International Immunopharmacology 19 (2014) 300–307

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5-Aminolevulinic acid combined with ferrous iron enhances the expression of heme oxygenase-1 Yoshiaki Nishio a,b, Masayuki Fujino a,c, Mingyi Zhao a, Takuya Ishii d, Masahiro Ishizuka d, Hidenori Ito d, Kiwamu Takahashi d, Fuminori Abe d, Motowo Nakajima d, Tohru Tanaka d, Shigeru Taketani e, Yukitoshi Nagahara b,⁎, Xiao-Kang Li a,⁎⁎ a

Division of Transplantation Immunology, National Research Institute for Child Health and Development, Tokyo, Japan Department of Biomedical Sciences, Tokyo Denki University, Saitama, Japan c AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan d SBI Pharmaceuticals Co., Ltd., Tokyo, Japan e Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan b

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Article history: Received 22 December 2013 Received in revised form 1 February 2014 Accepted 3 February 2014 Available online 13 February 2014 Keywords: 5-Aminolevulinic acid (ALA) Heme Heme oxygenase-1 (HO-1) Mitogen-activated protein kinases (MAPKs) Nrf2 Bach1

a b s t r a c t 5-Aminolevulinic acid (5-ALA) is the naturally occurring metabolic precursor of heme. Heme negatively regulates the Maf recognition element (MARE) binding- and repressing-activity of the Bach1 transcription factor through its direct binding to Bach1. Heme oxygenase (HO)-1 is an inducible enzyme that catalyzes the rate-limiting step in the oxidative degradation of heme to free iron, biliverdin and carbon monoxide. These metabolites of heme protect against apoptosis, inflammation and oxidative stress. Monocytes and macrophages play a critical role in the initiation, maintenance and resolution of inflammation. Therefore, the regulation of inflammation in macrophages is an important target under various pathophysiological conditions. In order to address the question of what is responsible for the anti-inflammatory effects of 5-ALA, the induction of HO-1 expression by 5-ALA and sodium ferrous citrate (SFC) was examined in macrophage cell line (RAW264 cells). HO-1 expression induced by 5-ALA combined with SFC (5-ALA/SFC) was partially inhibited by MEK/ERK and p38 MAPK inhibitor. The NF-E2-related factor 2 (Nrf2) was activated and translocated from the cytosol to the nucleus in response to 5-ALA/SFC. Nrf2-specific siRNA reduced the HO-1 expression. In addition, 5-ALA/SFC increased the intracellular levels of heme in cells. The increased heme indicated that the inactivation of Bach1 by heme supports the upregulation of HO-1 expression. Taken together, our data suggest that the exposure of 5-ALA/SFC to RAW264 cells enhances the HO-1 expression via MAPK activation along with the negative regulation of Bach1. © 2014 Elsevier B.V. All rights reserved.

1. Introduction 5-Aminolevulinic acid (5-ALA), a natural amino acid, is synthesized through the condensation of glycine and succinyl–CoA by the catalytic effect of 5-ALA synthase. In the cytosol, 5-ALA sequentially generates porphobilinogen, hydroxymethylbilane, uroporphyrinogen III and finally coproporphyrinogen III. In the mitochondrion, coproporphyrinogen III is metabolized to coproporphyrinogen III, protoporphyrinogen IX and protoporphyrin IX, into which iron is inserted via a ferrochelatase-catalyzed reaction, the latter resulting in the formation of heme [1–3]. Recent studies reported that 5-ALA induced the upregulation of heme oxygenase (HO)-1 mRNA levels [4,5]. HO is the rate-limiting enzyme in heme catabolism. It catalyzes the degradation of heme, thereby ⁎ Correspondence to: Y. Nagahara, Division of Life Science and Engineering, Tokyo Denki University, Ishizaka, Hatoyama, Hiki-gun, Saitama 350-0394, Japan. Tel.: +81 49 296 5949; fax: +81 49 296 5162. ⁎⁎ Correspondence to: X.-K. Li, Division of Transplantation Immunology, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan. Tel.: +81 3 3416 0181; fax: +81 3 3417 2864. E-mail addresses: [email protected] (Y. Nagahara), [email protected] (X.-K. Li). 1567-5769/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2014.02.003

