EFFECT OF Hey1 EXPRESSION ON OSTEOGENIC DIFFERENTIATION AND PROLIFERATION OF C3H10T1/2 CELLS INDUCED BY BONE MORPHOGENETIC PROTEIN 9_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANGMiao , WANGNan , QUANZhengxue ,  LUOXiaoji

Department of Orthopaedics, First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, P.R.China;

Corresponding?author：

LUOXiaoji, Email: cy2982@163.com

Keywords：

Hey1; Bone morphogenetic protein 9; C3H10T1/2 cells; Osteogenic differentiation; Proliferation

DOI：

10.7507/1002-1892.20160056

Video：

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Objective To investigate the effect of Notch signaling pathway important target Hey1 expression on the differentiation and proliferation of C3H10T1/2 cells induced by bone morphogenetic protein 9 (BMP-9). Methods Hey1 lentivirus and Hey1 short hairpin RNA lentivirus were constructed and used to infect C3H10T1/2 cells to change the expression level of Hey1 in C3H10T1/2 cells. C3H10T1/2 cells infected with LV-Blank (empty plasmid) as control. The Hey1 expression levels of different groups were detected by fluorescence microscope, real-time fluorescence quantitative PCR, and Western blot. The C3H10T1/2 cells with different Hey1 expression level were induced by BMP-9 conditioned medium (BMP-9+C3H10T1/2 group, BMP-9+C3H10T1/2-Hey1 group, and BMP-9+C3H10T1/2-shHey1 group); the cells of control groups (C3H10T1/2 group and C3H10T1/2-Blank group) were cultured with normal medium. The mRNA and protein expression levels of osteogenesis related transcription factors (Runx2, osteopontin, and osteocalcin) were detected at 48 hours by real-time fluorescence quantitative PCR and Western blot assay. The cells proliferation and cycles were detected by MTT assay at 4, 5, 6, and 7 days and flow cytometry at 4, 5, and 10 days. The alkaline phosphatase (ALP) activity was analyzed by ELISA and observed by ALP staining at 4 and 7 days. Results C3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In osteogenesis compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 enhanced the mRNA and protein expressions of transcription factors (Runx2, osteopontin, and osteocalcin), and the expression of osteogenic differentiation marker (ALP) (P < 0.05); however, inhibition of Hey1 expression significantly decreased the above indexes (P < 0.05). In cell proliferation activity compared with BMP-9+C3H10T1/2 group, overexpression of Hey1 increased absorbance (A) value in MTT assay and pecentage of G2+S cells in cytometry assay, but inhibition of Hey1 expression significantly decreased the indexes (P < 0.05). Conclusion Expression of Hey1 is the important link in the osteogenic differentiation process of C3H10T1/2 cells induced by BMP-9, and plays an important role in the regulation of early cell proliferation.

Citation： WANGMiao, WANGNan, QUANZhengxue, LUOXiaoji. EFFECT OF Hey1 EXPRESSION ON OSTEOGENIC DIFFERENTIATION AND PROLIFERATION OF C3H10T1/2 CELLS INDUCED BY BONE MORPHOGENETIC PROTEIN 9. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(3): 279-285. doi: 10.7507/1002-1892.20160056 Copy

