Receptor Gamma Research Articles

56. Age dependant effects of the PPAR-gamma agonist rosiglitazone on the brain and periphery during systemic LPS-induced inflammation in rats

Aging is associated with altered immune responses to stimuli such as lipopolysaccharide (LPS) and a mitochondrial decline is proposed to be a main underlying factor in age-related diseases. Recently, rosiglitazone (RZG), a peroxisome proliferator-activated receptor (PPAR) gamma agonist was shown to exert anti-inflammatory properties and to induce a key regulator of mitochondrial biogenesis, namely, PPARgamma co-activator-1-alpha (PGC-1alpha). Here, we aimed to further elucidate age-dependent changes in signaling pathways and sickness response during LPS-induced inflammation and to test potential beneficial effects of rosiglitazone. Pretreatment of old (24 months) and young (2 months) rats with RZG or solvent was followed by intraperitoneal injection of LPS (100 μg/kg) or saline. 24 h after stimulation blood, brain and liver were withdrawn for further analysis. We found age-dependent differences in fever and severity of other sickness symptoms after LPS-stimulation. Pretreatment with rosiglitazone, however, did not alter this response in young or old rats. Nevertheless, analysis of the liver by RT-PCR revealed increased mRNA-levels of markers of mitochondrial biogenesis in saline-injected and RZG-pretreated animals. Interestingly, several plasma cytokines including interleukin (IL)-6, IL-10, and IL-1ra and brain/liver inflammatory mediators such as nuclear factor IL-6 showed age-dependent changes independent of RZG-injections. Overall, beneficial effects of RZG could not be revealed in the current experimental paradigm. Nonetheless, our results reveal further insights into changes of brain and peripheral inflammatory mediators during aging and LPS-induced inflammation.

Comparison of three humanized mouse models for adoptive T cell transfer

Humanized mouse models for adoptive T cell transfer are important for preclinical efficacy and toxicity studies. However, common xenograft models using immunodeficient mice have so far failed to efficiently support the homing of human T cells to secondary lymphoid tissues. We established a new mouse model for the adoptive transfer of genetically-modified (gm) T cells using conditioned BALB/c mice. Conditioning involved cyclophosphamide injections, lethal irradiation and radioprotection with bone marrow from immunodeficient mice. We compared repopulation kinetics and the quality of grafts in these modified Trimera (mT3) mice with immunodeficient BALB/c Rag2(-/-) interleukin (IL)2 receptor gamma (rg) knockout (DKO) and NOD/LtSz-scid IL2rg(-/-) (NSG) recipient mouse strains. DKO mice showed only marginal engraftment until onset of graft-versus-host disease, whereas mT3 and NSG were repopulated with comparable kinetics. However, T cell repertoire and human cytokine profiles suggest a xenoreactivity-driven gm T cell expansion in mT3 mice, whereas NSG mice were characterized by an initial homeostatic proliferation. Morphological analysis revealed high levels of human gm T cell infiltration in the spleen and liver of all three mouse strains. However, mT3 mice provided the strongest homing of human gm T cells to mucosal sites. Additionally, mT3 mice were the only model with macroscopically visible superficial inguinal lymph nodes. These lymph nodes strongly supported the homing of gm T cells. In the present study, we give proof-of-concept that wild-type mice can accept gm T cell grafts while providing secondary lymphoid structures. Despite limitations, mT3 mice are a valid alternative for applications that specifically rely on improved secondary lymphoid structures.

