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Published as doi: 10.1096/fj.06-6346fje.
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(The FASEB Journal. 2006;20:2651-2653.)
© 2006 FASEB

Role of Bach1 and Nrf2 in up-regulation of the heme oxygenase-1 gene by cobalt protoporphyrin

Ying Shan*,{dagger},1, Richard W. Lambrecht{dagger},{ddagger}, Susan E. Donohue*,{dagger} and Herbert L. Bonkovsky*,{dagger},§

* Department of Medicine,

{dagger} the Liver-Biliary-Pancreatic Center, Departments of

{ddagger} Pharmacology and

§ Molecular, Microbial and Structural Biology of the University of Connecticut Health Center, Farmington, Connecticut, USA

1Correspondence: 263 Farmington Ave. MC-1119, Farmington, CT 06030, USA. E-mail: shan{at}uchc.edu

ABSTRACT

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is highly inducible by a large number of physical and chemical factors. CoPP is known to be a potent and effective inducer of HO-1 activity in many tissues. Here we report that CoPP up-regulates HO-1 via Bach1 and Nrf2 in human liver cells. CoPP did not influence hepatic Bach1 or Nrf2 mRNA levels, but markedly reduced Bach1 protein levels by increasing degradation of Bach1 protein (t1/2 from 19 h to 2.8 h), and increased Nrf2 by decreasing degradation of Nrf2 protein (t1/2 from 2.5 h to 9 h). Silencing Bach1 by Bach1-siRNA significantly increased levels of HO-1 mRNA and protein, and addition of CoPP up-regulated HO-1 mRNA and protein further. However, silencing Nrf2 mRNA by Nrf2-siRNA did not significantly change baseline HO-1 mRNA or protein levels, but significantly decreased 5–10 µM CoPP-mediated up-regulation of HO-1 mRNA levels compared with CoPP alone. Transfection with equal amounts of non-Bach1 or non-Nrf2 related control siRNA did not reduce Bach1 or Nrf2 mRNA or protein, confirming the specificity of Bach1- and Nrf2-siRNA in Huh-7 cells. We conclude that the pathway of CoPP-mediated induction of HO-1 involves the repression of Bach1 and up-regulation of the Nrf2 protein by post-transcriptional site(s) of action. Because CoPP, unlike heme, is neither a prooxidant nor a substrate for HO-1, it might be considered as a potential therapeutic agent in situations where up-regulation of HO-1 is desired.—Shan, Y., Lambrecht, R. W., Donohue, S. E., Bonkovsky, H. L. Role of Bach1 and Nrf2 in up-regulation of the heme oxygenase-1 gene by cobalt protoporphyrin.


Key Words: HO-1 • siRNA

HEME OXYGENASE-1 (HO-1) is the highly inducible rate-controlling enzyme of heme catabolism (1 2 3 4 5) . It catalyzes the breakdown of heme to biliverdin, iron, and carbon monoxide (CO). HO-1 has cytoprotectant, antioxidant, anti-inflammatory, and immunomodulatory properties (4 5 6) . These are believed to be related to its ability to decrease excessive levels of potentially toxic heme within cells and to convert heme to less reactive iron (which can be stored in the nontoxic form of ferritin) and to the antioxidant properties of biliverdin and bilirubin, as well as the anti-inflammatory and immunosuppressant properties of CO (4 5 6) . An infant with severe deficiency of HO-1 born to parents, each of whom had a partial deficiency of HO-1 without clinical manifestations, showed a severely affected phenotype, which proved fatal (7) . Increased expression of HO-1 has been shown to be protective in ischemia/reperfusion injury, organ transplantation, protection against renal and pulmonary injury, and amelioration of adverse hemodynamic effects resulting from liver disease and portal hypertension (5 , 8 , 9) . HO-1 also plays a vital role in the inhibition of vascular-occlusion in mice expressing the sickle cell transgene (10) . Treatment of such mice with heme further increases HO-1 expression and inhibits hypoxia/reoxygenation-induced stasis, leukocyte-endothelium interactions and NF-{kappa}B, vascular cell adhesion molecule (VCAM)-1, and ICAM-1 expression. In contrast, HO-1 inhibition by tin protoporphyrin exacerbates stasis in this murine model of sickle cell disease (10) .

