Amelotin Research Articles

Amelotin Promotes Mineralization and Adhesion in Collagen-Based Systems.

Periodontitis is characterized by the destruction of tooth-supporting tissues including the alveolar bone. Barrier membranes are used in dentistry for tissue regenerative therapy. Nevertheless, conventional membranes have issues related to membrane stability and direct induction of bone mineralization. Amelotin (AMTN), an enamel matrix protein, regulates hydroxyapatite crystal nucleation and growth. To apply an AMTN membrane in clinical practice, we investigated the mineralizing and adhesive effects of recombinant human (rh) AMTN in vitro using a collagen-based system. Collagen hydrogel incorporated with rhAMTN (AMTN gel) and rhAMTN-coated dentin slices were prepared. AMTN gel was then applied on a commercial membrane (AMTN membrane). Samples were incubated for up to 24 h in mineralization buffer, and the structures were observed. The peak adhesive tensile strength between the dentin and AMTN membrane was measured. Using an enzyme-linked immunosorbent assay, the release kinetics of rhAMTN from the membrane were investigated. The AMTN gel resulted in the formation of hydroxyapatite deposits both onto and within the collagen matrix. Furthermore, coating the dentin surface with rhAMTN promoted the precipitation of mineral deposits on the surface. Interestingly, site-specific mineralization was observed in the AMTN membrane. Only 1% of rhAMTN was released from the membrane. Hence, the AMTN membrane adhered to the dentin surface with more than twofold greater tensile strength than that detected for a rhAMTN-free barrier membrane. RhAMTN can accelerate mineralization and adhesion in collagen-based systems. Furthermore, the AMTN membrane could inform the optimal design of calcified tissue regenerative materials. The online version contains supplementary material available at 10.1007/s12195-022-00722-2.

Promoting mineralization at biological interfaces Ex vivo with novel amelotin-based bio-nano complexes

Interfacial failure at the resin-dentin interface is a significant disadvantage of resin-based dental restoration. In this study, we created bio-inspired bio-nano complexes using the enamel protein amelotin (AMTN) or AMTN with an engineered collagen-binding site (AMTN-Col) to coat hydroxyapatite nanoparticles (HANP). The resulting nano-bio complexes, AMTN-HANP and AMTN-Col–HANP, were evaluated for their ability to promote collagen mineralization. Our study comprises three separate phases.In phase I, developing a method for functionalizing HANP with AMTN/AMTN-Col was explored. HANP were synthesized and characterized using TEM, SAED-TEM, XRD and ATR-FTIR. The nanoparticles were functionalized with AMTN or AMTN-Col. The successful coating of the nanoparticles with the proteins was confirmed using a TEM image of immunogold-labelled samples.In phase II of the study, the mineralization potential of the synthesized bio-nano complexes was studied using model systems consisting of simulated body fluid (SBF), polymerized collagen gels, and dentin disks prepared from human extracted molars. Mineral formation in SBF was recorded with a light scattering assay using a microplate reader on 8 replicates of each sample per study time point. Statistical analysis was performed using one-way ANOVA and the Tukey test. Significance was assigned at P ​< ​0.01. The extent of mineral formation on collagen gel and remineralization of demineralized dentin was studied with SEM. Accelerated mineral formation collagen mineralization of bio-nano complexes treated samples were observed in all model systems.In phase III of the study, the clinical utilization of AMTN/AMTN-Col coated HANP in bio-integration and enhancing the bond strength of a resin-based dental restoration and the dentin interface was investigated. The bio-nano complexes were applied as a pretreatment on dentin disks prepared from human extracted molars prior to the composite resin restoration. The micro-shear bond strength test was done on 8 samples per treatment group (a total of 32 samples). Statistical analysis on shear bond strength was performed using one-way ANOVA and the Tukey test. Significance was assigned at P ​< ​0.01. Shear bond strength values indicated that pretreatment of dentin with the bio-nano complexes before adhesive application significantly improved shear bond strength.ConclusionWe have shown that AMTN based bio-nano complexes promote mineral formation on collagenous interfaces. Our findings can be the basis of new bio-inspired, bio-nano materials that may improve dental restoration longevity by enhancing the stability and integrity of the dentin-composite resin interface.

