Bone morphogenetic proteins-2/Osterix signaling pathway regulates the differentiation of preosteoblasts

doi:10.3969/j.issn.2095-4344.2016.24.013

Abstract

Abstract:

BACKGROUND: Bone formation and development are reported to be regulated by bone morphogenetic protein 2 (BMP2)-induced Osterix expression.
OBJECTIVE: To investigate the regulatory effect of BMP2/Osterix signaling pathway on differentiation of preosteoblasts in mice.
METHODS: mRNA and protein expression of Osterix was determined by real-time RT-PCR and western blot assay, respectively at various time points after mouse preosteoblasts were treated with BMP2. pcDNA3.1/myc-Osterix eukaryotic expression vector was constructed and transducted into preosteoblasts, and then mRNA expression of alkaline phosphatase, bone sialoprotein, and matrix extracellular phosphoglycoprotein was detected by real-time RT-PCR after transduction and BMP2 treatment.
RESULTS AND CONCLUSION: Osterix mRNA expression was up-regulated when treated with BMP2 in mouse preosteoblasts, and reached the peak at 24 hours. In addition, the protein expression of Osterix was increased after BMP2 treatment. Alkaline phosphatase, bone sialoprotein, and matrix extracellular phosphoglycoprotein mRNA expression was up-regulated after transfection of mouse preosteoblasts with pcDNA3.1/myc-Osterix eukaryotic expression vector and BMP2 treatment. Our results indicate that BMP2 regulates the synthesis of genetic markers of osteogenesis, such as alkaline phosphatase, matrix extracellular phosphoglycoprotein via up-regulating Osterix expression in mouse preosteoblasts, suggesting BMP2/Osterix signaling pathway plays a critical role in bone development.

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

Key words: Bone Morphogenetic Proteins, Signal Transduction, Osteoblasts, Tissue Engineering

CLC Number:

R318

Li Cheng-lin, Chen Shu-lan, Ren Wei-wei. Bone morphogenetic proteins-2/Osterix signaling pathway regulates the differentiation of preosteoblasts[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(24): 3581-3587.

Figures/Tables 1

2.1 实时定量RT-PCR检测骨形态发生蛋白2调控小鼠前成骨细胞中Osterix mRNA的表达根据实时定量RT-PCR检测结果发现，骨形态发生蛋白2上调小鼠前成骨细胞中Osterix基因的mRNA表达，并且在刺激细胞 24 h后Osterix基因的mRNA表达最为显著(图1，P < 0.05)。 2.2 免疫印迹法检测骨形态发生蛋白2调控小鼠前成骨细胞中Osterix蛋白的表达根据免疫印迹法检测结果发现，骨形态发生蛋白2上调小鼠前成骨细胞中Osterix基因的蛋白表达水平，72 h的蛋白表达量比48 h升高(图2)。 2.3 转录因子Osterix基因的克隆及重组质粒pcDNA3.1/myc-Osterix的构建结果测序成功后，将pcDNA3.1/myc-Hisa真核载体和经测序正确的重组质粒pMD18-T Simple-Osterix。分别用EcoRⅣ及Hind Ⅲ进行双酶切后割胶回收。割胶回收后的pcDNA3.1/ myc-Hisa真核载体与Osterix基因片段进行连接。连接产物转化感受态细胞E.coli DH5α，挑取单克隆，37 ℃振荡培养，小量质粒提取，经EcoR Ⅳ及Hind Ⅲ进行酶切鉴定正确者，将构建的重组真核质粒命名为pcDNA3.1/myc-Osterix(图3)。 2.4 实时定量RT-PCR检测转染质粒pcDNA3.1/ myc-Osterix后小鼠前成骨细胞中碱性磷酸酶、骨涎蛋白、细胞外基质磷酸化糖蛋白的mRNA表达水平根据实时定量RT-PCR检测结果发现，转染质粒pcDNA3.1/myc-Osterix后小鼠前成骨细胞中碱性磷酸酶、骨涎蛋白、细胞外基质磷酸化糖蛋白的mRNA表达水平上调，其中碱性磷酸酶、骨涎蛋白的mRNA表达水平在Osterix转染100 ng时上调最为显著(图4A，B，P < 0.05)，细胞外基质磷酸化糖蛋白的mRNA表达水平在Osterix转染50 ng时上调最为显著(图4C，P < 0.05)。 2.5 实时定量RT-PCR检测骨形态发生蛋白2作用下小鼠前成骨细胞中碱性磷酸酶、骨涎蛋白、细胞外基质磷酸化糖蛋白的mRNA表达水平根据实时定量RT-PCR检测结果发现，相对于空白对照组，碱性磷酸酶、骨涎蛋白、细胞外基质磷酸化糖蛋白的mRNA表达水平在骨形态发生蛋白2作用下得到显著上调(图5，P < 0.05)。"

