Triphlorethol-A Induces Heme Oxygenase-1 Via Activation of ERK and NF-E2 Related Factor 2 Transcription Factor

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FEBS Letters 581 (2007) 2000–2008

Triphlorethol-A induces heme oxygenase-1 via activation of ERK and NF-E2 related factor 2 transcription factor

Kyoung Ah Kanga, Kyoung Hwa Leea, Jae Woo Parkb, Nam Ho Leec, Hye Kyung Nad, Young Joon Surhd, Ho Jin Youe, Myung Hee Chungf, Jin Won Hyuna,* a Department of Biochemistry, College of Medicine and Applied Radiological Science Research Institute, Cheju National University, Jeju 690-756, Republic of Korea b Department of Nuclear and Energy Engineering and Applied Radiological Science Research Institute, Cheju National University, Jeju 690-756, Republic of Korea c Department of Chemistry, College of Natural Sciences, Cheju National University, Jeju 690-756, Republic of Korea d Laboratory of Biochemistry and Molecular Toxicology, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea e Department of Pharmacology, College of Medicine, Chosun University, 375 Seosuk-dong, Gwangju 501-759, Republic of Korea f Department of Pharmacology, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea

Received 2 November 2006; revised 7 April 2007; accepted 10 April 2007

Available online 20 April 2007

Edited by Lukas Huber

ates transcriptional activation of Nrf2 in response to oxidative Abstract Triphlorethol-A, phlorotannin found in Ecklonia cava, induced heme oxygenase-1 (HO-1) expression at mRNA stress [15]. ARE is found in the promoters of genes that encode and protein levels, leading to increased HO-1 activity. Transcrip- HO-1 [16] and other cellular antioxidant enzymes, such as c- tion factor NF-E2 related factor 2 (Nrf2) regulates antioxidant glutamylcysteine synthetase [17]. Therefore, genes regulated response element (ARE) of phase 2 detoxifying and antioxidant by the ARE encode proteins that help control of the cellular enzymes. Triphlorethol-A increased nuclear translocation, ARE redox status and protect the cells from oxidative damage binding, and transcriptional activity of Nrf2. Triphlorethol-A [18]. Mitogen activated protein kinase (MAPK) family is exhibited activation of ERK and U0126, inhibitor of ERK ki- important signaling intermediates involved transducing a vari- nase, suppressed triphlorethol-A induced activation of Nrf2, ﬁ- ety of signals from the cell surface to the nucleus. MAPKs nally decreased HO-1 protein level. Taken together, these data pathway associates with the modulation of ARE driven gene suggest that triphlorethol-A augments cellular antioxidant de- expression via Nrf2 activation. ERK and p38 MAPK path- fense capacity through induction of HO-1 via ERK-Nrf2-ARE signaling pathway, thereby protecting cells from oxidative stress. ways involved the nucleus binding of Nrf2 to the ARE [19]. 2007 Federation of European Biochemical Societies. Published Inhibition of ERK and p38 abrogated binding of Nrf2 and by Elsevier B.V. All rights reserved. induction of ARE-driven c-glutamylcysteine synthetase [20]. In view of the increasing evidence explaining the importance Keywords: Triphlorethol-A; Heme oxygenase-1; NF-E2 related of carbon monoxide and bilirubin in counteracting cellular factor 2; Antioxidant response element dysfunction [21–24], attention has recently been focused on upregulation of HO-1 by natural compounds [25–30]. Ecklonia cava is a brown alga (Laminariaceae) that is abundant in the subtidal regions of Jeju island in Korea. Ecklonia species exhibits radical scavenging activity [31,32], antiplasmin activity 1. Introduction [33–35], antimutagenic activity [36–38], bacteriocidal activity [39], HIV-1 reverse transcriptase and protease inhibiting activ- Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in ity [40], and tyrosinase inhibitory activity [41]. Triphlorethol-A heme catabolism, leading to the formation of biliveridin, free (Fig. 1), an open-chain trimer of phloroglucinol, is a phloro- iron and carbon monoxide. In the presence of biliveridin tannin component isolated from E. cava. Recently, we re- reductase, biliveridin is further converted to bilirubin, which ported that triphlorethol-A protected cells from H2O2 is a potent antioxidant [1–3]. HO-1 expression is induced in re- induced oxidative stress via radical quenching and catalase sponse to various chemically or physiologically produced oxi- activation [42] and from ionizing radiation induced cellular dative stresses in cells and tissues [4–7]. The HO-1 pathway damage [43]. represents a prime cellular defense system against oxidative In the present study, we investigated the capability of triph- stress [4,8–13]. Recently, a new class of activator protein-1 lorethol-A to induce the antioxidant enzyme HO-1 via activa- (AP-1) related sequences has been shown to mediate the oxidation of the Nrf2 transcription factor. tive stress responsiveness of the HO-1 gene. These regions, termed stress responses elements (StRE) or antioxidant responses elements (ARE), are tightly regulated by the redox- 2. Materials and methods sensitive transcription factor, NF-E2 regulated factor 2 (Nrf2) [14]. ARE is a cis-acting enhancer sequence that medi- 2.1. Preparation of triphlorethol-A Dried E. cava (4 kg), collected from Jeju Island in Korea, was im- mersed in 80% methanol at room temperature for 2 days. The aqueous *Corresponding author. Fax: +82 064 726 4152. methanol was removed in vaccuo to give a brown extract (1 kg), which E-mail address: [email protected] (J.W. Hyun). was partitioned between ethyl acetate and water. The ethyl acetate

