Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress

doi:10.7518/hxkq.2023.2022382

Abstract

Abstract:

Objective This work aimed to investigate the molecular mechanism of cyclic tensile stress (CTS) stimulating autophagy in human periodontal ligament cells (hPDLCs). Methods hPDLCs were isolated and cultured from normal periodontal tissues. hPDLCs were loaded with tensile stress by force four-point bending extender to simulate the autophagy of hPDLCs induced by orthodontic force du-ring orthodontic tooth movement. XMU-MP-1 was used to inhibit the Hippo signaling pathway to explore the role of the Hippo-YAP signaling pathway in activating hPDLC autophagy by tensile stress. The expression levels of autophagy-related genes (Beclin-1, LC3, and p62) in hPDLCs were detected by real-time quantitative polymerase chain reaction. Western blot was used to detect the expression levels of autophagy-related proteins (Beclin-1, LC3-Ⅱ/LC3-Ⅰ, and p62) and Hippo-YAP pathway proteins (active-YAP and p-YAP) in hPDLCs. Immunofluorescence was used to locate autophagy-related proteins (LC3-Ⅱand p62) and Hippo-YAP pathway proteins (active-YAP) of hPDLCs. Results CTS-activated autophagy in hPDLCs and expression of autophagy-related proteins initially increased and then decreased; it began to increase at 30 min, peaked at 3 h, and decreased (P<0.05). CTS increased the expression of active-YAP protein and decreased the expression of p-YAP protein (P<0.05). When XMU-MP-1 inhibited the Hippo-YAP signaling pathway (P<0.05), active-YAP protein was promoted to enter the nucleus and autophagy expression was enhanced (P<0.05). Conclusion The Hippo-YAP signaling pathway is involved in the regulation of autophagy activation in hPDLCs under CTS.

Key words: autophagy, Hippo-YAP signaling pathway, tensile stress, human periodontal ligament cells

CLC Number:

R 783.5

Wan Xiaofang, He Haiyan, Jialing Lü, Wu Yujie, Zhong Guannan, Xu Xiaomei. Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress[J]. West China Journal of Stomatology, 2023, 41(3): 260-268.

