Search

Enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. Venous thrombosis and thromboembolism risks are associated with increased plasma levels of TAFI (Thrombin Activatable Fibrinolysis Inhibitor) as well as its active form TAFIa. A new TAFIa inhibitor, namely S62798 has been identified. Its ability to enhance fibrinolysis was investigated both in vitro and in vivo in a mouse model of pulmonary thromboembolism, as well as its effect on bleeding. S62798 is a highly selective human, mouse and rat TAFIa inhibitor (IC50 = 11; 270; 178 nmol/L, respectively). It accelerates lysis of a human clot in vitro, evaluated by thromboelastometry (EC50 = 27 nmol/L). In a rat tail bleeding model, no effect of S62798 treatment was observed up to 20 mg/kg. Enhancement of endogenous fibrinolysis by S62798 was investigated in a mouse model of Tissue Factor-induced pulmonary thromboembolism. Intravenous administration of S62798 decreased pulmonary fibrin clots with a minimal effective dose of 0.03 mg/kg. Finally, effect of S62798 in combination with heparin was evaluated. When treatment of heparin was done in a curative setting, no effect was observed whereas a significantly decreased pulmonary fibrin deposition was observed in response to S62798 alone or in combination with heparin. This study demonstrates that S62798 is a potent TAFIa inhibitor with minimal risk of bleeding. In vivo, curative S62798 intravenous treatment, alone or associated with heparin, accelerated clot lysis by potentiating endogenous fibrinolysis and thus decreased pulmonary fibrin clots. S62798 is expected to be a therapeutic option for pulmonary embolism patients on top of anticoagulants.

Background Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, which is associated with features of metabolic syndrome. NAFLD may progress in a subset of patients into nonalcoholic steatohepatitis (NASH) with liver injury resulting ultimately in cirrhosis and potentially hepatocellular carcinoma. Today, there is no approved treatment for NASH due to, at least in part, the lack of preclinical models recapitulating features of human disease. Here, we report the development of a dietary model of NASH in the Gottingen minipig. Methods First, we performed a longitudinal characterization of diet-induced NASH and fibrosis using biochemical, histological, and transcriptional analyses. We then evaluated the pharmacological response to Obeticholic acid (OCA) treatment for 8 weeks at 2.5mg/kg/d, a dose matching its active clinical exposure. Results Serial histological examinations revealed a rapid installation of NASH driven by massive steatosis and inflammation, including evidence of ballooning. Furthermore, we found the progressive development of both perisinusoidal and portal fibrosis reaching fibrotic septa after 6 months of diet. Histological changes were mechanistically supported by well-defined gene signatures identified by RNA Seq analysis. While treatment with OCA was well tolerated throughout the study, it did not improve liver dysfunction nor NASH progression. By contrast, OCA treatment resulted in a significant reduction in diet-induced fibrosis in this model. Conclusions These results, taken together, indicate that the diet-induced NASH in the Gottingen minipig recapitulates most of the features of human NASH and may be a model with improved translational value to prioritize drug candidates toward clinical development

Most research on human pancreatic islets is conducted on samples obtained from normoglycaemic or diseased brain-dead donors and thus cannot accurately describe the molecular changes of pancreatic islet beta cells as they progress towards a state of deficient insulin secretion in type 2 diabetes (T2D). Here, we conduct a comprehensive multi-omics analysis of pancreatic islets obtained from metabolically profiled pancreatectomized living human donors stratified along the glycemic continuum, from normoglycemia to T2D. We find that islet pools isolated from surgical samples by laser-capture microdissection display remarkably more heterogeneous transcriptomic and proteomic profiles in patients with diabetes than in non-diabetic controls. The differential regulation of islet gene expression is already observed in prediabetic individuals with impaired glucose tolerance. Our findings demonstrate a progressive, but disharmonic, remodelling of mature beta cells, challenging current hypotheses of linear trajectories toward precursor or transdifferentiation stages in T2D. Furthermore, through integration of islet transcriptomics with preoperative blood plasma lipidomics, we define the relative importance of gene coexpression modules and lipids that are positively or negatively associated with HbA1c levels, pointing to potential prognostic markers. Wigger, Barovic and Brunner et al. perform a multidimensional analysis of islets from metabolically characterized patients who had undergone pancreatectomy, observing remarkable heterogeneity between samples from individuals with type 2 diabetes, thus arguing against models of linear beta-cell dedifferentiation in diabetes.

Maturation of fibrillar collagen is known to play a crucial role in the pathophysiology of myocardial fibrosis. Procollagen C-proteinase enhancer 1 (PCPE1) has a key role in procollagen maturation and collagen fibril formation. The phenotype of both male and female PCPE1 knock-out mice was investigated under basal conditions to explore the potential of PCPE1 as a therapeutic target in heart failure. Global constitutive PCPE1-/- mice were generated. Serum procollagen I C-terminal propeptide, organ histology, and cutaneous wound healing were assessed in both wild type (WT) and PCPE1-/- mice. In addition, the cardiac expression of genes involved in collagen metabolism was investigated and the total and insoluble cardiac collagen contents determined. Cardiac function was evaluated by echocardiography. No differences in survival, clinical chemistry, or organ histology were observed in PCPE1-/- mice compared with WT. Serum procollagen I C-terminal propeptide was lower in PCPE1-/- mice. Cardiac mRNA expression of Bmp1, Col1a1, Col3a1, and Loxl2 was similar, whereas Tgfb and Loxl1 mRNA levels were decreased in PCPE1-/- mice compared with sex-matched WT. No modification of total or insoluble cardiac collagen content was observed between the 2 strains. Ejection fraction was slightly decreased in PCPE1-/- male mice, but not in females. Finally, wound healing was not altered in PCPE1-/- mice. PCPE1 deficiency does not trigger any major liabilities and does not affect cardiac collagen content nor its function under basal conditions. Further studies are required to evaluate its role under stressed conditions and determine its suitability as a therapeutic target for heart failure.

Existing studies do not sufficiently describe the molecular changes of pancreatic islet beta cells leading to their deficient insulin secretion in type 2 diabetes (T2D). Here we address this deficiency with a comprehensive multi-omics analysis of metabolically profiled pancreatectomized living human donors stratified along the glycemic continuum from normoglycemia to T2D. Islet pools isolated from surgical samples by laser-capture microdissection had remarkably heterogeneous transcriptomic and proteomic profiles in diabetics, but not in non-diabetic controls. Transcriptomics analysis of this unique cohort revealed islet genes already dysregulated in prediabetic individuals with impaired glucose tolerance. Our findings demonstrate a progressive but disharmonic remodeling of mature beta cells, challenging current hypotheses of linear trajectories toward precursor or trans-differentiation stages in T2D. Further, integration of islet transcriptomics and pre-operative blood plasma lipidomics data enabled us to define the relative importance of gene co-expression modules and lipids positively or negatively associated with HbA1c levels, pointing to potential prognostic markers.

