Erythropoietin induces the osteogenesis of periodontal mesenchymal stem cells from healthy and periodontitis sources via activation of the p38 MAPK pathway

Liying Wang, Fan Wu, Yang Song, Yinzhong Duan, Zoulin Jin, Liying Wang, Fan Wu, Yang Song, Yinzhong Duan, Zoulin Jin

Abstract

Erythropoietin (Epo), a hematopoietic hormone, has multiple biological functions. Recently, the positively osteogenic effects of Epo on mesenchymal stem cells (MSCs) have attracted broad interest. However, the effects of Epo on the osteogenesis of human periodontal ligament tissue‑derived mesenchymal stem cells (hPDLSCs) and periodontitis mesenchymal stem cells (pPDLSCs) from patients with periodontitis remain unknown. In the present study, osteogenic effects of Epo on hPDLSCs and pPDLSCs were investigated, and the results suggested that the effects were mediated by promoting the expression of runt related transcription factor 2, alkaline phosphatase (ALP) and osteocalcin. Using Alizarin Red and ALP staining, it was demonstrated that Epo exerted positive osteogenic effects on hPDLSCs and pPDLSCs. Additionally, Epo upregulated the proliferation of hPDLSCs and pPDLSCs, based on flow cytometric analyses of the cell cycle. To determine the underlying mechanism, the role of the p38 mitogen‑activated protein kinase (MAPK) pathway, which is associated with the osteogenesis of hPDLSCs and pPDLSCs, was investigated further. Epo increases p38 phosphorylation (the target of the MAPK pathway) in hPDLSCs and pPDLSCs. Furthermore, when the cells were treated with SB203580, an inhibitor of the p38 MAPK pathway, the osteogenic effects of Epo on hPDLSCs and pPDLSCs were attenuated. In conclusion, Epo may upregulate the bone formation ability of hPDLSCs and pPDLSCs via the p38 MAPK pathways.

Figures

Figure 1
Figure 1
Effects of Epo on the proliferation of hPDLSCs and pPDLSCs. (A) Percentage of cells in G2 + S phase was measured by flow cytometry at day 5. (B) Quantitative data of the cell cycle analysis, PI of hPDLSCs and pPDLSCs with or without Epo induced (left) and fold upregulation (right) of PI in hPDLSCs and pPDLSCs induced by Epo. Data represent mean ± standard deviation. **P<0.01. Epo(-), cells cultured without Epo; Epo(+), cells cultured with Epo; Epo, erythropoietin; hPDLSC, human periodontal ligament tissue-derived mesenchymal stem cells; pPDLSCs, periodontitis mesenchymal stem cells; PI, proliferation index; NS, not significant.
Figure 2
Figure 2
Effects of Epo on the osteogenic differentiation of hPDLSCs and pPDLSCs. (A) Osteogenic differentiation was determined by ALP staining at day 7 after osteogenic differentiation induced. (B) ALP activity was measured by ALP activity assay at day 7 after osteogenic differentiation induced and fold upregulation of ALP activity in hPDLSCs and pPDLSCs induced by Epo. (C) Expression levels of the osteogenic genes ALP, Runx2 and OCN were measured by reverse transcription-quantitative polymerase chain reaction at day 7 after osteogenic differentiation induced. (D) Osteogenic differentiation was determined by Alizarin Red S staining at day 21 after osteogenic differentiation induced. (E) Calcium concentration determined by Alizarin Red S and fold upregulation of calcium concentration in hPDLSCs and pPDLSCs induced by Epo. Data are presented as the mean ± standard deviation.*P<0.05, **P<0.01. Scale bar, 100 µm. Epo(-), cells cultured without Epo; Epo(+), cells cultured with Epo; Epo, erythropoietin; hPDLSC, human periodontal ligament tissue-derived mesenchymal stem cells; pPDLSCs, periodontitis mesenchymal stem cells; Runx2, runt related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin; OD, optical density.
Figure 3
Figure 3
Effects of Epo on the p38 mitogen-activated protein kinase pathway. (A) p-p38 level was examined by western blot analysis and (B) scanning densitometry. Actin was used as the internal control. All tests were measured 7 days after cells cultured. Data are presented as the mean ± standard deviation. **P<0.01. hPDL SC, human periodontal ligament tissue‑derived mesenchymal stem cells; pPDL SCs, periodontitis mesenchymal stem cells; Epo, erythropoietin; p-p38, phospho-p38.
Figure 4
Figure 4
Effects of the p38 mitogen-activated protein kinase pathway on the processes of Epo regulating the osteogenic differentiation of hPDLSCs and pPDLSCs. (A) Osteogenic differentiation was determined by Alizarin Red S staining at day 21 after osteogenic differentiation induced. (B) Alizarin Red S staining was used to determine calcium levels. (C) Osteogenic differentiation was determined by alkaline phosphatase (ALP) staining at day 7 after osteogenic differentiation induced. (D) ALP activity was measured by ALP activity assay at day 7 after osteogenic differentiation induced. (E) Expression levels of the osteogenic genes ALP, Runx2 and OCN were measured by reverse transcription-quantitative polymerase chain reaction at day 7 after osteogenic differentiation induced. All all analyses were performed after 7 days in culture. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01. Scale bar, 100 µm. hPDLSC, human periodontal ligament tissue-derived mesenchymal stem cells; pPDLSCs, periodontitis mesenchymal stem cells; Epo, erythropoietin; Epo(-), cells cultured without Epo; Epo(+), cells cultured with Epo; SB203580(-), cells cultured without SB203580; SB203580(+), cells cultured with SB203580; ALP, alkaline phosphatase; OD, optical density; NS, not significant; Runx2, runt related transcription factor 2; OCN, osteocalcin.