1.	Selvamurugan N, Kwok S, Vasilov A, et al. Effects of BMP-2 and pulsed electromagnetic field (PEMF) on rat primary osteoblastic cell proliferation and gene expression. J Orthop Res, 2007, 25(9): 1213-1220.
2.	唐大剛, 王淼, 張艷亮, 等. Hey1基因參與調控BMP9誘導的C3H10T1/2細胞成骨分化.中國生物工程雜志, 2015, 35(6): 14-20.
3.	Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2, -4, and-6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res, 2005, 320(2): 269-276.
4.	Qian X, Zhang C, Chen G, et al. Effects of BMP-2 and FGF2 on the osteogenesis of bone marrow-derived mesenchymal stem cells in hindlimb-unloaded rats. Cell Biochem Biophys, 2014, 70(2): 1127-1136.
5.	Zhang X, Guo WG, Cui H, et al. In vitro and in vivo enhancement of osteogenic capacity in a synthetic BMP-2 derived peptide-coated mineralized collagen composite. J Tissue Eng Regen Med, 2013. [Epub ahead of print].
6.	Wegman F, Bijenhof A, Schuijff L, et al. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo. Eur Cell Mater, 2011, 21: 230-242.
7.	Zhang Y, Madhu V, Dighe AS, et al. Osteogenic response of human adipose-derived stem cells to BMP-6, VEGF, and combined VEGF plus BMP-6 in vitro. Growth Factors, 2012, 30(5): 333-343.
8.	Cheng H, Jiang W, Phillips FM, et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg (Am), 2003, 85-A(8): 1544-1552.
9.	Sharff KA, Song WX, Luo X, et al. Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. J Biol Chem, 2009, 284(1): 649-659.
10.	Lavery K, Hawley S, Swain P, et al. New insights into BMP-7 mediated osteoblastic differentiation of primary human mesenchymal stem cells. Bone, 2009, 45(1): 27-41.
11.	Ugarte F, Ryser M, Thieme S, et al. Notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human bone marrow stromal cells. Exp Hematol, 2009, 37(7): 867-875.
12.	Weber D, Wiese C, Gessler M. Hey bHLH transcription factors. Curr Top Dev Biol, 2014, 110: 285-315.
13.	張曉艷, 楊倫韻, 謝佳瑛, 等.攜帶siRbpj重組腺病毒載體的構建及其對BMP9誘導骨髓間充質干細胞成骨分化作用影響.重慶醫科大學學報, 2014, 39(5): 601-606.
14.	Moellering RE, Cornejo M, Davis TN, et al. Direct inhibition of the NOTCH transcription factor complex. Nature, 2009, 462(7270): 182-188.
15.	Tezuka K, Yasuda M, Watanabe N, et al. Stimulation of osteoblastic cell differentiation by Notch. J Bone Miner Res, 2002, 17(2): 231-239.
16.	McLarren KW, Lo R, Grbavec D, et al. The mammalian basic helix loop helix protein HES-1 binds to and modulates the transactivating function of the runt-related factor Cbfa1. J Biol Chem, 2000, 275(1): 530-538.
17.	Suh JH, Lee HW, Lee JW, et al. Hes1 stimulates transcriptional activity of Runx2 by increasing protein stabilization during osteoblast differentiation. Biochem Biophys Res Commun, 2008, 367(1): 97-102.
18.	Lin GL, Hankenson KD. Integration of BMP, Wnt, and notch signaling pathways in osteoblast differentiation. J Cell Biochem, 2011, 112(12): 3491-3501.
19.	Nobta M, Tsukazaki T, Shibata Y, et al. Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling. J Biol Chem, 2005, 280(16): 15842-15848.
20.	Tezuka K, Yasuda M, Watanabe N, et al. Stimulation of osteoblastic cell differentiation by Notch. J Bone Miner Res, 2002, 17(2): 231-239.
21.	趙艷芳, 宋濤, 劉躍亮, 等. RUNX2對BMP9誘導的間充質干細胞C3H10T1/2成骨分化的影響.中國生物工程雜志, 2013, 33(2): 21-26.
22.	Sharff KA, Song WX, Luo X, et al. Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. J Biol Chem, 2009, 284(1): 649-659.
23.	Salie R, Kneissel M, Vukevic M, et al. Ubiquitous overexpression of Hey1 transcription factor leads to osteopenia and chondrocyte hypertrophy in bone. Bone, 2010, 46(3): 680-694.