Earlier work from our own and other laboratories established that HO-1 could be up-regulated markedly by a variety of stressful stimuli, as well as by heme or certain other metalloporphyrins, particularly cobalt protoporphyrin (CoPP) (11 12 13 14 15) . The primary mechanism for up-regulation of the HO-1 gene is by increased transcription of the gene (16) . Induction by such stressors as sodium arsenite or other arsenicals (which produce a chemical oxidative stress), by transition metal ions such as cadmium or cobalt, by hydrogen peroxide or other reactive oxygen species (ROS), or by heat shock are clearly different in mechanism from the up-regulation produced by metalloporphyrins (14 , 15 , 17 18 19 20 21) . For example, earlier work from our laboratory on the avian HO-1 promoter showed that cMyc/Max and USF elements in the 5'-untranslated region (UTR) of the HO-1 gene played the key role in inductions by cadmium chloride or cobalt chloride (12) . In contrast, inductions of HO-1 by sodium arsenite or phenylarsine oxide depend primarily on activation of the MAP kinases leading to increased levels of activating protein (AP) –1 proteins, which bind to several AP-1-consensus elements found in the HO-1 promoter (19 , 22 , 23) . The up-regulation of the HO-1 gene produced by heme or cobalt protoporphyrin is not mediated by these classic stress pathways or kinase cascades, but rather depends on several heme-responsive elements (and a metalloporphyrin-responsive element) found in the 5'-UTR of rodent, human, and avian HO-1 (13) that are distinct from other consensus promoter elements.

Bach1, one of the family of basic leucine zipper transcription factors, is a heme binding protein (24 25 26) . It is highly conserved, and Bach1 proteins have been described in avian and mammalian species. Recent work indicates that Bach1, under baseline conditions, forms heterodimers with small proteins of the Maf family, and these heterodimers repress transcription of the HO-1 gene by binding to the heme-responsive elements (HeRE) in the 5'-UTR of the HO-1 promoter. Under conditions of excess heme, increased binding of heme to Bach1 leads to a conformational change and a decrease in DNA binding activity. This permits Maf-Maf, Nrf2-Maf, and other activating heterodimers to occupy the HeRE sites in the HO-1 promoter and leads to increased transcription and up-regulation of expression of the gene (26 27 28) . We recently reported that heme-mediated induction of HO-1 gene expression occurs via Bach1 in human Huh-7 cells. By using Bach1-siRNA to silence the Bach1 gene, we demonstrated that up-regulation of HO-1 gene expression by heme was markedly increased in these cells (29) .

The transcription factor Nrf2 is also a basic leucine zipper transcription factor (30 31 32 33 34 35) . Nrf2 is involved in cellular protection against oxidative stress through antioxidant response element (ARE) -directed induction of several phase 2 detoxifying and antioxidant enzymes, including HO-1 (36) . In a recent study of immortalized proximal tubular epithelial cells, heme up-regulated Nrf2 by stabilizing Nrf2 proteins without affecting Nrf2 mRNA levels (37) .

CoPP is known to be a potent and effective inducer of HO-1 activity in many tissues. The cytoprotective function of HO-1 activity during CoPP-mediated cellular injury is now readily apparent and has been verified in multiple studies (38 39 40 41 42 43) ; however, the mechanisms by which CoPP activates the HO-1 gene in the liver or other organs are less well understood. In the work described here, we demonstrate that the transcription factors Bach1 and Nrf2 are both involved in CoPP up-regulation of HO-1 gene expression in human hepatoma cells. Additional studies indicated a CoPP-mediated mechanism for regulation of Bach1 and Nrf2 and subsequent HO-1 gene activation, namely, posttranscriptional destabilization of the Bach1 protein and stabilization of the Nrf2 protein in response to CoPP.