IntroductionA potential role for the orphan G protein-coupled receptor, GPR21, in linking immune cell infiltration into tissues and obesity-induced insulin resistance has been proposed, although limited studies in mice are complicated by non-selective deletion of Gpr21.Research design and methodsWe hypothesized that a Gpr21-selective knockout mouse model, coupled with type 2 diabetes patient samples, would clarify these issues and enable clear assessment of GPR21 as a potential therapeutic target.ResultsHigh-fat feeding studies in Gpr21−/− mice revealed improved glucose tolerance and modest changes in inflammatory gene expression. Gpr21−/− monocytes and intraperitoneal macrophages had selectively impaired chemotactic responses to monocyte chemoattractant protein (MCP)-1, despite unaltered expression of Ccr2. Further genotypic analysis revealed that chemotactic impairment was due to dysregulated monocyte polarization. Patient samples revealed elevated GPR21 expression in peripheral blood mononuclear cells in type 2 diabetes, which was correlated with both %HbA1c and fasting plasma glucose levels.ConclusionsCollectively, human and mouse data suggest that GPR21 influences both glucose homeostasis and MCP-1/CCL2-CCR2-driven monocyte migration. However, a Gpr21−/− bone marrow transplantation and high-fat feeding study in mice revealed no effect on glucose homeostasis, suggesting that there is no (or limited) overlap in the mechanism involved for monocyte-driven inflammation and glucose homeostasis.

In type 2 diabetes (T2D), both muscle and liver are severely resistant to insulin action. Muscle insulin resistance accounts for more than 80% of the impairment in total body glucose disposal in T2D patients and is often characterized by an impaired insulin signaling. Mitsugumin 53 (MG53), a muscle-specific TRIM family protein initially identified as a key regulator of cell membrane repair machinery has been suggested to be a critical regulator of muscle insulin signaling pathway by acting as ubiquitin E3 ligase targeting both the insulin receptor and insulin receptor substrate 1 (IRS1). Here, we show using in vitro and in vivo approaches that MG53 is not a critical regulator of insulin signaling and glucose homeostasis. First, MG53 expression is not consistently regulated in skeletal muscle from various preclinical models of insulin resistance. Second, MG53 gene knock-down in muscle cells does not lead to impaired insulin response as measured by Akt phosphorylation on Serine 473 and glucose uptake. Third, recombinant human MG53 does not alter insulin response in both differentiated C2C12 and human skeletal muscle cells. Fourth, ectopic expression of MG53 in HEK293 cells lacking endogenous MG53 expression fails to alter insulin response as measured by Akt phosphorylation. Finally, both male and female mg53 −/− mice were not resistant to high fat induced obesity and glucose intolerance compared to wild-type mice. Taken together, these results strongly suggest that MG53 is not a critical regulator of insulin signaling pathway in skeletal muscle.

FFA2, also called GPR43, is a G-protein coupled receptor for short chain fatty acids which is involved in the mediation of inflammatory responses. A class of azetidines was developed as potent FFA2 antagonists. Multiparametric optimization of early hits with moderate potency and suboptimal ADME properties led to the identification of several compounds with nanomolar potency on the receptor combined with excellent pharmacokinetic (PK) parameters. The most advanced compound, 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid 99 (GLPG0974), is able to inhibit acetate-induced neutrophil migration strongly in vitro and demonstrated ability to inhibit a neutrophil-based pharmacodynamic (PD) marker, CD11b activation-specific epitope [AE], in a human whole blood assay. All together, these data supported the progression of 99 toward next phases, becoming the first FFA2 antagonist to reach the clinic.

G protein-coupled receptors (GPCRs) continue to be important discovery targets for the treatment of type 2 diabetes mellitus (T2DM). Many GPCRs are directly involved in the development of insulin resistance and β-cell dysfunction, and in the etiology of inflammation that can lead to obesity-induced T2DM. This review summarizes the current literature describing a number of well-validated GPCR targets, but also outlines several new and promising targets for drug discovery. We highlight the importance of understanding the role of these receptors in the disease pathology, and their basic pharmacology, which will pave the way to the development of novel pharmacological probes that will enable these targets to fulfill their promise for the treatment of these metabolic disorders.

Monocyte-like cell lines (MCLCs), including THP-1, HL-60 and U-937 cells, are used routinely as surrogates for isolated human peripheral blood mononuclear cells (PBMCs). To systematically evaluate these immortalised cells and PBMCs as model systems to study inflammation relevant to the pathogenesis of type II diabetes and immuno-metabolism, we compared mRNA expression of inflammation-relevant genes, cell surface expression of cluster of differentiation (CD) markers, and chemotactic responses to inflammatory stimuli. Messenger RNA expression analysis suggested most genes were present at similar levels across all undifferentiated cells, though notably, IDO1, which encodes for indoleamine 2,3-dioxygenase and catabolises tryptophan to kynureninase (shown to be elevated in serum from diabetic patients), was not expressed in any PMA-treated MCLC, but present in GM-CSF-treated PBMCs. There was little overall difference in the pattern of expression of CD markers across all cells, though absolute expression levels varied considerably and the correlation between MCLCs and PBMCs was improved upon MCLC differentiation. Functionally, THP-1 and PBMCs migrated in response to chemoattractants in a transwell assay, with varying sensitivity to MCP-1, MIP-1α and LTB-4. However, despite similar gene and CD expression profiles, U-937 cells were functionally impaired as no migration was observed to any chemoattractant. Our analysis reveals that the MCLCs examined only partly replicate the genotypic and phenotypic properties of human PBMCs. To overcome such issues a universal differentiation protocol should be implemented for these cell lines, similar to those already used with isolated monocytes. Although not perfect, in our hands the THP-1 cells represent the closest, simplified surrogate model of PBMCs for study of inflammatory cell migration.

Objective— We previously reported that a systemic liver X receptor (LXR) agonist promoted macrophage reverse-cholesterol transport (mRCT) in vivo. Because LXR are expressed in multiple tissues involved in RCT (macrophages, liver, intestine), we analyzed the effect of tissue-specific LXR agonism on mRCT. Methods and Results— In initial studies, the systemic LXR agonist GW3965 failed to promote mRCT in a setting in which LXR was expressed in macrophages but not in liver or intestine. To evaluate the effect of LXR activation specifically in small intestine on mRCT, wild-type mice were treated with either intestinal-specific LXR agonist (GW6340) or systemic LXR agonist (GW3965). Both GW3965 and GW6340 significantly promoted excretion of [ 3 H]-sterol in feces by 162% and 52%, respectively. To evaluate the requirement for macrophage LXR activation, we assessed the ability of GW3965 to promote mRCT in wild-type mice using primary macrophages deficient in LXRα/β vs wild-type macrophages. Whereas GW3965 treatment promoted fecal excretion compared with vehicle, its overall ability to promote mRCT was significantly attenuated using LXRα/β knockout macrophages. Conclusion— We demonstrate that intestinal-specific LXR agonism promotes macrophage RCT in vivo and that macrophage LXR itself plays an important, but not predominant, role in promoting RCT in response to an LXR agonist.

