Role of Bach-1 in Regulation of Heme Oxygenase-1 in Human Liver Cells

Abstract

Heme oxygenase-1 is an antioxidant defense enzyme that converts heme to biliverdin, iron, and carbon monoxide. Bach-1 is a bZip protein that forms heterodimers with small Maf proteins and was reported recently to down-regulate the HO-1 gene in mice. Using small interfering RNAs targeted to human Bach-1 mRNA, we investigated whether modulation of human hepatic Bach-1 expression by small interfering (si)RNA technology influences heme oxygenase-1 gene expression. We found that Bach-1 siRNAs transfected into Huh-7 cells significantly reduced Bach-1 mRNA and protein levels ϳ80%, compared with non siRNA-treated cells. In contrast, transfection with the same amounts of nonspecific control duplexes or LaminB2-duplex did not reduce Bach-1 mRNA or protein levels, confirming the specificity of Bach-1 siRNA. Expression of the heme oxygenase-1 gene in Bach-1 siRNA-transfected cells was up-regulated 7-fold, compared with cells without Bach-1 siRNA. The effect of increasing concentrations of heme to up-regulate levels of heme oxygenase-1 was more pronounced when Bach-1 siRNA was present. Taken together, these results indicated that Bach-1 has a specific and selective ability to repress expression of human hepatic heme oxygenase-1. Silencing of Bach-1 by siRNAs is a useful method for up-regulating HO-1 gene expression. Exogenous heme produces additional up-regulation, beyond that produced by Bach-1 siRNAs, suggesting that heme does not act solely through its effects on Bach-1.Heme oxygenase (HO, 1 E.C. 1.14.99.3) is the rate-controlling enzyme of heme catabolism (1-5). It carries out the specific cleavage of the ␣-methene bridge of the macrocycle with the liberation of one molecule of carbon monoxide, iron, and biliverdin. Recent studies (4 -6) have highlighted important biological effects of these HO reaction products, which display antioxidant, anti-inflammatory, and anti-apoptotic functions. Three isoforms of HO, termed HO-1, -2, and -3, have been described (7)(8)(9). Among the three isoforms of HO, only HO-1 is highly inducible. Earlier work from our 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 (10 -14). The primary mechanism for up-regulation of the HO-1 gene is by increased transcription of the gene (15), and the induction by such stressors as sodium arsenite or other arsenicals (which produce a chemical oxidative stress), by transition metals, such as cadmium or cobalt, hydrogen peroxide, other reactive oxygen species, or heat shock are clearly different in mechanism from the up-regulation produced by metalloporphyrins (13, 14, 16 -19). For example, earlier work from our laboratory showed that cMyc/Max and upstream stimulatory factor elements in the 5Ј-untranslated region of the HO-1 gene played the key role in inductions by cadmium or cobalt (11). In contrast, inductions by sodium arsenite or phenylarsene oxide depend primarily upon activation of the mitogen-activat...

Reciprocal Effects of Micro-RNA-122 on Expression of Heme Oxygenase-1 and Hepatitis C Virus Genes in Human Hepatocytes

“…Total RNA from treated cells was extracted and quantitative RT-PCR was performed as described before (45). Primers used for HCV core, HO-1, Bach1, and GAPDH quantitative PCR were described before (45;56;58).…”

“…Protein preparation and Western blots were as described before (45). In brief, total proteins (30-50 μg) were separated on 4-15% gradient SDS-polyacrylamide gels (Bio-Rad).…”

“…We more recently reported that heme-mediated induction of HO-1 gene expression occurs via Bach1 and Nrf2 (a basic leucine zipper transcription factor) 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 (45).…”

Bach1 Repression of Ferritin and Thioredoxin Reductase1 Is Heme-sensitive in Cells and in Vitro and Coordinates Expression with Heme Oxygenase1, β-Globin, and NADP(H) Quinone (Oxido) Reductase1

“…Heme also inactivates Bach1 by inducing nuclear export (24) and polyubiquitination followed by proteasome-mediated degradation (25). Manipulating Bach1 expression changed expression of MARE/ARE genes: (i) Mouse Bach1 knock-outs displayed significant increases in ho1 RNA in several tissues, which were diminished by concomitant knock out of Nrf2 (4); and (ii) Bach1 siRNA treatment of liver cells increased ho1 expression 7-fold (26).…”

The role and clinical implications of microRNAs in hepatocellular carcinoma

“…Additionally, miR-196 has been demonstrated to block HCV replication through its action on NS5A gene in the JFH1 region of the HCV genome [31]. Similar downregulation of HCV NS3 protein expression by miR-196 has also been confirmed in an HCV J6/JFH1 cell culture system [47]. Moreover, miR-196 is able to repress oxidative damage by targeting the transcription regulator protein Bach1 [48].…”