Modulation of hormone-dependent glucocorticoid receptor function using a tetracycline-regulated expression system.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor capable of stimulating and inhibiting the expression of target genes. To better understand the biological action of glucocorticoids and the function of GR, we have utilized the tetracycline (Tc)-regulated mammalian expression system to develop a novel cell line, E8.2/GR3, derived from GR null mouse L929 fibroblasts, that exhibits conditional expression of rat GR. The intracellular concentration of rGR in E8.2/GR3 cells--from undetectable levels to levels more than 10-fold greater than that observed in wild-type L929 cells--could be manipulated by varying the Tc concentration in the culture media. Similarly, dexamethasone (DEX)-dependent transactivation of the mouse mammary tumor virus long terminal repeat and transrepression of the cadmium-induced activity of the mouse heme oxygenase-1 gene enhancer, SX2, were strictly dependent on the presence of rGR, and the levels of these activities could be modulated by Tc. Similar levels of Tc, and thus rGR, were required for half-maximal transactivation and transrepression whereas a 6-fold lower concentration of DEX was required for half-maximal transrepression than for transactivation. RU486 inhibited both DEX-dependent transactivation and transrepression. DEX decreased the steady-state level of rGR mRNA and protein in a Tc dependent manner. DEX also induced morphological changes in E8.2/GR3 cells that were dependent on rGR as no alterations were observed in the presence of Tc. These cells provide a powerful system for examining the various activities of GR, particularly as a function of different intracellular receptor concentrations.

The heme-responsive element of the mouse heme oxygenase-1 gene is an extended AP-1 binding site that resembles the recognition sequences for MAF and NF-E2 transcription factors.

Jun and Fos (AP-1) transcription factors were recently proposed to mediate induction of the mouse heme oxygenase-1 gene by different agents including heme and cadmium. In this report we show that the AP-1 binding sequence, TGAGTCA, is necessary but insufficient for gene activation in response to heme or cadmium. The minimal heme response element was identified as an extended AP-1 binding site, (T/C)GCTGAGTCA. In addition to the AP-1 heptad, this element also contains an interdigitated antioxidant response element, GCnnnGTCA. Specific antioxidant response elements from the NAD(P)H:quinone oxidoreductase-1 and the glutathione S-transferase Ya subunit genes were in fact responsive to heme but not all sequences that conform to the consensus antioxidant response element were activated by this agent. The heme response element resembles the consensus binding sites for the product of the maf oncogene and for the transcription factor NF-E2. The potential role of these and related transcription factors and the implication of the composite heme response element in heme oxygenase-1 gene regulation are discussed.

Parallel induction of heme oxygenase-1 and chemoprotective phase 2 enzymes by electrophiles and antioxidants: regulation by upstream antioxidant-responsive elements (ARE).

BACKGROUND: Heme oxygenase (HO; EC 1.14.99.3) catalyzes the conversion of heme to biliverdin, which is reduced enzymatically to bilirubin. Since bilirubin is a potent antioxidant and heme a pro-oxidant, HO may protect cells against oxidative damage. HO-1 is highly inducible by diverse chemical agents, resembling those evoking induction of phase 2 enzymes (i.e., Michael reaction acceptors, heavy metals, trivalent arsenicals, and sulfhydryl reagents). Phase 2 enzymes (glutathione transferases; NAD (P)H:quinone reductase; glucuronosyltransferases) are regulated by antioxidant-responsive elements (ARE), and their induction protects against chemical carcinogenesis. Is HO-1 regulated by chemical agents and enhancer elements similar to those controlling phase 2 enzymes? MATERIALS AND METHODS: Induction of HO-1 by phorbol ester and heavy metals is transcriptionally controlled through a 268-bp SX2 fragment, containing two phorbol ester-responsive (TRE) sites (TGAC/GT C/AA) which overlap ARE consensus sequences (TGACNNNGC). Therefore, mutations of the SX2 element designed to distinguish ARE from TRE were inserted into chloramphenicol acetyltransferase (CAT) reporter plasmids, and the response of the CAT activity of murine hepatoma cells stably transfected with these constructs was examined with a wide range of inducers of phase 2 enzymes. RESULTS: All compounds raised HO-1 mRNA and CAT expression constructs containing wild-type SX2. When the SX2 region was mutated to alter TRE consensus sequences without destroying the ARE consensus, full inducibility was preserved. Conversely, when the ARE consensus was disturbed, inducibility was abolished. CONCLUSION: Induction of heme oxygenase-1 is regulated by several chemically distinct classes of inducers (mostly electrophiles), which also induce phase 2 enzymes, and these inductions are mediated by similar AREs. These findings support the importance of HO-1 as a protector against oxidative damage and suggest that HO-1 induction is part of a more generalized protective cellular response that involves phase 2 enzymes.

Genetic polymorphism of apolipoprotein A-IV in the chimpanzee: common deletion of a conserved 12-nucleotide tandem repeat.