The liver receptor homolog-1 (LRH-1) is an orphan nuclear receptor believed to play a key role in bile acid metabolism, cholesterol homeostasis, and intestinal cell crypt renewal. LRH-1 has recently been reported to negatively regulate the hepatic acute phase response by antagonizing, at least in part, the CCAAT/enhancer-binding protein signaling pathway. Here we have shown, using adenovirus-mediated LRH-1 overexpression and gene-silencing experiments, that the interleukin-1 receptor antagonist (IL-1RA) gene is a novel LRH-1 target gene in hepatic cells. Promoter mapping and chromatin immunoprecipitation experiments revealed that LRH-1 regulates IL-1RA gene expression under inflammatory conditions at the transcriptional level via the binding to an LRH-1 response element. Interestingly, IL-1RA induction by an intraperitoneal injection of lipopolysaccharide is significantly lower in LRH-1 heterozygous compared with wild-type mice, demonstrating the contribution of LRH-1 in IL-1RA gene regulation. Finally, RNA interference experiments indicate that LRH-1 blocks the hepatic acute phase response by, at least in part, inducing IL-1RA expression. Taken together, these results lead to the identification of IL-1RA as a novel LRH-1 target gene and demonstrate the existence of multiple mechanisms contributing to the overall anti-inflammatory properties of LRH-1 in hepatic cells.

We report the identification of substituted cis-bicyclo[3.3.0]-oct-2-enes as small molecule agonists of subfamily V orphan nuclear receptors (NR5A), liver receptor homolog-1 (LRH-1) and steroidogenic factor-1 (SF-1). Using fluorescence resonance energy transfer (FRET)-based biochemical assays, compound 5a (GSK8470) was identified as a high-affinity ligand for LRH-1 and SF-1. In liver cells, 5a increased the expression of the LRH-1 target gene small heterodimer partner (SHP). Synthesis of analogues modified at three positions led to the development of compounds with functional selectivity between LRH-1 and SF-1.

The orphan nuclear receptor liver receptor homolog 1 (LRH-1) has been reported to play an important role in bile acid biosynthesis and reverse cholesterol transport. Here, we show that LRH-1 is a key player in the control of the hepatic acute-phase response. Ectopic expression of LRH-1 with adenovirus resulted in strong inhibition of both interleukin-6 (IL-6)- and IL-1beta-stimulated haptoglobin, serum amyloid A, and fibrinogen beta gene expression in hepatocytes. Furthermore, induction of the hepatic inflammatory response was significantly exacerbated in HepG2 cells expressing short hairpin RNA targeting LRH-1 expression. Moreover, transient-transfection experiments and electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that LRH-1 regulates this cytokine-elicited inflammatory response by, at least in part, antagonizing the CCAAT/enhancer binding protein beta signaling pathway. Finally, we show, by using LRH-1 heterozygous mice, that LRH-1 is involved in the control of the inflammatory response at the hepatic level in vivo. Taken together, our results outline an unexpected role for LRH-1 in the modulation of the hepatic acute-phase response.

Whereas the role of thyroid hormone is clearly established in the regulation of cholesterol homeostasis, its involvement in the control of serum triglyceride (TG) levels remains largely debated. Angiopoietin-like proteins 3 and 4 have recently been characterized as potent lipoprotein lipase inhibitors and therefore as important components of plasma triglyceride homeostasis. In the present study, the role of thyroid hormone in the regulation of both ANGPTL4 and ANGPTL3 gene expression was investigated. In vivo studies revealed that thyroid hormone down-regulates ANGPTL3 but not ANGPTL4 gene expression in hypothyroid rats. Using thyroid hormone receptor (TR)-deficient mice, we show that thyroid hormone regulates ANGPTL3 gene expression in a TRbeta-dependent manner. Transfection studies revealed that this inhibition occurs at the transcriptional level in a DNA binding-independent fashion and requires the proximal (-171 to +66) region of the ANGPTL3 gene promoter. Moreover, site-directed mutagenesis experiments indicate that the HNF1 site within this proximal region mediates this TRbeta-dependent repression. Finally, co-transfection studies and electrophoretic mobility shift assays suggest that TRbeta antagonizes the HNF1alpha signaling pathway by inhibiting its transcriptional activity without interfering with its DNA-binding capacity. Taken together, our results lead to the identification of ANGPTL3 as a novel TRbeta target gene and provide a new potential mechanism to explain the hypotriglyceridemic properties of TRbeta agonists in vivo.

Endothelin is a potent vasoconstrictor peptide isolated from endothelial cells and it induces smooth muscle cell proliferation. Endothelin-1 secretion is increased in atheroma and induces deleterious effects such as vasospasm and atherosclerosis. Oxidized low-density lipoproteins (LDLs) induce atherosclerosis in the vascular wall, as well as endothelin-1 secretion in endothelial cells and are activators of both peroxisome proliferator-activated receptor-alpha (PPAR-alpha) and PPAR-gamma. PPAR-alpha (fibric acids) and PPAR-gamma (glitazones) activators are used to treat dyslipoproteinemias and type 2 diabetes, respectively. Furthermore, these drugs induce numerous pleiotropic effects, such as inhibiting thrombin-induced endothelin-1 secretion in endothelial cells. This study shows that both PPAR-alpha (Wy 14643) and PPAR-gamma activation (rosiglitazone) partially inhibit oxidized LDL-induced protein kinase C activity and endothelin-1 secretion in endothelial cells at the transcriptional levels and suggests that synthetic PPAR activators are stronger PPAR activators than oxidized LDL. This study also suggests that fibrate and glitazone treatments should have beneficial effects on the vascular wall by reducing endothelin-1 secretion and the resulting vasospasm and atherosclerosis.

The nuclear receptor superfamily comprises a large number of ligand-activated transcription factors that are involved in numerous biological processes such as cell proliferation, differentiation, and homeostasis. ROR(alpha) (NR1F1) and Reverb(alpha) (NR1D1) are two members of this family whose biological functions are largely unknown. In addition, no ligand has been yet identified for these two receptors; therefore, they are referred as orphan receptors. Here, we show that ROR(alpha) and Reverb(alpha) are expressed with a similar tissue distribution and are both induced during the differentiation of rat L6 myoblastic cells. Ectopic expression of ROR(alpha)1 in L6 cells significantly induces Reverb(alpha) expression as demonstrated by Northern blot analysis. Using reverse transcription-PCR to analyze Reverb(alpha) gene expression from staggerer mice, we found that there was a significant reduction of Reverb(alpha) mRNA in the skeletal muscle comparing it with the wild-type mice, which suggests that ROR(alpha) is involved in the regulation of Reverb(alpha) gene expression. Transient transfection assays using the Reverb(alpha) promoter demonstrate that ROR(alpha) regulates the Reverb(alpha) gene at the transcriptional level. Furthermore, mutagenesis experiments indicate that ROR(alpha) regulates Reverb(alpha) transcription via a monomeric ROR response element located in the Reverb(alpha) gene promoter. Electrophoretic mobility shift assays show that ROR(alpha) binds strongly to this site in a specific-manner. Finally, overexpression of GRIP-1/TIF-2, but not SRC-1, potentiates ROR(alpha)-stimulated Reverb(alpha) promoter activity in transient transfection experiments. Together, our results identify Reverb(alpha) as a novel target gene for ROR(alpha).

