[1]谢映春,涂小林.体外激活骨细胞Notch信号对骨髓基质细胞成骨分化的影响[J].第三军医大学学报,2020,42(09):891-899.
 XIE Yingchun,TU Xiao.Impact of activating Notch signaling in osteocytes on osteogenic differentiation of bone marrow stromal cells in vitro[J].J Third Mil Med Univ,2020,42(09):891-899.
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体外激活骨细胞Notch信号对骨髓基质细胞成骨分化的影响(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第09期
页码:
891-899
栏目:
基础医学
出版日期:
2020-05-15

文章信息/Info

Title:
Impact of activating Notch signaling in osteocytes on osteogenic differentiation of bone marrow stromal cells in vitro
作者:
谢映春涂小林
重庆医科大学生命科学研究院骨发育与再生平台
Author(s):
XIE Yingchun TU Xiao

linBone Development and Regeneration Platform, Life Sciences Institute, Chongqing Medical University, Chongqing, 400016, China

关键词:
骨细胞Notch信号骨髓基质细胞成骨分化
Keywords:
osteocytes Notch signaling bone marrow stromal cells osteogenic differentiation
分类号:
R322.71; R329.2; R394-33
文献标志码:
A
摘要:

目的探究体外激活骨细胞Notch信号对骨髓基质细胞(bone marrow stromal cells, BMSCs)成骨分化的影响。方法设置Cre重组腺病毒(Ad-Cre)及对照腺病毒GFP(Ad-GFP),分别转染RosaNotch小鼠骨细胞后与野生C57BL/6小鼠BMSCs共培养为Ad-Cre组及Ad-GFP组,设置仅BMSCs为Blank组。碱性磷酸酶染色及生化定量测定碱性磷酸酶(ALP);qPCR检测转染后骨细胞Notch信号靶基因及共培养产物成骨相关标志物、Notch信号配体及成血管相关标志物,茜素红染色检测成骨诱导21 d钙盐沉积水平。结果Ad-Cre成功激活RosaNotch小鼠骨细胞Notch信号,ALP染色及生化定量结果显示Ad-Cre组ALP表达相较Ad-GFP组及Blank组显著降低(P<0.05),Ad-GFP组与Blank组间无统计学差异;qPCR结果显示Ad-Cre组共培养产物成骨标志物ALP、核心结合因子a1(Runx2)、特异性骨转化因子(Ostrix)及Notch配体Dll4 mRNA转录水平相较Ad-GFP组及Blank组显著降低(P<0.05),Jag1 mRNA 转录水平显著降低(P<0.05);血管内皮生长因子(VEGF)、低氧诱导因子(hypoxia-inducible factor 1α,HIF1α)、血小板内皮细胞黏附分子(platelet endothelial cell adhesion molecule-1, CD31/PECAM-1)及内皮粘蛋白(endomucin, EMCN)mRNA转录水平相较Ad-GFP组及Blank组显著增高(P<0.05);茜素红染色结果显示Ad-Cre组在成骨诱导培养基作用21 d后钙盐沉积水平相较Ad-GFP组及Blank组显著降低。结论体外激活骨细胞Notch信号抑制骨髓基质细胞成骨分化。

Abstract:

ObjectiveTo investigate the effect and underlying mechanism of activating Notch signaling in osteocytes on osteogenic differentiation of bone marrow stromal cells (BMSCs). MethodsThe osteocytes derived from RosaNotch mice were infected with recombinant adenovirus expressing Cre or GFP respectively, and then the obtained cells were co-cultured with BMSCs isolated from wild-type C57BL/6 mice, and named as Ad-Cre group and Ad-GFP group, with a BMSCs group without any treatment as blank control. Alkaline phosphatase (ALP) staining and detection of ALP relative activity were used to measure the expression of ALP. The mRNA expression levels of the target genes in osteocyte of RosaNotch and ligands of Notch signaling, osteogenic markers, and angiogenic makers in the co-cultured product were detected by qPCR. Alizarin red staining was applied to test the matrix mineralization on day 21. ResultsInfection of recombinant adenovirus Ad-Cre could successfully activate Notch signaling. ALP staining and detection of ALP activity showed that the expression level of ALP was significantly lower in the Ad-Cre group than the Ad-GFP group and Blank group (P<0.05), and there was no statistical difference in the level between the latter 2 groups. The results of qPCR indicated that the mRNA levels of osteogenic makers ALP, Osterix, Runx2 and Notch ligand Dll4 were deceased significantly, while those of Jag1, VEGF, hypoxia-inducible factor 1α (HIF1α), platelet endothelial cell adhesion molecule-1 (CD31/PECAM-1), and endomucin (EMCN) were statistically increased in the Ad-Cre group when compared with the Ad-GFP group and Blank group (P<0.05). Alizarin red staining displayed that the Ad-Cre group had more calcium deposition in 21 d after co-culture than the Ad-GFP group and blank group (P<0.05). ConclusionActivating Notch signaling in osteocytes inhibits osteogenic differentiation of BMSCs in vitro.

