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

Pillai HK, Fang M, Beglov D, Kozakov D, Vajda S, Stapleton HM, Webster TF, Schlezinger JJ - Environ. Health Perspect. (2014)

Bottom Line: Our findings suggest that FM550 components bind and activate PPARγ.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health, Boston University, Boston, Massachusetts, USA.

ABSTRACT

Background: 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.

Objectives: We tested the hypothesis that components of FM550 are biologically active peroxisome proliferator-activated receptor γ (PPARγ) ligands and estimated indoor exposure to TPP.

Methods: FM550 and its components were assessed for ligand binding to and activation of human PPARγ. Solvent mapping was used to model TPP in the PPARγ 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.

Results: FM550 bound human PPARγ, and binding appeared to be driven primarily by TPP. Solvent mapping revealed that TPP interacted with binding hot spots within the PPARγ ligand binding domain. FM550 and its organophosphate components increased human PPARγ1 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.

Conclusions: Our findings suggest that FM550 components bind and activate PPARγ. 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.

Show MeSH

Related in: MedlinePlus

Assessment of effects of FM550 (top) and TPP (bottom) on osteogenesis in vitro. Primary bone marrow cultures were established, and osteogenesis was initiated with the addition of ascorbate, β-glycerol phosphate, insulin, and dexamethasone, except for naive wells. Cells were treated with vehicle (Veh; DMSO), FM550 (0.1–10 μg/mL; 0.2–20 μM), TPP (0.1–10 μM), or rosiglitazone (Rosi; 0.1 μM) and cultured for 7 days (gene expression) or 12 days (phenotype). (A) Lipid accumulation. (B) Fabp4 mRNA expression. (C) Alkaline phosphatase activity. (D) Mineralization. (E) Sp7 mRNA expression. Data are presented as mean ± SE of 4–6 independent bone marrow preparations.*p < 0.05, and **p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with Veh treatment.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4216168&req=5

f5: Assessment of effects of FM550 (top) and TPP (bottom) on osteogenesis in vitro. Primary bone marrow cultures were established, and osteogenesis was initiated with the addition of ascorbate, β-glycerol phosphate, insulin, and dexamethasone, except for naive wells. Cells were treated with vehicle (Veh; DMSO), FM550 (0.1–10 μg/mL; 0.2–20 μM), TPP (0.1–10 μM), or rosiglitazone (Rosi; 0.1 μM) and cultured for 7 days (gene expression) or 12 days (phenotype). (A) Lipid accumulation. (B) Fabp4 mRNA expression. (C) Alkaline phosphatase activity. (D) Mineralization. (E) Sp7 mRNA expression. Data are presented as mean ± SE of 4–6 independent bone marrow preparations.*p < 0.05, and **p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with Veh treatment.

Mentions: Analysis of effects of FM550 and TPP on bone differentiation in vitro. To test the hypothesis that FM550 and TPP are negative regulators of bone formation, we examined the effect of FM550 and TPP on adipogenic and osteogenic differentiation in primary bone marrow cultures prepared from female C57BL/6J mice. Established bone marrow cultures were induced to undergo osteogenesis and treated with vehicle, FM550, TPP, or rosiglitazone. FM550 induced significant lipid accumulation at a concentration of 5 μg/mL (10 μM), and TPP induced lipid accumulation at a concentration of 10 μM (Figure 5A). Activation of PPARγ by FM550 and TPP was reflected in the significantly increased mRNA expression of Fabp4, a PPARγ-target gene (Tontonoz et al. 1994) (Figure 5B). Although FM550 significantly suppressed both alkaline phosphatase activity and mineralization at 5 μg/mL (10 μM), TPP significantly suppressed only alkaline phosphatase activity (Figure 5C,D). Suppression of the transcriptional program of Runx2, the master regulator of osteogenesis, by FM550 and TPP was indicated by the significant decrease in mRNA expression of Sp7, a Runx2-target gene (Bonewald 2011) (Figure 5E). Taken together, these results suggest that FM550 and TPP can divert MSC differentiation away from osteogenesis and toward adipogenesis and that the FM550 mixture, as a whole, may have a greater effect than TPP.