Expression and localization of amelotin, laminin γ2 and odontogenesis-associated phosphoprotein (ODAPH) on the basal lamina and junctional epithelium.

At maturation stage of enamel development, a specialized basal lamina (sBL) was built between ameloblasts and enamel. After the teeth eruption, the ameloblasts transform into the inner cell layer of junctional epithelium. The inner cell layer forms the internal basal lamina of junctional epithelium. However, the composition of the sBL and internal basal lamina was not clarified. The objective of our study was to make a description of the localization of amelotin (AMTN), laminin γ2 (LAMC2) and Odontogenesis-associated phosphoprotein (ODAPH) on the sBL and internal basal lamina. In immunohistochemical study, AMTN, LAMC2 and ODAPH were detected on the sBL at maturation stage. AMTN was also detected in ameloblasts at maturation stage. The expression of AMTN decreased from early-to-late maturation stage. In contrast, the expression of LAMC2 and ODAPH was stable. Immunofluorescence double-staining showed the localization of AMTN was close to enamel surface. However, the localization of ODAPH was close to ameloblasts. LAMC2 and ODAPH were observed on internal basal lamina of junctional epithelium. In contrast, no expression of AMTN was detected on internal basal lamina of junctional epithelium. Our results suggested that ODAPH might participate in enamel maturation and periodontal health, which might provide a better understanding of enamel defects and periodontal disease in clinic.

Hydroxyapatite Growth on Amelogenin‐Amelotin Recombinamers

AbstractStrategies to improve hydroxyapatite mineralization for enamel repair are essential for tissue regeneration. Amelogenin and amelotin (AMTN) are enamel matrix proteins playing critical roles in enamel formation. Amelogenin acts as a scaffold for hydroxyapatite, while AMTN (specifically its ‘SSEEL’ domain) is necessary for proper enamel mineralization. The functional relationship between recombinant AMTN and amelogenin, and their combined ability to guide uniaxial hydroxyapatite growth in vitro has been investigated recently. However, incorporation of the active domain of AMTN within recombinant amelogenins has not been studied yet. Here we describe the synthesis of modified amelogenin by inserting, internally and at its C‐terminus, the mineralizing AMTN‐derived motif SSEEL. C‐terminus modified amelogenin promoted hydroxyapatite formation, whereas internal incorporation of the motif initially resulted in amorphous calcium phosphate formation. Here we show that modified amelogenin with the mineralizing AMTN‐motif SSEEL can promote hydroxyapatite growth. These results give new insights for mineralized tissue regeneration using recombinamer proteins.

MiR-200b suppresses TNF-α-induced AMTN production in human gingival epithelial cells.

Amelotin (AMTN) is an enamel protein that is localized in junctional epithelium (JE) of gingiva and suggested to be involved in the attachment between JE and tooth enamel. MicroRNA is a small non-coding RNA that regulates gene expression at post-transcriptional level by binding to the 3'-untranslated region (3'-UTR) of target mRNAs. In this study, we have analyzed the effects of miR-200b on the expression of AMTN in human gingival epithelial (Ca9-22) cells. Total RNAs and proteins were extracted from Ca9-22 cells transfected with miR-200b expression plasmid or miR-200b inhibitor and stimulated by TNF-α (10ng/ml, 12h). AMTN and inhibitor of kappa-B kinase beta (IKKβ) mRNA and protein levels were measured by qPCR and Western blot. Human AMTN 3'-UTR that contains putative miR-200b target sites were cloned downstream of -353AMTN luciferase (LUC) plasmid. Ca9-22 cells were transfected with -353AMTN 3'-UTR LUC constructs and miR-200b expression plasmid, and LUC activities were measured with or without stimulation by TNF-α. TNF-α-induced AMTN mRNA levels were partially inhibited by miR-200b overexpression and enhanced by miR-200b inhibitor. TNF-α-induced IKKβ mRNA and protein levels were almost completely inhibited by miR-200b. Transcriptional activities of -353AMTN 3'-UTR LUC constructs were induced by TNF-α and partially inhibited by miR-200b. IKKβ inhibitor IMD0354 and NF-κB inhibitor triptolide decreased TNF-α-induced LUC activities. Furthermore, both inhibitors reduced AMTN mRNA levels in the presence or absence of TNF-α. These results suggest that miR-200b suppresses AMTN expression by targeting to AMTN and IKKβ mRNAs in the human gingival epithelial cells.