Dose‐ and time‐dependent effects of recombinant human bone morphogenetic protein‐2 on the osteogenic and adipogenic potentials of alveolar bone‐derived stromal cells

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a well-known growth factor that can induce robust bone formation, and recent studies have shown that rhBMP-2-induced osteogenesis is closely related to adipogenesis. The aim of the present study was to determine the dose- and time-dependent effects of rhBMP-2 on the osteogenic and adipogenic differentiation of human alveolar bone-derived stromal cells (hABCs) in vivo and in vitro. hABCs were isolated and cultured, and then transplanted using a carrier treated either with or without rhBMP-2 (100 μg/mL) into an ectopic subcutaneous mouse model. Comprehensive histologic and histometric analyses were performed after an 8-wk healing period. To further understand the dose-dependent (0, 10, 50, 200, 500 and 1000 ng/mL) and time-dependent (0, 3, 5, 7 and 14 d) effects of rhBMP-2 on osteogenic and adipogenic differentiation, in vitro osteogenic and adipogenic differentiation of hABCs were evaluated, and the expression of related mRNAs, including those for alkaline phosphatase, osteocalcin, bone sialoprotein, peroxisome-proliferator-activated receptor gamma-2 and lipoprotein lipase, were assessed using quantitative RT-PCR. rhBMP-2 significantly promoted the osteogenic and adipogenic differentiation of hABCs in vivo, and gradually increased both the osteogenic and adipogenic potential in a dose- and time-dependent manner with minimal deviation in vitro. The expression of osteogenesis- and adipogenesis-associated mRNAs were concomitantly up-regulated by rhBMP-2. The findings of the present study showed that rhBMP-2 significantly enhanced the adipogenic as well as the osteogenic potential of hABCs in dose- and time-dependent manner. The control of adipogenic differentiation of hABCs should be considered when regenerating the alveolar bone using rhBMP-2.

Chemical Proteomics‐Based Analysis of Off‐target Binding Profiles for Rosiglitazone and Pioglitazone: Clues for Assessing Potential of Cardiotoxicity

The insulin‐sensitizing drugs rosiglitazone and pioglitazone are currently the only two thiazolidinedione (TZD)‐based drugs on the market to treat type II diabetes in the U.S. Both drugs are thought to be selective for the peroxisome proliferator‐activated receptor gamma. Despite their chemical and mechanistic similarities, there are large cohort studies suggesting rosiglitazone causes congestive heart failure and peripheral edema to greater extent than pioglitazone. This study was performed to complete a mechanistic comparison between the two drugs. The common functional core between the drugs, glitazone, was used as a privileged scaffold upon which rat heart proteins were bound and eluted with glitazone, rosiglitazone, or pioglitazone. Liquid chromatography tandem mass spectrometry was used to determine the drug binding profiles. Results suggest that both drugs bind specific ion channels and modulators, with implications in congestive heart failure, arrhythmia, and peripheral edema. Additionally, numerous proteins involved in fatty acid metabolism, glycolysis/gluconeogenesis, neuronal synaptic transduction, and energy production were detected. While there were slight differences in off‐target binding profiles between rosiglitazone and pioglitazone, there were more similarities in individual protein and pathway comparisons, suggesting similar mechanisms of action and risk profile.

Effect of the peroxisome proliferator‐activated receptor gamma (PPARγ) agonist Rosiglitazone on the hypothalamic GT1‐7 cell line

Polycystic ovary syndrome (PCOS) is a major cause of infertility in women and is linked to obesity and insulin resistance. Rosiglitazone, a PPARγ agonist, increases insulin sensitivity and improves PCOS and diabetes. PPARγ ligands have also been found to affect the hypothalamus‐pituitary‐gonadal axis, although their site and mechanism of action are not entirely known. The aim of this study was to determine the effects of rosiglitazone on insulin and kisspeptin stimulation of GT1‐7 hypothalamic GnRH neurons. Timecourse and dose response experiments were conducted using 24 hour pretreatment of rosiglitazone or vehicle, and with either insulin or kisspeptin stimulation. Akt and ERK phosphorylation were analyzed with Western Blot and GnRH mRNA expression was analyzed with RT‐QPCR. Preliminary results indicated that treatment with rosiglitazone increased insulin‐induced phosphorylation of Akt in GT1‐7 cells. Maximal stimulation occurred between 10 and 15 minutes, and an insulin concentration of 10 nM. Kisspeptin alone increased GnRH mRNA, with maximal stimulation at 1 nM, as reported by others. Furthermore, rosiglitazone appears to increase kisspeptin induced GnRH mRNA. In conclusion, rosiglitazone appears to have a direct role in regulating hypothalamic GT1‐7 neurons by acting as an insulin sensitizer and modulating the effects of kisspeptin.Supported by NIH grant U54HD12303. VH was supported by Julia Brown Undergraduate Research Scholarship.

