Effect of GLUD1 on proliferation, osteogenic differentiation and mineralization of human dental pulp stem cells

doi:10.19439/j.sjos.2017.04.002

Abstract

Abstract: To investigate the effect of glutamate dehydrogenase 1 (GLUD1) on proliferation, osteogenic differentiation and mineralization of human dental pulp stem cells (hDPSCs). METHODS: hDPSCs were isolated by tissue-explant method in vitro, and shGLUD1 lentivirus was transfected to knock down the expression of GLUD1. RT-PCR and Western blot were performed to detect the expression of GLUD1. CCK8 assay was used to evaluate cell proliferation. After culture with osteogenic inducing medium for 14 days, alizarin red staining was used to detect the formation of mineralization nodules, and RT-PCR and immunofluorescence staining were performed to detect the expression of Runx2 and OCN, respectively. The data were analyzed with SPSS 20.0 software package. RESULTS: The expression of GLUD1 was significantly increased in hDPSCs after osteogenic induction compared with the control. After transfection with shGLUD1 lentivirus, GLUD1 expression was significantly decreased (P<0.05). Compared with the control group, mineralization nodule formation was significantly decreased in shGLUD1 group after osteogenic induction. The expression of OCN (late-staged markers for osteogenic differentiation) were significantly decreased both in mRNA and protein levels, while the expression of Runx2 (early-staged markers for osteoblast differentiation) was up-regulated. CONCLUSIONS: shGLUD1 inhibits the proliferation, mineralization and the late stage of osteogenic differentiation of hDPSCs in vitro. GLUD1 may play an important role in osteogenic differentiation of hDPSCs.

Key words: GLUD1, Human dental pulp stem cells, Osteogenic differentiation, Proliferation, Mineralization

CLC Number:

R781.3

LU Xi, LIU Shang-feng, ZHAO Shou-liang. Effect of GLUD1 on proliferation, osteogenic differentiation and mineralization of human dental pulp stem cells[J]. Shanghai Journal of Stomatology, 2017, 26(4): 358-362.

References

[1] Karner CM, Esen E, Okunade AL, et al. Increased glutamine catabolism mediates bone anabolism in response to WNT signaling [J]. J Clin Invest, 2015, 125(2): 551-562.
[2] Brown PM, Hutchison JD, Crockett JC. Absence of glutamine supplementation prevents differentiation of murine calvarial osteoblasts to a mineralizing phenotype[J]. Calcif Tissue Int, 2011, 89(6): 472-482.
[3] Lin TH, Yang RS, Tang CH, et al. Regulation of the maturation of osteoblasts and osteoclastogenesis by glutamate[J]. Eur J Pharmacol, 2008, 589(1-3): 37-44.
[4] Hensley CT, Wasti AT, Deberardinis RJ. Glutamine and cancer: cell biology, physiology, and clinical opportunities [J]. J Clin Invest, 2013, 123(9): 3678-3684.
[5] Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo [J]. P Natl Acad Sci USA, 2000, 97(25): 13625-13630.
[6] Ledesma-Martinez E, Mendoza-Nunez VM, Santiago-Osorio E. Mesenchymal stem cells derived from dental pulp: a review [J]. Stem Cells Int, 2016, 2016: 4709572.
[7] Tae SK, Lee SH, Park JS, et al. Mesenchymal stem cells for tissue engineering and regenerative medicine [J]. Biomed Mater, 2006, 1(2): 63-71.
[8] Liu YK, Zhou ZY, Liu F. Transcriptome changes during TNF-α promoted osteogenic differentiation of dental pulp stem cells (DPSCs) [J]. Biochem Biophs Res Commun, 2016, 476(4): 426-430.
[9] D'alimonte I, Nargi E, Lannutti A, et al. Adenosine A1 receptor stimulation enhances osteogenic differentiation of human dental pulp-derived mesenchymal stem cells via WNT signaling [J]. Stem Cell Res, 2013, 11(1): 611-624.
[10] Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine [J]. J Dent Res, 2009, 88(9): 792-806.
[11] Aralali M, Ishikawa M, Nakamura T, et al. Role of epithelial-stem cell interactions during dental cell differentiation [J]. J Biol Chem, 2012, 287(13): 10590-10601.
[12] Liu G, Zhu J, Yu M, et al. Glutamate dehydrogenase is a novel prognostic marker and predicts metastases in colorectal cancer patients [J]. J Trans Med, 2015, 13: 144.
[13] Lorin S, Tol MJ, Bauvy C, et al. Glutamate dehydrogenase contributes to leucine sensing in the regulation of autophagy [J]. Autophagy, 2013, 9(6): 850-860.
[14] Singha UK, Jiang Y, Yu S, et al. Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells[J]. J Cell Biochem, 2008, 103(2): 434-446.
[15] Lee KW, Yook JY, Son MY, et al. Rapamycin promotes the osteoblastic differentiation of human embryonic stem cells by blocking the mTOR pathway and stimulating the BMP/Smad pathway [J]. Stem Cells Dev, 2010, 19(4): 557-568.
[16] Chen J, Long F. mTORC1 signaling promotes osteoblast differentiation from preosteoblasts[J]. PLoS ONE, 2015, 10(6): e0130627.
[17] Huang B, Wang Y, Wang W, et al. mTORC1 prevents preosteoblast differentiation through the notch signaling pathway [J]. PLoS Genet, 2015, 11(8): e1005426.
[18] Kim JK, Baker J, Nor JE, et al. mTor plays an important role in odontoblast differentiation [J]. J Endodont, 2011, 37(8): 1081-1085.