在Prrx1~+骨髓间充质干细胞中过表达肝素结合表皮生长因子样生长因子抑制小鼠骨发育
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摘要
背景:肝素结合表皮生长因子样生长因子对骨骼的发育十分重要,但目前尚未报道过体内骨髓间充质干细胞来源的肝素结合表皮生长因子样生长因子对骨发育的影响。目的:探究在骨髓间充质干细胞中过表达肝素结合表皮生长因子样生长因子对骨发育的影响并初步分析相关机制。方法:利用Cre-LoxP系统构建在Prrx1~+骨髓间充质干细胞中过表达肝素结合表皮生长因子样生长因子(ROSA26~(tm1(HB-EGF)); Prrx1-Cre)的小鼠模型,取骨组织切片染色进行组织学分析。取小鼠骨髓间充质干细胞进行体外培养,进行分化能力检测和分子机制研究。实验于2015年经上海交通大学实验动物管理、学术、伦理与使用委员会批准,批准号为A2015027。结果和结论:①Prrx1在成年小鼠股骨关节头、生长板、骨小梁表面和骨膜上均有表达;②ROSA26~(tm1(HB-EGF));Prrx1-Cre小鼠股骨变短,骨量明显减少,软骨形态和结构异常,出现骨关节炎样表型;③ROSA26~(tm1(HB-EGF));Prrx1-Cre小鼠骨髓间充质干细胞体外成骨、软骨分化减弱。结果提示在骨髓间充质干细胞中过表达HB-EGF可能通过削弱骨髓间充质干细胞成骨、软骨分化抑制小鼠骨发育。
BACKGROUND: Heparin-binding epidermal growth factor-like growth factor(HB-EGF) plays an important role in bone development. However, there is no report regarding the effects of HB-EGF derived from bone marrow mesenchymal stem cells on skeletal development.OBJECTIVE: To study the effects of HB-EGF overexpression in bone marrow mesenchymal stem cells on skeletal development and to preliminarily explore the underlying mechanisms.METHODS: The mouse model of HB-EGF overexpression in Prrx1~+ bone marrow mesenchymal stem cells(ROSA26~(tm1(HB-EGF)); Prrx1-Cre) was constructed using Cre-LoxP system. Bone sections were processed and stained for histological analysis. Mouse bone marrow mesenchymal stem cells were cultured in vitro and their differentiation potential and relevant molecular mechanism were assessed. The experiment was approved by the Experimental Animal Management, Academics, Ethics and Use Committee of Shanghai Jiao Tong University in 2015. The approval number is A2015027.RESULTS AND CONCLUSION:(1) Prrx1 was expressed in the articular cartilage, growth plate, trabecular bone and periosteum of adult mice.(2) The femurs of ROSA26~(tm1(HB-EGF)); Prrx1-Cre mice were shortened, the bone mass decreased significantly, and the cartilage morphology and structure were abnormal, all of which indicated osteoarthritic phenotype.(3) Overexpression of HB-EGF inhibited the osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells from ROSA26~(tm1(HB-EGF)); Prrx1-Cre mice in vitro. These results suggest that overexpression of HB-EGF in Prrx1~+ bone marrow mesenchymal stem cells may influence the skeletal development by inhibiting osteogenic and chondrogenic differentiation.
BACKGROUND: Heparin-binding epidermal growth factor-like growth factor(HB-EGF) plays an important role in bone development. However, there is no report regarding the effects of HB-EGF derived from bone marrow mesenchymal stem cells on skeletal development.OBJECTIVE: To study the effects of HB-EGF overexpression in bone marrow mesenchymal stem cells on skeletal development and to preliminarily explore the underlying mechanisms.METHODS: The mouse model of HB-EGF overexpression in Prrx1~+ bone marrow mesenchymal stem cells(ROSA26~(tm1(HB-EGF)); Prrx1-Cre) was constructed using Cre-LoxP system. Bone sections were processed and stained for histological analysis. Mouse bone marrow mesenchymal stem cells were cultured in vitro and their differentiation potential and relevant molecular mechanism were assessed. The experiment was approved by the Experimental Animal Management, Academics, Ethics and Use Committee of Shanghai Jiao Tong University in 2015. The approval number is A2015027.RESULTS AND CONCLUSION:(1) Prrx1 was expressed in the articular cartilage, growth plate, trabecular bone and periosteum of adult mice.(2) The femurs of ROSA26~(tm1(HB-EGF)); Prrx1-Cre mice were shortened, the bone mass decreased significantly, and the cartilage morphology and structure were abnormal, all of which indicated osteoarthritic phenotype.(3) Overexpression of HB-EGF inhibited the osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells from ROSA26~(tm1(HB-EGF)); Prrx1-Cre mice in vitro. These results suggest that overexpression of HB-EGF in Prrx1~+ bone marrow mesenchymal stem cells may influence the skeletal development by inhibiting osteogenic and chondrogenic differentiation.