This study was conducted to evaluate whether treatment of normal and diabetic rat hearts with rosiglitazone, a high-affinity ligand of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) used for the treatment of type 2 diabetes, improves postischemic functional recovery. The effects of acute rosiglitazone administration were investigated using working hearts isolated from normal rat or rats diabetic for 4 weeks after streptozotocin (STZ) injection. Hearts were subjected to 30 min of normothermic, zero-flow ischemia followed by 30-min reperfusion. Rosiglitazone (1 micromol/l) administered before ischemia had no effect on cardiac function during baseline perfusion, but it significantly improved aortic flow during reperfusion in both normal and diabetic hearts. In a chronic protocol in which rosiglitazone was given by daily gavage (10 micromol/kg body wt) immediately after STZ injection, rosiglitazone also prevented postischemic injury and significantly improved functional recovery. Using Western immunoblotting, it was demonstrated that the acute cardioprotective effect of rosiglitazone is associated with an inhibition of Jun NH(2)-terminal kinase phosphorylation in both normal and diabetic rat hearts. Furthermore, rosiglitazone also inhibited activating protein-1 DNA-binding activity. These data, demonstrating that rosiglitazone limits postischemic injury in isolated hearts, suggest an important function for PPAR-gamma in the heart.

PPARs are ligand-activated transcription factors that regulate energy homeostasis. In addition, PPARs furthermore control the inflammatory response by antagonizing the nuclear factor-κB (NF-κB) signaling pathway. We recently demonstrated that PPARα activators increase IκBα mRNA and protein levels in human aortic smooth muscle cells. Here, we studied the molecular mechanisms by which PPARα controls IκBα expression. Using transient transfection assays, it is demonstrated that PPARα potentiates p65-stimulated IκBα transcription in a ligand-dependent manner. Site-directed mutagenesis experiments revealed that PPARα activation of IκBα transcription requires the NF-κB and Sp1 sites within IκBα promoter. Chromatin immunoprecipitation assays demonstrate that PPARα activation enhances the occupancy of the NF-κB response element in IκBα promoter in vivo. Overexpression of the oncoprotein E1A failed to inhibit PPARα-mediated IκBα promoter induction, suggesting that cAMP response element binding protein-binding protein/p300 is not involved in this mechanism. By contrast, a dominant-negative form of VDR-interacting protein 205 (DRIP205) comprising its two LXXLL motifs completely abolished PPARα ligand-mediated activation. Furthermore, cotransfection of increasing amounts of DRIP205 relieved this inhibition, suggesting that PPARα requires DRIP205 to regulate IκBα promoter activity. By contrast, DRIP205 is not involved in PPARα-mediated NF-κB transcriptional repression. Taken together, these data provide a molecular basis for PPARα-mediated induction of IκBα and demonstrate, for the first time, that PPARα may positively regulate gene transcription in the absence of functional PPAR response elements.

Ligand-dependent gene transcription mediated by the nuclear receptors involves the recruitment of transcriptional coactivators to the ligand-binding domain (LBD), which leads to interaction with the basal transcription machinery, and ultimately with RNA polymerase II. Although most of these coactivators are ubiquitously expressed, a tissue-selective coactivator, PGC-1, has recently been characterized. Because PGC-1 and the retinoid X receptors (RXRs) possess an overlapping tissue distribution, we investigated whether PGC-1 is a coactivator for the retinoid X receptors. In a transient transfection assay, PGC-1 augments ligand-stimulated RXR transcription. Furthermore, PGC-1 efficiently enhances the RXR element-driven reporter gene transcription by all three RXR isoforms. An immunoprecipitation assay reveals that PGC-1 and RXRalpha interact in vivo. In addition, a glutathione S-transferase pull-down assay showed that this interaction requires the presence of the LXXLL motif of PGC-1. We demonstrate further, in a mammalian two-hybrid assay, that this physical interaction also requires the presence of the AF-2 region of RXR to interact with the LXXLL motif of PGC-1, which is consistent with our protein-protein interaction results. A time-resolved fluorescence assay shows that a peptide within the NR box of PGC-1 is efficiently recruited by a ligand-bound RXRalpha in vitro. Finally, PGC-1 and TIF2 synergistically enhance ligand-activated RXRalpha transcriptional activity. Taken together, these results indicate that PGC-1 is a bona fide coactivator for RXRalpha.

Peroxisome proliferator-activated receptor gamma (PPARgamma ), a member of the nuclear receptor superfamily, has recently been described as a modulator of macrophage functions and as an inhibitor of T cell proliferation. Here, we investigated the role of PPARgamma in dendritic cells (DC), the most potent antigen-presenting cells. We showed that PPARgamma is highly expressed in immature human monocyte-derived DC (MDDC) and that it may affect the immunostimulatory function of MDDC stimulated with lipopolysaccharide (LPS) or via CD40 ligand (CD40L). We found that the synthetic PPARgamma agonist rosiglitazone (as well as pioglitazone and troglitazone) significantly increases on LPS- and CD40L-activated MDDC, the surface expression of CD36 (by 184% and 104%, respectively) and CD86 (by 54% and 48%), whereas it reduces the synthesis of CD80 (by 42% and 42%). Moreover, activation of PPARgamma resulted in a dramatic decreased secretion of the Th1-promoting factor IL-12 in LPS- and CD40L-stimulated cells (by 47% and 62%), while the production of IL-1beta, TNF-alpha, IL-6 and IL-10 was unaffected. Finally, PPARgamma ligands down-modulate the synthesis of IFN-gamma -inducible protein-10 (recently termed as CXCL10) and RANTES (CCL5), both chemokines involved in the recruitment of Th1 lymphocytes (by 49% and 30%), but not the levels of the Th2 cell-attracting chemokines,macrophage-derived chemokine (CCL22) and thymus and activation regulated chemokine (CCL17), in mature MDDC. Taken together, our data suggest that activation of PPARgamma in human DC may have an impact in the orientation of primary and secondary immune responses by favoring type 2 responses.

