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Ligand binding and activation of PPAR? by Firemaster® 550: effects on adipogenesis and osteogenesis in vitro.

ABSTRACT: The use of alternative flame retardants has increased since the phase out of pentabromodiphenyl ethers (pentaBDEs). One alternative, Firemaster® 550 (FM550), induces obesity in rats. Triphenyl phosphate (TPP), a component of FM550, has a structure similar to that of organotins, which are obesogenic in rodents.We tested the hypothesis that components of FM550 are biologically active peroxisome proliferator-activated receptor ? (PPAR?) ligands and estimated indoor exposure to TPP.FM550 and its components were assessed for ligand binding to and activation of human PPAR?. Solvent mapping was used to model TPP in the PPAR? binding site. Adipocyte and osteoblast differentiation were assessed in bone marrow multipotent mesenchymal stromal cell models. We estimated exposure of children to TPP using a screening-level indoor exposure model and house dust concentrations determined previously.FM550 bound human PPAR?, and binding appeared to be driven primarily by TPP. Solvent mapping revealed that TPP interacted with binding hot spots within the PPAR? ligand binding domain. FM550 and its organophosphate components increased human PPAR?1 transcriptional activity in a Cos7 reporter assay and induced lipid accumulation and perilipin protein expression in BMS2 cells. FM550 and TPP diverted osteogenic differentiation toward adipogenesis in primary mouse bone marrow cultures. Our estimates suggest that dust ingestion is the major route of exposure of children to TPP.Our findings suggest that FM550 components bind and activate PPAR?. In addition, in vitro exposure initiated adipocyte differentiation and antagonized osteogenesis. TPP likely is a major contributor to these biological actions. Given that TPP is ubiquitous in house dust, further studies are warranted to investigate the health effects of FM550.

SUBMITTER: Pillai HK

PROVIDER: S-EPMC4216168 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Ligand binding and activation of PPARγ by Firemaster® 550: effects on adipogenesis and osteogenesis in vitro.

Pillai Hari K HK Fang Mingliang M Beglov Dmitri D Kozakov Dima D Vajda Sandor S Stapleton Heather M HM Webster Thomas F TF Schlezinger Jennifer J JJ

Environmental health perspectives 20140725 11

<h4>Background</h4>The use of alternative flame retardants has increased since the phase out of pentabromodiphenyl ethers (pentaBDEs). One alternative, Firemaster® 550 (FM550), induces obesity in rats. Triphenyl phosphate (TPP), a component of FM550, has a structure similar to that of organotins, which are obesogenic in rodents.<h4>Objectives</h4>We tested the hypothesis that components of FM550 are biologically active peroxisome proliferator-activated receptor γ (PPARγ) ligands and estimated in ...[more]

PMID: 25062436

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Project description:Exposure to flame retardants has been associated with negative health outcomes including metabolic effects. As polybrominated diphenyl ether flame retardants were pulled from commerce, human exposure to new flame retardants such as Firemaster® 550 (FM550) has increased. Although previous studies in murine systems have shown that FM550 and its main components increase adipogenesis, the effects of FM550 in human models have not been elucidated.The objectives of this study were to determine if FM550 and its components are active in human preadipocytes, and to further investigate their mode of action.Human primary preadipocytes were differentiated in the presence of FM550 and its components. Differentiation was assessed by lipid accumulation and expression of peroxisome proliferator-activated receptor ? (PPARG), fatty acid binding protein (FABP) 4 and lipoprotein lipase (LPL). mRNA was collected for Poly (A) RNA sequencing and was used to identify differentially expressed genes (DEGs). Functional analysis of DEGs was undertaken in Ingenuity Pathway Analysis.FM550 triphenyl phosphate (TPP) and isopropylated triphenyl phosphates (IPTP), increased adipogenesis in human primary preadipocytes as assessed by lipid accumulation and mRNA expression of regulators of adipogenesis such as PPAR?, CCAAT enhancer binding protein (C/EBP) ? and sterol regulatory element binding protein (SREBP) 1 as well as the adipogenic markers FABP4 LPL and perilipin. Poly (A) RNA sequencing analysis revealed potential modes of action including liver X receptor/retinoid X receptor (LXR/RXR) activation, thyroid receptor (TR)/RXR, protein kinase A, and nuclear receptor subfamily 1 group H members activation.We found that FM550, and two of its components, induced adipogenesis in human primary preadipocytes. Further, using global gene expression analysis we showed that both TPP and IPTP likely exert their effects through PPARG to induce adipogenesis. In addition, IPTP perturbed signaling pathways that were not affected by TPP. https://doi.org/10.1289/EHP1318.