0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.04.022 K.A. Kang et al. / FEBS Letters 581 (2007) 2000–2008 2001

OH ERK, phospho ERK, Nrf2, and b-actin antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Primary mouse monoclonal HO-1 antibody was purchased from Stressgen Corp., Victoria, Canada. U0126 was provided from Calbiochem, San Diego, CA, USA. The ARE-luciferase reporter gene was kindly OH O OH provided by Dr. Young-Joon Surh (Seoul National University, Seoul, Korea). The sequences of ARE construct was followed: OH O 50-CTCAGCCTTCCAAATCGCAGTCACAGTGACTCAGCAGA- ATC-30. The other chemicals and reagents were of analytical grade.

HO OH 2.3. Cell culture OH The Chinese hamster lung fibroblast cells (V79-4) were obtained from the American Type Culture Collection. The V79-4 cells were Fig. 1. Chemical structure of triphlorethol-A. maintained at 37 C in an incubator with a humidified atmosphere of 5% CO2 and cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal calf serum, streptomycin (100 lg/ml) fraction (230 g) was mixed with celite. The mixed celite was dried and and penicillin (100 U/ml). packed into a glass column, and eluted in the order of hexane, methy- lene chloride, diethyl ether and methanol. The obtained diethyl ether 2.4. Cell viability fraction (14 g) was subjected to Sephadex LH-20 chromatography The effect of triphlorethol-A on the viability of the V79-4 cells was using a CHCl3–MeOH gradient solvent (2/1 fi 0/1). The triphlore- determined by the MTT assay, which is based on the reduction of a tet- thol-A (220 mg) was obtained from these fractions and was identified razolium salt by mitochondrial dehydrogenase in the viable cells [45]. according to the previously reported method [44]. The purity of triph- The cells were seeded in a 96 well plate at a density of 1 · 105 cells/ lorethol-A assessed by HPLC was >90%. Triphlorethol-A was freshly ml and treated with 5, 15 and 30 lM triphlorethol-A, followed 1 h later dissolved in dimethyl sulfoxide (DMSO); the final concentration of by 1 mM of H2O2 or c-irradiation at 5 Gy. After incubated for 48 h at which did not exceed 0.1%. 37 C, 50 ll of the MTT stock solution (2 mg/ml) was then added to each well to attain a total reaction volume of 200 ll. After incubation 2.2. Reagents for 4 h, the plate was centrifuged at 800 · g for 5 min and the superna- [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium]bromide(MTT) tants were aspirated. The formazan crystals in each well were dissolved and zinc protoporphyrin (ZnPP) were purchased from Sigma in 150 ll DMSO, and the A540 was read on a scanning multi-well spec- Chemical Company, St. Louis, MO, USA. Primary rabbit polyclonal trophotometer.

