CDC20通过泛素蛋白酶体途径降解p65调控人脂肪间充质干细胞成骨向分化

doi:10.13701/j.cnki.kqyxyj.2022.06.015

口腔医学研究 ›› 2022, Vol. 38 ›› Issue (6): 565-571.DOI: 10.13701/j.cnki.kqyxyj.2022.06.015

CDC20通过泛素蛋白酶体途径降解p65调控人脂肪间充质干细胞成骨向分化

杜杨格, 程雅雯, 郭倩, 张萍^*, 周永胜^*

北京大学口腔医学院·口腔医院修复科国家口腔医学中心国家口腔疾病临床医学研究中心口腔生物材料和数字诊疗装备国家工程研究中心口腔数字医学北京市重点实验室北京 100081

收稿日期:2022-01-06 出版日期:2022-06-28 发布日期:2022-06-23
通讯作者: * 张萍,E-mail: zhangping332@hsc.pku.edu.cn;周永胜,E-mail: kqzhouysh@hsc.pku.edu.cn
作者简介:杜杨格(1994~),女,湖北人,博士,研究方向:干细胞与骨再生。
基金资助:
国家自然科学基金(编号:81970911)；北京市自然科学基金(编号:7182183、7202233)

CDC20 Regulates the Osteogenic Differentiation of hASCs through Proteasome-dependent Degradation of p65

DU Yangge, CHENG Yawen, GUO Qian, ZHANG Ping^*, ZHOU Yongsheng^*

Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China

Received:2022-01-06 Online:2022-06-28 Published:2022-06-23

摘要/Abstract

摘要： 目的:探究细胞分裂周期蛋白20 (cell division cycle 20, CDC20)对人脂肪间充质干细胞(human adipose-derived stem cells, hASCs)成骨分化的调控作用及其机制。方法:将hASCs分别进行成骨向诱导0、7、14 d后,通过实时荧光定量PCR和Western blot检测CDC20表达量的变化。对稳定敲低CDC20的hASCs进行成骨向诱导后,分别通过碱性磷酸酶(alkaline phosphatase, ALP)染色定量实验,Western blot检测成骨相关标志物的表达。将稳定敲低CDC20的hASCs与β-磷酸三钙(β-tricalcium phosphate,β-TCP)材料混合植入裸鼠皮下,8周后取材,HE、MASSON染色检测骨胶原的形成。对过表达CDC20的hASCs进行成骨向诱导后,通过ALP染色定量实验检测hASCs成骨向分化。利用免疫共沉淀和Western blot探索CDC20和p65的关系。构建CDC20和p65双敲细胞系,检测p65在CDC20调控骨再生中的作用。结果:在hASCs成骨向分化过程中,CDC20的表达显著升高(P＜0.05)。稳定敲低CDC20的hASCs在成骨诱导后ALP活性为(7.31±0.25)、(11.01±0.49) U/gprot,低于对照组(16.00±0.35) U/gprot,差异有统计学意义(P＜0.05);Western blot结果显示敲低CDC20组RUNX2的蛋白水平降低。稳定敲低CDC20的hASCs与β-TCP材料混合物植入裸鼠皮下8周后其形成的骨胶原较对照组减少。过表达CDC20的hASCs在成骨诱导后ALP活性为(20.74±0.53) U/gprot,高于对照组(12.58±0.42) U/gprot,差异有统计学意义(P＜0.05)。CDC20可结合p65;敲低CDC20后,p65的表达量升高;过表达CDC20后,p65的表达量降低,且在加入蛋白酶体抑制剂MG132后回升。CDC20和p65双敲细胞系成骨诱导后,ALP染色及定量结果表明CDC20对hASCs成骨向分化的调控依赖于p65。结论:CDC20通过泛素蛋白酶体途径降解p65促进人脂肪间充质干细胞成骨向分化,为骨再生提供全新的潜在治疗靶点。

