The PPARgamma agonist pioglitazone modifies the vascular sodium-angiotensin II relationship in insulin-resistant rats.

Glitazones are efficient insulin sensitizers that blunt the effects of angiotensin II (ANG II) in the rat. Sodium chloride is another important modulator of the systemic and renal effects of ANG II. Whether glitazones interfere with the interaction between sodium and the response to ANG II is not known. Therefore, we investigated the effects of pioglitazone on the relationship between sodium and the systemic and renal effects of ANG II in rats. Pioglitazone, or vehicle, was administered for 4 wk to 8-wk-old obese Zucker rats. Animals were fed a normal-sodium (NS) or a high-sodium (HS) diet. Intravenous glucose tolerance tests, systemic and renal hemodynamic responses to ANG II, and the renal ANG II binding and expression of ANG II type 1 (AT(1)) receptors were measured. The results of our study were that food intake and body weight increased, whereas blood pressure, heart rate, filtration fraction, and insulin levels decreased significantly with pioglitazone in obese rats on both diets. Pioglitazone blunted the systemic response to ANG II and abolished the increased responsiveness to ANG II induced by a HS diet. Pioglitazone modified the renal hemodynamic response to changes in salt intake while maintaining a lower filtration fraction with ANG II perfusion. These effects were associated with a decrease in the number and expression of the AT(1) receptor in the kidney. In conclusion, these data demonstrate that the peroxisome proliferator-activated receptor-gamma agonist pioglitazone modifies the physiological relationship between sodium chloride and the response to ANG II in insulin-resistant rats.

Functional role of RXRs and PPARgamma in mature adipocytes.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is abundantly expressed in adipocytes, and plays an important role in adipocyte differentiation and fat accretion. It is a heterodimeric partner of the retinoid X receptors alpha, beta and gamma, which are also expressed in the adipose tissue. As lethality of PPARgamma(-/-) and RXRalpha(-/-) mouse fetuses precluded the analysis of PPARgamma and RXRalpha functions in mature adipocytes, we generated RXRalpha(ad-/-) and PPARgamma(ad-/-) mice, in which RXRalpha and PPARgamma are selectively ablated in adult adipocytes, respectively. Even though the adiposity of RXRalpha(ad-/-) mice is similar to that of control mice when fed a regular diet, they are resistant to chemically and dietary-induced obesity. However, mature adipocytes lacking either both RXRalpha and RXRgamma or PPARgamma die, and are replaced by newly formed adipocytes. Thus, in adipocytes, RXRalpha is essential for lipogenesis, but RXRgamma can functionally replace RXRalpha for the adipocyte vital functions exerted by PPARgamma/RXR heterodimers.

Scaffold attachment factor B1 directly interacts with nuclear receptors in living cells and represses transcriptional activity.

Transcriptional activity relies on coregulators that modify the chromatin structure and serve as bridging factors between transcription factors and the basal transcription machinery. Using the DE domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) as bait in a yeast two-hybrid screen of a human adipose tissue library, we isolated the scaffold attachment factor B1 (SAFB1/HET/HAP), which was previously shown to be a corepressor of estrogen receptor alpha. We show here that SAFB1 has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis. SAFB1 interacts in pull-down assays not only with PPARgamma but also with all nuclear receptors tested so far, albeit with different affinities. The association of SAFB1 and PPARgamma in vivo is further demonstrated by fluorescence resonance energy transfer (FRET) experiments in living cells. We finally show that SAFB1 is a rather general corepressor for nuclear receptors. Its change in expression during the early phases of adipocyte and enterocyte differentiation suggests that SAFB1 potentially influences cell proliferation and differentiation decisions.

Lack of hypotriglyceridemic effect of gemfibrozil as a consequence of age-related changes in rat liver PPARalpha.

