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Taurine chloramine induces heme oxygenase-1 expression via Nrf2 activation in murine macrophages
International Immunopharmacology 10 (2010) 440–446
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
Taurine chloramine induces heme oxygenase-1 expression via Nrf2 activation in murine macrophages Chaekyun Kim a,b,c,d,⁎, Jin Sun Jang a,b,c, Mi-Ran Cho a, Santosh R. Agarawal c, Young-Nam Cha b a
Laboratory for Leukocyte Signaling Research, Inha University School of Medicine, Incheon 400-712, Republic of Korea Department of Pharmacology, Inha University School of Medicine, Incheon 400-712, Republic of Korea BK21 Program, Inha University School of Medicine, Incheon 400-712, Republic of Korea d Inha Research Institute for Medical Science, Inha University School of Medicine, Incheon 400-712, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 26 October 2009 Received in revised form 11 December 2009 Accepted 29 December 2009 Keywords: Taurine chloramine Heme oxygenase Macrophages Nrf2 Reactive oxygen species
a b s t r a c t Taurine chloramine (TauCl) is produced abundantly in activated neutrophils by a reaction between the stored taurine and the newly produced HOCl by the myeloperoxidase system, and is much less oxidizing or toxic than HOCl. TauCl has been shown to provide cytoprotection against inflammatory tissue injury by inhibiting the overproduction of inflammatory mediators. The result of this study shows that TauCl upregulated the expression of heme oxygenase (HO)-1 and increased HO activity in RAW 264.7 macrophages, while taurine had no effect. TauCl by itself generated reactive oxygen species (ROS) in macrophages and diminished total glutathione (GSH) level initially. TauCl increased the nuclear translocation of NF-E2-related factor 2 (Nrf2) and enhanced its binding to the anti-oxidant response element (ARE). This, in turn, was responsible for the upregulation of HO-1 expression. In summary, TauCl generated ROS in RAW 264.7 macrophages and decreased cellular GSH level initially. This was responsible for the nuclear translocation of Nrf2 and its binding to ARE promoted the expression of HO-1 and increased HO activity. Thus, TauCl-derived elevation of HO activity may play an essential role in the adaptive cytoprotection of inflammatory tissues. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Taurine, the decarboxylation product of cysteine, is one of the most abundant free amino acids in phagocytic cells and is not incorporated into proteins [1–3]. Taurine, stored in neutrophils up to 50 mM, reacts stoichiometrically with HOCl, an antibacterial oxidant produced from H2O2 by the myeloperoxidase (MPO) in activated neutrophils. This results in the generation of taurine chloramine (TauCl). The production of TauCl by activated neutrophils has been well established in vitro, so that 2 × 106 of human neutrophils are known to produce up to 100 μM TauCl in the presence of high taurine content [4,5]. Taurine is reported to protect phagocytic cells against the injury caused by inflammatory overproduction of HOCl. Cytoprotection of phagocytic cells provided by exogenously added taurine results primarily from its efficient elimination of highly toxic HOCl and generation of less toxic TauCl. Furthermore, TauCl is also known to exert anti-inflammatory effects on its own. TauCl has been demonstrated to suppress the production of many inflammatory intermedi⁎ Corresponding author. Laboratory for Leukocyte Signaling Research, Inha University School of Medicine, 7-206 3rd St, Shinheung-dong, Jung-gu, Incheon 400-712, Republic of Korea. Tel.: +82 32 890 0976; fax: +82 32 887 7488. E-mail address: [email protected] (C. Kim). 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.12.018
ates such as superoxide anion (O− 2 ), nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, -2, -6, -8, and -10, prostaglandin E2 (PGE2), macrophage inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1 and -2, and matrix metalloproteinase (MMP) in the stimulated phagocytes [6–11]. In spite of increasing evidences showing that TauCl diminishes the production of inflammatory mediators and protects cells at the inflammatory site from inflammatory cytotoxicity, its underlying cytoprotective mechanism has not yet been elucidated. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme and provides potent anti-inflammatory and anti-oxidant functions. Cells that overexpress HO-1 are protected from oxidative injury and can resist subsequent cytotoxic oxidative challenges. HO catalyzes the rate-limiting step in the oxidative degradation of potentially damaging free-heme into equimolar amounts of biliverdin/bilirubin, carbon monoxide (CO) and free iron [12]. Biliverdin and bilirubin can scavenge peroxy radicals and serve as strong anti-oxidants [13], and the CO arising from heme degradation inhibits inflammatory responses by blocking the expression of pro-inflammatory genes and protects cells against apoptosis and necrosis caused by oxidants [14]. The increased HO-1 expression occurring widely in response to oxidative stress can thus contribute to the adaptive increase of cellular anti-oxidant and chemoprotective defense systems. Upregulation of
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HO-1 is mediated by activation of relevant redox-sensitive cytoplasmic transcriptional regulators, such as nuclear factor E2-related factor 2 (Nrf2), activator protein (AP)-1, nuclear factor kappa B (NFκB), and cAMP-responsive element-binding protein (CREB) [15,16]. Nrf2 which belongs to the CNC-bZIP (cap‘n'collar subfamily of basic leucine-zipper) is kept inactive in association with Keap1 (Kelch-like ECH associating protein 1) in normal conditions. Upon oxidative stimulation, Nrf2 dissociates from Keap1, translocates into the nucleus and binds to the anti-oxidant response element (ARE) in the promoter region of phase II detoxifying or anti-oxidant enzymes such as NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferase (GST), glutathione peroxidase (GPx), peroxiredoxin I (Prx-1) and HO-1 [15,16]. Upon inflammatory stimulation of phagocytic cells, O− 2 , NO and CO are overproduced in sequence. In previous studies, we demonstrated that O− 2 overproduction triggered by LPS treatment enhanced the expression of iNOS and overproduction of NO in macrophages [17]. Such overproduction of O− 2 and NO promoted production of peroxynitrite (ONOO−), a highly cytotoxic oxidant that is known to release heme from intracellular heme proteins. On the other hand, the ONOO− also increased the expression of HO-1 and elevated HO activity. This leads to enhance oxidative degradation of free-heme to overproduce CO. This overproduced CO has been shown to inhibit the production of O− 2 in the PMA-stimulated neutrophils and also in the LPS-stimulated macrophages by inhibiting the NADPH oxidase activity [18–20]. Furthermore, the CO derived from elevated HO activity also inhibited the upregulation of inducible nitric oxide synthase (iNOS) expression as well as the overproduction of NO in the LPS-stimulated macrophages [18]. Thus, induction of HO-1 and elevation of HO activity and the resulting overproduction of CO afforded protection against the cytotoxicity caused by excessive oxidative stress in activated phagocytes [21]. In this context, the ability of TauCl to induce the expression of HO-1 in macrophages [22] provided the basis to explore the molecular mechanisms involved in the cytoprotective effect of TauCl in phagocytic cells. Here we show that TauCl induces the expression of HO-1 and elevates HO activity by producing ROS, and thus stimulating the nuclear translocation of Nrf2 and enhancing its binding to ARE. Our results suggest that upregulation of HO-1 expression and elevation of HO activity induced by TauCl is essential for the adaptive cytoprotection of inflammatory tissues by suppressing additional overproduction of NO and O− 2 from activated phagocytic cells. 2. Materials and methods 2.1. Antibodies and reagents Antibodies against Nrf2 (Santa Cruz, Santa Cruz, CA), iNOS (Transduction Laboratories, Lexington, KY), and HO-1 (Stressgen, Victoria, Canada) were purchased from commercial sources. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from HyClone (Logan, UT). The oligonucleotides were purchased from Bioneer (Daejeon, Korea) and Santa Cruz Biotechnology (Santa Cruz, CA), and [γ-32P]ATP was from NEN (Boston, MA). Other routinely used chemicals were purchased from Sigma (St. Louis, MO). TauCl was synthesized freshly on the day of use by adding 400 mM NaOCl (Aldrich, Milwaukee, MI) to 410 mM taurine. The authenticity of TauCl formation was monitored by its UV absorption at 200–400 nm [23].
