The Experts below are selected from a list of 537 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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Enamel proteins and proteases in MMP20 and Klk4 null and double-null mice.
European journal of oral sciences, 2020Co-Authors: Yasuo Yamakoshi, Amelia S Richardson, John D. Bartlett, Jan C.-c. Hu, Stephanie M Nunez, Fumiko Yamakoshi, Rachel N Milkovich, 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.
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Kallikrein-related peptidase 4, matrix metalloproteinase 20, and the maturation of murine and porcine enamel.
European journal of oral sciences, 2020Co-Authors: Yuanyuan Hu, John D. Bartlett, Jan C.-c. Hu, Charles E. Smith, James P. SimmerAbstract:The crowns of matrix metalloproteinase 20 (MMP20) null mice fracture at the dentino-enamel junction (DEJ), whereas the crowns of kallikrein-related peptidase 4 (Klk4) null mice fracture in the deep enamel just above the DEJ. We used backscatter scanning electron microscopy to assess enamel mineralization in incisors from 9-wk-old wild-type, Klk4 null, and MMP20 null mice, and in developing pig molars. We observed a line of hypermineralization along the DEJ in developing wild-type mouse and pig teeth. This line was discernible from the early secretory stage until the enamel in the maturation stage reached a similar density. The line was apparent in Klk4 null mice, but absent in MMP20 null mice. Enamel in the Klk4 null mice matured normally at the surface, but was progressively less mineralized with depth. Enamel in the MMP20 null mice formed as a mineral bilayer, with neither layer looking like true enamel. The most superficial mineral layer expanded during the maturation stage and formed irregular surface nodules. A surprising finding was the observation of electron backscatter from mid-maturation wild-type ameloblasts, which we attributed to the accumulation and release of iron. We conclude that enamel breaks in the deep enamel of Klk4 null mice because of decreasing enamel maturation with depth, and at the DEJ in MMP20 null mice because of hypomineralization at the DEJ.
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Matrix Metalloproteinase-20 and Ameloblast Cell Movement in Rows
Interface Oral Health Science 2011, 2020Co-Authors: John D. BartlettAbstract:Ameloblasts move in rows that slide by one another to form the decussating enamel rod pattern characteristic of rodent teeth. Each rod is formed by one ameloblast and each rod preserves a complete record of the migratory path of the ameloblast that formed it. MMP20 null mouse enamel has at best a grossly malformed rod pattern and the maturation stage ameloblasts of the enamel organ overlap and grow atop one another. This suggests that ameloblast cell movement is restrained and that cell–cell attachment is amplified in the MMP20 null mouse enamel organ. Cadherins are a family of proteins that span the ameloblast cell membrane mediating attachment to identical cadherins present on adjacent ameloblasts. Herein we postulate that MMP20 cleaves the extracellular domains of ameloblast cadherins to allow ameloblasts to move synchronously in rows to form the characteristic and complex decussating enamel rod patterns.
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MMP20 overexpression disrupts molar ameloblast polarity and migration
Journal of Dental Research, 2018Co-Authors: M Shin, M B Chavez, A Ikeda, Brian L Foster, 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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Murine matrix metalloproteinase-20 overexpression stimulates cell invasion into the enamel layer via enhanced Wnt signaling
Scientific Reports, 2016Co-Authors: Masashi Shin, Charles E. Smith, Maiko Suzuki, Xiaomu Guan, 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.
Henry C. Margolis - One of the best experts on this subject based on the ideXlab platform.
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proteolysis by MMP20 prevents aberrant mineralization in secretory enamel
Journal of Dental Research, 2019Co-Authors: Hajime Yamazaki, Henry C. Margolis, S Y Kwak, B Tran, Elia BeniashAbstract:The present study was conducted to investigate the role of proteolysis by matrix metalloproteinase 20 (MMP20) in regulating the initial formation of the enamel mineral structure during the secretor...
