Association between LTF/MMP20/CA6/TAS1R2 polymorphisms and susceptibility to dental caries.

OBJECTIVES: The aim was to assess the associations between the LTF, MMP20, CA6, and TAS1R2 polymorphisms and caries in the Zhuang population and explore the underlying mechanism of the impact of lactoferrin on caries susceptibility. METHODS: A case-control study of 315 adolescents was conducted in Guangxi, China, from May-November 2022. Data were collected through oral examinations and questionnaires. Buccal mucosa cells and DNA samples were collected using the SNPscan technique. Saliva and supragingival plaque samples were taken from 69 subjects with various LTF rs10865941 genotypes. The relationships among the LTF rs10865941 polymorphism, lactoferrin, Streptococcus mutans, and caries were investigated by using the ELISA and qRT-PCR, along with logistic regression analysis. RESULTS: The genotype distribution of the LTF gene were significantly different between the case and control groups (p = 0.018). The case group had lower C allele and greater T allele frequencies than the control group (p = 0.006). The LTF rs10865941 polymorphism was associated with caries in the codominant, dominant, and additive models (p < 0.05). MMP20 rs1784418, CA6 rs2274328, and TAS1R2 rs35874116 were not significantly different between the two groups (p > 0.05). A greater quantity of S. mutans. in the supragingival plaque was found in the case group (p = 0.03). There were significant differences between the two groups in both the codominant model and the dominant model (p < 0.05). CONCLUSIONS: The LTF rs10865941 polymorphism may be associated with caries susceptibility in the Zhuang population of China. The LTF rs10865941 T allele may be a potential risk factor for dental caries.

Molecular evolutionary analyses of tooth genes support sequential loss of enamel and teeth in baleen whales (Mysticeti).

The loss of teeth and evolution of baleen racks in Mysticeti was a profound transformation that permitted baleen whales to radiate and diversify into a previously underutilized ecological niche of bulk filter-feeding on zooplankton and other small prey. Ancestral state reconstructions suggest that postnatal teeth were lost in the common ancestor of crown Mysticeti. Genomic studies provide some support for this hypothesis and suggest that the genetic toolkit for enamel production was inactivated in the common ancestor of living baleen whales. However, molecular studies to date have not provided direct evidence for the complete loss of teeth, including their dentin component, on the stem mysticete branch. Given these results, several questions remain unanswered: (1) Were teeth lost in a single step or did enamel loss precede dentin loss? (2) Was enamel lost early or late on the stem mysticete branch? (3) If enamel and dentin/tooth loss were decoupled in the ancestry of baleen whales, did dentin loss occur on the stem mysticete branch or independently in different crown mysticete lineages? To address these outstanding questions, we compiled and analyzed complete protein-coding sequences for nine tooth-related genes from cetaceans with available genome data. Seven of these genes are associated with enamel formation (ACP4, AMBN, AMELX, AMTN, ENAM, KLK4, MMP20) whereas two other genes are either dentin-specific (DSPP) or tooth-specific (ODAPH) but not enamel-specific. Molecular evolutionary analyses indicate that all seven enamel-specific genes have inactivating mutations that are scattered across branches of the mysticete tree. Three of the enamel genes (ACP4, KLK4, MMP20) have inactivating mutations that are shared by all mysticetes. The two genes that are dentin-specific (DSPP) or tooth-specific (ODAPH) do not have any inactivating mutations that are shared by all mysticetes, but there are shared mutations in Balaenidae as well as in Plicogulae (Neobalaenidae + Balaenopteroidea). These shared mutations suggest that teeth were lost at most two times. Shared inactivating mutations and dN/dS analyses, in combination with cetacean divergence times, were used to estimate inactivation times of genes and by proxy enamel and tooth phenotypes at ancestral nodes. The results of these analyses are most compatible with a two-step model for the loss of teeth in the ancestry of living baleen whales: enamel was lost very early on the stem Mysticeti branch followed by the independent loss of dentin (and teeth) in the common ancestors of Balaenidae and Plicogulae, respectively. These results imply that some stem mysticetes, and even early crown mysticetes, may have had vestigial teeth comprised of dentin with no enamel. Our results also demonstrate that all odontocete species (in our study) with absent or degenerative enamel have inactivating mutations in one or more of their enamel genes.

