Free
Meeting Abstracts  |   December 2006
Heme Oxygenase 1, Nuclear Factor E2–related Factor 2, and Nuclear Factor κB Are Involved in Hemin Inhibition of Type 2 Cationic Amino Acid Transporter Expression and l-Arginine Transport in Stimulated Macrophages
Author Affiliations & Notes
  • Pei-Shan Tsai, M.D.
    *
  • Chien-Chuan Chen, M.D.
  • Pei-Shan Tsai, Ph.D.
  • Lin-Cheng Yang, M.D.
    §
  • Wan-Yu Huang, M.D.
  • Chun-Jen Huang, M.D., Ph.D.
    #
  • * Lecturer, Department of Anesthesiology, Mackay Memorial Hospital. † Department of Anesthesiology, Mackay Memorial Hospital; Mackay Medicine, Nursing and Management College, Taipei, Taiwan, Republic of China. ‡ Associate Professor, College of Nursing, Taipei Medical University, Taipei, Taiwan, Republic of China. § Associate Professor, Department of Anesthesiology, E-DA Hospital/I-Shou University, Kaohsiung, Taiwan, Republic of China. ∥ Department of Anesthesiology, Mackay Memorial Hospital. # Assistant Professor, Department of Anesthesiology, Mackay, Memorial Hospital; Mackay Medicine, Nursing and Management College; Graduate Institute of Pharmacology, Taipei Medical University, Taipei, Taiwan, Republic of China.
Article Information
Meeting Abstracts   |   December 2006
Heme Oxygenase 1, Nuclear Factor E2–related Factor 2, and Nuclear Factor κB Are Involved in Hemin Inhibition of Type 2 Cationic Amino Acid Transporter Expression and l-Arginine Transport in Stimulated Macrophages
Anesthesiology 12 2006, Vol.105, 1201-1210. doi:
Anesthesiology 12 2006, Vol.105, 1201-1210. doi:
UP-REGULATION of inducible nitric oxide (iNOS) and subsequent nitric oxide overproduction has been reported to play an essential role in initiating systemic inflammatory responses during sepsis.1,2 Previous studies indicated that inhibition of iNOS and reducing nitric oxide production may be beneficial during sepsis.3,4 Cellular uptake of l-arginine, the sole substrate of iNOS, has been identified as one crucial mechanism that regulates nitric oxide biosynthesis via  iNOS.5 Previous reports clearly demonstrated that cellular uptake of l-arginine transport is mainly mediated by type 2 cationic amino acid transporter (CAT-2).6 The crucial role of CAT-2 was clearly demonstrated by previous data showing that inhibition of CAT-2 significantly inhibits nitric oxide production.7 This CAT-2/l-arginine pathway thus constitutes part of the downstream regulatory pathways of iNOS activity. In addition, this downstream pathway offers an alternative therapeutic target against sepsis, even after the induction of iNOS.
Heme oxygenases (HOs) catalyze the first and rate-limiting step in the oxidative degradation of heme to carbon monoxide, biliverdin, bilirubin, and iron.8 To date, at least two isoenzymes of HO, HO-1 and HO-2, have been identified. HO-2 is expressed constitutively, whereas HO-1 is highly induced by heme and oxidative stress.8 HO-1 induction has been shown to increase antioxidant defenses in rats.8 Furthermore, HO-1 has been reported to have antiinflammatory, antiapoptotic, and antiproliferative effects.8,9 It is now known that HO-1 induction has beneficial effects in diseases such as atherosclerosis and sepsis.8,9 
Heme oxygenase 1 is significantly induced by lipopolysaccharide.10 In addition, overproduced nitric oxide from up-regulated iNOS further increases HO-1 expression, which, in turn, limits further expression of iNOS and nitric oxide biosynthesis.10 Previous data further demonstrated that only “superinduction” (i.e.  , further enhancement) of HO-1 expression could significantly inhibit iNOS expression and nitric oxide biosynthesis in lipopolysaccharide-stimulated macrophages.11 We recently have shown that HO-1 accounts for the therapeutic effect of hyperbaric oxygen therapy against lipopolysaccharide-induced acute lung injury.12 In that study, we also found that hyperbaric oxygen pretreatment superinduced HO-1 expression and significantly inhibited iNOS expression and nitric oxide production in lipopolysaccharide-stimulated rat lung.12 
As mentioned above, superinduction of HO-1 significantly inhibited iNOS expression.11 However, effects of HO-1 superinduction on CAT-2 expression and l-arginine transport during sepsis remained unstudied. To explore further, we conducted this cellular study to examine the hypothesis that HO-1 superinduction significantly attenuates CAT-2 expression and l-arginine transport in lipopolysaccharide-stimulated macrophages. In addition, previous data indicated that HO-1 induction involves the activation of nuclear factor E2–related factor 2 (Nrf2).13 Previous data also indicated that induction of CAT-2 involves nuclear factor κB (NF-κB).14 Therefore, this study was also conducted to investigate the effects of HO-1 induction on Nrf2 and NF-κB.
Materials and Methods
Cell Culture
Immortalized murine macrophages (RAW264.7 cells) were plated in cell culture dishes (60 × 15 mm) and grown in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Life Technologies), as we previously reported.15 RAW264.7 cells were incubated in a humidified chamber at 37°C in a mixture of 95% air and 5% CO2.
Experimental Protocols
According to our previous report,15 we used bacterial lipopolysaccharide (100 ng/ml; Escherichia coli  serotype 0127:B8; Sigma-Aldrich, St. Louis, MO) to induce CAT-2 expression and l-arginine transport. Hemin (an HO-1 inducer; Sigma-Aldrich),16 tin protoporphyrin (SnPP, an HO-1 inhibitor; Sigma-Aldrich),17 and hemoglobin (a carbon monoxide scavenger; Sigma-Aldrich)18 were used to elucidate the effects of HO-1 induction and carbon monoxide.