MATERIALS AND METHODS

Materials and cell culture
The human hepatoma cell line, Huh-7, was purchased from the Japan Health Research Resources Bank (Osaka, Japan). Ferric (Fe+3)-protoporphyrin IX·Cl (heme) and cobalt protoporphyrin (CoPP) were purchased from Frontier Scientific (Logan, UT, USA). Dimethyl sulfoxide (DMSO) was purchased from FisherBiotech (Fair Lawn, NJ, USA). RNAzol was from Biotecx (Houston, TX, USA). Goat anti-human Bach1, goat anti-human GAPDH polyclonal antibodies, rabbit anti-human Nrf2, mouse anti-rabbit IgG, and rabbit anti-goat IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-human HO-1 polyclonal antibody (pAb) was purchased from StressGen (Victoria, BC, Canada). Enhanced chemiluminescence (ECL)-Plus was purchased from Amersham Biosciences Corp (Piscataway, NJ, USA). Nonspecific control duplexes-XIII (NSCD), LaminB2 duplex, Bach1-siRNA, and Nrf2-siRNA were purchased from Dharmacon (Lafayette, CO, USA).

Cell culture and treatment
Huh-7 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 100 U/ml penicillin, 100 µg /ml streptomycin, and 10% (v/v) FBS (Invitrogen, Carlsbad, CA, USA) and were routinely passaged twice a week. Heme and CoPP were dissolved in DMSO and stored at –20°C until use. Cycloheximide was dissolved in DMSO and stored at 4–8°C until use. Addition of DMSO to the cultures did not exceed 1 µl of DMSO per milliliter of media.

siRNAs preparation and transfection
Bach1 and Nrf2-siRNAs were synthesized and annealed at Dharmacon. We used Bach1-siRNAs, targeting the four positions of the human Bach1 mRNA (accession no. NM_001186) as described (29) . In brief, Huh-7 cells were plated the day before transfections and grown up to 50% confluence in 24-well plates. Transfections were carried out with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Transfected cells were grown for 48 h in a 37°C incubator with 5% CO2 and changed to serum-free medium just before treatments were added. After treatment with indicated CoPP concentrations for 4–16 h, cells were harvested. Total RNA and protein were extracted and stored at –80°C for subsequent quantitative RT-polymerase chain reaction (RT-PCR) or Western blot measurements. Each treatment used at least triplicate samples.

Quantitative RT-PCR
Total RNA from treated cells was extracted and quantitative RT-PCR was performed as described before (29) . Nrf2 sense primer was 5'-AGC ACA CCC AGT CAG AAA CCA G; Nrf2 antisense primer was 5'-TCT ACA AAC GGG AAT GTC G. We included no-template and no-reverse transcriptase as negative controls, which were expected to produce negligible signals (Ct values>35). Standard curves of HO-1, Bach1, Nrf2, and GAPDH were constructed with results of parallel polymerase chain reaction (PCR) reactions performed on serial dilutions of a standard DNA (from one of the controls). Fold change values were calculated by comparative Ct analysis after normalizing for the quantity of GAPDH mRNA in samples.

Western blot
Protein preparation and Western blot were as described before (29) . In brief, total proteins (30–50 µg) were separated on 4–15% gradient SDS-PAGE (Bio-Rad). After electrophoretic transfer onto Immune-Blot PVDF membrane (Bio-Rad), membranes were blocked for 1 h in PBS containing 5% nonfat dry milk and 0.1% Tween-20, then incubated for 1 h with primary antibody (Ab) at room temperature. Dilutions of the primary antibodies were as follows: 1:500 for anti-HO-1 Ab, and 1:1000 for anti-Bach1, anti-Nrf2, and anti-GAPDH antibodies. The membranes were then incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (dilution 1:10,000). Finally, the bound antibodies were visualized with the ECL-Plus chemiluminescence system according to the manufacturer’s protocol (Amersham, Piscataway, NJ, USA). A Kodak 1DV3.6 computer-based imaging system (Eastman-Kodak, Rochester, NY, USA) was used to measure the relative optical density (OD) of each specific band obtained after Western blot. Data are expressed as percentage of the control (corresponding to the value obtained with the vehicle-treated cells or nontransfected cells), which were assigned values of 100% or one.

Statistical analyses of data
Experiments were performed at least three times with similar results. Except for Western blots, all experiments included at least triplicate samples for each treatment group. Representative results from single experiments are presented. Statistical analyses were performed with JMP 4.0.4 software (SAS Institute, Cary, NC, USA). For normally distributed data, differences in mean values were assessed by analysis-of-variance techniques, with the Tukey-Kramer correction for multiple pairwise comparisons, or Dunnett’s test vs. a control. Nonlinear regression was analyzed with Sigmaplot 2001 (Systat, Point Richmond, CA, USA). Values of P < 0.05 were considered significant.