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2.3. Preparation of bone marrow-derived macrophages Murine bone marrow-derived macrophages (BMDM) were harvested from BALB/c mice (Orient Bio, Seoul, Korea) as described [24]. Following lysis of red blood cells, murine bone marrow cells were suspended in macrophage culture medium (α-MEM containing 20% FBS, 20 ng/ml recombinant human macrophage colony stimulating factor (M-CSF), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) and cultured in 5% CO2 incubator kept at 37 °C. Adherent cells were discarded twice at 4 and 16 h by transferring the suspended cells to new dishes. At 3 and 6 days during the culture, the medium was replaced with fresh medium. BMDM were harvested at 7th day by removing the adherent cells from culture dishes with cell dissociation buffer. For experiments, BMDM were treated with 1 μg/ml LPS and TauCl (0.5 and 0.7 mM) at 12 h before harvest. 2.4. RT-PCR Total RNA was extracted using the TRI reagent (MRC, Cincinnati, OH) according to the manufacturer's instructions and reverse transcription of 500 ng total RNA was performed according to the instructions provided by TaKaRa (Shuzo, Shiga, Japan). PCR amplification of mRNA was carried out using the following primers (forward and reverse, respectively), HO-1, 5′-TGA AGG AGG CCA CCA AGG AGG-3′ and 5′-AGA GGT CAC CCA GGT AGC GGG-3′ (375 bp); iNOS, 5′AGA CTG GAT TTG GCT GGT CCC TCC-3′ and 5′-AGA ACT GAG GGT ACA TGC TGG AGC C-3′ (527 bp); GAPDH, 5′-GTC GGT GTG AAC GGA TTT G-3′ and 5′-ACA AAC ATG GGG GCA TCA G-3′ (386 bp). 2.5. Western blot analysis Cell lysates were prepared from RAW 264.7 cells as described previously [25] and 20–30 μg of total proteins were electrophoresed on SDS-PAGE. The resolved proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (BioRad, Hercules, CA), and the blots were probed with specific antibodies and developed by using the ECL method (Amersham, Arlington Heights, IL). 2.6. Heme oxygenase activity assay The HO activity was determined by measuring the amount of bilirubin produced from hemin added as the substrate as described previously [17]. The amount of bilirubin formed was quantitated from the difference in absorbance between 464 nm and 530 nm (extinction coefficient; 40 mM− 1 cm− 1 for bilirubin). 2.7. Measurement of ROS production using H2-DCFDA Production of ROS in RAW 264.7 cells treated with TauCl and taurine was monitored by employing 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCFDA). The cell permeable H2-DCFDA rapidly diffuses through the cell membrane and then is hydrolyzed by esterases to H2-DCF. The oxidation of H2-DCF by ROS leads to a fluorescent DCF. Cells that adhered on the cover slide were rinsed with Krebs ringer solution and loaded with 10 μM H2-DCFDA. After 15 min of incubation at 37 °C, the cells were examined under a fluorescence microscope set at 488 nm for excitation and 530 nm for emission (Karl Zeiss, Oberkochen, Germany), and analyzed on FACScan cytometer (BD Bioscience, San Jose, CA) using CellQuest software (BD Bioscience).
2.2. RAW 264.7 cell culture 2.8. Total GSH level RAW 264.7 cells, a murine macrophage cell line obtained from ATCC (Manassas, VA), were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin and maintained at 37 °C in a 5% CO2 incubator.
RAW 264.7 cells were collected after addition of 5% 5-sulfosalicylic acid and 0.2% Triton X-100. Cells were then freeze–thawed three times, sonicated and centrifuged. The total amount of GSH present in
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the acidic supernatant was determined as described by Srisook et al [17]. 2.9. Electrophoretic mobility shift assay (EMSA) to detect Nrf2–ARE binding Nuclear extracts were prepared by sequential cell lysis and nuclear lysis as reported previously [26]. Protein concentration in the nuclear extract was determined by using the Bradford method (BioRad, Hercules, CA). DNA binding activity for Nrf2, NFκB, AP-1 and CREB was determined. The DNA–protein complexes were resolved by 5% nondenaturing PAGE as reported previously [26] to detect the changes of intensity for the labeled oligonucleotides binding to specific transcription factors. The oligonucleotides used to detect the activities of Nrf2, NFκB, AP-1 and CREB are as follows: Nrf2, 5′-TGG GGA ACC TGT GCT GAG TCA CTG GAG-3′; NFκB, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′; AP-1, 5′-CTA GTG ATG AGT CAG CCG GAT C-3′; and CREB, 5′-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3′. 2.10. Nrf2 siRNA knockdown To knockdown the Nrf2 expression, RAW 264.7 cells were transfected with 100 nM siRNA directed against murine Nrf2 (Dharmacon, Lafayette, CO) or a non-targeting scRNA by using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the
manufacturer's instructions. Seventy-two hours after transfection with Nrf2 siRNA, the lysates were prepared from cells and the knockdown of Nrf2 was confirmed by immunoblot analysis. 2.11. Statistical analysis The two-tailed Student's t-test (paired) was performed using Microsoft Excel software (Redmond, WA). Data were expressed as mean ± SD, and a p value b 0.05 was considered significant. 3. Results 3.1. Induction of HO-1 expression and HO activity The induction of HO-1 expression has been demonstrated to decrease the inflammatory response as well as the apoptotic death of phagocytic cells via rapid removal of the potentially toxic free-heme released from diverse heme-containing proteins by oxidative stress. To determine the effect of TauCl on HO-1 expression, RAW 264.7 cells were treated with TauCl for various time periods. TauCl enhanced expression of HO-1 mRNA beginning at 2 h and continued through 20 h, while taurine had no effect (Fig. 1A, B). Induction of HO-1 mRNA expression by TauCl was started earlier than that induced by LPS. While LPS enhanced mRNA expression of both iNOS and HO-1, TauCl did not induce the expression of iNOS mRNA. TauCl dose-dependently increased the expression of HO-1 protein beginning at 6 h and reached
Fig. 1. TauCl induces HO-1 expression in RAW 264.7 cells. (A) RT-PCR analysis of HO-1 and iNOS mRNA was performed. RAW 264.7 cells were incubated with 1 μg/ml LPS or 0.5 and 0.7 mM TauCl for 0, 2, 4, 6, 8, 12 and 20 h (n = 3). (B) HO-1 mRNA expression by various concentrations of TauCl at 12 h was analyzed (n = 5). GAPDH was used as a control for equal loading of RNA. (C and D) The effect of TauCl on HO-1 protein expression in RAW 264.7 cells was determined (n = 5). (E) The effect of TauCl on HO-1 expression was determined in murine BMDM (n = 3). The immunoblots were re-probed with β-actin antibody to show equal loading.
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Fig. 2. TauCl increases HO activity in RAW 264.7 cells. (A) The effect of TauCl on HO activity was determined. A microsomal fraction was isolated at 12 h of TauCl treatment and HO activity was determined by the production of bilirubin (n = 3). (B) A microsomal fraction was isolated at 4, 12 and 24 h after treatment (n = 3). Data are expressed as the mean ± SD, ⁎p b 0.05 and ⁎⁎p b 0.01 compared to control.
to a maximum at 12 h in RAW 264.7 cells (Fig. 1C, D). To further confirm the HO-1 inducing effect of TauCl in murine BMDM, mouse BMDM were treated with TauCl. Consistent with that observed in RAW 264.7 cells, TauCl increased the expression of HO-1 in murine BMDM in a dose-dependent manner (Fig. 1E). HO catalyzes the oxidative degradation of free-heme to yield equimolar amount of ferrous iron, CO and biliverdin which is then converted to bilirubin. HO activity was determined by measuring the bilirubin production. TauCl dose-dependently increased the produc-
tion of bilirubin (Fig. 2A). The production of bilirubin reached a maximum level at 12 h after TauCl treatment, and it was increased slightly more by costimulation with LPS (Fig. 2B). 3.2. TauCl triggers ROS production To test whether TauCl induces HO-1 expression via ROS production, we measured ROS production by employing the H2-DCFDA fluorescence staining assay. As shown in Fig. 3A and B, TauCl enhanced
Fig. 3. TauCl generates ROS and reduces total glutathione level. (A) RAW 264.7 cells were treated with 1 μg/ml LPS, 0.7 mM taurine and TauCl for 10 min. ROS production was determined by the H2-DCFDA assay as described in Section 2. The representative picture of DCF-derived fluorescence in RAW 264.7 cells (n = 5). (B) Flow cytometric analysis was performed after H2-DCFDA staining (n = 3). (C) The effect of NAC treatment on TauCl-induced HO-1 expression (n = 3). (D) The total glutathione level was determined at the indicated times after 0.7 mM TauCl treatment (n = 3). Data are expressed as the mean ± SD, ⁎p b 0.05 compared to the glutathione level at 0 h.