References

    1. Holstein JH, Orth M, Scheuer C, Tami A, Becker SC, Garcia P, Histing T, Mörsdorf P, Klein M, Pohlemann T, et al. Erythropoietin stimulates bone formation, cell proliferation, and angiogenesis in a femoral segmental defect model in mice. Bone. 2011;49:1037–1045. doi: 10.1016/j.bone.2011.08.004.
    1. Shiozawa Y, Jung Y, Ziegler AM, Pedersen EA, Wang J, Wang Z, Song J, Wang J, Lee CH, Sud S, et al. Erythropoietin couples hematopoiesis with bone formation. PLoS One. 2010;5:e10853. doi: 10.1371/journal.pone.0010853.
    1. Kim J, Jung Y, Sun H, Joseph J, Mishra A, Shiozawa Y, Wang J, Krebsbach PH, Taichman RS. Erythropoietin mediated bone formation is regulated by mTOR signaling. J Cell Biochem. 2012;113:220–228. doi: 10.1002/jcb.23347.
    1. Chen S, Li J, Peng H, Zhou J, Fang H. Administration of erythropoietin exerts protective effects against glucocorticoid-induced osteonecrosis of the femoral head in rats. Int J Mol Med. 2014;33:840–848. doi: 10.3892/ijmm.2014.1644.
    1. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet. 2005;366:1809–1820. doi: 10.1016/S0140-6736(05)67728-8.
    1. Nanci A, Bosshardt DD. Structure of periodontal tissues in health and disease. Periodontol. 2006;40:11–28. doi: 10.1111/j.1600-0757.2005.00141.x.
    1. Chen FM, Zhang J, Zhang M, An Y, Chen F, Wu ZF. A review on endogenous regenerative technology in periodontal regenerative medicine. Biomaterials. 2010;31:7892–7927. doi: 10.1016/j.biomaterials.2010.07.019.
    1. Izumi Y, Aoki A, Yamada Y, Kobayashi H, Iwata T, Akizuki T, Suda T, Nakamura S, Wara-Aswapati N, Ueda M, et al. Current and future periodontal tissue engineering. Periodontol. 2011;56:166–187. doi: 10.1111/j.1600-0757.2010.00366.x.
    1. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S. Investigation of multi-potent postnatal stem cells from human periodontal ligament. Lancet. 2004;364:149–155. doi: 10.1016/S0140-6736(04)16627-0.
    1. Akiyama K, Chen C, Gronthos S, Shi S. Lineage differentiation of mesenchymal stem cells from dental pulp, apical papilla, and periodontal ligament. Methods Mol Biol. 2012;887:111–121. doi: 10.1007/978-1-61779-860-3_11.
    1. Washio K, Iwata T, Mizutani M, Ando T, Yamato M, Okano T, Ishikawa I. Assessment of cell sheets derived from human periodontal ligament cells: a pre-clinical study. Cell Tissue Res. 2010;341:397–404. doi: 10.1007/s00441-010-1009-1.
    1. Gault P, Black A, Romette JL, Fuente F, Schroeder K, Thillou F, Brune T, Berdal A, Wurtz T. Tissue-engineered ligament: implant constructs for tooth replacement. J Clin Periodontol. 2010;37:750–758.
    1. Yang H, Gao LN, An Y, Hu CH, Jin F, Zhou J, Jin Y, Chen FM. Comparison of mesenchymal stem cells derived from gingival tissue and periodontal ligament in different incubation conditions. Biomaterials. 2013;34:7033–7047. doi: 10.1016/j.biomaterials.2013.05.025.
