John D Bartlett

Harvard University, Cambridge, Massachusetts, United States

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Publications (111)290.27 Total impact

  • J D Bartlett · J P Simmer
    Journal of dental research 05/2015; 94(5):642-4. DOI:10.1177/0022034515572442 · 4.14 Impact Factor
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    ABSTRACT: Purpose: The purposes of this study were to: (1) investigate adhesion through shear bond strength (SBS) testing of a resin composite bonded with a self-etching bonding system (SEB) to amelogenesis imperfecta (AI)-affected deproteinized mouse enamel or dentin; and (2) compare wild-type (WT), amelogenin null (AmelxKO), and matrix metalloproteinase-20 null (Mmp20KO) enamel and dentin phenotypes using micro-CT and nanoindentation. Methods: Enamel incisor surfaces of WT, AmelxKO, and Mmp20KO mice were treated with SEB with and without sodium hypochlorite and tested for SBS. Incisor dentin was also treated with SEB and tested for SBS. These surfaces were further examined by scanning electron miscroscopy. Micro-CT and nanoindentation analyses were performed on mouse dentin and enamel. Data were analyzed for significance by analysis of variance. Results: Deproteinization did not improve SBS of SEB to these AI-affected enamel surfaces. SBS of AmelxKO teeth was similar in dentin and enamel; however, it was higher in Mmp20KO dentin. The nanohardness of knockout enamel was significantly lower than WT, while knockout dentin nanohardness was not different from WT. Conclusions: Using animal amelogenesis imperfecta models, enamel sodium hypochlorite deproteinization of hypoplastic and hypoplastic-hypomaturation enamel did not increase shear bond strength, while removal of the defective enamel allowed optimal dentin bonding.
    Pediatric dentistry 10/2014; 36(5). · 0.56 Impact Factor
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    ABSTRACT: Abstract Dental fluorosis is characterized by subsurface hypomineralization and retention of enamel matrix proteins. Fluoride (F(-)) exposure generates reactive oxygen species (ROS) that can cause endoplasmic reticulum (ER)-stress. We therefore screened oxidative stress arrays to identify genes regulated by F(-) exposure. Vitamin E is an antioxidant so we asked if a diet high in vitamin E would attenuate dental fluorosis. Maturation stage incisor enamel organs (EO) were harvested from F(-)-treated rats and mice were assessed to determine if vitamin E ameliorates dental fluorosis. Uncoupling protein-2 (Ucp2) was significantly up-regulated by F(-) (∼1.5 & 2.0 fold for the 50 or 100 ppm F(-) treatment groups, respectively). Immunohistochemical results on maturation stage rat incisors demonstrated that UCP2 protein levels increased with F(-) treatment. UCP2 down-regulates mitochondrial production of ROS, which decreases ATP production. Thus, in addition to reduced protein translation caused by ER-stress, a reduction in ATP production by UCP2 may contribute to the inability of ameloblasts to remove protein from the hardening enamel. Fluoride-treated mouse enamel had significantly higher quantitative fluorescence (QF) than the untreated controls. No significant QF difference was observed between control and vitamin E-enriched diets within a given F(-) treatment group. Therefore, a diet rich in vitamin E did not attenuate dental fluorosis. We have identified a novel oxidative stress response gene that is up-regulated in vivo by F(-) and activation of this gene may adversely affect ameloblast function.
    Connective Tissue Research 08/2014; 55(S1):25-28. DOI:10.3109/03008207.2014.923854 · 1.98 Impact Factor
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    M Suzuki · M Shin · J P Simmer · J D Bartlett
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    ABSTRACT: Dental fluorosis is caused by chronic high-level fluoride (F(-)) exposure during enamel development, and fluorosed enamel has a higher than normal protein content. Matrix metalloproteinase 20 cleaves enamel matrix proteins during the secretory stage, and KLK4 further cleaves these proteins during the maturation stage so that the proteins can be reabsorbed from the hardening enamel. We show that transforming growth factor β1 (TGF-β1) can induce Klk4 expression, and we examine the effect of F(-) on TGF-β1 and KLK4 expression. We found that in vivo F(-) inhibits Klk4 but not Mmp20 transcript levels. LacZ-C57BL/6-Klk4(+/LacZ) mice have LacZ inserted in frame at the Klk4 translation initiation site so that the endogenous Klk4 promoter drives LacZ expression in the same temporal/spatial way as it does for Klk4. KLK4 protein levels in rat enamel and β-galactosidase staining in LacZ-C57BL/6-Klk4(+/LacZ) mouse enamel were both significantly reduced by F(-) treatment. Since TGF-β1 induces KLK4 expression, we tested and found that F(-) significantly reduced Tgf-β1 transcript levels in rat enamel organ. These data suggest that F(-)-mediated downregulation of TGF-β1 expression contributes to reduced KLK4 protein levels in fluorosed enamel and provides an explanation for why fluorosed enamel has a higher than normal protein content.