Amelotin is expressed in retinal pigment epithelium and localizes to hydroxyapatite deposits in dry age-related macular degeneration

Deposition of hydroxyapatite (HAP) basal to the retinal pigment epithelium (RPE) is linked to the progression of age-related macular degeneration (AMD). Serum-deprivation of RPE cells in culture mimics some features of AMD. We now show that serum-deprivation also leads to the induction of amelotin (AMTN), a protein involved in hydroxyapatite mineralization in enamel. HAP is formed in our culture model and is blocked by siRNA inhibition of AMTN expression. In situ hybridization and immunofluorescence imaging of human eye tissue show that AMTN is expressed in RPE of donor eyes with geographic atrophy ("dry" AMD) in regions with soft drusen containing HAP spherules or nodules. AMTN is not found in hard drusen, normal RPE, or donor eyes diagnosed with wet AMD. These findings suggest that AMTN is involved in formation of HAP spherules or nodules in AMD, and as such provides a new therapeutic target for slowing disease progression.

Phosphorylated/Nonphosphorylated Motifs in Amelotin Turn Off/On the Acidic Amorphous Calcium Phosphate-to-Apatite Phase Transformation.

Amelotin (AMTN) as a matrix protein exerts a direct effect on biomineralization by modulating apatite (HAP) formation during the dental enamel maturation stage through the specific interaction of a potentially phosphorylated Ser-Ser-Glu-Glu-Leu (SSEEL) peptide fragment with calcium phosphate (Ca-P) surfaces. However, the roles of (non)phosphorylation of this evolutionarily conserved subdomain within AMTN remain poorly understood. Here, we show, by time-resolved atomic force microscopy (AFM) imaging of in situ HAP crystallization via the HPO42--rich amorphous calcium phosphate (acidic ACP), the on/off switching of the phase transformation process through a nonphosphorylation-to-phosphorylation transition of the SSEEL motif. Using high-resolution transmission electron microscopy (HRTEM), we observed that the acidic ACP phase is stabilized by the phosphorylated SSEEL motif, delaying its transformation to HAP, whereas the nonphosphorylated counterpart promotes HAP formation by accelerating the dissolution-recrystallization of the acidic ACP substrate. Dynamic force spectroscopy measurements demonstrate greater binding energies of nonphosphorylated SSEEL to the acidic ACP substrate by the formation of molecular peptide-ACP bonding, explaining the enhanced dissolution of the acidic ACP substrate by stronger complexion with surface Ca2+ ions. Our findings demonstrate direct evidence for the switching role of (non)phosphorylation of an evolutionarily conserved subdomain within AMTN in controlling the phase transition of growing enamel and designing tissue regeneration biomaterials.

Organized Assembly of Fluorapatite Nanorods Controlled by Amelotin: Implications for Enamel Regeneration

Dental enamel possesses remarkable mechanical properties because of the hierarchical structural organization and hybridization of organic and inorganic components. Amelotin (AMTN), as an enamel mat...

Selective bacterial degradation of the extracellular matrix attaching the gingiva to the tooth.