To investigate the effects of HO-1 induction on lipopolysaccharide-induced CAT-2 expression and l-arginine transport, confluent cells were randomized to one of the 14 groups. Each group contained six culture dishes (n = 6). Among them, two groups of cell cultures that received either 1× phosphate-buffered saline (Life Technologies; denoted as the PBS group) or lipopolysaccharide (denoted as the LPS group) served as the negative or positive control, respectively. To elucidate the effects of HO-1, another six groups of cell cultures were treated with one of the three different doses of hemin (i.e.  , low dose: 5 μm; moderate dose: 50 μm; or high dose: 500 μm) or hemin (5, 50, or 500 μm) plus SnPP (50 μm) immediately after lipopolysaccharide administration [denoted as the LPS + Hemin (5), LPS + Hemin (50), LPS + Hemin (500), LPS + Hemin (5) + SnPP, LPS + Hemin (50) + SnPP, and LPS + Hemin (500) + SnPP groups, respectively]. To elucidate the role of carbon monoxide, another three groups of cell cultures were treated with hemin (5, 50, or 500 μm) and hemoglobin (10 μm) immediately after lipopolysaccharide [denoted as the LPS + Hemin (5) + Hb, LPS + Hemin (50) + Hb, and LPS + Hemin (500) + Hb groups, respectively]. Another three groups of cell cultures that received only hemin (500 μm), SnPP (50 μm), or hemoglobin (10 μm) served as the control of the effects of hemin, SnPP, or hemoglobin (denoted as the Hemin, SnPP, and Hb groups, respectively). The dose and administration timing of lipopolysaccharide, hemin, SnPP, and hemoglobin were chosen according to previously published articles15–18 and further validated by a series of preliminary studies performed in our laboratory. After exposure to lipopolysaccharide for 18 h or comparable duration in groups without lipopolysaccharide, the cell cultures were harvested.
To elucidate the effects of hemin on Nrf2 and NF-κB pathways, confluent cells were randomized to one of the eight groups: the PBS, Hemin (50 μm), SnPP, Hb, LPS, LPS + Hemin, LPS + Hemin + SnPP (50 μm), or LPS + Hemin + Hb (10 μm) groups. Hemin, SnPP, and hemoglobin were administered immediately after lipopolysaccharide. Each group contained 18 culture dishes (n = 18). Three culture dishes from each group were then harvested after they were exposed to lipopolysaccharide for 0, 15, 30, 45, 60, and 120 min or comparable duration in groups without lipopolysaccharide, respectively.
Preparation of Whole Cell Lysates, Nuclear Extracts, and Cytosolic Extracts
To elucidate the effects of each additive on the induction of HO-1, whole cell lysates were prepared according to our previous report.19 To elucidate the effects of HO-1 induction on Nrf2 and NF-κB pathway, the nuclear and cytosolic extracts of the harvested cell cultures were prepared according to protocols that were modified from a previously published article.20 In brief, cells were washed, scraped, and centrifuged at 1,500g  for 5 min. The cell pellet was resuspended in 5 ml cell lysis buffer (10 mm HEPES [pH 7.9], 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, and 0.2 mm phenylmethylsulfonyl fluoride) and centrifuged again at 1,500g  for 5 min. Cells were resuspended again in cell lysis buffer and allowed to swell on ice for 10 min followed by homogenization. Homogenates were centrifuged at 3,300g  for 15 min at 4°C. The supernatants were saved for cytosolic extracts and the pellets for nuclear extracts. The pellets were resuspended and homogenized in three volumes of nuclear extraction buffer (20 mm HEPES [pH 7.9], 1.5 mm MgCl2, 400 mm KCl, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, and 25% glycerol). After being stirred on ice for 30 min and centrifuged at 89,000g  for 30 min, the supernatant from the nuclear suspensions were collected and concentrated in a Microcon 10 concentrator (Millipore Corporation, Burlington, MA) by centrifugation at 14,000g  for 3 h at 4°C. For preparation of cytosolic extracts, the supernatant obtained after removal of nuclei was mixed with cytoplasmic extraction buffer (30 mm HEPES [pH 7.9] at 4°C, 140 mm KCl, 3 mm MgCl2) and then centrifuged at 89,000g  for 1 h. The supernatants were collected and also concentrated in a Microcon 10 concentrator by centrifugation at 14,000g  for 1 h at 4°C. The protein concentration of each sample (including whole cell lysates, nuclear extracts, and cytosolic extracts) was measured using a BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL).