The energetic basis for hydroxyapatite mineralization by amelogenin variants provides insights into the origin of amelogenesis imperfecta.

Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta, enamel that is defective and easily damaged. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer-oligomer binding energies. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization.

Spectrum of pathogenic variants and founder effects in amelogenesis imperfecta associated with MMP20.

Amelogenesis imperfecta (AI) describes a heterogeneous group of developmental enamel defects that typically have Mendelian inheritance. Exome sequencing of 10 families with recessive hypomaturation AI revealed four novel and one known variants in the matrix metallopeptidase 20 (MMP20) gene that were predicted to be pathogenic. MMP20 encodes a protease that cleaves the developing extracellular enamel matrix and is necessary for normal enamel crystal growth during amelogenesis. New homozygous missense changes were shared between four families of Pakistani heritage (c.625G>C; p.(Glu209Gln)) and two of Omani origin (c.710C>A; p.(Ser237Tyr)). In two families of UK origin and one from Costa Rica, affected individuals were homozygous for the previously reported c.954-2A>T; p.(Ile319Phefs*19) variant. For each of these variants, microsatellite haplotypes appeared to exclude a recent founder effect, but elements of haplotype were conserved, suggesting more distant founding ancestors. New compound heterozygous changes were identified in one family of the European heritage: c.809_811+12delinsCCAG; p.(?) and c.1122A>C; p.(Gln374His). This report further elucidates the mutation spectrum of MMP20 and the probable impact on protein function, confirms a consistent hypomaturation phenotype and shows that mutations in MMP20 are a common cause of autosomal recessive AI in some communities.

Effects of DSPP and MMP20 Silencing on Adhesion, Metastasis, Angiogenesis, and Epithelial-Mesenchymal Transition Proteins in Oral Squamous Cell Carcinoma Cells.

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 αvß3 and αvß6, 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 αvß3, αvß6, 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).

MMP20 Single-Nucleotide Polymorphisms Correlate with Susceptibility to Alcohol-Induced Osteonecrosis of the Femoral Head in Chinese Males.

BACKGROUND Alcohol-induced osteonecrosis of the femoral head (ONFH) is caused by the interaction of genetic and environmental factors. Genetic variations of matrix metalloproteinase (MMP) system are associated with ONFH development and progression. In this study, we aimed to evaluate the relationships between MMP20 gene polymorphisms and the risk of alcohol-induced ONFH in Chinese Han males. MATERIAL AND METHODS In this case-control study, genotypes of 14 selected SNPs in the MMP20 gene were assayed using MassARRAY in 299 male cases with alcohol-induced ONFH and in 197 healthy males. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the influence of gene polymorphism on occurrence of alcohol-induced ONFH by allelic model analysis, genotype model analysis and haplotype analysis. RESULTS After allelic model analysis, the minimum alleles of rs10895322, rs1784424, rs3781788, and rs1573954 correlated with an increased risk of alcohol-induced ONFH (P<0.05). Genetic model analysis revealed significant associations of 9 SNPs with alcohol-induced ONFH occurrence even after adjustment for age (P<0.05): 2 protective SNPs (rs1711423 and rs1784418) and 7 high-risk SNPs (rs10895322, rs1784424, rs3781788, rs7126560, rs1573954, rs1711399, and rs2292730). Moreover, 8 SNPs showed a statistically significant association with different clinical phenotypes (P<0.05). Beyond that, haplotype "CGGTTCCA" in MMP20 was discovered to correlate with a 1.63-fold increased risk of alcohol-induced ONFH (OR: 1.63, 95% CI: 1.15-2.30, P=0.0058). CONCLUSIONS Our data sheds new light on the associations of MMP20 gene polymorphisms with alcohol-induced ONFH predisposition in Chinese Han males.