The junctional epithelium (JE) is a specialized portion of the gingiva that seals off the tooth‐supporting tissues from the oral environment. This relationship is achieved via a unique adhesive extracellular matrix that is, in fact, a specialized basal lamina (sBL). Three unique proteins – amelotin (AMTN), odontogenic ameloblast‐associated (ODAM), and secretory calcium‐binding phosphoprotein proline‐glutamine rich 1 (SCPPPQ1) – together with laminin‐332 structure the supramolecular organization of this sBL and determine its adhesive capacity. Despite the constant challenge of the JE by the oral microbiome, little is known of the susceptibility of the sBL to bacterial degradation. Assays with trypsin‐like proteases, as well as incubation with Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola, revealed that all constituents, except SCPPPQ1, were rapidly degraded. Porphyromonas gingivalis was also shown to alter the supramolecular network of reconstituted and native sBLs. These results provide evidence that proteolytic enzymes and selected gram‐negative periodontopathogenic bacteria can attack this adhesive extracellular matrix, intimating that its degradation could contribute to progression of periodontal diseases.

An Evolutionarily Conserved Subdomain in Amelotin Promotes Amorphous Calcium Phosphate-to-Hydroxyapatite Phase Transition

Amelotin (AMTN) has been shown to promote hydroxyapatite (HAP) formation in the late stages of tooth enamel biomineralization, but the molecular mechanisms that control this process are poorly understood. Here, by the use of high-resolution Raman spectroscopy and atomic force microscopy to directly observe time-resolved in situ crystallization, we show that AMTN facilitates the in vitro formation of HAP crystals by promoting phase transformation from amorphous calcium phosphate (ACP) as a naturally occurring HAP precursor in enamel. In the presence of a near-physiological enamel fluid, mineral conversions of ACP proceed through dissolution and recrystallization at the ACP–fluid interface, where the adsorbed AMTN promotes the ACP dissolution through a strong complexation of the AMTN protein with calcium ions, occurring even in supersaturated solutions. Single-molecule measurements of equilibrium rupture force and the binding free-energy of AMTN adsorbed to four calcium phosphate phases identify a highly co...

C/EBPβ and YY1 bind and interact with Smad3 to modulate lipopolysaccharide-induced amelotin gene transcription in mouse gingival epithelial cells.

Junctional epithelium (JE) develops from reduced enamel epithelium during tooth formation and is critical for the maintenance of healthy periodontal tissue through ensuring appropriate immune responses and the rapid turnover of gingival epithelial cells. We have previously shown a relationship between inflammatory cytokines and expression of JE‐specific genes, such as amelotin (AMTN), in gingival epithelial cells. Here, we elucidated the effects of Porphyromonas gingivalis‐derived lipopolysaccharide (Pg LPS) on Amtn gene transcription and the interaction of transcription factors. To determine the molecular basis of transcriptional regulation of the Amtn gene by Pg LPS, we performed real‐time PCR and carried out luciferase assays using a mouse Amtn gene promoter linked to a luciferase reporter gene in mouse gingival epithelial GE1 cells. Gel mobility shift and chromatin immunoprecipitation assays were performed to identify response elements bound to LPS‐induced transcription factors. Next, we analyzed protein levels of the LPS‐induced transcription factors and the interaction of transcription factors by western blotting and immunoprecipitation. LPS increased Amtn mRNA levels and elevated luciferase activities of constructs containing regions between −116 and −238 of the mouse Amtn gene promoter. CCAAT/enhancer‐binding protein (C/EBP) 1–, C/EBP2– and Ying Yang 1 (YY1)–nuclear protein complexes were increased by LPS treatment. Furthermore, we identified LPS‐modulated interactions with C/EBPβ, YY1 and Smad3. These results demonstrate that Pg LPS regulates Amtn gene transcription via binding of C/EBPβ–Smad3 and YY1–Smad3 complexes to C/EBP1, C/EBP2 and YY1 response elements in the mouse Amtn gene promoter.

Transcriptional regulation of human amelotin gene by interleukin-1β.