Immunoblotting Assay
Equal amounts of protein (65 μg) were loaded into each well of a 7.5% Tris-glycine precast polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and separated by gel electrophoresis. The proteins were then transferred from the gel to nitrocellulose membranes (Bio-Rad). For whole cell lysates, nitrocellulose membranes were incubated overnight at 4°C in primary antibody solution of HO-1 (1:1,000 dilution, polyclonal anti-HO-1 antibody; Stressgen Bioreagents, Ann Arbor, MI), HO-2 (1:1,000 dilution, polyclonal anti-HO-2 antibody; Stressgen), CAT-2 (1:500 dilution, polyclonal anti-CAT-2 antibody, provided by Lin-Cheng Yang, M.D., Associate Professor, Department of Anesthesiology, E-DA Hospital/I-Shou University, Kaohsiung, Taiwan, Republic of China), or β-actin (as an internal standard, 1:5,000 dilution, monoclonal antiactin antibody; Chemicon International, Inc., Temecula, CA). For cytosolic extracts, the nitrocellulose membranes were incubated overnight at 4°C in primary antibody solution of phosphorylated inhibitor κBα (I-κBα), the product of I-κB degradation (1:1,000 dilution, monoclonal anti–phosphorylated I-κBα antibody; Cell Signaling Technology, Inc., Danvers, MA) or β-actin (Chemicon). For nuclear extracts, the nitrocellulose membranes were incubated overnight at 4°C in primary antibody solution of NF-κB (1:500 dilution, polyclonal anti-NF-κB p65 antibody; Cell Signaling Technology, Inc.), Nrf2 (1:250 dilution, polyclonal anti-Nrf2 antibody; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or β-actin (Chemicon). β-Actin was used as an internal standard. Horseradish peroxidase–conjugated sheep anti-mouse immunoglobulin G antibody (Amersham Pharmacia Biotec, Inc., Piscataway, NJ) was used as a secondary antibody. Bound antibody was detected by chemiluminescence (ECL plus kit; Amersham) and high-performance chemiluminescence film (Hyperfilm; Amersham). Densitometry was used to quantify the protein band densities using National Institutes of Health software (Scion Corp., Frederic, MD).
HO Activity Assay
We chose to measure the bilirubin concentrations of the culture media to determine the activity of HO, as reported by Turcanu et al.  21 In brief, culture supernatant (500 μl) was collected. After being mixed with barium chloride (250 mg) and benzene (750 μl), the sample was vortexed vigorously and then centrifuged at 13,000g  for 30 min. The upper benzene layer containing bilirubin was collected. Bilirubin was determined spectrophotometrically as a difference in absorbance between 450 and 600 nm using an excitation coefficient of 27.3 mm −1cm−1. HO activity was expressed as picomoles of bilirubin formed per milligram of cell protein. Protein content level was determined by a BCA protein assay kit (Pierce).
l-[3H]Arginine Uptake Studies
We chose to measure l-[3H]arginine uptake to determine l-arginine transport in macrophages, as we previously reported.15 Macrophages were cultured for 18 h in the absence or presence of test substances. Then the cellular uptake of l-arginine was determined using previously published protocols.15,22 In brief, macrophages were incubated at 37°C for 2 min in uptake solutions (137 mm NaCl, 5.4 mm KCl, 2.8 mm CaCl2, 1.2 mm MgSO4, 10.0 mm HEPES-Tris [pH 7.4]) supplemented with 0.1 mm l-arginine containing 1.0 μCi/ml l-[3H]arginine. Uptake was stopped with ice-cold stop solution (137 mm NaCl, 14 mm Tris-HCl [pH 7.4]). Cells were then lysed in 0.5 ml Tris-Triton (0.1%) solution followed by determination of the cellular radioactivity. l-Arginine transport was expressed as picomoles of l-arginine uptake per milligram of cell protein. Protein content level was also determined by a BCA protein assay kit (Pierce).
Statistical Analysis
To determine the intergroup differences, one-way analysis of variance was used. The Tukey test was used for multiple comparisons. All data were presented as mean ± SD. The significance level was set at 0.05. A commercial software package (SigmaStat for Windows; SPSS Science, Chicago, IL) was used for data analysis.
Results
Hemin Enhanced Lipopolysaccharide-induced HO-1 Expression and HO Activity
Our data revealed that HO-1 protein concentration in the PBS group was low (fig. 1). Exposure to both hemin (an HO-1 inducer) and hemoglobin (a carbon monoxide scavenger) significantly increased HO-1 expression because the HO-1 protein concentrations in the Hemin and Hb groups were significantly higher than that in the PBS group (approximately 1.8-fold and 1.4-fold higher; data not shown). In contrast, SnPP (an HO-1 inhibitor) posted no significant effect on HO-1 expression (data not shown). Exposure to lipopolysaccharide significantly increased HO-1 expression because the HO-1 protein concentrations in the LPS group were significantly higher than that in the PBS group (approximately fourfold higher; fig. 1). In a dose-dependent manner, we found that hemin significantly enhanced the lipopolysaccharide-induced increases in HO-1 expression (fig. 1). Data of the HO activity assay revealed that lipopolysaccharide also significantly increased HO activity (fig. 1). Hemin significantly enhanced the effects of lipopolysaccharide on HO activity in a dose-dependent manner (fig. 1). In addition, our data revealed that the HO-1 expression induced by lipopolysaccharide plus a low or moderate dose of hemin (i.e.  , 5 or 50 μm) was further enhanced by SnPP or hemoglobin (fig. 2). In contrast, the HO-1 expression induced by lipopolysaccharide plus a high dose of hemin (i.e.  , 500 μm) was not affected by either SnPP or hemoglobin (fig. 2). However, in contrast to the HO-1 data, we found that the increases in HO activity induced by lipopolysaccharide plus a low or moderate dose of hemin (i.e.  , 5 μm or 50 μm) were significantly inhibited by both SnPP and hemoglobin (fig. 2). In addition, the HO activity increase induced by lipopolysaccharide plus a high dose of hemin (i.e.  , 500 μm) was not affected by SnPP or hemoglobin (fig. 2).
Fig. 1. Effects of hemin on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
Fig. 1. Effects of hemin on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
Fig. 1. Effects of hemin on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
×
Fig. 2. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. 
Fig. 2. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. 
Fig. 2. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. 
×
Our data also revealed that the expression of HO-2, the constitutively expressed HO isozyme, was not affected by lipopolysaccharide, hemin, SnPP, or hemoglobin because there were no between-group differences regarding the HO-2 protein concentrations measured by immunoblotting assay (figs. 1 and 2).
