Schnurri3 regulates osteogenic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2

doi:10.12307/2023.707

Abstract

Abstract: BACKGROUND: This study is based on the evidence that bone morphogenetic protein 2 plays an important role in osteogenesis and bone repair, and Schnurri3 can regulate the transforming growth factor-β signal transduction.

OBJECTIVE: To investigate the effect of inhibiting Schnurri3 on bone morphogenetic protein 2-induced osteogenic differentiation of C3H10T1/2 cells.

METHODS: Mouse mesenchymal stem cell line C3H10T1/2 cells were used as research materials. Four groups were set in this study: adenovirus-green fluorescent protein, adenovirus-bone morphogenetic protein 2, adenovirus-Simshn3 and adenovirus-bone morphogenetic protein 2+adenovirus-Simshn3. The expression of bone morphogenetic protein 2 was enhanced and the expression of Schnurri3 was inhibited. Alkaline phosphatase staining was used to detect the expression of alkaline phosphatase. The mRNA transcriptional levels of bone morphogenetic protein 2, Schnurri3, osteogenic and angiogenic markers were determined by RT-qPCR. Immunohistochemical staining was used to detect the expression of type I collagen, vascular endothelial growth factor and endothelial mucin. Alizarin red staining and semiquantitative analysis were used to detect calcium salt deposition levels. Subcutaneous stem cell implantation in nude mice was used to measure ectopic bone.

RESULTS AND CONCLUSION: (1) Recombinant adenovirus-bone morphogenetic protein 2 could induce osteogenic differentiation of C3H10T1/2 cells. Compared with the adenovirus-bone morphogenetic protein 2 group, mRNA transcription levels of osteogenic markers alkaline phosphatase, type I collagen, osteoblast-specific transcription factor, osteocalcin, Runt related transcription factor 2 and angiogenic markers neurite guidance factor, vascular endothelial growth factor, von Willebrand factor, angiopoietin and endothelial mucin were significantly increased in the adenovirus-bone morphogenetic protein 2+adenovirus-Simshn3 group (P < 0.001). Simultaneously, it significantly enhanced the expression of type I collagen, vascular endothelial growth factor and endothelial mucin at the protein level (P < 0.05), and the calcium salt deposition level (P < 0.05). (2) The application of bone morphogenetic protein 2 and Schnurri3 alone could act on C3H10T1/2 cells to form ectopic bone blocks subcutaneously in nude mice. Bone morphogenetic protein 2 combined with Schnurri3 could form more significant ectopic bone blocks. (3) The results showed that inhibition of Schnurri3 significantly enhanced the osteogenic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2, and enhanced the expression of angiogenesis factors mRNA and protein to regulate angiogenesis.

Key words: mesenchymal stem cell, bone morphogenetic protein 2, Schnurri3, osteogenic differentiation, angiogenic factor

CLC Number:

Xie Yingchun, Xu Wenjuan, Li Yuwan. Schnurri3 regulates osteogenic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(33): 5270-5276.