producing iron, carbon monoxide (CO) and biliverdin. HO-1 is the inducible isoform of HO [6] and HO-1 expression is induced in a number of cell types by a range of stress stimuli [7–14], including heme [10,15] and other metalloporphyrins [16,17]. Exposure of HO-1deficient mice to endotoxin leads to increased hepatocellular necrosis, the upregulation of splenic proinflammatory cytokine secretion and higher mortality from endotoxic shock compared with wild-type animals [18]. In addition, a recent study with humans with HO-1deficiency has strengthened the above finding that HO-1 plays an important role in counteracting the deleterious increase in inflammation and oxidative injury [19]. Similarly, an in vitro study confirmed that there is a reduction of stress resistance in HO-1-deficient cells [20]. The signaling mechanisms that activate transcription of HO-1 are poorly defined. Most studies have focused on the activation of the mitogen-activated protein kinases (MAPKs) related to cell growth and the stress response. Extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 pathways appear to be involved to some extent in the upregulation of HO-1 expression in response to diverse stimuli [21–23]. The MAPK signaling leads to the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus

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[23]. Nrf2 is one of the basic leucine zipper (bZip) transcription factor [24]. Additionally, recent work has indicated that under basal conditions, Bach1 which is also a bZip transcription factor [25] formed heterodimers with MafK, and these heterodimers repressed the transcription of HO-1 gene by binding to the MARE in the HO-1 promoter [26]. Induction of HO-1 expression by 5-ALA has been reported [4,5], whereas little is known about whether MAPKs and both of bZip transcription factors are involved in the induction of HO-1 expression by 5-ALA. Thus, in the present study, the mechanism underlying the 5ALA-induced HO-1 expression was investigated using a mouse macrophage cell line, RAW264. Our results demonstrated that the 5-ALA combined with SFC (5-ALA/SFC) induced HO-1 protein expression, and was partially related to the activation of ERK. In addition, the silencing of Nrf2 by specific siRNA reduced the levels of HO-1 protein expression induced by 5-ALA/SFC. On the other hand, the treatment of 5-ALA/ SFC increased the internal cellular heme levels.

2. Materials and methods 2.1. Cell culture and regents The mouse macrophage cell line, RAW264, was obtained from the RIKEN Cell Bank (Ibaraki, Japan). The 5-ALA/HCl (COSMO ALA Co., Ltd., Tokyo, Japan) and Fe2 + (SFC, sodium ferrous citrate) (Eisai Food & Chemical Co., Ltd., Tokyo, Japan) were dissolved in distilled water, and the molar ratio of the 5-ALA: Fe2+ was 1:0.5. The Fe2+ was diluted in distilled water immediately before use. Hemin and SB203580 were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). PD98059 were purchased from LC Laboratories (Woburn, MA). Stealth RNAi Negative Control Medium GC Duplex #2 was purchased from Life Technologies. Phospho-p44/42 MAPK (Thr202/Tyr204), Phospho-p38 MAPK (Thr180/Tyr182), Phospho-stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185), p44/42 MAPK, p38 MAPK and SAPK/JNK antibodies were purchased from Cell Signaling Technology Japan K.K. (Tokyo, Japan). The Nrf2, Bach1 and α-tubulin antibodies were purchased from

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Santa Cruz (San Diego, CA). The HO-1 antibody was purchased from Abcam (Cambridge, UK). 2.2. Protein preparation and Western blot analysis Protein preparation and western blot analysis were performed as described previously with modification [27]. In brief, cell lysates were prepared by RIPA buffer (Wako) and total proteins (20 μg) were separated on 4–10% SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membranes were incubated overnight with primary antibodies at room temperature with following dilutions; 1:4000 for the phospho-p44/42 MAPK (Thr202/ Tyr204), phospho-p38 MAPK (Thr180/Tyr182), phospho-SAPK/JNK (Thr183/Tyr185), p44/42 MAPK, p38 MAPK, SAPK/JNK and HO-1 antibodies, and 1:2000 for the α-tubulin antibody. The ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, UK) was used to measure the relative optical density of each specific band obtained after the Western blotting. 2.3. Cellular immunostaining After fixation with 4% paraformaldehyde in PBS for 10 min at room temperature, cells were permeated with 0.5% Triton X-100 in PBS for 1 h. The cells were incubated with primary antibody for 2 h at room temperature, followed by incubation with a secondary antibody conjugated to FITC for 1 h at room temperature, and subsequent counterstaining with 0.2 μg/ml propidium iodide (PI) for 2 min at room temperature. Images were obtained using a confocal laser scan microscope (Olympus, Tokyo, Japan). 2.4. siRNA preparation and transfection The siRNA specific for Nrf2 (Stealth siRNA MSS207018) and Bach1 (Stealth siRNA MSS202323) were purchased from Life Technologies. RAW264 cells were plated the day before transfections and grown to 30–40% confluence in 60-mm dishes. Transfections were carried

