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 (t(1/2) from 19 h to 2.8 h), and increased Nrf2 by decreasing degradation of Nrf2 protein (t(1/2) 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 microM 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.
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...
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.
Some patients with acute hereditary porphyrias have seizures and require anticonvulsant therapy, but many anticonvulsants induce exacerbations of the hepatic porphyrias. Recently, several new anticonvulsants have become available. Among these are gabapentin, vigabatrin, felbamate, lamotrigine, and tiagabine. Little is known about their potential for induction of porphyric attacks. We used a cell culture model of primary chicken embryo liver cells, which maintain intact heme synthesis and regulation, to study the effects of these new anticonvulsants on porphyrin accumulation. Treatment of the cells with deferoxamine (250 microM) led to a partial block in heme synthesis, simulating the conditions encountered in human beings with porphyria. Concomitant exposure of these cells to phenobarbital (2 mM) strongly induced accumulation of porphyrins, serving as a positive control in this model. Cells were treated for 20 hours with increasing doses (3.2 to 1,000 microM) of the newer anticonvulsants, with or without deferoxamine. For most of these anticonvulsants 5 to 100 microM is representative of the concentrations achieved in humans with therapeutic doses. Porphyrins were measured spectrofluorometrically as uro-, copro-, and protoporphyrins. Results were confirmed by high-pressure liquid chromatography. Neither vigabatrin nor gabapentin treatment, with or without deferoxamine, led to any increase in porphyrin accumulation. Similar doses of felbamate (with deferoxamine) led to a marked increase in (mainly proto-) porphyrin levels, qualitatively and quantitatively almost identical to the accumulation produced by phenobarbital. Lamotrigine or tiagabine (with deferoxamine) caused similar porphyrin accumulation. Tiagabine treatment up to 100 microM (with deferoxamine) also resulted in very high levels of predominantly proto-porphyrin. In contrast to the other anticonvulsants tested, tiagabine without deferoxamine led to mild porphyrin accumulation. In the presence of deferoxamine, phenobarbital, felbamate, lamotrigine, or tiagabine, but not gabapentin or vigabatrin, increased levels of the mRNA of ALA synthase, the first and rate-controlling enzyme of porphyrin synthesis. Such enzyme induction is a sine qua non for acute porphyric attacks. We conclude that neither vigabatrin nor gabapentin is porphyrogenic, whereas felbamate, lamotrigine, and, especially, tiagabine lead to much accumulation of porphyrins. The latter three anticonvulsants, therefore, may precipitate or exacerbate acute porphyric attacks in humans. We recommend use of vigabatrin or gabapentin, but not felbamate, lamotrigine, or tiagabine, in patients with acute porphyria and seizures.
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