Hemin Inhibited Lipopolysaccharide-induced CAT-2 Expression and CAT-2 Activity
Our data revealed that the CAT-2 protein concentration in the PBS group was low (fig. 3). Hemin, SnPP, or hemoglobin alone posted no significant effect on CAT-2 expression because the CAT-2 protein concentrations in the Hemin, SnPP, and Hb groups were similar to those in the PBS group (data not shown). As expected, lipopolysaccharide significantly increased the protein concentrations of CAT-2 (fig. 3). Our data also revealed that hemin, in a dose-dependent manner, significantly inhibited lipopolysaccharide-induced CAT-2 expression (fig. 3). Furthermore, the inhibitory effect of a low or moderate dose of hemin (i.e.  , 5 or 50 μm) on lipopolysaccharide-induced CAT-2 expression was significantly reversed by SnPP and hemoglobin (fig. 4). In contrast, the inhibitory effect of a high dose of hemin (i.e.  , 500 μm) on lipopolysaccharide-induced CAT-2 expression was not affected by SnPP and hemoglobin (fig. 4). In addition, the changes in CAT-2 activity paralleled the changes of CAT-2 expression (figs. 3 and 4).
Fig. 3. Effects of hemin on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
Fig. 3. Effects of hemin on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
Fig. 3. Effects of hemin on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
×
Fig. 4. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. ¥  P  < 0.05, LPS + Hemin (5) + SnPP group  versus  LPS + Hemin (5) + Hb group. 
Fig. 4. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. ¥  P  < 0.05, LPS + Hemin (5) + SnPP group  versus  LPS + Hemin (5) + Hb group. 
Fig. 4. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. ¥  P  < 0.05, LPS + Hemin (5) + SnPP group  versus  LPS + Hemin (5) + Hb group. 
×
Hemin Enhanced Lipopolysaccharide-induced Nrf2 Activation and, in Turn, Inhibited Lipopolysaccharide-induced NF-κB Activation
Our data revealed that the baseline protein concentrations of Nrf2 and NF-κB p65 in nuclear extracts and phosphorylated I-κBα in cytosolic extracts in all of the eight groups were almost undetectable because the protein concentrations of Nrf2, NF-κB p65, and phosphorylated I-κBα in culture dishes that were harvested at 0 min after lipopolysaccharide exposure (fig. 5) or comparable duration in the groups without lipopolysaccharide were low (data not shown). As expected, PBS did not activate Nrf2 and NF-κB because the protein concentrations of Nrf2 and NF-κB p65 in nuclear extracts and phosphorylated I-κBα in cytosolic extracts in the PBS group were low throughout the experiment (data not shown). Exposure to hemin or hemoglobin alone but not SnPP resulted in an early increase in Nrf2 protein concentrations in the nuclear extracts at 15, 30, and 45 min after the experiment began and then gradually returned to baseline level at 60 and 120 min after the experiment began (data not shown). In contrast, exposure to hemin, SnPP, or hemoglobin alone did not affect the protein concentrations of NF-κB p65 in the nuclear extracts or phosphorylated I-κBα in the cytosolic extracts (data not shown).
Fig. 5. Effects of lipopolysaccharide and lipopolysaccharide (LPS) plus hemin on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. #  P  < 0.05 compared with the LPS group. 
Fig. 5. Effects of lipopolysaccharide and lipopolysaccharide (LPS) plus hemin on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. #  P  < 0.05 compared with the LPS group. 
Fig. 5. Effects of lipopolysaccharide and lipopolysaccharide (LPS) plus hemin on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. #  P  < 0.05 compared with the LPS group. 
×
Exposure to lipopolysaccharide, however, resulted in prominent increases in the protein concentrations of Nrf2, NF-κB p65, and phosphorylated I-κBα (fig. 5). Hemin significantly enhanced this lipopolysaccharide-induced increase in Nrf2 protein concentrations and, in turn, inhibited the increases in NF-κB p65 and phosphorylated I-κBα protein concentrations (fig. 5). These effects of hemin were not affected by either SnPP or hemoglobin at 15–60 min after the experiment began. However, the effects of hemin on enhancing lipopolysaccharide-induced increases in Nrf2 protein concentrations in the delayed phase of the experiment (i.e.  , 120 min after lipopolysaccharide) were further accentuated by SnPP and hemoglobin (fig. 6). In addition, the effects of hemin on attenuating lipopolysaccharide-induced increases in NF-κB p65 and phosphorylated I-κBα protein concentrations in the delayed phase of the experiment (i.e.  , 120 min after lipopolysaccharide) were significantly reversed by SnPP and hemoglobin. (fig. 6).
Fig. 6. Effects of hemin, tin protoporphyrin, and hemoglobin (Hb) on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. Only data derived from samples that were harvested at 15 and 120 min after LPS exposure are illustrated. H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. #  P  < 0.05 compared with the L group. §  P  < 0.05, L + H group  versus  L + H + S group or L + H group  versus  L + H + Hb group. 
Fig. 6. Effects of hemin, tin protoporphyrin, and hemoglobin (Hb) on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. Only data derived from samples that were harvested at 15 and 120 min after LPS exposure are illustrated. H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. #  P  < 0.05 compared with the L group. §  P  < 0.05, L + H group  versus  L + H + S group or L + H group  versus  L + H + Hb group. 
Fig. 6. Effects of hemin, tin protoporphyrin, and hemoglobin (Hb) on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. Only data derived from samples that were harvested at 15 and 120 min after LPS exposure are illustrated. H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. #  P  < 0.05 compared with the L group. §  P  < 0.05, L + H group  versus  L + H + S group or L + H group  versus  L + H + Hb group. 