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Potential function of TGF-β isoforms in maturation-stage ameloblasts.
Journal of Oral Biosciences, 2019Co-Authors: Miu Okubo, Takahiko Niwa, Hajime Yamazaki, Takeo Karakida, Risako Chiba, Takatoshi Nagano, Ryuji Yamamoto, Henry C. Margolis, Saeko Kobayashi, Yasuo YamakoshiAbstract:Abstract Objectives To investigate potential functions of transforming growth factor-beta (TGF-β) isoforms in maturation-stage ameloblasts during amelogenesis. Methods In vivo activation of TGF-β was characterized by using matrix metalloproteinase 20 null (MMP20-/-) and wild-type (MMP20+/+) mice. Using mHAT9d cells cultured in the presence of each TGF-β isoform, (1) cell proliferation was determined by MTS assay, (2) immunostaining with anti-cleaved caspase-3 monoclonal antibody was performed and apoptotic indices were measured, (3) gene expression was analyzed by RT-qPCR, and (4) the uptake of amelogenin into mHAT9d cells was directly observed using a fluorescence microscope. Results TGF-β1 and TGF-β3 were present in the enamel matrix of developing teeth which were activated by MMP20 in vivo. A genetic study revealed that the three TGF-β isoforms upregulate kallikrein 4 (KLK4) mRNA levels but downregulate carbonic anhydrase II. Moreover, TGF-β1 and TGF-β2 significantly upregulated the mRNA level of amelotin, whereas TGF-β3 dramatically downregulated the mRNA levels of odontogenic ameloblast-associated protein (ODAM), family with sequence similarity 83 member H (FAM83H), and alkaline phosphatase (ALP). Immunostaining analysis showed that the apoptosis of mHAT9d cells is induced by three TGF-β isoforms, with TGF-β3 being most effective. Both TGF-β1 and TGF-β3 induced endocytosis of amelogenin. Conclusions We propose that TGF-β is regulated in an isoform-specific manner to perform multiple biological functions such as gene expression related to the structure of basal lamina/ameloblasts, mineral ion transport, apoptosis, and endocytosis in maturation-stage ameloblasts.
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The dynamics of TGF-β in dental pulp, odontoblasts and dentin.
Scientific Reports, 2018Co-Authors: Takahiko Niwa, Jan C.-c. Hu, James P. Simmer, Yasuo Yamakoshi, Hajime Yamazaki, Takeo Karakida, Risako Chiba, Takatoshi Nagano, Ryuji Yamamoto, Henry C. MargolisAbstract:Transforming growth factor-beta (TGF-β) is critical for cell proliferation and differentiation in dental pulp. Here, we show the dynamic mechanisms of TGF-β in porcine dental pulp, odontoblasts and dentin. The mRNA of latent TGF-β1 and TGF-β3 is predominantly expressed in odontoblasts, whereas the mRNA expression level of latent TGF-β2 is high in dental pulp. TGF-β1 is a major isoform of TGF-β, and latent TGF-β1, synthesized in dental pulp, is primarily activated by matrix metalloproteinase 11 (MMP11). Activated TGF-β1 enhances the mRNA expression levels of MMP20 and full-length dentin sialophosphoprotein (DSPP) in dental pulp cells, coinciding with the induction of odontoblast differentiation. Latent TGF-β1 synthesized in odontoblasts is primarily activated by MMP2 and MMP20 in both odontoblasts and dentin. The activity level of TGF-β1 was reduced in the dentin of MMP20 null mice, although the amount of latent TGF-β1 expression did not change between wild-type and MMP20 null mice. TGF-β1 activity was reduced with the degradation of DSPP-derived proteins that occurs with ageing. We propose that to exert its multiple biological functions, TGF-β1 is involved in a complicated dynamic interaction with matrix metalloproteinases (MMPs) and/or DSPP-derived proteins present in dental pulp, odontoblasts and dentin.