Apolipoprotein A-IV (apoA-IV protein; APOA4 gene) is structurally polymorphic in various mammalian species, including human, baboon, dog, horse, and mouse. To analyze the extent of genetic variation in the chimpanzee APOA4 gene, we screened 115 common chimpanzees (Pan troglodytes) (86 unrelated wild captured parents and 29 captive-born offspring) using isoelectric focusing followed by immunoblotting for protein polymorphism and using polymerase chain reaction (PCR) assay for DNA polymorphism. At the protein level the unrelated sample of chimpanzees is highly variable, having four alleles, APOA4*1, APOA4*2, APOA4*3, and APOA4*4, with frequencies of 0.192, 0.430, 0.331, and 0.047, respectively. The chimpanzee APOA4 locus, with four common alleles and a gene diversity of 67%, is more variable than previously reported variations in baboons (five alleles with 52% gene diversity) and humans (two alleles with 15% gene diversity). PCR amplification of chimpanzee DNAs, using a pair of human oligonucleotide primers covering a region of 300 nucleotides in the third exon, revealed a common 12-nucleotide deletion (allele frequency = 0.192) that correlates exactly with the APOA4*1 allele detected by isoelectric focusing and immunoblotting. DNA sequencing of the 300-nucleotide PCR amplified product revealed the deletion of 12 nucleotides near the carboxyl terminal region of the mature apoA-IV protein. This in-frame deletion, which codes for and eliminates four amino acids [glutamic acid (GAG), glutamine (CAG), glutamine (CAG), and glutamine (CAG)], occurs in a region that is evolutionarily conserved among rats, mice, chimpanzees, and humans. The partial DNA sequencing of the 3' end of the chimpanzee APOA4 gene revealed 99% identity with the human APOA4 gene.

A common deletion polymorphism in the apolipoprotein A4 gene and its significance in lipid metabolism.

Apolipoprotein A-IV (apoA-IV, protein; APOA4, gene) is a major constituent of high-density lipoprotein (HDL) and triglyceride-rich lipoprotein particles, but its precise function in lipid metabolism is still uncertain. We have determined APOA4 genetic polymorphism in 285 randomly selected Melanesians from the Solomon Islands and have evaluated its significance in lipid metabolism. By using isoelectric focusing and immunoblotting techniques, a variant pattern, indistinguishable from the APOA4*2 allele uniquely found in white populations at a frequency of about 8%, was detected at a relatively high frequency (19%) in the Melanesian sample. Polymerase chain reaction (PCR) amplification and DNA sequencing of the 3' end of the APOA4 gene revealed that the Melanesian mutation is distinct from the known APOA4*2 mutation and that it involves a four-amino acid deletion in the evolutionarily conserved carboxyl-terminal region in the apoA-IV protein, which consists of four repeats of four amino acids each. After adjustment for concomitant variables, we investigated the impact of the deletion polymorphism on plasma levels of cholesterol, triglycerides, apoA-I, apoA-II, and apoE. A significant (P = .02) and gene-dosage effect was observed on the plasma levels of apoA-I and apoA-II: these levels were lowest in individuals homozygous for the deletion allele (D), intermediate in heterozygotes (ND), and highest in homozygous individuals for the normal allele (N). The average effect of the APOA4*D allele was to lower apoA-I and apoA-II by 8 mg/dL and 2 mg/dL, respectively, and the APOA4 polymorphism accounted for about 3% of the phenotypic variance in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of common genetic polymorphisms in the LDL receptor gene in affecting plasma cholesterol levels in the general population.

A large number of rare mutations in the low-density lipoprotein (LDL) receptor gene cause the autosomal dominant disorder familial hypercholesterolemia. In addition, a number of common DNA polymorphisms have been identified in the LDL receptor gene, but their significance in affecting plasma cholesterol levels in the general population has not been studied widely. We investigated the role of two common DNA polymorphisms, Ava II (exon 13) and Nco I (exon 18), at the LDL receptor locus in affecting plasma lipid profiles in normolipidemic Hispanics (n = 385) and non-Hispanic whites (NHWs; n = 543) from the San Luis Valley, Colorado. While the distribution of the Nco I polymorphism was comparable between Hispanics and NHWs, the allele frequencies at the Ava II restriction site differed significantly between the two ethnic groups (P < .001). The Ava II and Nco I polymorphisms were in linkage disequilibrium (P < .05) in both Hispanics and NHWs. Both polymorphisms revealed a gender-specific effect on total and LDL cholesterol (LDL-C) confined to women only in both ethnic groups. The AVA II polymorphism was associated significantly with total cholesterol and LDL-C in NHW women (P = .001 and P = .014) and in Hispanic women (P = .011 and P = .057). The effect of the Nco I polymorphism was significant on total cholesterol and LDL-C (P = .019 and P = .035) in Hispanic women only. Although a similar trend was observed in NHW women, the effect was not significant at the 5% level.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of lipoprotein lipase gene variation with the physiological components of the insulin-resistance syndrome in the population of the San Luis Valley, Colorado.

