Foxc2 regulates osteogenesis and angiogenesis of bone marrow mesenchymal stem cells

BMC Musculoskeletal Disorders

Kunzheng Wang¹  Chunsheng Wang¹  Dapeng Duan¹  Lihong Fan¹  Hongwei Gao²  Wulin You¹ 

[1] Department of Orthopedics, The Second Affilliated Hospital of Xi’an Jiaotong University, Xiwu Road, Xi’an, Shaanxi Province, 710004, China;Department of Orthopedics, The Ninth Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi Province, 710000, China

关键词: Differentiation;    Angiogenesis;    Osteogenesis;    Foxc2;    Bone marrow mesenchymal stem cells;   

Others  :  1130376
DOI  :  10.1186/1471-2474-14-199

 received in 2013-04-05, accepted in 2013-06-28,  发布年份 2013

【 摘 要 】

Background

The Forkhead/Fox transcription factor Foxc2 is a critical regulator of osteogenesis and angiogenesis of cells. Bone marrow mesenchymal stem cells (BMSCs) have the capacity to differentiate into osteoblasts, chondrocytes, adipocytes, myocytes and fibroblasts. The present study investigates the role of Foxc2 overexpression in osteogenesis and angiogenesis of BMSCs in vitro.

Methods

BMSCs were isolated from SD rat femurs and tibias, and characterized by cell surface antigen identification and osteoblasts and adipocytes differentiation. The cells were transfected with lentiviral Foxc2 (Lv-Foxc2) or green fluorescent protein (Lv-GFP). Seventy hours later, Foxc2 expression was observed using real time-PCR and Western blot. The transfected cells were stained with Alizarin red S or alkaline phosphatase (ALP) after osteogenic induction. Meanwhile, the expression levels of osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), vascular endothelial growth factor (VEGF) and platelet-derived growth factor-β (PDGF-β) were measured by real time-PCR, Western blot and immunostaining.

Results

Results of cell characterization showed that the cells were positive to CD44 (99.56%) and negative to CD34 (0.44%), and could differentiate into osteoblasts and adipocytes. Foxc2 overexpression not only increased the numbers of mineralized nodes and ALP activity, but also enhanced the expressions of Runx2, OCN, VEGF and PDGF-β in transfected BMSCs after osteogenic induction. The effects of Foxc2 on osteogenesis and angiogenesis were significantly different between Lv-Foxc2 transfected BMSCs and Lv-GFP transfected BMSCs (P<0.05). In addition, the MAPK-specific inhibitors, PD98059 and LY294002, blocked the Foxc2-induced regulation of BMSC differentiation.

Conclusions

Foxc2 gene is successfully transfected into BMSCs with stable and high expression. The overexpression of Foxc2 acts on BMSCs to stimulate osteogenesis and angiogenesis. The effect of Foxc2 on angiogenesis of the cells is mediated via activating PI3K and ERK.