References

[1] Dorman LJ,Tucci M,Benghuzzi H.In vitro effects of bmp-2,bmp-7,and bmp-13 on proliferation and differentation of mouse mesenchymal stem cells. Biomed Sci Instrum.2012;48:81-87.
[2] GanssB,Jheon A.Zinc Finger Transcription Factors in Skeletal Development.Crit Rev Oral Biol Med.2004；15(5):282-297.
[3] Farley JR,Stilt-Coflfing B.Apoptosis may delermine the release of skeletal alkaline phosphatase activity from human osteoblast-line cell.Calcif Tissue Int.2001；6868(1):43.
[4] Nguyen H,Qiall JJ,Bhamagar RS,et al.Enhanced cell attachment and osteoblastic activity by P-15 peptide-coated matrix in hydrogels.Biochem Biophys Res Commun.2003;311(1):179.
[5] Saito Y,Yoshizawa T,Takizawa F,et al.A cell line with characteristics of the periodontal ligame- nt fibroblasts is negatively regulated for mineralization and Runx2/Cbfa1/Osf2 activity,part of which can be overcome by bone morphogenetic protein-2.J Cell Sci.2002;115(21):4191-4200.
[6] 王建,刘宗霞,王鹏,等.细胞外基质磷酸化糖蛋白在大鼠牙周组织缺损愈合过程中的表达[J]. 牙体牙髓牙周病学杂志,2011;21(5):259-262.
[7] Silverio KG,Davidson KC,James RG,et al. Wnt/β-catenin pathway regulates bone morphogene-tic protein(BMP2) mediated differentiation of dental follicle cells.J Periodontal Res.2012;47(3): 309-319.
[8] Kaewsrichan J,Wongwitwichot P,Chandarajoti K,et al.Sequential induction of marrow stromal cells by FGF2 and BMP2 improves their growth and differentiation potential in vivo.Arch Oral Biol.2011；56(1):90-101.
[9] Wu JY,Wang YH,Wang GJ,et al.Low-power GaAlAs laser irradiation promotes the prolifera- tion and osteogenic differentiation of stem cells via IGF1 and BMP2.Plos One.2012;7(9):e44027.
[10] Lv HZ,Huang XD,Jin SS,et al.Strontium ranelate promotes osteogenic differentiation protein-2/ smad signaling pathway.Nan Fang Yi Ke Da Xue Bao.2013; 33(3):376-381.
[11] Liu DD,Zhang JC,Zhang Q,et al.TGF-β/BMP signaling pathway is involved in cerium promo-ted osteogenic differentiation of mesenchymal stem cells.J Cell Biochem.2013;114(5): 1105-1114.
[12] Lowery JW,Pazin D,Intini G,et al.The role of BMP2 signaling in the skeleton.Crit Rev Eukaryot Gene Exp.2011;21(2):177-185.
[13] Bragdon BD,Angelo A,Gurski L,et al.Altered plasma membrance dynamics of bone morpho- genetic protein receptor type la in a low bone mass mouse model. Bone.2012;50(1): 198-199.
[14] Nakashima K,Zhou X,Kunkel G,et al.The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation.Cell. 2002;108:17-29.
[15] Baek WY,de-Crombrugghe B,Kim JE.Postnatally induced inactivation of Osterix in osteo- blasts results in the reduction of bone formation and maintenance. Bone.2010;46(4):920-928.
[16] Zhou X,Zhang Z,Feng JQ,et al.Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice.Proc Natl Acad Sci USA.2010;107 (29): 12919-12924.
[17] Matsubara T,Kida K,Yamaguchi A,et al.BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation.J Biol Chem.2008;283(43): 29119-29125.
[18] 王朝元,段友容，Boban Markovic C,等.羟基磷灰石陶瓷诱导成骨细胞的研究[J].四川大学学报:自然科学版,2003, 40(6): 1110-1113.
[19] Rowe PS, de Zoysa PA, Dong R, et al. MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia.Genomics.2000;67(1):54-60.
[20] 王建,刘宗霞,唐开亮,等.细胞外基质磷酸化糖蛋白在小鼠牙齿发育过程中的表达[J].牙体牙髓牙周病学杂志,2009, 19(6):314-317.
[21] Hou C, Liu ZX, Tang KL, et al. Developmental changes and regional localization of Dspp, Mepe, Mimecan and Versican in postnatal developing mouse teeth. 2011; Epub ahead of print.