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ROS production in RAW 264.7 cells, while taurine had no such effect. The production of ROS by 0.7 mM TauCl was greater than that produced by LPS (Fig. 3B). The addition of an NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) and catalase diminished the accumulation of ROS (data not shown). To confirm that the increased ROS production influences on HO-1 expression, we treated cells with 2.5 mM N-acetyl L-cysteine (NAC) for 1 h before the TauCl treatment. NAC completely abolished TauCl-induced HO-1 expression (Fig. 3C). This suggests that TauCl induces HO-1 expression through ROS. 3.3. TauCl alters GSH level The intracellular GSH level was determined following treatment with TauCl. Acidic protein-free supernatants were prepared from the harvested cells at different time points following treatment with TauCl. The intracellular level of total GSH decreased significantly at 30 min (70% of control level). Subsequently, the depleted GSH level began to recover at 1 h, reaching the control level by 2 h, and further increasing above the control level at 4 h (Fig. 3D). TauCl lowered the GSH/GSSG ratio at 30 min and this lowering peaked at 1 h, subsequently recovering to control level at 2 h (data not shown). 3.4. TauCl upregulates HO-1 expression via activation of Nrf2 The nuclear translocation and DNA binding of redox-sensitive transcription factor are important mechanisms that contribute toward HO-1 expression. The promoter region of HO-1 gene possesses a number of binding sites for transcription factors like Nrf2, NFκB, AP1 and CREB [27]. To explore whether TauCl stimulated the nuclear translocation of HO-1-related transcription factors and enhanced their DNA binding, Western blot analysis of cytosolic and nuclear extracts as well as the gel shift assays were conducted. As shown in Fig. 4A and B, TauCl increased the nuclear accumulation of Nrf2, which reached at a maximum at 30 min after the TauCl treatment and decreased thereafter. TauCl also increased the cytosolic Nrf2 expression with time. However, the nuclear accumulation of Nrf2 did not quite reach statistical significance, comparing directly using the paired Student's t-test (Fig. 4B). In the EMSA conducted using a binding sequence for Nrf2, no binding complex was detected in the untreated RAW 264.7 cells, but the nuclear translocation and DNA binding of Nrf2 was increased by TauCl treatment. The ARE-binding complex appeared at 20 min of TauCl treatment and was maintained through 60 min (Fig. 4C). DNA binding activity for NFκB, AP-1 and CREB was also determined, and there was no difference after TauCl treatment (Fig. 4C, and data not shown). To further verify the involvement of Nrf2 in the TauCl-induced upregulation of HO-1 expression, we knocked down Nrf2 expression using its specific siRNA and examined HO-1 expression (Fig. 5). TauClinduced HO-1 expression was reduced to 29% of control by transfection with Nrf2-siRNA (Fig. 5B). This finding confirmed that TauCl induced HO-1 expression through Nrf2 activation in RAW 264.7 cells. 4. Discussion When neutrophils are stimulated, they undergo a respiratory burst − to produce O− 2 and about 30% of the produced O2 is converted to HOCl by the MPO system [28]. The highly toxic HOCl reacts with free amino acids such as lysine, glycine, histamine and taurine to form Nchloramines. TauCl is the primary N-chloramine produced in neutrophils [29,30], and the released TauCl is transported into surrounding cells in a Na+ and Cl− dependent manner [31,32]. Taurine protects neutrophils from the toxicity of HOCl by stoichiometric reaction and results in the generation of TauCl. TauCl was previously thought to be eliminated in urine. However, TauCl has recently been shown to protect the inflammatory tissues by suppres-
Fig. 4. TauCl induces nuclear translocation and ARE binding of Nrf2. The RAW 264.7 cells were stimulated with 0.5 mM TauCl for various time periods and cytosolic and nuclear lysates were prepared. (A) Immunoblots of cytosolic and nuclear lysates were probed with the Nrf2-specific antibody. The blots are representative of three independent experiments. (B) Bar graphs represent the relative level of nuclear Nrf2 based on densitometry of immunoblot signals normalized to lamin B (n = 3). (C) The nuclear extract isolated from TauCl-treated cells was subjected to EMSA and Nrf2 and NFκB activation was detected (n = 4).
sing the production of various inflammatory mediators such as, O− 2 , NO, TNF-α, ILs and prostaglandins. However, the mechanisms underlying these cytoprotective effects afforded by TauCl have not been fully understood. Heme oxygenase catalyzes the first and rate-limiting step in the oxidative degradation of potentially toxic free-heme to yield ferrous iron, CO and also biliverdin which is subsequently converted to the anti-oxidant bilirubin [33]. Between the two HO isozymes expressed in mammalian cells, HO-2 is expressed constitutively while the inducible HO-1 is expressed upon exposure to a free-heme, cell permeable substrate of HO, or a wide variety of stimuli that cause oxidative stress. The induction of HO-1 expression and the resulting elevation of HO activity increase CO production but decrease the production of inflammatory mediators. Enhancement of HO activity leads to protect the inflammatory tissues from the cytotoxicity of oxidative stress via rapid removal of free-heme to produce the antioxidants like biliverdin/bilirubin and CO. In particular, CO binds avidly to the reduced iron (Fe++) contained in heme-containing enzymes such as NADPH oxidase and NOS and inhibits the ironcatalyzed electron transfer required to produce O− 2 and NO, respectively. In addition, CO is also known to modulate many other cellular functions such as leukocyte adhesion, apoptosis, and production of cytokines like TNF-α, IL-1β, MIP-1 and IL-10 [34,35]. Therefore, enhanced production of CO and bilirubin by TauCl-derived induction of HO-1 and elevation of HO activity can protect cells from the cytotoxicity of ROS by inhibiting further production of O− 2 and NO.
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Fig. 5. The targeted disruption of Nrf2 diminishes TauCl-induced HO-1 expression. RAW 264.7 cells were transfected with Nrf2 specific siRNA prior to 0.5 mM TauCl treatment. (A) Nrf2-dependent HO-1 expression by siRNA was determined by immunoblotting. Representative blots are shown (n = 3). (B) HO-1 bands were quantitated using densitometry and values are expressed as the ratio of HO-1 to β-actin (n = 3). Data are expressed as the mean ± SD, ⁎⁎p b 0.01.
