Structurally-diverse, PPARγ-activating Environmental Toxicants Induce Adipogenesis and Suppress Osteogenesis in Bone Marrow Mesenchymal Stromal Cells

Abstract

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 γ (PPARγ), 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 PPARγ, and ligands may selectively alter these activities. It currently is unknown whether suppression of osteogenesis is a common toxic endpoint of environmental PPARγ ligands. Using a primary mouse bone marrow culture model, we tested the hypothesis that environmental toxicants acting as PPARγ 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 PPARγ1 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 PPARγ ligands represent an emerging threat to human bone health.

Keywords: Mono-(2-ethylhexyl) phthalate; Mono-(2-ethylhexyl) tetrabromophthalate; Organotin; Osteoblast; PPARγ; Tetrabromobisphenol-a.

Figure 1. Environmental toxicantss activate PPARγ1 and PPARγ2 with differing potencies and efficacies

Cos-7 cells transfected with mouse PPARγ1 or PPARγ2 and a PPRE-luciferase reporter plasmid were treated with the indicated compounds. Luminescence normalized to GFP fluorescence was divided by the normalized luminescence of untreated cultures to calculate fold change from untreated. n = 4–6 independent transfections.

Figure 2. Known modulators of adipogenesis increase lipid accumulation and expression of adipogenic genes in mouse BM-MSCs

Primary bone marrow cultures were established from male C57BL/6J mice and treated with vehicle (Vh, DMSO), rosiglitazone (Rosi, 100 nM) or TBT (100 nM) in the presence of osteoinductive media for 7 (gene expression) or 11 days (lipid accumulation). (A) Lipid accumulation was quantified by Nile Red staining. (B–D) mRNA expression was quantified by RT-qPCR. Data are presented as means ± SE (n = 4–8 independent bone marrow preparations). *p < 0.05, **p < 0.01 compared to Vh-treated cultures (ANOVA, Dunnett’s). Vehicle-treated cells showed increases in expression of adipocyte-related genes relative to undifferentiated cells (1.9-fold for PPARγ, 7.7-fold for Fabp4, and 144.4-fold for Plin1).

Figure 3. Structurally distinct environmental PPARγ ligands induce lipid accumulation and adipogenic gene expression in mouse BM-MSCs

Primary bone marrow cultures were established from male C57BL/6J mice and treated with Vh (DMSO), TPhT (10–80 nM; A), MEHP (10–20 µM; B), METBP (10–20 µM; C) or TBBPA (10–20 µM; D) in the presence of osteoinductive media for 7 (gene expression) or 11 days (lipid accumulation). Lipid accumulation was quantified by Nile Red staining. mRNA expression was quantified by RT-qPCR. Data are presented as means ± SE (n = 4–8 independent bone marrow preparations). *p < 0.05, **p < 0.01 compared to Vh-treated cultures (ANOVA, Dunnett’s).

Figure 4. Environmental PPARγ ligands induce perilipin protein expression in mouse BM–MSCs

Primary bone marrow cultures were established from male C57BL/6J mice and treated with Vh (DMSO), rosiglitazone (Rosi, 100 nM), TBT (100 nM), TPhT (50 nM), MEHP, TBBPA, or METBP (20 µM) in the presence of osteoinductive media. Cells were harvested after 7 days. Perilipin and β-actin expression were determined in whole cell lysates by immunoblot. Image is representative of 4 separate experiments.

Figure 5. Rosiglitazone and TBT suppress osteogenesis in mouse BM-MSCs

Primary bone marrow cultures were established from male C57BL/6J mice and treated with vehicle (Vh, DMSO), rosiglitazone (Rosi, 100 nM) or TBT (100 nM) in the presence of osteoinductive media for 7 (gene expression) or 11 days (osteogenesis assays). (A) Osteogenesis was assessed via alkaline phosphatase activity, alizarin staining and bone nodule counting. (B) mRNA expression was quantified by RT-qPCR. Data are presented as means ± SE (n = 5–8 independent bone marrow preparations). *p < 0.05, **p < 0.01 compared to Vh-treated cultures (ANOVA, Dunnett’s).

Figure 6. Structurally distinct environmental PPARγ ligands suppress osteogenesis in mouse BM-MSCs

Primary bone marrow cultures were established from male C57BL/6J mice and treated with Vh (DMSO), TPhT (10–80 nM; A), MEHP (10–20 µM; B), METBP (10–20 µM; C), or TBBPA (10–20 µM; D), in the presence of osteoinductive media for 11 days. Osteogenesis was assessed by alkaline phosphatase activity, alizarin staining and bone nodule counting. Data are presented as means ± SE (n = 5–8 independent bone marrow preparations). *p < 0.05, **p < 0.01 compared to Vh-treated cultures (ANOVA, Dunnett’s).

Figure 7. Structurally distinct environmental PPARγ ligands suppress osteogenic gene expression in mouse BM-MSCs

Primary bone marrow cultures were established from male C57BL/6J mice and treated with Vh (DMSO), TPhT (10–80 nM; A), MEHP (10–20 µM; B), METBP (10–20 µM; C), TBBPA (10–20 µM; D) in the presence of osteoinductive media for 7 days. mRNA expression was quantified by RT-qPCR. Data are presented as means ± SD (n = 4–7 independent bone marrow preparations). *p < 0.05, **p < 0.01 compared to Vh-treated cultures (ANOVA, Dunnett’s).

Figure 8. Adipogenesis is inversely correlated with osteogenesis in PPARγ-ligand treated mouse BMMSCs

Data points are representative of mean endpoint values for individual treatments and concentrations reported in Figs 2, 3, 5, 6, 7. (A) Fold change in alkaline phosphatase activity vs. lipid accumulation (Nile Red fluorescence) (All data Pearson’s r = −0.51). (B) Bone nodule number vs. lipid accumulation (All data Pearson’s r = −0.01). (C) Osx mRNA expression vs. Fabp4 mRNA expression (All data Pearson’s r = −0.57). ■ – Vh (DMSO); □ – Rosiglitazone (100 nM); ● – METBP; ○ – MEHP; Δ – TBBPA;×– TBT or TPhT. Linear fit excludes organotin data points (x). r = Pearson’s correlation coefficient (excluding organotins), number of pairs = 8.

Figure 9. Differential suppression of osteogenic Wnt10b mRNA does not explain efficacious effect of organotins on osteogenesis

Primary bone marrow cultures were established from male C57BL/6J mice and treated with Vh (DMSO), rosiglitazone (100 nM), TBT (100 nM), TPhT (10–80 nM), MEHP (10–20 µM) in the presence of osteoinductive media for 7 days. mRNA expression was quantified by RT-qPCR. Data are presented as means ± SE (n = 4–10 independent bone marrow preparations). *p < 0.05, **p < 0.01 compared to Vh-treated cultures (ANOVA, Dunnett’s).