Synergistic Up-Regulation of Heme Oxygenase-1 in Human Liver by Heme and siRNA to Bach1:Possible Therapeutic Implications.

Background: Heme oxygenase-1 (HO-1) is an antioxidant defense enzyme that converts toxic heme into antioxidants. HO-1 is strongly up-regulated by its physiologic substrate, heme, which is currently the treatment of choice for acute attacks of porphyria and which may have other therapeutic uses, as well (e.g., for cytoprotection or amelioration of ischemia/reperfusion injury by increasing supply of carbon monoxide, biliverdin, or bilirubin). Up-regulation of HO-1 expression has been associated with increased resistance to tissue injury. Bach1 is a bZip protein which forms heterodimers with small Maf proteins. HO-1 is expressed at higher levels in tissues of Bach1-deficient mice, indicating that Bach1 acts as a negative regulator of the mouse HO-1 gene. The molecular mechanism that confers repression of HO-1 by Bach1, and whether there are similar effects in human cells, has remained elusive. The aim of this study was to assess whether modulation of human hepatic Bach1 expression by siRNA technology influences HO-1 gene expression and whether such gene silencing would enhance the inducing effects of heme on HO-1. Methods: siRNAs targeted 4 different positions of human Bach1 mRNA were designed and synthesized. We transfected Bach1-siRNA (25–200 nM) into Huh-7 cells using Lipofectamine for 24–72 h, after which, cells were treated with or without heme. We quantified HO-1 and Bach1 mRNA and protein levels by quantitative RT-PCR and western blotting, respectively. Effects and specificity of Bach1-siRNA were analyzed and compared with those of non-Bach1 related siRNAs (non-specific control-duplex (NSCD) and LaminB2-siRNA). Results: Bach1-siRNAs (25–200 nM) transfected into Huh-7 cells for 24–72 h significantly reduced Bach1 mRNA and protein levels approximately 80%, compared with non siRNA treated cells. In contrast, transfection with same amounts of NSCD or LaminB2 siRNA did not reduce Bach1 mRNA or protein levels, confirming the specificity of Bach1-siRNA in Huh-7 cells. A significant finding of these studies was the 7-fold up-regulation of the HO-1 gene in Bach1-siRNA transfected cells, compared to cells without Bach1-siRNA or those transfected with NSCD or LaminB2. Bach1, NSCD, and LaminB2 siRNAs had no effect on HO-2 or 5-aminolevulinate synthase-1 mRNA levels (two genes that are not induced by heme). The effects of increasing concentrations of heme (up to 10 μM) in the presence or absence of Bach1-siRNA on the levels of HO-1 mRNA expression are shown in the Figure. For all of the heme concentrations tested, the levels of HO-1 mRNA were greater when Bach1 siRNA was present. Conclusions: Bach1 has a specific and selective effect to repress expression of human hepatic HO-1. Silencing of the Bach1 gene by siRNAs may be a useful method for up-regulating HO-1 gene expression. The combination of intravenous heme and Bach1 silencing may be useful for therapy of acute porphyrias in relapse or other conditions in which up-regulation of HO-1 may be beneficial. (Supported by grants from NIH [DK38825] and Ovation Pharmaceuticals, Inc.) Figure Figure