Regulation of the expression of platelet-activating factor (PAF) receptor by atherogenic lipoproteins might contribute to atherogenesis. We show that progressive oxidation of low-density lipoprotein (LDL) gradually inhibits PAF receptor expression on the macrophage cell surface. We tested the effect of oxidized LDL (oxLDL) on PAF receptor expression in human monocytes that do not contain peroxisome-proliferator-activated receptor γ (PPARγ), a nuclear receptor activated by oxLDL. OxLDL decreased by 50% (P ⩽0.001) and by 29% (P⩽0.05) the binding of PAF and the expression of PAF receptor mRNA respectively. Next we demonstrated that progressive oxidation of LDLs significantly activated PPARα-dependent transcription in transfected mouse aortic endothelial cells. Finally we demonstrated, in mature macrophages, that fenofibrate (20µM), a specific PPARα agonist, but not the specific PPARγ agonist BRL49653 (20nM), significantly decreased both PAF binding and PAF receptor mRNA expression, by 65% and 40% (P⩽0.001) respectively. Additionally, another PPARα agonist, Wy14,643, decreased PAF receptor promoter activity by 70% (P⩽0.05) in transfected THP-1 cells, suggesting the involvement of the proximal promoter region (-980 to -500) containing a series of four nuclear factor (NF)-κB motifs. Thus PPARα might be involved in the down-regulation of PAF receptor gene expression by oxLDLs in human monocytes/macrophages. The oxidation of one or more lipid components of LDLs might result in the formation of natural activators of PPARα. It is hypothesized that such activators might modulate inflammation and apoptosis upon atherogenesis by decreasing the expression of PAF receptor.

Retinoid-related orphan receptor alpha (ROR alpha) (NR1F1) is a member of the nuclear receptor superfamily whose biological functions are largely unknown. Since staggerer mice, which carry a deletion in the ROR alpha gene, suffer from immune abnormalities, we generated an adenovirus encoding ROR alpha1 to investigate its potential role in control of the inflammatory response. We demonstrated that ROR alpha is expressed in human primary smooth-muscle cells and that ectopic expression of ROR alpha1 inhibits TNFalpha-induced IL-6, IL-8 and COX-2 expression in these cells. ROR alpha1 negatively interferes with the NF-kappaB signalling pathway by reducing p65 translocation as demonstrated by western blotting, immunostaining and electrophoretic mobility shift assays. This action of ROR alpha1 on NF-kappaB is associated with the induction of IkappaB alpha, the major inhibitory protein of the NF-kappaB signalling pathway, whose expression was found to be transcriptionally upregulated by ROR alpha1 via a ROR response element in the IkappaB alpha promoter. Taken together, these data identify ROR alpha1 as a potential target in the treatment of chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis.

Abstract —Oncostatin M (OSM), a cytokine first identified from activated monocytes and T lymphocytes, is one of the most potent autocrine growth factor for AIDS and Kaposi’s sarcoma. Little is known about the effects of OSM on normal vascular cells. We thus exposed human aortic smooth muscle cells (hASMCs) to OSM, examined cell proliferation and morphology, and determined interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) expression. OSM had a weak antiproliferative effect. After a 4-day incubation with 100 ng/mL OSM, cell count decreased to 69±3% of control. However, OSM induced striking changes in hASMC morphology, characterized by a polyclonal shape, in contrast to the spindle morphological feature of control hASMCs. OSM stimulated the release of IL-6 by hASMCs in a dose-dependent way; after a 48-hour exposure, values were 8.5±0.7, 29.7±3.5, 50.9±4.4, and 73.8±7.6×10 3 U/mL (n=6) at OSM concentrations of 0, 1, 10, and 100 ng/mL, respectively. OSM induced marked expression of COX-2 protein and mRNA. Leukemia inhibitory factor had no effect on hASMCs, indicating that OSM effects on hASMCs were mediated by the OSM type II receptor and not by the leukemia inhibitory factor receptor. OSM used the JAK/STAT signaling pathway, as demonstrated by rapid phosphorylation of JAK1 and specific activation of STAT1. Interestingly, OSM acted in synergy with IL-1β on IL-6 production and COX-2 expression. In conclusion, OSM is a novel regulator of human smooth muscle cell functions, acting in concert with IL-1β, and OSM may play a role in major vascular diseases such as atherosclerosis.

The recruitment of immune cells into the lungs is a key step in protection against murine schistosomiasis. In this phenomenon, pulmonary (micro)vascular endothelial cells (EC) probably play a central role, by expressing specific adhesion molecules on their surface. Recently, we have shown that Schistosoma mansoni schistosomula, the parasitic stage which resides in the lungs, could activate microvascular EC to acquire an anti-inflammatory phenotype. In the present study, we tested the hypothesis that schistosomula could also regulate the expression of adhesion molecules in vitro by human lung microvascular EC (HMVEC-l) in the present of the pro-inflammatory cytokine TNF-alpha. We found that lipophilic substance(s) present in the excretory/secretory products from schistosomula selectively reduce the TNF-alpha-induced synthesis of E-selectin and VCAM-1 mRNA and proteins without affecting ICAM-1. This inhibitory effect appears to be mediated by a cyclic AMP/protein kinase A (cAMP/PKA) pathway that probably interferes with the NF-kappaB pathway induced by TNF-alpha at the level of the E-selectin promoter, whereas a cAMP-independent pathway appears to operate in VCAM-1 down-modulation. Finally, schistosomula also significantly reduce the VLA-4/VCAM-1-dependent adherence of leukocytes to TNF-alpha-stimulated HMVEC-l. We speculate that this mechanism could represent a new stratagem that parasites may use to escape the immune system by controlling leukocyte recruitment to the lungs.

Interleukin-6 (IL-6) is a pleiotropic cytokine, whose plasma levels are elevated in inflammatory diseases such as atherosclerosis. We have previously reported that peroxisome proliferator-activated receptor alpha (PPARalpha) ligands (fibrates) lower elevated plasma concentrations of IL-6 in patients with atherosclerosis and inhibit IL-1-stimulated IL-6 secretion by human aortic smooth muscle cells (SMC). Here, we show that aortic explants isolated from PPARalpha-null mice display an exacerbated response to inflammatory stimuli, such as lipopolysaccharide (LPS), as demonstrated by increased IL-6 secretion. Furthermore, fibrate treatment represses IL-6 mRNA levels in LPS-stimulated aortas of PPARalpha wild-type, but not of PPARalpha-null mice, demonstrating a role for PPARalpha in this fibrate action. In human aortic SMC, fibrates inhibit IL-1-induced IL-6 gene expression. Furthermore, activation of PPARalpha represses both c-Jun- and p65-induced transcription of the human IL-6 promoter. Transcriptional interference between PPARalpha and both c-Jun and p65 occurs reciprocally, since c-Jun and p65 also inhibit PPARalpha-mediated activation of a PPAR response element-driven promoter. This transcriptional interference occurs independent of the promoter context as demonstrated by cotransfection experiments using PPARalpha, p65, and c-Jun Gal4 chimeras. Overexpression of the transcriptional coactivator cAMP-responsive element-binding protein-binding protein (CBP) does not relieve PPARalpha-mediated transcriptional repression of p65 and c-Jun. Finally, glutathione S-transferase pull-down experiments demonstrate that PPARalpha physically interacts with c-Jun, p65, and CBP. Altogether these data indicate that fibrates inhibit the vascular inflammatory response via PPARalpha by interfering with the NF-kappaB and AP-1 transactivation capacity involving direct protein-protein interaction with p65 and c-Jun.