    Journal of Dental Research 07/2014; 93(10). DOI:10.1177/0022034514545629 · 4.14 Impact Factor
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    ABSTRACT: Ameloblasts are ectoderm-derived cells that produce an extracellular enamel matrix that mineralizes to form enamel. The development and use of immortalized cell lines, with a stable phenotype, is an important contribution to biological studies as it allows for the investigation of molecular activities without the continuous need for animals. In this study we compare the expression profiles of enamel-specific genes in two mouse derived ameloblast-like cell lines: LS8 and ALC cells. Quantitative PCR analysis indicates that, relative to each other, LS8 cells express greater mRNA levels for genes that define secretory-stage activities (Amelx, Ambn, Enam, and Mmp20), while ALC express greater mRNA levels for genes that define maturation-stage activities (Odam and Klk4). Western blot analyses show that Amelx, Ambn, and Odam proteins are detectable in ALC, but not LS8 cells. Unstimulated ALC cells form calcified nodules, while LS8 cells do not. These data provide greater insight as to the suitability of both cell lines to contribute to biological studies on enamel formation and biomineralization, and highlight some of the strengths and weaknesses when relying on enamel epithelial organ-derived cell lines to study molecular activities of amelogenesis.
    Frontiers in Physiology 07/2014; 5:277. DOI:10.3389/fphys.2014.00277 · 3.50 Impact Factor
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    Xiaomu Guan · Felicitas B Bidlack · Nicole Stokes · John D Bartlett
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    ABSTRACT: Background N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development. Methodology/Principal Findings The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. μCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ. Conclusions/Significance The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous increase in E-cadherin expression due to N-cadherin ablation in a healthy developing tissue.
    PLoS ONE 07/2014; 9(7):e102153. DOI:10.1371/journal.pone.0102153 · 3.23 Impact Factor
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    John D Bartlett · James P Simmer
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    ABSTRACT: Enamel development occurs in stages. During the secretory stage, a soft protein rich enamel layer is produced that expands to reach its final thickness. During the maturation stage, proteins are removed and the enamel matures into the hardest substance in the body. KLK4 is expressed during the transition from secretory to the maturation stage and its expression continues throughout maturation. KLK4 is a glycosylated chymotrypsin-like serine protease that cleaves enamel matrix proteins prior to their export out of the hardening enamel layer. Mutations in KLK4 can cause autosomal recessive, non-syndromic enamel malformations in humans and mice. Klk4 ablated mice initially have normal-looking teeth with enamel of full thickness. However, the enamel is soft and protein-rich. Three findings are notable from Klk4 ablated mice: first, enamel rods fall from the interrod enamel leaving behind empty holes where the enamel fractures near the underlying dentin surface. Second, the ~10,000 crystallites that normally fuse to form a solid enamel rod fail to grow together in the ablated mice and can fall out of the rods. Third, and most striking, the crystallites grow substantially in width and thickness (a- and b-axis) in the ablated mice until they almost interlock. The crystallites grow in defined enamel rods, but interlocking is prevented presumably because too much protein remains. Conventional thought holds that enamel proteins bind specifically to the sides of enamel crystals to inhibit growth in width and thickness so that the thin, ribbon-like enamel crystallites grow predominantly in length. Results from Klk4 ablated mice demonstrate that this convention requires updating. An alternative mechanism is proposed whereby enamel proteins serve to form a mold or support structure that shapes and orients the mineral ribbons as they grow in length. The remnants of this support structure must be removed by KLK4 so that the crystallites can interlock to form fully hardened enamel.