A 0 5 15 30 μM D 0 12 24 48h

HO-1 HO-1

ß-Actin ß-Actin

B 6000 E 6000 * *

4000 * 4000 *

2000 * 2000 * Heme oxygenase activity Heme oxygenase activity (pmole of bilirubin/mg protein/h) (pmole of bilirubin/mg protein/h) 0 0 051530[μM] 0122448h

C 0 6 12 24 48 h

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Fig. 2. Effect of triphlorethol-A on heme oxygenase-1 mRNA, protein expression and activity in V79-4 cells. (A) Cell lysates treated by various concentration of triphlorethol-A at 48 h were electrophoresed and the expression of HO-1 expression was detected by a HO-1 specific antibody. Control cells were incubated in 0.1% DMSO in medium, which is the same concentration used to dissolve triphlorethol-A. (B) HO-1 activity was measured at 48 h after exposure to various concentration of triphlorethol-A. Each bar represents the mean ± S.E. in triplicate experiments. *Significantly different from control (P < 0.05). (C) Cells were exposed to 30 lM of triphlorethol-A and total RNA was extracted at the times indicated. HO-1 mRNA expression was analyzed at indicated times by RT-PCR. The b-actin band is shown to confirm integrity and equal loading of RNA. (D) Cell lysates were electrophoresed and the expression of HO-1 protein was detected at indicated times by a HO-1 specific antibody. (E) Cells were exposed to 30 lM triphlorethol-A and HO-1 activity was measured at indicated times. Data are expressed as the means ± S.E. in triplicate experiments. *Significantly different from control (P < 0.05). 2002 K.A. Kang et al. / FEBS Letters 581 (2007) 2000–2008

2.5. HO-1 assay and protein concentrations were determined. Aliquots of the HO-1 enzyme activity was measured as described previously [46]. lysates (40 lg of protein) were boiled for 5 min and electrophoresed Cells were homogenized in 0.5 ml ice-cold 0.25 M sucrose solution con- on a 10% SDS–polyacrylamide gel. Blots in the gels were transferred taining 50 mM potassium phosphate buffer (pH 7.4). Homogenates onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were centrifuged at 200 · g for 10 min. The supernatants were centri- were then incubated rabbit polyclonal ERK, phospho ERK, Nrf2, b- fuged at 9000 · g for 20 min, and further centrifuged at 30000 · g for actin or mouse monoclonal HO-1 antibodies. The membranes were 60 min. The pellet was then resuspended in 50 mM potassium phos- further incubated with secondary antibody-horseradish peroxidase phate buffer (pH 7.4) and the amount of protein was determined by conjugates (Pierce, Rockland, IL, USA), and exposed to X-ray film. Bradford method [47]. The reaction mixture (200 ll) containing Protein bands were detected using an enhanced chemiluminescence 0.2 mM of the substrate hemin, 500 lg/ml of cell lysate, 0.5 mg/ml Western blotting detection kit (Amersham, Little Chalfont, Bucking- rat liver cytosol as a source of biliveridin reductase, 0.2 mM MgCl2, hamshire, UK). 2 mM glucose-6-phosphate, 1 U/ml glucose-6-phosphate dehydrogenase, 1 mM NADPH and 50 mM potassium phosphate buffer (pH 2.7. Reverse transcriptase polymerase chain reaction 7.4) was incubated at 37 C for 2 h. The reaction was stopped with Total RNA was isolated from V79-4 cells using Trizol (GibcoBRL, 0.6 ml of chloroform and after extraction, the chloroform layer was Grand Island, NY, USA). Reverse transcriptase polymerase chain measured spectrophotometrically. Bilirubin formation was calculated reaction (RT-PCR) was performed as described previously [48]. PCR from the difference in absorption between 464 and 530 nm. conditions for HO-1 and for the housekeeping gene, b-actin, were: 35 cycles of 94 C for 45 s; 53 C for 45 s; and 72 C for 60 s. The pri- 2.6. Western blot mer pairs (Bionics, Seoul, Korea) were follows (forward and reverse, The V79-4 cells were plated at 1 · 105 cells/ml, and 16 h after plating, respectively): HO-1, 50-GAGAATGCTGAGTTCATG-30 and 50- the cells were treated with 30 lM triphlorethol-A. The cells were ATGTTGAGCAGGAAGGC-30; and b-actin, 50-GTGGGCCGCCC- harvested at the indicated times, and washed twice with PBS. The TAGGCACCAGG-30; and 50-GGAGGAAGAGGATGCGGCA- harvested cells were then lysed on ice for 30 min in 100 ll of lysis buffer GTG-30. Amplified products were resolved by 1% agarose gel [120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP 40] and centrifuged at electrophoresis, stained with ethidium bromide, and photographed 13000 · g for 15 min. Supernatants were collected from the lysates under ultraviolet light.