The polyunsaturated fatty acids (PUFAs) of the omega 3 series are known to modulate adrenergic functions in ventricular myocytes. This study evaluated the influence of hypoxia duration and PUFA composition on the ability of cultured rat cardiomyocytes in producing alpha- and beta-adrenergic messengers (IPs and cAMP). After hypoxia (1.5, 2.5 or 3.5 h) followed by reoxygenation (1h). IP and cAMP production was induced by phenylephrine or isoproterenol stimulation, respectively. Hypoxia did not affect the basal level of messenger production in unstimulated cells, but decreased the cAMP production elicited by isoproterenol stimulation (up to 50%). The decrease in IP production after phenylephrine stimulation was observed only after long-term hypoxia duration close to irreversible cellular damages. The use of modified culture media supplemented with either arachidonic acid (AA) or docosahexaenoic acid (DHA) induced cardiomyocytes displaying either an arachidonic acid membrane profile (35% AA and 2% DHA in the phospholipids) or a docosahexaenoic acid membrane profile (15% AA and 20% DHA). These modifications did not alter the basal level of either messenger production in unstimulated cells nor the IP released after alpha-adrenergic stimulation. Conversely, the decrease in cAMP production was significantly more pronounced in docosahexaenoic acid-enriched cells than in arachidonic acid-enriched cells. This study suggests that hypoxia alters the beta-adrenergic messenger production, and that the alpha-system may balance the depression of the beta-system. The depression of the beta-adrenergic function induced by the incorporation of docosahexaenoic acid in membrane phospholipids may contribute to the beneficial effect of this fatty acid in the reperfused heart.

Peroxisome proliferator-activated receptors (PPARs) are key players in lipid and glucose metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as dyslipidaemia and diabetes. Whereas PPARgamma promotes lipid storage by regulating adipocyte differentiation, PPARalpha stimulates the beta-oxidative degradation of fatty acids. PPARalpha-deficient mice show a prolonged response to inflammatory stimuli, suggesting that PPARalpha is also a modulator of inflammation. Hypolipidaemic fibrate drugs are PPARalpha ligands that inhibit the progressive formation of atherosclerotic lesions, which involves chronic inflammatory processes, even in the absence of their atherogenic lipoprotein-lowering effect. Here we show that PPARalpha is expressed in human aortic smooth-muscle cells, which participate in plaque formation and post-angioplasty re-stenosis. In these smooth-muscle cells, we find that PPARalpha ligands, and not PPARgamma ligands, inhibit interleukin-1-induced production of interleukin-6 and prostaglandin and expression of cyclooxygenase-2. This inhibition of cyclooxygenase-2 induction occurs transcriptionally as a result of PPARalpha repression of NF-kappaB signalling. In hyperlipidaemic patients, fenofibrate treatment decreases the plasma concentrations of interleukin-6, fibrinogen and C-reactive protein. We conclude that activators of PPARalpha inhibit the inflammatory response of aortic smooth-muscle cells and decrease the concentration of plasma acute-phase proteins, indicating that PPARalpha in the vascular wall may influence the process of atherosclerosis and re-stenosis.

International audience; BACKGROUND: Atherosclerosis and non-alcoholic fatty liver disease (NAFLD) are complex pathologies characterized by lipid accumulation, chronic inflammation and extensive tissue remodelling. Microparticles (MPs), small membrane vesicles produced by activated and apoptotic cells, might not only be biomarkers, but also functional actors in these pathologies. The apoE2-KI mouse is a model of atherosclerosis and NAFLD. Activation of the nuclear receptor PPARα decreases atherosclerosis and components of non-alcoholic steatohepatitis (NASH) in the apoE2-KI mouse. OBJECTIVES: (1) To determine whether MPs are present in atherosclerotic lesions, liver and plasma during atherosclerosis and NASH progression in apoE2-KI mice, and (2) to study whether PPARα activation modulates MP concentrations. METHODS: ApoE2-KI mice were fed a Western diet to induce atherosclerosis and NASH. MPs were isolated from atherosclerotic lesions, liver and blood and quantified by flow cytometry. RESULTS: An increase of MPs was observed in the atherosclerotic lesions and in the liver of apoE2-KI mice upon Western diet feeding. PPARα activation with fenofibrate decreased MP levels in the atherosclerotic lesions in a PPARα-dependent manner, but did not influence MP concentrations in the liver. CONCLUSION: Here we report that MPs are present in atherosclerotic lesions and in the liver of apoE2-KI mice. Their concentration increased during atherosclerosis and NASH development. PPARα activation differentially modulates MP levels in a tissue-specific manner.

Western diet is characterized by a hypercaloric and hyperlipidic intake, enriched in saturated fats, that is associated with the increased occurrence of metabolic diseases. To cope with this overload of dietary lipids, the intestine, which delivers dietary lipids to the body, has to adapt its capacity in lipid absorption and lipoprotein synthesis. We have studied the early effects of a high-fat diet (HFD) on intestinal lipid metabolism in mice. After 7 days of HFD, mice displayed normal fasting triglyceridemia but postprandial hypertriglyceridemia. HFD induced a decreased number of secreted chylomicrons with increased associated triglycerides. Secretion of larger chylomicrons was correlated with increased intestinal microsomal triglyceride transfer protein (MTP) content and activity. Seven days of HFD induced a repression of genes involved in fatty acid synthesis (FAS, ACC) and an increased expression of genes involved in lipoprotein assembly (apoB, MTP, and apoA-IV), suggesting a coordinated control of intestinal lipid metabolism to manage a high-fat loading. Of note, the mature form of the transcription factor SREBP-1c was increased and translocated to the nucleus, suggesting that it could be involved in the coordinated control of gene transcription. Activation of SREBP-1c was partly independent of LXR. Moreover, HFD induced hepatic insulin resistance whereas intestine remained insulin sensitive. Altogether, these results demonstrate that a short-term HFD is sufficient to impact intestinal lipid metabolism, which might participate in the development of dyslipidemia and metabolic diseases.

Liver X receptor-alpha (LXRalpha) and LXRbeta are ligand-activated transcription factors belonging to the nuclear receptor superfamily. They have been identified as key players in cholesterol homeostasis and lipid and glucose metabolism as well as immune and inflammatory responses. In the small intestine, LXRs have been shown not only to regulate cholesterol absorption and excretion but also to promote high-density lipoprotein biogenesis via the ATP-binding cassette A1 signaling pathway. Here, using gene expression assays, we identified PPARalpha as an intestine-specific LXR target gene. Chronic administration of LXR synthetic agonists led to a significant increase of PPARalpha mRNA levels in the small intestine but not in the liver. In addition, this specific PPARalpha gene up-regulation occurred in the duodenum, jejunum, and ileum in a dose-dependent manner and translated at the protein level as demonstrated by Western blot analysis. Furthermore, PPARalpha gene induction was completely abolished in LXR-deficient mice. Finally, the physiological relevance of LXR-mediated PPARalpha up-regulation in the small intestine was assessed in PPARalpha-deficient mice. Administration of a synthetic LXR agonist to wild-type mice led to the induction of several PPARalpha target genes including PDK4 and CPT1. Those effects were completely abolished in PPARalpha-deficient mice, demonstrating the biological relevance of this LXR-PPARalpha transcriptional cascade. Taken together, these results demonstrate that PPARalpha is an intestine-specific LXR target gene and suggest the existence of a transcriptional cross talk between those members of the nuclear receptor superfamily.

