ROLE OF FORKHEAD/FOX TRANSCRIPTION FACTOR 2 OVER-EXPRESSION IN REGULATING OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS BY Wnt SIGNALING PATHWAYS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YOUWulin ¹ ,  WANGJianwei ¹ , HUANGGuicheng ² , ZHANGYafeng ¹

1. Department of Orthopedics, Wuxi Affilliated Hospital of Nanjing University of Chinese Medicine, Wuxi Jiangsu, 214071, P. R. China;
2. Department of Orthopedics, Nanjing University of Chinese Medicine;

Corresponding author：

WANGJianwei, Email: osteowjw@163.com

Keywords：

Forkhead/Fox transcription factor 2; Wnt pathway; Bone marrow mesenchymal stem cells; Osteogenic differentiation; Rabbit

DOI：

10.7507/1002-1892.20160260

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To investigate the role of the forkhead/Fox transcription factor 2 (Foxc2) over-expression in regulating osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by Wnt-β-catenin signaling pathways in vitro so as to provide the experimental basis for repairing osteonecrosis of the femoral head. Methods The recombinant lentivirus carrying green fluorescent protein (group A) or Foxc2 (group B) were used to transfect the fifth generation rabbit BMSCs, and untransfected BMSCs served as a control (group C). The cell viability was measured with water soluble tetrazolium-1 (WST-1) regent at 72 hours after transfection. After 2 weeks of transfection, the expression of β-catenin in BMSCs was detected by real time fluorescence quantitative PCR, Western blot, and immunofluorescence staining. Meanwhile, the β-catenin inhibitors XAV-939 (0, 0.1, and 1.0 μmol/L) was added in group B; at 2 weeks after osteogenic and adipogenic induction, the gene and protein expressions of collagen type I (COL I), osteocalcin (OCN), and peroxisome proliferator activated receptor gamma 2 (PPARγ-2) were detected by real time PCR and Western blot. Results WST-1 results showed that the cell viability of group B (130.85%±0.15%) was significantly higher than that of group A (100.45%±0.35%) (t=7.500, P=0.004) at 72 hours after transfection. At 2 weeks after transfection, the gene and protein expressions of β-catenin in group B were significantly higher than those in group A (P<0.01). After XAV-939 was added in group B, the mRNA and protein expressions of OCN and COL I gradually decreased; the mRNA and protein expressions of PPARγ-2 significantly increased (P<0.05), showing a dose-dependent manner. Conclusion The over-expression of Foxc2 gene in BMSCs may promote osteogenic differentiation by Wnt-β-catenin signaling pathway.

Citation： YOU Wulin, WANG Jianwei, HUANG Guicheng, ZHANG Yafeng. ROLE OF FORKHEAD/FOX TRANSCRIPTION FACTOR 2 OVER-EXPRESSION IN REGULATING OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS BY Wnt SIGNALING PATHWAYS. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(10): 1276-1281. doi: 10.7507/1002-1892.20160260 Copy

1.	Bäckesjö CM, Li Y, Lindgren U, et al. Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells. J Bone Miner Res, 2006, 21(7):993-1002.
2.	Zhang JC, Zheng GF, Wu L, et al. Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats. Braz J Med Biol Res, 2014, 47(10):886-894.
3.	Gutiérrez-Fernández M, Rodríguez-Frutos B, Ramos-Cejudo J, et al. Effects of intravenous administration of allogenic bone marrow and adipose tissue derived mesenchymal stem cells on functional recovery and brain repair markers in experimental ischemic stroke. Stem Cell Res Ther, 2013, 4(1):11.
4.	Hayashi H, Kume T. Forkhead transcription factors regulate expression of the chemokine receptor CXCR4 in endothelial cells and CXCL12-induced cell migration. Biochemo Biophys Res Commun, 2008, 367(3):584-589.
5.	De Val S, Chi NC, Meadows SM, et al. Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors. Cell, 2008, 135(6):1053-1064.
6.	Kim SH, Cho K, Choi HS, et al. The forkhead transcription factor Foxc2 stimulates osteoblast differentiation. Biochem Biophys Res Commun, 2009, 386(3):532-536.
7.	Wong RW, Rabie AB. Statin-induced osteogenesis uses in orthodontics:a scientific review. World J Orthod, 2006, 7(1):35-40.
8.	Johnson ML, Kamel MA. The Wnt signaling pathway and bone metabolism. Curr Opin Rheumatol, 2007, 19(4):376-382.
9.	尤武林, 王坤正, 段大鹏, 等. 插头/翼状螺旋转录因子C2基因慢病毒载体构建及其在BMSCs中的表达. 中国修复重建外科杂志, 2013, 27(5):535-540.
10.	Phinney DG. Biochemical heterogeneity of mesenchymal stem cell populations:clues to their therapeutic efficacy. Cell Cycle, 2007, 6(23):2884-2889.
11.	You WL, Fan LH, Duan DP, et al. Foxc2 over-expression in bone marrow mesenchymal stem cells stimulates osteogenic differentiation and inhibits adipogenic differentiation. Mol Cell Biochem, 2014, 386(1-2):125-134.
12.	Cederberg A, Gronning LM, Ahren B, et al. FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance. Cell, 2001, 106(5):563-573.
13.	Nishimura R, Yoneda T. Role of Wnt in bone formation. Clin Calcium, 2006, 16(5):817-822.
14.	Zeiter S, Lezuo P, Ito K. Effect of TGF beta1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells. Biorheology, 2009, 46(1):45-55.
15.	Eivers E, Demagny H, De Robertis EM. Integration of BMP and Wnt signaling via vertebrate Smad1/5/8 and Drosophila Mad. Cytokine Growth Factor Rev, 2009, 20(5-6):357-365.
16.	Cho SW, Yang JY, Sun HJ, et al. Wnt inhibitory factor (WIF)-1 inhibits osteoblastic differentiation in mouse embryonic mesenchymal cells. Bone, 2009, 44(6):1069-1077.
17.	Ashihara E, Takada T, Maekawa T. Targeting the canonical Wnt/β-catenin pathway in hematological malignancies. Cancer Sci, 2015, 106(6):665-671.
18.	Isaeva AV, Zima AP, Shabalova IP, et al. β-Catenin:structure, function and role in malignant transformation of epithelial cells. Vestn Ross Akad Med Nauk, 2015, (4):475-483.

