IL-13 induces expression of CD36 in human monocytes through PPARgamma activation.

The class B scavenger receptor CD36 is a component of the pattern recognition receptors on monocytes that recognizes a variety of molecules. CD36 expression in monocytes depends on exposure to soluble mediators. We demonstrate here that CD36 expression is induced in human monocytes following exposure to IL-13, a Th2 cytokine, via the peroxisome proliferator-activated receptor (PPAR)gamma pathway. Induction of CD36 protein was paralleled by an increase in CD36 mRNA. The PPARgamma pathway was demonstrated using transfection of a PPARgamma expression plasmid into the murine macrophage cell line RAW264.7, expressing very low levels of PPARgamma, and in peritoneal macrophages from PPARgamma-conditional null mice. We also show that CD36 induction by IL-13 via PPARgamma is dependent on phospholipase A2 activation and that IL-13 induces the production of endogenous 15-deoxy-Delta12,14-prostaglandin J2, an endogenous PPARgamma ligand, and its nuclear localization in human monocytes. Finally, we demonstrate that CD36 and PPARgamma are involved in IL-13-mediated phagocytosis of Plasmodium falciparum-parasitized erythrocytes. These results reveal a novel role for PPARgamma in the alternative activation of monocytes by IL-13, suggesting that endogenous PPARgamma ligands, produced by phospholipase A2 activation, could contribute to the biochemical and cellular functions of CD36.

The Interleukin-1 receptor antagonist is a direct target gene of PPARalpha in liver.

BACKGROUND/AIMS: The Peroxisome Proliferator-Activated Receptor (PPAR) alpha belongs to the superfamily of Nuclear Receptors and plays an important role in numerous cellular processes, including lipid metabolism. It is known that PPARalpha also has an anti-inflammatory effect, which is mainly achieved by down-regulating pro-inflammatory genes. The objective of this study was to further characterize the role of PPARalpha in inflammatory gene regulation in liver. RESULTS: According to Affymetrix micro-array analysis, the expression of various inflammatory genes in liver was decreased by treatment of mice with the synthetic PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. In contrast, expression of Interleukin-1 receptor antagonist (IL-1ra), which was acutely stimulated by LPS treatment, was induced by PPARalpha. Up-regulation of IL-1ra by LPS was lower in PPARalpha -/- mice compared to Wt mice. Transactivation and chromatin immunoprecipitation studies identified IL-1ra as a direct positive target gene of PPARalpha with a functional PPRE present in the promoter. Up-regulation of IL-1ra by PPARalpha was conserved in human HepG2 hepatoma cells and the human monocyte/macrophage THP-1 cell line. CONCLUSIONS: In addition to down-regulating expression of pro-inflammatory genes, PPARalpha suppresses the inflammatory response by direct up-regulation of genes with anti-inflammatory properties.

Species differences in the response of liver drug-metabolizing enzymes to (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949) in vivo and in vitro.

Induction of drug-metabolizing enzymes (DMEs) is highly species-specific and can lead to drug-drug interaction and toxicities. In this series of studies we tested the species specificity of the antidiabetic drug development candidate and mixed peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949, EMD) with regard to the induction of gene expression and activities of DMEs, their regulators, and typical PPAR target genes. EMD clearly induced PPARalpha target genes in rats in vivo and in rat hepatocytes but lacked significant induction of DMEs, except for cytochrome P450 (P450) 4A. CYP2C and CYP3A were consistently induced in livers of EMD-treated monkeys. Interestingly, classic rodent peroxisomal proliferation markers were induced in monkeys after 17 weeks but not after a 4-week treatment, a fact also observed in human hepatocytes after 72 h but not 24 h of EMD treatment. In human hepatocyte cultures, EMD showed similar gene expression profiles and induction of P450 activities as in monkeys, indicating that the monkey is predictive for human P450 induction by EMD. In addition, EMD induced a similar gene expression pattern as the PPARalpha agonist fenofibrate in primary rat and human hepatocyte cultures. In conclusion, these data showed an excellent correlation of in vivo data on DME gene expression and activity levels with results generated in hepatocyte monolayer cultures, enabling a solid estimation of human P450 induction. This study also clearly highlighted major differences between primates and rodents in the regulation of major inducible P450s, with evidence of CYP3A and CYP2C inducibility by PPARalpha agonists in monkeys and humans.

Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids

Studies in animal models and humans suggest anti-inflammatory roles on the N acylethanolamide (NAE)-peroxisome proliferators activated receptor alpha (PPARα) system in inflammatory bowel diseases. However, the presence and function of NAE-PPARα signaling system in the ulcerative colitis (UC) of humans remain unknown as well as its response to active anti-inflammatory therapies such as 5-aminosalicylic acid (5-ASA) and glucocorticoids. Expression of PPARα receptor and PPARα ligands-biosynthetic (NAPE-PLD) and -degrading (FAAH and NAAA) enzymes were analyzed in untreated active and 5-ASA/glucocorticoids/immunomodulators-treated quiescent UC patients compared to healthy human colonic tissue by RT-PCR and immunohistochemical analyses. PPARα, NAAA, NAPE-PLD and FAAH showed differential distributions in the colonic epithelium, lamina propria, smooth muscle and enteric plexus. Gene expression analysis indicated a decrease of PPARα, PPARγ and NAAA, and an increase of FAAH and iNOS in the active colitis mucosa. Immunohistochemical expression in active colitis epithelium confirmed a PPARα decrease, but showed a sharp NAAA increase and a NAPE-PLD decrease, which were partially restored to control levels after treatment. We also characterized the immune cells of the UC mucosa infiltrate. We detected a decreased number of NAAA-positive and an increased number of FAAH-positive immune cells in active UC, which were partially restored to control levels after treatment. NAE-PPARα signaling system is impaired during active UC and 5-ASA/glucocorticoids treatment restored its normal expression. Since 5-ASA actions may work through PPARα and glucocorticoids through NAE-producing/degrading enzymes, the use of PPARα agonists or FAAH/NAAA blockers that increases endogenous PPARα ligands may yield similar therapeutics advantages.

Peroxisome proliferator-activated receptors: finding the orphan a home.

The peroxisome proliferator-activated receptor (PPAR) is a member of the steroid hormone receptor superfamily and is activated by a variety of fibrate hypolipidaemic drugs and non-genotoxic rodent hepatocarcinogens that are collectively termed peroxisome proliferators. A key marker of peroxisome proliferator action is the peroxisomal enzyme acyl CoA oxidase, which is elevated about ten fold in the livers of treated rodents. Additional peroxisome proliferator responsive genes include other peroxisomal beta-oxidation enzymes and members of the cytochrome P450 IVA family. A peroxisome proliferator response element (PPRE), consisting of an almost perfect direct repeat of the sequence TGACCT spaced by a single base pair, has been identified in the upstream regulatory sequences of each of these genes. The retinoid X receptor (RXR) forms a heterodimer with PPAR and binds to the PPRE. Furthermore, the RXR ligand, 9-cis retinoic acid, enhances PPAR action. Retinoids may therefore modulate the action of peroxisome proliferators and PPAR may interfere with retinoid action, perhaps providing one mechanism to explain the toxicity of peroxisome proliferators. Interestingly, a variety of fatty acids can activate PPAR supporting the suggestion that fatty acids, or their acyl CoA derivatives, may be the natural ligands of PPAR and that the physiological role of PPAR is to regulate fatty acid homeostasis. Taken together, the discovery of PPAR has opened up new opportunities in understanding how lipid homeostasis is regulated, how the fibrate hypolipidaemic drugs may act and should lead to improvements in the assessment of human risk from peroxisome proliferators based upon a better understanding of their mechanism of action.

GW501516-activated PPARβ/δ promotes liver fibrosis via p38-JNK MAPK-induced hepatic stellate cell proliferation.