Analysis of Polymorphisms in Genes Differentially Expressed in the Enamel of Mice with Different Genetic Susceptibilities to Dental Fluorosis.

Genes expressed during amelogenesis are candidates to increase the risk of dental fluorosis (DF). Thus, this study aimed to evaluate the association between polymorphisms in enamel development genes and susceptibility to DF in mice. Mice of both sexes, representing strains 129P3/J (n = 20; resistant to DF) and A/J (n = 20; susceptible to DF), were divided into 2 groups. Each strain received a diet with a low concentration of fluoride (F) and drinking water containing 0 or 50 mg/L of F for 6 weeks. Clinical evaluation and analysis of Vickers enamel microhardness of the incisors were performed. Livers were collected for genomic DNA extraction. Seventeen genetic polymorphisms in Amelx, Ambn, Ambn, Col14a1, Col1a1, Col5a2, Enam, Fam20a, Fam83h, Foxo1, Klk4, Mmp20, Serpinf1, Serpinh1, Smad3, Tuft1, and Wdr72 were genotyped by real-time PCR using Taqman chemistry. Overrepresentation of alleles and genotypes in DF was evaluated using the χ2 test with an alpha of 5%. The clinical aspects of the enamel and the surface enamel microhardness confirmed the DF condition. In the polymorphisms rs29569969, rs13482592, and rs13480057 in Ambn, Col14a1, and Mmp20, respectively, genotype and allele distributions were statistically significantly different between A/J and 129P3/J strains (p < 0.05). In conclusion, polymorphisms in Ambn, Col14a1, and Mmp20 are associated with the susceptibility to DF.

DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells.

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.

Effects of Er:YAG and Diode Laser Irradiation on Dental Pulp Cells and Tissues.

Vital pulp therapy (VPT) is to preserve the nerve and maintain healthy dental pulp tissue. Laser irradiation (LI) is beneficial for VPT. Understanding how LI affects dental pulp cells and tissues is necessary to elucidate the mechanism of reparative dentin and dentin regeneration. Here, we show how Er:YAG-LI and diode-LI modulated cell proliferation, apoptosis, gene expression, protease activation, and mineralization induction in dental pulp cells and tissues using cell culture, immunohistochemical, genetic, and protein analysis techniques. Both LIs promoted proliferation in porcine dental pulp-derived cell lines (PPU-7), although the cell growth rate between the LIs was different. In addition to proliferation, both LIs also caused apoptosis; however, the apoptotic index for Er:YAG-LI was higher than that for diode-LI. The mRNA level of odontoblastic gene markers-two dentin sialophosphoprotein splicing variants and matrix metalloprotease (MMP)20 were enhanced by diode-LI, whereas MMP2 was increased by Er:YAG-LI. Both LIs enhanced alkaline phosphatase activity, suggesting that they may help induce PPU-7 differentiation into odontoblast-like cells. In terms of mineralization induction, the LIs were not significantly different, although their cell reactivity was likely different. Both LIs activated four MMPs in porcine dental pulp tissues. We helped elucidate how reparative dentin is formed during laser treatments.

MMP20 rs1784418 Protects Certain Populations against Caries.

This work aimed to further evaluate the association of MMP20 rs1784418 C>T and dental caries experience with the hypothesis that MMP20 rs1784418 C>T is a risk factor for dental caries. 184 children 4-7 years of age had their caries experience determined and buccal cheek swabs collected for DNA extraction to test for association with the MMP20 rs1784418 C>T using standard statistical approaches. A meta-analytic approach was also implemented to compile previous discrepant reports of the same association. We found an association between MMP20 rs1784418 C>T and dental caries experience in primary dentition (p = 0.01). The meta-analysis showed that this association appears to favor individuals born in Brazil and not Turkey. MMP20 rs1784418 C>T appears to protect against dental caries, but its effects are likely to be more marked in certain populations.

