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Signaling Crosstalk between PPARγ and BMP2 in Mesenchymal Stem Cells.

Takada I, Yogiashi Y, Kato S - PPAR Res (2012)

Bottom Line: Among these factors, BMP2 strongly induces bone formation, but the effect of BMP2 on PPARγ function remains unclear.We examined the effect of BMP2 and PPARγ in ST2 cells and found that PPARγ activation affected BMP2's signaling pathway through epigenetic regulation.These results suggest that the order of treatment with BMP2 and a PPARγ ligand is critical for adipogenesis and osteoblastogenesis switching in MSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Tissue Biology, School of Medicine, Keio University, Tokyo 160-8582, Japan ; Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo 160-8582, Japan.

ABSTRACT
Recent studies have revealed that PPARγ's transactivation function is regulated by extracellular signals. In particular, cytokines and Wnt family proteins suppress the ligand-inducible transactivation function of PPARγ and attenuate adipogenesis/osteoblastogenesis switching in mesenchymal stem cells (MSCs). For example, Wnt5a suppresses PPARγ transcriptional activity through the NLK/SETDB1/CHD7 pathway. Among these factors, BMP2 strongly induces bone formation, but the effect of BMP2 on PPARγ function remains unclear. We examined the effect of BMP2 and PPARγ in ST2 cells and found that PPARγ activation affected BMP2's signaling pathway through epigenetic regulation. Although BMP2 did not interfere with PPARγ-mediated adipogenesis, BMP2 increased mRNA expression levels of PPARγ target genes (such as Fabp4 and Nr1h3) when cells were first treated with troglitazone (TRO). Moreover, PPARγ activation affected BMP2 through enhancement of histone activation markers (acetylated histone H3 and trimethylated Lys4 of histone H3) on the Runx2 promoter. After TRO treatment for three hours, BMP2 enhanced the levels of active histone marks on the promoter of a PPARγ target gene. These results suggest that the order of treatment with BMP2 and a PPARγ ligand is critical for adipogenesis and osteoblastogenesis switching in MSCs.

No MeSH data available.


Related in: MedlinePlus

Adipocyte and osteoblast marker gene mRNA levels were altered by changing the order of BMP2 and TRO treatment. (a) Scheme of experiments used in Figures 2 and 3. We examined five conditions. (-) Untreated ST2 cells were cultured in αMEM supplemented with 10% FBS and penicillin/streptomycin. (TRO) ST2 cells were incubated with one μM TRO for three hr. (BMP) ST2 cells were treated with 50 ng/mL BMP2 for three hr. (TRO-BMP) ST2 cells were treated with TRO for three hr, the medium was changed, and the cells were then incubated with BMP2 for three hr. (BMP-TRO) ST2 cells were treated with BMP2 for three hr and the medium was changed; the cells were then incubated with TRO for three hr. (b) RT-qPCR analyses of adipocyte or osteoblast differentiation marker genes. After ST2 cells were cultured under the conditions described above, cells were collected and RNAs were extracted. Then RT-qPCR experiments were performed and normalized to Gapdh mRNA. Each experiment was performed at least three times. Student's t-test was performed. *P < 0.05.
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fig2: Adipocyte and osteoblast marker gene mRNA levels were altered by changing the order of BMP2 and TRO treatment. (a) Scheme of experiments used in Figures 2 and 3. We examined five conditions. (-) Untreated ST2 cells were cultured in αMEM supplemented with 10% FBS and penicillin/streptomycin. (TRO) ST2 cells were incubated with one μM TRO for three hr. (BMP) ST2 cells were treated with 50 ng/mL BMP2 for three hr. (TRO-BMP) ST2 cells were treated with TRO for three hr, the medium was changed, and the cells were then incubated with BMP2 for three hr. (BMP-TRO) ST2 cells were treated with BMP2 for three hr and the medium was changed; the cells were then incubated with TRO for three hr. (b) RT-qPCR analyses of adipocyte or osteoblast differentiation marker genes. After ST2 cells were cultured under the conditions described above, cells were collected and RNAs were extracted. Then RT-qPCR experiments were performed and normalized to Gapdh mRNA. Each experiment was performed at least three times. Student's t-test was performed. *P < 0.05.