RESULTS

CoPP-mediated induction of HO-1 gene expression in human hepatoma cells
We first examined whether HO-1 mRNA and protein levels are up-regulated by CoPP in Huh-7 cells. As in other systems (39 40 41 42 43) , CoPP induced HO-1 mRNA and protein levels in these cells in a dose-dependent fashion (Fig. 1 A, B). A concentration of CoPP as low as 0.5 µM significantly up-regulated HO-1 mRNA by 3.5-fold (P<0.05), and the maximum (35- to 40-fold) was reached with 10–25 µM CoPP treatment for 4 h (Fig. 1A ). Huh-7 cells treated with 25 µM CoPP failed to grow well, and the total RNA yield was lower than for the other concentrations of CoPP tested, indicating that this concentration of CoPP was toxic to the cells. Consequently, 10 µM CoPP was the highest concentration used in subsequent experiments. The lowest concentration of CoPP that led to detectable up-regulation of HO-1 protein after 16 h of treatment was 1 µM, with the maximum effect at 5–10 µM (Fig. 1B ). Up-regulation of HO-1 mRNA and protein by CoPP was also time related. When Huh-7 cells were treated with 10 µM CoPP for 2, 4, 6, and 8 h, increased HO-1 mRNA was detectable at 2 h and was maximal at 4–6 h. CoPP increased HO-1 protein starting at 6 h with a peak at 16 h (data not shown). Therefore, in subsequent studies we treated cells with CoPP for 4 h for measures of mRNA and for 16 h for measures of proteins.


Figure 1
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  		Figure 1. CoPP up-regulates hepatic HO-1 gene expression in Huh-7 cells. Huh-7 cells were treated with the same amount of vehicle-only (DMSO, 0 µM) and the indicated concentrations of CoPP, after which cells were harvested, total RNA and protein assays were carried out, and HO-1 mRNA and protein levels were quantified by qRT-polymerase chain reaction and Western blot as described in Materials and Methods. Relative amounts of HO-1 mRNA were normalized to GAPDH mRNA, which did not vary with treatment. Values for cells treated with vehicle-only were set equal to 1. A) Dose-dependent up-regulation of hepatic HO-1 mRNA by exposure to CoPP for 4 h. Data are presented as means ± SE from 3 samples. B) Dose-dependent up-regulation of hepatic HO-1 protein by exposure to CoPP for 16 h. Proteins were separated by 4–15% SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with anti-human HO-1 and GAPDH specific antibodies. Representative results from 1 of 3 experiments are shown.

Effects of CoPP on Bach1 and Nrf2 mRNA or protein levels in human hepatoma cells
To assess whether CoPP affected Bach1 or Nrf2 mRNA levels, Huh-7 cells were treated with 0, 1, 5, or 10 µM CoPP for 4 h. Bach1 and Nrf2 mRNA were measured by quantitative RT-PCR. CoPP had no significant effects on Bach1 or Nrf2 mRNA levels (Fig 2 A). We also investigated the effects of CoPP treatment on protein levels of Bach1 and Nrf2. Huh-7 cells were treated with 0, 1, 5, or 10 µM CoPP for 6 and 16 h, total protein was extracted, and Bach1 and Nrf2 proteins were detected by Western blot as described in Materials and Methods. We found that CoPP-mediated regulation of Bach1 and Nrf2 proteins were dose dependent and time related. There were no detectable effects of 10 µM CoPP on Bach1 or Nrf2 proteins after 6 h (data not shown). However, there was down-regulation of Bach1 protein after exposure of Huh-7 cells to 5–10 µM CoPP for 16 h (Fig. 2B ). Nrf2 protein was increased with as little as 1 µM CoPP treatment, with peak up-regulation at 10 µM CoPP for 16 h in Huh-7 cells (Fig. 2B ). These results indicate that CoPP exerts reciprocal effects on Bach1 and Nrf2 by posttranscriptional mechanism(s).


