双调蛋白经PI3K/AKT通路促进ATDC5细胞成骨分化

doi:10.13701/j.cnki.kqyxyj.2024.07.006

口腔医学研究 ›› 2024, Vol. 40 ›› Issue (7): 599-604.DOI: 10.13701/j.cnki.kqyxyj.2024.07.006

双调蛋白经PI3K/AKT通路促进ATDC5细胞成骨分化

王心怡, 王心茹, 王硕, 王家伟^*

口颌系统重建与再生全国重点实验室,口腔生物医学教育部重点实验室,口腔医学湖北省重点实验室,武汉大学口腔医(学)院湖北武汉 430079

收稿日期:2024-03-13 出版日期:2024-07-28 发布日期:2024-07-24
通讯作者: *王家伟,E-mail:wb000238@whu.edu.cn
作者简介:王心怡(1998~ ),女,安徽人,硕士在读,主要从事软骨内成骨相关的分子机制研究。
基金资助:
国家自然科学基金(编号:82170930、82201025、81870744)及中央高校基本科研业务费

Amphiregulin Regulates Proliferation and Differentiation of ATDC5 Cells through PI3K/AKT Signaling Pathway

WANG Xinyi, WANG Xinru, WANG Shuo, WANG Jiawei^*

State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, China.

Received:2024-03-13 Online:2024-07-28 Published:2024-07-24

摘要/Abstract

摘要： 目的:探究双调蛋白(amphiregulin,Areg)对ATDC5细胞增殖和成骨分化的影响。方法:实时荧光定量逆转录聚合酶链反应(quantitative real time polymerase chain reaction,qRT-PCR)检测ATDC5细胞诱导成骨分化过程中Areg的表达;免疫组织化学染色分析胫骨生长板Areg表达量。Areg过表达或敲低慢病毒感染ATDC5细胞,CCK-8检测Areg对ATDC5细胞增殖的影响;qRT-PCR和Western blot分别从mRNA和蛋白水平检测Areg对ATDC5细胞诱导成骨分化7 d或14 d相关标志物Sox9、Ⅱ型胶原蛋白(collagentype Ⅱ alpha1chain,Col2α1)、Ⅹ型胶原蛋白(collagentype Ⅹ alpha1chain,ColⅩα1)、骨桥蛋白(osteopontin,OPN)和Runt相关转录因子2(Runt-related transcription factor 2,Runx2)的影响;甲苯胺蓝、阿尔新蓝和茜素红染色检测细胞外基质形成。Western blot和染色检测ATDC5细胞诱导成骨分化7 d及加入p-蛋白激酶B(p-protein kinase B,p-AKT)抑制剂MK2206后磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/AKT通路的磷酸化水平和细胞外基质变化。结果:Areg在ATDC5细胞成骨分化过程中始终表达,并于7 d达到峰值;胫骨生长板可见软骨细胞高表达Areg;过表达Areg后细胞活力,细胞成骨分化能力和细胞外基质形成显著增加;敲低Areg后则表现为相反的结果;Areg过表达激活了PI3K/AKT通路,p-AKT抑制剂部分逆转了PI3K/AKT通路的激活和Areg过表达对软骨细胞外基质生成的促进作用。结论:Areg促进 ATDC5细胞增殖,且部分经PI3K/AKT通路促进ATDC5细胞成骨分化。

关键词: 软骨细胞, 细胞增殖, 成骨向分化, 磷脂酰肌醇3-激酶/蛋白激酶B信号通路

Abstract: Objective: To explore the impact of amphiregulin (Areg) on the proliferation and osteogenic differentiation of ATDC5 cells. Methods: Quantitative real time polymerase chain reaction (qRT-PCR) was used to assess Areg expression in ATDC5 cells. The expression of Areg in tibial growth plates underwent analysis via immunohistochemical staining. Lentivirus-mediated Areg overexpression or knockdown was conducted in ATDC5 cells, and the impact on cell proliferation was evaluated using CCK-8 assay. qRT-PCR and Western blot analyses evaluated the impact of Areg on osteogenic differentiation in ATDC5 cells on markers associated with 7-day, such as Sox9 and collagentype Ⅱ alpha1chain (Col2α1) or 14-day, such as collagentype Ⅹ alpha1chain (ColⅩα1), osteopontin (OPN), and Runt-related transcription factor 2 (Runx2). Cartilage matrix formation was assessed using staining techniques. Western blot analysis was conducted 7 days after initiating ATDC5 cells osteogenic differentiation to assess the phosphorylation of the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) pathway. The phosphorylation of PI3K/AKT was attenuated following the administration of the p-AKT inhibitor MK2206. Results: Areg expression persisted throughout the osteogenic differentiation of ATDC5 cells, peaking on day 7. Areg was highly expressed in chondrocytes of tibial growth plates. Furthermore, Areg overexpression significantly enhanced cell viability, chondrogenic differentiation and extracellular matrix formation. While the opposite effect was observed when Areg was knockdown. Areg overexpression activated PI3K/AKT pathway, and the addition of p-AKT inhibitor MK2206 reversed the activation. Conclusion: Areg promotes the proliferation of ATDC5 cells, and partially stimulates the osteogenic differentiation of ATDC5 cells via the PI3K/AKT pathway.