ABSTRACT: BACKGROUND: After liver injury, the repair process comprises activation and proliferation of hepatic stellate cells (HSCs), which produce extracellular matrix (ECM) proteins. Peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is highly expressed in these cells, but its function in liver repair remains incompletely understood. This study investigated whether activation of PPARβ/δ with the ligand GW501516 influenced the fibrotic response to injury from chronic carbon tetrachloride (CCl4) treatment in mice. Wild type and PPARβ/δ-null mice were treated with CCl4 alone or CCl4 co-administered with GW501516. To unveil mechanisms underlying the PPARβ/δ-dependent effects, we analyzed the proliferative response of human LX-2 HSCs to GW501516 in the presence or absence of PPARβ/δ. RESULTS: We found that GW501516 treatment enhanced the fibrotic response. Compared to the other experimental groups, CCl4/GW501516-treated wild type mice exhibited increased expression of various profibrotic and pro-inflammatory genes, such as those involved in extracellular matrix deposition and macrophage recruitment. Importantly, compared to healthy liver, hepatic fibrotic tissues from alcoholic patients showed increased expression of several PPAR target genes, including phosphoinositide-dependent kinase-1, transforming growth factor beta-1, and monocyte chemoattractant protein-1. GW501516 stimulated HSC proliferation that caused enhanced fibrotic and inflammatory responses, by increasing the phosphorylation of p38 and c-Jun N-terminal kinases through the phosphoinositide-3 kinase/protein kinase-C alpha/beta mixed lineage kinase-3 pathway. CONCLUSIONS: This study clarified the mechanism underlying GW501516-dependent promotion of hepatic repair by stimulating proliferation of HSCs via the p38 and JNK MAPK pathways.

