PPARs: nuclear receptors for fatty acids, eicosanoids, and xenobiotics.

The peroxisome proliferator-activated receptors have enjoyed the spotlight for many reasons. These transcription factors are ligand-inducible nuclear receptors that modulate gene expression in response to a broad spectrum of compounds. The recognition that PPARs are indeed nuclear receptors for polyunsaturated fatty acids, some eicosanoids and also lipid-lowering and antidiabetic drugs, has opened many exciting avenues of research and drug discovery. Recent studies on the PPAR function have extended the role of these transcription factors beyond energy homeostasis to master gene in adipogenesis and also determinants in inflammation control. While rapid advances have been made, it is clear that we are far from a global understanding of the mechanisms and functions of PPARs.

The major transcription initiation site of the SV40 late promoter is a potent thyroid hormone response element.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily, which act as transcription factors upon binding to specific DNA sequences called thyroid hormone (T3) response elements (TREs). Such elements are found in the upstream regulatory region of promoters as well as in intragenic sequences of T3-responsive genes. In this report, we demonstrate that SV40 late (SVL) promoter activity is strongly down-regulated by TR in the absence of ligand. Addition of T3 releases this repression, but does not further induce SVL promoter activity. Electrophoretic mobility shift analyses reveal a TR binding element that overlaps with the SV40 major late transcription initiation site. This element closely fits the consensus TRE, formed of two hexanucleotides organized in a tandem repeat separated by 4 nt, and is able to confer T3 responsiveness on a heterologous promoter. We further show that, although the presence of TR leads to quantitatively modified expression of an SVL-driven reporter gene, neither displacement of the site of transcription initiation nor modification of the splicing pattern of the primary transcripts occur.

PPARbeta regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation.

Activation of cultured hepatic stellate cells correlated with an enhanced expression of proteins involved in uptake and storage of fatty acids (FA translocase CD36, Acyl-CoA synthetase 2) and retinol (cellular retinol binding protein type I, CRBP-I; lecithin:retinol acyltransferases, LRAT). The increased expression of CRBP-I and LRAT during hepatic stellate cells activation, both involved in retinol esterification, was in contrast with the simultaneous depletion of their typical lipid-vitamin A (vitA) reserves. Since hepatic stellate cells express high levels of peroxisome proliferator activated receptor beta (PPARbeta), which become further induced during transition into the activated phenotype, we investigated the potential role of PPARbeta in the regulation of these changes. Administration of L165041, a PPARbeta-specific agonist, further induced the expression of CD36, B-FABP, CRBP-I, and LRAT, whereas their expression was inhibited by antisense PPARbeta mRNA. PPARbeta-RXR dimers bound to CRBP-I promoter sequences. Our observations suggest that PPARbeta regulates the expression of these genes, and thus could play an important role in vitA storage. In vivo, we observed a striking association between the enhanced expression of PPARbeta and CRBP-I in activated myofibroblast-like hepatic stellate cells and the manifestation of vitA autofluorescent droplets in the fibrotic septa after injury with CCl4 or CCl4 in combination with retinol.

Transcriptional regulatory patterns of the myelin basic protein and malic enzyme genes by the thyroid hormone receptors alpha1 and beta1.

While there is evidence that the two ubiquitously expressed thyroid hormone (T3) receptors, TRalpha1 and TRbeta1, have distinct functional specificities, the mechanism by which they discriminate potential target genes remains largely unexplained. In this study, we demonstrate that the thyroid hormone response elements (TRE) from the malic enzyme and myelin basic protein genes (METRE and MBPTRE) respectively, are not functionally equivalent. The METRE, which is a direct repeat motif with a 4-base pair gap between the two half-site hexamers binds thyroid hormone receptor as a heterodimer with 9-cis-retinoic acid receptor (RXR) and mediates a high T3-dependent activation in response to TRalpha1 or TRbeta1 in NIH3T3 cells. In contrast, the MBPTRE, which consists of an inverted palindrome formed by two hexamers spaced by 6 base pairs, confers an efficient transactivation by TRbeta1 but a poor transactivation by TRalpha1. While both receptors form heterodimers with RXR on MBPTRE, the poor transactivation by TRalpha1 correlates also with its ability to bind efficiently as a monomer. This monomer, which is only observed with TRalpha1 bound to MBPTRE, interacts neither with N-CoR nor with SRC-1, explaining its functional inefficacy. However, in Xenopus oocytes, in which RXR proteins are not detectable, the transactivation mediated by TRalpha1 and TRbeta1 is equivalent and independent of a RXR supply, raising the question of the identity of the thyroid hormone receptor partner in these cells. Thus, in mammalian cells, the binding characteristics of TRalpha1 to MBPTRE (i.e. high monomer binding efficiency and low transactivation activity) might explain the particular pattern of T3 responsiveness of MBP gene expression during central nervous system development.

A gut feeling of the PXR, PPAR and NF-kappaB connection.

Bowel diseases reveal the complex interplay of sensing and signalling pathways in maintaining healthy homeostasis of the intestine. Recent studies of the xenobiotic nuclear receptor, pregnane X receptor and the inflammatory mediator nuclear transcription factor kappaB (NF-kappaB) reveal a functional link between xenobiotic neutralization and inflammation and explain how certain xenobiotics can affect the immune response. Furthermore, another nuclear receptor, peroxisome proliferator-activated receptor gamma (PPAR gamma) has been shown to produce beneficial effects in experimental inflammatory bowel diseases by repression of NF-kappaB thereby reducing inflammation, whilst its close relative PPAR beta/delta appears at a central position in signalling pathways involved in the progression of colon cancer. Recently accumulated knowledge on the action of these nuclear receptors and NF-kappaB in intestinal homeostasis may provide the rationale for the development of innovative treatment strategies with selective receptor modulators.

The biochemistry of drug metabolism-an introduction: part 7. Intra-individual factors affecting drug metabolism.

This review on intra-individual factors affecting drug metabolism completes our series on the biochemistry of drug metabolism. The article presents the molecular mechanisms causing intra-individual differences in enzyme expression and activity. They include enzyme induction by transcriptional activation and enzyme inhibition on the protein level. The influencing factors are of physiological, pathological, or external origin. Tissue characteristics and developmental age strongly influence enzyme-expression patterns. Further influencing factors are pregnancy, disease, or biological rhythms. Xenobiotics, drugs, constituents of herbal remedies, food constituents, ethanol, and tobacco can all influence enzyme expression or activity and, hence, affect drug metabolism.

Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.

Pharmacogenetic Study on Antidementia Drugs

Differences in efficacy and safety of drugs among patients are a recognized problem in pharmacotherapy. The reasons are multifactorial and, therefore, the choice of a drug and its dosage for a particular patient based on different clinical and genetic factors is suggested to improve the clinical outcome. Four drugs are currently used for the treatment of Alzheimer's disease: three acetylcholinesterase inhibitors (donepezil, galantamine, rivastigmine) and the N-methyl-D-aspartate-antagonist memantine. For these drugs, a high interindividual variability in plasma levels was observed, which might influence the response to treatment. The main objective of this thesis was to provide a better understanding of clinical and genetic factors affecting the plasma levels of antidementia drugs. Furthermore, the relationship between plasma levels, genetic variations and side effects was assessed. For this purpose, a pharmacogenetic study was conducted including 300 patients from a naturalistic clinical setting. Analytical methods for the simultaneous measurement of antidementia drugs in plasma have been developed and validated using liquid chromatography methods coupled with mass spectrometry detection. Presently, these methods are used in the therapeutic drug monitoring service of our laboratory. The routine use of therapeutic drug monitoring for antidementia drugs cannot yet be recommended with the available data, but it may be beneficial for some patients in special clinical cases such as insufficient treatment response, side effects or drug interactions. Donepezil and galantamine are extensively metabolized by the liver enzymes cytochromes P450 (CYP) 2D6 and 3A and are substrates of the drug transporter P-glycoprotein. The relationship of variations in genes affecting the activity of these metabolic enzymes and drug transporter (CYP2D6, CYP3A, POR, NR1I2, ABCB1) with donepezil and galantamine plasma levels was investigated. The CYP2D6 genotype appeared to be the major genetic factor involved in the pharmacokinetics of these two drugs. Thus, CYP2D6 poor metabolizers demonstrated significantly higher drug plasma levels than extensive metabolizers. Additionally, in the donepezil study population, the frequency of side effects was significantly increased in poor metabolizers. Lower donepezil plasma levels were observed in ultra rapid metabolizers, which might expose those patients to the risk of non-response. Memantine is mainly eliminated unchanged by the kidney, with implication of tubular secretion by renal transporters. A population pharmacokinetic model was developed to quantify the effects of clinical factors and genetic variations in renal cation transporters (SLC22A1/2/5, SLC47A1, ABCB1), and nuclear receptors (NR1I2, NR1I3, PPARG) involved in transporter expression, on memantine plasma levels. In addition to the renal function and gender, a genetic variation in the nuclear receptor Pregnane-X-Receptor (NR1I2) significantly affected memantine elimination. These findings suggest that an individualized therapy approach for antidementia drugs, taking into account clinical characteristics and genetic background of a patient, might increase efficacy and safety of the treatment. - Les différences interindividuelles dans l'efficacité et la tolérance des médicaments sont un problème connu en pharmacothérapie. Les raisons sont multiples, et le choix du médicament et de la dose, basé sur des facteurs cliniques et génétiques spécifiques au patient, peut contribuer à améliorer la réponse clinique. Quatre médicaments sont couramment utilisés dans le traitement de la maladie d'Alzheimer : trois inhibiteurs de l'acétylcholinestérase (donépézil, galantamine, rivastigmine) et un antagoniste du récepteur N-méthyl-D-aspartate, la mémantine. Une forte variabilité interindividuelle dans les taux plasmatiques de ces quatre composés a été observée, ce qui pourrait influencer la réponse au traitement. L'objectif principal de ce travail de thèse est de mieux comprendre les facteurs cliniques et génétiques influençant les taux des médicaments pro-cognitifs. En outre, des associations entre les taux, la variabilité génétique et les effets secondaires ont été recherchées. Dans ce but, 300 patients sous traitement avec un médicament pro-cognitif ont été recrutés pour une étude pharmacogénétique. Des méthodes de dosage simultané de médicaments pro-cognitifs par chromatographie liquide couplée à la spectrométrie de masse ont été développées et validées. Ces méthodes sont actuellement utilisées dans le service de suivi thérapeutique de notre unité. Malgré le fait qu'un suivi des taux sanguins des pro-cognitifs ne puisse pas encore être recommandé en routine, un dosage peut être utile dans des cas cliniques spécifiques, comme une réponse insuffisante, une intolérance ou une interaction médicamenteuse. Le donépézil et la galantamine sont fortement métabolisés par les cytochromes P450 (CYP) 2D6 et 3A, et sont également substrats du transporteur P-glycoprotéine. Les associations entre les polymorphismes génétiques de ces enzymes, cofacteur, récepteur nucléaire et transporteur (CYP2D6, CYP3A, POR, NR1I2, ABCB1) et les taux de donépézil et de galantamine ont été étudiées. Le génotype du CYP2D6 a été montré comme le facteur génétique majeur impliqué dans la pharmacocinétique de ces deux médicaments. Ainsi, les métaboliseurs déficients du CYP2D6 ont démontré des taux plasmatiques significativement plus élevés comparé aux bons métaboliseurs. De plus, dans la population traitée avec le donépézil, la fréquence des effets secondaires était plus élevée chez les métaboliseurs déficients. Des taux plasmatiques bas ont été mesurés chez les métaboliseurs ultra-rapides traités avec le donépézil, ce qui pourrait être un facteur de risque à une non-réponse au traitement. La mémantine est principalement éliminée sous forme inchangée par les reins, et partiellement par sécrétion tubulaire grâce à des transporteurs rénaux. Un modèle de cinétique de population a été développé pour quantifier les effets des différents facteurs cliniques et de la variabilité génétique des transporteurs rénaux (SLC22A1/2/5, SLC47A1, ABCB1) et des récepteurs nucléaires (NR1I2, NR1I3, PPARG, impliqués dans l'expression des transporteurs) sur les taux plasmatiques de mémantine. En plus de la fonction rénale et du genre, une variation génétique dans le récepteur nucléaire Pregnane-X-Receptor (NR1I2) a montré une influence significative sur l'élimination de la mémantine. Ces résultats suggèrent qu'une approche thérapeutique individualisée, prenant en compte des facteurs cliniques et génétiques du patient, pourrait améliorer l'efficacité et la sécurité du traitement pro-cognitif.

Hepatic glucose sensing is required to preserve β cell glucose competence.

Liver glucose metabolism plays a central role in glucose homeostasis and may also regulate feeding and energy expenditure. Here we assessed the impact of glucose transporter 2 (Glut2) gene inactivation in adult mouse liver (LG2KO mice). Loss of Glut2 suppressed hepatic glucose uptake but not glucose output. In the fasted state, expression of carbohydrate-responsive element-binding protein (ChREBP) and its glycolytic and lipogenic target genes was abnormally elevated. Feeding, energy expenditure, and insulin sensitivity were identical in LG2KO and control mice. Glucose tolerance was initially normal after Glut2 inactivation, but LG2KO mice exhibited progressive impairment of glucose-stimulated insulin secretion even though β cell mass and insulin content remained normal. Liver transcript profiling revealed a coordinated downregulation of cholesterol biosynthesis genes in LG2KO mice that was associated with reduced hepatic cholesterol in fasted mice and reduced bile acids (BAs) in feces, with a similar trend in plasma. We showed that chronic BAs or farnesoid X receptor (FXR) agonist treatment of primary islets increases glucose-stimulated insulin secretion, an effect not seen in islets from Fxr-/- mice. Collectively, our data show that glucose sensing by the liver controls β cell glucose competence and suggest BAs as a potential mechanistic link.

The retinoid-related orphan receptor RORα promotes keratinocyte differentiation via FOXN1.

RORα is a retinoid-related orphan nuclear receptor that regulates inflammation, lipid metabolism, and cellular differentiation of several non-epithelial tissues. In spite of its high expression in skin epithelium, its functions in this tissue remain unclear. Using gain- and loss-of-function approaches to alter RORα gene expression in human keratinocytes (HKCs), we have found that this transcription factor functions as a regulator of epidermal differentiation. Among the 4 RORα isoforms, RORα4 is prominently expressed by keratinocytes in a manner that increases with differentiation. In contrast, RORα levels are significantly lower in skin squamous cell carcinoma tumors (SCCs) and cell lines. Increasing the levels of RORα4 in HKCs enhanced the expression of structural proteins associated with early and late differentiation, as well as genes involved in lipid barrier formation. Gene silencing of RORα impaired the ability of keratinocytes to differentiate in an in vivo epidermal cyst model. The pro-differentiation function of RORα is mediated at least in part by FOXN1, a well-known pro-differentiation transcription factor that we establish as a novel direct target of RORα in keratinocytes. Our results point to RORα as a novel node in the keratinocyte differentiation network and further suggest that the identification of RORα ligands may prove useful for treating skin disorders that are associated with abnormal keratinocyte differentiation, including cancer.

Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity.

AIMS: Aldosterone plays a crucial role in cardiovascular disease. 'Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the 'endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. METHODS AND RESULTS: C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high 'endogenous' aldosterone) and in 'exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. CONCLUSION: Obesity-induced endothelial dysfunction depends on the 'endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Profiling of T-cell receptor signaling complex assembly in human CD4 T-lymphocytes using RP protein arrays.

The RP protein (RPP) array approach immobilizes minute amounts of cell lysates or tissue protein extracts as distinct microspots on NC-coated slide. Subsequent detection with specific antibodies allows multiplexed quantification of proteins and their modifications at a scale that is beyond what traditional techniques can achieve. Cellular functions are the result of the coordinated action of signaling proteins assembled in macromolecular complexes. These signaling complexes are highly dynamic structures that change their composition with time and space to adapt to cell environment. Their comprehensive analysis requires until now relatively large amounts of cells (>5 x 10(7)) due to their low abundance and breakdown during isolation procedure. In this study, we combined small scale affinity capture of the T-cell receptor (TCR) and RPP arrays to follow TCR signaling complex assembly in human ex vivo isolated CD4 T-cells. Using this strategy, we report specific recruitment of signaling components to the TCR complex upon T-cell activation in as few as 0.5 million of cells. Second- to fourth-order TCR interacting proteins were accurately quantified, making this strategy specially well-suited to the analysis of membrane-associated signaling complexes in limited amounts of cells or tissues, e.g., ex vivo isolated cells or clinical specimens.