The C-terminus of the amelogenin peptide promotes the
proliferation of ALC ameloblasts through accelerating cell cycle

doi:10.3969/j.issn.2095-4344.1858

Abstract

Abstract:

BACKGROUND: C-terminus of the amelogenin peptide (AMG-CP) is a small molecular endogenous peptide that is highly conserved among species. It is involved in important physiological processes during tooth development. Some studies have shown that AMG-CP can regulate the proliferation and differentiation of cementoblasts, bone marrow mesenchymal stem cells and periodontal ligament fibroblasts, but the biological function of AMG-CP on ameloblasts has not been elucidated.

OBJECTIVE: To investigate the effects of different concentrations of AMG-CP on the proliferation of ALC ameloblasts and its underlying mechanisms.

METHODS: AMG-CP was successfully synthesized and determinated by liquid chromatography and mass spectrometry. The effects of AMG-CP at 0, 0.5, 1, 2 mg/L on the proliferation of ALC ameloblasts were observed by xCELLigence RTCA cell analysis system in real time. The effect of AMG-CP at 0, 1, 2 mg/L on cell cycle of ALC was detected by flow cytometry. Real-time PCR was used to detect the expression of cyclin D1, CDK4, MCM2, MCM5 mRNA in ALC cells treated with AMG-CP at 0, 1, 2 mg/L. Western blot was carried out to evaluate the effect of AGM-CP at 0, 1 mg/L on MAPK-ERK1/2 pathway by detecting the expression of phosphorylated ERK1/2 and total ERK1/2 in ALC cells. Pathway blockade assay was performed by using ERK1/2 blocker U0126 to pretreat ALC cells. Then cell proliferation ability as well as phosphorylated ERK1/2 expression was analyzed by xCELLigence RTCA cell analysis system and western blot.

RESULTS AND CONCLUSION: Compared with the control group, AMG-CP promoted the proliferation of ALC cells, and decreased the population doubling time in a dose-depending manner. Flow cytometry detected the acceleration of cell cycle after treatment with AMG-CP. The results of Real-time PCR showed that AMG-CP upregulated cell cycle-related genes (cyclin D1, CDK4, MCM2, MCM5) expression. Western blot results showed that AMG-CP could upregulate the expression of phosphorylated ERK1/2 and activate MAPK-ERK1/2 signaling pathway in ALC cells. After U0126 was used to inhibit the MAPK-ERK1/2 pathway, the ability of AMG-CP promoting ALC proliferation was inhibited. These results suggest that AMG-CP has a potential to activate MAPK-ERK1/2 pathway, accelerate the process of cell cycle, and then promote the proliferation of ALC cells, all of which indicate that AMG-CP has the potential to promote the proliferation of ameloblasts.

Key words: C-terminus of the amelogenin peptide, amelogenin, ameloblast, cell proliferation, cell cycle, MAPK-ERK signaling pathway, enamel development, National Natural Science Foundation of China

CLC Number:

Liu Min, Wang Ruijie, Song Danyang, Yang Sui, Tan Tao, Wang Lei, Wang Yixiang. The C-terminus of the amelogenin peptide promotes the proliferation of ALC ameloblasts through accelerating cell cycle[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(1): 99-105.