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MMP20 proteolysis of native amelogenin regulates mineralization in vitro
Journal of Dental Research, 2016Co-Authors: James P. Simmer, Yasuo Yamakoshi, Henry C. Margolis, S Y KwakAbstract: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...
Charles E. Smith - One of the best experts on this subject based on the ideXlab platform.
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Kallikrein-related peptidase 4, matrix metalloproteinase 20, and the maturation of murine and porcine enamel.
European journal of oral sciences, 2020Co-Authors: Yuanyuan Hu, John D. Bartlett, Jan C.-c. Hu, Charles E. Smith, James P. SimmerAbstract:The crowns of matrix metalloproteinase 20 (MMP20) null mice fracture at the dentino-enamel junction (DEJ), whereas the crowns of kallikrein-related peptidase 4 (Klk4) null mice fracture in the deep enamel just above the DEJ. We used backscatter scanning electron microscopy to assess enamel mineralization in incisors from 9-wk-old wild-type, Klk4 null, and MMP20 null mice, and in developing pig molars. We observed a line of hypermineralization along the DEJ in developing wild-type mouse and pig teeth. This line was discernible from the early secretory stage until the enamel in the maturation stage reached a similar density. The line was apparent in Klk4 null mice, but absent in MMP20 null mice. Enamel in the Klk4 null mice matured normally at the surface, but was progressively less mineralized with depth. Enamel in the MMP20 null mice formed as a mineral bilayer, with neither layer looking like true enamel. The most superficial mineral layer expanded during the maturation stage and formed irregular surface nodules. A surprising finding was the observation of electron backscatter from mid-maturation wild-type ameloblasts, which we attributed to the accumulation and release of iron. We conclude that enamel breaks in the deep enamel of Klk4 null mice because of decreasing enamel maturation with depth, and at the DEJ in MMP20 null mice because of hypomineralization at the DEJ.
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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, Charles E. Smith, Maiko Suzuki, Xiaomu Guan, 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, John D. Bartlett, Jan C.-c. Hu, Charles E. Smith, 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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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, JOSHUA D BARTLETT, Amelia S Richardson, Jan C.-c. Hu, Charles E. Smith, 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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matrix metalloproteinase 20 over expression is detrimental to enamel development a mus musculus model
PLOS ONE, 2014Co-Authors: Masashi Shin, Jerry V. Antone, James P. Simmer, Charles E. Smith, Yuanyuan Hu, Xiaomu Guan, Craig C Deagle, 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.
James P. Simmer - One of the best experts on this subject based on the ideXlab platform.
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Proteomics and genetics of dental enamel.
Cells tissues organs, 2020Co-Authors: Jan C.-c. Hu, Yasuo Yamakoshi, Fumiko Yamakoshi, Paul H Krebsbach, James P. SimmerAbstract:The initiation of enamel crystals at the dentino-enamel junction is associated with the expression of dentin sialophosphoprotein (DSPP, a gene normally linked with dentin formation), three 'structural' enamel proteins--amelogenin (AMELX), enamelin (ENAM), and ameloblastin (AMBN)--and a matrix metalloproteinase, enamelysin (MMP20). Enamel formation proceeds with the steady elongation of the enamel crystals at a mineralization front just beneath the ameloblast distal membrane, where these proteins are secreted. As the crystal ribbons lengthen, enamelysin processes the secreted proteins. Some of the cleavage products accumulate in the matrix, others are reabsorbed back into the ameloblast. Once crystal elongation is complete and the enamel layer reaches its final thickness, kallikrein 4 (KLK4) facilitates the breakdown and reabsorption of accumulated enamel matrix proteins. The importance of the extracellular matrix proteins to proper tooth development is best illustrated by the dramatic dental phenotypes observed in the targeted knockouts of enamel matrix genes in mice (Dspp, Amelx, Ambn, MMP20) and in human kindreds with defined mutations in the genes (DSPP, AMELX, ENAM, MMP20, KLK4) encoding these matrix proteins. However, ablation studies alone cannot give specific mechanistic information on how enamel matrix proteins combine to catalyze the formation of enamel crystals. The best approach for determining the molecular mechanism of dental enamel formation is to reconstitute the matrix and synthesize enamel crystals in vitro. Here, we report refinements to the procedures used to isolate porcine enamel and dentin proteins, recent advances in the characterization of enamel matrix protein posttranslational modifications, and summarize the results of human genetic studies that associate specific mutations in the genes encoding matrix proteins with a range of dental phenotypes.
