The Experts below are selected from a list of 61959 Experts worldwide ranked by ideXlab platform
John D. Bartlett - One of the best experts on this subject based on the ideXlab platform.
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mmp20 overexpression disrupts molar ameloblast polarity and migration
Journal of Dental Research, 2018Co-Authors: Masashi Shin, M B Chavez, Brian L. Foster, A Ikeda, John D. BartlettAbstract:Ameloblasts responsible for enamel formation express Matrix Metalloproteinase 20 (MMP20), an enzyme that cleaves enamel Matrix proteins, including amelogenin (AMELX) and ameloblastin (AMBN). Previously, we showed that continuously erupting incisors from transgenic mice overexpressing active MMP20 had a massive cell infiltrate present within their enamel space, leading to enamel mineralization defects. However, effects of MMP20 overexpression on mouse molars were not analyzed, although these teeth more accurately represent human odontogenesis. Therefore, MMP20-overexpressing mice ( Mmp20+/+Tg+) were assessed by multiscale analyses, combining several approaches from high-resolution micro-computed tomography to enamel organ immunoblots. During the secretory stage at postnatal day 6 (P6), Mmp20+/+Tg+ mice had a discontinuous ameloblast layer and, unlike incisors, molar P12 maturation stage ameloblasts abnormally migrated away from the enamel layer into the stratum intermedium/stellate reticulum. TOPflash assays performed in vitro demonstrated that MMP20 expression promoted β-catenin nuclear localization and that MMP20 expression promoted invasion through Matrigel-coated filters. However, for both assays, significant differences were eliminated in the presence of the β-catenin inhibitor ICG-001. This suggests that MMP20 activity promotes cell migration via the Wnt pathway. In vivo, the unique molar migration of amelogenin-expressing ameloblasts was associated with abnormal deposition of ectopic calcified nodules surrounding the adherent enamel layer. Enamel content was assessed just prior to eruption at P15. Compared to wild-type, Mmp20+/+Tg+ molars exhibited significant reductions in enamel thickness (70%), volume (60%), and mineral density (40%), and MMP20 overexpression resulted in premature cleavage of AMBN, which likely contributed to the severe defects in enamel mineralization. In addition, Mmp20+/+Tg+ mouse molar enamel organs had increased levels of inactive p-cofilin, a protein that regulates cell polarity. These data demonstrate that increased MMP20 activity in molars causes premature degradation of ameloblastin and inactivation of cofilin, which may contribute to pathological Wnt-mediated cell migration away from the enamel layer.
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mmp20 overexpression disrupts molar ameloblast polarity and migration
Journal of Dental Research, 2018Co-Authors: M. Shin, M B Chavez, Brian L. Foster, A Ikeda, John D. BartlettAbstract:Ameloblasts responsible for enamel formation express Matrix Metalloproteinase 20 (MMP20), an enzyme that cleaves enamel Matrix proteins, including amelogenin (AMELX) and ameloblastin (AMBN). Previously, we showed that continuously erupting incisors from transgenic mice overexpressing active MMP20 had a massive cell infiltrate present within their enamel space, leading to enamel mineralization defects. However, effects of MMP20 overexpression on mouse molars were not analyzed, although these teeth more accurately represent human odontogenesis. Therefore, MMP20-overexpressing mice (Mmp20+/+Tg+) were assessed by multiscale analyses, combining several approaches from high-resolution micro–computed tomography to enamel organ immunoblots. During the secretory stage at postnatal day 6 (P6), Mmp20+/+Tg+ mice had a discontinuous ameloblast layer and, unlike incisors, molar P12 maturation stage ameloblasts abnormally migrated away from the enamel layer into the stratum intermedium/stellate reticulum. TOPflash assay...