A 0 6 12 24 48 h Nrf2(nucleus)

ß-Actin

B Nrf2-FITC DAPI Merge

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C 0 6 12 24 48h D 4

Nrf2 3 *

(Relative intensity) 1 ARE-luciferase activity

0 Control Triphlorethol-A

Fig. 3. Effect of triphlorethol-A on Nrf2 expression, translocalization into nucleus, and its transcriptional activity in V79-4 cells. (A) Effect of triphlorethol-A on the protein levels of nuclear Nrf2. Nuclear extract from V79-4 cells was prepared from after treatment with 30 lM triphlorethol-A for the indicated times. Immunoblots for nuclear lysates from treated V79-4 cells were detected with a Nrf2 specific antibody. (B) Effect of triphlorethol-A on nuclear localization of Nrf2. Confocal image shows that FITC-conjugated secondary antibody staining indicates the location of Nrf2 (green) by anti- Nrf2 antibody, DAPI staining indicates the location of the nucleus (blue), and merged image in triphlorethol-A treated cells indicates the nuclear location of Nrf2 protein. (C) Effect of triphlorethol-A on the ARE-binding activity of Nrf2 in V79-4 cells. Nuclear extracts prepared from V79-4 cells were treated with 30 lM of triphlorethol-A for the indicated times. (D) Effect of triphlorethol-A on the transcriptional activity of ARE in V79-4 cells. V79-4 cells were transfected with an ARE-luciferase construct (1 lg per well) or control vector (1 lg per well). After overnight, cells were treated with triphlorethol-A, cell lysates were mixed with a luciferase substrate, and the luciferase activity was measured by the luminometer. Data are expressed as the means ± S.E. in triplicate experiments. *Significantly different from control (P < 0.05). K.A. Kang et al. / FEBS Letters 581 (2007) 2000–2008 2003

2.8. Nuclear extract preparation and electrophoretic mobility shift assay 2.9. Transient transfection and luciferase assay The cells were harvested at the indicated times, and were then lysed A day before transfection, cells were sub-cultured at a density of on ice with 1 ml of lysis buffer (10 mM Tris–HCl, pH 7.9, 10 mM 1 · 106 cells in 60 mm dish to maintain approximately 60–80% conflu- NaCl, 3 mM MgCl2, and 1% NP-40) for 4 min. After 10 min of centri- ent. The cells were transiently transfected with the plasmid harboring fugation at 3000 · g, the pellets were re-suspended in 50 ll of extrac- the ARE-promoter using the transfection reagent DOTAP according tion buffer (20 mM HEPES, pH 7.9, 20% glycerol, 1.5 mM MgCl2, to the manufacturer’s instructions (Roche, Mannheim, Germany). 300 mM NaCl, 0.2 mM EDTA, 1 mM DTT, and 1 mM PMSF), incu- After overnight transfection, cell were treated with 30 lM triphlore- bated on ice for 30 min, and centrifuged at 13000 · g for 5 min. The thol-A. Cell were then washed twice with PBS and lysed with reporter supernatant was then harvested as nuclear protein extracts and stored lysis buffer. After vortex-mixing and centrifugation at 12000 · g for at 70 C after determination of protein concentration. Synthetic dou- 1 min at 4 C, the supernatant was stored at 70 C for the luciferase ble strand oligonucleotide containing the Nrf2-binding domain (ARE) assay. After 20 ll of the cell extract was mixed with 100 ll of the lucif- was labeled with [c-32P] ATP using T4 polynucleotide kinase, and used erase assay reagent at room temperature, the mixture was placed in a as probes. The sequence of oligonucleotides in double strand was 50- luminometer to measure the light produced. TTTTCTGCTGAGTCAAGGGTCCG-30 and 30-AAAAGACGACT- 0 CAGTTCCAGGC-5 . The probes (50000 cpm) were incubated with 2.10. Immunocytochemistry 6 lg of the nuclear extracts at 4 C for 30 min in a final volume of Cells plated on coverslips were fixed with 4% paraformaldehyde for 20 ll containing 12.5% glycerol, 12.5 mM HEPES (pH 7.9), 4 mM 30 min and permeabilized with 0.1% Triton X-100 in PBS for 2.5 min. Tris–HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, and 1 mM DTT with Cells were treated with blocking medium (3% bovine serum albumin in 1 lg of poly(dI-dC). Binding products were resolved on a 5% PBS) for 1 h and incubated with Nrf2 antibody diluted in blocking polyacrylamide gel and the bands were visualized by autoradiography medium for 2 h. Immuno-reacted primary Nrf2 antibody was detected [49]. by a 1:500 dilution of FITC-conjugated secondary antibody (Jackson