Fig. 1. 5-ALA/SFC induced HO-1 expression in RAW264 cells. A) RAW264 cells were treated with or without 1000 μM 5-ALA and/or 500 μM SFC for 6 h. B) RAW264 cells were treated with 0–1000 μM 5-ALA and 0–500 μM SFC, as shown, and were cultured for 6 h. C) RAW264 cells were treated with 1000 μM 5-ALA and 500 μM SFC for the indicated periods. After treatment, the cells were harvested, total proteins were prepared and the levels of HO-1 expression were quantified by a Western blot analysis, as described in Materials and methods. The data are presented as the means ± SEM from three independent experiments.

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out with Lipofectamine RNAiMAX (Life technologies) following the manufacturer's instructions. 2.5. RNA preparation and quantitative RT-PCR Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's instructions with DNase digestion steps. The concentration and purity of the RNA were spectrophotometrically determined by measuring the absorbance at 260/280 nm. The minimal required value of this ratio was 2.0. Total RNA (800 ng) was reverse transcribed into cDNA in a total volume of 25 μl using the PrimeScript RT reagent kit (TaKaRa, Sigma, Japan), according to the manufacturer's instructions. Quantitative PCR was performed in a 25 μl reaction mixture containing 6.5 μl of cDNA obtained as described above, 0.25 μM Probe (Nrf2, 5′-AGTTGCCACCGC CAGGACTACAGTCC-3′; Bach1, 5′-TGCAGCTT CTCGATTTCCGACTCAAGGT-3′; HO-1, 5′-TCCTGCT CAACATTGAGCTGTTT GAGGA-3′ and 18s, 5′-ATCCAT TGGAGGGCAAGTCTG GTGC-3′), 0.9 μM primers (Nrf2, 5′-GCCCTCAGCATGAT GGACTTG-3′ (forward) and 5′TGCCTCCAAAGGATGTCAATCAA-3′ (reverse); Bach1, 5′-CAACGCTGTC GCAAGAGGAA-3′ (forward) and 5′-TGGTCTCGCTC CTTCAGCAA-3′ (reverse); HO-1, 5′-CAGGGTGACAGAAGAGGCTAAGAC-3′ (forward) and 5′-TTGTGTTC CTCTGTCAGCATCAC-3′ (reverse) and 18s, 5′ATGAGTCCACTTTAAATCCTTT AACGA-3′ (forward) and 5′-CTTTAA

TATAC GCTATTGGAGCTGGAA-3′ (reverse)), dNTPs, 10 × buffer and Premix Ex Taq polymerase (TaKaRa). Amplification was conducted in a 7900HT Fast Real-Time PCR System (Life Technologies). The PCR conditions were as follows: 50 °C for 2 min, 95 °C for 15 min, 50 cycles at 95 °C for 30 s and 60 °C for 1 min, followed by 25 °C for 2 min. The expression levels of Nrf2, Bach1 and HO-1 were normalized to the 18s expression levels.

2.6. Protein preparation and immunoprecipitation Cells were washed twice with cold PBS and lysed in 1% SDS. The lysed cells were homogenized by a 25 gauge needle 10 times, followed by centrifugation for 5 min at 16,000 g at 4 °C. The experiment was carried out with 1.5 mg of protein in 1.0 ml 1% SDS. To remove the antibodies present in the cell lysates, samples were pre-cleaned with 20 μl nProtein G Sepharose (GE Healthcare) for 1 h at 4 °C with rotation. A total of 1.5 μg of Bach1 antibody was bound to 20 μl of nProtein G Sepharose in 1.0 ml 1% SDS overnight at 4 °C while rotating the samples. The beads were washed three times with 0.1% SDS and once with wash buffer (50 mM Tris, pH 8.0). The bound protein was eluted in 20 μl freshly prepared sample buffer (1% SDS, 100 mM dithiothreitol (DTT); Nacalai tesque, 50 mM Tris, pH 7.5) for 5 min at 95 °C.

