University of Birmingham The histone-deacetylase-inhibitor suberoylanilide hydroxamic acid promotes dental pulp repair mechanisms through modulation of matrix metalloproteinase-13 activity Duncan, Henry; Smith, Anthony; Fleming, Garry James P.; Partridge, Nicola C.; Shimizu, Emi; Moran, Gary P.; Cooper, Paul DOI: 10.1002/jcp.25128 License: None: All rights reserved Document Version Peer reviewed version Citation for published version (Harvard): Duncan, H, Smith, A, Fleming, GJP, Partridge, NC, Shimizu, E, Moran, GP & Cooper, P 2016, 'The histone- deacetylase-inhibitor suberoylanilide hydroxamic acid promotes dental pulp repair mechanisms through modulation of matrix metalloproteinase-13 activity', Journal of Cellular Physiology, vol. 231, no. 4, pp. 798-816. https://doi.org/10.1002/jcp.25128 Link to publication on Research at Birmingham portal Publisher Rights Statement: This is the peer reviewed version of the following article: Duncan, H. F., Smith, A. J., Fleming, G. J. P., Partridge, N. C., Shimizu, E., Moran, G. P. and Cooper, P. R. (2016), The Histone-Deacetylase-Inhibitor Suberoylanilide Hydroxamic Acid Promotes Dental Pulp Repair Mechanisms Through Modulation of Matrix Metalloproteinase-13 Activity. J. Cell. Physiol., 231: 798–816., which has been published in final form at http://dx.doi.org/10.1002/jcp.25128. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. Checked Jan 2016 General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. 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Oct. 2021 Journal of Cellular Physiology The his tone -deacetylase -inhibitor suberoylanilide hydroxamic acid promotes dental pulp repair mechanisms through modulation of matrix metalloproteinase-13 activity For Peer Review Journal: Journal of Cellular Physiology Manuscript ID: Draft Wiley - Manuscript type: Original Research Article Date Submitted by the Author: n/a Complete List of Authors: Duncan, Henry; Dublin Dental University Hospital, Trinity College Dublin, University of Dublin, Restorative Dentistry Smith, Anthony J; Oral Biology, School of Dentistry, University of Birmingham, Birmingham B4 6NN, UK, Fleming, Garry; Dublin Dental University Hospital, Division of Biosciences Partridge, Nicola; New York University, Shimizu, Emi; NYU, Craniofacial Biology Moran, Gary; Dublin Dental University Hospital, Division of Biosciences Cooper, Paul R; Oral Biology, School of Dentistry, University of Birmingham, Birmingham B4 6NN, UK, Epigenetics, Histone deacetylase inhibitor, Cellular differentiation, Key Words: Mineralization, Gene expression John Wiley & Sons, Inc. Page 1 of 88 Journal of Cellular Physiology 1 2 3 4 5 ORIGINAL RESEARCH ARTICLE 6 7 8 9 The histone-deacetylase-inhibitor suberoylanilide hydroxamic acid promotes 10 dental pulp repair mechanisms through modulation of matrix 11 12 metalloproteinase-13 activity 13 14 1 2 3 4 15 Henry F. Duncan , Anthony J. Smith , Garry J. P. Fleming , Nicola C. Partridge , Emi 4 5 2 16 Shimizu , Gary P. Moran , Paul R. Cooper 17 18 1 Division of RestorativeFor Dentistry Peer & Periodontology, Review Dublin Dental University Hospital, Trinity 19 College Dublin, Lincoln Place, Dublin 2, Ireland 20 2Oral Biology, School of Dentistry, University of Birmingham, Birmingham, B4 6NN, UK 21 3 22 Material Science Unit, Dublin Dental University Hospital, Trinity College Dublin, Ireland 4 23 Department of Basic Science and Craniofacial Biology, New York University College of 24 Dentistry, USA 25 5Division of Oral Biosciences, Dublin Dental University Hospital, Trinity College Dublin, 26 Ireland 27 28 29 30 31 Running head: HDACi promotes mineralization by modulating MMP-13 expression 32 33 34 Corresponding author : Henry F. Duncan; Division of Restorative Dentistry & Periodontology, 35 36 Dublin Dental University Hospital, Trinity College Dublin, University of Dublin, Lincoln Place, 37 Dublin 2, Ireland 38 Tel.: +353 (1) 612 7356; Fax: +353 (1) 612 7297; Email: [email protected] 39 40 41 Keywords: 42 43 • Histone-deacetylase-inhibitor 44 • Epigenetics 45 • Matrix metalloproteinase 46 • Cellular differentiation , 47 48 • Mineralization 49 • Gene expression 50 51 52 Total number of text figures: seven Total number of tables : seven 53 54 Grant support: 55 56 1. Contract grant sponsor: Irish Endodontic Society; Contract Grant Number 2008/1 57 58 2. Contract grant sponsor: European Society of Endodontology; Contract Grant Number ARG 2012/2 59 60 1 John Wiley & Sons, Inc. Journal of Cellular Physiology Page 2 of 88 1 2 3 Abstract 4 5 6 Direct application of histone-deacetylase-inhibitors (HDACis) to dental pulp cells (DPCs) 7 8 induces chromatin changes, promoting gene expression and cellular-reparative events. We have 9 10 previously demonstrated that HDACis (Valproic acid, Trichostatin A) increase mineralization in dental 11 12 papillae-derived cell-lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we 13 14 investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify 15 16 new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only 17 18 For Peer Review 19 at relatively high concentrations (5µM); while low concentrations (1µM) SAHA did not increase 20 21 apoptosis. HDACi-exposure for 24h induced mineralization-per-cell dose-dependently after 2 weeks; 22 23 however, constant 14d SAHA-exposure inhibited mineralization. Microarray analysis (24h and 14d) of 24 25 SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0-fold change at 24h, 26 27 which reduced to 36 genes at 14d. 59% of genes were down-regulated at 24h and 36% at 14d, 28 29 respectively. Pathway analysis indicated SAHA increased expression of members of the matrix 30 31 32 metalloproteinase (MMP) family. Furthermore, SAHA-supplementation increased MMP-13 protein 33 34 expression (7d, 14 d) and enzyme activity (48h, 14d). Selective MMP-13-inhibition (MMP-13i) dose- 35 36 dependently accelerated mineralization in both SAHA-treated and non-treated cultures. MMP-13i- 37 38 supplementation promoted expression of several mineralization-associated markers, however, HDACi- 39 40 induced cell migration and wound healing were impaired. Data demonstrate that short-term low-dose 41 42 SAHA-exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. 43 44 45 MMP-13i further increased mineralization-associated events, but decreased HDACi cell migration 46 47 indicating a specific role for MMP-13 in pulpal repair processes. Pharmacological inhibition of HDAC 48 49 and MMP may provide novel insights into pulpal repair processes with significant translational benefit. 50 51 52 53 54 55 56 57 58 59 60 2 John Wiley & Sons, Inc. Page 3 of 88 Journal of Cellular Physiology 1 2 3 Introduction 4 5 6 The balance between the cellular enzymes, histone deacetylases (HDACs) and histone 7 8 9 acetyltransferases (HATs), controls chromatin conformation and regulates transcription. Predominant 10 11 HDAC activity results in the removal of acetyl groups from the histone tails within the nucleosome, 12 13 leading to a condensed chromatin conformation and reduced transcription, while HAT activity has the 14 15 opposite effect leading to an open, transcriptionally active chromatin structure (Bolden et al., 2006). 16 17 There are 18 identified mammalian HDACs, which are categorized into four classes functioning via zinc- 18 For Peer Review 19 dependent or independent mechanisms (Gregoretti et al., 2004). Class I (-1, -2, -3, -8) are zinc-dependent, 20 21 22 ubiquitously distributed and expressed in the cell nucleus (Marks and Dokmanovic, 2005), while class II 23 24 (-4, -5, -6, -9, -10) are also zinc-dependent, but demonstrate tissue-restricted expression and shuttle 25 26 between the nucleus and cytoplasm (Verdin et al., 2003; Marks, 2010). Class III HDACs, known as 27 28 sirtuins, are not zinc-dependent, instead requiring coenzyme nicotinamide adenine dinucleotide (NAD+) 29 30 for function (Haigis and Guarente, 2006), while there is currently only one class IV member, HDAC -11 31 32 (Villagra et al., 2009). A recent analysis of HDAC expression in human dental pulp tissue demonstrated