Figures/Tables 5

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References 34

1	Chang M, Lin H, Luo M, et al. Integrated miRNA and mRNA expression profiling of tension force-induced bone formation in periodontal ligament cells[J]. In Vitro Cell Dev Biol Anim, 2015, 51(8): 797-807.
2	Xu J, Zhao X, Zeng J, et al. Role of autophagy in the periodontal ligament reconstruction during orthodontic tooth movement in rats[J]. J Dent Sci, 2020, 15(3): 351-363.
3	Dohmen M, Krieg S, Agalaridis G, et al. AMPK-dependent activation of the Cyclin Y/CDK16 complex controls autophagy[J]. Nat Commun, 2020, 11(1): 1032.
4	Blawat K, Mayr A, Hardt M, et al. Regulation of auto-phagic signaling by mechanical loading and inflammation in human PDL fibroblasts[J]. Int J Mol Sci, 2020, 21(24): E9446.
5	Deretic V, Levine B. Autophagy balances inflammation in innate immunity[J]. Autophagy, 2018, 14(2): 243-251.
6	Pedro JMB, Sica V, Madeo F, et al. Acyl-CoA-binding protein (ACBP): the elusive ‘hunger factor’ linking autophagy to food intake[J]. Cell Stress, 2019, 3(10): 312-318.
7	Feng X, Zhang H, Meng LB, et al. Hypoxia-induced acetylation of PAK1 enhances autophagy and promotes brain tumorigenesis via phosphorylating ATG5[J]. Autophagy, 2021, 17(3): 723-742.
8	Keith SA, Maddux SK, Zhong Y, et al. Graded proteasome dysfunction in caenorhabditis elegans activates an adaptive response involving the conserved SKN-1 and ELT-2 transcription factors and the autophagy-lysosome pathway[J]. PLoS Genet, 2016, 12(2): e1005823.
9	Vidoni C, Ferraresi A, Secomandi E, et al. Autophagy drives osteogenic differentiation of human gingival mesenchymal stem cells[J]. Cell Commun Signal, 2019, 17(1): 98.
10	Elbediwy A, Vincent-Mistiaen ZI, Thompson BJ. YAP and TAZ in epithelial stem cells: a sensor for cell polarity, mechanical forces and tissue damage[J]. Bioessays, 2016, 38(7): 644-653.
11	Fu HD, Wang BK, Wan ZQ, et al. Wnt5a mediated canonical Wnt signaling pathway activation in orthodontic tooth movement: possible role in the tension force-induced bone formation[J]. J Mol Histol, 2016, 47(5): 455-466.
12	Gong Y, Li SJ, Liu R, et al. Inhibition of YAP with si-RNA prevents cartilage degradation and ameliorates osteoarthritis development[J]. J Mol Med (Berl), 2019, 97(1): 103-114.
13	Wang D, He J, Huang B, et al. Emerging role of the Hippo pathway in autophagy[J]. Cell Death Dis, 2020, 11(10): 880.
14	Elosegui-Artola A, Andreu I, Beedle AEM, et al. Force triggers YAP nuclear entry by regulating transport across nuclear pores[J]. Cell, 2017, 171(6): 1397-1410.e14.
15	Piccolo S, Dupont S, Cordenonsi M. The biology of YAP/TAZ: Hippo signaling and beyond[J]. Physiol Rev, 2014, 94(4): 1287-1312.
16	Pei T, Huang X, Long Y, et al. Increased expression of YAP is associated with decreased cell autophagy in the eutopic endometrial stromal cells of endometriosis[J]. Mol Cell Endocrinol, 2019, 491: 110432.
17	Sun B, Wen Y, Wu X, et al. Expression pattern of YAP and TAZ during orthodontic tooth movement in rats[J]. J Mol Histol, 2018, 49(2): 123-131.
18	Pan X, Wu B, Fan X, et al. YAP accelerates vascular senescence via blocking autophagic flux and activating mTOR[J]. J Cell Mol Med, 2021, 25(1): 170-183.
19	Pei T, Luo B, Huang W, et al. Increased expression of YAP inhibited the autophagy level by upregulating m-TOR signal in the eutopic ESCs of endometriosis[J]. Front Endocrinol (Lausanne), 2022, 13: 813165.
20	Jin L, Chen Y, Cheng D, et al. YAP inhibits autophagy and promotes progression of colorectal cancer via upregulating Bcl-2 expression[J]. Cell Death Dis, 2021, 12(5): 457.
21	Ge MK, Zhou CC, Li H, et al. Lithium chloride atte-nuates suppressed differentiation induced by mechanical strain in cementoblasts[J]. Connect Tissue Res, 2019, 60(5): 444-451.
22	朱庆党, 刘丽, 杨艳丽. 张应力刺激下人牙周膜成纤维细胞纤连蛋白、整合素、细胞骨架的变化[J]. 上海口腔医学, 2010, 19(6): 601-606.
	Zhu QD, Liu L, Yang YL. Effect of tensile stress on fibronectin-integrins-cytoskeleton in cultured human pe-riodontal ligament fibroblasts[J]. Shanghai J Stomatol, 2010, 19(6): 601-606.
23	朱庆党, 巢永烈, 陈新民, 等. 机械应力对人牙周膜成纤维细胞整合素β1 mRNA表达的调节[J]. 华西口腔医学杂志, 2008, 26(2): 194-197.
	Zhu QD, Chao YL, Chen XM, et al. Regulation of integrin beta1 mRNA expression by mechanical stress in human periodontal ligament fibroblasts[J]. West China J Stomatol, 2008, 26(2): 194-197.
24	Memmert S, Damanaki A, Weykopf B, et al. Autophagy in periodontal ligament fibroblasts under biomechanical loading[J]. Cell Tissue Res, 2019, 378(3): 499-511.
25	Chen L, Mo S, Hua Y. Compressive force-induced autophagy in periodontal ligament cells downregulates osteoclastogenesis during tooth movement[J]. J Periodontol, 2019, 90(10): 1170-1181.
26	Zhang J, Zhou Y, Tang PMK, et al. Mechanotransduction and cytoskeleton remodeling shaping YAP1 in gastric tumorigenesis[J]. Int J Mol Sci, 2019, 20(7): 1576.
27	Yang Y, Wang BK, Chang ML, et al. Cyclic stretch enhances osteogenic differentiation of human periodontal ligament cells via YAP activation[J]. Biomed Res Int, 2018, 2018: 2174824.
28	Zhao M, Zhang Y, Jiang Y, et al. YAP promotes autophagy and progression of gliomas via upregulating HMGB1[J]. J Exp Clin Cancer Res, 2021, 40(1): 99.
29	Zhou X, Wang H, Li D, et al. MST1/2 inhibitor XMU-MP-1 alleviates the injury induced by ionizing radiation in haematopoietic and intestinal system[J]. J Cell Mol Med, 2022, 26(5): 1621-1628.
30	van Soldt BJ, Cardoso WV. Hippo-Yap/Taz signaling: complex network interactions and impact in epithelial cell behavior[J]. Wiley Interdiscip Rev Dev Biol, 2020, 9(3): e371.
31	Zheng Y, Pan D. The Hippo signaling pathway in development and disease[J]. Dev Cell, 2019, 50(3): 264-282.
32	Pavel M, Park SJ, Frake RA, et al. α-Catenin levels determine direction of YAP/TAZ response to autophagy perturbation[J]. Nat Commun, 2021, 12(1): 1703.
33	Liang N, Zhang C, Dill P, et al. Regulation of YAP by mTOR and autophagy reveals a therapeutic target of tuberous sclerosis complex[J]. J Exp Med, 2014, 211(11): 2249-2263.
34	Wang P, Gong Y, Guo T, et al. Activation of Aurora A kinase increases YAP stability via blockage of autophagy[J]. Cell Death Dis, 2019, 10(6): 432.