In the present study, we attempted to explore the possible molecular mechanisms underlying the cytoprotective effects provided by TauCl, focusing on the upregulation of HO-1 expression and increase of HO activity. Exposure of RAW 264.7 cells up to 0.7 mM TauCl enhanced the expression of HO-1 mRNA, HO-1 protein and HO activity in a dose-dependent manner without causing significant toxicity. This result is consistent with a recent study by Olszanecki and Marcinkiewicz [22], who reported that HO-1 is induced by TauCl and taurine bromamine in J774.2 macrophages. Thus, TauCl elevates HO activity and removes toxic free-heme and at the same time, produces more antioxidants like biliverdin/bilirubin. In previous studies, we showed that CO inhibited overproduction of O− 2 and NO [17,18], and TauCl inhibited the overproduction of O− 2 and NO in the PMA or LPSstimulated phagocytes [26,36]. Thus, induction of HO-1 expression afforded protection of activated phagocytes against cytotoxicity − caused by O− [37]. Here, we 2 , NO and highly reactive ONOO demonstrate for the first time that TauCl generates ROS in macrophages and that TauCl depletes the intracellular GSH level initially and subsequently leads to increase HO-1 expression via activation of Nrf2/ ARE system. In support of these results, the increased HO-1 expression caused by TauCl was abolished by exposure to NAC (Fig. 3C). This result suggests that the ROS derived from TauCl causes oxidation and depletion of GSH to drive the activation of Nrf2/ARE system for the induction of HO-1 expression. In this connection, TauCl protects cells from H2O2-induced cell death by inducing anti-oxidant enzymes including HO-1, peroxiredoxin and thioredoxin [21]. Many reports suggest that HO-1 induction is dependent on the activation or nuclear translocation of Nrf2. The promoter region of the HO-1 gene contains multiple copies of ARE that bind the translocated Nrf2 and promote the upregulation of HO-1 expression. Thus, we tested whether TauCl increased HO-1 expression via enhancing the Nrf2 translocation to the nucleus and increased the DNA binding. As shown in Fig. 4, TauCl enhanced the nuclear translocation of Nrf2 and
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its binding to ARE. It also has been reported that HOCl, a highly oxidizing cytotoxic precursor of TauCl, enhances the nuclear translocation of Nrf2 [38]. The activation of Nrf2 was sustained between 20– 60 min of TauCl treatment and declined to basal level thereafter (data not shown). This differs from the results published by other authors showing that Nrf2 binding to ARE was detected at 4–7 h following treatment with ONOO− or LPS [37,39,40]. TauCl did not increase the activation of NFκB, AP-1 and CREB (Fig. 4A, and data not shown), albeit the promoter region of HO-1 gene is known to possess the binding sites for these transcription factors [27]. Several upstream signaling pathways such as MAPKs, PI3K/Akt, and Ras are known to activate Nrf2 and increase HO-1 expression [37,41–44]. Kontny et al. [45] reported that TauCl had no effect on ERK activation in Jurkat cells, while Midwinter et al. [46] reported that TauCl induced ERK activation in HUVEC. TauCl showed no effect on the phosphorylation of MAPKs and Akt in the present study (data not shown). Combined, these results suggest that TauCl induces HO-1 expression through mechanisms other than the MAPKs and Akt signaling pathways in macrophages. In summary, TauCl, produced following detoxification of HOCl by the stored taurine and released by the activated neutrophils, generated ROS that could oxidize and deplete intracellular GSH level initially in macrophages. Perhaps this lowered GSH level caused stimulation of Nrf2/ARE system and induced HO-1 expression and enhanced HO activity. Thus, TauCl-induced HO-1 diminished the production of additional inflammatory mediators to protect the surrounding macrophages and tissues at the inflammatory site from cytotoxicity caused by inflammation. Acknowledgments We are grateful to Dr. Y.J. Surh at Seoul National University for his helpful support and Mr. M. Azam for his technical assistance. This study was supported by The Korea Research Foundation Grant (KRF2008-531-C00051) and Nano R&D program through the KOSEF (M10642040001-07N4204-00110). References [1] Vinton NE, Laidlaw SA, Ament ME, Kopple JD. Taurine concentrations in plasma and blood cells of patients undergoing long-term parenteral nutrition. Am J Clin Nutr 1986;44:398–404. [2] Learn DB, Fried VA, Thomas EL. Taurine and hypotaurine content of human leukocytes. J Leukoc Biol 1990;48:174–82. [3] Fukuda K, Hirai Y, Yoshida H, Nakajima T, Usui T. Free amino acid content of lymphocytes and granulocytes compared. Clin Chem 1982;28:1758–61. [4] Thomas EL, Grisham MB, Jefferson MM. Myeloperoxidase-dependent effect of amines on functions of isolated neutrophils. J Clin Invest 1983;72:441–54. [5] Pero RW, Sheng Y, Olsson A, Bryngelsson C, Lund-Pero M. Hypochlorous acid/Nchloramines are naturally produced DNA repair inhibitors. Carcinogenesis 1996;17:13–8. [6] Park E, Schuller-Levis G, Quinn MR. Taurine chloramine inhibits production of nitric oxide and TNF-alpha in activated RAW 264.7 cells by mechanisms that involve transcriptional and translational events. J Immunol 1995;154:4778–84. [7] Marcinkiewicz J, Grabowska A, Bereta J, Stelmaszynska T. Taurine chloramine, a product of activated neutrophils, inhibits in vitro the generation of nitric oxide and other macrophage inflammatory mediators. J Leukoc Biol 1995;58:667–74. [8] Kim C, Park E, Quinn MR, Schuller-Levis G. The production of superoxide anion and nitric oxide by cultured murine leukocytes and the accumulation of TNF-alpha in the conditioned media is inhibited by taurine chloramine. Immunopharmacology 1996;34:89–95. [9] Park E, Jia J, Quinn MR, Schuller-Levis G. Taurine chloramine inhibits lymphocyte proliferation and decreases cytokine production in activated human leukocytes. Clin Immunol 2002;102:179–84. [10] Kim KS, Park EK, Ju SM, Jung HS, Bang JS, Kim C, et al. Taurine chloramine differentially inhibits matrix metalloproteinase 1 and 13 synthesis in interleukin1beta stimulated fibroblast-like synoviocytes. Arthritis Res Ther 2007;9:R80. [11] Chorazy M, Kontny E, Marcinkiewicz J, Maslinski W. Taurine chloramine modulates cytokine production by human peripheral blood mononuclear cells. Amino Acids 2002;23:407–13. [12] Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 1997;37:517–54. [13] Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science 1987;235:1043–6.
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[14] Wagener FA, Volk HD, Willis D, Abraham NG, Soares MP, Adema GJ, et al. Different faces of the heme–heme oxygenase system in inflammation. Pharmacol Rev 2003;55:551–71. [15] Jeong WS, Jun M, Kong AN. Nrf2: a potential molecular target for cancer chemoprevention by natural compounds. Antioxid Redox Signal 2006;8:99–106. [16] Lee JS, Surh YJ. Nrf2 as a novel molecular target for chemoprevention. Cancer Lett 2005;224:171–84. [17] Srisook K, Cha YN. Biphasic induction of heme oxygenase-1 expression in macrophages stimulated with lipopolysaccharide. Biochem Pharmacol 2004;68: 1709–20. [18] Srisook K, Han SS, Choi HS, Li MH, Ueda H, Kim C, et al. CO from enhanced HO activity or from CORM-2 inhibits both O− 2 and NO production and downregulates HO-1 expression in LPS-stimulated macrophages. Biochem Pharmacol 2006;71: 307–18. [19] Taille C, El-Benna J, Lanone S, Boczkowski J, Motterlini R. Mitochondrial respiratory chain and NAD(P)H oxidase are targets for the antiproliferative effect of carbon monoxide in human airway smooth muscle. J Biol Chem 2005;280: 25350–60. [20] Nakahira K, Kim HP, Geng XH, Nakao A, Wang X, Murase N, et al. Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts. J Exp Med 2006;203:2377–89. [21] Sun Jang J, Piao S, Cha YN, Kim C. Taurine chloramine activates Nrf2, increases HO1 expression and protects cells from death caused by hydrogen peroxide. J Clin Biochem Nutr 2009;45:37–43. [22] Olszanecki R, Marcinkiewicz J. Taurine chloramine and taurine bromamine induce heme oxygenase-1 in resting and LPS-stimulated J774.2 macrophages. Amino Acids 2004;27:29–35. [23] Thomas EL, Grisham MB, Jefferson MM. Preparation and characterization of chloramines. Methods Enzymol 1986;132:569–85. [24] Yamauchi A, Kim C, Li S, Marchal CC, Towe J, Atkinson SJ, et al. Rac2-deficient murine macrophages have selective defects in superoxide production and phagocytosis of opsonized particles. J Immunol 2004;173:5971–9. [25] Kim C, Dinauer MC. Rac2 is an essential regulator of neutrophil nicotinamide adenine dinucleotide phosphate oxidase activation in response to specific signaling pathways. J Immunol 2001;166:1223–32. [26] Kim JW, Kim C. Inhibition of LPS-induced NO production by taurine chloramine in macrophages is mediated through Ras-ERK-NF-kappaB. Biochem Pharmacol 2005;70: 1352–60. [27] Inamdar NM, Ahn YI, Alam J. The heme-responsive element of the mouse heme oxygenase-1 gene is an extended AP-1 binding site that resembles the recognition sequences for MAF and NF-E2 transcription factors. Biochem Biophys Res Commun 1996;221:570–6. [28] Kettle AJ, Winterbourn CC. Mechanism of inhibition of myeloperoxidase by antiinflammatory drugs. Biochem Pharmacol 1991;41:1485–92. [29] Carr AC, Hawkins CL, Thomas SR, Stocker R, Frei B. Relative reactivities of Nchloramines and hypochlorous acid with human plasma constituents. Free Radic Biol Med 2001;30:526–36. [30] Peskin AV, Midwinter RG, Harwood DT, Winterbourn CC. Chlorine transfer between glycine, taurine, and histamine: reaction rates and impact on cellular reactivity. Free Radic Biol Med 2005;38:397–405.
