The ability of pollutants to affect human health is a major concern, justified by the wide demonstration that reproductive functions are altered by endocrine disrupting chemicals. The definition of endocrine disruption is today extended to broader endocrine regulations, and includes activation of metabolic sensors, such as the peroxisome proliferator-activated receptors (PPARs). Toxicology approaches have demonstrated that phthalate plasticizers can directly influence PPAR activity. What is now missing is a detailed molecular understanding of the fundamental basis of endocrine disrupting chemical interference with PPAR signaling. We thus performed structural and functional analyses that demonstrate how monoethyl-hexyl-phthalate (MEHP) directly activates PPAR␥ and promotes adipogenesis, albeit to a lower extent than the full agonist rosiglitazone. Importantly, we demonstrate that MEHP induces a selective activation of different PPAR␥ target genes. Chromatin immunoprecipitation and fluorescence microscopy in living cells reveal that this selective activity correlates with the recruitment of a specific subset of PPAR␥ coregulators that includes Med1 and PGC-1␣, but not p300 and SRC-1. These results highlight some key mechanisms in metabolic disruption but are also instrumental in the context of selective PPAR modulation, a promising field for new therapeutic development based on PPAR modulation.
Adipose tissue is not an inert cell mass contributing only to the storage of fat, but a sophisticated ensemble of cellular components with highly specialized and complex functions. In addition to managing the most important energy reserve of the body, it secretes a multitude of soluble proteins called adipokines, which have beneficial or, alternatively, deleterious effects on the homeostasis of the whole body. The expression of these adipokines is an integrated response to various signals received from many organs, which depends heavily on the integrity and physiological status of the adipose tissue. One of the main regulators of gene expression in fat is the transcription factor peroxisome proliferatoractivated receptor g (PPARg), which is a fatty acid-and eicosanoid-dependent nuclear receptor that plays key roles in the development and maintenance of the adipose tissue. Furthermore, synthetic PPARg agonists are therapeutic agents used in the treatment of type 2 diabetes.This review discusses recent knowledge on the link between fat physiology and metabolic diseases, and the roles of PPARg in this interplay via the regulation of lipid and glucose metabolism. Finally, we assess the putative benefits of targeting this nuclear receptor with still-to-be-identified highly selective PPARg modulators.
Macrophages play a central role in the pathogenesis of atherosclerosis by accumulating cholesterol through increased uptake of oxidized low-density lipoproteins by scavenger receptor CD36, leading to foam cell formation. Here we demonstrate the ability of hexarelin, a GH-releasing peptide, to enhance the expression of ATP-binding cassette A1 and G1 transporters and cholesterol efflux in macrophages. These effects were associated with a transcriptional activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma in response to binding of hexarelin to CD36 and GH secretagogue-receptor 1a, the receptor for ghrelin. The hormone binding domain was not required to mediate PPARgamma activation by hexarelin, and phosphorylation of PPARgamma was increased in THP-1 macrophages treated with hexarelin, suggesting that the response to hexarelin may involve PPARgamma activation function-1 activity. However, the activation of PPARgamma by hexarelin did not lead to an increase in CD36 expression, as opposed to liver X receptor (LXR)alpha, suggesting a differential regulation of PPARgamma-targeted genes in response to hexarelin. Chromatin immunoprecipitation assays showed that, in contrast to a PPARgamma agonist, the occupancy of the CD36 promoter by PPARgamma was not increased in THP-1 macrophages treated with hexarelin, whereas the LXRalpha promoter was strongly occupied by PPARgamma in the same conditions. Treatment of apolipoprotein E-null mice maintained on a lipid-rich diet with hexarelin resulted in a significant reduction in atherosclerotic lesions, concomitant with an enhanced expression of PPARgamma and LXRalpha target genes in peritoneal macrophages. The response was strongly impaired in PPARgamma(+/-) macrophages, indicating that PPARgamma was required to mediate the effect of hexarelin. These findings provide a novel mechanism by which the beneficial regulation of PPARgamma and cholesterol metabolism in macrophages could be regulated by CD36 and ghrelin receptor downstream effects.
Estrogen regulates target gene expression by binding to specific nuclear receptors that function as ligand-dependent transcription factors. Estrogen receptors (ERs) 1 contain two transcription activation domains, AF1 at the N terminus and AF2 in the C-terminal ligand-binding domain (1-3). Several proteins interact with AF2 in the presence of estrogen, some of which have the properties of transcriptional coactivators (4 -6).For instance, the p160 family of coactivators, which in humans includes the three proteins SRC1/N-CoA1 (7, 8), TIF2/GRIP1 (9, 10), and AIB1/ACTR/RAC3 (11-13), can interact with most nuclear receptors in a ligand-dependent manner and potentiate transcription of their target genes.The crystal structures of several nuclear receptor ligandbinding domains (LBDs) have now been determined (14 -19) and have revealed a striking conservation despite modest sequence homology (20). The LBD folds into a structure described as a sandwich of ␣-helices with a central hydrophobic ligandbinding pocket. In the presence of ligand, helices 3, 5, and 12 form a hydrophobic groove (21-24) important for interaction with the LXXLL motifs (9, 25-27) found in the p160 family members and also in other coactivators. However, the crystal structure of estrogen receptor ␣ (ER␣) revealed that helix 12 is repositioned in the presence of the antagonists tamoxifen (Tam) or raloxifene (Ral), thereby disrupting the surface of interaction with coactivators (16, 23). The side chain of these antiestrogens plays an important role in displacing helix 12. This suggests that amino acids of the ligand-binding domain that interact with the antiestrogen side chain play an important role in the transcriptionally inactive conformation of this domain. It has been suggested that integrity of aspartate 351, which forms a hydrogen bond with the tertiary amine present at the end of the side chains of Tam and Ral, is the key to the antiestrogenic character of these analogs (28). Indeed, a mutation of Asp-351 to tyrosine was isolated from an MCF7 tumor variant that was not inhibited but rather stimulated by Tam (29,30). Both Tam and Ral also behaved as agonists for expression of the estrogen target gene transforming growth factor-␣ in MDA-MB-231 cells stably transfected with this mutant of ER␣, while the full antiestrogen ICI182,780 remained inactive (28).Here we have introduced several mutations at position 351 and tested the functional consequences of these changes on ER␣ transactivation properties in the presence of estrogen and of antiestrogens. Our results demonstrate that Asp-351 can be mutagenized to Gly, Ala, or Val without diminishing the antagonist activity of antiestrogens in HeLa cells. However, we provide evidence for a stabilizing effect of Asp-351 on the active conformation of the wild-type ER LBD in the absence of hormones. EXPERIMENTAL PROCEDURESMaterials-Cell culture media and fetal bovine serum were purchased from Life Technologies, Inc. Estradiol, 4-hydroxytamoxifen (OHT), and ICI182,780 were purchased from Sigma. RU39,411 and RU...
The bulky side chains of antiestrogens hinder folding of the ligand binding domain (LBD) of estrogen receptors (ERs) into a transcriptionally active conformation. The presence of a tertiary amine in the side chain of raloxifene, which interacts with a negatively charged residue in helix H3 of the ER LBD [Asp351 in human (h)ER␣], is important for antiestrogenicity in animal and cellular models. To better understand the molecular basis of the differential activity of tamoxifen and raloxifene, we have examined the influence of tertiary amine substituents and of mutations at position 351 in hER␣ on the activity profiles of tamoxifen derivatives. Results obtained in several cellular model systems suggest that the degree of antagonist activity of tamoxifen derivatives does not strictly correlate with the basicity of the side chain but depends on an optimal spatial relationship between the tertiary amine of these antiestrogens and the negative charge at position 351. Although altering the position of the negative charge at residue 351 (mutation D351E) had little effect on transcriptional activity in the presence of tamoxifen, it drastically increased the partial agonist activity of a tamoxifen derivative with improved antagonist activity as well as that of raloxifene. Our results suggest that contrary to raloxifene, tamoxifen and most of its derivatives do not interact with Asp351 in an optimal manner, although this can be improved by modifying tertiary amine substituents.
The peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in fat tissue development and physiology. Mutations in the gene encoding this receptor have been associated to disorders in lipid metabolism. A thorough investigation of mice in which one PPARgamma allele has been mutated reveals that male PPARgamma heterozygous (PPARgamma +/-) mice exhibit a reduced body size associated with decreased body weight, reflecting lean mass reduction. This phenotype is reproduced when treating the mice with a PPARgamma- specific antagonist. Monosodium glutamate treatment, which induces weight gain and alters body growth in wild-type mice, further aggravates the growth defect of PPARgamma +/- mice. The levels of circulating GH and that of its downstream effector, IGF-I, are not altered in mutant mice. However, the IGF-I mRNA level is decreased in white adipose tissue (WAT) of PPARgamma +/- mice and is not changed by acute administration of recombinant human GH, suggesting an altered GH action in the mutant animals. Importantly, expression of the gene encoding the suppressor of cytokine signaling-2, which is an essential negative regulator of GH signaling, is strongly increased in the WAT of PPARgamma +/- mice. Although the relationship between the altered GH signaling in WAT and reduced body size remains unclear, our results suggest a novel role of PPARgamma in GH signaling, which might contribute to the metabolic disorder affecting insulin signaling in PPARgamma mutant mice.
The new CD-LFIA rapid screening test shows good diagnostic accuracy, sensitivity and specificity, and may rule out CD in patients with CD-related symptoms.
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