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Matrix Metalloproteinase-20 Over-Expression Is Detrimental to Enamel Development: A Mus musculus Model
2016Co-Authors: Masashi Shin, Jerry V. Antone, Xiaomu Guan, Craig C Deagle, James P. Simmer, Coralee E. Tye, Charles E. Smith, John D. BartlettAbstract: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 (Mmp202/2 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
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Murine Matrix Metalloproteinase-20 overexpression stimulates cell invasion into the enamel layer via enhanced Wnt signaling
Scientific Reports, 2016Co-Authors: Masashi Shin, Xiaomu Guan, Maiko Suzuki, John D. BartlettAbstract:Matrix Metalloproteinase-20 (MMP20) is expressed by ameloblasts in developing teeth and MMP20 mutations cause enamel malformation. We established a stably transfected Tet-Off Mmp20-inducible ameloblast-lineage cell line and found that MMP20 expression promoted cell invasion. Previously, we engineered transgenic mice (Tg) that drive Mmp20 expression and showed that Mmp20+/+Tg mice had soft enamel. Here we asked if Mmp20 overexpression disrupts ameloblast function. Incisors from Mmp20+/+ mice expressing the Mmp20 Tg had a striking cell infiltrate which nearly replaced the entire enamel layer. A thin layer of enamel-like material remained over the dentin and at the outer tooth surface, but between these regions were invading fibroblasts and epithelial cells that surrounded ectopic bone-like calcifications. Mmp20+/+Tg mice had decreased enamel organ cadherin levels compared to the Mmp20 ablated and WT mice and, instead of predominantly locating adjacent to the ameloblast cell membrane, β-catenin was predominantly present within the nuclei of invading cells. Our data suggest that increased cadherin cleavage by transgenic MMP20 in the WT background releases excess β-catenin, which translocates to ameloblast nuclei to promote cell migration/invasion. Therefore, we conclude that MMP20 plays a role in normal ameloblast migration through tightly controlled Wnt signaling and that MMP20 overexpression disrupts this process.
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mmp20 klk4 and mmp20 klk4 double null mice define roles for Matrix proteases during dental enamel formation
Molecular Genetics & Genomic Medicine, 2016Co-Authors: Yuanyuan Hu, Amelia S Richardson, Jan C C Hu, John D. Bartlett, James P. SimmerAbstract:Matrix Metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are secreted proteinases that are essential for proper dental enamel formation. We characterized and compared enamel formed in wild-type, Mmp20−/−, Klk4−/−, Mmp20+/−Klk4+/−, and Mmp20−/−Klk4−/− mice using dissecting and light microscopy, backscattered scanning electron microscopy (bSEM), SEM, microcomputed tomography (μCT), and energy-dispersive X-ray analysis (EDX). Following eruption, fractures were observed on Mmp20−/−, Klk4−/−, Mmp20+/−Klk4+/−, and Mmp20−/−Klk4−/− molars. Failure of the enamel in the Mmp20+/−Klk4+/− molars was unexpected and suggested that digenic effects could contribute to the etiology of amelogenesis imperfecta in humans. Micro-CT analyses of hemimandibles demonstrated significantly reduced high-density enamel volume in the Mmp20−/− and Klk4−/− mice relative to the wild-type, which was further reduced in Mmp20−/−Klk4−/− mice. bSEM images of 7-week Mmp20−/− and Mmp20−/−Klk4−/− mandibular incisors showed rough, pitted enamel surfaces with numerous indentations and protruding nodules. The Mmp20+/− and Mmp20+/−Klk4+/− incisors showed prominent, evenly spaced, horizontal ridges that were more distinct in Mmp20+/−Klk4+/− incisors relative to Mmp20+/− incisors due to the darkening of the valleys between the ridges. In cross sections, the Mmp20−/− and Mmp20−/−Klk4−/− exhibited three distinct layers. The outer layer exhibited a disturbed elemental composition and an irregular enamel surface covered with nodules. The Mmp20 null enamel was apparently unable to withstand the sheer forces associated with eruption and separated from dentin during development. Cells invaded the cracks and interposed between the dentin and enamel layers. MMP20 and KLK4 serve overlapping and complementary functions to harden enamel by removing protein, but MMP20 potentially serves multiple additional functions necessary for the adherence of enamel to dentin, the release of intercellular protein stores into the enamel Matrix, the retreat of ameloblasts to facilitate thickening of the enamel layer, and the timely transition of ameloblasts to maturation.
James P. Simmer - One of the best experts on this subject based on the ideXlab platform.
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mmp20 proteolysis of native amelogenin regulates mineralization in vitro
Journal of Dental Research, 2016Co-Authors: Yasuo Yamakoshi, S. Y. Kwak, Henry C. Margolis, James P. SimmerAbstract:Recent studies have shown that native phosphorylated full-length porcine amelogenin (P173) and its predominant cleavage product (P148) can inhibit spontaneous calcium phosphate formation in vitro by stabilizing an amorphous calcium phosphate (ACP) precursor phase. Since full-length amelogenin undergoes proteolysis by Matrix Metalloproteinase 20 (MMP20, enamelysin) soon after secretion, the present study was conducted to assess the effect of amelogenin proteolysis on calcium phosphate formation. Calcium and phosphate were sequentially added to protein solutions without and with added MMP20 (ratio = 200:1) under physiological-like conditions of ionic strength (163 mM) in 50 mM Tris-HCl (pH 7.4) at 37 °C. Protein degradation with time was assessed by gel-electrophoresis, and mineral products formed were characterized by transmission electron microscopy (TEM). MMP20 was found to cleave P173 to primarily generate P148, along with P162, P46-148, and P63/64-148. In sharp contrast, MMP20 did not cleave P148. In a...