Nuclear receptor PPARS function in stem cell differentiation and bone physiology

In adult, bone remodeling is a permanent process, reaching an annual turnover of about 10% of the skeleton. Bone remodeling requires the sequential and coordinated actions of the hematopoietic origin osteoclasts, to remove bone and the mesenchymal origin osteoblasts to replace it. An increased level of bone resorption is the primary cause of age-related bone loss often resulting in osteopenia, and is the major cause of osteoporosis.¦Peroxisome proliferator-activated receptors (PPARs), which are expressed in three isotypes, PPARa, PPARp and PPARy, are ligand-activated transcription factors that control many cellular and metabolic processes, more particularly linked to lipid metabolism. In bone, previous works has shown that PPARy inhibits osteogenesis by favoring adipogenesis from common mesenchymal progenitors. In addition, the pro-osteoclastogenesis activity of PPARy results in an increased bone resorption. Accordingly, treatment with PPARy agonist such as the anti-diabetic drug TZD causes bone loss and accumulation of marrow adiposity in mice as well as in postmenopausal women. The aim of the present thesis work was to elucidate the PPARs functions in bone physiology.¦The initial characterization of the PPARP" bone phenotype mainly revealed a decreased BMD. In vitro studies exploring the potency of mesenchymal stem cells to differentiate in osteoblast showed no differences depending on the genotype. However, we could demonstrate an effect of PPARp in partially inhibiting osteoclastogenesis. These results are further sustained by a study made in collaboration with the group of Dr Kronke, which showed an impressive protection against ovariectomy-generated bone loss when the females are treated with a PPARp agonist.¦Observations in PPARy null mice are more complex. The lab has recently been able to generate mice carrying a total deletion of PPARy. Intriguingly, the exploration of the bone phenotype of these mice revealed paradoxical findings. Whereas short bones such as vertebrae exhibit an elevated BMD as expected, long bones (tibia and femur) are clearly osteoporotic. According to their activity when set in culture, osteoblast differentiation normally occurs. Indeed the phenotype can be mainly attributed to a high density of osteoclasts in the cortical bone of PPARy null mice, associated to large bone resorption areas.¦Our explorations suggest a mechanism that involves regulatory processes linking osteoclastogenesis to adipogenesis, the latter being totally absent in PPARy null mice. Indeed, the lack of adipose tissue creates a favorable niche for osteoclastogenesis since conditioned medium made from differentiated adipocyte 3T3L1 inhibited osteoclastogenesis from both PPARy-/- and WT cells. Thus, adipokines deficiency in PPARy-/- mice contributes to de- repress osteoclastogenesis. Using specific blocking antibody, we further identified adiponectin as the major player among dozens of adipokines. Using flow cytometry assay, we explored the levels at which the osteoclastic commitment was perturbed in the bone marrow of PPARy-/- mice. Intriguingly, we observe a general decrease for hematopoietic stem cell and lineage progenitors but increased proportion of osteoclast progenitor in PPARy-/- bone marrow. The general decrease of HSC in the bone marrow is however largely compensated by an important extra-medullary hematopoeisis, taking place in the liver and in the spleen.¦These specific characteristics emphasize the key role of PPARy on a cross road of osteogenesis, adipogenesis and hematopoiesis/osteoclastogenesis. They underline the complexity of the bone marrow niche, and demonstrate the inter-dependance of different cell types in defining bone homeostasis, that may be overseen when experimental design single out pure cell populations.¦Chez l'adulte, même après la fin de la croissance, le renouvellement des os se poursuit et porte sur environ 10% de l'ensemble du squelette adulte, par année. Ce renouvellement implique à la fois des mécanismes séquentiels et coordonnés des ostéoclastes d'origine hématopoïetique, qui dégradent l'os, et des ostéoblastes d'origine mésenchymale, qui permettent la régénération de l'os. La perte en densité osseuse due à l'âge entraîne un fort niveau de résorption, conduisant souvent à une ostéopénie, elle-même cause de l'ostéoporose.¦Les trois isotypes PPAR (Peroxisome proliferator-activated receptor, PPARa, PPARp, et PPARy) sont des récepteurs nucléaires qui contrôlent de nombreux mécanismes cellulaires et métaboliques, plus particulièrement liés au métabolisme lipidique. Au niveau osseux, des travaux précédents ont montré que PPARy inhibe l'ostéoblastogenèse en favorisant la formation d'adipocytes à partir de la cellule progénitrice commune. De plus, l'activité pro- ostéoclastogénique de PPARy induit une résorption osseuse accrue. Condormément à ces observations, les patients diabétiques traités par les thiazolidinediones qui agissent sur PPARy, ont un risque accrue d'ostéoporose liée à une perte osseuse accrue et un accroissement de l'adiposité au niveau de la moelle osseuse. Dans ce contexte, l'objectif de mon travail de thèse a été d'élucider le rôle des PPAR dans la physiologie osseuse, en s'appuyant sur le phénotype des souris porteuses de mutation pour PPAR.¦La caractérisation initiale des os des souris porteuses d'une délétion de ΡΡΑΕφ a principalement révélé une diminution de la densité minérale osseuse (DMO). Alors que l'ostéogenèse n'est pas significativement altérée chez ces souris, l'ostéoclastogenèse est elle augmentée, suggérant un rôle modérateur de ce processus par ΡΡΑΕΙβ. Ces résultats sont par ailleurs soutenus par une étude menée par le groupe du Dr Krônke en collaboration avec notre groupe, et qui monte une protection très importante des souris traitées par un activateur de PPARP contre l'ostéoporose provoquée par l'ovariectomie.¦Les observations concernant PPARy donnent des résultats plus complexes. Le laboratoire a en effet été capable récemment de générer des souris portant une délétion totale de PPARy. Alors que les os courts chez ces souris présentent une augmentation de la DMO, comme attendu, les os longs sont clairement ostéoporotiques. Ce phénotype corrèle avec une densité élevée d'ostéoclastes dans l'os cortical de ces os longs. Deux processus semblent contribuer à ce phénotype. En premier lieu, nous démontrons qu'un milieu conditionné provenant de cultures de cellules 3T3-L1 différenciées en adipocytes contiennent une forte activité inhibitrice d'osteoclastogenesis. L'utilisation d'anticorps neutralisant permet d'identifier l'adiponectine comme l'un des facteurs principaux de cette inhibition. Les souris PPARy étant totalement dépourvues d'adipocytes et donc de tissu adipeux, la sécrétion locale d'adiponectine dans la moelle osseuse est donc également absente, entraînant une désinhibition de l'ostéoclastogenèse. En second lieu, des analyses par FACS révèle une proportion accrue des cellules progénitrices d'ostéoclastes dans la moelle osseuse. Cela s'accompagne par une diminution globale des cellules souches hématopoïétiques, qui est cependant largement compensée par une importante hématopoëise extra-médullaire, dans le foie comme dans la rate.¦L'ensemble de notre travail montre toute l'importance de PPARy au carrefour de l'ostéogenèse, adipogenèse, et hématopoëise/osteoclastogenèse. Il souligne la complexité de la niche que représente la moelle osseuse et démontre l'inter-dépendance des différents types cellulaires définissant l'homéostasie osseuse, complexité qui peut facilement être masqué lorsque le travail expérimental se concentre sur le comportement d'un type cellulaire donné.