Egr‐1 mediates the suppressive effect of IL‐1 on PPARg expression in human OA chondrocytes

BackgroundPeroxisome proliferator‐activated receptor gamma (PPARg) is a ligand activated transcription factor. Several lines of evidence indicate that PPARg have protective effects in osteoarthritis (OA). We have previously shown that IL‐1 down‐regulated PPARg expression in OA chondrocytes. In the present study we will investigate the mechanisms underlying this effect of IL‐1.MethodsChondrocytes were stimulated with IL‐1, and the level of PPARg and Egr‐1 protein and mRNA were evaluated using WB and Real Time‐RT‐PCR, respectively. The PPARg promoter activity was analyzed in transient transfection experiments. Egr‐1 recruitment to the PPARg promoter was evaluated using ChIP assays. Small interfering RNA (siRNA) approaches were used to silence Egr‐1 expression.ResultsWe demonstrated that the suppressive effect of IL‐1 on PPARg expression requires de novo protein synthesis. ChIP analyses revealed that IL‐1 induced Egr‐1 recruitment at the PPARg promoter. IL‐1 inhibited the activity of PPARg promoter and overexpression of Egr‐1 potentiated the inhibitory effect of IL‐1, suggesting that Egr‐1 may mediate the suppressive effect of IL‐1. Finally, Egr‐1 silencing with siRNA blocked IL‐1‐mediated down‐regulation of PPARg expression.ConclusionThese results indicate that Egr‐1 contributes to IL‐1‐mediated down‐regulation of PPARg expression in OA chondrocytes.

Tetranectin Promotes Adipogenesis and Lipogenesis and is Negatively Regulated by Polyunsaturated Fatty Acids in Human Adipocytes

Adipose tissues secrets factors, or adipokines, regulate body energy homeostasis. To search for novel adipokines, we employed 2‐D protein gel electropherosis and LC‐MS/MS to analyze human primary adipocyte culture medium, in which tetranectin (TN). We found that the mRNA expression of TN is positively correlated with the differentiation and lipid accumulation of human preadipocytes, as well as the body mass index. Overexpression and knockdown of TN in human primary adipocytes by lentivirus prove that TN increases the expression of adipogenic and lipogenic genes, such as peroxisome proliferator‐activated receptor gamma, lipoprotein lipase and fatty acid synthesis, but decreases those related to lipolysis, including hormone sensitive lipase, interleukin 6 and serum amyloid A along with altered profiles of proteins responsible primarily for lipid metabolism, as analyzed by quantitative proteomic analysis and quantitative real‐time PCR. In addition, treatments of polyunsaturated fatty acids (PUFA) suppressed TN expression. Taken together, our data suggest that not only TN potentially serves as a novel adipokine contributing to the regulation of energy homeostasis and lipid metabolism, but also components of the TN‐associated signaling pathways the therapeutic targets for treating obesity and associated metabolic diseases.

The PPAR‐γ agonist Rosiglitazone increases angiotensin‐converting enzyme 2 (ACE2) promoter activity in neurons