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Matrix Metalloproteinase-20 Over-Expression Is Detrimental to Enamel Development: A Mus musculus Model
2016Co-Authors: Masashi Shin, Jerry V. Antone, Xiaomu Guan, Craig C Deagle, James P. Simmer, Coralee E. Tye, Charles E. Smith, John D. BartlettAbstract: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 (Mmp202/2 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
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mmp20 klk4 and mmp20 klk4 double null mice define roles for Matrix proteases during dental enamel formation
Molecular Genetics & Genomic Medicine, 2016Co-Authors: Yuanyuan Hu, Amelia S Richardson, Jan C C Hu, John D. Bartlett, James P. SimmerAbstract:Matrix Metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are secreted proteinases that are essential for proper dental enamel formation. We characterized and compared enamel formed in wild-type, Mmp20−/−, Klk4−/−, Mmp20+/−Klk4+/−, and Mmp20−/−Klk4−/− mice using dissecting and light microscopy, backscattered scanning electron microscopy (bSEM), SEM, microcomputed tomography (μCT), and energy-dispersive X-ray analysis (EDX). Following eruption, fractures were observed on Mmp20−/−, Klk4−/−, Mmp20+/−Klk4+/−, and Mmp20−/−Klk4−/− molars. Failure of the enamel in the Mmp20+/−Klk4+/− molars was unexpected and suggested that digenic effects could contribute to the etiology of amelogenesis imperfecta in humans. Micro-CT analyses of hemimandibles demonstrated significantly reduced high-density enamel volume in the Mmp20−/− and Klk4−/− mice relative to the wild-type, which was further reduced in Mmp20−/−Klk4−/− mice. bSEM images of 7-week Mmp20−/− and Mmp20−/−Klk4−/− mandibular incisors showed rough, pitted enamel surfaces with numerous indentations and protruding nodules. The Mmp20+/− and Mmp20+/−Klk4+/− incisors showed prominent, evenly spaced, horizontal ridges that were more distinct in Mmp20+/−Klk4+/− incisors relative to Mmp20+/− incisors due to the darkening of the valleys between the ridges. In cross sections, the Mmp20−/− and Mmp20−/−Klk4−/− exhibited three distinct layers. The outer layer exhibited a disturbed elemental composition and an irregular enamel surface covered with nodules. The Mmp20 null enamel was apparently unable to withstand the sheer forces associated with eruption and separated from dentin during development. Cells invaded the cracks and interposed between the dentin and enamel layers. MMP20 and KLK4 serve overlapping and complementary functions to harden enamel by removing protein, but MMP20 potentially serves multiple additional functions necessary for the adherence of enamel to dentin, the release of intercellular protein stores into the enamel Matrix, the retreat of ameloblasts to facilitate thickening of the enamel layer, and the timely transition of ameloblasts to maturation.
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fluoride affects enamel protein content via tgf β1 mediated klk4 inhibition
Journal of Dental Research, 2014Co-Authors: Maiko Suzuki, Masashi Shin, James P. Simmer, John D. BartlettAbstract: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.
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Matrix Metalloproteinase 20 over expression is detrimental to enamel development a mus musculus model
PLOS ONE, 2014Co-Authors: Masashi Shin, Jerry V. Antone, Yuanyuan Hu, Xiaomu Guan, Craig C Deagle, James P. Simmer, John D. BartlettAbstract: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−/− 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.
Kalu U.e. Ogbureke - One of the best experts on this subject based on the ideXlab platform.