Figures/Tables 7

References

[1] KOSTENUIK P, MIRZA FM. Fracture healing physiology and the quest for therapies for delayed healing and nonunion. J Orthop Res. 2017;35(2):213-223.
[2] 王钊,闫石.应用Masquelet技术治疗大段骨缺损减少自体骨用量的可能性[J].中国组织工程研究,2020,24(24):3862-3869.
[3] 彭鑫,彭中华,谭奇超,等.骨组织工程学中复合支架及其应用研究进展[J].医学综述, 2022,28(13):2548-2554.
[4] 程永刚,宋晓阳,刘浩,等.股骨头缺血性坏死髋关节外科脱位入路打压植骨术后截骨端不愈合1例[J].中国骨与关节损伤杂志,2021,36(12):1340-1341.
[5] KATAGIRI T, WATABE T. Bone Morphogenetic Proteins. Cold Spring Harb Perspect Biol. 2016; 8(6):a021899.
[6] PARK SH, KWON JS, LEE BS, et al. BMP2-modified injectable hydrogel for osteogenic differentiation of human periodontal ligament stem cells. Sci Rep. 2017;7(1):6603.
[7] 从凯,李善龙,王飞,等.骨形态发生蛋白2,7治疗骨不连的效果评价[J].中国组织工程研究,2020,24(26):4243-4250.
[8] SHEIKH Z, JAVAID MA, HAMDAN N, et al. Bone Regeneration Using Bone Morphogenetic Proteins and Various Biomaterial Carriers. Materials (Basel). 2015;8(4):1778-1816.
[9] PENG Y, KANG Q, CHENG H, et al. Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling. J Cell Biochem. 2003;90(6):1149-1165.
[10] ZHANG L, LUO Q, SHU Y, et al. Transcriptomic landscape regulated by the 14 types of bone morphogenetic proteins (BMPs) in lineage commitment and differentiation of mesenchymal stem cells (MSCs). Genes Dis. 2019;6(3):258-275.
[11] RAHMAN MS, AKHTAR N, JAMIL HM, et al. TGF-β/BMP signaling and other molecular events: regulation of osteoblastogenesis and bone formation. Bone Res. 2015;3:15005.
[12] RAUNER M, BASCHANT U, ROETTO A, et al. Transferrin receptor 2 controls bone mass and pathological bone formation via BMP and Wnt signaling. Nat Metab. 2019;1(1):111-124.
[13] CAHILL KS, CHI JH, DAY A, et al. Prevalence, complications, and hospital charges associated with use of bone-morphogenetic proteins in spinal fusion procedures. JAMA. 2009;302(1):58-66.
[14] FU M, BLACKSHEAR PJ. RNA-binding proteins in immune regulation: a focus on CCCH zinc finger proteins. Nat Rev Immunol. 2017;17(2):130-143.
[15] MACKEH R, MARR AK, FADDA A,et al. C2H2-Type Zinc Finger Proteins: Evolutionarily Old and New Partners of the Nuclear Hormone Receptors. Nucl Recept Signal. 2018;15: 1550762918801071.
[16] JIN W, TAKAGI T, KANESASHI SN, et al. Schnurri-2 controls BMP-dependent adipogenesis via interaction with Smad proteins. Dev Cell. 2006;10(4):461-471.
[17] CASSANDRI M, SMIRNOV A, NOVELLI F, et al. Zinc-finger proteins in health and disease. Cell Death Discov. 2017;3:17071.
[18] WEI S, ZHANG L, ZHOU X, et al. Emerging roles of zinc finger proteins in regulating adipogenesis. Cell Mol Life Sci. 2013;70(23):4569-4584.
[19] UPADHYAY A, MOSS-TAYLOR L, KIM MJ, et al. TGF-β Family Signaling in Drosophila. Cold Spring Harb Perspect Biol. 2017;9(9):a022152.
[20] STROEBELE E, ERIVES A. Integration of Orthogonal Signaling by the Notch and Dpp Pathways in Drosophila. Genetics. 2016;203(1):219-240.
[21] AFFOLTER M, BASLER K. The Decapentaplegic morphogen gradient: from pattern formation to growth regulation. Nat Rev Genet. 2007;8(9):663-674.
[22] VAN BORTLE K, PETERSON AJ, TAKENAKA N, et al. CTCF-dependent co-localization of canonical Smad signaling factors at architectural protein binding sites in D. melanogaster. Cell Cycle. 2015;14(16):2677-2687.