引文
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[14]Krampera M,Pasini A,Rigo A,et al.HB-EGF/HER-1 signaling in bone marrow mesenchymal stem cells:inducing cell expansion and reversibly preventing multilineage differentiation.Blood.2005;106(1):59-66.
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[16]Madisen L,Zwingman TA,Sunkin SM,et al.A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci.2010;13(1):133-140.
[17]Logan M,Martin JF,Nagy A,et al.Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer.Genesis.2002;33(2):77-80.
[18]Buch T,Heppner FL,Tertilt C,et al.A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration.Nat Methods.2005;2(6):419-426.
[19]常晓朋,陈涛,赵寅,等.骨形态发生蛋白2和转化生长因子β2协同促进骨髓间充质干细胞成骨分化[J].中国组织工程研究,2019,23(1):1-6.
[20]张文.SkyScan 1176 Micro-CT在骨科研究中的应用[J].数字化用户,2017,23(51):272,266.
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[22]Taylor SR,Markesbery MG,Harding PA.Heparin-binding epidermal growth factor-like growth factor(HB-EGF)and proteolytic processing by a disintegrin and metalloproteinases(ADAM):a regulator of several pathways.Semin Cell Dev Biol.2014;28:22-30.
[23]Iwamoto R,Mekada E.HB-EGF(Heparin-Binding EGF-Like Growth Factor).In:Choi S,ed.Encyclopedia of Signaling Molecules.Cham:Springer International Publishing.2018:2318-2329.
[24]Adam RM.Role of HB-EGF in cancer.Atlas Genet Cytogenet Oncol Haematol.2011;15(7):610-619.
[25]Dao DT,Anez-Bustillos L,Adam RM,et al.Heparin-Binding Epidermal Growth Factor-Like Growth Factor as a Critical Mediator of Tissue Repair and Regeneration.Am J Pathol.2018;188(11):2446-2456.
[26]Hanoun M,Frenette PS.This niche is a maze;an amazing niche.Cell Stem Cell.2013;12(4):391-392.d
[27]Greenbaum A,Hsu YM,Day RB,et al.CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance.Nature.2013;495(7440):227-230.
[28]Mehta VB,Besner GE.HB-EGF promotes angiogenesis in endothelial cells via PI3-kinase and MAPK signaling pathways.Growth Factors.2007;25(4):253-263.
[29]Jackson LF,Qiu TH,Sunnarborg SW,et al.Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling.EMBO J.2003;22(11):2704-2716.
[30]Mine N,Iwamoto R,Mekada E.HB-EGF promotes epithelial cell migration in eyelid development.Development.2005;132(19):4317-4326.
[31]Berendsen AD,Olsen BR.Bone development.Bone.2015;80:14-18.
[32]Ono N,Kronenberg HM.Bone repair and stem cells.Curr Opin Genet Dev.2016;40:103-107.
[33]Nakamura T,Toita H,Yoshimoto A,et al.Potential involvement of Twist2 and Erk in the regulation of osteoblastogenesis by HB-EGF-EGFR signaling.Cell Struct Funct.2010;35(1):53-61.
[34]Chan SY,Wong RW.Expression of epidermal growth factor in transgenic mice causes growth retardation.J Biol Chem.2000;275(49):38693-38698.
[35]Loeser RF,Olex AL,McNulty MA,et al.Microarray analysis reveals age-related differences in gene expression during the development of osteoarthritis in mice.Arthritis Rheum.2012;64(3):705-717.