PPARgamma in placental angiogenesis.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in diverse biological processes including adipocyte differentiation, glucose homeostasis, and inflammatory responses. Analyses of PPARγ knockout animals have been so far preempted by the early embryonic death of PPARγ-/- embryos as a consequence of the severe alteration of their placental vasculature. Using Sox2Cre/PPARγL2/L2 mice, we obtained fully viable PPARγ-null mice through specific and total epiblastic gene deletion, thereby demonstrating that the placental defect is the unique cause of PPARγ-/- embryonic lethality. The vasculature defects observed in PPARγ-/- placentas at embryonic d 9.5 correlated with an unsettled balance of pro- and antiangiogenic factors as demonstrated by increased levels of proliferin (Prl2c2, PLF) and decreased levels of proliferin-related protein (Prl7d1, PRP), respectively. To analyze the role of PPARγ in the later stage of placental development, when its expression peaks, we treated pregnant wild-type mice with the PPARγ agonist rosiglitazone. This treatment resulted in a disorganization of the placental layers and an altered placental microvasculature, accompanied by the decreased expression of proangiogenic genes such as Prl2c2, vascular endothelial growth factor, and Pecam1. Together our data demonstrate that PPARγ plays a pivotal role in controlling placental vascular proliferation and contributes to its termination in late pregnancy.

Functional interaction between peroxisome proliferator-activated receptors-alpha and Mef-2C on human carnitine palmitoyltransferase 1beta (CPT1beta) gene activation

Muscle-type carnitine palmitoyltransferase 1 (CPT1β) is considered to be the gene that controls fatty acid mitochondrial β-oxidation. A functional peroxisome proliferator-activated receptor (PPAR) responsive element (PPRE) and a myocite-specific (MEF2) site that binds MEF2A and MEF2C in the promoter of this gene had been previously identified. We investigated the roles of the PPRE and the MEF2 binding sites and the potential interaction between PPARα and MEF2C regulating the CPT1β gene promoter. Mutation analysis indicated that the MEF2 site contributed to the activation of the CPT1β promoter by PPAR in C2C12 cells. The reporter construct containing the PPRE and the MEF2C site was synergistically activated by co-expression of PPAR, retinoid X receptor (RXR) and MEF2C in non-muscle cells. Moreover, protein-binding assays demonstrated that MEF2C and PPAR specifically bound to one another in vitro. Also for the synergistic activation of the CPT1β gene promoter by MEF2C and PPARα-RXRα, a precise arrangement of its binding sites was essential.

Expression and secretion of the atrial natriuretic peptide in human adipose tissue and preadipocytes.

OBJECTIVE: Atrial natriuretic peptide (ANP) is a secretory hormone displaying diuretic, natriuretic, and vasorelaxant activities. Recently, its lipolytic activity has been reported. Since the expression of ANP in adipose tissue has not been documented, we used real-time reverse transcriptase polymerase chain reaction (RT-PCR) to investigate the expression of ANP in human adipose tissue and preadipocytes. RESEARCH METHODS AND PROCEDURES: RNA was extracted from the human adipose tissue of severely obese premenopausal women as well as from human preadipocytes. For human preadipocytes, two cell systems were investigated: the human preadipose immortalized (Chub-S7) cells, a well-characterized human preadipose cell line, and primary preadipocytes derived from the stromal vascular fraction of the human adipose tissue. We measured the mRNA of ANP, of corin (a transmembrane serine protease involved in the conversion of pro-ANP to ANP) and of uncoupling protein 2 (UCP2; a control gene known to be ubiquitously expressed). The expression of ANP was also investigated using immunofluorescence and radioimmunoassay in Chub-S7 cells and human primary preadipocytes in culture. RESULTS: Our results indicate that ANP and corin are expressed at the mRNA level in human adipose tissue and preadipocytes. Immunofluorescence experiments demonstrated that pro-ANP was expressed in Chub-S7 cells. In addition, ANP secretion could be measured in Chub-S7 cells and human primary preadipocytes in culture. Rosiglitazone, a selective peroxisome proliferator-activated receptor type gamma (PPAR-gamma) agonist promoting adipocyte differentiation, was found to modulate both ANP expression and secretion in preadipocytes. DISCUSSION: Our findings suggest the existence of an autocrine/paracrine system for ANP in the human adipose tissue whose implications in lipolysis and cardiovascular function need to be further explored.