    1. Ma Z, Li S, Song Y, Tang L, Ma D, Liu B, Jin Y. The biological effect of dentin noncollagenous proteins (DNCPs) on the human periodontal ligament stem cells (HPDLSCs) in vitro an in vivo. Tissue Eng Part A. 2008;14:2059–2068. doi: 10.1089/ten.tea.2008.0021.
    1. Chen FM, Zhao YM, Wu H, Deng ZH, Wang QT, Zhou W, Liu Q, Dong GY, Li K, Wu ZF, et al. Enhancement of periodontal tissue regeneration by locally controlled delivery of insulin-like growth factor-I from dextran-co-gelatin microspheres. J Control Release. 2006;114:209–222. doi: 10.1016/j.jconrel.2006.05.014.
    1. Ramseier CA, Abramson ZR, Jin Q, Giannobile WV. Gene therapeutics for periodontal regenerative medicine. Dent Clin North Am. 2006;50:245–263. doi: 10.1016/j.cden.2005.12.001.
    1. Wu RX, Bi CS, Yu Y, Zhang LL, Chen FM. Age-related decline in the matrix contents and functional properties of human periodontal ligament stem cell sheets. Acta Biomater. 2015;22:70–82. doi: 10.1016/j.actbio.2015.04.024.
    1. Zhang J, An Y, Gao LN, Zhang YJ, Jin Y, Chen FM. The effect of aging on the pluripotential capacity and regenerative potential of human periodontal ligament stem cells. Biomaterials. 2012;33:6974–6986. doi: 10.1016/j.biomaterials.2012.06.032.
    1. Zhou Y, Fan W, Xiao Y. The effect of hypoxia on the stemness and differentiation capacity of PDLC and DPC. BioMed Res Int. 2014;890675 doi: 10.1155/2014/890675.
    1. Kim SY, Kang KL, Lee JC, Heo JS. Nicotinic acetylcholine receptor α7 and β4 subunits contribute nicotine-induced apoptosis in periodontal ligament stem cells. Mol Cells. 2012;33:343–350. doi: 10.1007/s10059-012-2172-x.
    1. Liu W, Konermann A, Guo T, Jäger A, Zhang L, Jin Y. Canonical Wnt signaling differently modulates osteogenic differentiation of mesenchymal stem cells derived from bone marrow and from periodontal ligament under inflammatory conditions. Biochim Biophys Acta. 2014;1840:1125–1134. doi: 10.1016/j.bbagen.2013.11.003.
    1. Zheng W, Wang S, Wang J, Jin F. Periodontitis promotes the proliferation and suppresses the differentiation potential of human periodontal ligament stem cells. Int J Mol Med. 2015;36:915–922. doi: 10.3892/ijmm.2015.2314.
    1. Liu N, Shi S, Deng M, Tang L, Zhang G, Liu N, Ding B, Liu W, Liu Y, Shi H, et al. High levels of β-catenin signaling reduce osteogenic differentiation of stem cells in inflammatory microenvironments through inhibition of the noncanonical Wnt pathway. J Bone Miner Res. 2011;26:2082–2095. doi: 10.1002/jbmr.440.
    1. Liu N, Tian J, Cheng J, Zhang J. Effect of erythropoietin on the migration of bone marrow-derived mesenchymal stem cells to the acute kidney injury microenvironment. Exp Cell Res. 2013;319:2019–2027. doi: 10.1016/j.yexcr.2013.04.008.
    1. Park SL, Won SY, Song JH, Kambe T, Nagao M, Kim WJ, Moon SK. EPO gene expression promotes proliferation, migration and invasion via the p38M/APK/AP-1/MMP-9 pathway by p21WAF1 expression in vascular smooth muscle cells. Cell Signal. 2015;27:470–478. doi: 10.1016/j.cellsig.2014.12.001.