We have investigated if changes in hepatic lipid metabolism produced by old age are related to changes in liver peroxisome proliferator-activated receptor alpha (PPARalpha). Our results indicate that 18-month-old rats showed a marked decrease in the expression and activity of liver PPARalpha, as shown by significant reductions in PPARalpha mRNA, protein and binding activity, resulting in a reduction in the relative mRNA levels of PPARalpha target genes, such as liver-carnitine-palmitoyl transferase-I (CPT-I) and mitochondrial medium-chain acyl-CoA dehydrogenase (MCAD). Further, in accordance with a liver PPARalpha deficiency in old rats, treatment of old animals with a therapeutic dose of gemfibrozil (GFB) (3mg/kg per day, 21 days) was ineffective in reducing plasma triglyceride concentrations (TG), despite attaining a 50% reduction in TG when GFB was administered to young animals at the same dose and length of treatment. We hypothesize that the decrease in hepatic PPARalpha can be related to a state of leptin resistance present in old animals.

Differential inhibition of TRAIL-mediated DR5-DISC formation by decoy receptors 1 and 2.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that induces cancer cell death by apoptosis with some selectivity. TRAIL-induced apoptosis is mediated by the transmembrane receptors death receptor 4 (DR4) (also known as TRAIL-R1) and DR5 (TRAIL-R2). TRAIL can also bind decoy receptor 1 (DcR1) (TRAIL-R3) and DcR2 (TRAIL-R4) that fail to induce apoptosis since they lack and have a truncated cytoplasmic death domain, respectively. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated, TRAIL-induced apoptosis and we demonstrate here that this occurs through distinct mechanisms. While DcR1 prevents the assembly of the death-inducing signaling complex (DISC) by titrating TRAIL within lipid rafts, DcR2 is corecruited with DR5 within the DISC, where it inhibits initiator caspase activation. In addition, DcR2 prevents DR4 recruitment within the DR5 DISC. The specificity of DcR1- and DcR2-mediated TRAIL inhibition reveals an additional level of complexity for the regulation of TRAIL signaling.

Selective effects of PPARgamma agonists and antagonists on human pre-adipocyte differentiation.

Aim: The insulin sensitizer rosiglitazone (RTZ) acts by activating peroxisome proliferator and activated receptor gamma (PPAR gamma), an effect accompanied in vivo in humans by an increase in fat storage. We hypothesized that this effect concerns PPARgamma(1) and PPARgamma(2) differently and is dependant on the origin of the adipose cells (subcutaneous or visceral). To this aim, the effect of RTZ, the PPARgamma antagonist GW9662 and lentiviral vectors expressing interfering RNA were evaluated on human pre-adipocyte models. Methods: Two models were investigated: the human pre-adipose cell line Chub-S7 and primary pre-adipocytes derived from subcutaneous and visceral biopsies of adipose tissue (AT) obtained from obese patients. Cells were used to perform oil-red O staining, gene expression measurements and lentiviral infections. Results: In both models, RTZ was found to stimulate the differentiation of pre-adipocytes into mature cells. This was accompanied by significant increases in both the PPARgamma(1) and PPARgamma(2) gene expression, with a relatively stronger stimulation of PPARgamma(2). In contrast, RTZ failed to stimulate differentiation processes when cells were incubated in the presence of GW9662. This effect was similar to the effect observed using interfering RNA against PPARgamma(2). It was accompanied by an abrogation of the RTZ-induced PPARgamma(2) gene expression, whereas the level of PPARgamma(1) was not affected. Conclusions: Both the GW9662 treatment and interfering RNA against PPARgamma(2) are able to abrogate RTZ-induced differentiation without a significant change of PPARgamma(1) gene expression. These results are consistent with previous results obtained in animal models and suggest that in humans PPARgamma(2) may also be the key isoform involved in fat storage.

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.

Genetic polymorphisms of the main transcription factors for adiponectin gene promoter in regulation of adiponectin levels: association analysis in three European cohorts.