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effects of dspp and mmp20 silencing on adhesion metastasis angiogenesis and epithelial mesenchymal transition proteins in oral squamous cell carcinoma cells
International Journal of Molecular Sciences, 2020Co-Authors: Jaya Aseervatham, Kalu U.e. OgburekeAbstract:Recent reports highlight the potential tumorigenic role of Dentin Sialophosphoprotein (DSPP) and its cognate partner Matrix Metalloproteinase 20 (MMP-20) in Oral Squamous Cell Carcinomas (OSCCs). However, the function/mechanism of these roles is yet to be fully established. The present study aimed to investigate the effects of DSPP and MMP20 silencing on specific proteins involved in oral cancer cell adhesion, angiogenesis, metastasis, and epithelial-mesenchymal transition (EMT). Stable lines of DSPP/MMP20 silenced OSCC cell line (OSC2), previously established via lentiviral-mediated shRNA transduction, were analyzed for the effects of DSPP, MMP20, and combined DSPP-MMP20 silencing on MMP2, MMP9, integrins αvβ3 and αvβ6, VEGF, Kallikerin- 4,-5,-8,-10, E-cadherin, N-cadherin, Vimentin, met, src, snail, and Twist by Western blot. Results show a significant decrease (p < 0.05) in the expression of MMP2, MMP9, integrin αvβ3, αvβ6, VEGF, Kallikerins -4, -5, -8, -10, N-cadherin, vimentin met, src, snail and twist following DSPP and MMP20 silencing, individually and in combination. On the other hand, the expression of E-cadherin was found to be significantly increased (p < 0.05). These results suggest that the tumorigenic effect of DSPP and MMP20 on OSC2 cells is mediated via the upregulation of the genes involved in invasion, metastasis, angiogenesis, and epithelial-mesenchymal transition (EMT).
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survey of dentin sialophosphoprotein and its cognate Matrix Metalloproteinase 20 in human cancers
Cancer Medicine, 2019Co-Authors: Jaya Aseervatham, Komal Koli, Saxena Geetu, C C Anunobi, Kalu U.e. OgburekeAbstract:Background Matrix Metalloproteinases-20 (MMP20) expression is widely regarded as tooth specific, with expression limited to dental hard tissues. Recently, we reported MMP20 expression and interaction with dentin sialophosphoprotein (DSPP), a member of the Small Integrin Binding Ligand N-linked Glycoproteins (SIBLINGs), in human oral squamous cell carcinoma (OSCC) and dysplastic oral premalignant lesions (OPLs), suggesting a role for MMP20-DSPP interaction in oral carcinogenesis. Methods This study aimed to survey the expression of MMP20 and its cognate DSPP partner in the breast, colon, prostate, thyroid, and cervical neoplasms. Using commercially available tissue microarrays (TMAs) and cell lines, we performed immunohistochemistry, immunofluorescence, proximity ligation assay, and western blot experiments to determine the expressions of MMP20 and DSPP in the breast, colon, prostate, thyroid, cervical neoplasms, and their normal counterparts. Results Significantly high expression levels of MMP20 and DSPP were observed in the malignant breast, colon, prostate, thyroid, and cervical neoplasms compared with their benign and normal counterparts. Furthermore, MMP20 levels increased with advanced stages of colon and thyroid cancers. DSPP expression increased significantly with tumor stage in all cancers examined. Conclusions The co-localization and potential MMP20-DSPP interaction previously reported in oral cancers are present in other cancers. These results suggest MMP20-DSPP pairing as a potential marker of disease activity in some epithelial cancers with diagnostic and prognostic implications.
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Survey of dentin sialophosphoprotein and its cognate Matrix Metalloproteinase‐20 in human cancers
Cancer Medicine, 2019Co-Authors: Jaya Aseervatham, Komal Koli, Saxena Geetu, C C Anunobi, Kalu U.e. OgburekeAbstract:BACKGROUND: Matrix Metalloproteinases-20 (MMP20) expression is widely regarded as tooth specific, with expression limited to dental hard tissues. Recently, we reported MMP20 expression and interaction with dentin sialophosphoprotein (DSPP), a member of the Small Integrin Binding Ligand N-linked Glycoproteins (SIBLINGs), in human oral squamous cell carcinoma (OSCC) and dysplastic oral premalignant lesions (OPLs), suggesting a role for MMP20-DSPP interaction in oral carcinogenesis. METHODS: This study aimed to survey the expression of MMP20 and its cognate DSPP partner in the breast, colon, prostate, thyroid, and cervical neoplasms. Using commercially available tissue microarrays (TMAs) and cell lines, we performed immunohistochemistry, immunofluorescence, proximity ligation assay, and western blot experiments to determine the expressions of MMP20 and DSPP in the breast, colon, prostate, thyroid, cervical neoplasms, and their normal counterparts. RESULTS: Significantly high expression levels of MMP20 and DSPP were observed in the malignant breast, colon, prostate, thyroid, and cervical neoplasms compared with their benign and normal counterparts. Furthermore, MMP20 levels increased with advanced stages of colon and thyroid cancers. DSPP expression increased significantly with tumor stage in all cancers examined. CONCLUSIONS: The co-localization and potential MMP20-DSPP interaction previously reported in oral cancers are present in other cancers. These results suggest MMP20-DSPP pairing as a potential marker of disease activity in some epithelial cancers with diagnostic and prognostic implications.