One of the major causes of tooth loss is chronic inflammation of the periodontium, the tissues surrounding the tooth. Amelotin (AMTN) is a tooth enamel protein which is expressed in maturation‐stage ameloblasts and also in the internal basal lamina of junctional epithelium, a unique epithelial structure attached to the tooth surface which protects against the constant microbiological challenge to the periodontium. Localization of AMTN suggests that its function could be involved in the dentogingival attachment. The purpose of this study was to investigate the effect of interleukin‐1β (IL‐1β) on AMTN gene transcription in human gingival epithelial Ca9‐22 cells. IL‐1β increased AMTN mRNA and protein levels at 3 h, and the levels reached maximum at 6 and 12 h. IL‐1β induced luciferase activities of human AMTN gene promoter constructs (−211, −353, −501, −769, and −950AMTN), but these activities were partially inhibited in −353AMTN constructs that included 3‐bp mutations in CCAAT/enhancer binding protein 1 (C/EBP1), C/EBP2, and Ying Yang 1 (YY1) elements. Transcriptional activities induced by IL‐1β were abrogated by protein kinase A (PKA), tyrosine kinase, mitogen‐activated protein kinase kinase (MEK1/2), and phosphatidylinositol 3‐kinase (PI3K) inhibitors. Gel shift and ChIP assays showed that IL‐1β increased C/EBPβ binding to C/EBP1 and C/EBP2, and YY1 binding to YY1 elements after 3 h, and that these DNA–protein interactions were inhibited by PKA, tyrosine kinase, MEK1/2, and PI3K inhibitors. These results demonstrated that IL‐1β increases AMTN gene transcription in human gingival epithelial cells mediated through C/EBP1, C/EBP2, and YY1 elements in the human AMTN gene promoter.

The enamel protein ODAM promotes mineralization in a collagen matrix

ABSTRACTPurpose/aim of the study: Odontogenic ameloblast-associated protein (ODAM) is predominantly expressed during the maturation stage of enamel formation and interacts strongly with amelotin (AMTN). AMTN is involved in enamel mineralization, but the effect of ODAM on mineralization has not been investigated. This study determined whether ODAM was able to induce hydroxyapatite (HA) mineralization in modified simulated body fluid (SBF) and in a collagen matrix in vitro. Materials and methods: To monitor the kinetics of calcium phosphate mineralization, recombinant human (rh) ODAM protein in SBF buffer was incubated at 37°C and a light-scattering assay was conducted at intervals. To investigate the nucleation of ODAM in collagen matrix, the ODAM-impregnated collagen hydrogel was incubated in SBF buffer for 24 hours. Bovine serum albumin (BSA) was used as negative control. Mineral deposits were visualized using electron microscopy. Results: The presence of rh-ODAM protein in SBF resulted in higher light-scattering values after 18–24 hours. Calcium phosphate precipitates were observed on the surface of the ODAM-treated, but not BSA-treated collagen hydrogel after 24 hours in SBF. TEM and SAED analyses showed that these crystals consisted of needle-like HA. Conclusion: Similar to AMTN, ODAM is able to promote HA nucleation in a dose-dependent manner in SBF, and even outside of its biological context in vitro.

A Highly Conserved Motif within the Amelotin Protein Controls the Surface Growth of Brushite

Amelotin (AMTN) protein exerts a direct role on enamel biomineralization likely due to its binding affinity with calcium phosphates (Ca-Ps). However, the kinetics and molecular mechanisms of the AMTN–Ca-P interaction remain largely unknown. Here we used in situ atomic force microscopy (AFM) to directly image the surface growth of brushite (dicalcium phosphate dihydrate, DCPD, CaHPO4·2H2O) in the presence of recombinant human AMTN. Measured step movement velocities of the DCPD (010) face show that AMTN protein promotes crystal face growth only within a limited concentration range, whereas inhibition occurs outside of this range. A peptide derived from a highly conserved and potentially phosphorylated motif (SSEEL) within the AMTN protein inhibits crystal growth similar to that of the AMTN protein at low concentration. By the use of single-molecule force spectroscopy (SMFS), we directly measure the binding of the full-length AMTN and SSEEL to the DCPD (010) face. Similar rupture forces reveal that this acti...