ABTriphlorethol-A - + - + - - + + U0126 0 1/2 3 6 12h Phospho ErK Phospho ErK

ErK ErK

Triphlorethol-A CDTriphlorethol-A - + - + - + - + - - + + U0126 U0126 - - + +

HO-1 Nrf-2 ß-Actin ß-Actin

EFNrf2-FITC DAPI Merge 4

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Triphlorethol-A (Relative intensity)

ARE-luciferase activity 1

0 Triphlorethol-A+ Triphlorethol-A - + - + U0126 U0126 - - + +

Fig. 4. Induction of HO-1 and activation of Nrf2 by triphlorethol-A via phosphorylation of ERK. (A) Effect of triphlorethol-A on the phosphorylation of ERK. Cells were exposed to 30 lM triphlorethol-A for the time indicated. Cell lysates were electrophoresed, ERK and phospho- ERK were detected by their respective specific antibodies. (B) After treatment with U0126, cell lysates were electrophoresed and ERK and phospho- ERK were detected by their respective specific antibodies. (C) Effect of U0126 on triphlorethol-A induced HO-1 expression. Cells were incubated with 30 lM triphlorethol-A in the absence or presence of U0126 for 3 h. Cell lysates were electrophoresed and HO-1 was detected by a HO-1 specific antibody. (D) Nuclear extract were prepared from V79-4 cells treated with 30 lM of triphlorethol-A in the absence or presence of U0126 for 3 h and subjected to Western blot for the measurement of Nrf2 protein expression. (E) V79-4 cells treated with 30 lM triphlorethol-A in the absence or presence of U0126 for 3 h, and immunostained to detect the nuclear localization of Nrf2. (F) V79-4 cells were transfected with the ARE-luciferase construct (1 lg per well). After overnight, cells were treated with triphlorethol-A in the absence or presence of U0126, cell lysates were mixed with a luciferase substrate, and the luciferase activity was measured by the luminometer. Data are expressed as the means ± S.E. in triplicate experiments. *Significantly different from triphlorethol-A treated cells (P < 0.05). 2004 K.A. Kang et al. / FEBS Letters 581 (2007) 2000–2008

ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h. After lated with increased HO-1 activity (Fig. 2B). Treatment of cells washing with PBS, stained cells were mounted onto microscope slides with 30 lM triphlorethol-A resulted in a time-dependent in- in mounting medium with DAPI (Vector, Burlingame, CA, USA). crease in HO-1 mRNA and protein expression (Fig. 2C and Images were collected using the LSM 510 program on a Zeiss confocal microscope. D), which was accompanied by a gradual increase in HO-1 activity (Fig. 2E). 2.11. Statistical analysis All the measurements were made in triplicate and all values are rep- 3.2. Triphlorethol-A increased the nuclear translocation, ARE- resented as means ± S.E. The results were subjected to an analysis of binding, and transcriptional activity of Nrf2 the variance (ANOVA) using the Tukey test to analyze the difference. P < 0.05 was considered significant. Most of the genes encoding phase II detoxifying and antioxidant enzymes have an ARE sequence in their promoter re- gion. Nrf2 is an important transcription factor that regulates 3. Results ARE-driven HO-1 gene expression. Subsequently, we examined whether triphlorethol-A could activate Nrf2 in associa- 3.1. Effect of triphlorethol-A on HO-1 expression and activity tion with HO-1 upregulation in V79-4 cells. Triphlorethol-A As HO-1 is an important component of the cellular defense treatment resulted in increased Nrf2 accumulation in the nu- against oxidative stress, we assessed whether triphlorethol-A cleus (Fig. 3A and B). Nrf2 activation in triphlorethol-A trea- could induce HO-1 expression in relation to its antioxidant ted cells was assessed by EMSA with an oligonucleotide activity. V79-4 cells exposed to triphlorethol-A for 48 h harboring a consensus Nrf2 binding element. Triphlorethol- showed a concentration-dependent increase in HO-1 protein A treated cells exhibited a high level of Nrf2 binding expression (Fig. 2A). The enhanced HO-1 expression corre- (Fig. 3C). The transcriptional activity of Nrf2 was also as-