    Frontiers in Physiology 07/2014; 5:240. DOI:10.3389/fphys.2014.00240 · 3.50 Impact Factor
  • J.D. BARTLETT
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • M. SUZUKI · J.D. BARTLETT
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    ABSTRACT: Objectives: Sirtuin1 (SIRT1) is the NAD+-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum stress and oxidative stress. Previously, we reported that fluoride induces endoplasmic reticulum (ER) stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Methods: The mouse ameloblast-derived cell line (LS8) was treated with fluoride in the presence or absence of resveratrol (RES) and Sprague-Dawley rats (6-week-old) were provided water containing fluoride (0-125 ppm) ad libitum for 6 weeks. In vitro and in vivo analyses included qPCR and immunoblot results and in vivo, immunohistochemistry was performed to determine if ameloblasts participated in the stress response. Results: Fluoride treatment of LS8 cells significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used RES to augmented SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride in drinking water had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Conclusion: The SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis.
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • X. GUAN · F.B. BIDLACK · J. ANTONE · N. STOKES · E. FUCHS · J.D. BARTLETT
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    ABSTRACT: N-cadherin is an adhesion molecule present on the surface of a variety of cells including neural, muscle, and mesenchymal cells. Deletion of N-cadherin in mice is embryonic lethal. N-cadherin tissue-specific knockout studies have shown that N-cadherin plays a critical role in maintaining the structural integrity of the heart, but that cardiac specific expression of E-cadherin rescues this cell adhesion defect. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically upregulated in ameloblasts when ameloblasts slide across each other to form the rodent decussating enamel rod (prism) pattern. Objectives: Since N-cadherin promotes directional chain migration of cerebellar granule neurons, we asked if N-cadherin is essential for ameloblast cell movement necessary to form the decussating enamel prism pattern. Methods: The enamel organ and its ameloblasts are epithelial tissues. Therefore, a mouse strain with N-cadherin ablated from epithelium was generated using Cre/loxP technology. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed by Scanning Electron Microscopy (SEM), Vickers hardness, and immunohistochemistry. Results: N-cadherin deletion in enamel organ and ameloblasts was confirmed by qPCR and immunohistochemistry. N-cadherin cKO mice were viable and fertile and SEM showed that the enamel thickness, surface structure, and prism pattern of the cKO mice looked identical to WT. Vickers micro-hardness measurements demonstrated that no significant difference in enamel hardness existed between the WT and cKO mice. Interestingly, immunohistochemistry revealed that the WT and cKO ameloblasts expressed approximately equal amounts of total cadherins. We discovered that ameloblast E-cadherin was not down-regulated during the secretory stage in the cKO mice suggesting that E- and N-cadherins are functionally interchangeable during enamel development. Conclusion: Expression of N-cadherin during the secretory stage of enamel development is not essential for formation of the decussating enamel prism pattern because E-cadherin can functionally compensate for the loss of N-cadherin.
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • J.D. BARTLETT
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    ABSTRACT: Our understanding of dental enamel development has undergone a profound change in the last twenty years. Twenty years ago we knew that amelogenin was a bulk component of the enamel matrix along with some other minor proteins. These less abundant proteins were generically termed “non-amelogenin proteins” or “enamelins” and, for the most part, were not considered as important for enamel formation. Since that time, in addition to amelogenin, we have discovered that the enamel matrix is composed of proteins named: ameloblastin, enamelin, matrix metalloproteinase-20 (MMP20, enamelysin) and kallikrein-related peptidase-4 (KLK4). We now know that each of these five enamel proteins is absolutely essential for dental enamel development. Malfunction of any one results in non-syndromic enamel malformations classified as Amelogenesis Imperfecta (AI). We also know from mammals that once had teeth with enamel, but through evolution lost either their enamel or their teeth (such as baleen whales), that the only non-overlapping function of these five enamel proteins is in dental enamel development. Except for KLK4, every gene encoding these proteins are pseudogenes in specific species of enamel-less or toothless mammals. While Klk4 was not examined in these mammals, its loss of function in mice and humans results in AI with no other observed phenotype. This talk focuses on recent discoveries and discusses what is known about each of these proteins and proteinases. Recent results from genetically ablated mice and from humans with AI serve as a guide-post for the understanding of each protein’s function. Finally, based on recent results, a revised theory is offered about how each of these enamel matrix proteins serves to promote enamel development. Support by NIDCR grant R01DE016276 is gratefully acknowledged.