IL-1β and TNF-α regulate mouse amelotin gene transcription in gingival epithelial cells

Amelotin (AMTN) is an enamel protein expressed in maturation-stage ameloblasts and junctional epithelium. To clarify the transcriptional regulation of the AMTN gene by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with luciferase constructs including various lengths of the mouse AMTN gene promoter, and gel shift and chromatin immunoprecipitation assays using mouse gingival epithelial GE1 cells. The levels of AMTN mRNA and protein in GE1 cells were increased after 6 h of stimulation with IL-1β (1 ng/mL) and TNF-α (10 ng/mL). IL-1β and TNF-α induced luciferase activities of the constructs between -116AMTN and -705AMTN including the mouse AMTN gene promoter. Transcriptional activation by IL-1β and TNF-α was partially inhibited in -460AMTN including 3-bp mutations in the CCAAT-enhancer-binding protein 1 (C/EBP1), C/EBP2 and Yin Yang 1 (YY1) elements. Transcriptional activities induced by IL-1β and TNF-α were inhibited by tyrosine kinase, MEK1/2 and PI3-kinase inhibitors. Results of ChIP assays showed that IL-1β and TNF-α increased C/EBPβ and YY1 binding to the C/EBP1, C/EBP2 and YY1 elements. These results demonstrate that IL-1β and TNF-α increase AMTN gene transcription via the C/EBP1, C/EBP2 and YY1 elements in the mouse AMTN gene promoter.

TGFβ1-induced Amelotin gene expression is downregulated by Bax expression in mouse gingival epithelial cells.

Amelotin (AMTN) is induced upon initiation of apoptosis by transforming growth factor beta1 (TGFβ1) and is mediated by Smad3 in gingival epithelial cells (GE1 cells). This upregulation of AMTN gene expression is temporary, and the mechanism responsible is still unclear. The present study investigated the transcriptional downregulation of TGFβ1-induced AMTN gene expression in GE1 cells during the progression of apoptosis. To examine time-dependent changes in the levels of AMTN, Smad3 and Bax mRNA induced by TGFβ1, real-time PCR analyses were performed. Immunocytochemistry was carried out to detect the expression of Smad3 and Bax. Transient transfection analyses were performed using mouse AMTN gene promoter constructs of various lengths including Smad response elements (SBEs), in the presence or absence of TGFβ1. Changes in Smad3 binding to SBEs resulting from overexpression of Bax were examined using ChIP assays. Overexpression of Bax dramatically downregulated the levels of TGFβ1-induced AMTN mRNA and transcription of the AMTN gene. Smad3 binding to SBEs in the mouse AMTN gene promoter was induced by overexpression of Smad3 or TGFβ1, and this was inhibited by Bax overexpression. These results show that the levels of AMTN mRNA induced by TGFβ1 and Smad3 are decreased by robust expression of Bax in gingival epithelial cells.

Tumor necrosis factor-α stimulates human amelotin gene transcription in gingival epithelial cells.

Amelotin (AMTN) is an enamel protein that is localized in the basal lamina of ameloblasts in their maturation stage and the internal basal lamina of junctional epithelium (JE) and it is suggested that AMTN could be involved in the dentogingival attachment. To elucidate the transcriptional regulation of human AMTN gene in inflamed gingiva, we have analyzed the effect of tumor necrosis factor-α (TNF-α) on the expression of AMTN gene in Ca9-22 and Sa3 human gingival epithelial cells. Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10ng/ml). AMTN mRNA and protein levels were measured by real-time PCR and Western blotting. Transient transfection analyses were completed using the various lengths of human AMTN gene promoter constructs with or without TNF-α. Gel mobility shift and chromatin immunoprecipitation assays were performed to investigate the transcription factors bindings to the human AMTN gene promoter by TNF-α. TNF-α (10ng/ml) increased AMTN mRNA and protein levels after 12h. TNF-α induced luciferase activities of human AMTN gene promoter constructs (- 211AMTN, - 353AMTN, and - 501AMTN). TNF-α-induced luciferase activities were partially inhibited in the mutation - 353AMTN constructs that included 3-bp mutations in CCAAT enhancer-binding protein 1 (C/EBP1), C/EBP2 and Ying Yang 1 (YY1) elements. Transcriptional activities induced by TNF-α were inhibited by protein kinase A, Src-tyrosine kinase, MEK1/2, p38 kinase, NF-κB, and PI3-kinase inhibitors. Gel shift assays showed that TNF-α increased nuclear proteins binding to two types of C/EBP elements (C/EBP1 and C/EBP2) and YY1 element. The results of the chromatin immunoprecipitation assays showed that C/EBPβ binding to C/EBP1 and C/EBP2, and YY1 binding to YY1 were increased by TNF-α. These findings demonstrated that TNF-α stimulates AMTN gene transcription in human gingival epithelial cells via C/EBP1, C/EBP2, and YY1 elements in the human AMTN gene promoter.

