Role of the GRP78-c-Src signaling pathway on osteoblast differentiation of periodontal ligament fibroblasts induced by cyclic mechanical stretch

doi:10.7518/hxkq.2024.2023354

Abstract

Abstract:

Objective This study aims to investigate the influence of glucose regulated protein (GRP) 78 on osteoblast differentiation in periodontal ligament fibroblasts (PDLFs) under cyclic mechanical stretch and determine the underlying mechanism. Methods FlexCell 5000 cell mechanical device was applied to simulate the stress environment of orthodontic teeth. GRP78High and GRP78Low subpopulation were obtained by flow sorting. Gene transfection was performed to knockdown GRP78 and c-Src expression and overexpress c-Src. Western blot analysis was used to detect the protein expression of Runt-related gene 2 (RUNX2), Osterix, osteocalcin (OCN), and osteopontin (OPN). Immunoprecipitation assay was used to determine the interaction of GRP78 with c-Src. The formation of cellular mineralized nodules was determined by alizarin red staining. Results GRP78 was heterogeneously expressed in PDLFs, and GRP78High and GRP78Low subpopulations were obtained by flow sorting. The osteogenic differentiation ability and phosphorylation level of c-Src kinase in the GRP78High subpopulation were significantly increased compared with those in GRP78Low subpopulation after cyclic mechanical stretch (P<0.05). GRP78 interacted with c-Src in PDLFs. The overexpression c-Src group showed significantly increased osteogenic differentiation ability than the vector group (P<0.05), and the sic-Src group showed significantly decreased osteogenic differentiation ability (P<0.05) after cyclic mechanical stretch. Conclusion GRP78 upregulates c-Src expression by interacting with c-Src kinase and promotes osteogenic differentiation under cyclic mechanical stretch in PDLFs.

Key words: cyclic mechanical stretch, osteogenic differentiation, periodontal ligament fibroblasts, glucose regulated protein 78

CLC Number:

Hu Jing, Cui Zhihua, Huang Keqiang, Su Rongjian, Zhao Song. Role of the GRP78-c-Src signaling pathway on osteoblast differentiation of periodontal ligament fibroblasts induced by cyclic mechanical stretch[J]. West China Journal of Stomatology, 2024, 42(3): 304-312.

