Bone morphogenetic protein 2-induced C2C12 and MC3T3-E1 osteoblast differentiation and autophagy

doi:10.3969/j.issn.2095-4344.2014.20.022

Abstract

Abstract:

BACKGROUND: A series of studies indicate that autophagy is closely linked with differentiation. Bone morphogenetic protein 2 (BMP-2) is the classical pathway for C2C12 and MC3T3-E1 osteoblast differentiation, and the ideal model to study osteogenic differentiation process.
OBJECTIVE: To observe the relationship between autophagy and BMP-2-induced cell lines C2C12, MC3T3-E1 osteoblast differentiation.
METHODS: Real-Time PCR was applied to detect osteogenic differentiation and autophagy related index after C2C12 and MC3T3-E1 were induced with BMP-2 (100 μg/L) for 72 hours. The osteogenic index alkaline phosphatase in BMP-2-induced MC3T3-E1 and C2C12 cultured with different concentrations of 3-methyladenine (0, 1, 5, 10 mmol/L) was determined with alkaline phosphatase staining. Western blot analysis was applied to detect LC3-I/II expression levels in C2C12 and MC3T3-E1 induced with BMP-2 for different time points (0, 12, 24, 48, 72, 96 hours).
RESULTS AND CONCLUSION: The autophagy-related mRNA and protein expression showed an increasing tendency and autophagy-related protein LC3 levels was increased, which was associated with the time, during the BMP-2-induced cell lines C2C12, MC3T3-E1 osteoblast differentiation. Meanwhile, alkaline phosphatase expression levels were inhibited by autophagy in the process of osteogenic differentiation. Therefore, there is a close relationship between autophagy and the BMP-2-induced cell lines C2C12, MC3T3-E1 osteoblast differentiation.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: autophagy, bone morphogenetic proteins, cell line, myoblasts, osteoblasts

CLC Number:

R318

Gao Yan, Cheng Chen, Li Jing, Zhang Yue, Xiao Wei-fan, Pan Qiu-hui. Bone morphogenetic protein 2-induced C2C12 and MC3T3-E1 osteoblast differentiation and autophagy[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(20): 3236-3241.

Figures/Tables 2

2.1 骨形态发生蛋白2诱导成骨分化以C2C12与MC3T3-E1细胞为研究对象，骨形态发生蛋白2(100 μg/L)诱导7 d，成骨分化经典指标碱性磷酸酶染色观察成骨分化水平。结果表明，与未诱导对照组相比，骨形态发生蛋白2诱导时碱性磷酸酶表达水平显著增高(图1A，B)。为观察其他骨分化相关指标，C2C12与MC3T3-E1在骨形态发生蛋白2(100 μg/L)中诱导3 d，进行RT-PCR 反应。与未诱导对照组比较，C2C12骨分化指标ALP、COL1A2、OC、RUNX2、SP7增高，成肌指标MYOG降低，分化抑制因子ID1增高(图1C)；MC3T3-E1骨分化指标ALP、IBSP、RUNX2、SP7增高，成肌指标MYOG降低，分化抑制因子ID1增高(P < 0.05，图1D)。 2.2 成骨分化过程诱导自噬 LC3是自噬体膜的标记蛋白，LC3-Ⅰ经修饰形成LC3-Ⅱ， LC3-Ⅱ/LC3-Ⅰ值越大自噬作用越强，反之，自噬作用越弱。用Western Blot实验，以LC3为指标测不同时间点自噬水平。结果表明，C2C12与MC3T3-E1经骨形态发生蛋白2诱导后自噬水平均随时间先增强后减弱，在24 h与72 h之间自噬水平明显增强，其中C2C12自噬水平24 h时出现第1个表达峰，72 h时最强，出现第2个表达峰，MC3T3-E1自噬水平24 h时最强。因此，成骨分化过程中自噬作用增强且与时间相关(图2A，2B)。C2C12与MC3T3-E1经骨形态发生蛋白(100 μg/L)诱导3 d，设置未诱导对照组，进行RT-PCR反应，两组细胞自噬诱导指标ATG7、BECN1、LAMP1、PEA15均增高 (P < 0.05)，见图2C，D。 2.3 3-甲基腺嘌呤抑制成骨分化过程 C2C12与MC3T3-E1骨形态发生蛋白2(100 μg/L)诱导前2 h分别加入不同浓度自噬抑制剂3-甲基腺嘌呤，诱导7 d后碱性磷酸酶染色观察自噬改变对成骨分化的影响。原倍与400倍进行观察拍照。原倍观察发现C2C12在未用骨形态发生蛋白2与自噬抑制剂3-甲基腺嘌呤进行诱导时，碱性磷酸酶染色阴性，用骨形态发生蛋白2(100 μg/L)进行诱导，碱性磷酸酶染色阳性，自噬抑制剂3-甲基腺嘌呤浓度达到10 mmol/L时碱性磷酸酶活性开始下降；400倍观察发现C2C12在未用骨形态发生蛋白2与自噬抑制剂3-甲基腺嘌呤进行诱导时，细胞呈短梭形排列紧密散乱，骨形态发生蛋白2(100 μg/L)诱导后细胞呈长梭状排列成束，随着3-甲基腺嘌呤浓度增加细胞形状逐渐不规则排列散乱。MC3T3-E1与C2C12在骨形态发生蛋白2与自噬抑制剂3-甲基腺嘌呤进行诱导时有类似变化，原倍观察发现自噬抑制剂3-甲基腺嘌呤浓度达到5 mmol/L时碱性磷酸酶活性开始下降，400倍观察发现随着3-甲基腺嘌呤浓度增加细胞突起逐渐消失，细胞变大。因此，发现自噬抑制剂作用后抑制成骨分化，抑制程度与药物剂量正相关(图3)。"

