRNA or control synthetic RNA (Stealth RNAi; Invit- then incubated for 40 minutes at room temperature with a peroxidase- rogen) was transfected into 1 ϫ 106 NTM5 cells; 1.5 ϫ 105 cells conjugated secondary antibody and were visualized using an enhanced were used for luciferase assays, and 2.5 ϫ 103 cells were used for chemiluminescence kit (GE Healthcare Bio-Science, Uppsala, Sweden), the WST-8 assay, as described. The remaining cells were subjected and membranes were exposed to Kodak film (X-OMAT; Kodak, Roch- to Western blot analysis after 72-hour culture, as described. ester, NY). For the correlation assay, the intensity of each signal was quantified using ImageJ software (developed by Wayne Rasband, Na- Western Blot Analysis tional Institutes of Health, Bethesda, MD; available at http://rsb.info- The preparation of whole cell lysates and whole nuclear lysates has .nih.gov/ij/index.html). been described previously.29,30 The indicated amounts of whole cell lysate or whole nuclear lysate were separated by sodium dodecyl Luciferase Assay sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred Transient transfection and a luciferase assay were performed as de- to polyvinylidene difluoride microporous membranes (Millipore, Bil- scribed previously.29,30 Briefly, 1 ϫ 105 NTM5 cells per well were lerica, MA) using a semidry blotter. The blotted membranes were seeded onto 12-well plates. The following day, cells were cotrans- treated with 5% (wt/vol) skimmed milk in 10 mM Tris, 150 mM NaCl, fected with the indicated amount of reporter plasmid and expression and 0.2% (vol/vol) Tween 20 and were incubated for 1 hour at room plasmid using reagent (Superfect; Qiagen, Valencia, CA). For the lu- temperature with primary antibody. The following antibodies and ciferase assay using siRNA, siRNA-pretransfected 1.5 ϫ 105 NTM5 cells, dilutions were used: 1:500 dilution of anti-Nrf2, 1:5000 dilution of described above, were transfected with the indicated amounts of anti-PRDX2, 1:5000 dilution of anti-PRDX3, 1:1000 dilution of anti- reporter plasmid at intervals of 12 hours. Forty-eight hours after trans- PRDX4, 1:1000 dilution of anti-PRDX5, 1:1000 dilution of anti-Ets1, fection of reporter plasmid, cells were lysed with reporter lysis buffer 1:1000 dilution of anti-Ets2, 1:5000 dilution of anti-Foxo3a, 1:5000 (Promega). For quercetin treatment, 36 hours after transfection cells

FIGURE 2. (A) Schematic representa- tions of the PRDX luciferase constructs PRDX2-Luc, PRDX3-Luc, PRDX4-Luc, and PRDX5-Luc, with their CpG is- lands and transcription factor binding sites. (B) Transcriptional activity of the PRDX2-5 genes in response to quercetin treatment. The indicated reporter plasmids were transiently transfected into NTM5 cells. The following day, the cells were incubated for 48 hours in fresh medium or in medium containing 1 ␮M quercetin. These results shown are normalized to protein concentrations measured using the Bradford method and are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct under normal conditions corresponds to 1. Bars represent the SD.

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were further incubated under normal conditions or in the presence of Quercetin Enhances the Promoter Activity of 1 ␮M quercetin for 6 hours. Luciferase activity was detected using a Both the PRDX3 and PRDX5 Genes luciferase assay system (PicaGene; Toyo-Inki, Tokyo, Japan. The light intensity was measured using a luminometer (Luminescencer JNII We next investigated whether quercetin can activate the pro- RAB-2300; Atto, Tokyo, Japan). The results shown are normalized to moter activity of the PRDX genes using luciferase reporter the protein concentration measured using the Bradford method and assays. A schematic representation of the PRDX gene promoter 23 are representative of at least three independent experiments. is shown in Figure 2A. Promoter activity of both the PRDX3 and PRDX5 genes was signiﬁcantly enhanced approximately twofold to threefold by the quercetin treatment (Fig. 2B). We Cytotoxicity Analysis did a careful survey of the nucleotide sequences of the pro- The water-soluble tetrazolium salt (WST-8) assay was performed as moter of four PRDX genes and found several transcription described previously.24 Brieﬂy, 2.5 ϫ 103 NTM5 cells per well, trans- factor binding sites, as shown in Figure 2A. The NRF1 binding fected with siRNA as described, were seeded onto 96-well plates. The sites are found in the promoters of both PRDX3 and PRDX5 but following day, to induce oxidative stress, cells were incubated with the not in those of PRDX2 and PRDX4.