Postradiation Matrix Metalloproteinase-20 Expression and Its Impact on Dental Micromorphology and Radiation-Related Caries.

Recent evidence suggests that head-and-neck radiotherapy (HNRT) increases active forms of matrix metalloproteinase-20 (MMP-20) in human tooth crowns, degrading the dentin-enamel junction (DEJ) and leading to enamel delamination, which is a pivotal step in the formation of radiation-related caries (RRC). Additional participation of enzymatic degradation of organic matrix components in caries progression was attributed to MMP-20 in dentin. Therefore, the current study tested the hypothesis that MMP-20 is overexpressed in the DEJ, dentin-pulp complex components, and carious dentin of post-HNRT patients, leading to detectable micromorphological changes to the enamel and dentin. Thirty-six teeth were studied, including 19 post-HNRT specimens and 17 nonirradiated controls. Optical light microscopy was used to investigate the micromorphological components of the DEJ, dentin-pulp complex components, and carious dentin. The samples were divided into 2 subgroups: nondemineralized ground sections (n = 20) and demineralized histological sections (n = 16). In addition, immunohistochemical analysis using the immunoperoxidase technique was conducted to semiquantitatively assess MMP-20 expression in the DEJ, dentin-pulp complex components, and carious dentin. No apparent damage to the DEJ microstructure or other dentin-pulp complex components was observed and no statistically significant differences were detected in MMP-20 expression (p > 0.05) between the irradiated and control groups. This study rejected the hypothesis that MMP-20 is overexpressed in the DEJ, dentin-pulp complex components, and carious dentin of post-HNRT patients, leading to detectable micromorphological changes. Hence, direct effects of radiation may not be regarded as an independent factor to explain aggressive clinical patterns of RRC.

MMP20 and ARMS2/HTRA1 Are Associated with Neovascular Lesion Size in Age-Related Macular Degeneration.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of severe visual impairment. Despite treatment, a central scotoma often remains. The size of the scotoma depends on the lesion size of the choroidal neovascular membrane and significantly affects the patient's quality of life, and the lesion size of neovascularization also affects response to treatments. The aim of this study was to identify genes associated with the neovascular lesion size in neovascular AMD. DESIGN: A genome-wide association study (GWAS). PARTICIPANTS: We included 1146 Japanese patients with neovascular AMD. METHODS: We performed a 2-stage GWAS for the lesion size of AMD as a quantitative trait among 1146 (first stage: 727, second stage: 419) Japanese patients with neovascular AMD. Lesion size was determined by the greatest linear dimension measured with fluorescein angiography examination before treatment. We examined the association between the genotypic distribution of each single nucleotide polymorphism (SNP) and the trait using an additive model adjusted for age and sex. To evaluate the associations between AMD development and SNPs associated with lesion size, we also performed a case-control study by using the genotype data from these 1146 Japanese patients as case subjects and the fixed dataset from the Nagahama Study as control subjects. MAIN OUTCOME MEASURES: Genes associated with the lesion size in neovascular AMD. RESULTS: In the discovery stage, rs10895322 in MMP20 showed a genome-wide significant P value of 6.95×10(-8), and rs2284665 in ARMS2/HTRA1 showed a P value of 1.55×10(-7). The associations of these 2 SNPs were successfully replicated in the replication stage, and a meta-analysis of both stages showed genome-wide significant P values (2.80×10(-9) and 4.41×10(-9), respectively). In a case-control study using 3248 Japanese subjects as controls, we could not find contribution of MMP20 rs10895322 for AMD development. Although MMP20 has been thought to be expressed only in dental tissues, we confirmed MMP20 expression in the human retina and retinal pigment epithelium/choroid with polymerase chain reaction. CONCLUSIONS: The growth of choroidal neovascularization in AMD would be affected by 2 genes: MMP20, a newly confirmed gene expressed in the retina, and ARMS2/HTRA1, a well-known susceptibility gene for AMD.

