Homocysteine stimulates antioxidant response element-mediated expression of glutamate-cysteine ligase in mouse macrophages.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis. Uptake of homocysteine induces oxidative stress in macrophages. Antioxidant response elements (AREs) are regulatory elements within promoters of genes, which protect cells against oxidative stress. The current study investigated whether homocysteine induces transcription of glutamate-cysteine ligase (Gcl), via ARE driven gene expression in mouse macrophages. Gcl is the rate-limiting enzyme in the synthesis of glutathione, an important endogenous antioxidant. Gcl is heterodimeric and the genes encoding the subunits of Gcl contain several AREs within their 5'-promoter regions. Treatment of mouse macrophages with d-/l-homocysteine (50microM) induced depletion of intracellular glutathione and a compensatory increase in Gcl activity. Electro mobiliy shift assays demonstrated increased binding of nuclear proteins to ARE-containing oligonucleotides. Real-time RT-PCR revealed increased mRNA-expression of the catalytic subunit of Gcl (Gclc) after treatment with homocysteine, and this occurred via increased transcription as demonstrated with luciferase promoter reporter constructs for Gclc. Additional site directed mutagenesis demonstrated that ARE4 plays a direct role in mediating induction of Gclc by homocysteine. Supershift analysis and Western blotting revealed that Nrf2 signalling is critical in homocysteine-induced activation of ARE4. Inhibition of MAP kinase activity reduced binding of nuclear proteins to the AREs, nuclear expression of Nrf2 and mRNA expression of Gclc. Western blotting demonstrated phosporylation of ERK1/2 in homocysteine treated macrophages. These data suggest that ARE-driven gene expression of Gclc via a MEK/Nrf2 pathway could help to protect macrophages from oxidative stress due to hyperhomocysteinemia.

Over expression of glutamate cysteine ligase increases cellular resistance to H2O2-induced DNA single-strand breaks.

Hydrogen peroxide (H2O2) can cause single strand DNA breaks (ssDNA) in cells when the mechanisms normally in place to reduce it are overwhelmed. Such mechanisms include catalase, glutathione peroxidases (GPx), and peroxiredoxins. The relative importance of these enzymes in H2O2 reduction varies with cell and tissue type. The role of the GPx cofactor glutathione (GSH) in oxidative defense can be further understood by modulating its synthesis. The first and rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which has a catalytic subunit (Gclc) and a modifier subunit (Gclm). Using mouse hepatoma cells we evaluated the effects of GCL over expression on H2O2-induced changes in GSH and ssDNA break formation with the single cell gel electrophoresis assay (SCG or comet assay), and the acridine orange DNA unwinding flow cytometry assay (AO unwinding assay). Cells over expressing GCL had higher GSH content than control cells, and both SCG and AO unwinding assays revealed that cells over expressing GCL were significantly more resistant to H2O2-induced ssDNA break formation. Furthermore, using the AO unwinding assay, the prevalence of H2O2-induced breaks in different phases of the cell cycle was not different, and the degree of protection afforded by GCL over expression was also not cell cycle phase dependent. Our results support the hypothesis that GCL over expression enhanced GSH biosynthesis and protected cells from H2O2-induced DNA breaks. These results also suggest that genetic polymorphisms that affect GCL expression may be important determinants of oxidative DNA damage and cancer.

Glutamate cysteine ligase modifier subunit deficiency and gender as determinants of acetaminophen-induced hepatotoxicity in mice.

The analgesic and antipyretic drug acetaminophen (APAP) is bioactivated to the reactive intermediate N-acetyl-p-benzoquinoneimine, which is scavenged by glutathione (GSH). APAP overdose can deplete GSH leading to the accumulation of APAP-protein adducts and centrilobular necrosis in the liver. N-acetylcysteine (NAC), a cysteine prodrug and GSH precursor, is often given as a treatment for APAP overdose. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate cysteine ligase (GCL) a heterodimer composed of catalytic and modifier (GCLM) subunits. Previous studies have indicated that GCL activity is likely to be an important determinant of APAP toxicity. In this study, we investigated APAP toxicity, and NAC or GSH ethyl ester (GSHee)-mediated rescue in mice with normal or compromised GCLM expression. Gclm wild-type, heterozygous, and null mice were administered APAP (500 mg/kg) alone, or immediately following NAC (800 mg/kg) or GSHee (168 mg/kg), and assessed for hepatotoxicity 6 h later. APAP caused GSH depletion in all mice. Gclm null and heterozygous mice exhibited more extensive hepatic damage compared to wild-type mice as assessed by serum alanine aminotransferase activity and histopathology. Additionally, male Gclm wild-type mice demonstrated greater APAP-induced hepatotoxicity than female wild-type mice. Cotreatment with either NAC or GSHee mitigated the effects of APAP in Gclm wild-type and heterozygous mice, but not in Gclm null mice. Collectively, these data reassert the importance of GSH in protection against APAP-induced hepatotoxicity, and indicate critical roles for GCL activity and gender in APAP-induced liver damage in mice.

