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Biomedical subjects

T M Willson

Publications and source records attributed to T M Willson.

At least 19 recordsLinked to original sources
PubMed2006

Peroxisome proliferator-activated receptor-delta, a regulator of oxidative capacity, fuel switching and cholesterol transport.

Synthetic agonists of peroxisome proliferator-activated receptor (PPAR)-delta have shown a promising pharmacological profile in preclinical models of metabolic and cardiovascular disease. At present, the pharmaceutical development of these drugs exploits the potential to raise plasma HDL-cholesterol in animals and their insulin-sensitising and glucose-lowering properties. PPAR-delta agonists have also proven to be powerful research tools that have provided insights into the role of fatty acid metabolism in human physiology and disease. Activation of PPAR-delta induces the expression of genes important for cellular fatty acid combustion and an associated increase in whole-body lipid dissipation. The predominant target tissue in this regard is skeletal muscle, in which PPAR-delta activation regulates the oxidative capacity of the mitochondrial apparatus, switches fuel preference from glucose to fatty acids, and reduces triacylglycerol storage. These changes counter the characteristic derangements of insulin- resistant skeletal muscle but resemble the metabolic adaptation to regular physical exercise. Apart from effects on fuel turnover, there is evidence for direct antiatherogenic properties, because PPAR-delta activation increases cholesterol export and represses inflammatory gene expression in macrophages and atherosclerotic lesions. Whereas conclusions about the full potential of PPAR-delta as a drug target await the result of large scale clinical testing, ongoing investigation of this nuclear receptor has greatly improved our knowledge of the physiological regulation of whole-body fuel turnover and the interdependence of mitochondrial function and insulin sensitivity.

Atherosclerosis↗
PubMed2006

PPARbeta/delta selectively induces differentiation and inhibits cell proliferation.

Peroxisome proliferator-activated receptor (PPAR) beta-null mice exhibit exacerbated epithelial cell proliferation and enhanced sensitivity to skin carcinogenesis, suggesting that ligand activation of PPARbeta will inhibit keratinocyte proliferation. By using of a highly specific ligand (GW0742) and the PPARbeta-null mouse model, activation of PPARbeta was found to selectively induce keratinocyte terminal differentiation and inhibit keratinocyte proliferation. Additionally, GW0742 was found to be anti-inflammatory due to inhibition of myeloperoxidase activity, independent of PPARbeta. These data suggest that ligand activation of PPARbeta could be a novel approach to selectively induce differentiation and inhibit cell proliferation, thus representing a new molecular target for the treatment of skin disorders resulting from altered cell proliferation such as psoriasis and cancer.

Animals↗
PubMed2001

Synthesis and biological activity of L-tyrosine-based PPARgamma agonists with reduced molecular weight.

A series of PPARgamma agonists were synthesized from L-tyrosine that incorporated low molecular weight N-substituents. The most potent analogue, pyrrole (4e), demonstrated a K(i) of 6.9nM and an EC(50) of 4.7nM in PPARgamma binding and functional assays, respectively. Pyrrole (4e), which is readily synthesized from L-tyrosine methyl ester in four steps, also demonstrated in vivo activity in a rodent model of Type 2 diabetes.

Animals↗
PubMed2001

Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose, lipid, and cholesterol metabolism. We report the x-ray crystal structure of the ligand binding domain of PPAR alpha (NR1C1) as a complex with the agonist ligand GW409544 and a coactivator motif from the steroid receptor coactivator 1. Through comparison of the crystal structures of the ligand binding domains of the three human PPARs, we have identified molecular determinants of subtype selectivity. A single amino acid, which is tyrosine in PPAR alpha and histidine in PPAR gamma, imparts subtype selectivity for both thiazolidinedione and nonthiazolidinedione ligands. The availability of high-resolution cocrystal structures of the three PPAR subtypes will aid the design of drugs for the treatments of metabolic and cardiovascular diseases.

Amino Acid Sequence↗
PubMed2001

Peroxisome proliferator-activated receptor gamma inhibits transforming growth factor beta-induced connective tissue growth factor expression in human aortic smooth muscle cells by interfering with Smad3.

Activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) after balloon injury significantly inhibits VSMC proliferation and neointima formation. However, the precise mechanisms of this inhibition have not been determined. We hypothesized that activation of PPAR gamma in vascular injury could attenuate VSMC growth and matrix production during vascular lesion formation. Since connective tissue growth factor (CTGF) is a key factor regulating extracellular matrix production, abrogation of transforming growth factor beta (TGF-beta)-induced CTGF production by PPAR gamma activation may be one of the mechanisms through which PPAR gamma agonists inhibit neointima formation after vascular injury. In this study, we demonstrate that the PPAR gamma natural ligand (15-deoxyprostaglandin J(2)) and a synthetic ligand (GW7845) significantly inhibit TGF-beta-induced CTGF production in a dose-dependent manner in HASMCs. In addition, suppression of CTGF mRNA expression is relieved by pretreatment with an antagonist of PPAR gamma (GW9662), suggesting that the inhibition of CTGF expression is mediated by PPAR gamma. To elucidate further the molecular mechanism by which PPAR gamma inhibits CTGF expression, an approximately 2-kilobase pair CTGF promoter was cloned. We found that PPAR gamma activation inhibits TGF-beta-induced CTGF promoter activity in a dose-dependent manner, and suppression of CTGF promoter activity by PPAR gamma activation is completely rescued by overexpression of Smad3, but not by Smad4. Furthermore, PPAR gamma physically interacts with Smad3 but not Smad4 in vitro in glutathione S-transferase pull-down experiments. Taken together, the data suggest that PPAR gamma inhibits TGF-beta-induced CTGF expression in HASMCs by directly interfering with the Smad3 signaling pathway.

Aorta↗
PubMed2001

Identification of a series of oxadiazole-substituted alpha-isopropoxy phenylpropanoic acids with activity on PPARalpha, PPARgamma, and PPARdelta.

A series of oxadiazole-substituted alpha-isopropoxy phenylpropanoic acids with dual agonist activity on PPARalpha and PPARgamma is described. Several of these compounds also showed partial agonist activity on PPARdelta. Resolution of one analogue showed that PPARalpha and PPARgamma activity resided in mainly one enantiomer, whereas PPARdelta activity was retained in both enantiomers.

Chromatography, High Pressure Liquid↗
PubMed2001

Liver X receptor (LXR) regulation of the LXRalpha gene in human macrophages.

The nuclear oxysterol receptors LXRalpha (NR1H3) and LXRbeta (NR1H2) coordinately regulate the expression of genes involved in the transport and catabolism of cholesterol. In macrophages, LXR stimulates the transcription of genes encoding transporters involved in cholesterol efflux, which may limit the transformation of these cells into foam cells in response to lipid loading. Here, we report that natural and synthetic LXR ligands induce the expression of the LXRalpha gene in primary human macrophages and differentiated THP-1 macrophages. This regulation was not observed in primary human adipocytes or hepatocytes, a human intestinal cell line, or in any mouse tissue or cell line examined. The human LXRalpha gene was isolated, and the transcription initiation site delineated. Analysis of the LXRalpha promoter revealed a functional LXR/RXR binding site approximately 2.9 kb upstream of the transcription initiation site. We conclude that LXRalpha regulates its own expression in human macrophages and that this response is likely to amplify the effects of oxysterols on reverse cholesterol transport. These findings underscore the importance of LXR as a potential therapeutic target for the treatment of atherosclerosis.

Animals↗
PubMed2001

Comparison of complete nuclear receptor sets from the human, Caenorhabditis elegans and Drosophila genomes.

BACKGROUND: The availability of complete genome sequences enables all the members of a gene family to be identified without limitations imposed by temporal, spatial or quantitative aspects of mRNA expression. Using the nearly completed human genome sequence, we combined in silico and experimental approaches to define the complete human nuclear receptor (NR) set. This information was used to carry out a comparative genomic study of the NR superfamily. RESULTS: Our analysis of the human genome identified two novel NR sequences. Both these contained stop codons within the coding regions, indicating that both are pseudogenes. One (HNF4 gamma-related) contained no introns and expressed no detectable mRNA, whereas the other (FXR-related) produced mRNA at relatively high levels in testis. If translated, the latter is predicted to encode a short, non-functional protein. Our analysis indicates that there are fewer than 50 functional human NRs, dramatically fewer than in Caenorhabditis elegans and about twice as many as in Drosophila. Using the complete human NR set we made comparisons with the NR sets of C. elegans and Drosophila. Searches for the >200 NRs unique to C. elegans revealed no human homologs. The comparative analysis also revealed a Drosophila member of NR subfamily NR3, confirming an ancient metazoan origin for this subfamily. CONCLUSIONS: This work provides the basis for new insights into the evolution and functional relationships of NR superfamily members.

