The patterns of expression of glutathione S-transferases A1 and A2 in human liver (hGSTA1 and hGSTA2, respectively) are highly variable, notably in the ratio of hGSTA1/hGSTA2. We investigated if this variation had a genetic basis by sequencing the proximal promoters (-721 to -1 nucleotides) of hGSTA1 and hGSTA2, using 55 samples of human liver that exemplified the variability of hGSTA1 and hGSTA2 expression. Variants were found in the hGSTA1 gene: -631T or G, -567T, -69C, -52G, designated as hGSTA1*A; and -631G, -567G, -69T, -52A, designated as hGSTA1*B. Genotyping for the substitution -69C > T by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), showed that the polymorphism was widespread in Caucasians, African-Americans and Hispanics, and that it appeared to conform to allelic variation. Constructs consisting of the proximal promoters of hGSTA1*A, hGSTA1*B or hGSTA2, with luciferase as a reporter gene, showed differential expression when transfected into HepG2 cells: hGSTA1*A approximately hGSTA2 > hGSTA1*B. Similarly, mean levels of hGSTA1 protein expression in liver cytosols decreased significantly according to genotype: hGSTA1*A > hGSTA1-heterozygous > hGSTA1*B. Conversely, mean hGSTA2 expression increased according to the same order of hGSTA1 genotype. Consequently, the ratio of GSTA1/GSTA2 was highly hGSTA1 allele-specific. Because the polymorphism in hGSTA1 correlates with hGSTA1 and hGSTA2 expression in liver, and hGSTA1-1 and hGSTA2-2 exhibit differential catalysis of the detoxification of carcinogen metabolites and chemotherapeutics, the polymorphism is expected to be of significance for individual risk of cancer or individual response to chemotherapeutic agents.
The transcription of the progesterone receptor gene is induced by estrogens and decreased by progestins. Studies were performed to define the regions of the gene and the molecular mechanisms involved. No hormonal regulation could be observed using 5′ flanking regions of the gene up to −2762 in front of a heterologous gene. Estrogen and progestin regulation could be observed only when using fragments of the gene extending down to +788. Progressive deletions from the 5′ and 3′ ends, site‐directed mutagenesis and DNase protection experiments with purified estrogen receptor suggested that the biologically active estrogen responsive element (ERE) is present at +698/+723, overlapping the initiation of translation. An oligonucleotide was synthesized bearing this ERE and shown to impart estrogen inducibility to a heterologous gene. Its regulation by anti‐estrogens corresponded to that of the in situ progesterone receptor gene since tamoxifen was a partial agonist whereas ICI 164384 was a full antagonist. This ERE also mediated down‐regulation by progestins in the presence of the progesterone receptor, even though it has no progesterone receptor binding ability. DNase footprinting showed that this effect was not due to a decrease of estrogen receptor affinity for the ERE in the presence of progesterone receptor. Finally, use of deletion mutants of the progesterone receptor showed that the steroid binding and the DNA binding domains were necessary for down‐regulation whereas deletions of various parts of the N‐terminal domain were without effect.
Kappa class glutathione S-transferase (GST) cDNA sequences have been identified in rat, mouse, and human. In the present study, we determined the structure and chromosomal location of the human GST Kappa 1 (hGSTK1) gene, characterized the protein, and demonstrated its subcellular localization. The human gene spans ϳ5 kb, has 8 exons, and maps onto chromosome 7q34. The 5 -flanking region lacks TATA or CCAAT boxes, but there is an initiator element overlapping the transcription start site. hGSTK1 amino acid sequence showed homology to bacterial 2-hydroxychromene-2-carboxylate isomerase, an enzyme involved in naphthalene degradation pathway. hGSTK1 mRNA was expressed in all of the organs examined. Subcellular fractionation of HepG2 cells showed that the protein was located in peroxisomes and mitochondria and was not detectable in cytoplasm. The peroxisomal localization was confirmed by transfection of HepG2 cells with a plasmid coding a green fluorescent protein fused inframe to the N terminus of hGSTK1. The C terminus of hGSTK1 was essential for localization of the protein to peroxisomes, and the C-terminal sequence Ala-Arg-Leu represents a peroxisome targeting signal. This is the first time that a human GST has been found in peroxisomes, suggesting a new function for this family of enzymes.
Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1 and tumor necrosis factor-␣. In the present study, we demonstrated that the chemokine CCL1, notably involved in cardiovascular diseases and inflammatory or allergic processes, constitutes a new molecular target for PAHs. Indeed, exposure to PAHs such as benzo[a]pyrene (BP) markedly increased mRNA expression and secretion of CCL1 in primary human macrophage cultures. Moreover, intranasal administration of BP to mice enhanced mRNA levels of TCA3, the mouse orthologue of CCL1, in lung. CCL1 induction in cultured human macrophages was fully prevented by targeting the aryl hydrocarbon receptor (AhR) through chemical inhibition or small interfering RNA-mediated down-modulation of its expression. In addition, BP and the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin were found to enhance activity of a CCL1 promoter sequence containing a consensus xenobiotic-responsive element known to specifically interact with AhR. Moreover, 2,3,7,8-tetrachlorodibenzo-p-dioxin triggered AhR binding to this CCL1 promoter element as revealed by chromatin immunoprecipitation experiments and electrophoretic mobility shift assays. In an attempt to further characterize the mechanism of CCL1 induction, we demonstrated that BP was able to induce an early and transient increase of intracellular calcium concentration in human macrophages. Inhibition of this calcium increase, using the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetra(acetoxymethyl) ester or the calcium store-operated channel inhibitor 2-aminoethoxydiphenyl borate, fully blocked CCL1 up-regulation. Taken together, these results bring the first demonstration that PAHs induce expression of the chemokine CCL1 in an AhR-and calcium-dependent manner.
Oltipraz, a synthetic derivative of the cruciferous vegetable product 1,2-dithiole-3-thione, is considered as one of the most potent chemoprotectants. It modulates both cytochrome P-450 (CYP) and glutathione S-transferase expression and activities in rat tissues. Its effects, however, are variable according to the enzyme, tissue, and species. We show here that, as previously found in rat lung and kidney, CYP1A1 is inducible by oltipraz in both rat intestine and Caco-2 cells, a cell line originated from a human colon adenocarcinoma. In these cells, a 50 M oltipraz treatment increased CYP1A1 mRNA (ϳ30-fold), protein and activity. mRNA level was augmented as early as 2 h after the beginning of treatment, suggesting a transcriptional activation, and was maximal between 8 and 12 h. Transient transfection of Caco-2 cells with constructs containing different sizes of the 5-flanking region of the CYP1A1 gene upstream of the luciferase reporter gene showed an increase in luciferase activity in oltipraz-treated cells, which correlates with the presence of the xenobiotic responsive element (XRE). Furthermore we demonstrated that resveratrol, an antagonist of the aryl hydrocarbon (Ah) receptor, inhibited the induction of both CYP1A1 promoter activity and mRNA by oltipraz, supporting the involvement of the Ah receptor in this induction. In an attempt to further characterize the mechanism of CYP1A1 induction, we showed a rapid increase in intracellular calcium concentration upon treatment of Caco-2 cells with oltipraz. Moreover, the effect of this compound on CYP1A1 was strongly abolished in the presence of BAPTA-AM, a well known chelator of intracellular calcium, and 2-aminoethyl diphenylborate, an inhibitor of store-operated calcium channels. These results bring the first demonstration that oltipraz activates transcription of the CYP1A1 gene through the Ah receptor-XRE pathway in Caco-2 cells and that CYP1A1 induction relies upon an increase of intracellular calcium concentration.Oltipraz, a synthetic derivative of 1,2-dithiole-3-thione, a constituent of cruciferous vegetables, is considered as one of the most promising chemopreventive agents in development, based on preclinical studies (1-5) and a recent Phase IIa clinical trial in China (6, 7). This compound was first claimed to act as a chemopreventive agent by enhancing activities of phase II enzymes such as glutathione S-transferase, UDP-glucuronosyltransferases, NAD(P)H:quinone reductase, aflatoxin B 1 -aldehyde reductase, and epoxide hydrolase (for a review see Ref. 8). Biochemical and genetic studies showed that induction of these detoxifying enzymes is primarily due to transcriptional activation of the genes and is regulated by an enhancer, called an antioxidant-responsive element (ARE) 1 or electrophile-responsive element (9 -11). NF-E2-related factor 2 (Nrf2) has been implicated as an essential component of an ARE-binding transcriptional complex (12)(13)(14), but the signal transduction pathways that relay the chemical signals to the ARE-protein complex remain to...
Steroid hormone-receptor complexes regulate the transcription of specific genes. Recent studies of high-affinity interactions between the receptors and discrete regions of DNA, together with gene-transfer experiments, have led to the precise mapping of hormone regulatory elements. Nothing is known, however, about the mechanisms whereby DNA-bound receptors modulate gene transcription. At the start of transcription in prokaryotes two oligomeric molecules of several regulatory proteins must bind to two specific DNA sites and interact with one another to regulate the binding of RNA polymerase to DNA. Using electron microscopy to observe progesterone receptor binding to regulatory regions of uteroglobin and mouse mammary tumour virus genes, we demonstrate a similar binding between receptor oligomers at two DNA sites. DNA loops are formed when the hormone regulatory elements are at a distance from one another. Thus, in common with certain prokaryotic systems, protein-protein interactions may be important in steroid hormone regulation of gene transcription.
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