Osteogenic differentiation of human periodontal ligament stem cells under estrogen and crosstalk between the two pathways

doi:10.12307/2022.571

Abstract

Abstract: BACKGROUND: Periodontal tissue regeneration engineering based on stem cells provides a broad prospect for the treatment of periodontal diseases. Estrogen-mediated osteogenic differentiation in human periodontal ligament stem cells is closely related to the Wnt signaling pathway.
OBJECTIVE: To study the regulation of Wnt/β-catenin and Wnt/Ca2+ signaling pathways on estrogen-mediated osteogenic differentiation in human periodontal ligament stem cells, as well as the crosstalk between the two pathways.
METHODS: Premolars with healthy periodontal condition were collected to isolate and culture human periodontal ligament stem cells. Surface markers of the cells were detected by flow cytometry. The osteogenic/adipogenic differentiation potential was detected by Alizarin red S and Oil red O staining. The third-generation human periodontal ligament stem cells were randomly divided into blank group, control group, estrogen group, XAV939 (Wnt/β-catenin pathway inhibitor) group, L-690,330 (Wnt/Ca2+ pathway inhibitor) group, and XAV939+L-690,330 group. Except for the blank group, all other groups were cultured for osteogenic induction. qRT-PCR and western blot assay were performed to measure expression of osteogenic markers (Runx2, OCN) and key signal molecules of Wnt/β-catenin and Wnt/Ca2+ pathway. Intracellular Ca2+ concentration was evaluated by Fluo-4 fluorescent probe. Alkaline phosphatase assay was conducted to measure the alkaline phosphatase activity. Osteogenic differentiation potential was detected by Alizarin red S staining.
RESULTS AND CONCLUSION: (1) The human periodontal ligament stem cells isolated and purified by collagenase digestion were spindle shaped and polygonal. Flow cytometry proved that human periodontal ligament stem cells were of mesenchymal origin, and they also demonstrated the capacity to differentiate into osteogenic/adipogenic lineages. (2) Estrogen at 10-7 mol/L in the osteogenic medium can upregulate intracellular Ca2+ concentration, CaMKII and NLK gene expression and CaMKII protein expression of human periodontal ligament stem cells. (3) The usage of Wnt/β-catenin and Wnt/Ca2+ signaling pathway inhibitors can minimize osteogenic differentiation. (4) Inhibiting either of the two signaling pathways can increase crucial protein expression of the other pathway, which had no significant effect on the osteogenic differentiation of the cells. (5) In conclusion, estrogen through Wnt/Ca2+ and Wnt/β-catenin signaling pathways induces osteogenic differentiation of human periodontal ligament stem cells. In addition, there is mutual compensation between the two pathways, thus working together to regulate osteogenic differentiation.

Key words: stem cells, human periodontal ligament stem cells, estrogen, osteogenic differentiation, Wnt/β-catenin, Wnt/Ca2+, crosstalk, pathways

CLC Number:

Jin Ke, Wu Xiaoling, Luo Xiaoling, Xu Pengfei, Xu Xiaomei. Osteogenic differentiation of human periodontal ligament stem cells under estrogen and crosstalk between the two pathways[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(24): 3886-3891.