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Matrix Metalloproteinase 20 co expression with dentin sialophosphoprotein in human and monkey kidneys
Journal of Histochemistry and Cytochemistry, 2016Co-Authors: Kalu U.e. Ogbureke, Komal Koli, Geetu SaxenaAbstract:We recently reported the expression of Matrix Metalloproteinase 20 (MMP20), hitherto thought to be tooth specific, in the metabolically active ductal epithelial cells of human salivary glands. Furthermore, our report indicated that MMP20 co-expressed and potentially interacts with dentin sialophosphoprotein (DSPP), a member of the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Our earlier reports have shown the co-expression of three MMPs, MMP2, MMP3, and MMP9, with specific members of the SIBLING family: bone sialoprotein, osteopontin, and dentin Matrix protein 1, respectively. This study investigated the expression of MMP20 and verified its co-expression with DSPP in human and monkey kidney sections and human mixed renal cells by IHC, in situ proximity ligation assay, and immunofluorescence. Our results show that MMP20 is expressed in all segments of the human and monkey nephron with marked intensity in the proximal and distal tubules, and was absent in the glomeruli. Furthermore, MMP20 co-expressed with DSPP in the proximal, distal, and collecting tubules, and in mixed renal cells. Consistent with other SIBLING-MMP pairs, the DSPP-MMP20 pair may play a role in the normal turnover of cell surface proteins and/or repair of pericellular Matrix proteins of the basement membranes in the metabolically active duct epithelial system of the nephrons.
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expression of Matrix Metalloproteinase mmp 20 and potential interaction with dentin sialophosphoprotein dspp in human major salivary glands
Journal of Histochemistry and Cytochemistry, 2015Co-Authors: Komal Koli, Geetu Saxena, Kalu U.e. OgburekeAbstract:Matrix Metalloproteinase-20 (MMP-20) expression is widely regarded as tooth-specific, with expression limited to dental hard tissues. Necessary for sound enamel formation, MMP-20 and MMP-2 proteolytically process dentin sialophosphoprotein (DSPP) into dentin sialoprotein, dentin phosphoprotein, and dentin glycoprotein during tooth formation. In the mid-2000s, three members of the small integrin-binding ligand N-linked glycoproteins (SIBLINGs) were reported to bind specifically with high affinity (nM) to, and activate, three MMPs in vitro: bone sialoprotein with MMP-2; osteopontin with MMP-3; and dentin Matrix protein1 with MMP-9. The SIBLING-MMP interaction was confirmed in biological systems such as the ducts of salivary glands, where all five members of the SIBLINGs are expressed. Recently, we documented MMP-20 expression and interaction with DSPP (another member of the SIBLING family) in human oral squamous cell carcinoma. Here we report the expression of MMP-20, and confirm its co-expression and potential interaction with DSPP in human major salivary gland tissues and cell line using immunohistochemistry, immunofluorescence, western blot, quantitative RT-PCR, and proximity ligation assay. This report reinforces our earlier suggestion that the SIBLING-MMP complexes may be involved in the turnover of extracellular proteins damaged by oxidation byproducts in metabolically active duct epithelial systems.
Yasuo Yamakoshi - One of the best experts on this subject based on the ideXlab platform.
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TGF-β1 autocrine signalling and enamel Matrix components.
Scientific Reports, 2016Co-Authors: Saeko Kobayashi-kinoshita, Yasuo Yamakoshi, Ryuji Yamamoto, Kazuo Onuma, Yoshinobu AsadaAbstract:Transforming growth factor-β1 (TGF-β1) is present in porcine enamel extracts and is critical for proper mineralization of tooth enamel. Here, we show that the mRNA of latent TGF-β1 is expressed throughout amelogenesis. Latent TGF-β1 is activated by Matrix Metalloproteinase 20 (MMP20), coinciding with amelogenin processing by the same proteinase. Activated TGF-β1 binds to the major amelogenin cleavage products, particularly the neutral-soluble P103 amelogenin, to maintain its activity. The P103 amelogenin-TGF-β1 complex binds to TGFBR1 to induce TGF-β1 signalling. The P103 amelogenin-TGF-β1 complex is slowly cleaved by kallikrein 4 (KLK4), which is secreted into the transition- and maturation-stage enamel Matrix, thereby reducing TGF-β1 activity. To exert the multiple biological functions of TGF-β1 for amelogenesis, we propose that TGF-β1 is activated or inactivated by MMP20 or KLK4 and that the amelogenin cleavage product is necessary for the in-solution mobility of TGF-β1, which is necessary for binding to its receptor on ameloblasts and retention of its activity.