indicated concentration of H2O2 in serum-free medium for 40 minutes. Then the medium was changed to the normal culture medium. After 72 Quercetin Induces Expression of the hours, surviving cells were stained (TetraColor One; Seikagaku Corpo- Transcription Factor NRF1 ration, Tokyo, Japan) for 90 minutes at 37°C. Absorbance was then measured at 450 nm. Next, we examined whether quercetin can induce the expression of a transcription factor that regulates PRDX gene expres- Statistical Analysis sion. We initially investigated the cellular expression of NRF1 and found that the expression of NRF1 is localized primarily in Pearson correlation was used for statistical analysis, and signiﬁcance nuclei. As shown in Figure 3, nuclear NRF1 was markedly was set at the 5% level. increased after quercetin treatment in a time- and concentration-dependent manner. In contrast, there was no increase in the expression of the three transcription factors Ets1, Ets2, and Foxo3a. RESULTS

Quercetin Induces PRDX Expression in TM Cells NRF1 Regulates the Expression of Both PRDX3 and PRDX5 We have previously shown that the PRDX1 gene is not expressed in immortalized human TM NTM5 cells.24 To examine To confirm the NRF1-dependent expression of both PRDX3 whether quercetin can activate PRDX family gene expression, and PRDX5, we used specific siRNA for NRF1. Two indepen- NTM5 cells were treated with quercetin. As shown in Figure 1A, dent siRNAs (#1 and #2) for NRF1 could effectively downregu- both PRDX3 and PRDX5 were induced by 1 ␮M quercetin in a late NRF1 expression. As shown in Figure 4A, protein expres- time-dependent manner. We also found that both PRDX3 and sion of both PRDX3 and PRDX5 was significantly reduced by PRDX5 were induced by quercetin treatment in a dose-depen- the transfection of two siRNAs for NRF1. Furthermore, we dent manner (Fig. 1B). We next investigated the effects of performed cotransfection experiments using the NRF1 expres- quercetin on the expression of the peroxiredoxin family in sion plasmid with PRDX reporter plasmids. The reporter assays primary HTMCs. Although PRDX1 expression could not de- showed that NRF1 transactivated the promoter activity of both tected in immortalized TM cells,24 HTMCs express PRDX1.31 the PRDX3 and PRDX5 genes (Fig. 4B). On the other hand, the We again observed that the expression of both PRDX3 and promoter activity of both the PRDX3 and PRDX5 genes was PRDX5 was induced by the treatment of HTMCs with querce- significantly downregulated by NRF1-specific siRNA transfec- tin (Fig. 1C). tion (Fig. 4C).

FIGURE 3. (A) Quercetin treatment increases NRF1 but not Ets1, Ets2, or Foxo3a expression. NTM5 cells were incubated with 1 ␮M quercetin for the times indicated. Whole nuclear lysates (100 ␮g) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were for 12 hours cultured in the control medium or in medium containing the indicated concentrations of quercetin. Whole nuclear lysates (100 ␮g) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblot- ting of PCNA is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.

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FIGURE 4. (A) NRF1 regulates PRDX3 and PRDX5 gene expression. Control siRNA (100 pmol) or NRF1#1 and #2 siRNA (100 pmol) were transfected into NTM5 cells. Whole nuclear lysates (100 ␮g for NRF1) and whole cell lysates (50 ␮g for PRDXs) were subjected to SDS- PAGE. The transferred membrane was blotted with the indicated antibodies. Immunoblotting of ␤-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue. (B) NRF1 transactivates the promoter activity of the PRDX3 and PRDX5 genes. The indicated amount of NRF1 expression plasmid was transiently cotransfected with the indicated reporter plasmids into NTM5 cells. pGL3-P.V indicates the pGL3 promoter vector in which the luciferase gene is driven by the SV-40 promoter. Results were normalized to protein concentrations measured using the Bradford method. All values are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct with transfection of the Flag vector corresponds to 1. Bars represent the SD. (C) Knockdown of NRF1 downregulates the promoter activity of the PRDX3 and PRDX5 genes. NTM5 cells were transiently transfected with the indicated amounts of control siRNA or NRF1 siRNA, followed by transfection with 0.5 ␮g of the indicated reporter plasmids at intervals of 12 hours. The results shown are normalized to protein concentrations measured using the Bradford method and are representative of at least three independent experiments. The luciferase activity of each PRDX-Luc construct with transfection of control siRNA corresponds to 1. Bars represent the SD.