Thrombin-receptor antagonist vorapaxar in acute coronary syndromes

Background: Vorapaxar is a new oral protease-activated-receptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation. Methods: In this multinational, double-blind, randomized trial, we compared vorapaxar with placebo in 12,944 patients who had acute coronary syndromes without ST-segment elevation. The primary end point was a composite of death from cardiovascular causes, myocardial infarction, stroke, recurrent ischemia with rehospitalization, or urgent coronary revascularization. RESULTS: Follow-up in the trial was terminated early after a safety review. After a median follow-up of 502 days (interquartile range, 349 to 667), the primary end point occurred in 1031 of 6473 patients receiving vorapaxar versus 1102 of 6471 patients receiving placebo (Kaplan-Meier 2-year rate, 18.5% vs. 19.9%; hazard ratio, 0.92; 95% confidence interval [CI], 0.85 to 1.01; P = 0.07). A composite of death from cardiovascular causes, myocardial infarction, or stroke occurred in 822 patients in the vorapaxar group versus 910 in the placebo group (14.7% and 16.4%, respectively; hazard ratio, 0.89; 95% CI, 0.81 to 0.98; P = 0.02). Rates of moderate and severe bleeding were 7.2% in the vorapaxar group and 5.2% in the placebo group (hazard ratio, 1.35; 95% CI, 1.16 to 1.58; P<0.001). Intracranial hemorrhage rates were 1.1% and 0.2%, respectively (hazard ratio, 3.39; 95% CI, 1.78 to 6.45; P<0.001). Rates of nonhemorrhagic adverse events were similar in the two groups. Conclusions: In patients with acute coronary syndromes, the addition of vorapaxar to standard therapy did not significantly reduce the primary composite end point but significantly increased the risk of major bleeding, including intracranial hemorrhage. (Funded by Merck; TRACER ClinicalTrials.gov number, NCT00527943.)

PPARγ induces growth inhibition and apoptosis through upregulation of insulin-like growth factor-binding protein-3 in gastric cancer cells

Peroxisome proliferator activator receptor-gamma (PPARγ) is a ligand-activated transcriptional factor involved in the carcinogenesis of various cancers. Insulin-like growth factor-binding protein-3 (IGFBP-3) is a tumor suppressor gene that has anti-apoptotic activity. The purpose of this study was to investigate the anticancer mechanism of PPARγ with respect to IGFBP-3. PPARγ was overexpressed in SNU-668 gastric cancer cells using an adenovirus gene transfer system. The cells in which PPARγ was overexpressed exhibited growth inhibition, induction of apoptosis, and a significant increase in IGFBP-3 expression. We investigated the underlying molecular mechanisms of PPARγ in SNU-668 cells using an IGFBP-3 promoter/luciferase reporter system. Luciferase activity was increased up to 15-fold in PPARγ transfected cells, suggesting that PPARγ may directly interact with IGFBP-3 promoter to induce its expression. Deletion analysis of the IGFBP-3 promoter showed that luciferase activity was markedly reduced in cells without putative p53-binding sites (-Δ1755, -Δ1795). This suggests that the critical PPARγ-response region is located within the p53-binding region of the IGFBP-3 promoter. We further demonstrated an increase in PPARγ-induced luciferase activity even in cells treated with siRNA to silence p53 expression. Taken together, these data suggest that PPARγ exhibits its anticancer effect by increasing IGFBP-3 expression, and that IGFBP-3 is a significant tumor suppressor.