基因名称	引物序列（5'-3'）
GAPDH	F：CAATGACCCCTTCATTGACC R：GACAAGCTTCCCGTTCTCAG
Beclin-1	F：CGTGTCACCATCCAGGAACT R：ATCTCCAAACAGCGTCTGGCT
LC3	F：GAGAAGCAGCTTCCTGTTCTGG R：GTGTCCGTTCACCAACAGGAAG
p62	F：GGACCCGTCTACAGGTGAAC R：GAGAGGGACTCAATCAGCCG

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[2]	Wu Jie, Cui Zhanqin, Han Yu, Li Wenjing. Aging effect of osteoprotegerin and receptor activator of nuclear factor-κB ligand expression in human periodontal ligament cells under continuous static pressure [J]. West China Journal of Stomatology, 2022, 40(6): 654-661.
[3]	Liu Xinchen, Lu Jinjin, Chen Yumeng, Qiu Ying, Zheng Mengdan, Wang Zilin, Li Xiangwei. Roles of autophagy onself-renewal and differentiation of mesenchymal stem cells [J]. West China Journal of Stomatology, 2020, 38(6): 704-707.
[4]	Xu Zhi,Lü Fengyuan,Jiang Erhui,Zhao Xiaoping,Shang Zhengjun. Relationship among areca nut, intracellular reactive oxygen species, and autophagy [J]. West China Journal of Stomatology, 2020, 38(1): 80-85.
[5]	Longyi Mo,Xiaoyue Jia,Chengcheng Liu,Xuedong Zhou,Xin Xu. Role of autophagy in the pathogenesis of periodontitis [J]. West China Journal of Stomatology, 2019, 37(4): 422-427.
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[8]	Yang An, Huiyu Zhang, Junfeng Guo, Xin Li, Yang Yang, Gang Zhang, Yinghui. Tan. Effect of calcitonin gene-related peptide on MC3T3-E1 osteoblast apoptosis and autophagy induced by serum starvation [J]. West China Journal of Stomatology, 2017, 35(2): 133-138.
[9]	Yang Du, Shuai Yuan, Zhifei Zhou, Lizheng Wu, Lulu Wang, Xing’an Wu, Xiaojing Wang. A preliminary study on the autophagy level of human periodontal ligament cells regulated by nicotine [J]. West China Journal of Stomatology, 2017, 35(2): 198-202.
[10]	Ma Yue, Ren Aishu, Fu Gang.. Effects of salvianolic acid B on osteogenic differentiation of human periodontal ligament cells [J]. West China Journal of Stomatology, 2016, 34(5): 468-473.
[11]	Chen Zhanwei, Sun Dubin, Huang Shengyun, Wu Haiwei, Zhang Dongsheng. Analysis of BNIP3 expression and clinical research in salivary adenoid cystic carcinoma [J]. West China Journal of Stomatology, 2016, 34(4): 404-407.
[12]	Shi Shanwei, Li Yi. Autophagy and its relationship with tumor proliferation, invasion, and treatment [J]. West China Journal of Stomatology, 2015, 33(1): 98-103.
[13]	Zhan Xueling, Gao Jie, Liu Ying, Hu Jiao, Xue Yanxiang, Wu Buling. Lactoferrin downregulates the expression of Toll like receptor 4 stimulated by lipopolysaccharide in human periodontal ligament cells [J]. West China Journal of Stomatology, 2014, 32(2): 166-170.
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[15]	Fan Xiaofeng1, Wang Yu2，3, Li Yu4，5, Zhao Zhihe4，5.. Early proliferation changes and differences of gene expression in human periodontal ligament fibroblasts subjected to tensile and compressive stress [J]. West China Journal of Stomatology, 2012, 30(5): 463-467.