Interactions of AMTN, ODAM and SCPPPQ1 proteins of a specialized basal lamina that attaches epithelial cells to tooth mineral

A specialized basal lamina (sBL) mediates adhesion of certain epithelial cells to the tooth. It is distinct because it does not contain collagens type IV and VII, is enriched in laminin-332, and includes three novel constituents called amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1). The objective of this study was to clarify the structural organization of the sBL. Fluorescence and immunogold labeling showed that the three proteins co-localize. Quantitative analysis of the relative position of gold particles on the sBL demonstrates that the distribution of ODAM is skewed towards the cell while that of AMTN and SCPPPQ1 tends towards the tooth surface. Bacterial two-hybrid analysis and co-immunoprecipitation, gel filtration of purified proteins and transmission electron and atomic force microscopies highlight the propensity of AMTN, ODAM, and SCPPPQ1 to interact with and among themselves and form supramolecular aggregates. These data suggest that AMTN, ODAM and SCPPPQ1 participate in structuring an extracellular matrix with the distinctive capacity of attaching epithelial cells to mineralized surfaces. This unique feature is particularly relevant for the adhesion of gingival epithelial cells to the tooth surface, which forms a protective seal that is the first line of defense against bacterial invasion.

Localization and expression pattern of amelotin, odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva.

The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.

Amelotin gene expression is temporarily being upregulated at the initiation of apoptosis induced by TGFβ1 in mouse gingival epithelial cells.

Amelotin (AMTN) is expressed and secreted by ameloblasts in the maturation stage of amelogenesis and persist with low levels in the junctional epithelium (JE) of erupted teeth. The purpose of this study is to investigate the transcriptional regulation of the AMTN gene by transforming growth factor beta1 (TGFβ1) in gingival epithelial (GE1) cells in the apoptosis phase. Apoptosis was evaluated by the fragmentation of chromosomal DNA and TUNEL staining. A real-time PCR was carried out to examine the AMTN mRNA levels induced by TGFβ1 and Smad3 overexpression. Transient transfection analyses were completed using the various lengths of mouse AMTN gene promoter constructs with or without TGFβ1. Chromatin immunoprecipitation (ChIP) assays were performed to investigate the Smad3 bindings to the AMTN gene promoter by TGFβ1. TGFβ1-induced apoptosis in GE1 cells were detected at 24 and 48h by DNA fragmentation and TUNEL staining. AMTN mRNA levels increased at 6h and reached maximum at 24h in GE1 cells. Luciferase activities of the mouse AMTN gene promoter constructs were induced by TGFβ1. The results of the ChIP assays showed that there was an increase in Smad3 binding to Smad-binding element (SBE)#1 and SBE#2 after stimulation by TGFβ1. Immunohistochemical localization of AMTN was detected in the JE, and the AMTN protein levels in Smad3-deficient mice were decreased compared with wild-type mice. AMTN mRNA levels were induced at the initiation of apoptosis by TGFβ1, which mediated through the Smad3 bindings to SBEs in the mouse AMTN gene promoter.