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. 2009 May;18(4):545-59.
doi: 10.1089/scd.2008.0130.

A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells

Quan Kang  1 , Wen-Xin Song, Qing Luo, Ni Tang, Jinyong Luo, Xiaoji Luo, Jin Chen, Yang Bi, Bai-Cheng He, Jong Kyung Park, Wei Jiang, Yi Tang, Jiayi Huang, Yuxi Su, Gao-Hui Zhu, Yun He, Hong Yin, Zhenming Hu, Yi Wang, Liang Chen, Guo-Wei Zuo, Xiaochuan Pan, Jikun Shen, Tamara Vokes, Russell R Reid, Rex C Haydon, Hue H Luu, Tong-Chuan He
Affiliations

A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells

Quan Kang et al. Stem Cells Dev. 2009 May.

Abstract

Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of MSCs. Here, we conducted a comprehensive analysis of the 14 types of bone morphogenetic protein (BMPs) for their abilities to regulate multilineage specific differentiation of MSCs. We found that most BMPs exhibited distinct abilities to regulate the expression of Runx2, Sox9, MyoD, and PPARgamma2. Further analysis indicated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively induced both adipogenic and osteogenic differentiation in vitro and in vivo. BMP-induced commitment to osteogenic or adipogenic lineage was shown to be mutually exclusive. Overexpression of Runx2 enhanced BMP-induced osteogenic differentiation, whereas knockdown of Runx2 expression diminished BMP-induced bone formation with a decrease in adipocyte accumulation in vivo. Interestingly, overexpression of PPARgamma2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARgamma2 knockdown or mouse embryonic fibroblasts derived from PPARgamma2(-/-) mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARgamma2 may play a role in BMP-induced osteogenic and adipogenic differentiation. Thus, it is important to understand the molecular mechanism behind BMP-regulated lineage divergence during MSC differentiation, as this knowledge could help us to understand the pathogenesis of skeletal diseases and may lead to the development of strategies for regenerative medicine.