Evaluation of methods for oligonucleotide array data via quantitative real-time PCR.

BACKGROUND: There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is generally considered the "gold-standard" assay for measuring gene expression by biologists and is often used to confirm findings from microarray data. Here we use qRT-PCR measurements to validate methods for the components of processing oligo array data: background adjustment, normalization, mismatch adjustment, and probeset summary. An advantage of our approach over spike-in studies is that methods are validated on a real dataset that was collected to address a scientific question. RESULTS: We initially identify three of six popular methods that consistently produced the best agreement between oligo array and RT-PCR data for medium- and high-intensity genes. The three methods are generally known as MAS5, gcRMA, and the dChip mismatch mode. For medium- and high-intensity genes, we identified use of data from mismatch probes (as in MAS5 and dChip mismatch) and a sequence-based method of background adjustment (as in gcRMA) as the most important factors in methods' performances. However, we found poor reliability for methods using mismatch probes for low-intensity genes, which is in agreement with previous studies. CONCLUSION: We advocate use of sequence-based background adjustment in lieu of mismatch adjustment to achieve the best results across the intensity spectrum. No method of normalization or probeset summary showed any consistent advantages.

Chlamydia pneumoniae induces tissue factor expression in mouse macrophages via activation of Egr-1 and the MEK-ERK1/2 pathway.

Recent studies have suggested that infection with Chlamydia pneumoniae (C pneumoniae) may contribute to the instability of atherosclerotic plaques and thrombosis and is associated with acute coronary events. Tissue factor (TF), a potent prothrombotic molecule, is expressed by macrophages and other cell types within atherosclerotic lesions and plays an essential role in thrombus formation after plaque rupture. Therefore the effects of C pneumoniae on induction of TF expression in macrophages were investigated. Infection of RAW mouse macrophages with C pneumoniae induced a time-dependent increase in procoagulant activity, expression of TF protein, and TF mRNA. C pneumoniae infection stimulated increased binding of nuclear proteins to the consensus DNA sequence for Egr-1, a key response element within the TF promoter, and increased the expression of Egr-1 protein. Transient transfections of RAW cells with mutated TF promoter constructs showed that the Egr-1 binding region is an important transcriptional regulator of C pneumoniae-induced TF expression. Furthermore, C pneumoniae-stimulated phosphorylation of ERK1/2 and Elk-1 and pharmacological inhibition of mitogen-activated protein kinase activity reduced the expression of TF and Egr-1. Antibody and polymyxin B blocking of the Toll-like receptor 4 (TLR4) partially reduced the C pneumoniae-induced expression of TF and Egr-1. In conclusion, the C pneumoniae-induced increase in TF expression in macrophages is mediated in part by Egr-1, signaling through TLR4, and activation of the MEK-ERK1/2 pathway.

Induction of glutathione synthesis in macrophages by oxidized low-density lipoproteins is mediated by consensus antioxidant response elements.

The uptake of oxidized low-density lipoproteins (oxLDL) by macrophages leading to conversion into foam cells is a seminal event in atherogenesis. Excessive accumulation of oxLDL can cause oxidative stress in foam cells leading to cell death and the progression and destabilization of atherosclerotic lesions. Oxidative stress induces a protective compensatory increase in the synthesis of the endogenous antioxidant glutathione (GSH). Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in GSH synthesis and is composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM), which are products of separate genes. Treatment of RAW 264.7 mouse macrophages and mouse peritoneal macrophages with oxLDL (30 microg/mL) induces increased expression of both Gclc and Gclm in vitro. The increase in mRNA occurs in part via increased transcription as demonstrated with luciferase reporter constructs. The promoters for both GCLC and GCLM contain consensus antioxidant response elements (AREs). Electrophoretic mobility shift assays revealed induction of nuclear factor binding to these AREs after treatment of RAW 264.7 cells and mouse peritoneal macrophages with oxLDL. Nuclear factor binding to the AREs is diminished by a single base pair substitution in the core sequence. Site-directed mutagenesis of the AREs within the Gclc and Gclm promoters resulted in a decrease of oxLDL-induced luciferase activity. Supershift analyses revealed that oxLDL stimulates binding of the transcription factors Nrf1, Nrf2, and c-jun to the AREs. These data suggest that AREs play a direct role in mediating the induction of GSH synthesis by oxLDL and in protecting macrophages against oxidized lipid-induced oxidative stress.