The orphan nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) has been reported to play a crucial role in early development, in the control of the hepatic inflammatory response, in intestinal cell crypt renewal as well as in bile acid biosynthesis and reverse cholesterol transport (RCT). Here, we report the identification of apolipoprotein M (APOM) as a novel target gene for LRH-1. Using gene-silencing experiments, adenovirus-mediated overexpression, transient transfection, and chromatin immunoprecipitation (ChIP) assays, it is shown that LRH-1 directly regulates human and mouse APOM transcription by binding to an LRH-1 response element located in the proximal APOM promoter region. In addition, we demonstrate that bile acids suppress APOM expression in a SHP-dependent manner in vitro and in vivo by inhibiting LRH-1 transcriptional activity on the APOM promoter as demonstrated by in vivo ChIP assay. Taken together, our results demonstrate that LRH-1 is a novel regulator of APOM transcription and further extend the role of this orphan nuclear receptor in lipoprotein metabolism and cholesterol homeostasis.

The orphan nuclear receptor liver receptor homolog-1 (LRH-1) has been reported to play a role in bile acid biosynthesis and reverse cholesterol transport. In this study, we examined the role of LRH-1 in the regulation of the apolipoprotein AI (APOAI) gene. Using RNA interference and adenovirus-mediated overexpression, we show that LRH-1 directly regulates APOAI gene transcription. Transient transfection experiments and EMSAs revealed that LRH-1 directly regulates APOAI transcription by binding to an LRH-1 response element located in the proximal APOAI promoter region. Chromatin immunoprecipitation experiments revealed that LRH-1 binds to the human APO AI promoter in vivo. Finally, we show that the transcriptional repressor SHP (small heterodimer partner) suppressed APOAI gene expression by inhibiting LRH-1 transcriptional activity. Taken together, our results demonstrate that LRH-1 is a novel regulator of APOAI transcription and underscore the role of this receptor in cholesterol homeostasis.

The onset of inflammatory gene expression is driven by the transcription factor NF-kappaB, whose transcriptional activity is regulated at multiple levels. First, NF-kappaB activity is regulated by cytoplasmic degradation of the IkappaB inhibitor and nuclear translocation. Second, the nuclear p65 transactivation potential can be further influenced by posttranslational modifications, such as phosphorylation and/or acetylation. The p65 phosphorylation is a process highly regulated by both cell- and stimulus-dependent activating kinases. Ser276 phosphorylation seems to be highly important considering its crucial role in the interaction with and the engagement of the cofactor CBP/p300. We have identified MSK1 as an acting kinase in the TNF-signalling pathway, where it is responsible for p65 phosphorylation at Ser276, as well as for H3 phosphorylation of Ser10 in IL-6 promoter-associated chromatin (Fig. 1) (Saccani et al., 2002; Vermeulen et al., 2002, 2003). To our knowledge, this was the first report that identifies one particular kinase involved in transcription factor phosphorylation and histone modification at the level of a single promoter in order to establish gene activation. The question of which element takes the initial step to recruit and to assemble the activated transcription complex still remains unanswered (Vanden Berghe et al., 2002). PPAR alpha negatively interferes with inflammatory gene expression by up-regulation of the cytoplasmic inhibitor molecule IkappaB alpha, thus establishing an autoregulatory loop (Fig. 1). This induction takes place in the absence of a PPRE, but requires the presence of NF-kappaB and Sp1 elements in the IkappaB alpha promoter sequence as well as DRIP250 cofactors. The detailed mechanism how PPAR can activate genes in a non-DNA-binding way needs further investigation; moreover, it is at present not clear whether this upregulation, unlike the inhibitory effect of glucocorticoids, is a cell type- or a PPAR-specific phenomenon.

Inflammation is a rather general term for a variety of diseases, which appear as different affections depending on the tissue involved. They share a number of common characteristics, among which the expression and secretion of inflammatory cytokines and chemokines.

PPARs are ligand-activated transcription factors that regulate energy homeostasis. In addition, PPARs furthermore control the inflammatory response by antagonizing the nuclear factor-kappaB (NF-kappaB) signaling pathway. We recently demonstrated that PPARalpha activators increase IkappaBalpha mRNA and protein levels in human aortic smooth muscle cells. Here, we studied the molecular mechanisms by which PPARalpha controls IkappaBalpha expression. Using transient transfection assays, it is demonstrated that PPARalpha potentiates p65-stimulated IkappaBalpha transcription in a ligand-dependent manner. Site-directed mutagenesis experiments revealed that PPARalpha activation of IkappaBalpha transcription requires the NF-kappaB and Sp1 sites within IkappaBalpha promoter. Chromatin immunoprecipitation assays demonstrate that PPARalpha activation enhances the occupancy of the NF-kappaB response element in IkappaBalpha promoter in vivo. Overexpression of the oncoprotein E1A failed to inhibit PPARalpha-mediated IkappaBalpha promoter induction, suggesting that cAMP response element binding protein-binding protein/p300 is not involved in this mechanism. By contrast, a dominant-negative form of VDR-interacting protein 205 (DRIP205) comprising its two LXXLL motifs completely abolished PPARalpha ligand-mediated activation. Furthermore, cotransfection of increasing amounts of DRIP205 relieved this inhibition, suggesting that PPARalpha requires DRIP205 to regulate IkappaBalpha promoter activity. By contrast, DRIP205 is not involved in PPARalpha-mediated NF-kappaB transcriptional repression. Taken together, these data provide a molecular basis for PPARalpha-mediated induction of IkappaBalpha and demonstrate, for the first time, that PPARalpha may positively regulate gene transcription in the absence of functional PPAR response elements.

Although PGC-1 (peroxisome proliferator-activated receptor-gamma coactivator-1) has been previously shown to enhance thyroid hormone receptor (TR)/retinoid X receptor-mediated ucp-1 gene expression in a ligand-induced manner in rat fibroblast cells, the precise mechanism of PGC-1 modulation of TR function has yet to be determined. In this study, we show that PGC-1 can potentiate TR-mediated transactivation of reporter genes driven by natural thyroid hormone response elements both in a ligand-dependent and ligand-independent manner and that the extent of coactivation is a function of the thyroid hormone response element examined. Our data also show that PGC-1 stimulation of TR activity in terms of Gal4 DNA-binding domain fusion is strictly ligand-dependent. In addition, an E457A AF-2 mutation had no effect on the ligand-induced PGC-1 enhancement of TR activity, indicating that the conserved charged residue in AF-2 is not essential for this PGC-1 function. Furthermore, GST pull-down and mammalian two-hybrid assays demonstrated that the PGC-1 LXXLL motif is required for ligand-induced PGC-1/TR interaction. This agonist-dependent PGC-1/TR interaction also requires both helix 1 and the AF-2 region of the TR ligand-binding domain. Taken together, these results support the notion that PGC-1 is a bona fide TR coactivator and that PGC-1 modulates TR activity via a mechanism different from that utilized with peroxisome proliferator activator receptor-gamma.

