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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.

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Related in: MedlinePlus

Computational analysis of TPP and ITP interactions with the PPARγ LBD. (A) Mapping of the PPARγ LBD structure (PDB ID 2q5s). The protein is shown as a gray cartoon, and representatives of probe clusters within the various consensus clusters are shown as lines. The color code is as follows: CC1 (27 probe clusters), cyan; CC2 (17 probe clusters), magenta; CC3 (16 probe clusters), yellow; CC4 (13 probe clusters), salmon; and CC5 (9 probe clusters), blue. To allow the probe clusters to be seen, some parts of the PPARγ were removed. (B) Close-up of the mapping results. The bound pose of the partial agonist nTZDpa (shown as green sticks) is superimposed for reference; the three rings of nTZDpa are bound at the hot spots, defined by the consensus clusters CC1, CC4, and CC5, respectively, whereas the carboxylic acid of nTZDpa orients toward CC2. (C) Best docked pose of TPP (shown as magenta sticks); the rings in TPP interact with the same three hot spots at CC1, CC4, and CC5. (D) Best docked poses for two isopropylated derivatives of TPP.
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f3: Computational analysis of TPP and ITP interactions with the PPARγ LBD. (A) Mapping of the PPARγ LBD structure (PDB ID 2q5s). The protein is shown as a gray cartoon, and representatives of probe clusters within the various consensus clusters are shown as lines. The color code is as follows: CC1 (27 probe clusters), cyan; CC2 (17 probe clusters), magenta; CC3 (16 probe clusters), yellow; CC4 (13 probe clusters), salmon; and CC5 (9 probe clusters), blue. To allow the probe clusters to be seen, some parts of the PPARγ were removed. (B) Close-up of the mapping results. The bound pose of the partial agonist nTZDpa (shown as green sticks) is superimposed for reference; the three rings of nTZDpa are bound at the hot spots, defined by the consensus clusters CC1, CC4, and CC5, respectively, whereas the carboxylic acid of nTZDpa orients toward CC2. (C) Best docked pose of TPP (shown as magenta sticks); the rings in TPP interact with the same three hot spots at CC1, CC4, and CC5. (D) Best docked poses for two isopropylated derivatives of TPP.

Mentions: The interaction of TPP and ITP with the PPARγ LBD was assessed computationally. In a previous study, the FTMap solvent mapping program (Brenke et al. 2009) was used to identify two main ligand binding regions within PPARγ’s large binding site (Sheu et al. 2005). The first of these binding regions is located at the polar headgroup of thiazolidinediones (TZDs), interacting with the H12 helix of the LBD, and the second is between the distal end of the TZDs and the entrance of the ligand binding site. Because the first region is too narrow for the binding of TPP, we focused on the second site, which is known to bind selective partial agonists (Bruning et al. 2007). Mapping of the latter region shows four binding hot spots (Figure 3A). As shown in Figure 3B, the locations of these hot spots are in good agreement with the positions of the three rings and the carboxylic acid group in several selective partial agonists, such as 5-chloro-1-(4-chlorobenzyl)-3-(phenylthio)-1h-indole-2-carboxylic acid (also called nTZDpa), that bind at this site and activate PPARγ using an H12-independent mechanism (Bruning et al. 2007). In the best docked mode of TPP, the three rings overlap with the three hot spots, which also interact with the rings of nTZDpa (Figure 3C). Ligands that bind to a site generally overlap well with binding hot spots (Hall et al. 2012); therefore, on the basis of our results, we are convinced that TPP binds to PPARγ in a manner very similar to the binding of selective partial agonists. The hot spots extend beyond the rings, and thus PPARγ very likely also binds the various isopropylated derivatives of TPP (Figure 3D).


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)

Computational analysis of TPP and ITP interactions with the PPARγ LBD. (A) Mapping of the PPARγ LBD structure (PDB ID 2q5s). The protein is shown as a gray cartoon, and representatives of probe clusters within the various consensus clusters are shown as lines. The color code is as follows: CC1 (27 probe clusters), cyan; CC2 (17 probe clusters), magenta; CC3 (16 probe clusters), yellow; CC4 (13 probe clusters), salmon; and CC5 (9 probe clusters), blue. To allow the probe clusters to be seen, some parts of the PPARγ were removed. (B) Close-up of the mapping results. The bound pose of the partial agonist nTZDpa (shown as green sticks) is superimposed for reference; the three rings of nTZDpa are bound at the hot spots, defined by the consensus clusters CC1, CC4, and CC5, respectively, whereas the carboxylic acid of nTZDpa orients toward CC2. (C) Best docked pose of TPP (shown as magenta sticks); the rings in TPP interact with the same three hot spots at CC1, CC4, and CC5. (D) Best docked poses for two isopropylated derivatives of TPP.
© Copyright Policy - public-domain
Related In: Results  -  Collection

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

f3: Computational analysis of TPP and ITP interactions with the PPARγ LBD. (A) Mapping of the PPARγ LBD structure (PDB ID 2q5s). The protein is shown as a gray cartoon, and representatives of probe clusters within the various consensus clusters are shown as lines. The color code is as follows: CC1 (27 probe clusters), cyan; CC2 (17 probe clusters), magenta; CC3 (16 probe clusters), yellow; CC4 (13 probe clusters), salmon; and CC5 (9 probe clusters), blue. To allow the probe clusters to be seen, some parts of the PPARγ were removed. (B) Close-up of the mapping results. The bound pose of the partial agonist nTZDpa (shown as green sticks) is superimposed for reference; the three rings of nTZDpa are bound at the hot spots, defined by the consensus clusters CC1, CC4, and CC5, respectively, whereas the carboxylic acid of nTZDpa orients toward CC2. (C) Best docked pose of TPP (shown as magenta sticks); the rings in TPP interact with the same three hot spots at CC1, CC4, and CC5. (D) Best docked poses for two isopropylated derivatives of TPP.
Mentions: The interaction of TPP and ITP with the PPARγ LBD was assessed computationally. In a previous study, the FTMap solvent mapping program (Brenke et al. 2009) was used to identify two main ligand binding regions within PPARγ’s large binding site (Sheu et al. 2005). The first of these binding regions is located at the polar headgroup of thiazolidinediones (TZDs), interacting with the H12 helix of the LBD, and the second is between the distal end of the TZDs and the entrance of the ligand binding site. Because the first region is too narrow for the binding of TPP, we focused on the second site, which is known to bind selective partial agonists (Bruning et al. 2007). Mapping of the latter region shows four binding hot spots (Figure 3A). As shown in Figure 3B, the locations of these hot spots are in good agreement with the positions of the three rings and the carboxylic acid group in several selective partial agonists, such as 5-chloro-1-(4-chlorobenzyl)-3-(phenylthio)-1h-indole-2-carboxylic acid (also called nTZDpa), that bind at this site and activate PPARγ using an H12-independent mechanism (Bruning et al. 2007). In the best docked mode of TPP, the three rings overlap with the three hot spots, which also interact with the rings of nTZDpa (Figure 3C). Ligands that bind to a site generally overlap well with binding hot spots (Hall et al. 2012); therefore, on the basis of our results, we are convinced that TPP binds to PPARγ in a manner very similar to the binding of selective partial agonists. The hot spots extend beyond the rings, and thus PPARγ very likely also binds the various isopropylated derivatives of TPP (Figure 3D).

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