Adiponectin serum concentrations are an important biomarker in cardiovascular epidemiology with heritability etimates of 30-70%. However, known genetic variants in the adiponectin gene locus (ADIPOQ) account for only 2%-8% of its variance. As transcription factors are thought to play an under-acknowledged role in carrying functional variants, we hypothesized that genetic polymorphisms in genes coding for the main transcription factors for the ADIPOQ promoter influence adiponectin levels. Single nucleotide polymorphisms (SNPs) at these genes were selected based on the haplotype block structure and previously published evidence to be associated with adiponectin levels. We performed association analyses of the 24 selected SNPs at forkhead box O1 (FOXO1), sterol-regulatory-element-binding transcription factor 1 (SREBF1), sirtuin 1 (SIRT1), peroxisome-proliferator-activated receptor gamma (PPARG) and transcription factor activating enhancer binding protein 2 beta (TFAP2B) gene loci with adiponectin levels in three different European cohorts: SAPHIR (n = 1742), KORA F3 (n = 1636) and CoLaus (n = 5355). In each study population, the association of SNPs with adiponectin levels on log-scale was tested using linear regression adjusted for age, sex and body mass index, applying both an additive and a recessive genetic model. A pooled effect size was obtained by meta-analysis assuming a fixed effects model. We applied a significance threshold of 0.0033 accounting for the multiple testing situation. A significant association was only found for variants within SREBF1 applying an additive genetic model (smallest p-value for rs1889018 on log(adiponectin) = 0.002, β on original scale = -0.217 µg/ml), explaining ∼0.4% of variation of adiponectin levels. Recessive genetic models or haplotype analyses of the FOXO1, SREBF1, SIRT1, TFAPB2B genes or sex-stratified analyses did not reveal additional information on the regulation of adiponectin levels. The role of genetic variations at the SREBF1 gene in regulating adiponectin needs further investigation by functional studies.

The inhibition of fat cell proliferation by n-3 fatty acids in dietary obese mice.

ABSTRACT: BACKGROUND: Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) of marine origin exert multiple beneficial effects on health. Our previous study in mice showed that reduction of adiposity by LC n-3 PUFA was associated with both, a shift in adipose tissue metabolism and a decrease in tissue cellularity. The aim of this study was to further characterize the effects of LC n-3 PUFA on fat cell proliferation and differentiation in obese mice. METHODS: A model of inducible and reversible lipoatrophy (aP2-Cre-ERT2 PPARgammaL2/L2 mice) was used, in which the death of mature adipocytes could be achieved by a selective ablation of peroxisome proliferator-activated receptor gamma in response to i.p. injection of tamoxifen. Before the injection, obesity was induced in male mice by 8-week-feeding a corn oil-based high-fat diet (cHF) and, subsequently, mice were randomly assigned (day 0) to one of the following groups: (i) mice injected by corn-oil-vehicle only, i.e."control" mice, and fed cHF; (ii) mice injected by tamoxifen in corn oil, i.e. "mutant" mice, fed cHF; (iii) control mice fed cHF diet with 15% of dietary lipids replaced by LC n-3 PUFA concentrate (cHF+F); and (iv) mutant mice fed cHF+F. Blood and tissue samples were collected at days 14 and 42. RESULTS: Mutant mice achieved a maximum weight loss within 10 days post-injection, followed by a compensatory body weight gain, which was significantly faster in the cHF as compared with the cHF+F mutant mice. Also in control mice, body weight gain was depressed in response to dietary LC n-3 PUFA. At day 42, body weights in all groups stabilized, with no significant differences in adipocyte size between the groups, although body weight and adiposity was lower in the cHF+F as compared with the cHF mice, with a stronger effect in the mutant than in control mice. Gene expression analysis documented depression of adipocyte maturation during the reconstitution of adipose tissue in the cHF+F mutant mice. CONCLUSION: Dietary LC n-3 PUFA could reduce both hypertrophy and hyperplasia of fat cells in vivo. Results are in agreement with the involvement of fat cell turnover in control of adiposity.

Viral genotype-specific role of PNPLA3, PPARG, MTTP, and IL28B in hepatitis C virus-associated steatosis.