Angiotensin converting enzyme 2 (ACE2) converts angiotensin‐(Ang)‐II to the vasodilator peptide Ang‐(1–7). Our laboratory has previously observed that ACE2 expression and activity in brain regions involved in the central regulation of blood pressure (BP) and volume homeostasis (e.g. SFO, PVN, and RVLM) are reduced in mice with elevated brain Ang‐II levels. Other investigators have shown that Rosiglitazone, a peroxisome proliferator‐activator receptor gamma (PPAR‐γ) agonist, increases ACE2 mRNA in adipocytes. Here we assessed the potential of PPAR‐γ to activate the ACE2 gene promoter. A reporter plasmid containing the human ACE2 proximal promoter sequence was constructed, and luciferase activity was measured after transfection of mouse Neuro‐2a cells in the presence or absence of Rosiglitazone (1 μM) and Ang‐II (100 nM) for 24 hours. Human ACE2 promoter activity increased by 2‐fold (P<0.05) with Rosiglitazone treatment independently of Ang‐II treatment, suggesting that PPAR‐γ may bind the proximal promoter and induce expression of hACE2. On the other hand, our data suggest that Ang‐II regulation of the hACE2 gene expression is not dependent on its proximal promoter. Altogether, we show that PPAR‐γ positively regulates the hACE2 promoter activity, supporting a beneficial role in modulating Ang‐II levels in neurogenic hypertension and in the regulation of volume homeostasis. (Funding: HL093178)

Effect of n‐3 polyunsaturated fatty acids on peroxisome proliferator‐activated receptor gamma (PPARγ) expression in adults

BackgroundN‐3 fatty acids play a role in reducing the risk of cardiovascular disease via lowering blood lipids and suppressing inflammation. Whether or not the anti‐inflammatory effects are mediated through alteration in gene expression is not known. Our objective was to determine the effect of varying dietary ratio of n‐ 6: n‐3 fatty acids on the expression of PPAR‐γ.MethodsA factorial design approach was used to test the effect of 4 diets on gene expression: 10:1 and 2:1 n‐6: n‐3 fatty acid ratio diets with and without a supplement (S) of eicosapentaenoic and docosahexaenoic acids (EPA/DHA). This study was part of an original randomized cross‐over feeding study (8‐week period each with a washout between periods) on 24 healthy adults. Subjects consumed eucaloric diets [10:1 (Control), 2:1 (α‐linolenic acid, ALA; 6–7g/2400 kcal/d), 10:1+S (0.20/0.72g EPA/DHA per 2400 kcal/d), and 2:1+S. At the end of each diet period, samples of subcutaneous tissue was collected from the abdominal region and used for measurement of PPAR‐γ mRNA levels by real‐time polymerase chain reaction with actin as an internal standard.ResultsPreliminary analyses of the fold changes in PPAR‐γ expression between diets was not statistically significant.ConclusionOur initial analyses did not show an effect of dietary ratio of n‐6: n‐3 fatty acids with or without the supplement on the expression of PPAR‐γ for the 8‐week study period.Funded by the Center for Health and Nutrition Research, University of California, Davis.

A dual color fluorescent reporter system for the real time detection of promoter activity

Understanding the mechanisms controlling transcription of a gene requires the identification and characterization of its cis-acting regulatory elements. A highly useful approach to the identification and characterization of cis-acting elements has been the systematic coupling of genomic fragments to reporter constructs, so called "promoter bashing". The expression from such reporters must be normalized for differences in transient transfection efficiency between cells and replicates. A novel dual color fluorescent reporter system to assay the promoter activity of a genomic DNA fragment of interest was established by cloning a Discosoma red fluorescent protein gene and a green fluorescent protein gene into a single vector, giving a system in which the ratio between red and green fluorescence is proportional to promoter activity. This system allows real time quantitative monitoring of promoter activity. We validated this approach by assaying the cis-acting regulatory potential of the peroxisome proliferators-activated receptor gamma2 gene.

Idealized PPAR-Based Therapies: Lessons from Bench and Bedside

The incidence of type 2 (T2D) diabetes and other chronic conditions associated with insulin resistance is increasing at an alarming rate, underscoring the need for effective and safe therapeutic strategies. Peroxisome-proliferator-activated receptor gamma (PPARγ) has emerged as a critical regulator of glucose homeostasis, lipid homeostasis, and vascular inflammation. Currently marketed drugs targeting this receptor, the thiazolidinediones (TZDs), have proven benefits on insulin resistance and hyperglycemia associated with T2D. Unfortunately, they have been associated with long-term unfavorable effects on health, such as weight gain, plasma volume expansion, bone loss, cardiovascular toxicity, and possibly cancer, and these safety concerns have led to reduced interest for many PPARγ ligands. However, over the last years, data from human genetic studies, animal models, and studies with ligands have increased our understanding of PPARγ's actions and provided important insights into how ligand development strategies could be optimized to increase effectiveness and safety of PPARγ-based therapies.