[36]Loeser RF,Long DL,Olex AL,et al.A systems biology approach identifies heparin-binding EGF-like growth factor as a potential mediator in OA.Osteoarthr Cartil.2013;21:S234-S235.
[37]Zhang X,Zhu J,Liu F,et al.Reduced EGFR signaling enhances cartilage destruction in a mouse osteoarthritis model.Bone Res.2014;2:14015.
[38]Long DL,Ulici V,Chubinskaya S,et al.Heparin-binding epidermal growth factor-like growth factor(HB-EGF)is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities.Osteoarthritis Cartilage.2015;23(9):1523-1531.
[39]Sherwood J.Osteoarthritis year in review 2018:biology.Osteoarthr Cartil.2018;27(3):365-370.
[40]Vasheghani F,Monemdjou R,Fahmi H,et al.Adult cartilage-specific peroxisome proliferator-activated receptor gamma knockout mice exhibit the spontaneous osteoarthritis phenotype.Am J Pathol.2013;182(4):1099-1106.
[2]Miyamoto S,Yagi H,Yotsumoto F,et al.Heparin-binding epidermal growth factor-like growth factor as a novel targeting molecule for cancer therapy.Cancer Sci.2006;97(5):341-347
[3]Falls DL.Neuregulins:functions,forms,and signaling strategies.Exp Cell Res.2003;284(1):14-30.
[4]Ming YC,Chao HC,Chu SM,et al.Heparin-binding epidermal growth factor-like growth factor(HB-EGF)protected intestinal ischemia-reperfusion injury through JNK and p38/MAPK-dependent pathway for anti-apoptosis.Pediatr Neonatol.2018.doi:10.1016/j.pedneo.2018.08.003.[Epub ahead of print]
[5]Zhang X,Tamasi J,Lu X,et al.Epidermal growth factor receptor plays an anabolic role in bone metabolism in vivo.J Bone Miner Res.2011;26(5):1022-1034.
[6]Roskoski R Jr.The ErbB/HER family of protein-tyrosine kinases and cancer.Pharmacol Res.2014;79:34-74.
[7]Barry FP.Biology and clinical applications of mesenchymal stem cells.Birth Defects Res C Embryo Today.2003;69(3):250-256.
[8]Ohishi M,Schipani E.Bone marrow mesenchymal stem cells.JCell Biochem.2010;109(2):277-282.
[9]Lamplot JD,Denduluri S,Liu X,et al.Major signaling pathways regulating the proliferation and differentiation of mesenchymal stem cells.In:Zhao R C,ed.Essentials of Mesenchymal Stem Cell Biology and Its Clinical Translation,Dordrecht:Springer Netherlands.2013:75-100.
[10]Chien HH,Lin WL,Cho MI.Down-regulation of osteoblastic cell differentiation by epidermal growth factor receptor.Calcif Tissue Int.2000;67(2):141-150.
[11]Zhang X,Siclari VA,Lan S,et al.The critical role of the epidermal growth factor receptor in endochondral ossification.J Bone Miner Res.2011;26(11):2622-2633.
[12]Vary CP,Li V,Raouf A,et al.Involvement of Ets transcription factors and targets in osteoblast differentiation and matrix mineralization.Exp Cell Res.2000;257(1):213-222.
[13]Tsuritani K,Takeda J,Sakagami J,et al.Cytokine receptor-like factor 1 is highly expressed in damaged human knee osteoarthritic cartilage and involved in osteoarthritis downstream of TGF-beta.Calcif Tissue Int.2010;86(1):47-57.
[14]Krampera M,Pasini A,Rigo A,et al.HB-EGF/HER-1 signaling in bone marrow mesenchymal stem cells:inducing cell expansion and reversibly preventing multilineage differentiation.Blood.2005;106(1):59-66.
[15]Ding L,Morrison SJ.Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches.Nature.2013;495(7440):231-235.
[16]Madisen L,Zwingman TA,Sunkin SM,et al.A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.Nat Neurosci.2010;13(1):133-140.
[17]Logan M,Martin JF,Nagy A,et al.Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer.Genesis.2002;33(2):77-80.