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Figures

FIG. 1.
FIG. 1.
Quantitative analysis of the four lineage-specific regulators affected by the 14 types of BMPs in C3H10T1/2 MSCs. BMP-regulated expression of the lineage-specific regulators in MSCs was assessed with quantitative real-time PCR. The MSCs were infected with AdBMPs or AdGFP for 5 days prior to RNA isolation for real time-PCR analysis. (A) Expression of osteoblastic lineage regulator Runx2. (B) Expression of chondrogenic lineage regulator Sox9. (C) Expression of myogenic lineage regulator MyoD. (D) Expression of adipogenic lineage regulator PPARγ2. The dotted lines indicate 50% upregulation of gene expression.
FIG. 2.
FIG. 2.
BMP-induced osteogenic and adipogenic differentiation in MSCs. (A) Osteogenic differentiation. C3H10T1/2 cells were infected with AdBMPs or AdGFP for 7 days, followed by histochemical staining of early osteogenic marker ALP activity. (B) Adipogenic differentiation. C3H10T1/2 cells were infected with AdBMPs or AdGFP for 20 days, followed by Oil Red-O staining. PPARγ agonist Troglitazone (TRO, 10 μM) was used as a positive control.
FIG. 3.
FIG. 3.
BMP-induced osteogenic and adipogenic differentiation in MSCs is mutually exclusive. (A) ALP and Oil Red-O costaining. C3H10T1/2 cells were infected with AdBMP-2, −4, −6, −7, −9, or AdGFP. At Day 14, cells were fixed and first stained for ALP activity, followed by Oil Red-O staining. (B) Transmission electron microscope analysis of BMP-induced MSC osteogenic and adipogenic differentiation. C3H10T1/2 cells were infected with the indicated AdBMP-2, AdBMP-9, or AdGFP. At Day 7 and Day 14, cells were processed for transmission electron microscopy (TEM) analysis. Magnification, 12,000×. Abbreviations: AP, membrane-bound ALP activity; LP, accumulated lipid droplets.
FIG. 4.
FIG. 4.
BMP-2 and BMP-9 induce both osteogenic and adipogenic differentiation in vivo. (A) MicroCT analysis of the tissue masses formed by GFP, BMP-2, or BMP-9–transduced MSCs. Transduced cells were implanted subcutaneously (5 × 106 cells per injection) into the flanks of athymic nude mice. After 5 weeks, animals were sacrificed and subjected to microCT analysis and histologic evaluation. Arrows indicate injection sites. (B) Histology of the tissue masses formed by BMP2- or BMP-9–transduced MSCs. Serial sections of decalcified tissues were stained with H&E. Dotted lines indicate the margin of masses. Boxed regions indicate the subsequently magnified areas. (C) Cartilage formation assessed with Alcian Blue staining. Serial sections were stained with Alcian Blue. Cartilage matrix was stained blue. (D) Mineralized bone matrix assessed with Masson's Trichrome staining. Serial sections of the decalcified tissues were stained with Masson's Trichrome. Fully mineralized bone matrix was stained red, while unmineralized osteoid matrix was stained light or dark blue. (E) Adipocyte formation assessed with Oil Red-O staining. Decalcified tissues were frozen sectioned and stained with Oil Red-O. Lipid-containing/producing cells, including preadipocytes, were stained red. Abbreviations: Ac, adipocyte; Ch, chondrocyte; MB, mineralized bone matrix; OM, osteoid matrix; PAc, preadipocyte; PC, progenitor cells.
FIG. 5.
FIG. 5.
Effect of overexpression of Runx2 or PPARγ2 on BMP-induced osteogenic and adipogenic differentiation. (A) Synergistic effect of Runx2 on BMP-induced ALP activity. C3H10T1/2 cells were coinfected with BMP-2, −6, −9, GFP, and/or Runx2 adenoviral vectors. ALP activity was assayed at Day 7. (B) Runx2 exhibits no significant effect on BMP-induced adipogenic differentiation. C3H10T1/2 cells were coinfected with BMP-2, −6, −9, GFP, and/or Runx2 adenoviral vectors. Oil Red-O stain was carried out at Day 14. (C) PPARγ2 enhances BMP-induced ALP activity. C3H10T1/2 cells were coinfected with BMP-2, −6, −9, GFP, and/or PPARγ2 adenoviral vectors. ALP activity was analyzed at Day 7. (D) PPARγ2 augments BMP-induced adipogenic differentiation. C3H10T1/2 cells were coinfected with BMP-2, −6, −9, GFP, and/or PPARγ2 adenoviral vectors. Oil Red-O stain was conducted at Day 14. (E) PPARγ2 overexpression enhances both adipogenic and osteogenic differentiation in vivo. C3H10T1/2 cells were transduced with BMP-9, GFP, and/or PPARγ2 adenoviral vectors and implanted subcutaneously (5 × 106 cells per injection) into the flanks of athymic nude mice. After 5 weeks, animals were sacrificed and subjected to microCT analysis (see Fig. 7B) and histologic evaluation. Abbreviations: Ac, adipocyte; MB, mineralized bone matrix.
FIG. 6.
FIG. 6.
Effect of RNAi-mediated silencing of Runx2 and PPARγ2 on BMP-induced osteogenic and adipogenic differentiation. (A) Verification of silencing Runx2 expression in C3H10-Runx2-kd and empty vector stable lines by qPCR. (B) Runx2 knock-down inhibits BMP-induced ALP activity. C3H10-Runx2-kd and empty vector stable lines were infected with AdBMP-2, AdBMP-6, AdBMP-9, or AdGFP. ALP assays were carried out 7 days after infection. (C) Verification of silencing PPARγ2 expression in C3H10-PPARγ2-kd and empty vector stable lines by qPCR. (D) PPARγ2 knock-down inhibits BMP-induced ALP activity. C3H10- PPARγ2-kd and empty vector stable lines were infected with AdBMP-2, AdBMP-6, AdBMP-9, or AdGFP. ALP assays were carried out 7 days after infection. (E) Runx2 knock-down exerts no significant effect on BMP-induced adipogenic differentiation. C3H10-Runx2-kd and empty vector stable lines were infected with AdBMP-2, AdBMP-6, AdBMP-9, or AdGFP. Oil Red-O stain was carried out 14 days after infection. (F) PPARγ2 knock-down exerts no significant effect on BMP-induced adipogenic differentiation. C3H10-PPARγ2-kd and empty vector stable lines were infected with AdBMP-2, AdBMP-6, AdBMP-9, or AdGFP. Oil Red-O stain was carried out 14 days after infection.
FIG. 7.
FIG. 7.
The in vivo effect of Runx2 and PPARγ2 depletion on BMP-9–induced osteogenic and adipogenic differentiation. (A) MicroCT analysis of BMP-9–induced bone formation affected by PPARγ2 overexpression, PPARγ2 knock-down, and Runx2 knockdown. Cell lines were transduced with AdBMP-9, AdGFP, and AdBMP-9+AdPPARγ2 for 15 h and subjected to subcanteous implantation (5 × 106 cells per injection) into the flanks of athymic nude mice. After 5 weeks, animals were sacrificed and subjected to microCT analysis and histologic evaluation. Dotted lines indicate the margin of the ossified masses. (B) Histologic evaluation of the ossified masses retrieved from (A). (C) qPCR analysis of PPARγ2 expression in MEFs derived from PPARγ2−/− and PPARγ2−/− animals. (D) BMP-9–induced ALP activity and Oil Red-O stain in PPARγ2+/− and PPARγ2−/− MEFs. The MEFs were infected with AdBMP-9 and stained for ALP activity and Red Oil-O at Day 7 and Day 14, respectively. (E) Genetic deletion of PPARγ2 significantly inhibits BMP-9–induced ossification. BMP-9–transduced MEFs were subcutaneously implanted in athymic nude mice. MicroCT analysis was carried out 5 weeks after implantation. Dotted lines indicate the margin of the palpable masses. (F) Histologic evaluation of the samples retrieved from (E). Abbreviations: Ac, adipocyte; Ch, chondrocyte; MB, mineralized bone matrix; OM, osteoid matrix.
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