1. Selvamurugan N, Kwok S, Vasilov A, et al. Effects of BMP-2 and pulsed electromagnetic field (PEMF) on rat primary osteoblastic cell proliferation and gene expression. J Orthop Res, 2007, 25(9): 1213-1220.
2. 唐大剛, 王淼, 張艷亮, 等. Hey1基因參與調控BMP9誘導的C3H10T1/2細胞成骨分化.中國生物工程雜志, 2015, 35(6): 14-20.
3. Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2, -4, and-6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res, 2005, 320(2): 269-276.
4. Qian X, Zhang C, Chen G, et al. Effects of BMP-2 and FGF2 on the osteogenesis of bone marrow-derived mesenchymal stem cells in hindlimb-unloaded rats. Cell Biochem Biophys, 2014, 70(2): 1127-1136.
5. Zhang X, Guo WG, Cui H, et al. In vitro and in vivo enhancement of osteogenic capacity in a synthetic BMP-2 derived peptide-coated mineralized collagen composite. J Tissue Eng Regen Med, 2013. [Epub ahead of print].
6. Wegman F, Bijenhof A, Schuijff L, et al. Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo. Eur Cell Mater, 2011, 21: 230-242.
7. Zhang Y, Madhu V, Dighe AS, et al. Osteogenic response of human adipose-derived stem cells to BMP-6, VEGF, and combined VEGF plus BMP-6 in vitro. Growth Factors, 2012, 30(5): 333-343.
8. Cheng H, Jiang W, Phillips FM, et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg (Am), 2003, 85-A(8): 1544-1552.
9. Sharff KA, Song WX, Luo X, et al. Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. J Biol Chem, 2009, 284(1): 649-659.
10. Lavery K, Hawley S, Swain P, et al. New insights into BMP-7 mediated osteoblastic differentiation of primary human mesenchymal stem cells. Bone, 2009, 45(1): 27-41.
11. Ugarte F, Ryser M, Thieme S, et al. Notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human bone marrow stromal cells. Exp Hematol, 2009, 37(7): 867-875.
12. Weber D, Wiese C, Gessler M. Hey bHLH transcription factors. Curr Top Dev Biol, 2014, 110: 285-315.
13. 張曉艷, 楊倫韻, 謝佳瑛, 等.攜帶siRbpj重組腺病毒載體的構建及其對BMP9誘導骨髓間充質干細胞成骨分化作用影響.重慶醫科大學學報, 2014, 39(5): 601-606.
14. Moellering RE, Cornejo M, Davis TN, et al. Direct inhibition of the NOTCH transcription factor complex. Nature, 2009, 462(7270): 182-188.
15. Tezuka K, Yasuda M, Watanabe N, et al. Stimulation of osteoblastic cell differentiation by Notch. J Bone Miner Res, 2002, 17(2): 231-239.
16. McLarren KW, Lo R, Grbavec D, et al. The mammalian basic helix loop helix protein HES-1 binds to and modulates the transactivating function of the runt-related factor Cbfa1. J Biol Chem, 2000, 275(1): 530-538.
17. Suh JH, Lee HW, Lee JW, et al. Hes1 stimulates transcriptional activity of Runx2 by increasing protein stabilization during osteoblast differentiation. Biochem Biophys Res Commun, 2008, 367(1): 97-102.
18. Lin GL, Hankenson KD. Integration of BMP, Wnt, and notch signaling pathways in osteoblast differentiation. J Cell Biochem, 2011, 112(12): 3491-3501.
19. Nobta M, Tsukazaki T, Shibata Y, et al. Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling. J Biol Chem, 2005, 280(16): 15842-15848.
20. Tezuka K, Yasuda M, Watanabe N, et al. Stimulation of osteoblastic cell differentiation by Notch. J Bone Miner Res, 2002, 17(2): 231-239.
21. 趙艷芳, 宋濤, 劉躍亮, 等. RUNX2對BMP9誘導的間充質干細胞C3H10T1/2成骨分化的影響.中國生物工程雜志, 2013, 33(2): 21-26.
22. Sharff KA, Song WX, Luo X, et al. Hey1 basic helix-loop-helix protein plays an important role in mediating BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. J Biol Chem, 2009, 284(1): 649-659.
23. Salie R, Kneissel M, Vukevic M, et al. Ubiquitous overexpression of Hey1 transcription factor leads to osteopenia and chondrocyte hypertrophy in bone. Bone, 2010, 46(3): 680-694.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF Hey1 EXPRESSION ON OSTEOGENIC DIFFERENTIATION AND PROLIFERATION OF C3H10T1/2 CELLS INDUCED BY BONE MORPHOGENETIC PROTEIN 9

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