References

[1] Klionsky DJ, Emr SD.Autophagy as a regulated pathway of cellular degradation.Science. 2000;290(5497):1717-1721.
[2] Levine B.Cell biology: autophagy and cancer.Nature. 2007; 446(7137):745-747.
[3] Levine B, Kroemer G.Autophagy in the pathogenesis of disease.Cell. 2008;132(1):27-42.
[4] Mizushima N, Yoshimori T, Levine B.Methods in mammalian autophagy research.Cell. 2010;140(3):313-326.
[5] Scarlatti F, Bauvy C, Ventruti A,et al. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.J Biol Chem. 2004;279(18): 18384-18391.
[6] Qian W, Liu J, Jin J,et al.Arsenic trioxide induces not only apoptosis but also autophagic cell death in leukemia cell lines via up-regulation of Beclin-1.Leuk Res. 2007;31(3):329-339.
[7] Cuervo AM.Autophagy: in sickness and in health.Trends Cell Biol. 2004;14(2):70-77.
[8] Paludan C, Schmid D, Landthaler M, et al.Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science. 2005;307(5709):593-596.
[9] Ogawa M, Yoshimori T, Suzuki T,et al.Escape of intracellular Shigella from autophagy.Science. 2005;307(5710):727-731.
[10] Katic M, Kahn CR.The role of insulin and IGF-1 signaling in longevity.Cell Mol Life Sci. 2005;62(3):320-343.
[11] Iovino S, Oriente F, Botta G,et al. PED/PEA-15 induces autophagy and mediates TGF-beta1 effect on muscle cell differentiation.Cell Death Differ. 2012;19(7):1127-1138.
[12] Gioia R, Panaroni C, Besio R,et al. Impaired osteoblastogenesis in a murine model of dominant osteogenesis imperfecta: a new target for osteogenesis imperfecta pharmacological therapy. Stem Cells. 2012;30(7):1465-1476.
[13] Wang J, Lian H, Zhao Y,et al.Vitamin D3 induces autophagy of human myeloid leukemia cells.J Biol Chem. 2008;283(37): 25596-25605.
[14] Kamitsuji Y, Kuroda J, Kimura S,et al.The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemias.Cell Death Differ. 2008;15(11):1712-1722.
[15] Reddi AH.Bone morphogenetic proteins: from basic science to clinical applications.J Bone Joint Surg Am. 2001;83-A Suppl 1(Pt 1):S1-6.
[16] Riley EH, Lane JM, Urist MR,et al.Bone morphogenetic protein-2: biology and applications.Clin Orthop Relat Res. 1996;(324):39-46.
[17] Nilsson OS, Urist MR, Dawson EG,et al.Bone repair induced by bone morphogenetic protein in ulnar defects in dogs.J Bone Joint Surg Br. 1986;68(4):635-642.
[18] 潘秋辉,马纪,于永春,等. 转录因子Osterix对成骨细胞Col1a1基因的反式激活作用[J].中国生物化学与分子生物学报, 2007, 23(11):921-925.
[19] Hirata S, Kitamura C, Fukushima H,et al. Low-level laser irradiation enhances BMP-induced osteoblast differentiation by stimulating the BMP/Smad signaling pathway.J Cell Biochem. 2010;111(6):1445-1452.
[20] Nishimura M, Mikura M, Hirasaka K,et al.Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells.J Biochem. 2008;144(6): 717-724.
[21] Heining E, Bhushan R, Paarmann P,et al. Spatial segregation of BMP/Smad signaling affects osteoblast differentiation in C2C12 cells.PLoS One. 2011;6(10):e25163.
[22] Sorkin A, von Zastrow M.Endocytosis and signalling: intertwining molecular networks.Nat Rev Mol Cell Biol. 2009;10(9):609-622.
[23] Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy.Int J Biochem Cell Biol. 2004;36(12): 2503-2518.
[24] Meijer AJ, Codogno P.Regulation and role of autophagy in mammalian cells.Int J Biochem Cell Biol. 2004;36(12): 2445-2462.
[25] Kroemer G, Jäättelä M.Lysosomes and autophagy in cell death control.Nat Rev Cancer. 2005;5(11):886-897.
[26] Juhasz G, Neufeld TP.Autophagy: a forty-year search for a missing membrane source.PLoS Biol. 2006;4(2):e36.
[27] Connelly LB, Woolf A, Brooks P. Cost-effectiveness of interventions for musculoskeletal conditions. Disease control in developing countries. Washington, DC:World Bank. 2006.
[28] Connelly LB, Supangan R.The economic costs of road traffic crashes: Australia, states and territories.Accid Anal Prev. 2006;38(6):1087-1093.
[29] Masiero E, Agatea L, Mammucari C,et al. Autophagy is required to maintain muscle mass.Cell Metab. 2009;10(6): 507-515.