HDAC inhibitor LMK‑235 promotes the odontoblast differentiation of dental pulp cells

Liu,Zhao; Chen,Ting; Han,Qianqian; Chen,Ming; You,Jie; Fang,Fuchun; Peng,Ling; Wu,Buling

doi:10.3892/mmr.2017.8055

HDAC inhibitor LMK‑235 promotes the odontoblast differentiation of dental pulp cells

Authors:
- Zhao Liu
- Ting Chen
- Qianqian Han
- Ming Chen
- Jie You
- Fuchun Fang
- Ling Peng
- Buling Wu
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Affiliations: Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, P.R. China, Department of Periodontics, Stomatology Hospital of Guangdong Province, Guangzhou, Guangdong 510260, P.R. China
Published online on: November 14, 2017 https://doi.org/10.3892/mmr.2017.8055
Pages: 1445-1452
Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The role of dental pulp cells (DPCs) in hard dental tissue regeneration had received increasing attention because DPCs can differentiate into odontoblasts and other tissue‑specific cells. In recent years, epigenetic modifications had been identified to serve an important role in cell differentiation, and histone deacetylase (HDAC) inhibitors have been widely studied by many researchers. However, the effects of HDAC4 and HDAC5 on the differentiation of DPCs and the precise molecular mechanisms remain unclear. The present study demonstrated that LMK‑235, a specific human HDAC4 and HDAC5 inhibitor, increased the expression of specific odontoblastic gene expression levels detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) in dental pulp cells, and did not reduce cell proliferation tested by MTT assay after 3 days in culture at a low concentration. In addition, the mRNA and protein expression levels of dentin sialophosphoprotein, runt‑related transcription factor 2, alkaline phosphatase (ALP) and osteocalcin were evaluated by RT‑qPCR and western blotting, respectively. The increased gene and protein expression of specific markers demonstrated, indicating that LMK‑235 promoted the odontoblast induction of DPCs. ALP activity and mineralised nodule formation were also enhanced due to the effect of LMK‑235, detected by an ALP activity test and Alizarin Red S staining, respectively. Additionally, the vascular endothelial growth factor (VEGF)/RAC‑gamma serine/threonine‑protein kinase (AKT)/mechanistic target of rapamycin (mTOR) signalling pathway was tested to see if it takes part in the differentiation of DPCs treated with LMK‑235, and it was demonstrated that the mRNA expression levels of VEGF, AKT and mTOR were upregulated. These findings indicated that LMK‑235 may serve a key role in the proliferation and odontoblast differentiation of DPCs, and could be used to accelerate dental tissue regeneration.