[31] Kim C, Chung JK, Jeong JM, Chang YS, Lee YJ, Kim YJ, et al. Uptake of taurine and taurine chloramine in murine macrophages and their distribution in mice with experimental inflammation. Adv Exp Med Biol 1998;442:169–76. [32] Tallan HH, Jacobson E, Wright CE, Schneidman K, Gaull GE. Taurine uptake by cultured human lymphoblastoid cells. Life Sci 1983;33:1853–60. [33] Tenhunen R, Marver HS, Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci U S A 1968;61:748–55. [34] Petrache I, Otterbein LE, Alam J, Wiegand GW, Choi AM. Heme oxygenase-1 inhibits TNF-alpha-induced apoptosis in cultured fibroblasts. Am J Physiol Lung Cell Mol Physiol 2000;278:L312–9. [35] Otterbein LE, Bach FH, Alam J, Soares M, Lu HT, Wysk M, et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 2000;6:422–8. [36] Choi HS, Cha YN, Kim C. Taurine chloramine inhibits PMA-stimulated superoxide production in human neutrophils perhaps by inhibiting phosphorylation and translocation of p47(phox). Int Immunopharmacol 2006;6:1431–40. [37] Li MH, Jang JH, Na HK, Cha YN, Surh YJ. Carbon monoxide produced by heme oxygenase-1 in response to nitrosative stress induces expression of glutamate– cysteine ligase in PC12 cells via activation of phosphatidylinositol 3-kinase and Nrf2 signaling. J Biol Chem 2007;282:28577–86. [38] Pi J, Zhang Q, Woods CG, Wong V, Collins S, Andersen ME. Activation of Nrf2mediated oxidative stress response in macrophages by hypochlorous acid. Toxicol Appl Pharmacol 2008;226:236–43. [39] Balogun E, Hoque M, Gong P, Killeen E, Green CJ, Foresti R, et al. Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidantresponsive element. Biochem J 2003;371:887–95. [40] Srisook K, Cha YN. Super-induction of HO-1 in macrophages stimulated with lipopolysaccharide by prior depletion of glutathione decreases iNOS expression and NO production. Nitric Oxide 2005;12:70–9. [41] Chen CY, Jang JH, Li MH, Surh YJ. Resveratrol upregulates heme oxygenase-1 expression via activation of NF-E2-related factor 2 in PC12 cells. Biochem Biophys Res Commun 2005;331:993–1000. [42] Gong P, Hu B, Cederbaum AI. Diallyl sulfide induces heme oxygenase-1 through MAPK pathway. Arch Biochem Biophys 2004;432:252–60. [43] Kietzmann T, Samoylenko A, Immenschuh S. Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes. J Biol Chem 2003;278:17927–36. [44] Joung EJ, Li MH, Lee HG, Somparn N, Jung YS, Na HK, et al. Capsaicin induces heme oxygenase-1 expression in HepG2 cells via activation of PI3K–Nrf2 signaling: NAD (P)H:quinone oxidoreductase as a potential target. Antioxid Redox Signal 2007;9: 2087–98. [45] Kontny E, Maslinski W, Marcinkiewicz J. Anti-inflammatory activities of taurine chloramine: implication for immunoregulation and pathogenesis of rheumatoid arthritis. Adv Exp Med Biol 2003;526:329–40. [46] Midwinter RG, Peskin AV, Vissers MC, Winterbourn CC. Extracellular oxidation by taurine chloramine activates ERK via the epidermal growth factor receptor. J Biol Chem 2004;279:32205–11.
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p
Taurine chloramine induces heme oxygenase-1 expression via Nrf2 activation in murine macrophages Chaekyun Kim a,b,c,d,⁎, Jin Sun Jang a,b,c, Mi-Ran Cho a, Santosh R. Agarawal c, Young-Nam Cha b a
Laboratory for Leukocyte Signaling Research, Inha University School of Medicine, Incheon 400-712, Republic of Korea Department of Pharmacology, Inha University School of Medicine, Incheon 400-712, Republic of Korea BK21 Program, Inha University School of Medicine, Incheon 400-712, Republic of Korea d Inha Research Institute for Medical Science, Inha University School of Medicine, Incheon 400-712, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 26 October 2009 Received in revised form 11 December 2009 Accepted 29 December 2009 Keywords: Taurine chloramine Heme oxygenase Macrophages Nrf2 Reactive oxygen species
a b s t r a c t Taurine chloramine (TauCl) is produced abundantly in activated neutrophils by a reaction between the stored taurine and the newly produced HOCl by the myeloperoxidase system, and is much less oxidizing or toxic than HOCl. TauCl has been shown to provide cytoprotection against inflammatory tissue injury by inhibiting the overproduction of inflammatory mediators. The result of this study shows that TauCl upregulated the expression of heme oxygenase (HO)-1 and increased HO activity in RAW 264.7 macrophages, while taurine had no effect. TauCl by itself generated reactive oxygen species (ROS) in macrophages and diminished total glutathione (GSH) level initially. TauCl increased the nuclear translocation of NF-E2-related factor 2 (Nrf2) and enhanced its binding to the anti-oxidant response element (ARE). This, in turn, was responsible for the upregulation of HO-1 expression. In summary, TauCl generated ROS in RAW 264.7 macrophages and decreased cellular GSH level initially. This was responsible for the nuclear translocation of Nrf2 and its binding to ARE promoted the expression of HO-1 and increased HO activity. Thus, TauCl-derived elevation of HO activity may play an essential role in the adaptive cytoprotection of inflammatory tissues. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Taurine, the decarboxylation product of cysteine, is one of the most abundant free amino acids in phagocytic cells and is not incorporated into proteins [1–3]. Taurine, stored in neutrophils up to 50 mM, reacts stoichiometrically with HOCl, an antibacterial oxidant produced from H2O2 by the myeloperoxidase (MPO) in activated neutrophils. This results in the generation of taurine chloramine (TauCl). The production of TauCl by activated neutrophils has been well established in vitro, so that 2 × 106 of human neutrophils are known to produce up to 100 μM TauCl in the presence of high taurine content [4,5]. Taurine is reported to protect phagocytic cells against the injury caused by inflammatory overproduction of HOCl. Cytoprotection of phagocytic cells provided by exogenously added taurine results primarily from its efficient elimination of highly toxic HOCl and generation of less toxic TauCl. Furthermore, TauCl is also known to exert anti-inflammatory effects on its own. TauCl has been demonstrated to suppress the production of many inflammatory intermedi⁎ Corresponding author. Laboratory for Leukocyte Signaling Research, Inha University School of Medicine, 7-206 3rd St, Shinheung-dong, Jung-gu, Incheon 400-712, Republic of Korea. Tel.: +82 32 890 0976; fax: +82 32 887 7488. E-mail address: [email protected] (C. Kim). 1567-5769/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.12.018
ates such as superoxide anion (O− 2 ), nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, -2, -6, -8, and -10, prostaglandin E2 (PGE2), macrophage inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1 and -2, and matrix metalloproteinase (MMP) in the stimulated phagocytes [6–11]. In spite of increasing evidences showing that TauCl diminishes the production of inflammatory mediators and protects cells at the inflammatory site from inflammatory cytotoxicity, its underlying cytoprotective mechanism has not yet been elucidated. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme and provides potent anti-inflammatory and anti-oxidant functions. Cells that overexpress HO-1 are protected from oxidative injury and can resist subsequent cytotoxic oxidative challenges. HO catalyzes the rate-limiting step in the oxidative degradation of potentially damaging free-heme into equimolar amounts of biliverdin/bilirubin, carbon monoxide (CO) and free iron [12]. Biliverdin and bilirubin can scavenge peroxy radicals and serve as strong anti-oxidants [13], and the CO arising from heme degradation inhibits inflammatory responses by blocking the expression of pro-inflammatory genes and protects cells against apoptosis and necrosis caused by oxidants [14]. The increased HO-1 expression occurring widely in response to oxidative stress can thus contribute to the adaptive increase of cellular anti-oxidant and chemoprotective defense systems. Upregulation of