×
Discussion
Data from this study confirmed our hypothesis that superinduction of HO-1 significantly attenuated lipopolysaccharide-induced CAT-2 expression and l-arginine transport in stimulated macrophages. Our data also illustrated that inhibiting the activity of HO-1 reversed the effects of HO-1. These data clearly demonstrated the regulatory effects of HO-1 on CAT-2 expression and l-arginine transport during sepsis. The crucial pathophysiologic role of iNOS/nitric oxide pathway during sepsis is well established.1,2 As mentioned before, CAT-2-mediated l-arginine transport constitutes part of the downstream regulatory pathways of iNOS activity.5,6 Data from this study thus provide clear evidence to support the concept that HO-1 superinduction has significantly antiinflammatory effects against sepsis and warrants further investigation.
Our data confirmed that both hemin and lipopolysaccharide induced HO-1 expression.10,12 Hemin has been reported to induce nuclear translocation of Nrf2, a crucial step for activation of this key regulator of the antioxidant responsive element that mediates the induction of antioxidant enzymes.13 Nrf2 has also been reported to mediate lipopolysaccharide-induced HO-1 expression.23 These concepts were confirmed by data from this study. In addition, our data demonstrated that hemin plus lipopolysaccharide superinduced HO-1 expression. Our data also revealed that lipopolysaccharide-induced Nrf2 activation was enhanced by hemin. Judging from these data, we believe that Nrf2 activation plays a crucial role in mediating the hemin-induced HO-1 superinduction in lipopolysaccharide-stimulated macrophages.
It is well established that regulation of CAT-2 expression involves NF-κB,14 the essential pathway that regulates transcription of a wide array of proinflammatory molecules.24 Our data demonstrated that hemin significantly enhanced Nrf2 activation and HO-1 expression and, in turn, inhibited NF-κB activation and CAT-2 expression in lipopolysaccharide-stimulated macrophages. These data, in accord with a recent report,25 highlighted the interplay between Nrf2, HO-1, NF-κB, and CAT-2 during sepsis. These data also provide clear evidence to outline the mechanisms that accounted for the antiinflammatory capacity of HO-1.
Interestingly, our data revealed that SnPP, the HO-1 inhibitor, further increased the HO-1 protein concentrations but reversed the inhibitory effects of HO-1 on CAT-2 expression and l-arginine transport that were induced by lipopolysaccharide and hemin. These data seemed to contradict the concept that superinduction of HO-1 possesses antiinflammatory capacity against sepsis. However, our HO activity data clearly demonstrated that SnPP significantly inhibited the HO activity in macrophages stimulated with lipopolysaccharide plus hemin. Judging from these data, we speculate that this SnPP-induced increase in HO-1 protein concentrations in macrophages stimulated with lipopolysaccharide plus hemin may be the result of a compensatory mechanism secondary to a significant decrease in HO-1 activity.
Heme oxygenase 1 catalyzes the degradation of heme to carbon monoxide, biliverdin, bilirubin, and iron.8 These end products were once considered as toxic metabolic waste products. However, recent data seem to suggest that these HO-1 end products possess certain therapeutic potentials. For example, administration of biliverdin has been demonstrated to mitigate the dextran sodium sulfate-induced experimental colitis.26 Administration of bilirubin has been reported to lead to the long-term survival of allogeneic islets.27 In addition, HO-1–related endogenous carbon monoxide production was reported to protect organs against ischemia–reperfusion injury.28,29 Administration of exogenous carbon monoxide has also been reported to attenuate the endotoxin-induced inflammatory response in macrophages.30 Data from this study clearly demonstrated that the effect of HO-1 on inhibiting LPS-induced CAT-2 expression and l-arginine transport was significantly reversed by hemoglobin, a carbon monoxide scavenger. We also found that hemoglobin, similar to SnPP, further increased the HO-1 protein concentrations but inhibited the HO activity in macrophages stimulated with lipopolysaccharide plus hemin. These data seem to support the concept that carbon monoxide plays a crucial role in mediating the therapeutic effects of HO-1. However, because this study did not assay the concentration of carbon monoxide, no definitive conclusion can be drawn.
Our data clearly demonstrated that the regulatory effects of hemin on the expression of HO-1, CAT-2, Nrf2, and NF-κB in lipopolysaccharide-stimulated macrophages were dose dependent. In addition, these effects of hemin could be significantly reversed by SnPP (i.e.  , the HO-1 inhibitor) and hemoglobin (i.e.  , the carbon monoxide scavenger). However, our data also revealed that SnPP and hemoglobin did not inhibit the effects of hemin at the highest dose (i.e.  , 500 μm). One possible explanation is that the dose of SnPP (i.e.  , 50 μm) and the dose of hemoglobin (i.e.  , 10 μm) used in this study were not potent enough to reverse the effects of hemin at the dose of 500 μm. The other possibility is that the timing of SnPP and hemoglobin administration does not allow SnPP and hemoglobin to fully exhibit their inhibiting effects on 500 μm of hemin. More studies are needed before further conclusions can be drawn.
In summary, HO-1 induction significantly inhibited CAT-2 expression and l-arginine transport in lipopolysaccharide-stimulated macrophages. The mechanisms involved activation of Nrf2 and inhibition of NF-κB. In addition, carbon monoxide mediated, at least in part, the effects of HO-1 induction on inhibiting lipopolysaccharide-induced CAT-2 expression and l-arginine transport in stimulated macrophages. The proposed mechanisms are summarized in figure 7.