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mmp20 proteolysis of native amelogenin regulates mineralization in vitro
Journal of Dental Research, 2016Co-Authors: Yasuo Yamakoshi, S. Y. Kwak, Henry C. Margolis, James P. SimmerAbstract:Recent studies have shown that native phosphorylated full-length porcine amelogenin (P173) and its predominant cleavage product (P148) can inhibit spontaneous calcium phosphate formation in vitro by stabilizing an amorphous calcium phosphate (ACP) precursor phase. Since full-length amelogenin undergoes proteolysis by Matrix Metalloproteinase 20 (MMP20, enamelysin) soon after secretion, the present study was conducted to assess the effect of amelogenin proteolysis on calcium phosphate formation. Calcium and phosphate were sequentially added to protein solutions without and with added MMP20 (ratio = 200:1) under physiological-like conditions of ionic strength (163 mM) in 50 mM Tris-HCl (pH 7.4) at 37 °C. Protein degradation with time was assessed by gel-electrophoresis, and mineral products formed were characterized by transmission electron microscopy (TEM). MMP20 was found to cleave P173 to primarily generate P148, along with P162, P46-148, and P63/64-148. In sharp contrast, MMP20 did not cleave P148. In a...
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mmp20 proteolysis of native amelogenin regulates mineralization in vitro
Journal of Dental Research, 2016Co-Authors: Yasuo Yamakoshi, S. Y. Kwak, Henry C. Margolis, J P SimmerAbstract:Recent studies have shown that native phosphorylated full-length porcine amelogenin (P173) and its predominant cleavage product (P148) can inhibit spontaneous calcium phosphate formation in vitro by stabilizing an amorphous calcium phosphate (ACP) precursor phase. Since full-length amelogenin undergoes proteolysis by Matrix Metalloproteinase 20 (MMP20, enamelysin) soon after secretion, the present study was conducted to assess the effect of amelogenin proteolysis on calcium phosphate formation. Calcium and phosphate were sequentially added to protein solutions without and with added MMP20 (ratio = 200:1) under physiological-like conditions of ionic strength (163 mM) in 50 mM Tris-HCl (pH 7.4) at 37 °C. Protein degradation with time was assessed by gel-electrophoresis, and mineral products formed were characterized by transmission electron microscopy (TEM). MMP20 was found to cleave P173 to primarily generate P148, along with P162, P46-148, and P63/64-148. In sharp contrast, MMP20 did not cleave P148. In addition, the formation of well-aligned bundles of enamel-like hydroxyapatite (HA) crystals was promoted in the presence of P173 with added MMP20, while only ACP particles were seen in the absence of MMP20. Although P148 was found to have a somewhat lower capacity to stabilize ACP and prevent HA formation compared with P173 in the absence of MMP20, essentially no HA formation was observed in the presence of somewhat higher concentrations of P148 regardless of MMP20 addition, due to the lack of observed protein proteolysis. Present findings suggest that ACP transformation to ordered arrays of enamel crystals may be regulated in part by the proteolysis of full-length native amelogenin, while the predominant amelogenin degradation product in developing enamel (e.g., P148) primarily serves to prevent uncontrolled mineral formation during the secretory stage of amelogenesis.