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HO-1 is mediated by activation of relevant redox-sensitive cytoplasmic transcriptional regulators, such as nuclear factor E2-related factor 2 (Nrf2), activator protein (AP)-1, nuclear factor kappa B (NFκB), and cAMP-responsive element-binding protein (CREB) [15,16]. Nrf2 which belongs to the CNC-bZIP (cap‘n'collar subfamily of basic leucine-zipper) is kept inactive in association with Keap1 (Kelch-like ECH associating protein 1) in normal conditions. Upon oxidative stimulation, Nrf2 dissociates from Keap1, translocates into the nucleus and binds to the anti-oxidant response element (ARE) in the promoter region of phase II detoxifying or anti-oxidant enzymes such as NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferase (GST), glutathione peroxidase (GPx), peroxiredoxin I (Prx-1) and HO-1 [15,16]. Upon inflammatory stimulation of phagocytic cells, O− 2 , NO and CO are overproduced in sequence. In previous studies, we demonstrated that O− 2 overproduction triggered by LPS treatment enhanced the expression of iNOS and overproduction of NO in macrophages [17]. Such overproduction of O− 2 and NO promoted production of peroxynitrite (ONOO−), a highly cytotoxic oxidant that is known to release heme from intracellular heme proteins. On the other hand, the ONOO− also increased the expression of HO-1 and elevated HO activity. This leads to enhance oxidative degradation of free-heme to overproduce CO. This overproduced CO has been shown to inhibit the production of O− 2 in the PMA-stimulated neutrophils and also in the LPS-stimulated macrophages by inhibiting the NADPH oxidase activity [18–20]. Furthermore, the CO derived from elevated HO activity also inhibited the upregulation of inducible nitric oxide synthase (iNOS) expression as well as the overproduction of NO in the LPS-stimulated macrophages [18]. Thus, induction of HO-1 and elevation of HO activity and the resulting overproduction of CO afforded protection against the cytotoxicity caused by excessive oxidative stress in activated phagocytes [21]. In this context, the ability of TauCl to induce the expression of HO-1 in macrophages [22] provided the basis to explore the molecular mechanisms involved in the cytoprotective effect of TauCl in phagocytic cells. Here we show that TauCl induces the expression of HO-1 and elevates HO activity by producing ROS, and thus stimulating the nuclear translocation of Nrf2 and enhancing its binding to ARE. Our results suggest that upregulation of HO-1 expression and elevation of HO activity induced by TauCl is essential for the adaptive cytoprotection of inflammatory tissues by suppressing additional overproduction of NO and O− 2 from activated phagocytic cells. 2. Materials and methods 2.1. Antibodies and reagents Antibodies against Nrf2 (Santa Cruz, Santa Cruz, CA), iNOS (Transduction Laboratories, Lexington, KY), and HO-1 (Stressgen, Victoria, Canada) were purchased from commercial sources. Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from HyClone (Logan, UT). The oligonucleotides were purchased from Bioneer (Daejeon, Korea) and Santa Cruz Biotechnology (Santa Cruz, CA), and [γ-32P]ATP was from NEN (Boston, MA). Other routinely used chemicals were purchased from Sigma (St. Louis, MO). TauCl was synthesized freshly on the day of use by adding 400 mM NaOCl (Aldrich, Milwaukee, MI) to 410 mM taurine. The authenticity of TauCl formation was monitored by its UV absorption at 200–400 nm [23].
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2.3. Preparation of bone marrow-derived macrophages Murine bone marrow-derived macrophages (BMDM) were harvested from BALB/c mice (Orient Bio, Seoul, Korea) as described [24]. Following lysis of red blood cells, murine bone marrow cells were suspended in macrophage culture medium (α-MEM containing 20% FBS, 20 ng/ml recombinant human macrophage colony stimulating factor (M-CSF), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) and cultured in 5% CO2 incubator kept at 37 °C. Adherent cells were discarded twice at 4 and 16 h by transferring the suspended cells to new dishes. At 3 and 6 days during the culture, the medium was replaced with fresh medium. BMDM were harvested at 7th day by removing the adherent cells from culture dishes with cell dissociation buffer. For experiments, BMDM were treated with 1 μg/ml LPS and TauCl (0.5 and 0.7 mM) at 12 h before harvest. 2.4. RT-PCR Total RNA was extracted using the TRI reagent (MRC, Cincinnati, OH) according to the manufacturer's instructions and reverse transcription of 500 ng total RNA was performed according to the instructions provided by TaKaRa (Shuzo, Shiga, Japan). PCR amplification of mRNA was carried out using the following primers (forward and reverse, respectively), HO-1, 5′-TGA AGG AGG CCA CCA AGG AGG-3′ and 5′-AGA GGT CAC CCA GGT AGC GGG-3′ (375 bp); iNOS, 5′AGA CTG GAT TTG GCT GGT CCC TCC-3′ and 5′-AGA ACT GAG GGT ACA TGC TGG AGC C-3′ (527 bp); GAPDH, 5′-GTC GGT GTG AAC GGA TTT G-3′ and 5′-ACA AAC ATG GGG GCA TCA G-3′ (386 bp). 2.5. Western blot analysis Cell lysates were prepared from RAW 264.7 cells as described previously [25] and 20–30 μg of total proteins were electrophoresed on SDS-PAGE. The resolved proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (BioRad, Hercules, CA), and the blots were probed with specific antibodies and developed by using the ECL method (Amersham, Arlington Heights, IL). 2.6. Heme oxygenase activity assay The HO activity was determined by measuring the amount of bilirubin produced from hemin added as the substrate as described previously [17]. The amount of bilirubin formed was quantitated from the difference in absorbance between 464 nm and 530 nm (extinction coefficient; 40 mM− 1 cm− 1 for bilirubin). 2.7. Measurement of ROS production using H2-DCFDA Production of ROS in RAW 264.7 cells treated with TauCl and taurine was monitored by employing 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCFDA). The cell permeable H2-DCFDA rapidly diffuses through the cell membrane and then is hydrolyzed by esterases to H2-DCF. The oxidation of H2-DCF by ROS leads to a fluorescent DCF. Cells that adhered on the cover slide were rinsed with Krebs ringer solution and loaded with 10 μM H2-DCFDA. After 15 min of incubation at 37 °C, the cells were examined under a fluorescence microscope set at 488 nm for excitation and 530 nm for emission (Karl Zeiss, Oberkochen, Germany), and analyzed on FACScan cytometer (BD Bioscience, San Jose, CA) using CellQuest software (BD Bioscience).
2.2. RAW 264.7 cell culture 2.8. Total GSH level RAW 264.7 cells, a murine macrophage cell line obtained from ATCC (Manassas, VA), were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin and maintained at 37 °C in a 5% CO2 incubator.
RAW 264.7 cells were collected after addition of 5% 5-sulfosalicylic acid and 0.2% Triton X-100. Cells were then freeze–thawed three times, sonicated and centrifuged. The total amount of GSH present in
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the acidic supernatant was determined as described by Srisook et al [17]. 2.9. Electrophoretic mobility shift assay (EMSA) to detect Nrf2–ARE binding Nuclear extracts were prepared by sequential cell lysis and nuclear lysis as reported previously [26]. Protein concentration in the nuclear extract was determined by using the Bradford method (BioRad, Hercules, CA). DNA binding activity for Nrf2, NFκB, AP-1 and CREB was determined. The DNA–protein complexes were resolved by 5% nondenaturing PAGE as reported previously [26] to detect the changes of intensity for the labeled oligonucleotides binding to specific transcription factors. The oligonucleotides used to detect the activities of Nrf2, NFκB, AP-1 and CREB are as follows: Nrf2, 5′-TGG GGA ACC TGT GCT GAG TCA CTG GAG-3′; NFκB, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′; AP-1, 5′-CTA GTG ATG AGT CAG CCG GAT C-3′; and CREB, 5′-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3′. 2.10. Nrf2 siRNA knockdown To knockdown the Nrf2 expression, RAW 264.7 cells were transfected with 100 nM siRNA directed against murine Nrf2 (Dharmacon, Lafayette, CO) or a non-targeting scRNA by using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the
manufacturer's instructions. Seventy-two hours after transfection with Nrf2 siRNA, the lysates were prepared from cells and the knockdown of Nrf2 was confirmed by immunoblot analysis. 2.11. Statistical analysis The two-tailed Student's t-test (paired) was performed using Microsoft Excel software (Redmond, WA). Data were expressed as mean ± SD, and a p value b 0.05 was considered significant. 3. Results 3.1. Induction of HO-1 expression and HO activity The induction of HO-1 expression has been demonstrated to decrease the inflammatory response as well as the apoptotic death of phagocytic cells via rapid removal of the potentially toxic free-heme released from diverse heme-containing proteins by oxidative stress. To determine the effect of TauCl on HO-1 expression, RAW 264.7 cells were treated with TauCl for various time periods. TauCl enhanced expression of HO-1 mRNA beginning at 2 h and continued through 20 h, while taurine had no effect (Fig. 1A, B). Induction of HO-1 mRNA expression by TauCl was started earlier than that induced by LPS. While LPS enhanced mRNA expression of both iNOS and HO-1, TauCl did not induce the expression of iNOS mRNA. TauCl dose-dependently increased the expression of HO-1 protein beginning at 6 h and reached
Fig. 1. TauCl induces HO-1 expression in RAW 264.7 cells. (A) RT-PCR analysis of HO-1 and iNOS mRNA was performed. RAW 264.7 cells were incubated with 1 μg/ml LPS or 0.5 and 0.7 mM TauCl for 0, 2, 4, 6, 8, 12 and 20 h (n = 3). (B) HO-1 mRNA expression by various concentrations of TauCl at 12 h was analyzed (n = 5). GAPDH was used as a control for equal loading of RNA. (C and D) The effect of TauCl on HO-1 protein expression in RAW 264.7 cells was determined (n = 5). (E) The effect of TauCl on HO-1 expression was determined in murine BMDM (n = 3). The immunoblots were re-probed with β-actin antibody to show equal loading.