参考文献/References:

[1]BI P P, KUANG S H. Notch signaling as a novel regulator of metabolism[J]. Trends Endocrinol Metab, 2015, 26(5): 248-255. DOI:10.1016/j.tem.2015.02.006.
[2]CAMPBELL D P, CHRYSOSTOMOU E, DOETZLHOFER A. Canonical Notch signaling plays an instructive role in auditory supporting cell development[J]. Sci Rep, 2016, 6: 19484. DOI:10.1038/srep19484.
[3]CHEUNG L, LE TISSIER P, GOLDSMITH S G, et al. NOTCH activity differentially affects alternative cell fate acquisition and maintenance[J]. Elife, 2018, 7: e33318. DOI:10.7554/eLife.33318.
[4]COLOMBO M, THMMLER K, MIRANDOLA L, et al. Notch signaling drives multiple myeloma induced osteoclastogenesis[J]. Oncotarget, 2014, 5(21): 10393-10406. DOI:10.18632/oncotarget.2084.
[5]KUSHWAH R, GUEZGUEZ B, LEE J B, et al. Pleiotropic roles of Notch signaling in normal, malignant, and developmental hematopoiesis in the human[J]. EMBO Rep, 2014, 15(11): 1128-1138. DOI:10.15252/embr.201438842.
[6]WAHI K, BOCHTER M S, COLE S E. The many roles of Notch signaling during vertebrate somitogenesis[J]. Semin Cell Dev Biol, 2016, 49: 68-75. DOI:10.1016/j.semcdb.2014.11.010.
[7]COLOMBO M, GALLETTI S, GARAVELLI S, et al. Notch signaling deregulation in multiple myeloma: a rational molecular target[J]. Oncotarget, 2015, 6(29): 26826-26840. DOI:10.18632/oncotarget.5025.
[8]ZANOTTI S, CANALIS E. Notch signaling and the skeleton[J]. Endocr Rev, 2016, 37(3): 223-253. DOI:10.1210/er.2016-1002.
[9]NG A H, BAHT G S, ALMAN B A, et al. Bone marrow stress decreases osteogenic progenitors[J]. Calcif Tissue Int, 2015, 97(5): 476-486. DOI:10.1007/s00223-015-0032-3.
[10]BONEWALD L F. The role of the osteocyte in bone and nonbone disease[J]. Endocrinol Metab Clin N Am, 2017, 46(1): 1-18. DOI:10.1016/j.ecl.2016.09.003.
[11]ANSARI N, HO P W, CRIMEEN-IRWIN B, et al. Autocrine and paracrine regulation of the murine skeleton by osteocyte-derived parathyroid hormone-related protein[J]. J Bone Miner Res, 2018, 33(1): 137-153. DOI:10.1002/jbmr.3291.
[12]TOKARZ D, MARTINS J S, PETIT E T, et al. Hormonal regulation of osteocyte perilacunar and canalicular remodeling in the hyp mouse model of X-linked hypophosphatemia[J]. J Bone Miner Res, 2018, 33(3): 499-509. DOI:10.1002/jbmr.3327.
[13]DELGADO-CALLE J, ANDERSON J, CREGOR M D, et al. Bidirectional notch signaling and osteocyte-derived factors in the bone marrow microenvironment promote tumor cell proliferation and bone destruction in multiple myeloma[J]. Cancer Res, 2016, 76(5): 1089-1100. DOI:10.1158/0008-5472.CAN-15-1703.
[14]JHN K, KELKAR S, ZHAO H, et al. Osteocytes acidify their microenvironment in response to PTHrP in vitro and in lactating mice in vivo[J]. J Bone Miner Res, 2017, 32(8): 1761-1772. DOI:10.1002/jbmr.3167.
[15]LIU W, WANG Z Y, YANG J, et al. Osteocyte TSC1 promotes sclerostin secretion to restrain osteogenesis in mice[J]. Open Biol, 2019, 9(5): 180262. DOI:10.1098/rsob.180262.
[16]NAKASHIMA T, HAYASHI M, FUKUNAGA T, et al. Evidence for osteocyte regulation of bone homeostasis through RANKL expression[J]. Nat Med, 2011, 17(10): 1231-1234. DOI:10.1038/nm.2452.
[17]CANALIS E, ADAMS D J, BOSKEY A, et al. Notch signaling in osteocytes differentially regulates cancellous and cortical bone remodeling[J]. J Biol Chem, 2013, 288(35): 25614-25625. DOI:10.1074/jbc.M113.470492.
[18]XU Y, SHU B, TIAN Y, et al. Notch activation promotes osteoblast mineralization by inhibition of apoptosis[J]. J Cell Physiol, 2018, 233(10): 6921-6928. DOI:10.1002/jcp.26592.