Figures/Tables 5

References

[1] CHALISSERRY EP, NAM SY, PARK SH, et al. Therapeutic potential of dental stem cells. J Tissue Eng. 2017;8:2041731417702531.
[2] BASSIR SH, WISITRASAMEEWONG W, RAANAN J, et al. Potential for Stem Cell-Based Periodontal Therapy. J Cell Physiol. 2016;231(1):50-61.
[3] 李晓钰,王红,谷秀格,等.年龄因素对牙周膜干细胞多向分化能力的影响[J].口腔生物医学,2019,10(2):78-82.
[4] BOTELHO J, CAVACAS MA, MACHADO V, et al. Dental stem cells: recent progresses in tissue engineering and regenerative medicine. Ann Med. 2017;49(8):644-651.
[5] WANG P, WANG W, GENG T, et al. EphrinB2 regulates osteogenic differentiation of periodontal ligament stem cells and alveolar bone defect regeneration in beagles. J Tissue Eng. 2019;10:2041731419894361.
[6] 李微,张博,张雨薇,等.雌激素调节骨代谢作用的研究进展[J].中国骨质疏松杂志,2017,23(2):262-266.
[7] 刘云,罗晓婷,李崇,等.雌激素治疗绝经后骨质疏松症的研究进展[J].实用临床医学,2020,21(10):91-96.
[8] JI MX, YU Q. Primary osteoporosis in postmenopausal women. Chronic Dis Transl Med. 2015;1(1):9-13.
[9] 马刚,李理,覃永保,等.大鼠双侧卵巢切除术后不同时期血清骨生化标志物与骨小梁三维结构变化之间的研究[J].医学研究杂志,2016, 45(5):76-80.
[10] KHOSLA S, OURSLER MJ, MONROE DG. Estrogen and the skeleton. Trends Endocrinol Metab. 2012;23(11):576-581.
[11] 张文韬,陈勋,宋涛,等.缺氧诱导因子抑制剂对去卵巢大鼠骨质疏松症治疗效果的研究[J].中国骨质疏松杂志,2018,24(12):1601-1605.
[12] JIANG B, XU J, ZHOU Y, et al. Estrogen Enhances Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Activating the Wnt/β-Catenin Signaling Pathway. J Craniofac Surg. 2020;31(2):583-587.
[13] DE A. Wnt/Ca2+ signaling pathway: a brief overview. Acta Biochim Biophys Sin (Shanghai). 2011;43(10):745-756.
[14] ISHITANI T, KISHIDA S, HYODO-MIURA J, et al. The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling. Mol Cell Biol. 2003;23(1):131-139.
[15] MARTINEAU X, ABED É, MARTEL-PELLETIER J, et al. Alteration of Wnt5a expression and of the non-canonical Wnt/PCP and Wnt/PKC-Ca2+ pathways in human osteoarthritis osteoblasts. PLoS One. 2017;12(8):e0180711.
[16] LI S, HU C, LI J, et al. Effect of miR-26a-5p on the Wnt/Ca(2+) Pathway and Osteogenic Differentiation of Mouse Adipose-Derived Mesenchymal Stem Cells. Calcif Tissue Int. 2016;99(2):174-186.
[17] 张世蘋,张旭.Wnt信号通路在肿瘤调控方面的研究进展[J].中国药理学通报,2017,33(1):14-18.
[18] BARON R, GORI F. Targeting WNT signaling in the treatment of osteoporosis. Curr Opin Pharmacol. 2018;40:134-141.
[19] RANDALL RM, SHAO YY, WANG L, et al. Activation of Wnt Planar Cell Polarity (PCP) signaling promotes growth plate column formation in vitro. J Orthop Res. 2012;30(12):1906-1914.
[20] DIAZ DE BARBOZA G, GUIZZARDI S, TOLOSA DE TALAMONI N. Molecular aspects of intestinal calcium absorption. World J Gastroenterol. 2015;21(23): 7142-7154.
[21] HUANG SM, MISHINA YM, LIU S, et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature. 2009;461(7264):614-620.
[22] KÜHL M, SHELDAHL LC, PARK M, et al. The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet. 2000;16(7):279-283.
[23] TORRES MA, YANG-SNYDER JA, PURCELL SM, et al. Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development. J Cell Biol. 1996;133(5):1123-1137.
[24] MINASHIMA T, KIRSCH T. Annexin A6 regulates catabolic events in articular chondrocytes via the modulation of NF-κB and Wnt/ß-catenin signaling. PLoS One. 2018;13(5):e0197690.
[25] NARENDRA TALABATTULA VA, MORGAN P, FRECH MJ, et al. Non-canonical pathway induced by Wnt3a regulates β-catenin via Pyk2 in differentiating human neural progenitor cells. Biochem Biophys Res Commun. 2017;491(1):40-46.
[26] 曹文达,蒋世一,鲁晓杰.基于iTRAQ技术的无功能性垂体瘤血清蛋白质组学分析[J].临床神经外科杂志,2017,14(3):220-224.
[27] PASSOS-SOARES JS, VIANNA MIP, GOMES-FILHO IS, et al. Association between osteoporosis treatment and severe periodontitis in postmenopausal women. Menopause. 2017;24(7):789-795.
[28] LÓPEZ-MARCOS JF, GARCÍA-VALLE S, GARCÍA-IGLESIAS AA. Periodontal aspects in menopausal women undergoing hormone replacement therapy. Med Oral Patol Oral Cir Bucal. 2005;10(2):132-141.
[29] VIALE-GARRONE A, TAVITIAN P, TARDIVO D, et al. Relationship between menopause and tooth loss. Odontostomatol Trop. 2013;36(144):56-64.
[30] 姚丽,黄薇,刘莹.雌激素替代疗法对绝经期骨质疏松伴牙周病患者系统治疗的影响[J].中国骨质疏松杂志,2015,21(2):196-198.