SiRNA silencing Rsk2 gene expression inhibits proliferation and osteogenic differentiation of human periodontal ligament cells

doi:10.19439/j.sjos.2017.05.007

Abstract

Abstract: PURPOSE: To study the effect of ribosomal S6 kinase (Rsk2) gene on proliferation and osteogenic differentiation of human periodontal ligament cells (hPDLCs) and underlying mechanism. METHODS: Premolar surgically extracted were collected, the periodontal ligament was separated and hPDLCs were primarily cultured. Cells of 4 to 6 passage were used in the experiment. The silencing efficiency of small interfering RNA (siRNA) on Rsk2 in hPDLCs was detected by RT-PCR and Western blot. MTT assay was used to detect the effect of Rsk2 siRNA on cell proliferation. Alkaline phosphatase (ALP) kit was used to detect ALP activity. P38MAPK signal pathway inhibitor SB203580 was used to detect hPDLCs after transfection. Western blot was used to detect the effect of Rsk2 siRNA on MAPK signaling pathway p38 protein phosphorylation. The expressions of Runt-related transcription factor-2 (Runx2), osteocalcin (OCN) and osteogenic protein BMP2 were detected by RT-PCR and Western blot. The data were analyzed using SPSS18.0 software package. RESULTS: The expression of Rsk2 was down-regulated by hPDLCs transfected with Rsk2 siRNA, the difference was statistically significant (P<0.05). Rsk2 siRNA significantly reduced phosphorylation of p38 protein (P<0.05), inhibition of hPDLCs proliferation (P<0.05), decreased ALP activity (P<0.01); the expression of Runx2, OCN and BMP2 was different, and the difference was statistically significant (P<0.05). After SB203580 treatment, hPDLCs transfected with Rsk2 siRNA showed increased cell proliferation, ALP activity, Runx2, OCN and BMP2 expression; compared with Rsk2 siRNA transfected hPDLCs, the difference was statistically significant. CONCLUSIONS: Rsk2 siRNA inhibits hPDLCs proliferation and osteogenic differentiation through p38MAPK signaling pathway.

Key words: Rsk2, Human periodontal ligament cells, siRNA, Proliferation, Osteogenic differentiation

CLC Number:

R781.4

DU Li, CAO Wei-jing, WANG Yuan-ming. SiRNA silencing Rsk2 gene expression inhibits proliferation and osteogenic differentiation of human periodontal ligament cells[J]. Shanghai Journal of Stomatology, 2017, 26(5): 498-503.

References

[1] Isaka J, Ohazama A, Kobayashi M, et al. Participation of periodontal ligament cells with regeneration of alveolar bone [J]. J Periodontol,2001,72(3): 314-323.
[2] Yu N, Prodanov L, te Riet JT, et al. Regulation of periodontal ligament cell behavior by cyclic mechanical loading and substrate nanotexture[J]. J Periodontol, 2013, 84(10): 1504-1513.
[3] Cho YY. RSK2 and its binding partners in cell proliferation, transformation and cancer development [J]. Arch Pharm Res, 2017, 40(3): 291-303.
[4] Koehne T, Jeschke A, Petermann F, et al. Rsk2, the kinase mutated in coffin-lowry syndrome, controls cementum formation [J]. J Dent Res, 2016, 95(7): 752-760.
[5] Greenblatt MB, Shim JH, Zou W, et al. The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice [J]. J Clin Invest, 2010, 120(7): 2457-2473.
[6] Patil CS, Kirkwood KL. p38 MAPK signaling in oral-related diseases [J]. J Dent Res, 2007, 86(9): 812-825.
[7] Xu X, Han J, Ito Y, et al. Ectodermal Smad4 and p38 MAPK are functionally redundant in mediating TGF-β/BMP signaling during tooth and palate development [J]. Dev Cell, 2008, 15(2): 322-329.
[8] Greenblatt MB, Kim JM, Oh H, et al. p38α MAPK is required for tooth morphogenesis and enamel secretion [J]. J Biol Chem, 2015, 290(1): 284-295.
[9] Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament [J]. Lancet, 2004, 364(9429): 149-155.
[10] Tang X, Meng H, Han J, et al. Up-regulation of estrogen receptor β expression during osteogenic differentiation of human periodontal ligament cells [J]. J Periodontal Res, 2008, 43(3): 311-321.
[11] David JP, Mehic D, Bakiri L, et al. Essential role of RSK2 in c-Fos-dependent osteosarcoma development [J]. J Clin Invest, 2005, 115(3): 664-672.
[12] Rogers JE, Li F, Coatney DD, et al. A p38 mitogen-activated protein kinase inhibitor arrests active alveolar bone loss in a rat periodontitis model [J]. J Periodontol, 2007, 78(10): 1992-1998.
[13] Jönsson D, Nilsson J, Odenlund M, et al. Demonstration of mitochondrial oestrogen receptor beta and oestrogen-induced attenuation of cytochrome c oxidase subunit I expression in human periodontal ligament cells[J]. Arch Oral Biol, 2007, 52(7): 669-676.
[14] Zhang D, Zheng H, Zhao J, et al. Porphorymonas gingivalis induces intracellular adhesion molecule-1 expression in endothelial cells through the nuclear factor-kappaB pathway, but not through the p38 MAPK pathway [J]. J Periodontal Res, 2011, 46(1): 31-38.
[15] Ying X, Chen X, Feng Y, et al. Myricetin enhances osteogenic differentiation through the activation of canonical Wnt/β-catenin signaling in human bone marrow stromal cells[J]. Eur J Pharmacol, 2014, 738: 22-30.
[16] Franceschi RT, Xiao G. Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways [J]. J Cell Biochem, 2003, 88(3): 446-454.
[17] Watanabe T, Oyama T, Asada M, et al. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development [J]. PLoS Genet, 2012, 9(1): e1003132.
[18] James AW. Review of signaling pathways governing msc osteogenic and adipogenic differentiation [J]. Scientifica (Cairo), 2013, 2013: 684736.
[19] Lowery JW, Pazin D, Intini G, et al. The role of BMP2 signaling in the skeleton [J]. Crit Rev Eukaryot Gene Expr, 2011, 21(2): 177-185.
[20] Nakashima K, Zhou X, Kunkel G, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation [J]. Cell, 2002, 108(1): 17-29.