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1	Jo YY, Lee HJ, Kook SY, Choung HW, Park JY, Chung JH, Choung YH, Kim ES, Yang HC and Choung PH: Isolation and characterization of postnatal stem cells from human dental tissues. Tissue Eng. 13:767–773. 2007. View Article : Google Scholar : PubMed/NCBI
2	Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY and Shi S: Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 364:149–155. 2004. View Article : Google Scholar : PubMed/NCBI
3	Ji YM, Jeon SH, Park JY, Chung JH, Choung YH and Choung PH: Dental stem cell therapy with calcium hydroxide in dental pulp capping. Tissue Eng Part A. 16:1823–1833. 2010. View Article : Google Scholar : PubMed/NCBI
4	Arnsdorf EJ, Tummala P, Castillo AB, Zhang F and Jacobs CR: The epigenetic mechanism of mechanically induced osteogenic differentiation. J Biomech. 43:2881–2886. 2010. View Article : Google Scholar : PubMed/NCBI
5	Lehrmann H, Pritchard LL and Harel-Bellan A: Histone acetyltransferases and deacetylases in the control of cell proliferation and differentiation. Adv Cancer Res. 86:41–65. 2002. View Article : Google Scholar : PubMed/NCBI
6	Marks PA, Richon VM and Rifkind RA: Histone deacetylase inhibitors: Inducers of differentiation or apoptosis of transformed cells. J Natl Cancer Inst. 92:1210–1216. 2000. View Article : Google Scholar : PubMed/NCBI
7	Jeon EJ, Lee KY, Choi NS, Lee MH, Kim HN, Jin YH, Ryoo HM, Choi JY, Yoshida M, Nishino N, et al: Bone morphogenetic protein-2 stimulates Runx2 acetylation. J Biol Chem. 281:16502–16511. 2006. View Article : Google Scholar : PubMed/NCBI
8	Fu Y, Zhang P, Ge J, Cheng J, Dong W, Yuan H, Du Y, Yang M, Sun R and Jiang H: Histone deacetylase 8 suppresses osteogenic differentiation of bone marrow stromal cells by inhibiting histone H3K9 acetylation and RUNX2 activity. Int J Biochem Cell Biol. 54:68–77. 2014. View Article : Google Scholar : PubMed/NCBI
9	Huynh NC, Everts V, Pavasant P and Ampornaramveth RS: Inhibition of histone deacetylases enhances the osteogenic differentiation of human periodontal ligament cells. J Cell Biochem. 117:1384–1395. 2016. View Article : Google Scholar : PubMed/NCBI
10	Lee HW, Suh JH, Kim AY, Lee YS, Park SY and Kim JB: Histone deacetylase 1-mediated histone modification regulates osteoblast differentiation. Mol Endocrinol. 20:2432–2443. 2006. View Article : Google Scholar : PubMed/NCBI
11	Nissen-Meyer LS, Svalheim S, Taubøll E, Reppe S, Lekva T, Solberg LB, Melhus G, Reinholt FP, Gjerstad L and Jemtland R: Levetiracetam, phenytoin, and valproate act differently on rat bone mass, structure, and metabolism. Epilepsia. 48:1850–1860. 2007. View Article : Google Scholar : PubMed/NCBI
12	Paino F, La Noce M, Tirino V, Naddeo P, Desiderio V, Pirozzi G, De Rosa A, Laino L, Altucci L and Papaccio G: Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts: Evidence for HDAC2 involvement. Stem Cells. 32:279–289. 2014. View Article : Google Scholar : PubMed/NCBI
13	Jin H, Park JY, Choi H and Choung PH: HDAC inhibitor trichostatin A promotes proliferation and odontoblast differentiation of human dental pulp stem cells. Tissue Eng Part A. 19:613–624. 2013. View Article : Google Scholar : PubMed/NCBI
14	Kwon A, Park HJ, Baek K, Lee HL, Park JC, Woo KM, Ryoo HM and Baek JH: Suberoylanilide hydroxamic acid enhances odontoblast differentiation. J Dent Res. 91:506–512. 2012. View Article : Google Scholar : PubMed/NCBI
15	Lu J, Qu S, Yao B, Xu Y, Jin Y, Shi K, Shui Y, Pan S, Chen L and Ma C: Osterix acetylation at K307 and K312 enhances its transcriptional activity and is required for osteoblast differentiation. Oncotarget. 7:37471–37486. 2016. View Article : Google Scholar : PubMed/NCBI
16	Xia ZY, Hu Y, Xie PL, Tang SY, Luo XH, Liao EY, Chen F and Xie H: Runx2/miR-3960/miR-2861 positive feedback loop is responsible for osteogenic transdifferentiation of vascular smooth muscle cells. Biomed Res Int. 2015:6240372015. View Article : Google Scholar : PubMed/NCBI
17	Hansen FK, Sumanadasa SD, Stenzel K, Duffy S, Meister S, Marek L, Schmetter R, Kuna K, Hamacher A, Mordmüller B, et al: Discovery of HDAC inhibitors with potent activity against multiple malaria parasite life cycle stages. Eur J Med Chem. 82:204–213. 2014. View Article : Google Scholar : PubMed/NCBI