[23] 孙鹏宇,张艳玲,荆玉明,等.腺病毒滴度不同测定方法比较[J].南方医科大学学报, 2011,31(2):234-238.
[24] CHEN L, JIANG W, HUANG J, et al. Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation. J Bone Miner Res. 2010;25(11):2447-2459.
[25] 任明诗,丁羽,李子涵,等.成骨细胞与破骨细胞相互调节作用的研究进展[J].中国药理学通报,2022,38(6):822-827.
[26] 王布雨,张勇,李飞非,等.骨软骨缺损修复中骨形态发生蛋白2 的作用与应用[J].中国组织工程研究,2023,27(20):3259-3265.
[27] HO SS, VOLLMER NL, REFAAT MI, et al. Bone Morphogenetic Protein-2 Promotes Human Mesenchymal Stem Cell Survival and Resultant Bone Formation When Entrapped in Photocrosslinked Alginate Hydrogels. Adv Healthc Mater. 2016;5(19):2501-2509.
[28] JAMES AW, LACHAUD G, SHEN J, et al. A Review of the Clinical Side Effects of Bone Morphogenetic Protein-2. Tissue Eng Part B Rev. 2016;22(4):284-297.
[29] KRISHNAN L, PRIDDY LB, ESANCY C, et al. Delivery vehicle effects on bone regeneration and heterotopic ossification induced by high dose BMP-2. Acta Biomater. 2017;49:101-112.
[30] ZHANG C, MENG C, GUAN D, et al. BMP2 and VEGF165 transfection to bone marrow stromal stem cells regulate osteogenic potential in vitro. Medicine (Baltimore). 2018;97(5):e9787.
[31] MCBRIDE-GAGYI SH, MCKENZIE JA, BUETTMANN EG, et al. Bmp2 conditional knockout in osteoblasts and endothelial cells does not impair bone formation after injury or mechanical loading in adult mice. Bone. 2015;81:533-543.
[32] MANNION RJ, NOWITZKE AM, WOOD MJ. Promoting fusion in minimally invasive lumbar interbody stabilization with low-dose bone morphogenic protein-2--but what is the cost? Spine J. 2011;11(6):527-533.
[33] RAZZOUK S, SARKIS R. BMP-2: biological challenges to its clinical use. N Y State Dent J. 2012; 78(5):37-39.
[34] YANG YS, XIE J, WANG D, et al. Bone-targeting AAV-mediated silencing of Schnurri-3 prevents bone loss in osteoporosis. Nat Commun. 2019;10(1):2958.
[35] JONES DC, WEIN MN, OUKKA M, et al. Regulation of adult bone mass by the zinc finger adapter protein Schnurri-3. Science. 2006;312(5777):1223-1227.
[36] DIOMEDE F, MARCONI GD, FONTICOLI L, et al. Functional Relationship between Osteogenesis and Angiogenesis in Tissue Regeneration. Int J Mol Sci. 2020;21(9):3242.
[37] SARAN U, GEMINI PIPERNI S, CHATTERJEE S. Role of angiogenesis in bone repair. Arch Biochem Biophys. 2014;561:109-117.
[38] SHARMA S, XUE Y, XING Z, et al. Adenoviral mediated mono delivery of BMP2 is superior to the combined delivery of BMP2 and VEGFA in bone regeneration in a critical-sized rat calvarial bone defect. Bone Rep. 2019;10:100205.
[39] SHARMA S, SAPKOTA D, XUE Y, et al. Delivery of VEGFA in bone marrow stromal cells seeded in copolymer scaffold enhances angiogenesis, but is inadequate for osteogenesis as compared with the dual delivery of VEGFA and BMP2 in a subcutaneous mouse model. Stem Cell Res Ther. 2018;9(1):23.
[40] SHARMA S, SAPKOTA D, XUE Y, et al. Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation. PLoS One. 2016; 11(1):e0147507.
[41] KUSUMBE AP, RAMASAMY SK, ADAMS RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature. 2014;507(7492):323-328.
[42] ZHANG J, PAN J, JING W. Motivating role of type H vessels in bone regeneration. Cell Prolif. 2020;53(9):e12874.
[43] XU R, YALLOWITZ A, QIN A, et al. Targeting skeletal endothelium to ameliorate bone loss. Nat Med. 2018;24(6):823-833.