Project description:Firemaster® 550 (FM550) is a chemical mixture currently used as an additive flame retardant in commercial products, and is comprised of 2-ethylhexyl-2,3,4,5-tertrabromobenzoate (TBB), bis(2-ethylhexyl) tetrabromophthalate (TBPH), triphenyl phosphate (TPP), and isopropylated triphenyl phosphate (IPTP). Animal and in vitro studies suggest that FM550, TPP and IPTP may have adipogenic effects and may exert these effects through PPARγ activation. Using murine 3T3-L1 preadipocytes, we investigated the detailed expression of transcription factors and adipogenic markers in response to FM550 and its components. Further we investigated the mechanism of action of the peroxisome proliferator-activated receptor gamma (PPARγ) on downstream targets of the receptor by focussing on the mature adipocyte marker, adipocyte protein 2 (aP2). In addition, we set to elucidate the components responsible for the adipogenic effects seen in the FM550 mixture. We show that FM550 and its components TPP, IPTP, and TBPH, but not TBB induced lipid accumulation in a dose-dependent manner. Interestingly, despite displaying enhanced lipid accumulation, TBPH did not alter the mRNA or protein expression of terminal differentiation markers. In contrast, FM550, TPP, and IPTP treatment enhanced lipid accumulation, and mRNA and protein expression of terminal differentiation markers. To further delineate the mechanisms of action of FM550 and its components we focussed on aP2 promoter activity. For this purpose we used the enhancer region of the mouse aP2 promoter using a 584-bp reporter construct containing an active PPRE located 5.4 kb away from the transcription start site of aP2. Exposure to FM550, IPTP, and TPP significantly increased PPARγ mediated aP2 enhancer activity. Furthermore, we show that TPP- and IPTP-dependent upregulation of aP2 was significantly inhibited by the selective PPARγ antagonist GW9662. In addition, chromatin immunoprecipitation experiments showed that IPTP and TPP treatment led to the recruitment of PPARγ to the regulatory region of aP2.

Project description:IntroductionSkeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPAR?) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPAR? shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model.MethodsPPAR? shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPAR? shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPAR?, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K).ResultsIn vitro, PPAR? shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+osteoblasts and decrease in TRAP+multinucleated osteoclasts was observed with PPAR? shRNA treatment.DiscussionThese findings suggest that acute loss of PPAR? in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPAR? shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPAR? shRNA on OB and OC cell types, and it may help determine whether PPAR? antagonists are potential therapeutic agents for osteoporotic bone loss.

Project description:Environmental obesogens are a newly recognized category of endocrine disrupting chemicals that have been implicated in contributing to the rising rates of obesity in the United States. While obesity is typically regarded as an increase in visceral fat, adipocyte accumulation in the bone has been linked to increased fracture risk, lower bone density, and osteoporosis. Exposure to environmental toxicants that activate peroxisome proliferator activated receptor ? (PPAR?), a critical regulator of the balance of differentiation between adipogenesis and osteogenesis, may contribute to the increasing prevalence of osteoporosis. However, induction of adipogenesis and suppression of osteogenesis are separable activities of PPAR?, and ligands may selectively alter these activities. It currently is unknown whether suppression of osteogenesis is a common toxic endpoint of environmental PPAR? ligands. Using a primary mouse bone marrow culture model, we tested the hypothesis that environmental toxicants acting as PPAR? agonists divert the differentiation pathway of bone marrow-derived multipotent mesenchymal stromal cells towards adipogenesis and away from osteogenesis. The toxicants tested included the organotins tributyltin and triphenyltin, a ubiquitous phthalate metabolite (mono-(2-ethylhexyl) phthalate, MEHP), and two brominated flame retardants (tetrabromobisphenol-a, TBBPA, and mono-(2-ethylhexyl) tetrabromophthalate, METBP). All of the compounds activated PPAR?1 and 2. All compounds increased adipogenesis (lipid accumulation, Fabp4 expression) and suppressed osteogenesis (alkaline phosphatase activity, Osx expression) in mouse primary bone marrow cultures, but with different potencies and efficacies. Despite structural dissimilarities, there was a strong negative correlation between efficacies to induce adipogenesis and suppress osteogenesis, with the organotins being distinct in their exceptional ability to suppress osteogenesis. As human exposure to a mixture of toxicants is likely, albeit at low doses, the fact that multiple toxicants are capable of suppressing bone formation supports the hypothesis that environmental PPAR? ligands represent an emerging threat to human bone health.

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Ligand binding and activation of PPAR? by Firemaster® 550: effects on adipogenesis and osteogenesis in vitro.

Publications

Ligand binding and activation of PPARγ by Firemaster® 550: effects on adipogenesis and osteogenesis in vitro.

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