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Fig. 2. TauCl increases HO activity in RAW 264.7 cells. (A) The effect of TauCl on HO activity was determined. A microsomal fraction was isolated at 12 h of TauCl treatment and HO activity was determined by the production of bilirubin (n = 3). (B) A microsomal fraction was isolated at 4, 12 and 24 h after treatment (n = 3). Data are expressed as the mean ± SD, ⁎p b 0.05 and ⁎⁎p b 0.01 compared to control.
to a maximum at 12 h in RAW 264.7 cells (Fig. 1C, D). To further confirm the HO-1 inducing effect of TauCl in murine BMDM, mouse BMDM were treated with TauCl. Consistent with that observed in RAW 264.7 cells, TauCl increased the expression of HO-1 in murine BMDM in a dose-dependent manner (Fig. 1E). HO catalyzes the oxidative degradation of free-heme to yield equimolar amount of ferrous iron, CO and biliverdin which is then converted to bilirubin. HO activity was determined by measuring the bilirubin production. TauCl dose-dependently increased the produc-
tion of bilirubin (Fig. 2A). The production of bilirubin reached a maximum level at 12 h after TauCl treatment, and it was increased slightly more by costimulation with LPS (Fig. 2B). 3.2. TauCl triggers ROS production To test whether TauCl induces HO-1 expression via ROS production, we measured ROS production by employing the H2-DCFDA fluorescence staining assay. As shown in Fig. 3A and B, TauCl enhanced
Fig. 3. TauCl generates ROS and reduces total glutathione level. (A) RAW 264.7 cells were treated with 1 μg/ml LPS, 0.7 mM taurine and TauCl for 10 min. ROS production was determined by the H2-DCFDA assay as described in Section 2. The representative picture of DCF-derived fluorescence in RAW 264.7 cells (n = 5). (B) Flow cytometric analysis was performed after H2-DCFDA staining (n = 3). (C) The effect of NAC treatment on TauCl-induced HO-1 expression (n = 3). (D) The total glutathione level was determined at the indicated times after 0.7 mM TauCl treatment (n = 3). Data are expressed as the mean ± SD, ⁎p b 0.05 compared to the glutathione level at 0 h.
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ROS production in RAW 264.7 cells, while taurine had no such effect. The production of ROS by 0.7 mM TauCl was greater than that produced by LPS (Fig. 3B). The addition of an NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) and catalase diminished the accumulation of ROS (data not shown). To confirm that the increased ROS production influences on HO-1 expression, we treated cells with 2.5 mM N-acetyl L-cysteine (NAC) for 1 h before the TauCl treatment. NAC completely abolished TauCl-induced HO-1 expression (Fig. 3C). This suggests that TauCl induces HO-1 expression through ROS. 3.3. TauCl alters GSH level The intracellular GSH level was determined following treatment with TauCl. Acidic protein-free supernatants were prepared from the harvested cells at different time points following treatment with TauCl. The intracellular level of total GSH decreased significantly at 30 min (70% of control level). Subsequently, the depleted GSH level began to recover at 1 h, reaching the control level by 2 h, and further increasing above the control level at 4 h (Fig. 3D). TauCl lowered the GSH/GSSG ratio at 30 min and this lowering peaked at 1 h, subsequently recovering to control level at 2 h (data not shown). 3.4. TauCl upregulates HO-1 expression via activation of Nrf2 The nuclear translocation and DNA binding of redox-sensitive transcription factor are important mechanisms that contribute toward HO-1 expression. The promoter region of HO-1 gene possesses a number of binding sites for transcription factors like Nrf2, NFκB, AP1 and CREB [27]. To explore whether TauCl stimulated the nuclear translocation of HO-1-related transcription factors and enhanced their DNA binding, Western blot analysis of cytosolic and nuclear extracts as well as the gel shift assays were conducted. As shown in Fig. 4A and B, TauCl increased the nuclear accumulation of Nrf2, which reached at a maximum at 30 min after the TauCl treatment and decreased thereafter. TauCl also increased the cytosolic Nrf2 expression with time. However, the nuclear accumulation of Nrf2 did not quite reach statistical significance, comparing directly using the paired Student's t-test (Fig. 4B). In the EMSA conducted using a binding sequence for Nrf2, no binding complex was detected in the untreated RAW 264.7 cells, but the nuclear translocation and DNA binding of Nrf2 was increased by TauCl treatment. The ARE-binding complex appeared at 20 min of TauCl treatment and was maintained through 60 min (Fig. 4C). DNA binding activity for NFκB, AP-1 and CREB was also determined, and there was no difference after TauCl treatment (Fig. 4C, and data not shown). To further verify the involvement of Nrf2 in the TauCl-induced upregulation of HO-1 expression, we knocked down Nrf2 expression using its specific siRNA and examined HO-1 expression (Fig. 5). TauClinduced HO-1 expression was reduced to 29% of control by transfection with Nrf2-siRNA (Fig. 5B). This finding confirmed that TauCl induced HO-1 expression through Nrf2 activation in RAW 264.7 cells. 4. Discussion When neutrophils are stimulated, they undergo a respiratory burst − to produce O− 2 and about 30% of the produced O2 is converted to HOCl by the MPO system [28]. The highly toxic HOCl reacts with free amino acids such as lysine, glycine, histamine and taurine to form Nchloramines. TauCl is the primary N-chloramine produced in neutrophils [29,30], and the released TauCl is transported into surrounding cells in a Na+ and Cl− dependent manner [31,32]. Taurine protects neutrophils from the toxicity of HOCl by stoichiometric reaction and results in the generation of TauCl. TauCl was previously thought to be eliminated in urine. However, TauCl has recently been shown to protect the inflammatory tissues by suppres-
Fig. 4. TauCl induces nuclear translocation and ARE binding of Nrf2. The RAW 264.7 cells were stimulated with 0.5 mM TauCl for various time periods and cytosolic and nuclear lysates were prepared. (A) Immunoblots of cytosolic and nuclear lysates were probed with the Nrf2-specific antibody. The blots are representative of three independent experiments. (B) Bar graphs represent the relative level of nuclear Nrf2 based on densitometry of immunoblot signals normalized to lamin B (n = 3). (C) The nuclear extract isolated from TauCl-treated cells was subjected to EMSA and Nrf2 and NFκB activation was detected (n = 4).
sing the production of various inflammatory mediators such as, O− 2 , NO, TNF-α, ILs and prostaglandins. However, the mechanisms underlying these cytoprotective effects afforded by TauCl have not been fully understood. Heme oxygenase catalyzes the first and rate-limiting step in the oxidative degradation of potentially toxic free-heme to yield ferrous iron, CO and also biliverdin which is subsequently converted to the anti-oxidant bilirubin [33]. Between the two HO isozymes expressed in mammalian cells, HO-2 is expressed constitutively while the inducible HO-1 is expressed upon exposure to a free-heme, cell permeable substrate of HO, or a wide variety of stimuli that cause oxidative stress. The induction of HO-1 expression and the resulting elevation of HO activity increase CO production but decrease the production of inflammatory mediators. Enhancement of HO activity leads to protect the inflammatory tissues from the cytotoxicity of oxidative stress via rapid removal of free-heme to produce the antioxidants like biliverdin/bilirubin and CO. In particular, CO binds avidly to the reduced iron (Fe++) contained in heme-containing enzymes such as NADPH oxidase and NOS and inhibits the ironcatalyzed electron transfer required to produce O− 2 and NO, respectively. In addition, CO is also known to modulate many other cellular functions such as leukocyte adhesion, apoptosis, and production of cytokines like TNF-α, IL-1β, MIP-1 and IL-10 [34,35]. Therefore, enhanced production of CO and bilirubin by TauCl-derived induction of HO-1 and elevation of HO activity can protect cells from the cytotoxicity of ROS by inhibiting further production of O− 2 and NO.