Figure 2
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  		Figure 2. Effects of CoPP on hepatic Bach1 and Nrf2 mRNA and protein levels in Huh-7 cells. Huh-7 cells were treated with 1 µl/ml of DMSO (0 µM) or with the indicated concentration of CoPP, after which cells were harvested and total RNA and protein were extracted. Bach1, Nrf2, and GAPDH mRNA and protein levels were quantified by qRT-polymerase chain reaction and Western blot as described in Materials and Methods. Representative results from 1 of 3 experiments are shown. A) Corresponding results for Bach1 and Nrf2 mRNA levels after treatment with CoPP for 4 h. Data are presented as means ± SE from 3 samples. Relative amounts of Bach1 and Nrf2 mRNA were normalized to GAPDH, which did not vary with treatment. Values for cells treated with vehicle-only were set equal to 1. Addition of CoPP had no effect on Bach1 or Nrf2 mRNA levels. In contrast, B) Treatment with CoPP for 16 h decreased Bach1 protein but increased Nrf2 protein levels.

CoPP influences the rates of Bach1 and Nrf2 protein degradation
To gain further insight into the mechanism by which CoPP affects the levels of Bach1 and Nrf2 proteins, the stabilities of these proteins were examined. As shown in Fig. 3 A, B, Bach1 protein levels in cells treated with CoPP were greatly reduced after treatment with cycloheximide (CHX), an inhibitor of protein synthesis. Bach1 protein levels in cells not treated with CoPP were also decreased by CHX, but to a lesser extent. Specifically, CoPP decreased the Bach1 protein half-life (t1/2) from 19 to 2.8 h. In contrast, CoPP increased the t1/2 of Nrf2 protein from 2.5 to 9 h (Fig. 3C, D ).


Figure 3
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  		Figure 3. Posttranscriptional regulation of Bach1 and Nrf2 expression by CoPP in Huh-7 cells. Huh-7 cells were treated with or without 5 µM CoPP for 16 h, followed by treatment with 100 µg/ml cycloheximide (CHX) for the indicated times, after which cells were harvested and total protein was isolated as described in Materials and Methods. Proteins were separated by 4–15% SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with anti-human Bach1, Nrf2, and GAPDH specific antibodies. A, C) Bach1 and Nrf2 protein levels with or without CoPP. The relative amounts of Bach1 (B) and Nrf2 (D) proteins were normalized to GAPDH. Values for cells treated with vehicle-only were set equal to 1. Protein half-lives (t1/2) are shown. Representative results from 1 of 3 experiments are shown.

Specifically silencing Bach1 and Nrf2 genes with Bach1- or Nrf2-siRNA in Huh-7 cells
Using Bach1- and Nrf2-siRNAs, we reduced the expression of Bach1 and Nrf2 mRNA and protein in Huh-7 cells (Fig. 4 and Fig. 5 ). Effects were analyzed and compared with those from nontransfected cells and with cells transfected with nonspecific control duplexes (NSCD). Specifically, 100 nM Bach1-siRNA at 48 h reduced Bach1 mRNA levels by ~75% compared with nonsiRNA-treated cells (Fig. 4A ), and 20–150 nM Bach1-siRNA almost completely abrogated Bach1 protein expression at 72 h (Fig 4B, C ). Nrf2-siRNA (20–150 nM) reduced Nrf2 mRNA and protein levels by nearly 70% (Fig. 5) . We used 100 nM Bach1-siRNA and 20 nM Nrf2-siRNA in further experiments. To confirm the specificity of gene silencing by Bach1- or Nrf2-siRNAs, we tested Bach1 and Nrf2 gene expression from cells transfected with non-Bach1- or Nrf2-related-siRNA [i.e., nonspecific control duplexes (NSCD)]. We found there were no significant reductions of either Bach1 mRNA or Nrf2 mRNA levels after transfections with NSCD compared with cells that were not transfected (Figs. 4 , 5) . Bach1 and Nrf2 also did not change after transfection with 20 nM LaminB2-siRNA (data not shown). Thus, silencing of the human Bach1 and Nrf2 genes using siRNA targeted to Bach1 and Nrf2 mRNA was effective, specific, and selective.


Figure 4
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  		Figure 4. Specific silencing of Bach1 gene expression by Bach1-siRNA in Huh-7 cells. Huh-7 cells were transfected with Bach1-siRNA or nonspecific control duplex (NSCD as negative control siRNA) and cultured for 48–72 h, after which cells were harvested, mRNA levels were determined by quantitative RT-PCR and proteins were determined by Western blot as described in Materials and Methods. A) Dose-dependent down-regulation of Bach1 mRNA levels after transfection with Bach1-siRNA for 48 h but not by 20 nM NSCD or no-siRNA control. Data are presented as means ± SE from 3 samples. B) Down-regulation of Bach1 protein levels after transfection of Bach1-siRNA for 72 h, but not by 20 nM NSCD or no-siRNA. C) The relative amount of Bach1 protein normalized to GAPDH protein. Values for cells treated with vehicle-only were set equal to 1.