Mesenchymal Stem Cells in Infantile Hemangioma Reside in the Perivascular Region

Infantile hemangioma grows quickly in the first year of life and regresses slowly to fibrofatty tissue during childhood; mesenchymal stem cells (MSCs) have been reported to contribute to this adipogenesis. Recent studies have shown the perivascular origin of MSCs in multiple organs. We hypothesized that MSCs in hemangioma might also reside in the perivascular region. We isolated MSCs from proliferating hemangioma by their selective adhesion to plastic culture dishes. Mesenchymal stem cells from bone marrow (BM-MSCs) and foreskin-derived fibroblasts were used as controls. Flow cytometry and immunofluorescence staining were used to examine their antigen profiles; in vitro induction of multi-lineage differentiation was performed to test their pluripotency. Platelet-derived growth factor R-β (PDGFR-β), CD133, and peroxisome-proliferator-activated receptor gamma (PPAR-γ) were selected as the markers to observe MSCs in hemangioma by immunohistochemistry staining, with costaining of CD31 and alpha-smooth muscle actin (α-SMA). Hemangioma-derived MSCs (Hem-MSCs) had fibroblast-like morphology; they expressed the MSC markers CD105, CD90, CD29, and vimentin and did not express the hematopoietic/endothelial markers CD45, CD34, CD31, and flt-1; Hem-MSCs also expressed CD133 and PPAR-γ. Most Hem-MSCs expressed PDGFR-β and α-SMA; in contrast, the expression of PDGFR-β and α-SMA in BM-MSCs was very weak. The Hem-MSCs differentiated into adipocytes, osteoblasts, and chondroblasts in vitro. This confirmed their pluripotency. Immunohistochemistry showed the colocalization of PDGFR-β/α-SMA, CD133/α-SMA, and PPAR-γ/α-SMA in the perivascular region. MSCs were successfully obtained from proliferating hemangioma, revealing the perivascular origin of MSCs in hemangioma.

PPARγ Signaling Mediates the Evolution, Development, Homeostasis, and Repair of the Lung.

Epithelial-mesenchymal interactions mediated by soluble growth factors determine the evolution of vertebrate lung physiology, including development, homeostasis, and repair. The final common pathway for all of these positively adaptive properties of the lung is the expression of epithelial parathyroid-hormone-related protein, and its binding to its receptor on the mesenchyme, inducing PPARγ expression by lipofibroblasts. Lipofibroblasts then produce leptin, which binds to alveolar type II cells, stimulating their production of surfactant, which is necessary for both evolutionary and physiologic adaptation to atmospheric oxygen from fish to man. A wide variety of molecular insults disrupt such highly evolved physiologic cell-cell interactions, ranging from overdistention to oxidants, infection, and nicotine, all of which predictably cause loss of mesenchymal peroxisome-proliferator-activated receptor gamma (PPARγ) expression and the transdifferentiation of lipofibroblasts to myofibroblasts, the signature cell type for lung fibrosis. By exploiting such deep cell-molecular functional homologies as targets for leveraging lung homeostasis, we have discovered that we can effectively prevent and/or reverse the deleterious effects of these pathogenic agents, demonstrating the utility of evolutionary biology for the prevention and treatment of chronic lung disease. By understanding mechanisms of health and disease as an evolutionary continuum rather than as dissociated processes, we can evolve predictive medicine.

Peroxisome Proliferator-Activated ReceptorαAgonists Differentially Regulate Inhibitor of DNA Binding Expression in Rodents and Human Cells

Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.