BACKGROUND & AIMS: Steatosis is a prominent feature of hepatitis C, especially in patients infected with genotype 3. The analysis of genetic polymorphisms influencing steatosis in chronic hepatitis C has been limited by the studies' small sample size, and important single nucleotide polymorphisms (SNPs), such as those in the patatin-like phospholipase family 3 protein (PNPLA3), were never evaluated. METHODS: We analyzed the role of SNPs, from 19 systematically selected candidate genes, on steatosis in 626 Caucasian hepatitis C virus (HCV) infected patients. SNPs were extracted from a genome-wide association-generated dataset. Associations of alleles with the presence and/or different severity of steatosis were evaluated by univariate and multivariate logistic regression, accounting for all relevant covariates. RESULTS: The risk of steatosis was increased by carriage of I148M in PNPLA3, but only in patients with HCV genotypes non-3 (odds ratio [OR]=1.9, 95% confidence interval [CI]=1.6-2.3, p<0.001) and similar, albeit weaker associations were found for SNPs in peroxisome proliferator-activated receptor-γ (PPARG) and interleukin-28B (IL28B). Carriage of a SNP in the microsomal triglyceride transfer protein (MTTP) increased the risk of steatosis, but only in patients with HCV genotype 3 (rs1800803, OR=3.4, 95% CI=2.4-4.9, p=0.001). CONCLUSIONS: The rs738409 SNP in PNPLA3 is associated with an increased risk of steatosis in patients infected with HCV genotypes non-3. Host genes affect steatosis depending on the infecting HCV genotype, suggesting their interaction with viral factors.

Essential role of Smad3 in the inhibition of inflammation-induced PPARbeta/delta expression.

Wound healing proceeds by the concerted action of a variety of signals that have been well identified. However, the mechanisms integrating them and coordinating their effects are poorly known. Herein, we reveal how PPARbeta/delta (PPAR: peroxisome proliferator-activated receptor) follows a balanced pattern of expression controlled by a crosstalk between inflammatory cytokines and TGF-beta1. Whereas conditions that mimic the initial inflammatory events stimulate PPARbeta/delta expression, TGF-beta1/Smad3 suppresses this inflammation-induced PPARbeta/delta transcription, as seen in the late re-epithelialization/remodeling events. This TGF-beta1/Smad3 action involves an inhibitory effect on AP-1 activity and DNA binding that results in an inhibition of the AP-1-driven induction of the PPARbeta/delta promoter. As expected from these observations, wound biopsies from Smad3-null mice showed sustained PPARbeta expression as compared to those of their wild-type littermates. Together, these findings suggest a mechanism for setting the necessary balance between inflammatory signals, which trigger PPARbeta/delta expression, and TGF-beta1/Smad3 that governs the timely decrease of this expression as wound healing proceeds to completion.

Activation/division of lymphocytes results in increased levels of cytoplasmic activation/proliferation-associated protein-1: prototype of a new family of proteins.

We purified from activated T lymphocytes a novel, highly conserved, 116-kDa, intracellular protein that occurred at high levels in the large, dividing cells of the thymus, was up-regulated when resting T or B lymphocytes or hemopoietic progenitors were activated, and was down-regulated when a monocytic leukemia, M1, was induced to differentiate. Expression of the protein was highest in the thymus and spleen and lowest in tissues with a low proportion of dividing cells such as kidney or muscle, although expression was high in the brain. The protein was localized to the cytosol and was phosphorylated, which is consistent with a previous report that the Xenopus laevis ortholog was phosphorylated by a mitotically activated kinase (1 ). The cDNA was previously mischaracterized as encoding p137, a 137-kDa GPI-linked membrane protein (2 ). We propose that the authentic protein encoded by this cDNA be called cytoplasmic activation/proliferation-associated protein-1 (caprin-1), and show that it is the prototype of a novel family of proteins characterized by two novel protein domains, termed homology regions-1 and -2 (HR-1, HR-2). Although we have found evidence for caprins only in urochordates and vertebrates, two insect proteins exhibit well-conserved HR-1 domains. The HR-1 and HR-2 domains have no known function, although the HR-1 of caprin-1 appeared necessary for formation of multimeric complexes of caprin-1. Overexpression of a fusion protein of enhanced green fluorescent protein and caprin-1 induced a specific, dose-dependent suppression of the proliferation of NIH-3T3 cells, consistent with the notion that caprin-1 plays a role in cellular activation or proliferation.

PPARβ/δ attenuates palmitate-induced endoplasmic reticulum stress and induces autophagic markers in human cardiac cells.

BACKGROUND: Chronic endoplasmic reticulum (ER) stress contributes to the apoptotic cell death in the myocardium, thereby playing a critical role in the development of cardiomyopathy. ER stress has been reported to be induced after high-fat diet feeding in mice and also after saturated fatty acid treatment in vitro. Therefore, since several studies have shown that peroxisome proliferator-activated receptor (PPAR)β/δ inhibits ER stress, the main goal of this study consisted in investigating whether activation of this nuclear receptor was able to prevent lipid-induced ER stress in cardiac cells. METHODS AND RESULTS: Wild-type and transgenic mice with reduced PPARβ/δ expression were fed a standard diet or a high-fat diet for two months. For in vitro studies, a cardiomyocyte cell line of human origin, AC16, was treated with palmitate and the PPARβ/δ agonist GW501516. Our results demonstrate that palmitate induced ER stress in AC16 cells, a fact which was prevented after PPARβ/δ activation with GW501516. Interestingly, the effect of GW501516 on ER stress occurred in an AMPK-independent manner. The most striking result of this study is that GW501516 treatment also upregulated the protein levels of beclin 1 and LC3II, two well-known markers of autophagy. In accordance with this, feeding on a high-fat diet or suppression of PPARβ/δ in knockout mice induced ER stress in the heart. Moreover, PPARβ/δ knockout mice also displayed a reduction in autophagic markers. CONCLUSION: Our data indicate that PPARβ/δ activation might be useful to prevent the harmful effects of ER stress induced by saturated fatty acids in the heart by inducing autophagy.

Signalling pathways in skin homeostasis

SUMMARY The results presented here contribute to a better understanding of the crucial molecular relationships and signalling cues exchanged by several fundamental cell types (epidermal keratinocytes, dermal fibroblasts, immune and endothelial cells) of the skin. Importantly we provide evidence to directly implicate Wnt/ß-catenin signalling as a putative player in different cell types (keratinocytes and neutrophils) in mediation of the cutaneous inflammatory response (Fart A). Finally we highlight the importance of several molecules, specifically expressed in the hair follicle stem cell niche to the morphogenesis and homeostasis of the hair follicle (Part B). PART A Currently the body of work pertaining to Wnt signalling and immune cells largely focuses on Wnt signalling in the development of these cells. The data presented here suggests a novel mechanism in which Wnt signalling appears to modulate immune cell recruitment to the skin. Keratinocytes are major contributors to early inflammatory responses by the release of chemokines which recruit immune cells. The resultant inflammatory response is a dynamic process of sequentially infiltrating immune cells governed by a network of growth factors, chemokines and cytokines. In wild type mice the response is typified by a rapid and substantial infiltration of neutrophils followed at later time points by macrophages and Tcells. The expression of the canonical Wnt pathway activating ligand, Wnt3a, is able to induce a strong neutrophil infiltration in the dermis. This response originates in keratinocytes, as it is abrogated upon keratinocyte-specific ablation of ß-catenin. Notably, this suggests that the crucial cross talk between these resident cells and recruited immune cells is, in part, mediated by Wnt signalling. In corroboration of this role of Wnt-mediated recruitment of neutrophils, expression of the Wnt inhibitory ligand sFRPI during acute inflammation results in a dramatic 'dampening' of immune cell infiltration in particular of neutrophil chemoattraction. Importantly, an intrinsic Wnt signalling pathway is essential for neutrophil chemoattraction in response to inflammatory stimuli. There is a marked reduction of neutrophil infiltration in mice grafted with a ß-catenin deficient bone marrow upon TPA induced cutaneous inflammation. Additionally, neutrophils lacking Wnt/ß-catenin fail to respond to IFNγ, an early inflammatory cue, in vitro. In combination, these data indicate a potent function of Wnt signalling in immune cell recruitment and the modulation of the inflammatory response. PART B Tissue specific stem cells form the cellular base on which tissue homeostasis and repair of adult tissue relies. The maintenance of this stem cell pool is highly dependent on the immediate environment or niche. We have identified three genes, the fibroblast growth factor receptor 1 (FGFR1), serpin protease inhibitor (serpin F1) and the haematopoietic cell phosphatase (Hcph) to be specifically expressed in a small population of stromal cells which are in close contact to bulge stem cells. These specialized stromal cells might represent an essential mesenchymal component of the skin stem cell niche and may regulate stem cell proliferation and differentiation. Multiple FGFR1 isoforms are generated through alternate transcript splicing and are able to interact with both FGFs and cell adhesion molecules. Two predominant forms of the receptor are FGFR1-α and FGFR1-ß. Expression of a dominant negative form of the alpha isoform prevents hair follicle morphogenesis altogether. Given that FGFR1-ß signals principally through the FGF ligands, this data indicates that FGF signalling is dispensable for follicle morphogenesis. Moreover the loss of follicular morphogenesis upon suggests a requirement for signalling via cell adhesion molecule association with the receptor as FGFR1 α has a greater affinity for these molecules. The expression of the second candidate niche gene serpin f1, lead to the complete ablation of hair follicle morphogenesis. The serpin f1 product, pigment-epithelial derived factor (PEDF) has potent anti-angiogenic effects. Immunohistochemical analysis using CD31, a endothelial cell marker, revealed that although these cells are present, they have are disorganised and do not form vessels. Interestingly, endothelial cells have been found to contribute to the neuronal stem cell niche and our results suggest a similar mechanism in the skin. SHP1, the Hcph gene product, is a phosphatase which acts in the haematopoetic system. Motheaten mice carrying spontaneous mutations in the Hcph gene have patchy alopecia in their skin and severe defects in their haematopoietic system. However the haematopoietic rescue of the mouse does not result in normal follicular homeostasis. Additionally, ablation of Hcph in either the dermal or keratinocyte compartments of the skin produces hair follicles with abberant morphologies. This data indicates that although SHP1 is not essential for hair follicle morphogenesis it is required in both epidermal and dermal compartments to maintain follicular morphology. RÉSUMÉ PARTIE A Jusqu'à présent, les travaux dédiés à l'étude de la voie de signalisation Wnt dans le système immunitaire se sont essentiellement concentrés sur son rôle dans le développement des cellules immunitaires. Les données présentées ici suggèrent fortement et de manière nouvelle, l'existence d'un mécanisme par lequel la voie de signalisation Wnt/ß-caténine module le recrutement de cellules immunitaires dans un tissu périphérique, la peau, et ainsi la réponse inflammatoire cutanée. La réponse inflammatoire cutanée est un processus dynamique d'infiltration séquentielle de diverses cellules immunitaires, orchestré par un réseau de facteurs de croissance, chémokines et cytokines. Les kératinocytes sont des contributeurs majeurs à la réponse inflammatoire précoce par la libération de chémokines qui permettent ensuite de recruter les cellules immunitaires. Dans des souris sauvages, la réponse est d'abord caractérisée par une infiltration rapide et substantielle de neutrophiles, suivie par celle des macrophages et des lymphocytes T. L'expression d'un ligand activateur de le voie canonique de signalisation Wnt (après injection infra-dermique de fibroblastes sur-exprimant Wnt-3a) induit une infiltration dermique très marquée