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enamel proteins and proteases in mmp20 and klk4 null and double null mice
European Journal of Oral Sciences, 2011Co-Authors: Yasuo Yamakoshi, Amelia S Richardson, Stephanie M Nunez, Fumiko Yamakoshi, Rachel N Milkovich, Jan C C Hu, John D. Bartlett, James P. SimmerAbstract:Dental enamel forms in two stages. During the secretory stage mineral ribbons lengthen along a mineralization front at the enamel surface (1, 2). By the end of the secretory stage the enamel layer has already reached its final dimensions. In the rat incisor about 9% of the volume and 36% of the weight of the enamel Matrix is mineral, which equals only about 14% of the mineral present at eruption (3). During the maturation stage, crystals initially deposited during the secretory stage grow thicker and wider. By the end of the maturation stage the mineral comprises about 70% of the volume and over 90% of the weight of the enamel Matrix, which is more highly mineralized than other mineralized tissues, such as bone and dentin. Proteins are abundant in secretory-stage and early maturation-stage enamel, but are virtually absent from the late maturation-stage Matrix (4). Proteins secreted during the secretory stage are degraded during both the secretory and maturation stages. Enamel protein cleavage products are reabsorbed by ameloblasts and degraded (4–7). There are two major secreted enamel proteases: Matrix Metalloproteinase 20 (MMP20) (8) and kallikrein-related peptidase 4 (KLK4) (9). These enzymes are necessary for enamel formation, as mutations in MMP20 (10) and KLK4 (11) cause inherited enamel malformations. A major function of enamel proteases is to facilitate the removal of enamel proteins to free up space within the enamel Matrix for the enamel crystallites to grow in width and thickness (12). Several reviews on the roles of proteases in dental enamel formation are available (13–15). Enamel protein cleavage sites have been characterized for proteins that accumulate in secretory-stage pig enamel, and MMP20 is able to catalyze the same amelogenin (16, 17) and ameloblastin (18, 19) cleavages in vitro as occur in vivo. Kallikrein-related peptidase 4 cleaves amelogenin at many sites, but the cleavage pattern is different from that produced by MMP20 (20). The amelogenin C-terminus is highly charged relative to the rest of the protein and increases the affinity of amelogenin for hydroxyapatite (21). Matrix Metalloproteinase 20 removes the amelogenin C-terminus, suggesting that MMP20 may be necessary to dissociate amelogenin from the crystals. However, cleavages by KLK4 on the N-terminal side of amelogenin also decrease amelogenin binding to hydroxyapatite in vitro (22). Mmp20 (23, 24) and Klk4 (25) null mice both have dramatic enamel phenotypes in which the hypomineralized enamel undergoes rapid attrition. The enamel in the Mmp20 null mice breaks off at the dentino–enamel junction (DEJ), while the enamel in the Klk4 null mice breaks just above the DEJ, in the deep enamel (26). Mmp20 null mice cover dentin with a rough mineral layer that is generally thin but irregular, and lacks rod and inter-rod organization (27). The enamel in Klk4 null mice has normal thickness and rod organization and is hard at the surface but is progressively less mineralized with depth (27). The enamel layers of both types of null mice retain enamel proteins, but the state of degradation of these proteins has not been characterized. In this study we analyzed the enamel proteins and proteases in wild-type, Mmp20 null, Klk4 null, and Mmp20/Klk4 double-null mouse maxillary first molars during the secretory stage, the maturation stage, and just prior to tooth eruption.
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Enamel proteins and proteases in Mmp20 and Klk4 null and double-null mice.
European Journal of Oral Sciences, 2011Co-Authors: Yasuo Yamakoshi, Amelia S Richardson, Stephanie M Nunez, Fumiko Yamakoshi, Rachel N Milkovich, John D. Bartlett, James P. SimmerAbstract:Matrix Metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are thought to be necessary to clear proteins from the enamel Matrix of developing teeth. We characterized Mmp20 and Klk4 null mice to better understand their roles in Matrix degradation and removal. Histological examination showed retained organic Matrix in Mmp20, Klk4, and Mmp20/Klk4 double-null mouse enamel Matrix, but not in the wild-type. X-gal histostaining of Mmp20 null mice heterozygous for the Klk4 knockout/lacZ knockin showed that Klk4 is expressed normally in the Mmp20 null background. This finding was corroborated by zymogram and western blotting, which discovered a 40-kDa protease induced in the maturation stage of Mmp20 null mice. Proteins were extracted from secretory-stage or maturation-stage maxillary first molars from wild-type, Mmp20 null, Klk4 null, and Mmp20/Klk4 double-null mice and were analyzed by SDS-PAGE and western blotting. Only intact amelogenins and ameloblastin were observed in secretory-stage enamel of Mmp20 null mice, whereas the secretory-stage Matrix from Klk4 null mice was identical to the Matrix from wild-type mice. More residual Matrix was observed in the double-null mice compared with either of the single-null mice. These results support the importance of MMP20 during the secretory stage and of KLK4 during the maturation stage and show there is only limited functional redundancy for these enzymes.
Masashi Shin - One of the best experts on this subject based on the ideXlab platform.