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References 25

1	Huelter-Hassler D, Tomakidi P, Steinberg T, et al. Or-thodontic strain affects the Hippo-pathway effector YAP concomitant with proliferation in human periodontal li-gament fibroblasts[J]. Eur J Orthod, 2017, 39(3): 251-257.
2	Jin Y, Ding L, Ding Z, et al. Tensile force-induced PD-GF-BB/PDGFRβ signals in periodontal ligament fibroblasts activate JAK2/STAT3 for orthodontic tooth movement[J]. Sci Rep, 2020, 10(1): 11269.
3	Mohanakumar A, Vijay GL, Vijayaraghavan N, et al. Morphological alterations, activity, mRNA fold changes, and aging changes before and after orthodontic force application in young and adult human-derived periodontal ligament cells[J]. Eur J Orthod, 2021, 43(6): 690-696.
4	Jia R, Yi Y, Liu J, et al. Cyclic compression emerged dual effects on the osteogenic and osteoclastic status of LPS-induced inflammatory human periodontal ligament cells according to loading force[J]. BMC Oral Health, 2020, 20(1): 7.
5	Tantilertanant Y, Niyompanich J, Everts V, et al. Cyclic tensile force stimulates BMP9 synthesis and in vitro mi-neralization by human periodontal ligament cells[J]. J Cell Physiol, 2019, 234(4): 4528-4539.
6	Li T, Fu J, Cheng J, et al. New progresses on cell surface protein HSPA5/BiP/GRP78 in cancers and COVID-19[J]. Front Immunol, 2023, 14: 1166680.
7	麻庆乐, 卢德赵. 葡萄糖调节蛋白78的研究进展[J]. 生命科学, 2017, 29(4): 331-335.
	Ma QL, Lu DZ. Research progress on GRP78[J]. Chin Bullet Life Sci, 2017, 29(4): 331-335.
8	Kuo LJ, Huang CY, Cheng WL, et al. Glucose-regulated protein 78 mediates the anticancer efficacy of shikonin in hormone-refractory prostate cancer cells[J]. Tumour Biol, 2015, 36(7): 5063-5070.
9	Farshbaf M, Khosroushahi AY, Mojarad-Jabali S, et al. Cell surface GRP78: an emerging imaging marker and therapeutic target for cancer[J]. J Control Release, 2020, 328: 932-941.
10	Li L, Han M, Li S, et al. Cyclic tensile stress during physiological occlusal force enhances osteogenic diffe-rentiation of human periodontal ligament cells via ERK1/2-Elk1 MAPK pathway[J]. DNA Cell Biol, 2013, 32(9): 488-497.
11	屠腾, 赵萤, 牛静, 等. 力生长因子调控牙周膜干细胞向牙周膜成纤维细胞分化的作用及机制研究[J]. 医用生物力学, 2021, 36 (S1): 106.
	Tu T, Zhao Y, Niu J, et al. Role and mechanism of force growth factor in regulating the differentiation of perio-dontal membrane stem cells to periodontal membrane fibroblasts[J]. J Med Biomech, 2021, 36 (S1): 106.
12	代媛媛, 原工杰, 余桂戎, 等. 间接共培养诱导大鼠骨髓间充质干细胞向牙周膜成纤维样细胞分化[J]. 临床口腔医学杂志, 2010, 26(5): 275-277.
	Dai YY, Yuan GJ, Yu GR, et al. Induction effect on the differentiation of rat bone marrow mesenchymal stem cells co-cultured with periodontal ligament fibroblasts[J]. J Clin Stomatol, 2010, 26(5): 275-277.
13	宋京, 任大鹏, 颜世果, 等. 细胞外信号调节蛋白激酶1/2在介导周期性牵张力对牙周膜细胞成骨分化中的作用[J]. 华西口腔医学杂志, 2017, 35(5): 520-526.
	Song J, Ren DP, Yan SG, et al. The role of extracellular signal regulated kinase 1/2 in mediating osteodifferentiation of human periodontal ligament cells induced by cyclic stretch[J]. West China J Stomatol, 2017, 35(5): 520-526.
14	Diercke K, Kohl A, Lux CJ, et al. Strain-dependent up-regulation of ephrin-B2 protein in periodontal ligament fibroblasts contributes to osteogenesis during tooth mo-vement[J]. J Biol Chem, 2011, 286(43): 37651-37664.
15	任大鹏. ERK1/2-Runx2信号通路介导周期性张应力作用下牙周膜成纤维细胞成骨分化的机制研究[D]. 济南: 山东大学, 2016.
	Ren DP. Role of ERK1/2-Runx2 pathway in osteodiffe-rentiation of human periodontal ligament fibroblasts induced by cyclic stretch[D]. Jinan: Shandong Uuiversity, 2016.
16	张春艳, 郭大伟, 宋玲, 等. 人牙周膜成纤维细胞的生物力学研究[J]. 中国卫生产业, 2015, 12(24): 195-198.
	Zhang CY, Guo DW, Song L, et al. Biomechanical stu-dies of human periodontal membrane fibroblasts[J]. China Health Industry, 2015, 12(24): 195-198.
17	Canepa DD, Casanova EA, Arvaniti E, et al. Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry[J]. Stem Cell Res Ther, 2021, 12(1): 7.
18	Li J, Zhao M, Wang Y, et al. p75NTR optimizes the osteogenic potential of human periodontal ligament stem cells by up-regulating α1 integrin expression[J]. J Cell Mol Med, 2020, 24(13): 7563-7575.
19	Merkel A, Chen Y, George A. Endocytic trafficking of DMP1 and GRP78 complex facilitates osteogenic differentiation of human periodontal ligament stem cells[J]. Front Physiol, 2019, 10: 1175.
20	李光, 李杰, 张平. 微小RNA-30d-5p通过葡萄糖调节蛋白78调控骨髓基质细胞成骨分化的机制[J]. 解剖学报, 2023, 54(2): 195-201.
	Li G, Li J, Zhang P. Mechanism of microRNA-30d-5p regulating osteogenic differentiation of bone marrow stromal cells through glucose-regulated 78[J]. Acta Ana-tom Sinica, 2023, 54(2): 195-201.
21	Schreiber C, Saraswati S, Harkins S, et al. Loss of ASA-P1 in mice impairs adipogenic and osteogenic differen-tiation of mesenchymal progenitor cells through dysre-gulation of FAK/Src and AKT signaling[J]. PLoS Genet, 2019, 15(6): e1008216.
22	Wei HM, Wang JC, Xu ZM, et al. Corrigendum: hepa-toma cell-derived extracellular vesicles promote liver cancer metastasis by inducing the differentiation of bo-ne marrow stem cells through microRNA-181d-5p and the FAK/Src Pathway[J]. Front Cell Dev Biol, 2021, 9: 760373
23	Park HJ, Gholam-Zadeh M, Yoon SY, et al. Estrogen decreases cytoskeletal organization by forming an ERα/SHP2/c-Src complex in osteoclasts to protect against o-variectomy-induced bone loss in mice[J]. Antioxidants (Basel), 2021, 10(4): 619.
24	Id Boufker H, Lagneaux L, Najar M, et al. The Src inhi-bitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts[J]. BMC Cancer, 2010, 10: 298.
25	Choi YH, Han Y, Lee SH, et al. Src enhances osteogenic differentiation through phosphorylation of Osterix[J]. Mol Cell Endocrinol, 2015, 407: 85-97.

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