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Kallikrein-related peptidase 4, matrix metalloproteinase 20, and the maturation of murine and porcine enamel.
European journal of oral sciences, 2020Co-Authors: Yuanyuan Hu, John D. Bartlett, Jan C.-c. Hu, Charles E. Smith, James P. SimmerAbstract:The crowns of matrix metalloproteinase 20 (MMP20) null mice fracture at the dentino-enamel junction (DEJ), whereas the crowns of kallikrein-related peptidase 4 (Klk4) null mice fracture in the deep enamel just above the DEJ. We used backscatter scanning electron microscopy to assess enamel mineralization in incisors from 9-wk-old wild-type, Klk4 null, and MMP20 null mice, and in developing pig molars. We observed a line of hypermineralization along the DEJ in developing wild-type mouse and pig teeth. This line was discernible from the early secretory stage until the enamel in the maturation stage reached a similar density. The line was apparent in Klk4 null mice, but absent in MMP20 null mice. Enamel in the Klk4 null mice matured normally at the surface, but was progressively less mineralized with depth. Enamel in the MMP20 null mice formed as a mineral bilayer, with neither layer looking like true enamel. The most superficial mineral layer expanded during the maturation stage and formed irregular surface nodules. A surprising finding was the observation of electron backscatter from mid-maturation wild-type ameloblasts, which we attributed to the accumulation and release of iron. We conclude that enamel breaks in the deep enamel of Klk4 null mice because of decreasing enamel maturation with depth, and at the DEJ in MMP20 null mice because of hypomineralization at the DEJ.
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Enamel proteins and proteases in MMP20 and Klk4 null and double-null mice.
European journal of oral sciences, 2020Co-Authors: Yasuo Yamakoshi, Amelia S Richardson, John D. Bartlett, Jan C.-c. Hu, Stephanie M Nunez, Fumiko Yamakoshi, Rachel N Milkovich, 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.
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The dynamics of TGF-β in dental pulp, odontoblasts and dentin.
Scientific Reports, 2018Co-Authors: Takahiko Niwa, Jan C.-c. Hu, James P. Simmer, Yasuo Yamakoshi, Hajime Yamazaki, Takeo Karakida, Risako Chiba, Takatoshi Nagano, Ryuji Yamamoto, Henry C. MargolisAbstract:Transforming growth factor-beta (TGF-β) is critical for cell proliferation and differentiation in dental pulp. Here, we show the dynamic mechanisms of TGF-β in porcine dental pulp, odontoblasts and dentin. The mRNA of latent TGF-β1 and TGF-β3 is predominantly expressed in odontoblasts, whereas the mRNA expression level of latent TGF-β2 is high in dental pulp. TGF-β1 is a major isoform of TGF-β, and latent TGF-β1, synthesized in dental pulp, is primarily activated by matrix metalloproteinase 11 (MMP11). Activated TGF-β1 enhances the mRNA expression levels of MMP20 and full-length dentin sialophosphoprotein (DSPP) in dental pulp cells, coinciding with the induction of odontoblast differentiation. Latent TGF-β1 synthesized in odontoblasts is primarily activated by MMP2 and MMP20 in both odontoblasts and dentin. The activity level of TGF-β1 was reduced in the dentin of MMP20 null mice, although the amount of latent TGF-β1 expression did not change between wild-type and MMP20 null mice. TGF-β1 activity was reduced with the degradation of DSPP-derived proteins that occurs with ageing. We propose that to exert its multiple biological functions, TGF-β1 is involved in a complicated dynamic interaction with matrix metalloproteinases (MMPs) and/or DSPP-derived proteins present in dental pulp, odontoblasts and dentin.