The role of PPARgamma in cartilage growth and development using cartilage-specific PPARgamma knockout mice

Le cartilage est un tissu conjonctif composé d’une seule sorte de cellule nommée chondrocytes. Ce tissu offre une fondation pour la formation des os. Les os longs se développent par l'ossification endochondral. Ce processus implique la coordination entre la prolifération, la différenciation et l'apoptose des chondrocytes, et résulte au remplacement du cartilage par l'os. Des anomalies au niveau du squelette et des défauts liés à l’âge tels que l’arthrose (OA) apparaissent lorsqu’il y a une perturbation dans l’équilibre du processus de développement. À ce jour, les mécanismes exacts contrôlant la fonction et le comportement des chondrocytes pendant la croissance et le développement du cartilage sont inconnus. Le récepteur activateur de la prolifération des peroxysomes (PPAR) gamma est un facteur de transcription impliqué dans l'homéostasie des lipides. Plus récemment, son implication a aussi été suggérée dans l'homéostasie osseuse. Cependant, le rôle de PPARγ in vivo dans la croissance et le développement du cartilage est inconnu. Donc, pour la première fois, cette étude examine le rôle spécifique de PPARγ in vivo dans la croissance et le développement du cartilage. Les souris utilisées pour l’étude avaient une délétion conditionnelle au cartilage du gène PPARγ. Ces dernières ont été générées en employant le système LoxP/Cre. Les analyses des souris ayant une délétion au PPARγ aux stades embryonnaire et adulte démontrent une réduction de la croissance des os longs, une diminution des dépôts de calcium dans l’os, de la densité osseuse et de la vascularisation, un délai dans l’ossification primaire et secondaire, une diminution cellulaire, une perte d’organisation colonnaire et une diminution des zones hypertrophiques, une désorganisation des plaques de croissance et des chondrocytes déformés. De plus, la prolifération et la différenciation des chondrocytes sont anormales. Les chondrocytes et les explants isolés du cartilage mutant démontrent une expression réduite du facteur de croissance endothélial vasculaire (VEGF)-A et des éléments de production de la matrice extracellulaire. Une augmentation de l’expression de la métalloprotéinase matricielle (MMP)-13 est aussi observée. Dans les souris âgées ayant une délétion au PPARγ, y est aussi noté des phénotypes qui ressemblent à ceux de l’OA tel que la dégradation du cartilage et l'inflammation de la membrane synoviale, ainsi qu’une augmentation de l’expression de MMP-13 et des néoépitopes générés par les MMPs. Nos résultats démontrent que le PPARγ est nécessaire pour le développement et l’homéostasie du squelette. PPARγ est un régulateur essentiel pour la physiologie du cartilage durant les stades de croissance, de développement et de vieillissement.

Rat protease-activated receptor–1 (rPAR1) expression and characterization in Sf9 cells