Fig. 2. Activation of the ERK and p38 MAPK was involved in the induction of HO-1 expression by 5-ALA/SFC in RAW264 cells. RAW264 cells were treated with or without 1000 μM 5-ALA and 500 μM SFC for 6 h, then the cells were harvested and the total protein was quantified by a Western blot analysis, as described in Materials and methods. A) The phosphorylation of ERK by 5-ALA/SFC was detected by a Western blot analysis. B) RAW264 cells were pretreated with 0–100 μM of a MEK/ERK pathway-specific inhibitor, PD98059, as shown, and were cultured for 1 h. The inhibition of the 5-ALA/SFC-induced phosphorylation of ERK and HO-1 expression by PD98059 was detected by a Western blot analysis. C) The phosphorylation of p38 MAPK by 5ALA/SFC was detected by a Western blot analysis. D) RAW264 cells were pretreated with 0–20 μM of a p38 MAPK pathway-specific inhibitor, SB203580, as shown, and were cultured for 2 h. The inhibition of the 5-ALA/SFC-induced phosphorylation of p38 MAPK and HO-1 expression by SB203580 was detected by a Western blot analysis. The data are presented as the means ± SEM from three independent experiments.

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Fig. 3. 5-ALA/SFC induced the translocation of Nrf2 from the cytosol to the nucleus, and activated Nrf2 is involved in the induction of HO-1 expression by 5-ALA/SFC in RAW264 cells. RAW264 cells were treated with or without 1000 μM 5-ALA and 500 μM SFC for 6 h. A) After treatment, the cells were stained with an anti-Nrf2 antibody and then incubated with FITC-conjugated antirabbit IgG (green). Nuclei were stained with PI (red). The stained cells were visualized by a confocal laser scanning microscopic analysis, as described in Materials and methods. a–c: untreated (control) cells, d–f; 5-ALA/SFC-treated cells, a and d; staining with Nrf2, b and e; PI staining, c and f: merged images of Nrf2 and PI staining. B) RAW264 cells were transfected with 3.4 nM Nrf2 siRNA or 3.4 nM siRNA negative-control duplex and cultured for 24 h, then the cells were harvested, and the total mRNA was prepared and quantified by quantitative RT-PCR, as described in Materials and methods. C) RAW264 cells were transfected with 3.4 nM Nrf2 siRNA or 3.4 nM siRNA negative-control duplex and cultured for 24 h, then the culture media were changed, and cells were treated with 5-ALA/SFC as described above. After treatment, the cells were harvested, and the Nef2 and HO-1 protein expression was detected by a Western blot analysis, as described in Materials and methods. Data are presented as the means ± SEM from three independent experiments.

2.7. Heme assay The heme assay was designed to take advantage of heme peroxidase [28,29], and was carried out by dot blotting. Each protein sample obtained by immunoprecipitation was applied to each polyvinylidene difluoride membrane. The membrane was dried, and the bound heme proteins were visualized with an ECL Prime Western Blotting Detection System (GE Healthcare) according to the manufacturer's protocol. An ImageQuant LAS 4000 (GE Healthcare) was used to measure the relative optical density of each spot obtained after the dot blotting. 2.8. Statistical analysis Statistical analysis was done by one-way ANOVA–Dunnett's test. Values of P b 0.05 were considered to be statistically significant. 3. Results 3.1. 5-ALA/SFC enhanced the HO-1 expression in RAW264 cells To examine the effects of 5-ALA in the HO-1 expression in RAW264 macrophage, we first examined whether the HO-1 protein levels are upregulated by exposure of 5-ALA and/or SFC, which is a cofactor of 5-ALA

