Susan E. Donohue

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

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 posttranscriptional 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. FASEB J. 20, E2258–E2267 (2006)

Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria

Exposure to light precipitates the symptoms of several genetic disorders that affect both skin and internal organs. It is presumed that damage to non-cutaneous organs is initiated indirectly by light, but this is difficult to study in mammals. Zebrafish have an essentially transparent periderm for the first days of development. In a previous large-scale genetic screen we isolated a mutation, dracula (drc), which manifested as a light-dependent lysis of red blood cells [1]. We report here that protoporphyrin IX accumulates in the mutant embryos, suggesting a deficiency in the activity of ferrochelatase, the terminal enzyme in the pathway for heme biosynthesis. We find that homozygous drc(m248) mutant embryos have a G-->T transversion at a splice donor site in the ferrochelatase gene, creating a premature stop codon. The mutant phenotype, which shows light-dependent hemolysis and liver disease, is similar to that seen in humans with erythropoietic protoporphyria, a disorder of ferrochelatase.

Effects of silymarin on hepatitis C virus and haem oxygenase-1 gene expression in human hepatoma cells.

Background/Aims: Hepatitis C virus (HCV) infection is a global medical problem. The current standard treatment of chronic hepatitis C (CHC), pegylated interferon plus ribavirin, is prolonged, expensive, has serious side effects and, at best, is only 50% effective. Silymarin (SI) is a natural antioxidant often used by patients with CHC, although its efficacy for decreasing HCV levels or ameliorating CHC remains uncertain. HCV infection is associated with increased hepatic oxidative stress, and one of the antioxidant enzymes that protect cells against this stress is haem oxygenase‐1 (HO‐1).

Differential regulation of human ALAS1 mRNA and protein levels by heme and cobalt protoporphyrin

Abstract5-Aminolevulinic acid synthase 1 (ALAS1) is the first and rate-controlling enzyme of heme biosynthesis. This study was to determine the effects of heme and selected nonheme metalloporphyrins on human ALAS1 gene expression in hepatocytes. We found that, upon heme and cobalt protoporphyrin (CoPP) treatments, ALAS1 mRNA levels were down-regulated significantly by ca. 50% or more. Measurement of mRNA in the presence of actinomycin D showed that these down-regulations were due to the decreases in mRNA half-lives. Furthermore, the levels of mitochondrial mature ALAS1 protein were down-regulated by 60–70%, but those of the cytosolic precursor protein were up-regulated by 2–5-fold. Measurement of protein in the presence of cycloheximide (CHX) suggests that elevation of the precursor form is due to the increase in protein half-lives. These results provide novel insights into the mechanisms of heme repressional effects on ALAS1 and provide a rationale for further investigation of CoPP as a therapeutic agent for acute porphyric syndromes.

Zinc mesoporphyrin induces rapid and marked degradation of the transcription factor bach1 and up-regulates HO-1

Heme oxygenase 1 (HO-1) is the first and rate-controlling enzyme in heme degradation. Bach1 is a mammalian transcriptional repressor of HO-1. To understand how zinc mesoporphyrin (ZnMP) induces the expression of HO-1, we investigated the effects of ZnMP on Bach1 mRNA and protein levels in human hepatoma Huh-7 cells by quantitative RT-PCR and Western blots. We found that ZnMP markedly up-regulated HO-1 mRNA and protein levels, and rapidly and significantly decreased Bach1 protein levels by increasing degradation of Bach1 protein [half life (t(1/2)) from 19 h to 45 min], whereas ZnMP did not influence Bach1 mRNA levels. The proteasome inhibitors, epoxomicin and MG132, significantly inhibited degradation of Bach1 by ZnMP in a dose-dependent fashion, indicating that the degradation of Bach1 by ZnMP is proteasome-dependent. Purified Bach1 C-terminal fragment bound heme, but there was no evidence for binding of ZnMP to the heme-binding region of Bach1. In conclusion, ZnMP produces profound post-transcriptional down-regulation of Bach1 protein levels and transcriptional up-regulation of HO-1. Our results indicate that ZnMP up-regulates HO-1 gene expression by markedly increasing Bach1 protein degradation in a proteasome-dependent manner.