Quercetin Induces NRF1 Expression through dependent manner (Fig. 5A). Furthermore, we investigated Nrf2 Activation whether the Nrf2 transcription factor can regulate the ex- It has been shown that Nrf2 regulates NRF1 expression.32 pression of NRF1 under quercetin treatment. We conﬁrmed Nrf2 is a cytoplasmic protein translocated to the nuclei by that quercetin-dependent induction of NRF1 was completely oxidative stress.33 We therefore examined whether querce- abolished by the transfection of Nrf2-speciﬁc siRNA (Fig. tin can increase nuclear Nrf2 expression. As expected, quer- 5B). As a control, the expression of Foxo3a and PCNA was cetin did induce Nrf2 expression in both a time- and dose- not affected by the transfection of siRNA under quercetin

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FIGURE 5. (A) Effect of quercetin on expression of Nrf2. Left: NTM5 cells were incubated with 1 ␮M quercetin for the times indicated. Whole nuclear lysates (100 ␮g) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblot- ting of PCNA is shown as a loading control. Relative intensity is shown under each blot. Right: NTM5 cells were cultured for 12 hours in the control medium or in medium containing the indicated concentrations of quercetin. Whole nuclear lysates (100 ␮g) were subjected to SDS- PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of PCNA is shown as a loading control. Relative intensity is shown under each blot. (B) NTM5 cells were transiently transfected with 100 pmol control or Nrf2 siRNAs. The following day, NTM5 cells were cultured in the control medium or in medium containing 1 ␮M quercetin. After 48 hours, whole nuclear lysates (100 ␮g) were subjected to SDS-PAGE, and Western blot analysis was performed using the indicated antibodies. Immuno- blotting of PCNA is shown as a loading control. Relative intensity is shown at the bottom of the panel. (C) Quercetin treatment counteracts

H2O2 sensitivity in TM cells. Approx- imately 2.5 ϫ 103 NTM5 cells were cultured in the control medium or in medium containing the indicated concentration of quercetin. The following day, to induce oxidative stress, cells were incubated with the

indicated concentration of H2O2 in serum-free medium for 40 minutes After 48 hours, cell survival was analyzed using a WST-8 assay. All values are the means of at least three independent experiments. Bars represent the SD. CBB, Coomassie brilliant blue.

treatment. To examine the protective efﬁcacy of quercetin transcription pathway (Figs. 1–4). Quercetin is also a potent

treatment, TM cells were exposed to H2O2 in the presence free radical scavenger, suggesting that quercetin would be an of quercetin. Quercetin treatment significantly protected effective agent against oxidative stress-induced ocular diseases, the TM cells against the cytotoxic activity of H2O2 (Fig. 5C). including glaucoma. Several transcription factors are activated under oxidative NRF1 Expression Modulates Cellular Sensitivity stress induced by hydrogen peroxide and inflammatory cyto- to H2O2 kines, such as TNF-␣ and IL-1␤. Among them, both NF-␬B and PRDX3 localizes to mitochondria and may protect mitochon- Nrf2 are well-known transcription factors related to oxidative 33,41 drial DNA from ROS. NRF1 expression leads to the activation of stress. PRDXs can eliminate hydrogen peroxide efficiently genes concerned with mitochondrial biogenesis and protects and can participate in many physiological processes such as 42 cells from apoptosis.34 As shown in Figure 6A, the downregu- signal transduction and apoptosis. There are six distinct lation of PRDX3 and PRDX5 sensitized TM cells approximately members located in various subcellular compartments. PRDX1,

twofold against H2O2. On the other hand, the downregulation PRDX2, and PRDX6 are in the cytoplasm, and PRDX3 is found of NRF1 sensitized TM cells approximately ﬁvefold against in mitochondria. PRDX4 is in endoplasmic reticulum and is

H2O2 (Fig. 6B). Among NRF1-regulated genes, mtTFA is impor- secreted. PRDX5 is found in various compartments. As shown tant in protecting mitochondrial DNA from ROS-dependent in Figure 1C, the expression of ﬁve PRDXs was observed, and apoptosis.35 We showed that quercetin signiﬁcantly induces both PRDX3 and PRDX5 were induced by the treatment with mtTFA protein expression (Fig. 6C). quercetin in primary TM cells. We have previously shown that PRDX1 is not found in immortalized TM cells.24 This might be due to the epigenetic mechanism because cellular transforma- DISCUSSION tion often induces epigenetic changes such as DNA methyl- Quercetin has a wide variety of pharmacologic proper- ation.43 We have previously shown that oxidative stress in- ties.36–40 However, little is known about the mechanisms by duces PRDX1 and PRDX5 through the activation of Ets1.23 which quercetin protects cells against oxidative stress. In the Furthermore, PRDX2 expression is regulated by the transcrip- present study, we demonstrated that quercetin stimulates the tion factor Foxo3a.24 In this study, we found that the Nrf2/ antioxidant system through the activation of the Nrf2/NRF1 NRF1 transcription pathway is also involved in the expression