Key words: chondrocyte, cell proliferation, osteogenic differentiation, PI3K/AKT signaling pathway

王心怡, 王心茹, 王硕, 王家伟. 双调蛋白经PI3K/AKT通路促进ATDC5细胞成骨分化[J]. 口腔医学研究, 2024, 40(7): 599-604.

WANG Xinyi, WANG Xinru, WANG Shuo, WANG Jiawei. Amphiregulin Regulates Proliferation and Differentiation of ATDC5 Cells through PI3K/AKT Signaling Pathway[J]. Journal of Oral Science Research, 2024, 40(7): 599-604.

参考文献

[1] Scotti C, Piccinini E, Takizawa H, et al. Engineering of a functional bone organ through endochondral ossification [J]. Proc Natl Acad Sci U S A, 2013, 110(10):3997-4002.
[2] Zaiss DMW, Gause WC, Osborne LC, et al. Emerging functions of amphiregulin in orchestrating immunity, inflammation, and tissue repair [J]. Immunity, 2015, 42(2):216-226.
[3] Qin L, Tamasi J, Raggatt L, et al. Amphiregulin is a novel growth factor involved in normal bone development and in the cellular response to parathyroid hormone stimulation [J]. J Biol Chem, 2005, 280(5):3974-3981.
[4] Kimura H, Schubert D. Schwannoma-derived growth factor promotes the neuronal differentiation and survival of PC12 cells [J]. J Cell Biol, 1992, 116(3):777-783.
[5] Pasic L, Eisinger-Mathason TS, Velayudhan BT, et al. Sustained activation of the HER1-ERK1/2-RSK signaling pathway controls myoepithelial cell fate in human mammary tissue [J]. Genes Dev, 2011, 25(15):1641-1653.
[6] Ulici V, Hoenselaar KD, Gillespie JR, et al. The PI3K pathway regulates endochondral bone growth through control of hypertrophic chondrocyte differentiation [J]. BMC Dev Biol, 2008, 8:40.
[7] Kita K, Kimura T, Nakamura N, et al. PI3K/Akt signaling as a key regulatory pathway for chondrocyte terminal differentiation [J]. Genes Cells, 2008, 13(8):839-850.
[8] Li G, Wang L, Jiang Y, et al. Upregulation of Akt signaling enhances femoral fracture healing by accelerating atrophic quadriceps recovery [J]. Biochim Biophys Acta Mol Basis Dis, 2017, 1863(11):2848-2861.
[9] Shukunami C, Ishizeki K, Atsumi T, et al. Cellular hypertrophy and calcification of embryonal carcinoma-derived chondrogenic cell line ATDC5 in vitro [J]. J Bone Miner Res, 1997, 12(8):1174-1188.
[10] Kirimoto A, Takagi Y, Ohya K, et al. Effects of retinoic acid on the differentiation of chondrogenic progenitor cells, ATDC5 [J]. J Med Dent Sci, 2005, 52(3):153-162.
[11] Wuelling M, Vortkamp A. Transcriptional networks controlling chondrocyte proliferation and differentiation during endochondral ossification [J]. Pediatr Nephrol, 2010, 25(4):625-631.
[12] Berasain C, García-Trevijano ER, Castillo J, et al. Novel role for amphiregulin in protection from liver injury [J]. J Biol Chem, 2005, 280(19):19012-19020.
[13] Hsu D, Fukata M, Hernandez YG, et al. Toll-like receptor 4 differentially regulates epidermal growth factor-related growth factors in response to intestinal mucosal injury [J]. Lab Invest, 2010, 90(9):1295-1305.
[14] 杜小颖,丁威薇,陈悦,等.IGF-1通过PI3K/AKT信号通路促进牙髓细胞增殖及碱性磷酸酶活性的研究[J].口腔医学研究,2017,33(5):538-541.
[15] Zhang H, Chen X, Xue P, et al. FN1 promotes chondrocyte differentiation and collagen production via TGF-β/PI3K/Akt pathway in mice with femoral fracture [J]. Gene, 2021, 769:145253.

双调蛋白经PI3K/AKT通路促进ATDC5细胞成骨分化

Amphiregulin Regulates Proliferation and Differentiation of ATDC5 Cells through PI3K/AKT Signaling Pathway

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