18	Li A, Liu Z, Li M, Zhou S, Xu Y, Xiao Y and Yang W: HDAC5, a potential therapeutic target and prognostic biomarker, promotes proliferation, invasion and migration in human breast cancer. Oncotarget. 7:37966–37978. 2016. View Article : Google Scholar : PubMed/NCBI
19	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
20	Ganai SA: Histone deacetylase inhibitor sulforaphane: The phytochemical with vibrant activity against prostate cancer. Biomed Pharmacother. 81:250–257. 2016. View Article : Google Scholar : PubMed/NCBI
21	Fu M, Shi W, Li Z and Liu H: Activation of mPTP-dependent mitochondrial apoptosis pathway by a novel pan HDAC inhibitor resminostat in hepatocellular carcinoma cells. Biochem Biophys Res Commun. 477:527–533. 2016. View Article : Google Scholar : PubMed/NCBI
22	Liu G, Xu G, Gao Z, Liu Z, Xu J, Wang J, Zhang C and Wang S: Demineralized dentin matrix induces odontoblastic differentiation of dental pulp stem cells. Cells Tissues Organs. 201:65–76. 2016. View Article : Google Scholar : PubMed/NCBI
23	Suzuki S, Sreenath T, Haruyama N, Honeycutt C, Terse A, Cho A, Kohler T, Müller R, Goldberg M and Kulkarni AB: Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization. Matrix Biol. 28:221–229. 2009. View Article : Google Scholar : PubMed/NCBI
24	Chen Y, Zhang Y, Ramachandran A and George A: DSPP is essential for normal development of the dental-craniofacial complex. J Dent Res. 95:302–310. 2016. View Article : Google Scholar : PubMed/NCBI
25	Gu S, Liang J, Wang J and Liu B: Histone acetylation regulates osteodifferentiation of human dental pulp stem cells via DSPP. Front Biosci (Landmark Ed). 18:1072–1079. 2013. View Article : Google Scholar : PubMed/NCBI
26	Wang T, Liu H, Ning Y and Xu Q: The histone acetyltransferase p300 regulates the expression of pluripotency factors and odontogenic differentiation of human dental pulp cells. PLoS One. 9:e1021172014. View Article : Google Scholar : PubMed/NCBI
27	Zhang X, Yang M, Lin L, Chen P, Ma KT, Zhou CY and Ao YF: Runx2 overexpression enhances osteoblastic differentiation and mineralization in adipose-derived stem cells in vitro and in vivo. Calcif Tissue Int. 79:169–178. 2006. View Article : Google Scholar : PubMed/NCBI
28	Sun X, Wei L, Chen Q and Terek RM: HDAC4 represses vascular endothelial growth factor expression in chondrosarcoma by modulating RUNX2 activity. J Biol Chem. 284:21881–21890. 2009. View Article : Google Scholar : PubMed/NCBI
29	Li S, Kong H, Yao N, Yu Q, Wang P, Lin Y, Wang J, Kuang R, Zhao X, Xu J, et al: The role of runt-related transcription factor 2 (Runx2) in the late stage of odontoblast differentiation and dentin formation. Biochem Biophys Res Commun. 410:698–704. 2011. View Article : Google Scholar : PubMed/NCBI
30	Cutarelli A, Marini M, Tancredi V, D'Arcangelo G, Murdocca M, Frank C and Tarantino U: Adenosine Triphosphate stimulates differentiation and mineralization in human osteoblast-like Saos-2 cells. Dev Growth Differ. 58:400–408. 2016. View Article : Google Scholar : PubMed/NCBI
31	Tsukamoto Y, Fukutani S, Shin-Ike T, Kubota T, Sato S, Suzuki Y and Mori M: Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts. Arch Oral Biol. 37:1045–1055. 1992. View Article : Google Scholar : PubMed/NCBI
32	Tong Y, Feng W, Wu Y, Lv H, Jia Y and Jiang D: Mechano-growth factor accelerates the proliferation and osteogenic differentiation of rabbit mesenchymal stem cells through the PI3K/AKT pathway. BMC Biochem. 16:12015. View Article : Google Scholar : PubMed/NCBI
33	Dung TT, Yi YS, Heo J, Yang WS, Kim JH, Kim HG, Park JG, Yoo BC, Cho JY and Hong S: Critical role of protein L-isoaspartyl methyltransferase in basic fibroblast growth factor-mediated neuronal cell differentiation. BMB Rep. 49:437–442. 2016. View Article : Google Scholar : PubMed/NCBI
34	Bae WJ, Auh QS, Kim GT, Moon JH and Kim EC: Effects of sodium tri- and hexameta-phosphate in vitro osteoblastic differentiation in Periodontal Ligament and Osteoblasts, and in vivo bone regeneration. Differentiation. 92:257–269. 2016. View Article : Google Scholar : PubMed/NCBI