Figures/Tables 13

References

[1] MORADIAN-OLDAK J. Amelogenins: assembly, processing and control of crystal morphology. Matrix Biol. 2001;20(5-6): 293-305.
[2] BARTLETT JD, SIMMER JP. Proteinases in developing dental enamel. Crit Rev Oral Biol Med. 1999;10(4):425-441.
[3] ZHU L, TANIMOTO K, ROBINSIN S, et al. Comparative properties of recombinant human and bovine matrix metalloproteinase-20. Arch Oral Biol. 2008;53(8):785-790.
[4] VEIS A. Amelogenin gene splice products: potential signaling molecules. Cell Mol Life Sci. 2003;60(1):38-55.
[5] HAMMARSTRÖM L, HEIJL L, GESTRELIUS S. Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins. J Clin Periodontol. 1997;24(9 Pt 2):669-677.
[6] YOSHIMI Y, KUNIMATSU R, HIROSE N, et al. Effects of C-Terminal Amelogenin Peptide on Proliferation of Human Cementoblast Lineage Cells. J Periodontol. 2016;87(7):820-827.
[7] KUNIMATSU R, AWADA T, YOSHIMI Y, et al. The C-terminus of the amelogenin peptide influences the proliferation of human bone marrow mesenchymal stem cells. J Periodontol. 2018;89(4):496-505.
[8] ESPOSITO M, COULTHARD P, THOMSEN P, et al. Enamel matrix derivative for periodontal tissue regeneration in treatment of intrabony defects: a Cochrane systematic review. J Dent Educ. 2004;68(8): 834-844.
[9] BRETT PM, PARKAR M, OLSEN I, et al. Expression profiling of periodontal ligament cells stimulated with enamel matrix proteins in vitro: a model for tissue regeneration. J Dent Res. 2002;81(11): 776-783.
[10] GRAZIANI F, GENNAI S, PETRINI M, et al. Enamel matrix derivative stabilizes blood clot and improves clinical healing in deep pockets after flapless periodontal therapy: A Randomized Clinical Trial. J Clin Periodontol. 2019;46(2):231-240.
[11] HAMAMOTO Y, KAWASAKI N, JARNBRING F, et al. Effects and distribution of the enamel matrix derivative Emdogain in the periodontal tissues of rat molars transplanted to the abdominal wall. Dent Traumatol. 2002;18(1):12-23.
[12] KUNIMATSU R, TANIMOTO K, TANNE Y, et al. Amelogenin enhances the proliferation of cementoblast lineage cells. J Periodontol. 2011;82(11): 1632-1638.
[13] HUANG YC, TANIMOTO K, TANNE Y, et al. Effects of human full-length amelogenin on the proliferation of human mesenchymal stem cells derived from bone marrow. Cell Tissue Res. 2010;342(2): 205-212.
[14] TANIMOTO K, KUNIMATSU R, TANNE Y, et al. Differential effects of amelogenin on mineralization of cementoblasts and periodontal ligament cells. J Periodontol. 2012;83(5):672-679.
[15] ZOU Y, WANG H, SHAPIRO JL, et al. Determination of protein regions responsible for interactions of amelogenin with CD63 and LAMP1. Biochem J. 2007;408(3):347-354.
[16] YAN G, DU Q, WEI X, et al. Application of Real-Time Cell Electronic Analysis System in Modern Pharmaceutical Evaluation and Analysis. Molecules. 2018;23(12): E3280.
[17] ZHU C, YU J, PAN Q, et al. Hypoxia-inducible factor-2 alpha promotes the proliferation of human placenta-derived mesenchymal stem cells through the MAPK/ERK signaling pathway. Sci Rep. 2016;6:35489.
[18] RYU OH, FINCHAM AG, HU CC, et al. Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins. J Dent Res. 1999;78(3):743-750.
[19] BOABAID F, GIBSON CW, KUEHL MA, et al. Leucine-rich amelogenin peptide: a candidate signaling molecule during cementogenesis. J Periodontol. 2004;75(8):1126-1136.
[20] HATAKEYAMA J, PHILP D, HATAKEYAMA Y, et al. Amelogenin- mediated regulation of osteoclastogenesis, and periodontal cell proliferation and migration. J Dent Res. 2006;85(2):144-149.
[21] LI X, SHU R, LIU D, et al. Different effects of 25-kDa amelogenin on the proliferation, attachment and migration of various periodontal cells. Biochem Biophys Res Commun. 2010;394(3):581-586.
[22] AMIN HD, OLSEN I, KNOWLES JC, et al. Differential effect of amelogenin peptides on osteogenic differentiation in vitro: identification of possible new drugs for bone repair and regeneration. Tissue Eng Part A. 2012;18(11-12):1193-1202.