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mmp20 overexpression disrupts molar ameloblast polarity and migration
Journal of Dental Research, 2018Co-Authors: Masashi Shin, M B Chavez, Brian L. Foster, A Ikeda, John D. BartlettAbstract:Ameloblasts responsible for enamel formation express Matrix Metalloproteinase 20 (MMP20), an enzyme that cleaves enamel Matrix proteins, including amelogenin (AMELX) and ameloblastin (AMBN). Previously, we showed that continuously erupting incisors from transgenic mice overexpressing active MMP20 had a massive cell infiltrate present within their enamel space, leading to enamel mineralization defects. However, effects of MMP20 overexpression on mouse molars were not analyzed, although these teeth more accurately represent human odontogenesis. Therefore, MMP20-overexpressing mice ( Mmp20+/+Tg+) were assessed by multiscale analyses, combining several approaches from high-resolution micro-computed tomography to enamel organ immunoblots. During the secretory stage at postnatal day 6 (P6), Mmp20+/+Tg+ mice had a discontinuous ameloblast layer and, unlike incisors, molar P12 maturation stage ameloblasts abnormally migrated away from the enamel layer into the stratum intermedium/stellate reticulum. TOPflash assays performed in vitro demonstrated that MMP20 expression promoted β-catenin nuclear localization and that MMP20 expression promoted invasion through Matrigel-coated filters. However, for both assays, significant differences were eliminated in the presence of the β-catenin inhibitor ICG-001. This suggests that MMP20 activity promotes cell migration via the Wnt pathway. In vivo, the unique molar migration of amelogenin-expressing ameloblasts was associated with abnormal deposition of ectopic calcified nodules surrounding the adherent enamel layer. Enamel content was assessed just prior to eruption at P15. Compared to wild-type, Mmp20+/+Tg+ molars exhibited significant reductions in enamel thickness (70%), volume (60%), and mineral density (40%), and MMP20 overexpression resulted in premature cleavage of AMBN, which likely contributed to the severe defects in enamel mineralization. In addition, Mmp20+/+Tg+ mouse molar enamel organs had increased levels of inactive p-cofilin, a protein that regulates cell polarity. These data demonstrate that increased MMP20 activity in molars causes premature degradation of ameloblastin and inactivation of cofilin, which may contribute to pathological Wnt-mediated cell migration away from the enamel layer.
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Matrix Metalloproteinase-20 Over-Expression Is Detrimental to Enamel Development: A Mus musculus Model
2016Co-Authors: Masashi Shin, Jerry V. Antone, Xiaomu Guan, Craig C Deagle, James P. Simmer, Coralee E. Tye, Charles E. Smith, John D. BartlettAbstract: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 (Mmp202/2 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
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Murine Matrix Metalloproteinase-20 overexpression stimulates cell invasion into the enamel layer via enhanced Wnt signaling
Scientific Reports, 2016Co-Authors: Masashi Shin, Xiaomu Guan, Maiko Suzuki, John D. BartlettAbstract:Matrix Metalloproteinase-20 (MMP20) is expressed by ameloblasts in developing teeth and MMP20 mutations cause enamel malformation. We established a stably transfected Tet-Off Mmp20-inducible ameloblast-lineage cell line and found that MMP20 expression promoted cell invasion. Previously, we engineered transgenic mice (Tg) that drive Mmp20 expression and showed that Mmp20+/+Tg mice had soft enamel. Here we asked if Mmp20 overexpression disrupts ameloblast function. Incisors from Mmp20+/+ mice expressing the Mmp20 Tg had a striking cell infiltrate which nearly replaced the entire enamel layer. A thin layer of enamel-like material remained over the dentin and at the outer tooth surface, but between these regions were invading fibroblasts and epithelial cells that surrounded ectopic bone-like calcifications. Mmp20+/+Tg mice had decreased enamel organ cadherin levels compared to the Mmp20 ablated and WT mice and, instead of predominantly locating adjacent to the ameloblast cell membrane, β-catenin was predominantly present within the nuclei of invading cells. Our data suggest that increased cadherin cleavage by transgenic MMP20 in the WT background releases excess β-catenin, which translocates to ameloblast nuclei to promote cell migration/invasion. Therefore, we conclude that MMP20 plays a role in normal ameloblast migration through tightly controlled Wnt signaling and that MMP20 overexpression disrupts this process.
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fluoride affects enamel protein content via tgf β1 mediated klk4 inhibition
Journal of Dental Research, 2014Co-Authors: Maiko Suzuki, Masashi Shin, James P. Simmer, John D. BartlettAbstract: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.
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Matrix Metalloproteinase 20 over expression is detrimental to enamel development a mus musculus model
PLOS ONE, 2014Co-Authors: Masashi Shin, Jerry V. Antone, Yuanyuan Hu, Xiaomu Guan, Craig C Deagle, James P. Simmer, John D. BartlettAbstract: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−/− 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.