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • F.B. BIDLACK · J. DOBECK · J.D. BARTLETT
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    ABSTRACT: An unresolved problem in tooth enamel studies has been to analyze simultaneously and with sufficient spatial resolution both mineral and organic phases in their three dimensional (3D) organization in a given specimen. Objectives: This study aims to address this need using high-resolution imaging to analyze the 3D structural organization of the enamel matrix, especially amelogenin, in relationship to forming enamel crystals. We compare between WT and Mmp20 knock-out incisors to ultimately better understand how amelogenin guides the hierarchical arrangement of enamel crystals. Methods: Chemically fixed hemi-mandibles were LR White embedded, polished either in longitudinal or cross sectional planes and etched to expose defined stages of incisor enamel development. Amelogenin was labeled with specific antibodies and either 10nm immuno-gold, Q-dots, or fluorescent dyes. This allowed us to detect fluorescence via light microscopy and to use back scattered electron mode in scanning electron microscopy in a correlative microscopy approach. In addition, helium ion microscopy (HIM) was used for high-resolution imaging of uncoated samples to study the spatial organization of organic and mineral structures. Results: Wild type enamel in late secretory to early maturation stage reveals adjacent to ameloblasts a lengthwise parallel alignment of full-length amelogenin proteins which then transitions into a more heterogeneous appearance. The matrix flanking crystal bundles forms a smooth and lacey sheath, whereas between enamel prisms it is organized into spherical components that are interspersed with rod-shaped protein. In comparison, Mmp20 KO incisors show more abundant organic material lacking this heterogeneity. The spherical organization and the distinct smooth and lacey appearance of protein that form tubular spaces to accommodate crystal bundles are not observed. Conclusion: The use of correlative light and electron microscopy in combination with HIM analyses is a powerful approach to elucidate the role of matrix proteins, their distribution, and their 3D structural organization in nanometer resolution.
    AADR Annual Meeting & Exhibition 2014; 03/2014
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    ABSTRACT: 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(-/-) or Mmp20(+/+) ) in which the transgenes were expressed. 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. 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.
    PLoS ONE 01/2014; 9(1):e86774. DOI:10.1371/journal.pone.0086774 · 3.23 Impact Factor
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    Maiko Suzuki · John D Bartlett
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    ABSTRACT: Sirtuin1 (SIRT1) is an (NAD(+))-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum stress and oxidative stress. Previously, we reported that fluoride induces endoplasmic reticulum (ER) stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augmented SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50 and 100ppm) in drinking water for 6weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis.
    Biochimica et Biophysica Acta 11/2013; 1842(2). DOI:10.1016/j.bbadis.2013.11.023 · 4.66 Impact Factor
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    ABSTRACT: Enamelysin (MMP20) and kallikrein 4 (KLK4) are believed to be necessary to clear proteins from the enamel matrix of developing teeth. MMP20 is expressed by secretory stage ameloblasts, while KLK4 is expressed from the transition stage throughout the maturation stage. The aim of this study is to investigate the activation of KLK4 by MMP20 and the inactivation of MMP20 by KLK4. Native pig MMP20 (pMMP20) and KLK4 (pKLK4) were isolated directly from enamel scrapings from developing molars. Recombinant human proKLK4 (rh-proKLK4) was activated by incubation with pMMP20 or recombinant human MMP20 (rhMMP20), and the resulting KLK4 activity was detected by zymography. Reaction products were isolated by reverse-phase high performance liquid chromatography (RP-HPLC), and their N-termini characterized by Edman degradation. The pMMP20 was incubated with pKLK4 under mildly acidic or under physiologic conditions, and enzyme activity was analyzed by zymography. The catalytic domain of rhMMP20 was incubated with pKLK4 or recombinant human KLK4 (rhKLK4) and the digestion products were characterized by zymography and Edman degradation. Both pMMP20 and rhMMP20 activated rh-proKLK4 by cleaving at the propeptide-enzyme junction used in vivo. The pMMP20 was inactivated by pKLK4 under physiologic conditions, but not under mildly acidic conditions. Both pKLK4 and rhKLK4 cleaved MMP20 principally at two sites in the catalytic domain of MMP20. MMP20 activates proKLK4 and KLK4 inactivates MMP20 in vitro, and these actions are likely to occur during enamel formation in vivo.