de neutrophiles. De plus, la réponse est éliminée en l'absence de ß-caténine spécifiquement dans les kératinocytes, indiquant que ces cellules sont à l'origine de la réponse. De manière remarquable, ceci suggère qu'une signalisation cruciale entre ces cellules résidentes de la peau et les cellules immunitaires recrutées est, au moins en partie, médiée par la voie Wnt. Corroborant ce rôle de la voie Wnt/ß-caténine dans le recrutement des neutrophiles, l'expression d'un ligand inhibiteur de la voie (sFRP1) résulte au cours d'une inflammation aigüe en une réduction spectaculaire de l'infiltration des cellules immunitaires en général, et des neutrophiles en particulier. De manière importante, la voie de signalisation Wnt est intrinsèquement requise pour la chémoattraction des neutrophiles en réponse à un stimulus inflammatoire. En effet, suite à une inflammation cutanée induite par un ester de phorbol (TPA), une réduction notable de l'infiltration des neutrophiles est observée dans des souris préalablement greffées avec de la moelle osseuse constituée de cellules déficientes en ß-caténine. De plus, in vitro, les neutrophiles sans ß-caténine ne répondent pas à une stimulation par l'interféron γ, qui est pourtant un signal inflammatoire établi in vivo. En conclusion, nos données indiquent que la voie de signalisation Wnt/ß-caténine joue une fonction active dans le recrutement des cellules immunitaires vers un organe périphérique, la peau, ainsi que dans la modulation, à plusieurs niveaux, de la réponse inflammatoire cutanée. PARTIE B Les cellules souches tissu-spécifiques forment la base cellulaire sur laquelle repose l'homéostase et la réparation tissulaires chez l'adulte. La maintenance de ce réservoir de cellules souches est hautement dépendante de leur environnement cellulaire immédiat, encore appelé «niche des cellules souches». Dans la peau, ces cellules stromales spécialisées représentent un compartiment mésenchymateux essentiel de la niche des cellules souches en régulant leurs prolifération et différentiation. Nous avons identifié trois gènes, le «récepteur 1 àux facteurs de croissance des fibroblastes » (Fgfr1 ), l' «inhibiteur de protéase à sérine » (serpinf1 ou pedf) et la « phosphatase des cellules hématopoiétiques » (Hcph ou Ptpn6), comme spécifiquement exprimés par une petite population de cellules stromales qui sont étroitement associées aux cellules souches de la peau (localisées au niveau du bombement du follicule pileux). Pour analyser leur fonction dans ce contexte, nous avons utilisé un test de reconstitution complète de peau murine en combinaison à des. transductions géniques basées sur l'utilisation de lentivirus. Ce test repose sur le mélange de deux compartiments cellulaires, épidermique (kératinocytes) et dermique (fibroblastes), greffés sur une zone ouverte de peau du dos d'une souris pour ensemble reconstituer la peau. Des isoformes multiples de FGFR1 sont générées par épissage alternatif de transcrits et sont capables d'interagir à la fois avec les FGFs (facteurs de croissance des fibroblastes) et les molécules d'adhésion cellulaires. Les deux formes prédominantes du récepteur, FGFR1-α et FGFR1-ß, ne différent que par le «domaine ressemblant aux immunoglobulines 1 » (immunoglobulin-like 1 domain), absent de FGFR1-ß. De plus, FGFR1-ß a une affinité plus grande pour les FGFs et plus faible pour les molécules d'adhésion cellulaires telles que la Ncadhérine (connue pour activer FGFR). La sur-expression de l'une ou l'autre des formes n'empêche pas la morphogenèse folliculaire mais conduit à la formation de follicules aberrants. Toutefois, une différence phénotypique majeure est observée lorsqu'une forme «Dominant-Négatif » (DN) est exprimée dans le compartiment dermique. La sur-expression de FGFR1-ß DN conduit en effet à la formation de follicules petits et tronqués, avec des gaines épithéliales et un bulbe élargis ainsi qu'une petite papille dermique. Par contre, l'expression de FGFR1-α DN abolit complètement la morphogenèse folliculaire. Etant donné que la signalisation par FGFR1-ß est principalement dépendante des ligands FGFs, ces données indiquent que la signalisation par ceux-cì est non-nécessaire à la morphogenèse folliculaire. De plus, l'abolition du processus par la sur-expression de FGFR1-a DN suggëre une signalisation nécessaire entre le récepteur FGFR1 et une ou des molécules d'adhésion cellulaire. L'expression de notre second candidat comme gène spécifique de la niche des cellules souches de la peau, serpinf1, prévient la morphogenèse folliculaire. Seules de petites structures ressemblant à des cystes sont observées après reconstitution de la peau. De plus, dans ces transplants, aucune cellule CD34-positive (marqueur des cellules souches) n'est retrouvée associé à ces cystes. Le produit du gène serpin f1, le «facteur dérivé d'épithélium pigmentaire » (PEDF) est un puissant facteur anti-angiogénique. Nous avons donc analysé la vascularisation des transplants par immunohistochirnies utilisant CD31, un marqueur des cellules endothéliales. Nos résultats révèlent que les cellules endothéliales sont bien présentes, mais de manière désorganisée et ne formant pas de vaisseaux. De manière intéressante, les cellules endothéliales contribuent activement à la niche des cellules souches neuronales, et nos résultats suggèrent donc l'existence possible d'un mécanisme similaire dans la peau. SHP1, le produit du gène Hcph, est une phosphatase quì agit dans le système hématopoiétique. Les souris « motheaten »qui portent des mutations spontanées du gène ont une alopécie inégale au niveau de la peau et de sévères troubles du système hématopoiétique. Pour s'assurer que le phénotype observé au niveau de la peau n'est pas une conséquence d'un défaut du système hématopoiétique, nous avons transplanté des souris Hcph -/- avec de la moelle osseuse sauvage afin de restaurer la fonction de SHP 1 dans le système hématopoiétique. Toutefois, le défaut de morphologie folliculaire est maintenu. De plus, l'ablation d'Hcph dans le compartiment dermique ou épidermique d'essais de reconstitution de peau conduit à la production de follicules pileux avec des morphologies aberrantes. Ces données indiquent que SHP1 n'est pas essentiel à la morphogenèse folliculaire mais est toutefois requis à la fois dans les compartiments épidermiques et dermiques pour la maintenance de la forme du follicule.