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FIGURE 6. (A) Downregulation of PRDX3 and PRDX5 sensitizes TM cells to oxidative stress. Left: NTM5 cells were transiently transfected with 100 pmol control, PRDX2, or PRDX5 siRNA. After 72 hours, whole cell lysates (50 ␮g) were subjected to SDS-PAGE, and Western blot analysis was performed using the indicated antibodies. Immunoblotting of ␤-actin is shown as a loading control. Relative intensity is shown at the bottom of the panel. Right: approximately 2.5 ϫ 103 NTM5 cells were transfected with 40 nM control siRNA (ﬁlled squares), PRDX3 siRNA (open rhomboids), or PRDX5 siRNA (open circles). In- duction of the indicated concentra-

tions of H2O2 was described for Fig- ure 5C. All values are the means of at least three independent experiments. Bars represent the SD. (B) Downregulation of NRF1 sensitizes TM cells to oxidative stress. Ap- proximately 2.5 ϫ 103 NTM5 cells were transfected with 40 nM control siRNA (ﬁlled squares), NRF1#1 siRNA (open rhomboids), or NRF1#2 siRNA (open circles). In- duction of the indicated concentra-

tions of H2O2 was described for Fig- ure 5C. All values are the means of at least three independent experiments. Bars represent the SD. (C) Effect of quercetin on the expression of mtTFA. Left: NTM5 cells were incubated with 1 ␮M quercetin for the times indicated. Whole cell lysates (100 ␮g) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immuno- blotting of ␤-actin is shown as a loading control. Relative intensity is shown under each blot. Right: NTM5 cells were cultured for 12 hours in the control medium or in medium containing the indicated concentration of quercetin. Whole cell lysates (100 ␮g) were subjected to SDS-PAGE, and Western blot analysis was performed with the indicated antibodies. Immunoblotting of ␤-actin is shown as a loading control. Relative intensity is shown under each blot. CBB, Coomassie brilliant blue.

of both the PRDX3 and PRDX5 genes (Figs. 4, 5). Nrf2, a basic protect mtDNA, acting as a guardian of mitochondrial func- leucine zipper transcription factor, is essential for the induction.55,56 As shown in Figure 6C, quercetin induced mtTFA ible and constitutive expression of several phase 2 detoxifica- protein expression. This indicates that quercetin may protect tion proteins, including those required for mitochondrial re- mitochondria from oxidative stress through the induction of spiratory function.44,45 NRF1 was found to act on many nuclear both mtTFA and PRDX3. We also demonstrated the protective 46 genes required for mitochondrial respiratory function. This activity of quercetin against H2O2 toxicity (Fig. 5C). Quercetin primary function was confirmed by disrupting the Nrf1 gene in inhibits the activation of caspase 3 and abolishes the H2O2- mice, resulting in a phenotype of peri-implantation lethality dependent induction of apoptogenic proteins such as Bcl2.57 and a striking decrease in the mitochondrial DNA content of This also suggests that quercetin inhibits the mitochondrial Nrf1-null blastocysts.34 apoptotic pathway induced by various stresses. One specific ROS, hydrogen peroxide, is produced by mi- The endothelium plays a key role in the maintenance of tochondria. Because PRDXs can eliminate hydrogen peroxide anterior chamber homeostasis and also is involved in glaucoma efficiently, mitochondrial PRDX3 may protect mitochondrial pathogenesis.58 Expression of the PRDX family was investi- DNA from ROS.47–50 We have previously reported that a mem- gated in Fuchs’ endothelial dystrophy, and the expression of ber of the high-mobility group protein family mtTFA can rec- PRDX2, PRDX3, and PRDX5 was significantly downregulated ognize oxidatively damaged DNA.51,52 Furthermore, it has in this disease.59 It has been reported that PRDX3 oxidation is been shown that mtTFA binds to mitochondrial DNA (mtDNA) found in TNF-␣–treated cells and is the early event in apopto- in the same way that histones bind to nuclear DNA.53,54 Be- sis. This leads to an increase of hydrogen peroxide to modulate cause mtTFA is not wrapped by chromatin proteins such as the progression of apoptosis.60 These data indicate that the histones, it is highly sensitive to oxidative stress. mtTFA may expression of PRDXs in endothelial cells may be also related to

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