    Archives of oral biology 11/2013; 58(11):1569-1577. DOI:10.1016/j.archoralbio.2013.08.005 · 1.88 Impact Factor
  • M K Pugach · C Suggs · Y Li · J T Wright · A B Kulkarni · J D Bartlett · C W Gibson
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    ABSTRACT: Amelogenin (AMELX) and matrix metalloproteinase-20 (MMP20) are essential for proper enamel development. Amelx and Mmp20 mutations cause amelogenesis imperfecta. MMP20, a protease secreted by ameloblasts, is responsible for processing enamel proteins, including AMELX, during the secretory stage of enamel formation. Of at least 16 different amelogenin splice products, the most abundant isoform found in murine ameloblasts and developing enamel is the M180 protein. To understand the role of MMP20 processing of M180 AMELX, we generated AmelxKO/Mmp20KO (DKO) mice with an amelogenin (M180Tg) transgene. We analyzed the enamel phenotype by SEM to determine enamel structure and thickness, µCT, and by nanoindentation to quantify enamel mechanical properties. M180Tg/DKO mouse enamel had 37% of the hardness of M180Tg/AmelxKO teeth and demonstrated a complete lack of normal prismatic architecture. Although molar enamel of M180Tg/AmelxKO mice was thinner than WT, it had similar mechanical properties and decussating enamel prisms, which were abolished by the loss of MMP20 in the M180Tg/DKO mice. Retention of the C-terminus or complete lack of this domain is unable to rescue amelogenin null enamel. We conclude that among amelogenins, M180 alone is sufficient for normal enamel mechanical properties and prism patterns, but that additional amelogenin splice products are required to restore enamel thickness.
    Journal of dental research 09/2013; 92(12). DOI:10.1177/0022034513506444 · 4.14 Impact Factor
  • X Guan · J D Bartlett
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    ABSTRACT: Matrix metalloproteinase-20 (enamelysin, MMP20) is essential for dental enamel development. Seven different MMP20 mutations in humans cause non-syndromic enamel malformations, termed amelogenesis imperfecta, and ablation of Mmp20 in mice results in thin brittle enamel with a dysplastic rod pattern. Healthy enamel formation requires the sliding movement of ameloblasts in rows during the secretory stage of development. This is essential for formation of the characteristic decussating enamel rod pattern observed in rodents, and this is also when MMP20 is secreted into the enamel matrix. Therefore, we propose that MMP20 facilitates ameloblast movement by cleaving ameloblast cell-cell contacts. Here we show that MMP20 cleaves the extracellular domains of the E- and N-cadherin adherens junction proteins, that both E- and N-cadherin transcripts are expressed at significantly higher levels in Mmp20 null vs. wild-type (WT) mice, and that in Mmp20 ablated mice, high-level ameloblast N-cadherin expression persists during the maturation stage of development. Furthermore, we show that E-cadherin gene expression is down-regulated from the pre-secretory to the secretory stage, while N-cadherin levels are up-regulated. This E- to N-cadherin switch supports epithelial migration in other tissues and may be an important event necessary for the ameloblasts to start moving in rows that slide by one another.
    Journal of dental research 09/2013; 92(12). DOI:10.1177/0022034513506581 · 4.14 Impact Factor
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    John D Bartlett
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    ABSTRACT: This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These proteins and their respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromic enamel malformations termed amelogenesis imperfecta. Pertinent controversies are addressed. For example, do any of these proteins have a critical function in addition to their role in enamel development? Does amelogenin initiate crystallite growth, does it inhibit crystallite growth in width and thickness, or does it do neither? Detailed examination of the null mouse literature provides unmistakable clues and/or answers to these questions, and this data is thoroughly analyzed. Striking conclusions from this analysis reveal that widely held paradigms of enamel formation are inadequate. The final section of this review weaves the recent data into a plausible new mechanism by which these enamel matrix proteins support and promote enamel development.