catabolism. The HO-1 protein expression had significantly increased at 6 h by treatment with 1.0 mM 5-ALA and 0.5 mM SFC compared with 5ALA or SFC alone (Fig. 1A). These results suggest that 5-ALA metabolism is an important factor required for the induction of HO-1. This induction of HO-1 by 5-ALA/SFC increased in a dose- and a time-dependent manner (Fig. 1B, C). 3.2. The effects of the phosphorylation of ERK and p38 on the HO-1 expression by 5-ALA/SFC To identify the signaling pathways affected by 5-ALA/SFC to induce HO-1 expression, we analyzed the effects of 5-ALA/SFC on the three MAPK cascades leading to the activation of ERK, p38 and JNK. As shown in Fig. 2, 5-ALA/SFC activated the ERK (Fig. 2A) and the p38 (Fig. 2C) pathways, while the 5-ALA/SFC didn't activate JNK pathway (data not shown). Next, in order to confirm the potential role of these activated pathways in the induction of HO-1 by 5-ALA/SFC, cells were pretreated with a PD98059 before 5-ALA/SFC treatment. A western blot analysis demonstrated that PD98059 partially inhibited (31%) the 5-ALA/SFC-induced HO-1 protein expression in RAW264 cells (Fig. 2B). Furthermore, the pretreatment of SB203580 before stimulation with 5-ALA/SFC also slightly reduced the 5-ALA/SFC-induced HO-1 protein expression in RAW264 cells (Fig. 2D). Thus, these results suggest that ERK and p38, but not JNK, mediate the 5-ALA/SFC-induced HO-1 expression in RAW264 cells.

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3.3. 5-ALA/SFC stimulated the translocation of Nrf2 to the nucleus and the HO-1 expression by Nrf2 To examine the contribution of Nrf2 in the HO-1 expression by 5ALA/SFC, a confocal microscopic analysis was performed. As shown in Fig. 3A–C, under normal conditions Nrf2 was localized in cytoplasm, while after treatment with 5-ALA/SFC, Nrf2 was accumulated in the nuclei (Figs. 3A–F). Thus, it is suggesting that 5-ALA/SFC is able to activate Nrf2. We also examined whether the activation of Nrf2 by 5-ALA/SFC is involved in the induction of HO-1 using Nrf2-specific siRNA. As shown in Fig. 3C, the Nrf2 and HO-1 protein levels were reduced by the Nrf2specific siRNA compared with the cells treated with the vehicle or nonspecific control duplexes. These results suggest that Nrf2 is related to the induction of HO-1 expression by 5-ALA/SFC.

3.4. Upregulation of heme by 5-ALA/SFC, and the interaction between heme and Bach1 It was previously demonstrated that HO-1 transcriptional activity was repressed by Bach1, and the function of Bach1 was inactivated by heme [30]. Therefore, we investigated the intracellular levels of heme induced by 5-ALA/SFC. Treatment with 5-ALA/SFC did not affect the levels of heme, but a significant increase in the heme levels was detected by using zinc protoporphyrin IX (ZnPpIX), an inhibitor of HO-1 that can prevent the degradation of heme by HO-1 (Fig. 4A). This result indicated that the intracellular heme level is controlled by the HO-1 activity. Maximal upregulation of heme by 5-ALA/SFC and ZnPpIX was observed after 4 h of treatment (Fig. 4B), and the upregulation disappeared from 9 h onward (data not shown). In addition, this upregulation of heme by 5-ALA/SFC and ZnPpIX increased dose-dependently (Fig. 4B).

To further examine the interaction between Bach1 and heme, we performed immunoprecipitation to target Bach1. The peroxidative activity of heme was detected from the immunoprecipitates of Bach1, suggesting that Bach1 bound the heme protein. Furthermore, treatment with 5-ALA/SFC led to a significant increase in the Bach1-bound heme levels compared with untreated cells (Fig. 4C). Taken together, these findings indicate that the 5-ALA-derived heme bound Bach1 in RAW264 cells, and suggest that the inactivated Bach1 may allow the HO-1 expression in RAW264 cells to be induced by 5-ALA/SFC. 3.5. Induction of HO-1 by specifically silencing Bach1 genes with Bach1 siRNA It was reported that Bach1 suppression or deficiency induced HO-1 expression [31,32]. To examine the induction of HO-1 expression by Bach1, we investigated whether the downregulation of Bach1 by siRNA induces HO-1 expression. A Bach1 siRNA reduced the Bach1 mRNA and protein compared with vehicle-treated cells (Fig. 5A and C), and significantly increased the HO-1 mRNA levels (Fig. 5B). As shown in Fig. 5C, HO-1 protein expression was significantly induced by Bach1 siRNA in RAW264 cells. Most importantly, this induction of HO-1 by Bach1 downregulation didn't require stimulation of the activated signaling cascade. 4. Discussion In the present study, we have shown that treatment with 5-ALA/SFC enhanced the HO-1 expression, and the upregulation of HO-1 in response to 5-ALA metabolism involved the activated MAPKs and activation of the Nrf2 pathway. Furthermore, 5-ALA/SFC increased the