Fig. 7. Schematic representation of possible mechanisms involved in hemin inhibition of type 2 cationic amino acid transporter (CAT-2) and l-arginine transport in lipopolysaccharide-stimulated murine macrophages.  Solid lines  are relations confirmed by data directly observed.  Broken lines  are relations supported by inhibition of heme oxygenase 1 (HO-1) by tin protoporphyrin and scavenging carbon monoxide by hemoglobin. (+) = Increase/induce; (−) = decrease/inhibit; NF-κB = nuclear factor-κB; Nrf2 = nuclear factor E2–related factor 2. 
Fig. 7. Schematic representation of possible mechanisms involved in hemin inhibition of type 2 cationic amino acid transporter (CAT-2) and l-arginine transport in lipopolysaccharide-stimulated murine macrophages.  Solid lines  are relations confirmed by data directly observed.  Broken lines  are relations supported by inhibition of heme oxygenase 1 (HO-1) by tin protoporphyrin and scavenging carbon monoxide by hemoglobin. (+) = Increase/induce; (−) = decrease/inhibit; NF-κB = nuclear factor-κB; Nrf2 = nuclear factor E2–related factor 2. 
Fig. 7. Schematic representation of possible mechanisms involved in hemin inhibition of type 2 cationic amino acid transporter (CAT-2) and l-arginine transport in lipopolysaccharide-stimulated murine macrophages.  Solid lines  are relations confirmed by data directly observed.  Broken lines  are relations supported by inhibition of heme oxygenase 1 (HO-1) by tin protoporphyrin and scavenging carbon monoxide by hemoglobin. (+) = Increase/induce; (−) = decrease/inhibit; NF-κB = nuclear factor-κB; Nrf2 = nuclear factor E2–related factor 2. 
×
References
Kilbourn RG, Griffith OW: Overproduction of nitric oxide in cytokine-mediated and septic shock. J Natl Cancer Inst 1992; 84:827–31Kilbourn, RG Griffith, OW
Szabo C, Mitchell JA, Thiemermann C, Vane JR: Nitric oxide-mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock. Br J Pharmacol 1993; 108:786–92Szabo, C Mitchell, JA Thiemermann, C Vane, JR
Liu SF, Ye X, Malik AB: In vivo  inhibition of nuclear factor-kappa B activation prevents inducible nitric oxide synthase expression and systemic hypotension in a rat model of septic shock. J Immunol 1997; 159:3976–83Liu, SF Ye, X Malik, AB
Dickinson E, Tuncer R, Nadler E, Boyle P, Alber S, Watkins S, Ford H: NOX, a novel nitric oxide scavenger, reduces bacterial translocation in rats after endotoxin challenge. Am J Physiol 1999; 277:G1281–7Dickinson, E Tuncer, R Nadler, E Boyle, P Alber, S Watkins, S Ford, H
Bogle RG, Baydoun AR, Pearson JD, Moncada S, Mann GE: L-arginine transport is increased in macrophages generating nitric oxide. Biochem J 1992; 284:15–8Bogle, RG Baydoun, AR Pearson, JD Moncada, S Mann, GE
Mori M, Gotoh T: Regulation of nitric oxide production by arginine metabolic enzymes. Biochem Biophys Res Commun 2000; 275:715–9Mori, M Gotoh, T
Nicholson B, Manner CK, Kleeman J, MacLeod CL: Sustained nitric oxide production in macrophages requires the arginine transporter CAT2. J Biol Chem 2001; 276:15881–5Nicholson, B Manner, CK Kleeman, J MacLeod, CL
Morse D, Choi AM: Heme oxygenase-1: The “emerging molecule” has arrived. Am J Respir Cell Mol Biol 2002; 27:8–16Morse, D Choi, AM
Morse D, Pischke SE, Zhou Z, Davis RJ, Flavell RA, Loop T, Otterbein SL, Otterbein LE, Choi AM: Suppression of inflammatory cytokine production by carbon monoxide involves the JNK pathway and AP-1. J Biol Chem 2003; 2786:36993–98Morse, D Pischke, SE Zhou, Z Davis, RJ Flavell, RA Loop, T Otterbein, SL Otterbein, LE Choi, AM
Srisook K, Cha YN: Biphasic induction of heme oxygenase-1 expression in macrophages stimulated with lipopolysaccharide. Biochem Pharmacol 2004; 68:1709–20Srisook, K Cha, YN
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; 12:70–9Srisook, K Cha, YN
Huang TY, Tsai PS, Wang TY, Huang CL, Huang CJ: Hyperbaric oxygen attenuation of lipopolysaccharide-induced acute lung injury involves heme oxygenase-1. Acta Anaesthesiol Scand 2005; 49:1293–301Huang, TY Tsai, PS Wang, TY Huang, CL Huang, CJ
Alam J, Stewart D, Touchard C, Boinapally S, Choi AM, Cook JL: Nrf2, a Cap‘n’Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem 1999; 274:26071–8Alam, J Stewart, D Touchard, C Boinapally, S Choi, AM Cook, JL
Hammermann R, Dreissig MD, Mossner J, Fuhrmann M, Berrino L, Gothert M, Racke K: Nuclear factor-kappaB mediates simultaneous induction of inducible nitric-oxide synthase and up-regulation of the cationic amino acid transporter CAT-2B in rat alveolar macrophages. Mol Pharmacol 2000; 58:1294–302Hammermann, R Dreissig, MD Mossner, J Fuhrmann, M Berrino, L Gothert, M Racke, K
Lin WC, Tsai PS, Huang CJ: Catecholamines’ enhancement of inducible nitric oxide synthase-induced nitric oxide biosynthesis involves CAT-1 and CAT-2A. Anesth Analg 2005; 101:226–32Lin, WC Tsai, PS Huang, CJ
