Biomedical subjects

Richard W Lambrecht

Publications and source records attributed to Richard W Lambrecht.

9 recordsLinked to original sources

PubMed2006

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 (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.

Basic-Leucine Zipper Transcription Factors↗

PubMed2006

Roles of iron and HFE mutations on severity and response to therapy during retreatment of advanced chronic hepatitis C.

BACKGROUND & AIMS: Iron overload may cause or contribute to hepatic injury and fibrosis. Mutations in the HFE gene may influence development or progression of chronic liver disease by increasing iron stores or modulating immune responses. The aim of this work was to assess the influence of HFE mutations and serum and hepatic measures of iron status on baseline features and response to lead-in therapy in subjects with advanced chronic hepatitis C enrolled in the Hepatitis C Anti-viral Long-term Treatment to prevent Cirrhosis (HALT-C) Trial. METHODS: Entry criteria included an Ishak fibrosis score >2 and lack of iron overload (Scheuer iron grade <3+) according to local study pathologists. All baseline biopsy specimens were rescored by consensus of study pathologists, and detailed assessment of stainable iron was performed. Hepatic iron concentrations were measured on portions of 144 liver biopsy specimens. A total of 1051 out of 1145 subjects agreed to HFE mutational testing (C282Y, H63D, S65C). RESULTS: Thirty-five percent carried at least one HFE gene mutation. There were no significant differences in the prevalence of HFE gene mutations among subjects with fibrosis (35.5%) versus cirrhosis (32.9%). Thirty-three percent of subjects had end-of-treatment and 16% sustained virologic responses. Presence of HFE mutations, in particular the H63D variation, was associated with increased end-of-treatment (40% vs 29%, P = .0078) and sustained virologic responses (20% with HFE mutation vs 14% sustained virologic response without HFE mutation; P = .009). CONCLUSIONS: Although HFE mutations (especially the most frequent H63D mutation) are associated with increased iron loading, they are also associated with increased sustained virologic responses in US patients with advanced chronic hepatitis C.

Adult↗

PubMed2006

HCV proteins increase expression of heme oxygenase-1 (HO-1) and decrease expression of Bach1 in human hepatoma cells.

BACKGROUND/AIMS: Hepatitis C infection induces hepatic oxidative stress. Heme oxygenase (HO), the rate-controlling enzyme of heme catabolism, plays a key role as a protector against oxidative, and other stresses. Other recent work has implicated Bach1, a heme binding protein that represses gene expression, in the regulation of HO-1 gene expression. METHODS: We investigated the effects of HCV polyprotein expression on expression of HO-1 and Bach1 genes in human hepatoma cells (Huh-7 cells). RESULTS: HO-1 was up-regulated in the cell line expressing HCV proteins from core up to the aminoterminal domain of NS3. Addition of increasing concentrations of N-acetylcysteine (NAC) led to down-regulation of HO-1 in cells expressing HCV proteins. In contrast, Bach1 was significantly down-regulated in these cells. Sodium arsenite, a strong inducer of oxidative stress and HO-1, reduced Bach1 expression in wild type Huh-7 cells, and NAC partially abrogated this decrease. CONCLUSIONS: Huh-7 cells expressing HCV proteins show significant up-regulation of the HO-1 gene, and reciprocal down-regulation of the Bach1 gene. Exogenous oxidative stressors and anti-oxidants can modulate expression of these genes. These and other results suggest a key role of down-regulation of Bach1 and up-regulation of HO-1 in diminishing cytotoxic effects of HCV proteins in human hepatocytes.

Basic-Leucine Zipper Transcription Factors↗

PubMed2005

Association of hepatitis C virus infection with serum iron status: analysis of data from the third National Health and Nutrition Examination Survey.

BACKGROUND: There is growing evidence that mildly increased amounts of iron in the liver can increase hepatic injury, particularly if combined with other hepatotoxic factors, such as alcohol use, use of porphyrogenic drugs, or chronic viral hepatitis. In the present study, the association of hepatitis C virus (HCV) infection with serum measurements of iron status was assessed in the US population. METHODS: We analyzed data from a total of 14,462 participants in the Third National Health and Nutrition Examination Survey. We excluded subjects who were aged <12 years, subjects for whom measurements of serum levels of iron or ferritin or the results of liver function tests were missing, and subjects who had a serum transferrin saturation of > or =50% (to help exclude subjects with hemochromatosis). RESULTS: Mean serum levels of ferritin and iron (+/- standard error) were significantly higher among subjects with HCV infection (100+/-3 ng/mL and 229+/-17 microg/dL, respectively) than among subjects without liver disease (83+/-0.3 ng/mL and 101+/-2.1 microg/dL, respectively) (P<.0001). Serum levels of ferritin were directly and significantly correlated with serum levels of alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transpeptidase (r=0.25, r=0.24, and r=0.28, respectively; P<.0001), whereas platelet counts were inversely correlated with serum levels of ferritin (r=-0.12; P<.0001). CONCLUSION: HCV infection is significantly associated with higher serum levels of ferritin and iron in the US population.

Adolescent↗

PubMed2004

Role of Bach-1 in regulation of heme oxygenase-1 in human liver cells: insights from studies with small interfering RNAS.

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 approximately 80%, 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.

Animals↗

PubMed2004

Identification of key elements that are responsible for heme-mediated induction of the avian heme oxygenase-1 gene.