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Fig. 5. The targeted disruption of Nrf2 diminishes TauCl-induced HO-1 expression. RAW 264.7 cells were transfected with Nrf2 specific siRNA prior to 0.5 mM TauCl treatment. (A) Nrf2-dependent HO-1 expression by siRNA was determined by immunoblotting. Representative blots are shown (n = 3). (B) HO-1 bands were quantitated using densitometry and values are expressed as the ratio of HO-1 to β-actin (n = 3). Data are expressed as the mean ± SD, ⁎⁎p b 0.01.
In the present study, we attempted to explore the possible molecular mechanisms underlying the cytoprotective effects provided by TauCl, focusing on the upregulation of HO-1 expression and increase of HO activity. Exposure of RAW 264.7 cells up to 0.7 mM TauCl enhanced the expression of HO-1 mRNA, HO-1 protein and HO activity in a dose-dependent manner without causing significant toxicity. This result is consistent with a recent study by Olszanecki and Marcinkiewicz [22], who reported that HO-1 is induced by TauCl and taurine bromamine in J774.2 macrophages. Thus, TauCl elevates HO activity and removes toxic free-heme and at the same time, produces more antioxidants like biliverdin/bilirubin. In previous studies, we showed that CO inhibited overproduction of O− 2 and NO [17,18], and TauCl inhibited the overproduction of O− 2 and NO in the PMA or LPSstimulated phagocytes [26,36]. Thus, induction of HO-1 expression afforded protection of activated phagocytes against cytotoxicity − caused by O− [37]. Here, we 2 , NO and highly reactive ONOO demonstrate for the first time that TauCl generates ROS in macrophages and that TauCl depletes the intracellular GSH level initially and subsequently leads to increase HO-1 expression via activation of Nrf2/ ARE system. In support of these results, the increased HO-1 expression caused by TauCl was abolished by exposure to NAC (Fig. 3C). This result suggests that the ROS derived from TauCl causes oxidation and depletion of GSH to drive the activation of Nrf2/ARE system for the induction of HO-1 expression. In this connection, TauCl protects cells from H2O2-induced cell death by inducing anti-oxidant enzymes including HO-1, peroxiredoxin and thioredoxin [21]. Many reports suggest that HO-1 induction is dependent on the activation or nuclear translocation of Nrf2. The promoter region of the HO-1 gene contains multiple copies of ARE that bind the translocated Nrf2 and promote the upregulation of HO-1 expression. Thus, we tested whether TauCl increased HO-1 expression via enhancing the Nrf2 translocation to the nucleus and increased the DNA binding. As shown in Fig. 4, TauCl enhanced the nuclear translocation of Nrf2 and
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its binding to ARE. It also has been reported that HOCl, a highly oxidizing cytotoxic precursor of TauCl, enhances the nuclear translocation of Nrf2 [38]. The activation of Nrf2 was sustained between 20– 60 min of TauCl treatment and declined to basal level thereafter (data not shown). This differs from the results published by other authors showing that Nrf2 binding to ARE was detected at 4–7 h following treatment with ONOO− or LPS [37,39,40]. TauCl did not increase the activation of NFκB, AP-1 and CREB (Fig. 4A, and data not shown), albeit the promoter region of HO-1 gene is known to possess the binding sites for these transcription factors [27]. Several upstream signaling pathways such as MAPKs, PI3K/Akt, and Ras are known to activate Nrf2 and increase HO-1 expression [37,41–44]. Kontny et al. [45] reported that TauCl had no effect on ERK activation in Jurkat cells, while Midwinter et al. [46] reported that TauCl induced ERK activation in HUVEC. TauCl showed no effect on the phosphorylation of MAPKs and Akt in the present study (data not shown). Combined, these results suggest that TauCl induces HO-1 expression through mechanisms other than the MAPKs and Akt signaling pathways in macrophages. In summary, TauCl, produced following detoxification of HOCl by the stored taurine and released by the activated neutrophils, generated ROS that could oxidize and deplete intracellular GSH level initially in macrophages. Perhaps this lowered GSH level caused stimulation of Nrf2/ARE system and induced HO-1 expression and enhanced HO activity. Thus, TauCl-induced HO-1 diminished the production of additional inflammatory mediators to protect the surrounding macrophages and tissues at the inflammatory site from cytotoxicity caused by inflammation. Acknowledgments We are grateful to Dr. Y.J. Surh at Seoul National University for his helpful support and Mr. M. Azam for his technical assistance. This study was supported by The Korea Research Foundation Grant (KRF2008-531-C00051) and Nano R&D program through the KOSEF (M10642040001-07N4204-00110). References [1] Vinton NE, Laidlaw SA, Ament ME, Kopple JD. Taurine concentrations in plasma and blood cells of patients undergoing long-term parenteral nutrition. Am J Clin Nutr 1986;44:398–404. [2] Learn DB, Fried VA, Thomas EL. Taurine and hypotaurine content of human leukocytes. J Leukoc Biol 1990;48:174–82. [3] Fukuda K, Hirai Y, Yoshida H, Nakajima T, Usui T. Free amino acid content of lymphocytes and granulocytes compared. Clin Chem 1982;28:1758–61. [4] Thomas EL, Grisham MB, Jefferson MM. Myeloperoxidase-dependent effect of amines on functions of isolated neutrophils. J Clin Invest 1983;72:441–54. [5] Pero RW, Sheng Y, Olsson A, Bryngelsson C, Lund-Pero M. Hypochlorous acid/Nchloramines are naturally produced DNA repair inhibitors. Carcinogenesis 1996;17:13–8. [6] Park E, Schuller-Levis G, Quinn MR. Taurine chloramine inhibits production of nitric oxide and TNF-alpha in activated RAW 264.7 cells by mechanisms that involve transcriptional and translational events. J Immunol 1995;154:4778–84. [7] Marcinkiewicz J, Grabowska A, Bereta J, Stelmaszynska T. Taurine chloramine, a product of activated neutrophils, inhibits in vitro the generation of nitric oxide and other macrophage inflammatory mediators. J Leukoc Biol 1995;58:667–74. [8] Kim C, Park E, Quinn MR, Schuller-Levis G. The production of superoxide anion and nitric oxide by cultured murine leukocytes and the accumulation of TNF-alpha in the conditioned media is inhibited by taurine chloramine. Immunopharmacology 1996;34:89–95. [9] Park E, Jia J, Quinn MR, Schuller-Levis G. Taurine chloramine inhibits lymphocyte proliferation and decreases cytokine production in activated human leukocytes. Clin Immunol 2002;102:179–84. [10] Kim KS, Park EK, Ju SM, Jung HS, Bang JS, Kim C, et al. Taurine chloramine differentially inhibits matrix metalloproteinase 1 and 13 synthesis in interleukin1beta stimulated fibroblast-like synoviocytes. Arthritis Res Ther 2007;9:R80. [11] Chorazy M, Kontny E, Marcinkiewicz J, Maslinski W. Taurine chloramine modulates cytokine production by human peripheral blood mononuclear cells. Amino Acids 2002;23:407–13. [12] Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 1997;37:517–54. [13] Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science 1987;235:1043–6.
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