Nature of fatty acids in high fat diets differentially delineates obesity-linked metabolic syndrome components in male and female C57BL/6J mice

BackgroundAdverse effects of high-fat diets (HFD) on metabolic homeostasis are linked to adipose tissue dysfunction. The goal of this study was to examine the effect of the HFD nature on adipose tissue activity, metabolic disturbances and glucose homeostasis alterations in male mice compared with female mice.MethodsC57BL/6J mice were fed either a chow diet or HFD including vegetal (VD) or animal (AD) fat. Body weight, plasmatic parameters and adipose tissue mRNA expression levels of key genes were evaluated after 20 weeks of HFD feeding.ResultsHFD-fed mice were significantly heavier than control at the end of the protocol. Greater abdominal visceral fat accumulation was observed in mice fed with AD compared to those fed a chow diet or VD. Correlated with weight gain, leptin levels in systemic circulation were increased in HFD-fed mice in both sexes with a significant higher level in AD group compared to VD group. Circulating adiponectin levels as well as adipose tissue mRNA expression levels were significantly decreased in HFD-fed male mice. Although its plasma levels remained unchanged in females, adiponectin mRNA levels were significantly reduced in adipose tissue of both HFD-fed groups with a more marked decrease in AD group compared to VD group. Only HFD-fed male mice were diabetic with increased fasting glycaemia. On the other hand, insulin levels were only increased in AD-fed group in both sexes associated with increased resistin levels. VD did not induce any apparent metabolic alteration in females despite the increased weight gain. Peroxisome Proliferator-Activated Receptors gamma-2 (PPARγ2) and estrogen receptor alpha (ERα) mRNA expression levels in adipose tissue were decreased up to 70% in HFD-fed mice but were more markedly reduced in male mice as compared with female mice.ConclusionsThe nature of dietary fat determines the extent of metabolic alterations reflected in adipocytes through modifications in the pattern of adipokines secretion and modulation of key genes mRNA expression. Compared with males, female mice demonstrate higher capacity in controlling glucose homeostasis in response to 20 weeks HFD feeding. Our data suggest gender specific interactions between the diet's fatty acid source, the adipocyte-secreted proteins and metabolic disorders.

Association of bovine fatty acid composition with missense nucleotide polymorphism in exon7 of peroxisome proliferator‐activated receptor gamma gene

Association of bovine fatty acid composition with missense nucleotide polymorphism in exon7 of <i>peroxisome proliferator‐activated receptor gamma</i> gene

S17 * ALCOHOL AND GABAA RECEPTORS * S17.1 * EXTRASYNAPTIC GABAA-RS: PHARMACOLOGY AND REGULATION OF GENE EXPRESSION

S17 * ALCOHOL AND GABAA RECEPTORS * S17.1 * EXTRASYNAPTIC GABAA-RS: PHARMACOLOGY AND REGULATION OF GENE EXPRESSION

Thyroid disruption by Di-n-butyl phthalate (DBP) and mono-n-butyl phthalate (MBP) in Xenopus laevis.

BackgroundDi-n-butyl phthalate (DBP), a chemical widely used in many consumer products, is estrogenic and capable of producing seriously reproductive and developmental effects in laboratory animals. However, recent in vitro studies have shown that DBP and mono-n-butyl phthalate (MBP), the major metabolite of DBP, possessed thyroid hormone receptor (TR) antagonist activity. It is therefore important to consider DBP and MBP that may interfere with thyroid hormone system. Methodology/Principal Findings Nieuwkoop and Faber stage 51 Xenopus laevis were exposed to DBP and MBP (2, 10 or 15 mg/L) separately for 21 days. The two test chemicals decelerated spontaneous metamorphosis in X. laevis at concentrations of 10 and 15 mg/L. Moreover, MBP seemed to possess stronger activity. The effects of DBP and MBP on inducing changes of expression of selected thyroid hormone response genes: thyroid hormone receptor-beta (TRβ), retinoid X receptor gamma (RXRγ), alpha and beta subunits of thyroid-stimulating hormone (TSHα and TSHβ) were detected by qPCR at all concentrations of the compounds. Using mammalian two-hybrid assay in vitro, we found that DBP and MBP enhanced the interactions between co-repressor SMRT (silencing mediator for retinoid and thyroid hormone receptors) and TR in a dose-dependent manner, and MBP displayed more markedly. In addition, MBP at low concentrations (2 and 10 mg/L) caused aberrant methylation of TRβ in head tissue.ConclusionsThe current findings highlight potential disruption of thyroid signalling by DBP and MBP and provide data for human risk assessment.