Figure 5
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  		Figure 5. Specific silencing of Nrf2 gene expression by Nrf2-siRNA in Huh-7 cells. Huh-7 cells were transfected with Nrf2-siRNA or nonspecific control duplex (NSCD, as negative control siRNA) and cultured for 48–72 h, after which cells were harvested, mRNA levels were determined by quantitative RT-PCR, and proteins were detected by Western blot as described in Materials and Methods. A) Dose-dependent down-regulation of Nrf2 mRNA levels after transfection with Nrf2-siRNA for 48 h, but not by 20 nM NSCD or no-siRNA control. Data are presented as means ± SE from 3 samples. B) Down-regulation of Nrf2 protein levels after transfection of Nrf2-siRNA for 72 h, but not by 20 nM NSCD and no-siRNA. C) Relative amount of Nrf2 protein normalized to GAPDH. Values for cells treated with vehicle-only were set equal to 1.

Regulation of HO-1 expression by CoPP and Bach1-siRNA
Next we studied the effects of combinations of CoPP and Bach1-siRNA on HO-1 gene regulation in Huh-7 cells. Before testing HO-1, we confirmed that Bach1 mRNA was repressed by >70% by Bach1-siRNA (Fig. 4) . HO-1 mRNA expression levels were increased 5-fold by transfection with 20 nM Bach1-siRNA and further up-regulated by treatment with all concentrations of CoPP tested in the presence of Bach1-siRNA (Fig. 6 A). Using nonlinear regression analysis, HO-1 mRNA levels correlated with CoPP concentrations in the presence or absence of Bach1-siRNA (r=0.99, P<0.0001) (Fig. 6A ). These values significantly increased in magnitude when CoPP and Bach1-siRNA were both present. The two curves of Fig. 6A significantly differ from each other (P<0.01), implying an additive effect of Bach1-siRNA and CoPP on up-regulation of HO-1 gene expression. Bach1-siRNA did not affect Nrf2 mRNA levels in Huh-7 cells (data not shown). As expected, HO-1 protein levels were consistent with those of HO-1 mRNA in cells treated with CoPP in the presence or absence of Bach1-siRNA (Fig. 6B, C ).


Figure 6
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  		Figure 6. Effects of Bach1-siRNA and CoPP on hepatic HO-1 mRNA and protein levels in Huh-7 cells. Huh-7 cells were transfected with or without 100 nM Bach1-siRNA for 48 h, then the culture medium was changed to medium without Bach1-siRNA. Selected cells were next treated with the indicated concentrations of CoPP or an equal volume of vehicle (DMSO, 0 µM) for 4 h for RNA or 16 h for protein. HO-1 mRNA levels were measured by quantitative RT-PCR and proteins were detected by Western blot as described in Materials and Methods. A) Bach1-siRNA significantly increased CoPP up-regulated HO-1 mRNA. Each data point represents mean ± SE from 3 samples. For both lines, nonlinear regression analysis shows r = 0.99, and the data points are significantly different from one another. The two curves are also significantly different from one another using nonlinear regression analysis (P<0.001). B) Bach1-siRNA markedly increased CoPP up-regulated HO-1 protein. C) Relative amounts of HO-1 protein normalized to GAPDH protein are shown graphically. Values for cells treated with vehicle-only were set equal to 1.