AB100 100 * 80 * 80

60 60 * * 40 40 Cell viability (%) Cell viability (%)

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0 0 0 5 15 30 [μM] 0 5 15 30 [μM]

CD120 120

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0 0 Triphlorethol-A - - + + Triphlorethol-A - - + +

H2O2 - + + + H2O2 - + + +

ZnPP - - - + U0126 - - - +

Fig. 5. Protective effect of triphlorethol-A against oxidative stress induced cell damage. (A) V79-4 cells were treated with 5, 15 and 30 lM triphlorethol-A, followed 1 h later by c-irradiation at 5 Gy. After 48 h, cell viability was assessed by the MTT assay. *Significantly different from irradiated cells (P < 0.05). (B) V79-4 cells were treated with 5, 15 and 30 lM triphlorethol-A, followed 1 h later by 1 mM H2O2. After 48 h, cell * viability was assessed by the MTT assay. Significantly different from H2O2 treated cells (P < 0.05). (C) V79-4 cells were pre-incubated with 30 lM triphlorethol-A and HO-1 inhibitor (ZnPP) at 10 lM for 6 h, followed by exposure to 1 mM H2O2. Cell viability was assessed by the MTT assay after * H2O2 treatment. Significantly different from H2O2 treated cells (P < 0.05). (D) After treatment with U0126, triphlorethol-A or/and H2O2, the * viability of V79-4 cells was determined by MTT assay. Significantly different from triphlorethol-A plus H2O2 treated cells (P < 0.05). K.A. Kang et al. / FEBS Letters 581 (2007) 2000–2008 2005 sessed using an ARE promoter construct containing the Nrf2 (Fig. 5C), suggesting that the cytoprotective effect of triphlore- binding DNA consensus site linked to a luciferase reporter thol-A is partly mediated through HO-1 induction. Moreover, gene. As illustrated in Fig. 3D, triphlorethol-A increased the U0126 also attenuated the protective effect of triphlorethol-A transcriptional activity of Nrf2. on H2O2-induced cytotoxicity (Fig. 5D), suggesting the involvement of ERK signaling in triphlorethol-A mediated 3.3. Triphlorethol-A activates Nrf2 via phosphorylation of ERK HO-1 gene induction and cytoprotection. To further elucidate the upstream signaling pathway involved in triphlorethol-A mediated Nrf2 activation and induction of HO-1, we examined the activation of ERK, a 4. Discussion major signaling enzyme involved in cell survival against oxidative stress. Western blot analysis showed that triphlore- Phlorotannin components, which are present in some brown thol-A increased ERK phosphorylation (Fig. 4A) and that algae, are polymers of phloroglucinol [50]. They are phytoalex- this activation was abrogated by U0126, a specific ERK ki- ins that have defensive or protective functions against herbi- nase inhibitor (Fig. 4B). Pretreatment with U0126 effectively vores. Recently, we reported that phlorotannins isolated suppressed triphlorethol-A induced HO-1 induction from E. cave showed antioxidant and cytoprotective effects (Fig. 4C). The nuclear accumulation of Nrf2 was significantly against oxidative stress; triphlorethol-A protected cell damage inhibited by U0126 (Fig. 4D and E). Likewise, transcrip- from H2O2 and radiation induced oxidative stress via radical tional activity of Nrf2 was effectively blocked by U0126 quenching effect [42,43]; eckol attenuates oxidative stress in- (Fig. 4F). duced cell damage via ERK and nuclear factor kappa B activation [51]; phlorglucinol induced cytoprotection via catalase 3.4. Effect of triphlorethol-A on cell damage induced by and ERK activation [52]. oxidative stress In this study, we demonstrated that triphlorethol-A upregu- The protective effect of triphlorethol-A on oxidative stress lated HO-1 expression through activation of Nrf2. Most stud- induced in c-ray irradiated or H2O2 treated V79-4 cells was as- ies on the regulation of HO-1 gene expression have focused on sessed. Cells were treated with 5, 15 and 30 lM triphlorethol-A the role of the MAPK pathways. For instance, the glutathione for 1 h prior to the addition of the 5 Gy of radiation or 1 mM depletor pherone and arsenite promote JNK-dependent induc- of H2O2. Cell viability was determined 48 h later by the MTT tion of HO-1 gene expression [53,54]. Curcumin induces HO-1 assay. Treatment with triphlorethol-A increased cell survival at expression by promoting dissociation of the Nrf2-Keap1 com- dose dependent pattern after c-radiation or after H2O2 treat- plex in a p38 dependent manner [26]. Resveratrol activates ment (Fig. 5A and B). To determine whether the increased Nrf2-driven ARE activation and HO-1 expression via the HO-1 activity induced by triphlorethol-A could confer cyto- ERK pathway [30]. Our results indicate a requirement for protection against oxidative stress, V79-4 cells were pretreated ERK activation in the induction of HO-1 expression by triph- with the HO-1 inhibitor ZnPP. ZnPP attenuated the protective lorethol-A because an ERK inhibitor significantly reduced effect of triphlorethol-A on H2O2-induced cytotoxicity HO-1 protein expression in response to triphlorethol-A. The