【 授权许可】

   
2013 You et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Karsenty G, Wagner EF: Reaching a genetic and molecular understanding of skeletal development. Dev Cell 2002, 2:389-406.
• [2]Parhami F, Jackson SM, Tintut Y, Le V, Balucan JP, Territo M, Demer LL: Athergenic diet and minimally oxidized low density lipoprotein inhibit osteogenic and promote adipogenic differentiation of marrow stromal cells. J Bone Miner Res 1999, 14:2067-2078.
• [3]Deckers MM, Karperien M, Van Der BC, Yamashita Y, Papapoulos SE, Lowik CW: Expression of vascular endothelial growth factor and their receptors during osteoblast differentiation. Endocrinology 2000, 141:1667-1674.
• [4]Uchida S, Sakai A, Kudo H, et al.: Vascular endothelial growth factor is expressed along with its receptors during the healing process of bone and bone marrow after drill-hole injury in rats. Bone 2003, 32:491-501.
• [5]Ghajar CM, Blevins KS, Hughes CC, George SC, Putnam AJ: Mesenchymal stem cells enhance angiogenesis in mechanically viable prevascularized tissues via early matrix metalloproteinase upregulation. Tissue Eng 2006, 12:2875-2888.
• [6]Ghajar CM, Kachgal S, Kniazeva E, Mori H, Costes SV, George SC, Putnam AJ: Mesenchymal cells stimulate capillary morphogenesis via distinct proteolytic mechanisms. Exp Cell Res 2010, 316:813-825.
• [7]Altman GH, Horan RL, Martin I, et al.: Cell differentiation by mechanical stress. FASEB J 2002, 16(2):270-272.
• [8]Otto WR, Rao J: Tomorrow’s skeleton staff: mesenchymal stem cells and the repair of bone and cartilage. Cell Prolif 2004, 37(1):97-110.
• [9]Vats A, Tolley NS, Buttery LDK, Polak JM: The stem cell in orthopaedic surgery. J Bone Joint Surg 2004, 86(2):159-164.
• [10]Heino TJ, Hentunen TA: Differentiation of osteoblasts and osteocytes from mesenchymal stem cells. Curr Stem Cell Res Ther 2008, 3:131-145.
• [11]Nishimura R, Hata K, Ikeda F, Ichida F, Shimoyama A, Matsubara T, et al.: Signal transduction and transcriptional regulation during mesenchymal cell differentiation. J Bone Miner Metab 2008, 26:203-212.
• [12]Carlsson P, Mahlapuu M: Forkhead transcription factors: key players in development and metabolism. Dev Biol 2002, 250(1):1-23.
• [13]Stephen S, Myatt , Eric Lam W-F: The emerging roles of forkhead box(Fox) proteins in cancer. Nat Rev Cancer 2007, 7(11):847-859.
• [14]Winner GE, Hargett L, Hogan BL: The winged helix transcription factor MFH1 is required for proliferation and patterning of paraxial mesoderm in the mouse embryo. Genes Dev 1997, 11(7):926-940.
• [15]Furumoto TA, Miura N, Akasaka T, et al.: Notochord-dependent expression of MFH1 and PAX1 cooperates to maintain the proliferation of sclerotome cells during the vertebral column development. Dev Biol 1999, 210(1):15-29.
• [16]Davis KE, Moldes M, Farmer SR: The forkhead transcription factor Foxc2 inhibits white adipocyte differentiation. J Biol Chem 2004, 279:42453-42461.
• [17]Hayashi H, Kume T: Forkhead transcription factors regulate expression of the chemokine receptor CXCR4 in endothelial cells and CXCL12-induced cell migration. Biochem Biophys Res Commun 2008, 367:584-589.
• [18]Hayashi H, Sano H, Seo S, Kume T: The Foxc2 transcription factor regulates angiogenesis via induction of integrin beta3 expression. J Biol Chem 2008, 283:23791-23800.
• [19]Sano H, Leboeuf JP, Novitskiy SV, Seo S, Zaja-Milatovic S, Dikov MM: The Foxc2 transcription factor regulates tumor angiogenesis. Biochem Biophys Res Commun 2010, 392:201-206.
• [20]Mani SA, Yang J, Brooks M, Schwaninger G, Zhou A, Miura N: Mesenchyme Forkhead 1(FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci USA 2007, 104:10069-10074.
• [21]Panepucci RA, Siufi JL, Silva WA Jr, Proto-Siquiera R, Neder L, Orellana M, et al.: Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells. Stem Cells 2004, 22(7):1263-1278.
• [22]Sun H, Wu C, Dai K, Chang J, Tang T: Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite- bioactive ceramics. Biomaterials 2006, 27(33):5651-5657.
• [23]Dominici M, Le Blanc K, Mueller I, et al.: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006, 8(4):315-317.
• [24]Caplan AI, Correa D: PDGF in bone formation and regeneration: new insights into a novel mechanism involving MSCs. J Orthop Res 2011, 29:1795-1803.
• [25]Yang J, Weinberg RA: Epithelial-Mesenchymal Transition: At the Crossroads of Development and Tumor Metastasis. Dev Cell 2008, 14:818-829.
• [26]Battula VL, Mani SA: Epithelial-mesenchymal transition-derived cells exhibit multilineage differentiation potential similar to mesenchymal stem cells. Stem Cells 2010, 28(8):1435-1445.
• [27]Zeisberg Raghu Kalluri EM: Discovery of Endothelial to Mesenchymal Transition as a Source for Carcinoma-Associated Fibroblasts. Cancer Res 2007, 67(21):10123-10128.
• [28]Hader C, Marlier A, Cantley L: Mesenchymal–epithelial transition in epithelial response to injury: the role of Foxc2. Oncogene 2010, 29:1031-1040.
• [29]Kim SH, Cho K, Choi HS, Park SJ, Rhee Y, Jung H, et al.: The forkhead transcription factor Foxc2 stimulates osteoblast differentiation. Biochem Biophys Res Commun 2009, 386:532-536.
• [30]Park SJ, Gadi J, Cho KW, et al.: The forkhead transcription factor Foxc2 promotes osteoblastogenesis via up-regulation of integrin β1 expression. Bone 2011, 49:428-438.
• [31]Kume T, Jiang H, Topczewska JM, Hogan BL: The murine winged helix transcription factors, Foxc1 and Foxc2, are both required for cardiovascular development and somitogenesis. Genes Dev 2001, 15(18):2470-2482.
• [32]Seo S, Fujita H, Nakano A, Kang M, Duarte A, Kume T: The forkhead transcription factors, Foxc1 and Foxc2, are required for arterial specification and lymphatic sprouting during vascular development. Dev Biol 2006, 294(2):458-470.
• [33]Li D, Yan D, Ruan Q, et al.: Foxc2 overexpression enhances benefit of endothelial progenitor cells for inhibiting neointimal formation by promoting CXCR4-dependent homing. J Vasc Surg 2011, 53(6):1668-1678.
• [34]Mortazavi F, An JB, Dubinett S, Rettig M: p120-Catenin Is Transcriptionally Downregulated by FOXC2 in Non-Small Cell Lung Cancer Cells. Mol Cancer Res 2010, 8:762-774.
• [35]Burger A, Kipps TJ: CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment. Blood 2006, 107:1761-1767.
• [36]Hong CC, Kume T, Peterson RT: Role of Cross Talk between PI3-Kinase and ERK/MAP Kinase Pathways in Artery-Vein Specification. Circ Res 2008, 103(6):573-579.
• [37]Hayashi H, Kume T: Foxc Transcription Factors Directly Regulate Dll4 and Hey2 Expression by Interacting with the VEGF-Notch Signaling Pathways in Endothelial Cells. PLoS One 2008, 3(6):e2401.
• [38]Blum S, Issbruker K, Willuweit A, et al.: An inhibitory role for the phosphatidylinositol 3-kinase-signaling pathway in vascular endothelial growth factor-induced tissue factor expression. J Biol Chem 2001, 276:33428-33434.
• [39]Li Z, Liu C, Xie Z: Epigenetic dysregulation in mesenchymal stem cell aging and spontaneous differentiation. PLoS One 2011, 6(6):e20526.