Kim YC, Masutani H, Yamaguchi Y, Itoh K, Yamamoto M, Yodoi J: Hemin-induced activation of the thioredoxin gene by Nrf2: A differential regulation of the antioxidant responsive element by a switch of its binding factors. J Biol Chem 2001; 276:18399–406Kim, YC Masutani, H Yamaguchi, Y Itoh, K Yamamoto, M Yodoi, J
Hartsfield CL, McMurtry IF, Ivy DD, Morris KG, Vidmar S, Rodman DM, Fagan KA: Cardioprotective and vasomotor effects of HO activity during acute and chronic hypoxia. Am J Physiol Heart Circ Physiol 2004; 287:H2009–15Hartsfield, CL McMurtry, IF Ivy, DD Morris, KG Vidmar, S Rodman, DM Fagan, KA
Lee TS, Tsai HL, Chau LY: Induction of heme oxygenase-1 expression in murine macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-Delta 12,14-prostaglandin J2. J Biol Chem 2003; 278:19325–30Lee, TS Tsai, HL Chau, LY
Huang CJ, Stevens BR, Nielsen RB, Slovin PN, Fang X, Nelson DR, Skimming JW: Interleukin-10 inhibition of nitric oxide biosynthesis involves suppression of CAT-2 transcription. Nitric Oxide 2002; 6:79–84Huang, CJ Stevens, BR Nielsen, RB Slovin, PN Fang, X Nelson, DR Skimming, JW
Smirnova IV, Bittel DC, Ravindra R, Jiang H, Andrews GK: Zinc and cadmium can promote rapid nuclear translocation of metal response element-binding transcription factor-1. J Biol Chem 2000; 275:9377–84Smirnova, IV Bittel, DC Ravindra, R Jiang, H Andrews, GK
Turcanu V, Dhouib M, Poindron P: Determination of heme oxygenase activity in murine macrophages for studying oxidative stress inhibitors. Anal Biochem 1998; 263:251–3Turcanu, V Dhouib, M Poindron, P
Kakuda DK, Sweet MJ, MacLeod CL, Hume DA, Markovich D: CAT2-mediated L-arginine transport and nitric oxide production in activated macrophages. Biochem J 1999; 340:549–53Kakuda, DK Sweet, MJ MacLeod, CL Hume, DA Markovich, D
Rushworth SA, Chen XL, Mackman N, Ogborne RM, O’connell MA: Lipopolysaccharide-induced heme oxygenase-1 expression in human monocytic cells is mediated via  Nrf2 and protein kinase C. J Immunol 2005; 175:4408–15Rushworth, SA Chen, XL Mackman, N Ogborne, RM O’connell, MA
Blackwell TS, Christman JW: The role of nuclear factor-kappa B in cytokine gene regulation. Am J Respir Cell Mol Biol 1997; 17:3–9Blackwell, TS Christman, JW
Banning A, Brigelius-Flohe R: NF-kappaB, Nrf2, and HO-1 interplay in redox-regulated VCAM-1 expression. Antioxid Redox Signal 2005; 7:889–99Banning, A Brigelius-Flohe, R
Berberat PO, Rahim YI, Yamashita K, Warny MM, Csizmadia E, Robson SC, Bach FH: Heme oxygenase-1-generated biliverdin ameliorates experimental murine colitis. Inflamm Bowel Dis 2005; 11:350–9Berberat, PO Rahim, YI Yamashita, K Warny, MM Csizmadia, E Robson, SC Bach, FH
Wang H, Lee SS, Dell’Agnello C, Tchipashvili V, D’Avilla J, Czismadia E, Chin BY, Bach FH: Bilirubin can induce tolerance to islet allografts. Endocrinology 2006; 147:762–8Wang, H Lee, SS Dell’Agnello, C Tchipashvili, V D’Avilla, J Czismadia, E Chin, BY Bach, FH
Szabo ME, Gallyas E, Bak I, Rakotovao A, Boucher F, de Leiris J, Nagy N, Varga E, Tosaki A: Heme oxygenase-1-related carbon monoxide and flavonoids in ischemic/reperfused rat retina. Invest Ophthalmol Vis Sci 2004; 45:3727–32Szabo, ME Gallyas, E Bak, I Rakotovao, A Boucher, F de Leiris, J Nagy, N Varga, E Tosaki, A
Akamatsu Y, Haga M, Tyagi S, Yamashita K, Graca-Souza AV, Ollinger R, Czismadia E, May GA, Ifedigbo E, Otterbein LE, Bach FH, Soares MP: Heme oxygenase-1-derived carbon monoxide protects hearts from transplant associated ischemia reperfusion injury. FASEB J 2004; 18:771–2Akamatsu, Y Haga, M Tyagi, S Yamashita, K Graca-Souza, AV Ollinger, R Czismadia, E May, GA Ifedigbo, E Otterbein, LE Bach, FH Soares, MP
Sawle P, Foresti R, Mann BE, Johnson TR, Green CJ, Motterlini R: Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages. Br J Pharmacol 2005; 145:800–10Sawle, P Foresti, R Mann, BE Johnson, TR Green, CJ Motterlini, R
Fig. 1. Effects of hemin on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
Fig. 1. Effects of hemin on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
Fig. 1. Effects of hemin on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
×
Fig. 2. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. 
Fig. 2. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. 
Fig. 2. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on heme oxygenase (HO)-1 and HO-2 expression and HO activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The HO-1 and HO-2 protein concentrations were normalized by β-actin. The HO activity was determined by measuring the bilirubin concentrations of the culture media. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. 