The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions.

BACKGROUND: Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated. METHODS: Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS. RESULTS: It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents. CONCLUSION: These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization. GENERAL SIGNIFICANCE: This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.

Genetic comparisons yield insight into the evolution of enamel thickness during human evolution.

Enamel thickness varies substantially among extant hominoids and is a key trait with significance for interpreting dietary adaptation, life history trajectory, and phylogenetic relationships. There is a strong link in humans between enamel formation and mutations in the exons of the four genes that code for the enamel matrix proteins and the associated protease. The evolution of thick enamel in humans may have included changes in the regulation of these genes during tooth development. The cis-regulatory region in the 5' flank (upstream non-coding region) of MMP20, which codes for enamelysin, the predominant protease active during enamel secretion, has previously been shown to be under strong positive selection in the lineages leading to both humans and chimpanzees. Here we examine evidence for positive selection in the 5' flank and 3' flank of AMELX, AMBN, ENAM, and MMP20. We contrast the human sequence changes with other hominoids (chimpanzees, gorillas, orangutans, gibbons) and rhesus macaques (outgroup), a sample comprising a range of enamel thickness. We find no evidence for positive selection in the protein-coding regions of any of these genes. In contrast, we find strong evidence for positive selection in the 5' flank region of MMP20 and ENAM along the lineage leading to humans, and in both the 5' flank and 3' flank regions of MMP20 along the lineage leading to chimpanzees. We also identify putative transcription factor binding sites overlapping some of the species-specific nucleotide sites and we refine which sections of the up- and downstream putative regulatory regions are most likely to harbor important changes. These non-coding changes and their potential for differential regulation by transcription factors known to regulate tooth development may offer insight into the mechanisms that allow for rapid evolutionary changes in enamel thickness across closely-related species, and contribute to our understanding of the enamel phenotype in hominoids.

Transforming growth factor-ß1 regulates expression of the matrix metalloproteinase 20 (Mmp20) gene through a mechanism involving the transcription factor, myocyte enhancer factor-2C, in ameloblast lineage cells.

Matrix metalloproteinase-20 (Mmp20) plays an essential role in amelogenesis during tooth development and is regulated by transforming growth factor-ß1 (TGF-ß1) in mouse ameloblast lineage cells (ALCs). The objective of this study was to explore the role of myocyte enhancer factor-2C (MEF2C), a key transcription factor in craniofacial development, in TGF-ß1-induced Mmp20 gene expression. We investigated Mmp20 expression in ALCs over-expressing MEF2C and in ALCs with MEF2C knocked down. We also analyzed activity of the Mmp20 promoter using a transient reporter gene-expression assay in cultured ALCs. Putative transcription factor-binding sites for MEF2C and TGF-ß1 on the Mmp20 promoter were analyzed with bioinformatics tools and examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The expression of Mmp20 was induced, in a dose-dependent manner, by MEF2C over-expression, and TGF-ß1-induced Mmp20 expression was blocked by MEF2C knockdown in ALCs. There was a TGF-ß1/MEF2C-responsive region, including a putative MEF2-binding site, between base pairs -356 and -73 of the Mmp20 promoter. Mutation of the putative MEF2-binding site significantly reduced Mmp20 promoter activity upon activation with MEF2C or TGF-ß1. In conclusion, TGF-ß1-induced Mmp20 expression in ALCs was regulated through the MEF2-binding site on the Mmp20 promoter and thus mediated by the MEF2C signaling pathway.

Activation profiles of human kallikrein-related peptidases by matrix metalloproteinases.