1067 ADIPOCYT-FATTY ACID BINDING PROTEIN, A POTENTIAL PROGNOSTIC MARKER OF BLADDER TRANSITIONAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Few studies provided evidence that loss of adipocyte-fatty acid binding protein (A-FABP) expression is associated with progression of human bladder TCC and suggest that it could be a putative marker of progression of the disease. The re-expression of A-FABP could be a therapeutic approach in early stage bladder cancer to prevent disease progression. The aim of this study is first to determine whether A-FABP is a progression marker by analysing A-FABP mRNA expression within the tumor. Secondly, to demonstrate whether the expression of this potential prognostic marker could be up-regulated by Peroxisome Proliferator-Activated Receptor (PPAR) gamma agonists such as prostaglandins and thiazolidinediones (antidiabetic drugs) known to exhibit anti-neoplastic effects. METHODS: Total RNA was extracted from 26 samples of bladder TCC from clinical stages pTa to pT4 obtained from transurethral resection or cystectomy specimens. The relative A-FABP mRNA expression level was evaluated by RTqPCR. The regulation of AFABP expression was studied by RTqPCR and western-blotting in T24 cells derived from an undifferentiated grade III carcinoma of the bladder. RESULTS: We reported from bladder tumors that decrease of A-FABP transcript level significantly correlated to tumor stage and to histologic grade (p 0.05). Namely, in poor prognosis high grade pT1 tumors there was a loss of A-FABP expression compared to good prognosis tumors. We described for the first time the molecular mechanism by which this marker is up-regulated by PPAR gamma in T24 cells. This effect occurred through a PPARdependent transcriptional mechanism without modifying mRNA stability and interestingly required de novo protein synthesis. CONCLUSIONS: Data as a whole suggest a prognostic significance of A-FABP in bladder cancer outcome and the potential utility of overexpression of this protein by antidiabetic agents already in clinical use open up new perspectives in the treatment of bladder cancer by intravesical instillations of thiazolidinediones in order to inhibit malignant progression of bladder cancer.

Retinoid X Receptor Gamma Is Implicated in Docosahexaenoic Acid Modulation of Despair Behaviors and Working Memory in Mice

Omega-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA), have antidepressant and promnemonic functions. The mechanisms of such activities are still elusive and may involve retinoid X receptors (RXRs), transcription factors known to bind DHA in vitro. Promnemonic and antidespair activities of acute DHA treatment were tested in BALBcByJ mice using spontaneous alternation and forced swim test, respectively. The involvement of retinoid receptors in such DHA activities was investigated using RXR and/or retinoic acid receptor (RAR) agonists to mimic DHA activities or a synthetic pan-RXR antagonist to block them. Involvement of RXR isotypes was analyzed using the same tasks and delayed nonmatch to place for working memory in RXRγ knockout mice. Docosahexaenoic acid decreased despair behavior and improved working memory in BALBcByJ mice. Such effects were suppressed by co-treatment with BR1211, a pan-RXR antagonist, whereas a pan-RXR agonist, UVI2108, mimicked DHA activities. Retinoic acid (RA), a natural ligand of RXRs, also reduced despair behavior and improved working memory and such activities did not require activation of RARs, as RA effects were abolished by co-treatment with BR1211 and they were not reproduced by TTNPB, a pan-RAR agonist. The RXRγ knockout mice displayed increased despair and deficits in working memory, which were insensitive to DHA and pan-RXR agonist treatments, whereas DHA or UVI2108 reversed these deficits in RXRγ heterozygous mice. Our data suggest that RXRs are a converging point in mediating DHA and RA modulations of despair behavior and working memory and that RXRγ is the predominant RXR isotype in these regulations.