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

Pillai HK, Fang M, Beglov D, Kozakov D, Vajda S, Stapleton HM, Webster TF, Schlezinger JJ - Environ. Health Perspect. (2014)

Assessment of effects of FM550 (top) and TPP (bottom) on osteogenesis in vitro. Primary bone marrow cultures were established, and osteogenesis was initiated with the addition of ascorbate, β-glycerol phosphate, insulin, and dexamethasone, except for naive wells. Cells were treated with vehicle (Veh; DMSO), FM550 (0.1–10 μg/mL; 0.2–20 μM), TPP (0.1–10 μM), or rosiglitazone (Rosi; 0.1 μM) and cultured for 7 days (gene expression) or 12 days (phenotype). (A) Lipid accumulation. (B) Fabp4 mRNA expression. (C) Alkaline phosphatase activity. (D) Mineralization. (E) Sp7 mRNA expression. Data are presented as mean ± SE of 4–6 independent bone marrow preparations.*p < 0.05, and **p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with Veh treatment.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216168&req=5

f5: Assessment of effects of FM550 (top) and TPP (bottom) on osteogenesis in vitro. Primary bone marrow cultures were established, and osteogenesis was initiated with the addition of ascorbate, β-glycerol phosphate, insulin, and dexamethasone, except for naive wells. Cells were treated with vehicle (Veh; DMSO), FM550 (0.1–10 μg/mL; 0.2–20 μM), TPP (0.1–10 μM), or rosiglitazone (Rosi; 0.1 μM) and cultured for 7 days (gene expression) or 12 days (phenotype). (A) Lipid accumulation. (B) Fabp4 mRNA expression. (C) Alkaline phosphatase activity. (D) Mineralization. (E) Sp7 mRNA expression. Data are presented as mean ± SE of 4–6 independent bone marrow preparations.*p < 0.05, and **p < 0.01, by ANOVA and Dunnett’s multiple comparisons test, compared with Veh treatment.
Mentions: Analysis of effects of FM550 and TPP on bone differentiation in vitro. To test the hypothesis that FM550 and TPP are negative regulators of bone formation, we examined the effect of FM550 and TPP on adipogenic and osteogenic differentiation in primary bone marrow cultures prepared from female C57BL/6J mice. Established bone marrow cultures were induced to undergo osteogenesis and treated with vehicle, FM550, TPP, or rosiglitazone. FM550 induced significant lipid accumulation at a concentration of 5 μg/mL (10 μM), and TPP induced lipid accumulation at a concentration of 10 μM (Figure 5A). Activation of PPARγ by FM550 and TPP was reflected in the significantly increased mRNA expression of Fabp4, a PPARγ-target gene (Tontonoz et al. 1994) (Figure 5B). Although FM550 significantly suppressed both alkaline phosphatase activity and mineralization at 5 μg/mL (10 μM), TPP significantly suppressed only alkaline phosphatase activity (Figure 5C,D). Suppression of the transcriptional program of Runx2, the master regulator of osteogenesis, by FM550 and TPP was indicated by the significant decrease in mRNA expression of Sp7, a Runx2-target gene (Bonewald 2011) (Figure 5E). Taken together, these results suggest that FM550 and TPP can divert MSC differentiation away from osteogenesis and toward adipogenesis and that the FM550 mixture, as a whole, may have a greater effect than TPP.

Bottom Line: Our findings suggest that FM550 components bind and activate PPARγ.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Health, Boston University, Boston, Massachusetts, USA.

ABSTRACT

Background: 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.

Objectives: We tested the hypothesis that components of FM550 are biologically active peroxisome proliferator-activated receptor γ (PPARγ) ligands and estimated indoor exposure to TPP.

Methods: FM550 and its components were assessed for ligand binding to and activation of human PPARγ. Solvent mapping was used to model TPP in the PPARγ 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.

Results: FM550 bound human PPARγ, and binding appeared to be driven primarily by TPP. Solvent mapping revealed that TPP interacted with binding hot spots within the PPARγ ligand binding domain. FM550 and its organophosphate components increased human PPARγ1 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.

Conclusions: Our findings suggest that FM550 components bind and activate PPARγ. 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.

Show MeSH
Related in: MedlinePlus