关键词: 人脂肪间充质干细胞, 细胞分裂周期蛋白20, 成骨向分化, p65

Abstract: Objective: To investigate the role of cell division cycle 20 (CDC20) in the osteogenesis of human adipose-derived stem cells (hASCs) and its underlying mechanisms. Methods: After 0, 7, and 14 days of osteogenic induction, RT-qPCR and Western blot assays were used to determine CDC20 level. In CDC20 stable knockdown hASCs, ALP staining and Western blot assays were performed to examine the expressions of related markers of osteogenic differentiation. The CDC20 stable knockdown hASCs mixed with β-tricalcium phosphate (β-TCP) were implanted under the skin of nude mice. Eight weeks later, the mice were sacrificed and ectopic bone were measured. In CDC20 overexpressed hASCs, ALP staining and quantification were performed to examine the osteogenic differentiation of hASCs. Co-immunoprecipitation and western blot assays were conducted to explore the relationship of CDC20 and p65 in hASCs. The CDC20 and p65 double knockdown hASCs were constructed to evaluate the effect of p65 in osteogenesis manipulated by CDC20. Results: CDC20 was significantly induced in hASCs during osteogenic differentiation (P＜0.05). After the osteogenic induction, ALP quantification of CDC20 stable knockdown hASCs [(7.31±0.25)、(11.01±0.49) U/gprot] was significantly lower than that of control [(16.00±0.35) U/gprot] (P＜0.05). Western blot results showed the decreased RUNX2 protein level in CDC20 knockdown group. The CDC20 stable knockdown hASCs mixed with β-TCP and implanted under the skin of nude mice represented less bone formation compared to control cells. After the osteogenic induction, ALP quantification of CDC20 overexpression in hASCs [(20.74±0.53) U/gprot] was significantly higher than that of control [(12.58±0.42) U/gprot](P＜0.05). CDC20 interacted with p65. The expression of p65 increased after the knockdown of CDC20. Overexpression of CDC20 led to decreased p65, and the treatment of MG132 (the proteasome inhibitor) reversed the trend. After osteogenic differentiation of CDC20 and p65 double knockdown hASCs,the ALP staining and quantification results illustrated that CDC20 regulated osteogenic differentiation of hASCs in a p65-dependent manner. Conclusion: CDC20 promoted the osteogenic differentiation of hASCs through proteasome-dependent degradation of p65,thus providing a potential target for bone regeneration.

Key words: hASCs, CDC20, osteogenic differentiation, p65

杜杨格, 程雅雯, 郭倩, 张萍, 周永胜. CDC20通过泛素蛋白酶体途径降解p65调控人脂肪间充质干细胞成骨向分化[J]. 口腔医学研究, 2022, 38(6): 565-571.

DU Yangge, CHENG Yawen, GUO Qian, ZHANG Ping, ZHOU Yongsheng. CDC20 Regulates the Osteogenic Differentiation of hASCs through Proteasome-dependent Degradation of p65[J]. Journal of Oral Science Research, 2022, 38(6): 565-571.