Triphlorethol A

Cytosol ERK

P Nrf2 Keap1 P Keap1

P Nrf2 P

Nucleus 5 -TGACnnnGCA-3

ARE-driven HO-1gene expression

Cytoprotective effect

Fig. 6. A proposed pathway for triphlorethol-A induced HO-1 via upregulation of ERK and Nrf2, which explains cytoprotection against oxidative stress in V79-4 cells. 2006 K.A. Kang et al. / FEBS Letters 581 (2007) 2000–2008 transcription factor Nrf2 is a member of the Cap-N-Collar [8] Lee, P.J., Alam, J., Wiegand, G.W. and Choi, A.M. (1996) family of basic leucine transcription factors, and plays an Overexpression of heme oxygenase-1 in human pulmonary essential role in the ARE-mediated expression of HO-1 in re- epithelial cells results in cell growth arrest and increased resistance to hyperoxia. Proc. Natl. Acad. Sci. USA 93, 10393–10398. sponse to oxidative stress [55–60]. Under normal physiological [9] Dennery, P.A., Sridhar, K.J., Lee, C.S., Wong, H.E., Shokoohi, conditions, Nrf2 is sequestered by cytoskeleton associated V., Rodgers, P.A. and Spitz, D.R. (1997) Heme oxygenase- cytoplasmic ‘Kelch-like ECH-associated protein 1 (Keap 1)’ mediated resistance to oxygen toxicity in hamster fibroblasts. J. which hampers the nuclear translocation of Nrf2. However, Biol. Chem. 272, 14937–14942. [10] Yamada, N., Yamaya, M., Okinaga, S., Lie, R., Suzuki, T., upon stimulation by electrophilic agents or reactive oxygen Nakayama, K., Takeda, A., Yamaguchi, T., Itoyama, Y., species, Nrf2 dissociates from its cytoplasmic docking protein Sekizawa, K. and Sasaki, H. (1999) Protective effects of heme Keap1, translocates into the nucleus and binds to ARE site oxygenase-1 against oxidant-induced injury in the cultured human [61]. This leads to the de novo synthesis of antioxidant enzymes tracheal epithelium. Am. J. Respir. Cell. Mol. Biol. 21, 428–435. that efficiently protect cells from oxidative stress. The actual [11] Foresti, R. and Motterlini, R. 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