1. Bäckesjö CM, Li Y, Lindgren U, et al. Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells. J Bone Miner Res, 2006, 21(7):993-1002.
2. Zhang JC, Zheng GF, Wu L, et al. Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats. Braz J Med Biol Res, 2014, 47(10):886-894.
3. Gutiérrez-Fernández M, Rodríguez-Frutos B, Ramos-Cejudo J, et al. Effects of intravenous administration of allogenic bone marrow and adipose tissue derived mesenchymal stem cells on functional recovery and brain repair markers in experimental ischemic stroke. Stem Cell Res Ther, 2013, 4(1):11.
4. Hayashi H, Kume T. Forkhead transcription factors regulate expression of the chemokine receptor CXCR4 in endothelial cells and CXCL12-induced cell migration. Biochemo Biophys Res Commun, 2008, 367(3):584-589.
5. De Val S, Chi NC, Meadows SM, et al. Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors. Cell, 2008, 135(6):1053-1064.
6. Kim SH, Cho K, Choi HS, et al. The forkhead transcription factor Foxc2 stimulates osteoblast differentiation. Biochem Biophys Res Commun, 2009, 386(3):532-536.
7. Wong RW, Rabie AB. Statin-induced osteogenesis uses in orthodontics:a scientific review. World J Orthod, 2006, 7(1):35-40.
8. Johnson ML, Kamel MA. The Wnt signaling pathway and bone metabolism. Curr Opin Rheumatol, 2007, 19(4):376-382.
9. 尤武林, 王坤正, 段大鹏, 等. 插头/翼状螺旋转录因子C2基因慢病毒载体构建及其在BMSCs中的表达. 中国修复重建外科杂志, 2013, 27(5):535-540.
10. Phinney DG. Biochemical heterogeneity of mesenchymal stem cell populations:clues to their therapeutic efficacy. Cell Cycle, 2007, 6(23):2884-2889.
11. You WL, Fan LH, Duan DP, et al. Foxc2 over-expression in bone marrow mesenchymal stem cells stimulates osteogenic differentiation and inhibits adipogenic differentiation. Mol Cell Biochem, 2014, 386(1-2):125-134.
12. Cederberg A, Gronning LM, Ahren B, et al. FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance. Cell, 2001, 106(5):563-573.
13. Nishimura R, Yoneda T. Role of Wnt in bone formation. Clin Calcium, 2006, 16(5):817-822.
14. Zeiter S, Lezuo P, Ito K. Effect of TGF beta1, BMP-2 and hydraulic pressure on chondrogenic differentiation of bovine bone marrow mesenchymal stromal cells. Biorheology, 2009, 46(1):45-55.
15. Eivers E, Demagny H, De Robertis EM. Integration of BMP and Wnt signaling via vertebrate Smad1/5/8 and Drosophila Mad. Cytokine Growth Factor Rev, 2009, 20(5-6):357-365.
16. Cho SW, Yang JY, Sun HJ, et al. Wnt inhibitory factor (WIF)-1 inhibits osteoblastic differentiation in mouse embryonic mesenchymal cells. Bone, 2009, 44(6):1069-1077.
17. Ashihara E, Takada T, Maekawa T. Targeting the canonical Wnt/β-catenin pathway in hematological malignancies. Cancer Sci, 2015, 106(6):665-671.
18. Isaeva AV, Zima AP, Shabalova IP, et al. β-Catenin:structure, function and role in malignant transformation of epithelial cells. Vestn Ross Akad Med Nauk, 2015, (4):475-483.

Chinese Journal of Reparative and Reconstructive Surgery

ROLE OF FORKHEAD/FOX TRANSCRIPTION FACTOR 2 OVER-EXPRESSION IN REGULATING OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS BY Wnt SIGNALING PATHWAYS

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