Different transcriptional control of metabolism and extracellular matrix in visceral and subcutaneous fat of obese and rimonabant treated mice.

BACKGROUND: The visceral (VAT) and subcutaneous (SCAT) adipose tissues play different roles in physiology and obesity. The molecular mechanisms underlying their expansion in obesity and following body weight reduction are poorly defined. METHODOLOGY: C57Bl/6 mice fed a high fat diet (HFD) for 6 months developed low, medium, or high body weight as compared to normal chow fed mice. Mice from each groups were then treated with the cannabinoid receptor 1 antagonist rimonabant or vehicle for 24 days to normalize their body weight. Transcriptomic data for visceral and subcutaneous adipose tissues from each group of mice were obtained and analyzed to identify: i) genes regulated by HFD irrespective of body weight, ii) genes whose expression correlated with body weight, iii) the biological processes activated in each tissue using gene set enrichment analysis (GSEA), iv) the transcriptional programs affected by rimonabant. PRINCIPAL FINDINGS: In VAT, "metabolic" genes encoding enzymes for lipid and steroid biosynthesis and glucose catabolism were down-regulated irrespective of body weight whereas "structure" genes controlling cell architecture and tissue remodeling had expression levels correlated with body weight. In SCAT, the identified "metabolic" and "structure" genes were mostly different from those identified in VAT and were regulated irrespective of body weight. GSEA indicated active adipogenesis in both tissues but a more prominent involvement of tissue stroma in VAT than in SCAT. Rimonabant treatment normalized most gene expression but further reduced oxidative phosphorylation gene expression in SCAT but not in VAT. CONCLUSION: VAT and SCAT show strikingly different gene expression programs in response to high fat diet and rimonabant treatment. Our results may lead to identification of therapeutic targets acting on specific fat depots to control obesity.