Regulation of HO-1 expression by CoPP and Nrf2-siRNA
We also studied the effects of combinations of CoPP and Nrf2-siRNA on HO-1 gene regulation in Huh-7 cells. Before testing HO-1, we confirmed that Nrf2 mRNA was repressed by >70% by Nrf2-siRNA (Fig. 5) . HO-1 mRNA expression levels did not change significantly after transfection with 20 nM Nrf2-siRNA for 48 h. HO-1 mRNA levels were slightly up-regulated by treatment with lower concentrations of CoPP in the presence of Nrf2-siRNA, but significantly reduced at higher concentrations of CoPP (5–10 µM) (Fig. 7 A). Using nonlinear regression analysis, HO-1 mRNA levels were correlated with CoPP concentrations in the presence or absence of Bach1-siRNA (r=0.99, P<0.0001). The two curves of Fig. 7A are significantly different from each other (P<0.005), implying an additive effect of Nrf2-siRNA and CoPP on up-regulation of HO-1 gene expression. Nrf2-siRNA did not affect Bach1 mRNA levels in Huh-7 cells (data not shown). The levels of HO-1 protein were also consistent with levels of mRNA in the cells from CoPP treated with or without Nrf2-siRNA (Fig. 7B, C ).


Figure 7
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  		Figure 7. Effects of Nrf2-siRNA and CoPP on hepatic HO-1 mRNA and protein levels in Huh-7 cells. Huh-7 cells were transfected with or without 20 nM Nrf2-siRNA for 48 h, after which culture medium were changed and selected cells were treated with the indicated concentrations of CoPP or the equal volume of vehicle-only (0 µM). The HO-1 mRNA levels were measured by quantitative RT-PCR, and protein levels were detected by Western blot as described in Materials and Methods. A) The effects of Nrf2-siRNA on HO-1 mRNA levels in the presence or absence of the indicated concentrations of CoPP for 4 h. Data are presented as mean ± SE from 3 samples. Nonlinear regression analysis shows r = 0.99, P < 0.0001 in the cells with or without Nrf2-siRNA treatment. The two curves are significantly different from one another by nonlinear regression analysis (P=0.01). B) Inhibition of Nrf2-siRNA on CoPP up-regulated the levels of HO-1 protein. C) Relative amounts of HO-1 protein normalized to GAPDH protein. Values for cells treated with vehicle-only were set equal to 1.

DISCUSSION

The major findings of this paper are 1) CoPP up-regulates HO-1 mRNA and protein levels in a time-related and dose-dependent fashion in human Huh-7 cells; 2) CoPP has no effect on mRNA levels of Bach1 or Nrf2, but down-regulates Bach1 protein and up-regulates Nrf2 protein levels in Huh-7 cells; 3) CoPP destabilizes Bach1 protein and stabilizes Nrf2 protein, suggesting it regulates these proteins by post-transcriptional mechanisms; 4) silencing the Bach1 gene with specific siRNA significantly increases the baseline level of HO-1 mRNA and protein (5- to 7-fold), and these increased levels of HO-1 mRNA and protein were maintained with increasing concentrations of CoPP; and 5) silencing the Nrf2 gene with specific siRNA does not significantly change the baseline level of HO-1 mRNA and protein but does significantly reduce the amount of HO-1 mRNA and protein induced by higher concentrations of CoPP.

HO-1 is an enzyme with important physiologically relevant antioxidant and cytoprotective effects (44 , 45) . This protection stems not only from a reduction in heme concentrations, but also from the production of biliverdin and bilirubin, which have been shown to be potent antioxidants (4 5 6) . HO-1 is robustly induced in many cells by numerous stressful stimuli such as heat shock (46) , hypoxia (47 , 48) , various oxidative agents (12 , 18 , 20 , 49 , 50) , and by nonstress mediated pathways, heme (51 , 52) , and other metalloporphyrins (52) . In previous studies we characterized up-regulation of the chick HO-1 promoter activity by metalloporphyrins, especially by heme and CoPP (13 , 14) .

Bach1, a member of the basic leucine zipper family of proteins, forms heterodimers with the Maf-related oncoprotein family (25 , 27 , 28) . Recent data from Igarashi et al. indicate that the C-terminal cysteine-proline motifs in Bach1 are critical for heme binding and that this binding is the mechanism by which heme can derepress some genes (26 , 27) . We previously reported that Bach1 plays a critical role in heme-dependent up-regulation of the human HO-1 gene (29) . We identified four HeREs and one MPRE located in the 5'-UTR of the chick HO-1 promoter region (21) . Our results were consistent with the idea that heme-dependent up-regulation of HO-1 is due at least in part to a Bach1/heme interaction, which allows Maf/MAREs (HeREs) to regulate the gene. In the present work, we demonstrate that Bach1 also plays an important role in the CoPP-dependent up-regulation of the human HO-1 gene. However, our previous results showed that exogenous heme produces an additional up-regulation of HO-1 beyond that produced by Bach1-siRNAs (29) , suggesting that heme does not act solely through its effects on Bach1.