UNLABELLED: 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. In recent work, we had identified a metalloporphyrin-responsive element (MPRE) that localized at -3.7 kb upstream of the transcription start site of the chick HO-1 gene. Here, we identify four additional heme-responsive elements (HeREs), which are "expanded" AP-1 sites, in the 5'-flanking region of the chick HO-1 gene. These sites, located at -4675, -4599, -3660, and -3625 bp from the transcription start site of the gene, were necessary and sufficient for up-regulation of luciferase reporter gene expression in the presence of heme and cobalt protoporphyrin (CoPP), but not several other metalloporphyrins. Site-directed mutagenesis was carried out using pcHO7.1Luc or pcHO7.1-4.6Luc as templates. Single and multiple mutations of HeREs and MPRE significantly abrogated the heme- and CoPP-dependent up-regulation of reporter gene expression in transient or stable transfection experiments. CONCLUSIONS: The chick HO-1 promoter region contains "expanded" AP-1 sites that are important for up-regulation of the gene by heme and CoPP, but not other metalloporphyrins. These key regulatory elements consist of consensus AP-1 binding sites that have been extended by three base pairs.

Animals↗

PubMed2003

Iron as a co-morbid factor in nonhemochromatotic liver disease.

Heavy iron overload, in both primary and secondary hemochromatosis, may cause fibrosis of parenchymal organs, especially the liver. The toxicity of iron is believed to involve increased oxidative stress, with iron-catalyzed production of reactive oxygen species causing oxidative damage to lipids, proteins, and nucleic acids. Lesser degrees of hepatic iron deposition are also associated with, and seem to be risk factors for, certain nonhemochromatotic liver diseases. Porphyria cutanea tarda is associated with hepatic iron overload and responds to iron-reduction therapy. Results of recent studies have demonstrated high prevalences (about 60%-80%) of HFE gene mutations in patients with porphyria cutanea tarda. Chronic hepatitis C is another risk factor for porphyria cutanea tarda. Other recent evidence indicates that the prevalence of HFE gene mutations is increased in chronic viral hepatitis and that patients with chronic hepatitis C harboring especially the C282Y mutation are more likely to suffer from advanced hepatic fibrosis or cirrhosis and to do so at younger ages. A role for modest iron overload in increasing severity of alcohol-induced liver disease has been well established from results of experimental studies. However, it is currently unresolved whether mild-to-moderate hepatic iron deposition or heterozygosity for the C282Y mutation plays a role in human alcoholic liver disease or in nonalcoholic fatty liver disease or nonalcoholic steatohepatitis. There is persuasive evidence that iron reduction decreases insulin resistance, and it likely also decreases oxidative stress, two key pathogenic features of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Iron loading has also been described after portosystemic shunts and in end-stage liver disease.

Fatty Liver↗

PubMed2002

Mapping of the chick heme oxygenase-1 proximal promoter for responsiveness to metalloporphyrins.

Heme oxygenase (HO) catalyzes the rate-controlling step of physiologic heme catabolism, namely, the oxidation of the alpha-methene bridge of the macrocycle with formation of CO, Fe, and biliverdin. HO-1, the first isoform of HO to be identified, is highly inducible by a large number of physical and chemical factors. Many of these factors cause oxidative or other stresses to cells. In this work, we have studied the regulation of the chick HO-1 gene, using selected promoter--reporter constructs of the gene transiently or stably transfected into primary cultures of chick embryo liver cells or into the LMH line of chicken hepatoma cells. By use of deletional and mutational analyses, DNase protection, and electromobility shift DNA-binding assays, we identified a heretofore undefined regulatory region in the 5'-UTR of the chick HO-1 gene which confers up-regulation of reporter gene (luciferase) expression in the presence of heme and other selected metalloporphyrins. This new metalloporphyrin-responsive element (MPRE) was localized to a 200-bp region 3.8 to 3.6 kb upstream of the transcription starting point of the chick HO-1 gene. It responded particularly to heme and cobalt protoporphyrin with maximal inductions at 10-15 microM concentrations and 15-18 h of exposure. In contrast, sodium arsenite, a prototypical stress-type inducer of HO-1, led to down-regulation of the reporter gene down stream of MPRE. DNase analysis identified an 18-mer oligonucleotide that was required for the metalloporphyrin response (5'-(-3711)TATTGCAGCTGTGTGGGG-3'). Mutations at any of four sites within this oligonucleotide abrogated the metalloporphyrin-dependent up-regulation of reporter gene expression. Nuclear protein extracts of cells treated with heme or cobalt protoporphyrin showed specific enhanced binding to this 18-mer. We conclude that the chick HO-1 promoter region contains a unique sequence that subserves up-regulation of the gene by metalloporphyrins and propose the name "metalloporphyrin-responsive element" for this sequence.

Animals↗

PubMed2002

Hemochromatosis and porphyria.

A 52-year-old man presented to his primary care physician with blisters and sores on the backs of his hands. Laboratory studies supported a diagnosis of porphyria cutanea tarda, complicated by the presence of both the C282Y and H63D mutations in the HFE gene, with susequent iron over-load. This case illustrates the need to understand the pathogenesis of porphyria cutanea tarda, particularly the role of excess iron in the overproduction of uroporphyrin. Iron, by catalyzing the formation of reactive oxygen species, can enhance uroporphyrin formation by increasing the rate at which uroporphyrinogen is oxidized to urophophyrin. Iron may also act indirectly to inhibit uroporphyrinogen decarboxylase activity by enhancing the formation of non-porphyrin products of porphyrinogen oxidation that are themselves direct inhibitors of the enzyme. Finally, iron can act to increase urophorphyrin production by inducing delta-aminolevulinic acid synthase, thus increasing the amount of delta-aminolevulinic acid, the precursor to uroporphyrinogen, present in the cell. After the diagnosis, the patient underwent an aggressive series of therapeutic phlebotomies to reduce iron levels, and gradually the cutaneous manifestations of porphyria cutanea tarda improved.

Diagnosis, Differential↗