×
Fig. 3. Effects of hemin on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
Fig. 3. Effects of hemin on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
Fig. 3. Effects of hemin on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; PBS = phosphate-buffered saline. *  P  < 0.05 compared with the negative control group. #  P  < 0.05 compared with the positive control group. †  P  < 0.05, LPS + Hemin (500) or LPS + Hemin (50) group  versus  LPS + Hemin (5) group. ‡  P  < 0.05, LPS + Hemin (500) group  versus  LPS + Hemin (50) group. 
×
Fig. 4. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. ¥  P  < 0.05, LPS + Hemin (5) + SnPP group  versus  LPS + Hemin (5) + Hb group. 
Fig. 4. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. ¥  P  < 0.05, LPS + Hemin (5) + SnPP group  versus  LPS + Hemin (5) + Hb group. 
Fig. 4. Effects of tin protoporphyrin (SnPP) and hemoglobin (Hb) on type 2 cationic amino acid transporter (CAT-2) expression and CAT activity in lipopolysaccharide (LPS) plus hemin-stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The CAT-2 protein concentrations were normalized by β-actin. The CAT activity was determined by measuring the l-arginine transport using l-[3H]arginine uptake assay. Data are expressed as mean ± SD. Hemin (5) = 5 μm hemin; Hemin (50) = 50 μm hemin; Hemin (500) = 500 μm hemin; H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. ¶  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + SnPP group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + SnPP group. §  P  < 0.05, LPS + Hemin (5) group  versus  LPS + Hemin (5) + Hb group or LPS + Hemin (50) group  versus  LPS + Hemin (50) + Hb group. ¥  P  < 0.05, LPS + Hemin (5) + SnPP group  versus  LPS + Hemin (5) + Hb group. 
×
Fig. 5. Effects of lipopolysaccharide and lipopolysaccharide (LPS) plus hemin on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. #  P  < 0.05 compared with the LPS group. 
Fig. 5. Effects of lipopolysaccharide and lipopolysaccharide (LPS) plus hemin on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. #  P  < 0.05 compared with the LPS group. 
Fig. 5. Effects of lipopolysaccharide and lipopolysaccharide (LPS) plus hemin on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. #  P  < 0.05 compared with the LPS group. 
×
Fig. 6. Effects of hemin, tin protoporphyrin, and hemoglobin (Hb) on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. Only data derived from samples that were harvested at 15 and 120 min after LPS exposure are illustrated. H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. #  P  < 0.05 compared with the L group. §  P  < 0.05, L + H group  versus  L + H + S group or L + H group  versus  L + H + Hb group. 
Fig. 6. Effects of hemin, tin protoporphyrin, and hemoglobin (Hb) on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. Only data derived from samples that were harvested at 15 and 120 min after LPS exposure are illustrated. H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. #  P  < 0.05 compared with the L group. §  P  < 0.05, L + H group  versus  L + H + S group or L + H group  versus  L + H + Hb group. 
Fig. 6. Effects of hemin, tin protoporphyrin, and hemoglobin (Hb) on the activation of nuclear factor E2–related factor 2 (Nrf2) and nuclear factor κB (NF-κB) in lipopolysaccharide (LPS)–stimulated murine macrophages. Representative gel photography illustrates the products of immunoblotting assay. The Nrf2, NF-κB p65, and β-actin (the internal standard) protein concentrations in the nuclear extracts and the phosphorylated inhibitor κBα (phos-I-κBα) and β-actin protein concentrations in the cytosolic extracts were determined, and the Nrf2, NF-κB p65, and phos-I-κBα protein concentrations were normalized by β-actin. Data are expressed as mean ± SD. Only data derived from samples that were harvested at 15 and 120 min after LPS exposure are illustrated. H = hemin; L = lipopolysaccharide; S = tin protoporphyrin. #  P  < 0.05 compared with the L group. §  P  < 0.05, L + H group  versus  L + H + S group or L + H group  versus  L + H + Hb group. 
×
Fig. 7. Schematic representation of possible mechanisms involved in hemin inhibition of type 2 cationic amino acid transporter (CAT-2) and l-arginine transport in lipopolysaccharide-stimulated murine macrophages.  Solid lines  are relations confirmed by data directly observed.  Broken lines  are relations supported by inhibition of heme oxygenase 1 (HO-1) by tin protoporphyrin and scavenging carbon monoxide by hemoglobin. (+) = Increase/induce; (−) = decrease/inhibit; NF-κB = nuclear factor-κB; Nrf2 = nuclear factor E2–related factor 2. 
Fig. 7. Schematic representation of possible mechanisms involved in hemin inhibition of type 2 cationic amino acid transporter (CAT-2) and l-arginine transport in lipopolysaccharide-stimulated murine macrophages.  Solid lines  are relations confirmed by data directly observed.  Broken lines  are relations supported by inhibition of heme oxygenase 1 (HO-1) by tin protoporphyrin and scavenging carbon monoxide by hemoglobin. (+) = Increase/induce; (−) = decrease/inhibit; NF-κB = nuclear factor-κB; Nrf2 = nuclear factor E2–related factor 2. 
Fig. 7. Schematic representation of possible mechanisms involved in hemin inhibition of type 2 cationic amino acid transporter (CAT-2) and l-arginine transport in lipopolysaccharide-stimulated murine macrophages.  Solid lines  are relations confirmed by data directly observed.  Broken lines  are relations supported by inhibition of heme oxygenase 1 (HO-1) by tin protoporphyrin and scavenging carbon monoxide by hemoglobin. (+) = Increase/induce; (−) = decrease/inhibit; NF-κB = nuclear factor-κB; Nrf2 = nuclear factor E2–related factor 2. 
×