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,1,
,
* Department of Medicine,
Liver-Biliary-Pancreatic Center, Departments of
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
SPECIFIC AIMS
Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide (CO). HO-1 is highly inducible by physical and chemical factors. CoPP is known to be a potent and effective inducer of HO-1 activity in many tissues. The mechanisms by which CoPP activates the HO-1 gene in the liver and other organs are less well understood. The aims of this study were to assess whether transcription factors Bach1 and Nrf2 are involved in CoPP up-regulation of HO-1 gene expression in human hepatoma cells and to delineate the mechanism of CoPP-mediated regulation of HO-1 gene expression.
PRINCIPAL FINDINGS
1. CoPP-mediated expression of HO-1, Bach1, and Nrf2 genes in human hepatoma cells
CoPP up-regulates HO-1 mRNA and protein levels in Huh-7 cells in a dose-dependent and time-related fashion. 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. A CoPP-mediated increase in HO-1 mRNA was detectable at 2 h and maximal at 4–6 h. CoPP increased HO-1 protein levels starting at 6 h, with a peak at 16 h. In subsequent studies we treated cells with 10 µM CoPP for 4 h to measure mRNA and for 16 h to measure proteins.
To assess whether CoPP affected Bach1 or Nrf2 mRNA and protein levels, Huh-7 cells were treated with 0, 1, 5, or 10 µM CoPP for 4–16 h. Bach1 and Nrf2 mRNA levels were measured by quantitative RT-polymerase chain reaction (RT-PCR); protein levels were measured by Western blot. We found that CoPP had no significant effects on Bach1 or Nrf2 mRNA levels, but CoPP-mediated regulation of Bach1 and Nrf2 proteins were dose dependent and time related. There were no detectable effects of CoPP on Bach1 or Nrf2 proteins after 6 h. There was down-regulation of Bach1 protein after exposure of Huh-7 cells to 5–10 µM CoPP for 16 h. 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. These results indicate that CoPP exerts reciprocal effects on Bach1 and Nrf2 by post-transcriptional mechanism(s).
2. 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. 1
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. 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. 1C, D
).
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3. Specific silencing of 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. Bach1-siRNA (100 nM) for 48 h reduced Bach1 mRNA levels by 
75% compared with nonsiRNA treated cells, and 20–150 nM Bach1-siRNA almost completely abrogated Bach1 protein expression at 72 h. Nrf2-siRNA (20–150 nM) reduced Nrf2 mRNA and protein levels by nearly 70%. 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 no significant reductions of Bach1 or Nrf2 mRNA and protein levels after transfections with NSCD compared with untransfected cells. Bach1/Nrf2 mRNA and proteins did not change after transfection with 20 nM LaminB2-siRNA. Thus, silencing of the human Bach1 and Nrf2 genes using siRNA targeted to Bach1 and Nrf2 mRNA was effective, specific, and selective.
4. Regulation of HO-1 expression by CoPP and Bach1-siRNA
We studied the effects of combinations of CoPP and Bach1-siRNA on HO-1 gene regulation in Huh-7 cells. HO-1 mRNA expression levels were increased 5-fold by transfection with 20 nM Bach1-siRNA and further up-regulated by treatment with CoPP in the presence of Bach1-siRNA. Using nonlinear regression analysis, HO-1 mRNA levels were correlated with CoPP concentrations in the presence or absence of Bach1-siRNA (r=0.99). These values were significantly increased when CoPP and Bach1-siRNA were present. The two curves, from with or without Bach1-siRNA, are significantly different (P<0.001), implying an additive effect of Bach-1-siRNA and CoPP on up-regulation of HO-1 gene expression. Bach1-siRNA did not affect Nrf2 mRNA levels in Huh-7 cells. 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. 2
A, B).
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5. Regulation of HO-1 expression by CoPP and Nrf2-siRNA
We studied the effects of combinations of CoPP and Nrf2-siRNA on HO-1 gene regulation in Huh-7 cells. 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 from 2330-fold to 17-fold at higher concentrations (5–10 µM). Using nonlinear regression analysis, HO-1 mRNA levels were correlated with CoPP concentrations in the presence or absence of Bach1-siRNA (r=0.99), which 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. HO-1 protein levels were also consistent with those of mRNA in CoPP-treated cells from either with or without Nrf2-siRNA.
CONCLUSIONS AND SIGNIFICANCE
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 that CoPP regulates these proteins by post-transcriptional mechanisms; 4) silencing the Bach1 gene with specific siRNA significantly increases the baseline levels 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; 5) silencing the Nrf2 gene with specific siRNA does not significantly change baseline levels of HO-1 mRNA or protein, but significantly reduces the amount of HO-1 mRNA and protein induced by higher concentrations of CoPP.
HO-1 is an enzyme with important antioxidant and cytoprotective effects. This protection stems not only from a reduction in heme concentrations, but also from the production of biliverdin and bilirubin, both potent antioxidants. Bach1, a member of the basic leucine zipper family of proteins, forms heterodimers with the Maf-related oncoprotein family. Igarashi et al. indicated 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 HO-1 genes. We have reported that Bach1 plays a critical role in heme-dependent up-regulation of the human HO-1 gene. A recent study also showed that heme increases the stability of the Nrf2 protein, leading to accumulation of heterodimers of Nrf2/Maf that bind to the HeRE’s, thus activating the HO-1 gene.
Under baseline conditions, Bach1 forms heterodimers with small proteins of the Maf family; these heterodimers repress transcription of the HO-1 gene by binding to heme-responsive elements (HeREs) in the 5'-UTR of the HO-1. Nrf2 remains in the cytoplasm, where it is associated with the actin cytoskeleton through Keap1, which mediates its continuous degradation by a proteasome-dependent pathway. 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 Nrf2-Maf or Maf-Maf heterodimers to occupy HeRE sites in the HO-1 promoter and leads to increased transcription and up-regulation of expression of the gene (Fig. 3
). Due to similarities in structure and chemistry, CoPP may bind to the same regulatory sites as heme itself. We show that CoPP-mediated induction of the HO-1 gene is a function of both Bach1 and Nrf2 genes.
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Heme and CoPP are both potent inducers of HO-1 activity in many systems. CoPP is not a substrate for HO-1; it is unable to catalyze the formation of active oxygen species within cells, but is the most efficacious metalloporphyrin inducer of HO-1 identified so far. This induction seems to be due to the macrocycle itself and to occur by nonstress pathway(s). Administration of heme results in induction of HO activity with subsequent catabolism of the administered heme and a reduction in its 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 thus be unimpeded. 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 gene.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6346fje
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