Connue pour son rôle dans la cascade de coagulation, la thrombine, une protéase à sérine, peut également agir par l’intermédiaire de PAR1, un récepteur activé par protéase et couplé aux protéines G liant le GTP (GPCR). La thrombine se lie et clive l’extrémité N-terminale du PAR1 entre l’Arg41 et la Ser42, exposant une nouvelle extrémité terminale qui agit elle-même comme un ligand. La thrombine et une séquence peptidique de cinq acides aminés, composée des résidus Ser42 à Arg46, nommée PAR1-AP, déclenchent dans diverses cellules de mammifères une réponse intracellulaire comportant une composante calcique. Dans cette étude, le système d’expression par baculovirus dans les cellules Sf9 d'insecte nous a permis d'exprimer le récepteur PAR1 du rat à la surface de ces cellules et de réaliser son couplage fonctionnel à leur signalisation intracellulaire (modèle rPAR1-Sf9). La composante calcique de celle-ci, en réponse au PAR1-AP, a ensuite été étudiée en détail à l’aide de la sonde fluorescente Fura-2 et de plusieurs inhibiteurs agissant sur les canaux calciques ou d'autres éléments de la cascade de signalisation du calcium intracellulaire. Lorsque le milieu extracellulaire contient du calcium (Ca2+), la thrombine ou PAR1-AP déclenchent un signal calcique qui consiste en une augmentation rapide de [Ca2+]i suivi d’un plateau relativement soutenu, puis d'un retour lent vers le niveau de base initial. En l'absence de Ca2+ dans le milieu extracellulaire, l'augmentation initiale rapide de [Ca2+]i est suivie d'un retour rapide vers le [Ca2+]i de base. À l’aide d’inhibiteurs de canaux calciques, tels que le lanthane, la nifédipine et le D-600, l'entrée du calcium du milieu extracellulaire dans les cellules est inhibée, abolissant le plateau soutenu de [Ca2+]i. L’inhibition de la pompe Ca2+-ATPase par la thapsigargine supprime la réponse au PAR1-AP après épuisement des sites de stockage de Ca2+intracellulaire. Le TMB-8 agit de façon discordante quant à l’inhibition de la libération de Ca2+ des sites de stockage intracellulaires. La réponse à PAR1-AP n’est pas affectée par le D-609, un inhibiteur de la phospholipase β. L’inhibition de la protéine kinase C (PKC) par le bisindolylmaléimide induit des oscillations en présence de Ca2+ extracellulaire et atténue fortement le signal calcique en absence de Ca2+ extracellulaire. En présence de Ca2+ extracellulaire, l’activation de la PKC par le PBDu tronque le flux de [Ca2+]i tandis que la réponse calcique est abolie en absence de Ca2+ dans le milieu extracellulaire. Le H-89, un inhibiteur de la protéine kinase A (PKA), cause une prolongation de la durée du plateau de [Ca2+]i dans un milieu riche en calcium et la suppression de la réponse à PAR1-AP lorsque le milieu extracellulaire est dépourvu de Ca2+. Les résultats obtenus nous permettent de conclure que la PKC et possiblement la PKA jouent un rôle critique dans la mobilisation du Ca2+ induite par le PAR1-AP dans le modèle rPAR1-Sf9.

Interaction between allelic variations in vitamin D receptor and retinoid X receptor genes on metabolic traits

BACKGROUND: Low vitamin D status has been shown to be a risk factor for several metabolic traits such as obesity, diabetes and cardiovascular disease. The biological actions of 1, 25-dihydroxyvitamin D, are mediated through the vitamin D receptor (VDR), which heterodimerizes with retinoid X receptor, gamma (RXRG). Hence, we examined the potential interactions between the tagging polymorphisms in the VDR (22 tag SNPs) and RXRG (23 tag SNPs) genes on metabolic outcomes such as body mass index, waist circumference, waist-hip ratio (WHR), high- and low-density lipoprotein (LDL) cholesterols, serum triglycerides, systolic and diastolic blood pressures and glycated haemoglobin in the 1958 British Birth Cohort (1958BC, up to n = 5,231). We used Multifactor- dimensionality reduction (MDR) program as a non-parametric test to examine for potential interactions between the VDR and RXRG gene polymorphisms in the 1958BC. We used the data from Northern Finland Birth Cohort 1966 (NFBC66, up to n = 5,316) and Twins UK (up to n = 3,943) to replicate our initial findings from 1958BC. RESULTS: After Bonferroni correction, the joint-likelihood ratio test suggested interactions on serum triglycerides (4 SNP - SNP pairs), LDL cholesterol (2 SNP - SNP pairs) and WHR (1 SNP - SNP pair) in the 1958BC. MDR permutation model testing analysis showed one two-way and one three-way interaction to be statistically significant on serum triglycerides in the 1958BC. In meta-analysis of results from two replication cohorts (NFBC66 and Twins UK, total n = 8,183), none of the interactions remained after correction for multiple testing (Pinteraction >0.17). CONCLUSIONS: Our results did not provide strong evidence for interactions between allelic variations in VDR and RXRG genes on metabolic outcomes; however, further replication studies on large samples are needed to confirm our findings.