    1. Rafiee P, Shi Y, Su J, Pritchard KA, Jr, Tweddell JS, Baker JE. Erythropoietin protects the infant heart against ischemia-reperfusion injury by triggering multiple signaling pathways. Basic Res Cardiol. 2005;100:187–197. doi: 10.1007/s00395-004-0508-1.
    1. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262.
    1. Wu H, Liu X, Jaenisch R, Lodish HF. Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell. 1995;83:59–67. doi: 10.1016/0092-8674(95)90234-1.
    1. Broxmeyer HE. Erythropoietin: multiple targets, actions, and modifying influences for biological and clinical consideration. J Exp Med. 2013;210:205–208. doi: 10.1084/jem.20122760.
    1. Li C, Shi C, Kim J, Chen Y, Ni S, Jiang L, Zheng C, Li D, Hou J, Taichman RS, et al. Erythropoietin promotes bone formation through EphrinB2/EphB4 signaling. J Dent Res. 2015;94:455–463. doi: 10.1177/0022034514566431.
    1. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. 1997;89:747–754. doi: 10.1016/S0092-8674(00)80257-3.
    1. Liu Y, Wang L, Kikuiri T, Akiyama K, Chen C, Xu X, Yang R, Chen W, Wang S, Shi S. Mesenchymal stem cell-based tissue regeneration is governed by recipient T lymphocytes via IFN-γ and TNF-α. Nat Med. 2011;17:1594–1601. doi: 10.1038/nm.2542.
    1. Xu DJ, Zhao YZ, Wang J, He JW, Weng YG, Luo JY. Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells. BMB Rep. 2012;45:247–252. doi: 10.5483/BMBRep.2012.45.4.247.
    1. Zhao Y, Song T, Wang W, Wang J, He J, Wu N, Tang M, He B, Luo J. P38 and ERK1/2 MAPKs act in opposition to regulate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. PLoS One. 2012;7:e43383. doi: 10.1371/journal.pone.0043383.
    1. Wang Y, Ma J, Du Y, Miao J, Chen N. Human amnion-derived mesenchymal stem cells protect human bone marrow mesenchymal stem cells against oxidative stress-mediated dysfunction via ERK1/2 MAPK signaling. Mol Cells. 2016;39:186–194. doi: 10.14348/molcells.2016.2159.
    1. Greenblatt MB, Shim JH, Zou W, Sitara D, Schweitzer M, Hu D, Lotinun S, Sano Y, Baron R, Park JM, et al. The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. J Clin Invest. 2010;120:2457–2473. doi: 10.1172/JCI42285.
    1. Higuchi C, Myoui A, Hashimoto N, Kuriyama K, Yoshioka K, Yoshikawa H, Itoh K. Continuous inhibition of MAPK signaling promotes the early osteoblastic differentiation and mineralization of the extracellular matrix. J Bone Miner Res. 2002;17:1785–1794. doi: 10.1359/jbmr.2002.17.10.1785.
    1. Chang J, Sonoyama W, Wang Z, Jin Q, Zhang C, Krebsbach PH, Giannobile W, Shi S, Wang CY. Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in craniofacial defects through activation of p38 MAPK. J Biol Chem. 2007;282:30938–30948. doi: 10.1074/jbc.M702391200.
    1. Ye G, Li C, Xiang X, Chen C, Zhang R, Yang X, Yu X, Wang J, Wang L, Shi Q, et al. Bone morphogenetic protein-9 induces PDLSCs osteogenic differentiation through the ERK and p38 signal pathways. Int J Med Sci. 2014;11:1065–1072. doi: 10.7150/ijms.8473.

Source: PubMed

Patrocinadores e Colaboradores

Condições médicas

Intervenções de drogas

3
Se inscrever