Abstract-Peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are members of the intracellular receptor superfamily. PPARs bind to peroxisome proliferator-response elements (PPREs) as heterodimers with RXR and as such activate gene transcription in response to activators. Fibrates like gemfibrozil are well-known PPAR␣ activators and are used in the treatment of hyperlipidemia. We show that the RXR ligand LGD1069 (Targretin ™ ), like gemfibrozil, can activate the PPAR␣/RXR signal-transduction pathway, including transactivation of the bifunctional enzyme or acyl-CoA oxidase response elements in a cotransfection assay. The activation also occurs in vivo, whereby in rats treated with LGD1069 or gemfibrozil, bifunctional enzyme and acyl-CoA oxidase RNA are induced and the combination of LGD1069 and gemfibrozil leads to a greater induction. Importantly, in hypertriglyceridemic db/db mice treated with RXR or PPAR␣ agonists, triglyceride levels are lowered, and the combination again has significantly greater efficacy. RXR agonists also raise HDL cholesterol levels without changing apoA-I RNA expression. This observation suggests the use of RXR-selective agonists, "rexinoids," either alone or in combination with a fibrate as a new therapeutic approach to treating patients with high triglyceride and low HDL cholesterol levels. 10 -12 In fibrate-treated animals, there is a rapid increase in the expression of genes that encode enzymes for the -oxidation of fatty acids such as AOX and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (bifunctional enzyme).13 PPREs have been identified in the promoters of these genes, suggesting that activation of the peroxisomal fatty acid -oxidation pathway contributes to the lipid lowering observed with fibrates.We have recently shown that the RXR/PPAR␥ heterodimer is activated by RXR agonists. 4 This finding emphasizes the permissive nature of the RXR/PPAR heterodimer, whereby either partner can bind ligand and activate gene expression. RXR agonists have similar effects as thiazolidinediones; they induce adipocyte differentiation, 14 lower elevated glucose and insulin levels, and improve insulin resistance in ob/ob and db/db mice. 15 We refer to these RXRselective ligands as "rexinoids" because their pharmacology is clearly distinct from "retinoids," which are retinoic acid receptor activators that mimic the action of retinoic acid. 16 We hypothesized that rexinoids would mimic the effects of fibrates via activation of the RXR side of the RXR/PPAR␣ heterodimer. Here we demonstrate for the first time that rexinoids elicit similar responses as PPAR␣ activators in vivo. In particular, expression of the bifunctional enzyme and AOX gene is induced in rat livers by gemfibrozil or an RXRselective agonist LGD1069 (Targretin ™ ) treatment. 17 The combination of LGD1069 and gemfibrozil gives a much stronger induction. Further, in db/db mice, RXR activators like LGD1069 17 and LG100268 18 lower triglyceride levels. HDL-C levels are also raised in rexinoid-treated...
The use of reverse genetics has permitted a definition of the structural features within estrogen receptor required for its productive association with the transcription apparatus. These sequences, transactivation function 1 (TAF1) in the amino terminus and TAF2 at the carboxyl terminus, display distinct transcriptional functions. Using specific receptor mutations it has been shown that on some promoters both TAF1 and TAF2 are required for maximal transcriptional activity, whereas on others, additional factors bound to the target promoter can functionally substitute for TAF1 or TAF2. Estrogen functions as an ER agonist by promoting functional synergism between TAF1 and TAF2. Conversely, 4-OH-tamoxifen inhibits TAF2 activity and functions as an antagonist in cell contexts where TAF2 is required. Alternatively, if a 'TAF2 function' is supplied by another factor, 4-OH tamoxifen can manifest ER agonist activity. These data indicate that alterations in the cellular expression of proteins which mimic TAF1 or TAF2 activity can have a profound effect on the pharmacology of ER modulators. Thus the identification of the cellular proteins which interact with ER and its TAF regions will allow a definition of the mechanism used by the cell to distinguish between hormone- and antihormone-activated estrogen receptor.
The polymerase chain reaction is a recently described technique that uses flanking oligonucleotide primers and repeated cycles of enzymatic primer extension to amplify a short segment of DNA by >100,000-fold. By use of sequencing primers located internal to the amplification primers, direct genomic sequence was obtained from enzymatically amplified DNA by using the dideoxynucleotide chaintermination method. The method is relatively simple and offers significant advantages in identifying mutations in genes for which the normal sequence is known. Heterozygous and homozygous mutations in the human A-and y-globin loci were unambiguously identified in 3 days with <1 pg of genomic DNA.The identification of mutations and polymorphisms in human genes by DNA sequencing contributes substantially to our understanding of the molecular nature of disease and has a variety of practical applications in diagnosis. Sequence information is usually obtained by cloning the genes from individuals expected to harbor a mutation and then comparing the sequence of the cloned DNA to that of the normal allele. The cloning process is laborious, however, requiring several weeks of library construction and screening to obtain DNA clones. Moreover, if the individual is not homozygous, several clones may have to be studied to be certain that both alleles are detected.Several methods of screening for mutations have recently been developed that do not require cloning. Carefully controlled hybridization with mutation-specific oligonucleotide probes (1), for example, can detect the presence of single base changes in short segments ofDNA if the exact nature of the mutation is known in advance. The location of mutations in a few genes has also been successfully identified by ribonuclease cleavage of mismatches between a labeled normal antisense RNA and genomic DNA (2) or by cleavage of RNARNA duplexes of a labeled antisense RNA and cellular RNA from an affected tissue (3, 4). Mutations in genomic DNA have also been detected by denaturing gradient gel electrophoresis (5). These approaches, however, do not provide the actual sequence at the mutation site, and some mismatches are not detected. Methods have been reported for direct chemical sequencing of mammalian genomic DNA (6), but these have not yet been widely adopted due to the degree of technical difficulty. Direct dideoxynucleotide sequencing (7) of eukaryotic genomic DNA has previously only been achieved in yeast (8).The development of the polymerase chain reaction (PCR; ref. 9) to selectively amplify a short segment of DNA by >100,000-fold has reduced the problem of sequence complexity and signal strength as impediments to direct analysis of single-copy DNA sequences. The PCR has been used to enrich for segments of the human f3-globin (9-11), HLADQa (10, 11), and c-ras (12) genes and has recently been used to acquire direct sequence from human mitochondrial DNA (12). The misincorporation rate of PCR DNA synthesis is low [1 in 600 base pairs (bp) or less], but in a cloning study only 1-2...
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