The 15 human kallikrein-related peptidases (KLKs) are clinically important biomarkers and therapeutic targets of interest in inflammation, cancer, and neurodegenerative disease. KLKs are secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide, and this is a key step in their functional regulation. Physiologically relevant KLK regulatory cascades of activation have been described in skin desquamation and semen liquefaction, and work by a large number of investigators has elucidated pairwise and autolytic activation relationships among the KLKs with the potential for more extensive activation cascades. More recent work has asked whether functional intersection of KLKs with other types of regulatory proteases exists. Such studies show a capacity for members of the thrombostasis axis to act as broad activators of pro-KLKs. In the present report, we ask whether such functional intersection is possible between the KLKs and the members of the matrix metalloproteinase (MMP) family by evaluating the ability of the MMPs to activate pro-KLKs. The results identify MMP-20 as a broad activator of pro-KLKs, suggesting the potential for intersection of the KLK and MMP axes under pathological dysregulation of MMP-20 expression.

Excessive fluoride induces endoplasmic reticulum stress and interferes enamel proteinases secretion.

Protein retention in the enamel layer during tooth formation is well known to be associated with dental fluorosis but the underlying mechanism is unclear. The functions of the endoplasmic reticulum (ER) correlate directly with secreted protein metabolism. We used an ameloblast-derived cell line to determine whether excessive amounts of fluoride cause ER stress, and whether this interferes with the secretion of enamel matrix proteinases. ER stress activates a signaling network called the unfolded protein response (UPR). Here, we used real-time RT-PCR and immunofluorescence to study the effect of fluoride on the expression, translation, and secretion of UPR transcription factors in ameloblast-like cells. Measurement of both the gene and protein expression of UPR transcription factors indicated that high-dose fluoride increases the expression of UPR transcription factors in a dose-dependent manner. We also used ELISA to detect and quantify the enamel proteinases secreted by ameloblasts. We found a corresponding decrease in extracellular secretion of the enamel proteinases matrix metalloproteinase-20 and kallikrein-4, after exposure to fluoride. Furthermore, correlation analysis indicated that the expression of UPR transcription factors showed a strong inverse correlation with that of enamel proteinases. The results suggest that high-dose fluoride initiates an ER stress response in ameloblasts and induces the UPR, which interferes with the synthesis and secretion of enamel proteinases. Taken together, these results suggest that excessive ingestion of fluoride during tooth formation can decrease the secretion of proteinases, thus causing protein retention in the enamel layer, indicating that the ER stress response may be responsible for dental fluorosis.

Mobility gene expression differences among wild-type, Mmp20 null and Mmp20 over-expresser mice plus visualization of 3D mouse ameloblast directional movement.

Enamel forming ameloblasts move away from the dentino-enamel junction and also move relative to each other to establish enamel shape during the secretory stage of enamel development. Matrix metalloproteinase-20 (MMP20) is a tooth specific proteinase essential for proper enamel formation. We previously reported that MMP20 cleaves cadherins and may regulate ameloblast movement. Here, we used an Amelx promoter driven tdTomato reporter to label mouse ameloblasts. With these transgenic mice, we assessed ameloblast mobility group dynamics and gene expression. Three-dimensional imaging of mouse ameloblasts were observed in hemi-mandibles by using a tissue clearing technique. The three-dimensional ameloblast layer in Tg(Amelx-Mmp20) mice that overexpress MMP20 was uneven and the ameloblasts migrated away from this layer. Mouse ameloblast movement toward incisal tips was monitored by ex vivo time-lapse imaging. Gene expression related to cell migration and adhesion was analyzed in ameloblasts from wild-type mice, Mmp20-/- mice with no functional MMP20 and from Tg(Amelx-Mmp20) overexpressing mice. Gene expression was altered in Mmp20-/- and Tg(Amelx-Mmp20) mice compared to wild type. Among the genes assessed, those encoding laminins and a gap junction protein were upregulated in Mmp20-/- mice. New techniques and findings described in this study may lead to an improved understanding of ameloblast movement during enamel formation.

Transcriptomic Network Regulation of Rat Tooth Germ from Bell Differentiation Stage to Secretory Stage: MAPK Signaling Pathway Is Crucial to Extracellular Matrix Remodeling.