OBJECTIVE: To cross-sectionally evaluate the presence of clustering of the insulin-resistance syndrome components. Tests were conducted for association of the HindIII restriction site polymorphism at the lipoprotein lipase locus with clustering of the physiological components of the insulin resistance syndrome. RESEARCH DESIGN AND METHODS: DNA samples of 370 normoglycemic Hispanics and 520 normoglycemic non-Hispanic whites from the San Luis Valley, Colorado, were amplified by the polymerase chain reaction. Lipids and glucose were determined by the standard procedures. Cross-tabulation and chi 2 analysis were used. RESULTS: The insulin-resistance syndrome components (elevated fasting insulin, reduced high-density lipoprotein cholesterol, and elevated triglycerides) appeared together in individuals of this population sample more often than expected by chance. Individuals in the population with the (+/+) lipoprotein lipase-HindIII restriction of fragment-length polymorphism genotype were more likely to have elevated fasting insulin and triglycerides and a reduced high-density lipoprotein-cholesterol level than subjects with the (+/-) genotype (odds ratio = 2.3, 95% confidence interval 1.38-3.98). CONCLUSIONS: As expected from the physiological function of lipoprotein lipase, the primary association of lipoprotein lipase genotypes is with triglyceride and high-density lipoprotein-cholesterol levels. This appears to be the first reported genetic association with the insulin-resistance syndrome and may reflect genotype specific differences in the regulation of lipoprotein lipase by insulin.

Two DNA polymorphisms in the lipoprotein lipase gene and their associations with factors related to cardiovascular disease.

Lipoprotein lipase (LPL) plays a crucial role in plasma lipoprotein processing by catalyzing the hydrolysis of core triglycerides of chylomicrons and very low density lipoproteins. Several polymorphic restriction sites have been reported in the LPL gene, including those identified by the enzymes HindIII and PvuII. We have determined the HindIII and PvuII polymorphisms in diabetic (D) and non-diabetic (ND) Hispanics (D = 195; ND = 384) and non-Hispanic Whites (D = 76; ND = 539) from the San Luis Valley, Colorado. Both polymorphisms showed comparable gene frequencies between diabetics and non-diabetics, and between the two ethnic groups. The HindIII and PvuII polymorphisms were in strong linkage disequilibrium in both Hispanics and non-Hispanic Whites (P < 0.001). We estimated whether the two DNA polymorphisms have significant impact in determining interindividual differences in plasma levels of total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, fasting glucose, and fasting insulin. Plasma triglyceride levels varied significantly among the HindIII genotypes in the normoglycemic sample. There was a clear gene dosage effect among the three HindIII genotypes, with the (-/-) genotype having the lowest and the (+/+) genotype having the highest triglyceride levels; these levels were intermediate in the (+/-) genotype. The average effect of the (-) allele of the HindIII polymorphism was to lower triglycerides by 12.85 mg/dl in non-Hispanic White males, 8.06 mg/dl in non-Hispanic White females, 10.91 mg/dl in Hispanic males, and 12.47 mg/dl in Hispanic females. The HindIII polymorphism also showed a significant association with HDL-cholesterol levels in the normoglycemic sample.(ABSTRACT TRUNCATED AT 250 WORDS)

A new index of abdominal adiposity as an indicator of risk for cardiovascular disease. A cross-population study.

A new index of abdominal adiposity, the conicity index, and the waist-to-hip ratio (WHR) were compared as health indicators in seven European populations and two USA populations. The total sample included 1280 men and 960 women. Abdominal adiposity as detected by these indices is significantly associated with more cardiovascular risk indicators among women than it is among men. Both indices are equivalent as health indicators. However, the conicity index has several advantages over the WHR: (i) it has a theoretical (expected) range; (ii) it includes a built-in adjustment of waist circumference for height and weight, allowing direct comparisons of abdominal adiposity between individuals or even between populations; and (iii) it does not require the hip circumference to assess fat distribution.

DNA polymorphisms of the apolipoprotein AI/CIII/AIV gene cluster influence plasma cholesterol and triglyceride levels in the Mayans of the Yucatán Peninsula, Mexico.

The purpose of this work was to examine the influence of apolipoprotein gene variation on plasma lipid levels in a population of Mayan Indians of the Yucatán Peninsula, Mexico. Four restriction enzymes: XmnI, PstI, SstI, and PvuII, were used to detect restriction fragment length polymorphisms (RFLP) within the region of the apolipoprotein AI/CIII/AIV gene cluster. The frequencies of these polymorphisms in this Mayan population were similar to those reported for other Amerindian populations, but differed widely from those reported for Caucasian populations. The XmnI and SstI RFLPs were informative for association studies in this population, and we analyzed their influence on the quantitative variation of plasma cholesterol and triglycerides. Using a nonparametric analysis of variance, it is shown that the presence of the XmnI restriction site had a significant effect in lowering plasma cholesterol, whereas the presence of the restriction site for SstI had a significant effect in raising plasma triglycerides. Consequently, genetic indicators of both low and high risk for lipid-related diseases, such as atherosclerosis and coronary heart disease, seem to be present within the same gene region in this Mayan population.