参考文献

[1] 王松灵.干细胞在口腔组织再生中大有可为[J].中华口腔医学杂志,2017,52(10): 585-587.
[2] Crane GM, Ishaug SL, Mikos AG. Bone tissue engineering [J]. Nat Med, 1995, 1(12): 1322-1324.
[3] Shafaei H, Kalarestaghi H. Adipose-derived stem cells: An appropriate selection for osteogenic differentiation [J]. J Cell Physiol, 2020, 235(11):8371-8386.
[4] Mao SH, Chen CH, Chen CT. Osteogenic potential of induced pluripotent stem cells from human adipose-derived stem cells [J]. Stem Cell Res Ther, 2019, 10(1):303.
[5] Pickart CM. Mechanisms underlying ubiquitination [J]. Annu Rev Biochem, 2001, 70:503-533.
[6] Tang Y, Lv L, Li W, et al. Protein deubiquitinase USP7 is required for osteogenic differentiation of human adipose-derived stem cells [J]. Stem Cell Res Ther, 2017, 8(1): 186.
[7] Yang X, Hao D, He B. The regulation of E3 ubiquitin ligases Cbl and its cross talking in bone homeostasis [J]. Curr Stem Cell Res Ther, 2021, 16(6):683-687.
[8] Kimata Y, Baxter JE, Fry AM, et al. A role for the Fizzy/Cdc20 family of proteins in activation of the APC/C distinct from substrate recruitment [J]. Mol Cell, 2008, 32(4):576-583.
[9] Yang Y, Kim AH, Yamada T, et al. A Cdc20-APC ubiquitin signaling pathway regulates presynaptic differentiation [J]. Science, 2009, 326(5952):575-578.
[10] Wan L, Tan M, Yang J, et al. APC(Cdc20) suppresses apoptosis through targeting Bim for ubiquitination and destruction [J]. Dev Cell, 2014, 29(4):377-391.
[11] 张哲,杨枫,杨剑,等.PLK1与CDC20在乳腺癌中的表达及其临床意义[J].临床肿瘤学杂志,2017,22(7):622-627.
[12] Chang DZ, Ma Y, Ji B, et al. Increased CDC20 expression is associated with pancreatic ductal adenocarcinoma differentiation and progression [J]. J Hematol Oncol, 2012, 5:15.
[13] 刘云松,吕珑薇,周永胜,等.体内成骨过程中人脂肪基质细胞对新骨形成的促进作用[J].北京大学学报(医学版), 2012, 44(6):916-920.
[14] Geley S, Kramer E, Gieffers C, et al. anaphase-romoting complex/cyclosome-dependent proteolysis of human cyclin a starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint [J]. J Cell Biol, 2001, 153(1): 137-147.
[15] Hames RS, Wattam SL, Yamano H, et al. APC/C-mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-type D-box [J]. EMBO J, 2001, 20(24): 7117-7127.
[16] Kim AH, Puram SV, Bilimoria PM, et al. A centrosomal Cdc20-APC pathway controls dendrite morphogenesis in postmitotic neurons [J]. Cell, 2009, 136(2): 322-336.
[17] Hou Y, Moreau F, Chadee K. PPARγ is an E3 ligase that induces the degradation of NFκB/p65 [J]. Nat Commun, 2012, 3:1300.
[18] Maine GN, Mao X, Komarck CM, et al. COMMD1 promotes the ubiquitination of NF-κB subunits through a cullin-containing ubiquitin ligase [J]. EMBO J, 2007, 26(2):436-447.
[19] Li X, Huang M, Zheng H, et al. CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation [J]. J Cell Biol, 2008, 181(6): 959-972.
[20] Bruzzaniti A, Neff L, Sanjay A, et al. Dynamin forms a Src kinase-sensitive complex with Cbl and regulates podosomes and osteoclast activity[J]. Mol Biol Cell, 2005, 16(7):3301-3313.
[21] Jones DC, Wein MN, Oukka M, et al. Regulation of adult bone mass by the zinc finger adapter protein Schnurri-3[J]. Science, 2006, 312(5777): 1223-1227.
[22] Wan L, Tan M, Yang J, et al. APC(Cdc20) suppresses apoptosis through targeting Bim for ubiquitination and destruction [J]. Dev Cell, 2014, 29(4): 377-391.

CDC20通过泛素蛋白酶体途径降解p65调控人脂肪间充质干细胞成骨向分化

CDC20 Regulates the Osteogenic Differentiation of hASCs through Proteasome-dependent Degradation of p65

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 2

编辑推荐

Metrics

[1]	侯婷婷, 潘爽, 李艳萍, 何丽娜, 肖婉鲁, 牛玉梅. 载柚皮苷羟基磷灰石胶原支架对人牙髓干细胞体外增殖及成骨向分化的影响[J]. 口腔医学研究, 2019, 35(8): 744-747.
[2]	李斯翰,段建民,文军,李洪涛. 富血小板血浆对骨髓间充质干细胞成骨向分化的作用[J]. 口腔医学研究, 2015, 31(7): 735-738.