Matrix Metalloproteinase-20 Over-Expression Is Detrimental to Enamel Development: A Mus musculus Model Masashi Shin 1 , Yuanyuan Hu 2 , Coralee E. Tye 1 , Xiaomu Guan 1 , Craig C. Deagle 3 , Jerry V. Antone 1 , Charles E. Smith , James P. Simmer , John D. Bartlett 2 1 * 1 Department of Mineralized Tissue Biology and Harvard School of Dental Medicine, The Forsyth Institute, Cambridge Massachusetts, United States of America, 2 Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan, United States of America, 3 Program in Endodontics, Harvard School of Dental Medicine, Boston Massachusetts, United States of America, 4 Facility for Electron Microscopy Research, Department of Anatomy & Cell Biology, and Faculty of Dentistry, McGill University, Montreal, QC, Canada Abstract Background: Matrix metalloproteinase-20 (Mmp20) ablated mice have enamel that is thin and soft with an abnormal rod pattern that abrades from the underlying dentin. We asked if introduction of transgenes expressing Mmp20 would revert this Mmp20 null phenotype back to normal. Unexpectedly, for transgenes expressing medium or high levels of Mmp20, we found opposite enamel phenotypes depending on the genetic background (Mmp20 2/2 or Mmp20 +/+ ) in which the transgenes were expressed. Methodology/Principal Findings: Amelx-promoter-Mmp20 transgenic founder mouse lines were assessed for transgene expression and those expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Regardless of expression level, each transgene brought the null enamel back to full thickness. However, the high and medium expressing Mmp20 transgenes in the Mmp20 null background had significantly harder more mineralized enamel than did the low transgene expresser. Strikingly, when the high and medium expressing Mmp20 transgenes were present in the wild-type background, the enamel was significantly less well mineralized than normal. Protein gel analysis of enamel matrix proteins from the high and medium expressing transgenes present in the wild-type background demonstrated that greater than normal amounts of cleavage products and smaller quantities of higher molecular weight proteins were present within their enamel matrices. Conclusions/Significance: Mmp20 expression levels must be within a specific range for normal enamel development to occur. Creation of a normally thick enamel layer may occur over a wider range of Mmp20 expression levels, but acquisition of normal enamel hardness has a narrower range. Since over-expression of Mmp20 results in decreased enamel hardness, this suggests that a balance exists between cleaved and full-length enamel matrix proteins that are essential for formation of a properly hardened enamel layer. It also suggests that few feedback controls are present in the enamel matrix to prevent excessive MMP20 activity. Citation: Shin M, Hu Y, Tye CE, Guan X, Deagle CC, et al. (2014) Matrix Metalloproteinase-20 Over-Expression Is Detrimental to Enamel Development: A Mus musculus Model. PLoS ONE 9(1): e86774. doi:10.1371/journal.pone.0086774 Editor: Ted S. Acott, Casey Eye Institute, United States of America Received September 26, 2013; Accepted December 17, 2013; Published January 23, 2014 Copyright: ß 2014 Shin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research reported in this publication was supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health under award numbers R01DE019775 (JPS) and R01DE016276 (JDB). The Forsyth Institute micro-CT Core is partly supported by National Institute of Health grant 1S10RR027553-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jbartlett@forsyth.org Introduction Dental enamel is the hardest tissue of the body, but it does not start that way. Enamel development (amelogenesis) can be defined as consisting of three stages; the secretory, transition, and maturation stages [1]. During the secretory stage the ameloblasts adjacent to the forming enamel elongate and secrete large quantities of protein into the enamel matrix. Approximately 90% of this protein is amelogenin [2]. Amelogenin has only one post-translational modification whereby Ser16 is phosphorylated [3,4], but its transcripts undergo extensive alternative splicing [5,6,7] to generate as many as 16 X-chromosomal murine amelogenin mRNAs [8,9,10]. The secretory stage is when thin crystallite ribbons begin growing in length [11,12,13] and they stop elongating once ameloblasts have defined the full thickness of the enamel layer. Secretory stage enamel is very soft and has a cheese-like consistency. Once the ameloblasts progress to the transition stage, they shorten and greatly reduce protein secretion. During the subsequent maturation stage, the short columnar ameloblasts reabsorb the proteins they had previously secreted. This is when the enamel ribbons grow to their greatest amount in width and thickness [1]. Enamel consists of mineralized rods that are sometimes entwined (gnarled) in human molars [14] or may form groups of rods that pass across each other (decussating) as is PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e86774 2,4