John D Bartlett

The Forsyth Institute, Cambridge, Massachusetts, United States

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Publications (118)297.59 Total impact

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    Maiko Suzuki · Cheryl Bandoski · John D. Bartlett

    Full-text · Dataset · Nov 2015
  • D. Faibish · M. Suzuki · J.D. Bartlett
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    ABSTRACT: Objective: Quantitative real-time PCR (qPCR) is routinely performed for experiments designed to identify the molecular mechanisms involved in the pathogenesis of dental fluorosis. Expression of reference gene(s) is expected to remain unchanged in fluoride-treated cells or in rodents relative to the corresponding untreated controls. The aim of this study was to select optimal reference genes for fluoride experiments performed in vitro and in vivo. Design: Five candidate genes were evaluated: B2m, Eef1a1, Gapdh, Hprt and Tbp. For in vitro experiments, LS8 cells derived from mouse enamel organ were treated with 0, 1, 3 and/or 5mM sodium fluoride (NaF) for 6 or 18h followed by RNA isolation. For in vivo experiments, six-week old rats were treated with 0 or 100ppm fluoride as NaF for six weeks at which time RNA was isolated from enamel organs. RNA from cells and enamel organs were reverse-transcribed and stability of gene expression for the candidate reference genes was evaluated by qPCR in treated versus non-treated samples. Results: The most stably expressed genes in vitro according to geNorm were B2m and Tbp, and according to Normfinder were Hprt and Gapdh. The most stable genes in vivo were Eef1a1 and Gapdh. Expression of Ddit3, a gene previously shown to be induced by fluoride, was demonstrated to be accurately calculated only when using an optimal reference gene. Conclusions: This study identifies suitable reference genes for relative quantification of gene expression by qPCR after fluoride treatment both in cultured cells and in the rodent enamel organ.
    No preview · Article · Nov 2015 · Archives of oral biology
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    Maiko Suzuki · Cheryl Bandoski · John D. Bartlett
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    ABSTRACT: Fluoride is an effective caries prophylactic, but at high doses can also be an environmental health hazard. Acute or chronic exposure to high fluoride doses can result in dental enamel and skeletal and soft tissue fluorosis. Dental fluorosis is manifested as mottled, discolored, porous enamel that is susceptible to dental caries. Fluoride induces cell stress, including endoplasmic reticulum stress and oxidative stress, which leads to impairment of ameloblasts responsible for dental enamel formation. Recently we reported that fluoride activates SIRT1 and autophagy as an adaptive response to protect cells from stress. However, it still remains unclear how SIRT1/autophagy is regulated in dental fluorosis. In this study, we demonstrate that fluoride exposure generates reactive oxygen species (ROS) and the resulting oxidative damage is counteracted by SIRT1/autophagy induction through JUN-terminal kinase (JNK) signaling in ameloblasts. In the mouse-ameloblast-derived cell line LS8, fluoride induced ROS, mitochondrial damage including cytochrome-c release, up-regulation of UCP2, attenuation of ATP synthesis, and H2AX phosphorylation (γH2AX), which is a marker of DNA damage. We evaluated the effects of the ROS inhibitor N-acetylcysteine (NAC) and the JNK inhibitor SP600125 on fluoride-induced SIRT1/autophagy activation. NAC decreased fluoride-induced ROS generation and attenuated JNK and c-Jun phosphorylation. NAC decreased SIRT1 phosphorylation and formation of the autophagy marker LC3II, which resulted in an increase in the apoptosis mediators γH2AX and cleaved/activated caspase-3. SP600125 attenuated fluoride-induced SIRT1 phosphorylation, indicating that fluoride activates SIRT1/autophagy via the ROS-mediated JNK pathway. In enamel organs from rats or mice treated with 50, 100, or 125ppm fluoride for 6 weeks, cytochrome-c release and the DNA damage markers 8-oxoguanine, p-ATM, and γH2AX were increased compared to those in controls (0ppm fluoride). These results suggest that fluoride-induced ROS generation causes mitochondrial damage and DNA damage, which may lead to impairment of ameloblast function. To counteract this impairment, SIRT1/autophagy is induced via JNK signaling to protect cells/ameloblasts from fluoride-induced oxidative damage that may cause dental fluorosis.
    Full-text · Article · Sep 2015 · Free Radical Biology and Medicine
  • J D Bartlett · J P Simmer

    No preview · Article · May 2015 · Journal of dental research
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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.
    No preview · Article · Oct 2014 · Pediatric dentistry
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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.
    Full-text · Article · Aug 2014 · Connective Tissue Research
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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.
    Full-text · Article · Jul 2014 · Journal of Dental Research
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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.
    Full-text · Article · Jul 2014 · Frontiers in Physiology
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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.
    Full-text · Article · Jul 2014 · PLoS ONE
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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.
    Preview · Article · Jul 2014 · Frontiers in Physiology
  • J.D. BARTLETT

    No preview · Conference Paper · Mar 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.
    No preview · Conference Paper · Mar 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.
    No preview · Conference Paper · Mar 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.
    No preview · Conference Paper · Mar 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.
    No preview · Conference Paper · Mar 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.
    Full-text · Article · Jan 2014 · PLoS ONE
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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.
    Full-text · Article · Nov 2013 · Biochimica et Biophysica Acta
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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.
    No preview · Article · Nov 2013 · Archives of oral biology
  • 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.
    No preview · Article · Sep 2013 · Journal of dental research
  • 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.
    No preview · Article · Sep 2013 · Journal of dental research

Publication Stats

4k Citations
297.59 Total Impact Points

Institutions

  • 1999-2015
    • The Forsyth Institute
      • Department of Cytokine Biology
      Cambridge, Massachusetts, United States
  • 2003-2014
    • Harvard University
      • Department of Developmental Biology
      Cambridge, Massachusetts, United States
  • 2004
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 2002
    • University of Oulu
      • Institute of Dentistry
      Uleoborg, Northern Ostrobothnia, Finland
  • 1996-1998
    • University of Texas Health Science Center at San Antonio
      • Division of Pediatric Dentistry
      San Antonio, Texas, United States