Hard tissues make up the vast majority of teeth and are mineralized from the surrounding matrix. If the development of tooth germ is affected during mineralization, hypoplasia of the tooth tissue can occur. To better understand the mechanisms mediating hypoplasia, we need to first study normal development. Using a rodent model, we highlight the transcriptomic changes that occur from the differentiation to secretion stages of mandibular molar germs. The tooth germ was dissected from rats at postnatal day 1.5 or 3.5 for high-throughput sequencing. Combining transcriptome analysis and DNA methylation, we identified 590 differentially expressed genes (436 upregulated and 154 downregulated) and 551 differentially expressed lncRNAs (long noncoding RNA; 369 upregulated and 182 downregulated) which were linked to the biological processes of odontogenesis, amelogenesis, tooth mineralization, and the alteration of extracellular matrix (ECM), especially matrix metalloproteinases (MMPs) and elastin. We found DNA methylation changes in 32 selected fragments involved in 5 chromosomes, 26 targets, and 2 haplotypes. Finally, three novel genes were identified: MMP20, Tgfb3, and Dusp1. Further analysis revealed that MMP20 has a role in odontogenesis and amelogenesis by influencing Slc24a4 and DSPP; Tgfb3 is involved in epithelial cell proliferation, cellular component disassembly process, ECM cellular component, and decomposition of cell components. But lncRNA expression could affect DNA methylation and mRNA expression. Moreover, the degree of DNA methylation could also affect the transcriptome level. Thus, Tgfb3 had no difference in DNA methylation, and Dusp1 conferred no difference at the transcriptome level. These three genes were all enriched in the MAPK pathway and played an important role in ECM remodeling. These data suggest that during the period of the bell differentiation stage to the secretory stage, along with enamel/dentin matrix secretion and hard tissue occurrence, the ECM is remodeled via MAPK signaling.

Impact of tooth mineral tissues genes on dental caries: A birth-cohort study.

OBJECTIVE: We aimed to investigate whether Single Nucleotide Polymorphisms present in the genes of tooth mineral tissues influence dental caries trajectory across the life course, and if there is an epistatic (gene-gene) interaction between these SNPs. METHODS: A representative sample of all 5,914 births from the 1982 Pelotas birth cohort study was prospectively investigated. Dental caries trajectory across the life course was assessed at 15(n = 888), 24(n = 720), and 31 years old(n = 539). Group-based trajectory modeling was used to identify distinct subgroups of individuals whose caries measurements followed a similar pattern over time. Genetic material was collected, and individuals were genotyped [rs4970957(TUFT1), rs1711437(MMP20), rs1784418(MMP20), rs2252070(MMP13), rs243847(MMP2), rs2303466(DLX3), rs11656951(DLX3), rs7501477(TIMP2), rs388286(BMP7), and rs5997096(TFIP11)]. Analyzes were performed for allele and genotype using logistic regression and generalized multifactor dimensionality reduction for epistatic interactions. RESULTS: The analyses included 678 individuals, those with allele C (OR=0.74, CI95%[0.59-0.92]), genotype CC in the additive effect (OR=0.52, CI95%[0.31-0.89]), and the genotype TC/CC in dominant effect (OR=0.72, CI95%[0.53-0.98]) on the rs243847(MMP2) were associated with low caries trajectory. Individuals with the allele T (OR=0.79, CI95%[0.64-0.98]) and the genotype TC/CC in dominant effect (OR=0.66, CI95%[0.47-0.95]) on the rs5997096(TFIP11) were associated with low caries trajectory. Positive epistatic interactions were observed involving two (MMP2 and BMP7; p = 0.006) and three (TUFT1, MMP2, and TFIP11; p<0.001) loci and high caries trajectory. CONCLUSIONS: Some SNPs present in the genes of tooth mineral tissues were associated with caries trajectory and epistatic interactions increasing the network of SNPs involved in individual caries experience. CLINICAL SIGNIFICANCE: Single nucleotide polymorphisms in the pathway of tooth mineral tissues genes may contribute significantly to the individual caries experience across the life course.