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analyses of MMP20 missense mutations in two families with hypomaturation amelogenesis imperfecta
Frontiers in Physiology, 2017Co-Authors: Jenny Kang, Jan C.-c. Hu, Figen Seymen, Mine Koruyucu, Koray Gencay, Teo Jeon Shin, Hongkeun Hyun, James P. SimmerAbstract:Amelogenesis imperfecta is a group of rare inherited disorders that affect tooth enamel formation, quantitatively and/or qualitatively. The aim of this study was to identify the genetic etiologies of two families presented with hypomaturation amelogenesis imperfecta. DNA was isolated from peripheral blood samples of participating family members. Whole exome sequencing was performed using DNA samples from the two probands. Sequencing data was aligned to the NCBI human reference genome (NCBI build 37.2, hg19) and sequence variations were annotated with the dbSNP build 138. Mutations in MMP20 gene were identified in both probands. A homozygous missense mutation (c.678T>A; p.His226Gln) was identified in the consanguineous Family 1. Compound heterozygous MMP20 mutations (c.540T>A, p.Tyr180* and c.389C>T, p.Thr130Ile) were identified in the non-consanguineous Family 2. Affected persons in the family 1 showed hypomaturation AI with dark brown discoloration, which is similar to the clinical phenotype in a previous report with the same mutation. However, the dentition of the proband in the family 2 exhibited slight yellowish discoloration with reduced transparency. The functional analysis showed that the p.Thr130Ile mutant protein had reduced activity of MMP20, while there’s no functional MMP20 in the proband of family 1. These results expand the mutational spectrum of the MMP20 and broaden our understanding of genotype-phenotype correlations in amelogenesis imperfecta.
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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INTERFERON GAMMA (IFNΓ) ANTITUMOR EFFECTS ON ORAL CANCER CELLS ARE ACCOMPANIED BY ER STRESS RESPONSE MODULATION AND DSPP ACTIVITY SUPPRESSION
Oral Surgery Oral Medicine Oral Pathology and Oral Radiology, 2019Co-Authors: Nikolaos G. Nikitakis, Ioannis Gkouveris, Jaya Asservatham, Kalu U.e. OgburekeAbstract:Objectives Expression of proinflammatory cytokines in various malignant neoplasms is widely considered to represent a host immune response to control tumor development. Recently, the role of interferon gamma (IFNγ) in oral squamous cell carcinoma (OSCC) and its relation with endoplasmic reticulum (ER) stress pathways were investigated. Dentin sialophosphoprotein (DSPP) has been involved in malignant transformation, invasion and metastasis of OSCC. The present study examined the effects of IFNγ treatment on ER stress, Unfolded Protein Response (UPR) and calcium homeostasis regulating mechanisms and the potential interaction with DSPP in OSCC cells. Findings Oral cancer OSC2 cells were assessed following IFNγ treatment at specific time-points. DSPP and MMP20 mRNA expression levels, as well as ER stress, UPR and calcium homeostasis-related proteins, including GRP78, SERCA2b, IP3r, PERK and IRE1, were assayed by RT-PCR, while Bcl-2, Bax, PCNA and Cytochrome C protein expression levels were analyzed by Western blot. IFNγ treatment significantly downregulated mRNA levels of major ER stress regulator GRP78, and, to a lesser extent, UPR-related molecule IRE1, but without significant effect on PERK. Furthermore, IFNγ affected the mRNA expression levels of important ER calcium homeostasis molecules, downregulating SERCA2b and upregulating IP3r. Additionally, DSPP and MMP20 mRNA levels were significantly reduced by IFNγ. IFNγ treatment also hampered OSC2 migration (assessed by wound-healing assay), reduced cell viability (evaluated by MTT), and enhanced apoptosis (assayed by Annexin V/FITC flow cytometry). These changes were accompanied by induction of Bax and Cytochrome c and downregulation of PCNA and Bcl-2 protein levels. Conclusions IFNγ appears to inhibit oral cancer cell viability and migration, and drive apoptosis, possibly by regulating ER stress and UPR mechanisms. DSPP and MMP20 downregulation appears to correspond to the IFNγ-induced changes in ER calcium homeostasis in OSCC.