Fig. 4. 5-ALA/SFC increased the intracellular levels of heme, and induced the binding of heme to Bach1 in RAW264 cells. A) RAW264 cells were treated with a combination of 1000 μM 5-ALA, 500 μM SFC and 10 μM ZnPpIX and cultured for 4 h. B) RAW264 cells were treated with the combination of 0–1000 μM 5-ALA, 0–500 μM SFC and 10 μM ZnPpIX and cultured for 4 h. C) RAW264 cells were treated with or without 1000 μM 5-ALA and 500 μM SFC and with 10 μM ZnPpIX for 4 h. After treatment, the cells were harvested, total proteins were prepared and the levels of heme were quantified by a heme assay analysis, as described in Materials and methods. The data are presented as the means ± SEM from three independent experiments.

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Fig. 5. Specific silencing of Bach1 genes with Bach1 siRNA induced HO-1 expression in RAW264 cells. RAW264 cells were transfected with 3.4 nM Bach1 siRNA or 3.4 nM siRNA negative-control duplex and cultured for 24 h, then the cells were harvested, and the total mRNA and protein were prepared and quantified by a quantitative RT-PCR or a Western blot analysis, as described in Materials and methods. A) The Bach1 mRNA levels were measured by quantitative RT-PCR. B) The HO-1 mRNA levels were measured by quantitative RT-PCR. C) The Bach1 and HO-1 protein expression was detected by a Western blot analysis. The data are presented as the means ± SEM from three independent experiments.

intracellular heme levels. HO-1 expression exerts antioxidant and cytoprotective effects [33–35]. This protection stems from the production of metabolites by HO-1, including CO and bilirubin [36–38]. However, little has been reported on the mechanism of HO-1 induction by 5-ALA. One of the major inducers of HO-1 is heme [10,15], and during heme synthesis, iron is one of the components for heme synthesis. Therefore, we used SFC as well as 5-ALA to increase the metabolism of 5-ALA. Based on the previous reports and our data, we proposed a hypothetical model for the induction of HO-1 expression by 5-ALA metabolism, as shown in Fig. 6. Numerous studies on the regulation of gene expression have focused on the role of the MAPK pathways. Our results indicate that, although the ERK and p38 MAPK pathways are activated by 5-ALA/SFC, they are only partially responsible for the induction of HO-1 in RAW264 cells. A report by Rushworth et al. [7] was in agreement with our data showing the partial involvement of MAPKs. Additionally, a classical isoform of protein kinase C (PKC) [7] and PI3K/Akt [39] played an important role in the induction of HO-1 by LPS, the antioxidant phytochemical and carnosol. Several reports have indicated that, in response to various stimuli, Nrf2 translocated to the nucleus, which was followed by the subsequent induction of HO-1 expression [7,40]. Nrf2 heterodimerizes with small Maf proteins (sMaf) in induction of HO-1 through binding to the sequence of the Maf recognition element (MARE) [25,26,41–43]. sMaf which includes MafF, MafG and MafK possesses a characteristic bZip domain which mediates DNA binding and dimerization with other bZip

proteins such as Nrf2 [44]. Nrf2 resides in the cytosol bound to Keap 1, where it is known as a negative regulator of Nrf2 in the cytoplasm [45,46]. Upon activation, Nrf2 is released from Keap 1, and it enters the nucleus where it heterodimerizes with sMaf and binds to the MARE in the promoters of various target genes [42,45]. A recent study reported that hemin evoked the nuclear translocation of Nrf2, and that Nrf2-specific siRNA suppressed the induction of HO-1 by hemin in human monocytes [47]. Other metalloporphyrins were also confirmed to cause the nuclear localization of Nrf2 followed by Nrf2-mediated HO-1 induction [16,17]. Moreover, the HO-1 induction by LPS was also inhibited by Nrf2 downregulation [7]. Our present findings and these studies suggest that Nrf2 plays a key role in the induction of HO-1. Indeed, our findings demonstrated that Nrf2 is involved in the induction of HO-1 by 5-ALA/SFC in RAW264 cells, which is in agreement with these previous reports. In this study, we didn't examine the relationship between Nrf2 and MAPKs. However, Andreadi et al. [23] reported evidence that p38 MAPK is involved in the nuclear accumulation of Nrf2. In addition, several reports suggested that there was activation of Nrf2 through MAPKs [48–50]. Therefore, these reports support the idea that treatment with 5-ALA/SFC activates ERK and p38 MAPK, and that the phosphorylation of these MAPKs activates the post-transcriptional factor, Nrf2, which may then lead to HO-1 expression. In fact the MAPKs activated by 5-ALA/SFC induced HO-1 expression through the activation of Nrf2 in our study (Fig. 6, pathway A). Moreover, we found that the treatment with 5-ALA/SFC induced the upregulation of the intracellular levels of heme. Recent reports indicate

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HO-1 expression through MAPK-mediated pathways. We propose at least two pathways by which 5-ALA metabolism may induce the expression of HO-1. Therefore, 5-ALA metabolism is an effective inducer of HO-1, and may be useful for anti-inflammatory and anti-oxidant therapies. Acknowledgments This study was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grantsin-Aid 20390349, 21659310 and 2109739) and the grant of the National Center for Child Health and Development (22-10, 24-1:756). References

Fig. 6. 5-ALA metabolism induced HO-1 expression through (A) and (B) pathways. A model of HO-1 induction based on our observations. Treatment with 5-ALA combined with SFC (5ALA/SFC) induces the phosphorylation of ERK and p38 MAPK. These activated MAPKs lead to HO-1 expression through their effects on post-transcriptional factors, such as Nrf2. In this pathway, 5-ALA/SFC indirectly regulated HO-1 expression. On the other hand, exposure to 5-ALA/SFC increases the intracellular levels of heme. Under conditions with a higher concentration of heme, the HO-1 repressor, Bach1, is inactivated by direct binding to heme to Bach1, which allows for increased expression of HO-1. In this pathway, 5-ALA/ SFC directly regulated HO-1 expression.

that heme controls HO-1 expression [10,15,21,47]. The transcription factor Bach1 heterodimerizes with Maf proteins and binds to MAREs, then the resulting Bach1 heterodimers repress MARE-dependent transcription, including the transcription of HO-1. In contrast, the DNA binding activity of Bach1 is negatively regulated by heme binding [30]. In the presence of higher concentrations of heme, increased binding of heme to Bach1 leads to a conformational change and a decrease in DNA binding activity [30,51]. This derepression permits Nrf2-sMaf and other activating heterodimers to occupy the MARE sites in the HO-1 promoter, and thus leads to increased transcription and upregulation of the gene expression. Therefore, the increasing levels of intracellular heme in RAW264 cells suggested that the DNA-binding and repressor activity of Bach1 may be alleviated by heme, while the transcriptional activity of repressed genes, such as HO-1, is upregulated. In addition, our findings and those of others [31,32] demonstrated that there was induction of HO-1 expression by the disappearance of Bach1, supporting that Bach1 represses HO-1 expression. These results indicated that Bach1 is an important factor for the induction of HO-1 expression. 5-ALA/SFC treatment leads to the production of heme in the cells, and the intracellular heme subsequently binds to Bach1. This binding of heme to Bach1 leads to a decrease in the DNA-binding activity of Bach1 and the subsequent upregulation of HO-1 expression. In other words, heme, produced by 5-ALA metabolism, directly induces HO-1 expression to remove the HO-1 repressor, Bach1, in RAW264 cells (Fig. 6, pathway B). In summary, our observations provide evidence that the upregulation of HO-1 by 5-ALA combined with SFC involves the ERK and p38 MAPK pathways, and the increase of the intercellular levels of heme appears to result in the reducing of Bach1 and the induction of HO-1 expression. This inactivation of the repressive component may enhance

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5-Aminolevulinic acid combined with ferrous iron enhances the expression of heme oxygenase-1.

5-Aminolevulinic acid (5-ALA) is the naturally occurring metabolic precursor of heme. Heme negatively regulates the Maf recognition element (MARE) bin...
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