A recent study showed that heme increases the stability of the Nrf2 protein (37) , leading to accumulation of heterodimers of Nrf2/MafG that bind to the HeRE’s, thus activating the HO-1 gene. Under basal conditions, Nrf2 remains in the cytoplasm, where it is associated with the actin cytoskeleton through Keap1 (53) , which mediates its continuous degradation by a proteasome-dependent pathway (37 , 54 , 55) . Like HO-1, Nrf2 activation confers protection to different cell types against oxidative stress (36 , 56 , 57) .

Understanding the regulation of expression of HO-1 is complicated by the fact that regulatory responses are not restricted to heme. Due to similarities in structure and chemistry, CoPP may bind to the same regulatory sites as heme itself. Here we show that the heme- and CoPP-mediated induction of the HO-1 gene is a function of both Bach1 (via heme or CoPP binding with subsequent derepression of HO-1 gene expression) and of Nrf2 (via a heme- or CoPP-mediated increase in stability of Nrf2 protein with subsequent induction of HO-1 gene expression), and even greater effects of CoPP on up-regulation of the HO-1. This mechanism is independent of the ability of (many) metalloporphyrins to bind or activate oxygen, because such actions are not part of the panoply of CoPP effects on hepatocytes (or other cells) (14) . Di Noìa et al. recently reported that CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers (58) . Carnitine is known to reduce oxidative stress (59) . Thus, if anything, CoPP appears to decrease oxidative stress.

Cobalt-containing compounds have been used in experimental and therapeutic applications in humans and laboratory animals. Inorganic cobalt is used to stimulate erythropoiesis in patients suffering from anemia associated with renal disease (60) . In addition to its effects on heme-cytochrome P-450 metabolism, CoPP has been shown to affect endocrine status and weight gain in experimental animals (61 62 63 64) . In fact, experimental use of CoPP to suppress appetite and body weight gain in normal and genetically obese Zucker rats has been highly successful (65 , 66) .

Both heme and CoPP are potent inducers of HO-1 activity in many systems (39 40 41 42 43) . CoPP is not a substrate for HO-1 and is not able to catalyze the formation of active oxygen species within cells, but is the most efficacious metalloporphyrin inducer of HO-1 identified so far (14) . This induction seems to be due to the macrocycle itself and to occur by nonstress pathway(s) (14) . Administration of heme results in induction of HO activity with subsequent catabolism of the administered heme, and thus a reduction in its effective concentration, possibly to a level insufficient to enable diffusion to active regulatory sites. CoPP is not degraded by the enzyme, and its diffusion to, and retention at, sites of high HO-1 activity would therefore be unimpeded. Thus, CoPP may be a more promising therapeutic agent than heme to up-regulate HO-1 without increasing prooxidant/oxidative stress. CoPP would also be preferred to other metalloporphyrins, such as CrMP, ZnMP, SnMP, or SnPP, all of which inhibit HO activity and some of which (e.g., SnMP and SnPP) are highly photosensitizing.

In summary, CoPP markedly induces HO-1 mRNA and protein levels in human Huh-7 cells. The pathway of CoPP-dependent HO-1 induction involves changes in the levels of at least two transcription factors, namely, down-regulation of Bach1 and up-regulation of Nrf2 proteins by post-transcriptional mechanisms. At lower concentrations of CoPP, Bach1 appears to be mainly responsible for the induction of HO-1, but at higher concentrations the contribution from Nrf2 appears to be increased. Using siRNA technology, we demonstrate that Bach1 and Nrf2 play important roles in CoPP-dependent up-regulation of human hepatic HO-1.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service grant RO1-DK38825 and contracts NO-1 DK92326 and UO-1 DK065193. The authors thank Drs. Peter R. Sinclair and Judith M. Jacobs (Departments of Pharmacology/Toxicology and Microbiology/Immunology, Dartmouth Medical School, Hanover, NH, USA) and Dr. Henry M. Furneaux (Center for Vascular Biology, University of Connecticut Health Center, Farmington, CT, USA) for helpful comments and discussions.

Received for publication May 30, 2006. Accepted for publication July 31, 2006.

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