    09/2013; 2013:684607. DOI:10.1155/2013/684607
  • X. GUAN · J.D. BARTLETT
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    ABSTRACT: Amelogenesis Imperfecta (AI) is an inherited enamel development disorder with thin, soft, rough, and/or pigmented enamel. Four homozygous mutations of the human matrix metalloproteinase-20 gene (MMP20) were shown to cause AI, and knockout of Mmp20in mice resulted in thin brittle enamel with a dysplastic rod pattern. The formation of healthy enamel requires sliding movement of ameloblasts during the secretory stage of enamel development which results in a decussating enamel rod pattern, and this is when MMP20 is secreted into the enamel matrix. Therefore, we propose that MMP20 affects cell-cell interaction through hydrolysis of adherens junction proteins to facilitate ameloblast movement. Objective: To identify MMP20 cell surface substrates and to investigate how MMP20 regulates ameloblast cell-cell interaction. Method: In vitro MMP20 assays were performed with protein extracts from ameloblast-lineage cells (ALC) to identify its cadherin substrates. Molars from WT and Mmp20null mouse pups were collected and cadherin levels at different enamel developmental stages were quantified by qPCR and immunohistochemistry. Result: MMP20 cleaves the extracellular domain of adherens junction proteins E- and N-cadherin in vitro. qPCR results show that E-cadherin gene expression level is down-regulated from pre-secretory to secretory stage in mice, while N-cadherin is up-regulated. This E- to N-cadherin switch supports epithelial migration as is observed in normal developmental processes. In addition, Mmp20 null mouse secretory stage ameloblasts express higher levels of both cadherins than WT, which may impair ameloblast movement for proper enamel formation. Immunohistochemistry confirms that both cadherins present in the enamel organ are located in the ameloblast layer, and further demonstrates that they are retained at higher levels through maturation stage in Mmp20null mice. Conclusion: In addition to its essential role in cleaving enamel matrix proteins, MMP20 may also release cell-cell cadherin contacts to facilitate ameloblast movement. Supported by NIDCR grant R01DE016276
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
  • X. GUAN · C.E. TYE · Y.Y. HU · C. DEAGLE · M.L. SIERANT · J. ANTONE · J.P. SIMMER · J.D. BARTLETT
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    ABSTRACT: Mmp20 null mice have a striking enamel phenotype where the enamel is thin, has an abnormal rod pattern, abrades from the dentin and has a deteriorating tooth morphology as enamel development progresses. Objective: We sought to determine if introduction of a transgene expressing Mmp20 would revert, or partially revert, the severe enamel phenotype observed in the Mmp20 null mouse. Methods: An expression construct was designed such that the mouse amelogenin promoter drove expression of the mouse Mmp20 cDNA. This construct was microinjected into the pronuclei of fertilized mouse eggs. The resulting transgenic mice were assessed by qPCR for transgene expression and offspring of founders expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Transgenic and control mice were then assessed for enamel quality. Results: The Mmp20 transgenes brought the enamel back to full thickness. No significant difference in enamel thickness was observed among the low, medium or high expressing Mmp20 transgenes. However, the high and medium expressing Mmp20 transgenes in the Mmp20 null background had significantly harder 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 softer than normal. In contrast, enamel from wild-type mice with the low expressing transgene was of normal hardness. Conclusions: Mmp20 expression levels must be within a specific range for normal enamel development to occur. Achievement of a normally thick enamel layer may occur over a wide range of Mmp20 expression levels, but achievement 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 is essential for formation of a properly hardened enamel layer. Supported by NIDCR grant R01DE016276.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013

Publication Stats

3k Citations
290.27 Total Impact Points

Institutions

  • 2003–2014
    • Harvard University
      • Department of Developmental Biology
      Cambridge, Massachusetts, United States
  • 1999–2014
    • The Forsyth Institute
      • Department of Cytokine Biology
      Cambridge, Massachusetts, United States
  • 2004–2013
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 2011
    • McGill University
      • Department of Anatomy and Cell Biology
      Montréal, Quebec, Canada
  • 2007
    • Tokyo Medical and Dental University
      Edo, Tōkyō, Japan
  • 2006
    • University of Toronto
      Toronto, Ontario, Canada
  • 2002
    • University of Oulu
      • Institute of Dentistry
      Uleoborg, Northern Ostrobothnia, Finland
  • 1998–2001
    • Laval University
      • Faculté de Médecine Dentaire
      Québec, Quebec, Canada
  • 2000
    • National Institutes of Health
      Maryland, United States
  • 1996–2000
    • University of Texas Health Science Center at San Antonio
      • Division of Pediatric Dentistry
      San Antonio, TX, United States