The nature of the interactions between the intravascular parasite Schistosoma mansoni and the host pulmonary vasculature is critical in determining the outcome of infection. In this report, we show that lung schistosomula selectively induce the synthesis of IL-6 mRNA and protein in cultured human and mouse lung microvascular endothelial cells (EC) and that parasite excretory/secretory lipophilic compounds, particularly prostaglandin E(2), are responsible for this effect. In vivo, a striking increase of IL-6 expression is observed in the pulmonary microvasculature of S. mansoni-infected C57BL/6 mice suggesting that, in vivo, parasites also induce the synthesis of IL-6 in lung EC. In infected mice, IL-6 deficiency results in an accelerated mobilization of eosinophils into the lung tissue and in a dramatic increased number of recruited leukocytes, particularly eosinophils, in the airway. This effect is associated with an enhanced production of eotaxin (CCL11) and IL-5 in the lungs of IL-6 knockout (KO) animals. Finally, compared to wild-type mice, we detect a dramatic increased level of parasite mortality in the lungs of IL-6 KO mice. Taken together, we suggest that parasite larvae activate EC to produce IL-6 to escape the inflammatory reaction that develops in the lungs of infected hosts. Finally, we show that the parasite-induced IL-6 synthesis is mediated by a protein kinase A-dependent pathway that principally targets the cAMP-response element and the nuclear factor-kappaB sites from the -256/+20 region of the IL-6 promoter.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. PPARalpha is highly expressed in liver, skeletal muscle, kidney, heart and the vascular wall. PPARgamma is predominantly detected in adipose tissue, intestine and macrophages. PPARs are activated by fatty-acid derivatives and pharmacological agents such as fibrates and glitazones which are specific for PPARalpha and PPARgamma respectively. PPARs regulate lipid and lipoprotein metabolism, glucose homeostasis, cell proliferation and differentiation, and apoptosis. PPARalpha controls intra- and extracellular lipid metabolisms whereas PPARgamma triggers adipocyte differentiation and promotes lipid storage. In addition, PPARs also modulate the inflammatory response. PPAR activators have been shown to exert anti-inflammatory activities in various cell types by inhibiting the expression of proinflammatory genes such as cytokines, metalloproteases and acute-phase proteins. PPARs negatively regulate the transcription of inflammatory response genes by antagonizing the AP-1, nuclear factor-kappaB (NF-kappaB), signal transducer and activator of transcription and nuclear factor of activated T-cells signalling pathways and by stimulating the catabolism of proinflammatory eicosanoids. These recent findings indicate a modulatory role for PPARs in inflammation with potential therapeutical applications in chronic inflammatory diseases.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily. They are divided into three subtypes (alpha, beta or delta, and gamma) and are involved in lipid and glucose homeostasis and in the control of inflammation. In this study, we analyzed the expression of PPARs in murine dendritic cells (DCs), the most potent antigen presenting cells. We find that immature as well as mature spleen-derived DCs express PPARgamma, but not PPARalpha, mRNA and protein. We also show that the PPARgamma activator rosiglitazone does not interfere with the maturation of DCs in vitro nor modifies their ability to activate naive T lymphocytes in vivo. Finally, we present evidence that PPARgamma activators down-modulate the CD40-induced secretion of interleukin-12, a potent Th1-driving factor. These data suggest a possible role for PPARgamma in the regulation of immune responses.

Chronic inflammation is a hallmark of degenerative diseases such as atherosclerosis. Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily, which are expressed in the cells of the atherosclerosic lesion. PPARalpha ligands have been reported to exert anti-inflammatory activities in different cell types by antagonizing the transcriptional activity of NF-kappaB. In the present study, the influence of PPARalpha activators on the NF-kappaB signaling pathway was investigated. Our results show that fibrates, synthetic PPARalpha activators, induced the expression of the inhibitory protein IkappaBalpha in human aortic smooth muscle cells as well as in primary human hepatocytes, whereas neither IkappaB-kinase activity nor the degradation rate of IkappaBalpha were affected. Using PPARalpha-null mice, we demonstrated that fibrates induced IkappaBalpha in liver in vivo and that this action required PPARalpha. Furthermore, fibrate treatment induced IkappaBalpha protein expression in the cytoplasm and also enhanced IL-1beta-induced accumulation of IkappaBalpha protein in the nucleus. These actions of fibrates on IkappaBalpha expression were accompanied by a decrease in NF-kappaB DNA binding activity as demonstrated by electrophoretic mobility shift assays. Taken together, these data provide an additional molecular mechanism for the anti-inflammatory activity of PPARalpha agonists and reinforce their potential use in the treatment of inflammatory diseases.

Abstract —Endothelin-1 (ET-1), a 21-amino acid vasoactive peptide mainly produced by vascular endothelial cells, is involved in the regulation of vascular tone and smooth muscle cell proliferation. Peroxisome proliferator-activated receptors (PPARs), key players in lipid and glucose metabolism, have been implicated in metabolic disorders that are predisposing to atherosclerosis. Because of the potential role of ET-1 in vascular disorders such as hypertension and atherosclerosis, we investigated the regulation of ET-1 expression by PPAR activators. Western blot and reverse transcription–polymerase chain reaction analyses demonstrated that both PPARα and PPARγ are expressed in human coronary artery endothelial cells as well as in endothelial cell lines such as HMEC-1 and ECV304. In bovine aortic endothelial cells and HMEC-1 cells, both PPARα and PPARγ ligands inhibited thrombin-induced ET-1 secretion, whereas basal ET-1 secretion was only slightly suppressed. Reverse transcription–polymerase chain reaction experiments showed that this inhibition of ET-1 production occurs at the gene expression level. Using transient transfection assays, we demonstrated that PPARs downregulate thrombin-activated transcription of the human ET-1 promoter. Transactivation studies with c-Jun and c-Fos expression plasmids indicated that PPARs negatively interfere with the activator protein-1 signaling pathway, which mediates thrombin activation of ET-1 gene transcription. Furthermore, electrophoretic mobility shift assays demonstrated that PPAR activators reduce the thrombin-stimulated binding activity of bovine aortic endothelial cell nuclear extracts as well as c-Jun binding to an activator protein-1 consensus site. Taken together, these data indicate that (1) both PPARα and PPARγ are expressed in human vascular endothelial cells and (2) PPAR activators inhibit thrombin-induced ET-1 biosynthesis, indicating a novel role for PPARs in vascular endothelial function.