Amino Acid Sequence↗
PubMed2001

The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity.

The human nuclear pregnane X receptor (hPXR) activates cytochrome P450-3A expression in response to a wide variety of xenobiotics and plays a critical role in mediating dangerous drug-drug interactions. We present the crystal structures of the ligand-binding domain of hPXR both alone and in complex with the cholesterol-lowering drug SR12813 at resolutions of 2.5 and 2.75 angstroms, respectively. The hydrophobic ligand-binding cavity of hPXR contains a small number of polar residues, permitting SR12813 to bind in three distinct orientations. The position and nature of these polar residues were found to be critical for establishing the precise pharmacologic activation profile of PXR. Our findings provide important insights into how hPXR detects xenobiotics and may prove useful in predicting and avoiding drug-drug interactions.

Amino Acid Sequence↗
PubMed2001

Target genes of peroxisome proliferator-activated receptor gamma in colorectal cancer cells.

Activation of the nuclear hormone peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits cell growth and promotes differentiation in a broad spectrum of epithelial derived tumor cell lines. Here we utilized microarray technology to identify PPARgamma gene targets in intestinal epithelial cells. For each gene, the induction or repression was seen with two structurally distinct PPARgamma agonists, and the change in expression could be blocked by co-treatment with a specific PPARgamma antagonist. A majority of the genes could be regulated independently by a retinoid X receptor specific agonist. Genes implicated in lipid transport or storage (adipophilin and liver fatty acid-binding protein) were also activated by agonists of PPAR subtypes alpha and/or delta. In contrast, PPARgamma-selective targets included genes linked to growth regulatory pathways (regenerating gene IA), colon epithelial cell maturation (GOB-4 and keratin 20), and immune modulation (neutrophil-gelatinase-associated lipocalin). Additionally, three different genes of the carcinoembryonic antigen family were induced by PPARgamma. Cultured cells treated with PPARgamma ligands demonstrated an increase in Ca(2+)-independent, carcinoembryonic antigen-dependent homotypic aggregation, suggesting a potential role for PPARgamma in regulating intercellular adhesion. Collectively, these results will help define the mechanisms by which PPARgamma regulates intestinal epithelial cell biology.

Animals↗
PubMed2001

Adipose tissue resistin expression is severely suppressed in obesity and stimulated by peroxisome proliferator-activated receptor gamma agonists.

Elevated levels of the hormone resistin, which is secreted by fat cells, are proposed to cause insulin resistance and to serve as a link between obesity and type 2 diabetes. In this report we show that resistin expression is significantly decreased in the white adipose tissue of several different models of obesity including the ob/ob, db/db, tub/tub, and KKA(y) mice compared with their lean counterparts. Furthermore, in response to several different classes of antidiabetic peroxisome proliferator-activated receptor gamma agonists, adipose tissue resistin expression is increased in both ob/ob mice and Zucker diabetic fatty rats. These data demonstrate that experimental obesity in rodents is associated with severely defective resistin expression, and decreases in resistin expression are not required for the antidiabetic actions of peroxisome proliferator-activated receptor gamma agonists.

Adipocytes↗
PubMed2001

Identification of a subtype selective human PPARalpha agonist through parallel-array synthesis.

Using solid-phase, parallel-array synthesis, a series of urea-substituted thioisobutyric acids was synthesized and assayed for activity on the human PPAR subtypes. GW7647 (3) was identified as a potent human PPARalpha agonist with approximately 200-fold selectivity over PPARgamma and PPARdelta, and potent lipid-lowering activity in animal models of dyslipidemia. GW7647 (3) will be a valuable chemical tool for studying the biology of PPARalpha in human cells and animal models of disease.

Animals↗
PubMed2001

A selective peroxisome proliferator-activated receptor delta agonist promotes reverse cholesterol transport.