Mentions: BMP2 reportedly inhibits PPARγ-mediated adipogenesis [26]; furthermore, BMP2-dependent osteoblastogenesis is suppressed by PPARγ activation [27]. These reports contradicted our findings. We hypothesized that the order of treatment with BMP2 and PPARγ ligand may attenuate the activation of one or the other differentiation pathway. Thus, we treated ST2 cells with BMP2 and then TRO or with TRO and then BMP2 for three hr each (Figure 2(a)). We then performed RT-qPCR and ChIP analysis (Figure 2(b) and Figures 3(a) and 3(b)). Interestingly, we found that the expression levels of adipocyte or osteoblast marker genes varied according to the order in which TRO and BMP2 were added to the cells. For example, after treatment with BMP2 for three hr, TRO-dependent induction of Fabp4 and Nr1h3 mRNAs (PPARγ target genes in adipocytes [4, 28]) was repressed in ST2 cells (Figure 2(b) left, compare TRO (three hr) with BMP2 (three hr)-TRO (three hr)). Interestingly, after TRO treatment for three hr, BMP2 induced mRNA levels of adipocyte marker genes (Fabp4 and Gpd1). In addition, pretreatment with TRO for three hr enhanced BMP2-dependent Runx2 and alkaline phosphatase (Alpl) mRNA expression (Figure 3(b) right: compare BMP2 (three hr) with TRO (three hr)-BMP2 (three hr)). However, pre-treatment with BMP2 did not enhance TRO-dependent induction of their mRNAs (Figure 2(b)). These results indicated that PPARγ activation enhanced BMP2 activity and pretreatment with PPARγ ligand attenuated osteoblast-related gene expression.


Signaling Crosstalk between PPARγ and BMP2 in Mesenchymal Stem Cells.

Takada I, Yogiashi Y, Kato S - PPAR Res (2012)