[18]Buch T,Heppner FL,Tertilt C,et al.A Cre-inducible diphtheria toxin receptor mediates cell lineage ablation after toxin administration.Nat Methods.2005;2(6):419-426.
[19]常晓朋,陈涛,赵寅,等.骨形态发生蛋白2和转化生长因子β2协同促进骨髓间充质干细胞成骨分化[J].中国组织工程研究,2019,23(1):1-6.
[20]张文.SkyScan 1176 Micro-CT在骨科研究中的应用[J].数字化用户,2017,23(51):272,266.
[21]Higashiyama S,Abraham JA,Miller J,et al.A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF.Science.1991;251(4996):936-939.
[22]Taylor SR,Markesbery MG,Harding PA.Heparin-binding epidermal growth factor-like growth factor(HB-EGF)and proteolytic processing by a disintegrin and metalloproteinases(ADAM):a regulator of several pathways.Semin Cell Dev Biol.2014;28:22-30.
[23]Iwamoto R,Mekada E.HB-EGF(Heparin-Binding EGF-Like Growth Factor).In:Choi S,ed.Encyclopedia of Signaling Molecules.Cham:Springer International Publishing.2018:2318-2329.
[24]Adam RM.Role of HB-EGF in cancer.Atlas Genet Cytogenet Oncol Haematol.2011;15(7):610-619.
[25]Dao DT,Anez-Bustillos L,Adam RM,et al.Heparin-Binding Epidermal Growth Factor-Like Growth Factor as a Critical Mediator of Tissue Repair and Regeneration.Am J Pathol.2018;188(11):2446-2456.
[26]Hanoun M,Frenette PS.This niche is a maze;an amazing niche.Cell Stem Cell.2013;12(4):391-392.d
[27]Greenbaum A,Hsu YM,Day RB,et al.CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance.Nature.2013;495(7440):227-230.
[28]Mehta VB,Besner GE.HB-EGF promotes angiogenesis in endothelial cells via PI3-kinase and MAPK signaling pathways.Growth Factors.2007;25(4):253-263.
[29]Jackson LF,Qiu TH,Sunnarborg SW,et al.Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling.EMBO J.2003;22(11):2704-2716.
[30]Mine N,Iwamoto R,Mekada E.HB-EGF promotes epithelial cell migration in eyelid development.Development.2005;132(19):4317-4326.
[31]Berendsen AD,Olsen BR.Bone development.Bone.2015;80:14-18.
[32]Ono N,Kronenberg HM.Bone repair and stem cells.Curr Opin Genet Dev.2016;40:103-107.
[33]Nakamura T,Toita H,Yoshimoto A,et al.Potential involvement of Twist2 and Erk in the regulation of osteoblastogenesis by HB-EGF-EGFR signaling.Cell Struct Funct.2010;35(1):53-61.
[34]Chan SY,Wong RW.Expression of epidermal growth factor in transgenic mice causes growth retardation.J Biol Chem.2000;275(49):38693-38698.
[35]Loeser RF,Olex AL,McNulty MA,et al.Microarray analysis reveals age-related differences in gene expression during the development of osteoarthritis in mice.Arthritis Rheum.2012;64(3):705-717.
[36]Loeser RF,Long DL,Olex AL,et al.A systems biology approach identifies heparin-binding EGF-like growth factor as a potential mediator in OA.Osteoarthr Cartil.2013;21:S234-S235.
[37]Zhang X,Zhu J,Liu F,et al.Reduced EGFR signaling enhances cartilage destruction in a mouse osteoarthritis model.Bone Res.2014;2:14015.
[38]Long DL,Ulici V,Chubinskaya S,et al.Heparin-binding epidermal growth factor-like growth factor(HB-EGF)is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities.Osteoarthritis Cartilage.2015;23(9):1523-1531.
[39]Sherwood J.Osteoarthritis year in review 2018:biology.Osteoarthr Cartil.2018;27(3):365-370.
[40]Vasheghani F,Monemdjou R,Fahmi H,et al.Adult cartilage-specific peroxisome proliferator-activated receptor gamma knockout mice exhibit the spontaneous osteoarthritis phenotype.Am J Pathol.2013;182(4):1099-1106.