The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of the fibrate drugs and the antidiabetic effects of the glitazone drugs in humans are due to activation of the alpha (NR1C1) and gamma (NR1C3) subtypes, respectively. By contrast, the therapeutic potential of the delta (NR1C2) subtype is unknown, due in part to the lack of selective ligands. We have used combinatorial chemistry and structure-based drug design to develop a potent and subtype-selective PPARdelta agonist, GW501516. In macrophages, fibroblasts, and intestinal cells, GW501516 increases expression of the reverse cholesterol transporter ATP-binding cassette A1 and induces apolipoprotein A1-specific cholesterol efflux. When dosed to insulin-resistant middle-aged obese rhesus monkeys, GW501516 causes a dramatic dose-dependent rise in serum high density lipoprotein cholesterol while lowering the levels of small-dense low density lipoprotein, fasting triglycerides, and fasting insulin. Our results suggest that PPARdelta agonists may be effective drugs to increase reverse cholesterol transport and decrease cardiovascular disease associated with the metabolic syndrome X.

ATP Binding Cassette Transporter 1↗
PubMed2001

Differential involvement of peroxisome-proliferator-activated receptors alpha and delta in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine.

Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (alpha, gamma and delta) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARalpha-null mice, we have found that PPARalpha in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARdelta/alpha agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARalpha-null mice. These findings demonstrate that PPARdelta can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARdelta target gene in the small intestine. We propose that PPARdelta contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.

Animals↗
PubMed2001

Circumventing tamoxifen resistance in breast cancers using antiestrogens that induce unique conformational changes in the estrogen receptor.

Tamoxifen inhibits estrogen receptor (ER) transcriptional activity by competitively inhibiting estradiol binding and inducing conformational changes in the receptor that may prevent its interaction with coactivators. In bone, the cardiovascular system, and some breast tumors, however, tamoxifen exhibits agonist activity, suggesting that the tamoxifen-ER complex is not recognized identically in all cells. We used phage display to demonstrate that the antiestrogen GW5638 induces a unique structural change in the ER. The biological significance of this conformational change was revealed in studies that demonstrated that tamoxifen-resistant breast tumor explants are not cross-resistant to GW5638. Because of these properties, this drug is currently being developed as a potential therapeutic for tamoxifen-resistant breast cancers.

Amino Acid Sequence↗
PubMed2001

Pharmacophore analysis of the nuclear oxysterol receptor LXRalpha.

A cell-free assay was developed for the orphan nuclear receptor LXRalpha that measures the ligand-dependent recruitment of a peptide from the steroid receptor coactivator 1 (SRC1) to the nuclear receptor. Using this ligand-sensing assay (LiSA), the structural requirements for activation of the receptor by oxysterols and related compounds were studied. The minimal pharmacophore for receptor activation was shown to be a sterol with a hydrogen bond acceptor at C24. 24(S),25-Epoxycholesterol (1), which meets this criterion, is among the most efficacious of the oxysterols and is an attractive candidate as the LXRalpha natural hormone. Cholenic acid dimethylamide (14) showed increased efficacy compared to 1, whereas the unnatural oxysterol 22(S)-hydroxycholesterol (4) was shown to be an antagonist of 1 in the LiSA. The structural requirements for SRC1 recruitment in the LiSA correlated with the transcriptional activity of compounds in a cell-based reporter assay employing LXRalpha-GAL4 chimeric receptors. Site-directed mutagenesis identified Trp(443) as an amino acid critical for activation of LXRalpha by oxysterol ligands. This information was combined with the structure-activity relationship developed from the LiSA to develop a 3D homology model of LXRalpha. This model may aid the design of synthetic drugs targeted at this transcriptional regulator of cholesterol homeostasis.

Amino Acid Sequence↗
PubMed2001

The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity.

The pregnane X receptor (PXR) is the molecular target for catatoxic steroids such as pregnenolone 16alpha-carbonitrile (PCN), which induce cytochrome P450 3A (CYP3A) expression and protect the body from harmful chemicals. In this study, we demonstrate that PXR is activated by the toxic bile acid lithocholic acid (LCA) and its 3-keto metabolite. Furthermore, we show that PXR regulates the expression of genes involved in the biosynthesis, transport, and metabolism of bile acids including cholesterol 7alpha-hydroxylase (Cyp7a1) and the Na(+)-independent organic anion transporter 2 (Oatp2). Finally, we demonstrate that activation of PXR protects against severe liver damage induced by LCA. Based on these data, we propose that PXR serves as a physiological sensor of LCA, and coordinately regulates gene expression to reduce the concentrations of this toxic bile acid. These findings suggest that PXR agonists may prove useful in the treatment of human cholestatic liver disease.

Animals↗