[19]CANALIS E, BRIDGEWATER D, SCHILLING L, et al. Canonical Notch activation in osteocytes causes osteopetrosis[J]. Am J Physiol Endocrinol Metab, 2016, 310(2): E171-E182. DOI:10.1152/ajpendo.00395.2015.
[20]ZANOTTI S, CANALIS E. Activation of Nfatc2 in osteoblasts causes osteopenia[J]. J Cell Physiol, 2015, 230(7): 1689-1695. DOI:10.1002/jcp.24928.
[21]ZANOTTI S, SMERDEL-RAMOYA A, STADMEYER L, et al. Notch inhibits osteoblast differentiation and causes osteopenia[J]. Endocrinology, 2008, 149(8): 3890-3899. DOI:10.1210/en.2008-0140.
[22]DEREGOWSKI V, GAZZERRO E, PRIEST L, et al. Notch 1 overexpression inhibits osteoblastogenesis by suppressing wnt/β-catenin but not bone morphogenetic protein signaling[J]. J Biol Chem, 2006, 281(10): 6203-6210. DOI:10.1074/jbc.m508370200.
[23]SHAH K M, STERN M M, STERN A R, et al. Osteocyte isolation and culture methods[J]. Bonekey Rep, 2016, 5: 838. DOI:10.1038/bonekey.2016.65.
[24]MARIDAS D E, RENDINA-RUEDY E, LE P T, et al. Isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice[J]. J Vis Exp, 2018(131): 56750. DOI:10.3791/56750.
[25]ISO T, KEDES L, HAMAMORI Y. HES and HERP families: multiple effectors of the Notch signaling pathway[J]. J Cell Physiol, 2003, 194(3): 237-255. DOI:10.1002/jcp.10208.
[26]KRATZER R F, KREPPEL F. Production, purification, and titration of first-generation adenovirus vectors[J]. Methods Mol Biol, 2017, 1654: 377-388. DOI:10.1007/978-1-4939-7231-9_28.
[27]ENGIN F, YAO Z Q, YANG T, et al. Dimorphic effects of Notch signaling in bone homeostasis[J]. Nat Med, 2008, 14(3): 299-305. DOI:10.1038/nm1712.
[28]HILTON M J, TU X L, WU X M, et al. Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation[J]. Nat Med, 2008, 14(3): 306-314. DOI:10.1038/nm1716.
[29]SHAO J, ZHOU Y H, XIAO Y. The regulatory roles of Notch in osteocyte differentiation via the crosstalk with canonical Wnt pathways during the transition of osteoblasts to osteocytes[J]. Bone, 2018, 108: 165-178. DOI:10.1016/j.bone.2018.01.010.
[30]TU X L, DELGADO-CALLE J, CONDON K W, et al. Osteocytes mediate the anabolic actions of canonical Wnt/β-catenin signaling in bone[J]. Proc Natl Acad Sci USA, 2015, 112(5): E478-E486. DOI:10.1073/pnas.1409857112.
[31]任磊, 代光明, 林枭, 等. 骨细胞Wnt/β-Catenin通过Notch信号促进BMSCs成骨分化[J]. 中国骨质疏松杂志, 2018, 24(5): 600-605.
REN L, DAI G M, LIN X, et al. Wnt/β-Catenin induces osteoblastic differentiation of BMCS via Notch signaling pathway by osteocytes[J]. Chin J Osteoporos, 2018, 24(5): 600-605.
[32]WANG N, LIU W, TAN T, et al. Notch signaling negatively regulates BMP9-induced osteogenic differentiation of mesenchymal progenitor cells by inhibiting JunB expression[J]. Oncotarget, 2017, 8(65): 109661-109674. DOI:10.18632/oncotarget.22763.
[33]BIGAS A, ROBERT-MORENO A, ESPINOSA L. The Notch pathway in the developing hematopoietic system[J]. Int J Dev Biol, 2010, 54(6/7): 1175-1188. DOI:10.1387/ijdb.093049ab.
[34]FRASER H M, HASTINGS J M, ALLAN D, et al. Inhibition of delta-like ligand 4 induces luteal hypervascularization followed by functional and structural luteolysis in the primate ovary[J]. Endocrinology, 2012, 153(4): 1972-1983. DOI:10.1210/en.2011-1688.
[35]BENEDITO R, ROCA C, SRENSEN I, et al. The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis[J]. Cell, 2009, 137(6): 1124-1135. DOI:10.1016/j.cell.2009.03.025.

更新日期/Last Update: 2020-05-06