Adipocyte and osteoblast marker gene mRNA levels were altered by changing the order of BMP2 and TRO treatment. (a) Scheme of experiments used in Figures 2 and 3. We examined five conditions. (-) Untreated ST2 cells were cultured in αMEM supplemented with 10% FBS and penicillin/streptomycin. (TRO) ST2 cells were incubated with one μM TRO for three hr. (BMP) ST2 cells were treated with 50 ng/mL BMP2 for three hr. (TRO-BMP) ST2 cells were treated with TRO for three hr, the medium was changed, and the cells were then incubated with BMP2 for three hr. (BMP-TRO) ST2 cells were treated with BMP2 for three hr and the medium was changed; the cells were then incubated with TRO for three hr. (b) RT-qPCR analyses of adipocyte or osteoblast differentiation marker genes. After ST2 cells were cultured under the conditions described above, cells were collected and RNAs were extracted. Then RT-qPCR experiments were performed and normalized to Gapdh mRNA. Each experiment was performed at least three times. Student's t-test was performed. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Adipocyte and osteoblast marker gene mRNA levels were altered by changing the order of BMP2 and TRO treatment. (a) Scheme of experiments used in Figures 2 and 3. We examined five conditions. (-) Untreated ST2 cells were cultured in αMEM supplemented with 10% FBS and penicillin/streptomycin. (TRO) ST2 cells were incubated with one μM TRO for three hr. (BMP) ST2 cells were treated with 50 ng/mL BMP2 for three hr. (TRO-BMP) ST2 cells were treated with TRO for three hr, the medium was changed, and the cells were then incubated with BMP2 for three hr. (BMP-TRO) ST2 cells were treated with BMP2 for three hr and the medium was changed; the cells were then incubated with TRO for three hr. (b) RT-qPCR analyses of adipocyte or osteoblast differentiation marker genes. After ST2 cells were cultured under the conditions described above, cells were collected and RNAs were extracted. Then RT-qPCR experiments were performed and normalized to Gapdh mRNA. Each experiment was performed at least three times. Student's t-test was performed. *P < 0.05.
Mentions: BMP2 reportedly inhibits PPARγ-mediated adipogenesis [26]; furthermore, BMP2-dependent osteoblastogenesis is suppressed by PPARγ activation [27]. These reports contradicted our findings. We hypothesized that the order of treatment with BMP2 and PPARγ ligand may attenuate the activation of one or the other differentiation pathway. Thus, we treated ST2 cells with BMP2 and then TRO or with TRO and then BMP2 for three hr each (Figure 2(a)). We then performed RT-qPCR and ChIP analysis (Figure 2(b) and Figures 3(a) and 3(b)). Interestingly, we found that the expression levels of adipocyte or osteoblast marker genes varied according to the order in which TRO and BMP2 were added to the cells. For example, after treatment with BMP2 for three hr, TRO-dependent induction of Fabp4 and Nr1h3 mRNAs (PPARγ target genes in adipocytes [4, 28]) was repressed in ST2 cells (Figure 2(b) left, compare TRO (three hr) with BMP2 (three hr)-TRO (three hr)). Interestingly, after TRO treatment for three hr, BMP2 induced mRNA levels of adipocyte marker genes (Fabp4 and Gpd1). In addition, pretreatment with TRO for three hr enhanced BMP2-dependent Runx2 and alkaline phosphatase (Alpl) mRNA expression (Figure 3(b) right: compare BMP2 (three hr) with TRO (three hr)-BMP2 (three hr)). However, pre-treatment with BMP2 did not enhance TRO-dependent induction of their mRNAs (Figure 2(b)). These results indicated that PPARγ activation enhanced BMP2 activity and pretreatment with PPARγ ligand attenuated osteoblast-related gene expression.

Bottom Line: Among these factors, BMP2 strongly induces bone formation, but the effect of BMP2 on PPARγ function remains unclear.We examined the effect of BMP2 and PPARγ in ST2 cells and found that PPARγ activation affected BMP2's signaling pathway through epigenetic regulation.These results suggest that the order of treatment with BMP2 and a PPARγ ligand is critical for adipogenesis and osteoblastogenesis switching in MSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Tissue Biology, School of Medicine, Keio University, Tokyo 160-8582, Japan ; Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo 160-8582, Japan.

ABSTRACT
Recent studies have revealed that PPARγ's transactivation function is regulated by extracellular signals. In particular, cytokines and Wnt family proteins suppress the ligand-inducible transactivation function of PPARγ and attenuate adipogenesis/osteoblastogenesis switching in mesenchymal stem cells (MSCs). For example, Wnt5a suppresses PPARγ transcriptional activity through the NLK/SETDB1/CHD7 pathway. Among these factors, BMP2 strongly induces bone formation, but the effect of BMP2 on PPARγ function remains unclear. We examined the effect of BMP2 and PPARγ in ST2 cells and found that PPARγ activation affected BMP2's signaling pathway through epigenetic regulation. Although BMP2 did not interfere with PPARγ-mediated adipogenesis, BMP2 increased mRNA expression levels of PPARγ target genes (such as Fabp4 and Nr1h3) when cells were first treated with troglitazone (TRO). Moreover, PPARγ activation affected BMP2 through enhancement of histone activation markers (acetylated histone H3 and trimethylated Lys4 of histone H3) on the Runx2 promoter. After TRO treatment for three hours, BMP2 enhanced the levels of active histone marks on the promoter of a PPARγ target gene. These results suggest that the order of treatment with BMP2 and a PPARγ ligand is critical for adipogenesis and osteoblastogenesis switching in MSCs.

No MeSH data available.


Related in: MedlinePlus