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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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Interferon γ suppresses dentin sialophosphoprotein in oral squamous cell carcinoma cells resulting in antitumor effects, via modulation of the endoplasmic reticulum response.
International Journal of Oncology, 2018Co-Authors: Ioannis Gkouveris, Nikolaos G. Nikitakis, Jaya Asservatham, Kalu U.e. OgburekeAbstract:: The expression of proinflammatory cytokines in various malignant neoplasms is widely considered to represent the host immune response to tumor development. The role of interferon (IFN)γ in head and neck squamous cell carcinoma, and its association with endoplasmic reticulum (ER) stress pathways, remains a subject of ongoing investigation. Dentin sialophosphoprotein (DSPP), which is a member of the small integrin‑binding N‑linked glycoproteins family, has been implicated in malignant transformation and invasion of oral squamous cell carcinoma (OSCC). Recent studies have established matrix metalloproteinase (MMP)20 as the cognate MMP partner of DSPP. The present study examined the effects of IFNγ treatment on DSPP and MMP20 expression, ER stress, the unfolded protein response (UPR), and calcium (Ca) homeostasis regulatory mechanisms in OSCC cells. The OSC2 OSCC cell line was treated with IFNγ at specific time‑points. At each time‑point, the mRNA expression levels of DSPP and MMP20, and those of ER‑stress‑, UPR‑ and Ca homeostasis‑associated proteins [78‑kDa glucose‑regulated protein (GRP78), sarco/endoplasmic reticulum Ca2+‑ATPase (SERCA2b), inositol 1,4,5‑trisphosphate receptor (IP3r), protein kinase R‑like ER kinase (PERK) and inositol‑requiring enzyme 1 (IRE1)], were assessed by reverse transcription‑quantitative polymerase chain reaction. The protein expression levels of B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax), proliferating cell nuclear antigen (PCNA) and cytochrome c were analyzed by western blotting. Cell viability, apoptosis and migration were evaluated by MTT, Annexin V‑fluorescein isothiocyanate flow cytometry and wound‑healing assays, respectively. IFNγ treatment significantly downregulated the mRNA expression levels of the major ER stress regulator GRP78 and, to a lesser extent, the UPR‑associated molecule IRE1; however, IFNγ had no significant effect on PERK. With regards to ER Ca homeostasis molecules, treatment with IFNγ downregulated the mRNA expression levels of SERCA2b and upregulated those of IP3r. Furthermore, DSPP and MMP20 mRNA expression levels were significantly reduced following IFNγ treatment. Notably, treatment with IFNγ hampered OSC2 migration, reduced cell viability and PCNA protein expression, enhanced apoptosis, downregulated Bcl‑2, and upregulated Bax and cytochrome c. Overall, IFNγ inhibited OSCC cell viability and migration, and increased apoptosis, possibly by regulating ER stress and UPR mechanisms. In addition, IFNγ‑induced DSPP and MMP20 downregulation may correspond with alteration in ER Ca homeostasis.
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DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells
Cellular & Molecular Biology Letters, 2018Co-Authors: Nikolaos G. Nikitakis, Jaya Aseervatham, Ioannis Gkouveris, Kelvin Barahona, Kalu U.e. OgburekeAbstract:Background Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers. Methods The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined. Results DSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 μM of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~ 1%). Conclusions We conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin.