Ameloblastin Expression Research Articles

Ameloblastin is not implicated in bone remodelling and repair

Ameloblastin (AMBN) is an enamel matrix protein produced by ameloblasts. It has been suggested that AMBN might also be implicated in craniofacial bone formation. Our objective was to determine whether AMBN has an effect on osteogenic mineralisation and influences bone remodelling and repair. MC3T3-E1 cells were screened for endogenous expression of enamel proteins using real time PCR. Various osteogenic cells were infected with lentivirus encoding for AMBN and protein expression was verified using immunochemistry. Cultures were stained with alizarin red and mineralisation was quantified. Healing bone was probed for expression of AMBN by DNA microarray analysis. Tooth extraction, experimental tooth movement (ETM), and creation of a non-critical size bone defect in the tibia (BDT) were carried out in wild type and AMBN(Δ5-6) mutant mice. Tissues were processed for immunolabelling of AMBN and Bril, an osteoblast specific protein associated with active bone formation. MC3T3-E1 cells and healing bone showed no significant expression of AMBN. Overexpression of AMBN in osteogenic cultures induced no noticeable changes in mineralisation. In wild type mice, AMBN was immunodetected in ameloblasts and enamel, but not in normal bone, and at sites where bone remodelling and repair were induced. Bone remodelling during ETM and BDT repair in AMBN(Δ5-6) mice were not significantly different from that in wild type animals. Our results suggest that AMBN does not influence osteogenic activity in vitro under the conditions used, and does not participate in craniofacial bone remodelling under mechanical stress and in repair of non-critical size bone defects.

Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro

Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions.

Transgenic Rescue of Enamel Phenotype in Ambn Null Mice

Ameloblastin null mice fail to make an enamel layer, but the defects could be due to an absence of functional ameloblastin or to the secretion of a potentially toxic mutant ameloblastin. We hypothesized that the enamel phenotype could be rescued by the transgenic expression of normal ameloblastin in Ambn mutant mice. We established and analyzed 5 transgenic lines that expressed ameloblastin from the amelogenin (AmelX) promoter and identified transgenic lines that express virtually no transgene, slightly less than normal (Tg+), somewhat higher than normal (Tg++), and much higher than normal (Tg+++) levels of ameloblastin. All lines expressing detectable levels of ameloblastin at least partially recovered the enamel phenotype. When ameloblastin expression was only somewhat higher than normal, the enamel covering the molars and incisors was of normal thickness, had clearly defined rod and interrod enamel, and held up well in function. We conclude that ameloblastin is essential for dental enamel formation.

Establishment of human dental epithelial cell lines expressing ameloblastin and enamelin by transfection of hTERT and cdk4 cDNAs

An in vitro cell culture system of dental epithelium is useful for the investigations of cellular differentiation and function of ameloblast in amelogenesis and of regenerative therapy in human tooth. However, there have been no immortalized human dental epithelial ameloblastic-lineage cell lines, which proliferate indefinitely and additionally produce enamel matrix proteins. We transfected two retroviral constructs of human telomerase reverse transcriptase (hTERT) cDNA and mouse cyclin-dependent kinase 4 (cdk4) cDNA into the primary ameloblastoma cells and isolated immortalized human dental epithelial cell lines of HAM1, HAM2 and HAM3. The three cell lines were examined by electron microscopy, assay of senescence-associated β-galactosidase activity, mRNA expression and immuno-reactivity of dental epithelial marker cell molecules and enamel matrix proteins. They showed undifferentiated phenotypes in monolayer culture and did not have any β-galactosidase activity. The transcripts of dental epithelial cell markers of Msx2, Jagged1, Notch1, Sp3, Sp6, keratin 14 and keratin 18 were confirmed. In addition, mRNA and protein expression of ameloblastin and enamelin were also detected in three cell lines. All cells in the three cell lines were keratin 14- and 18-positive and some elongated cells were Jagged1-positive. Msx2-positive nuclei were noted in only HAM2 cells. We established three cell lines by transfection of hTERT and cdk4 cDNAs, which were characterized as dental epithelial progenitor cells containing ameloblast-lineage cell phenotype.

Ameloblastin expression and putative autoregulation in mesenchymal cells suggest a role in early bone formation and repair

Ameloblastin is mainly known as a dental enamel protein, synthesized and secreted into developing enamel matrix by the enamel-forming ameloblasts. The function of ameloblastin in tooth development remains unclear, but it has been suggested to be involved in processes varying from regulating crystal growth to activity as a growth factor or partaking in cell signaling. Recent studies suggest that some enamel matrix proteins also might have important functions outside enamel formation. In this context ameloblastin has recently been reported to induce dentin and bone repair, as well as being present in the early bone and cartilage extracellular matrices during embryogenesis. However, what cells express ameloblastin in these tissues still remains unclear. Thus, the expression of ameloblastin was examined in cultured primary mesenchymal cells and in vivo during healing of bone defects in a “proof of concept” animal study. Real time RT-PCR analysis revealed human ameloblastin (AMBN) mRNA expression in human mesenchymal stem cells and primary osteoblasts and chondrocytes. Expression of AMBN mRNA was also confirmed in human CD34 positive cells and osteoclasts. Western and dot blot analysis of cell lysates and medium confirmed the expression and secretion of ameloblastin from mesenchymal stem cells, primary human osteoblasts and chondrocytes. Expression of ameloblastin was also detected in newly formed bone in experimental bone defects in adult rats. Together these findings suggest a role for this protein in early bone formation and repair.

Regulation of ameloblastin expression via gap junctional communication

Ameloblastin is a tooth‐specific glycoprotein that represents the most abundant enamel matrix protein, and plays an important role in maintaining the differentiation state of ameloblasts, serves as a cell adhesion molecule, and inhibits the proliferation of the dental epithelium. Our aim was to analyze the regulatory mechanism of ameloblastin expression via gap junctional communication using Gja1 null‐mice. Gja1 is one of the gap junctional proteins and highly expressed in tooth germ. Gja1 null‐mice showed disturbance of tooth germ, salivary gland, and lung morphogenesis, as similar to the phenotype of oculodento‐digital dysplasia. Inner dental epithelium in Gja1‐null mutants formed multiple cell layers. This phenotype was similar to that of ameloblastin mutant mice. Further, ameloblastin expression was dramatically decreased in Gja1‐null ameloblasts. TGF‐β1 administration induced ameloblastin expression in dental epithelial cell culture. On the other hand, Gap junction inhibitor 18α‐Gry and Oleamide inhibited the expression of ameloblastin induced by TGF‐β1. These results suggest that Gap junctional communication via Gja1 is important for the expression of ameloblastin and the differentiation of ameloblasts.Grant Funding Source: Ministry of Education, Culture, Sports, Science and Technology of Japan

Ameloblastin expression and effects in hard tissue (in vitro and in vivo)

Ameloblastin expression and effects in hard tissue (in vitro and in vivo)

Nordic PhD Conference in Dentistry at Lillehammer, Norway

A Nordic PhD conference was arranged at Lillehammer in the south-eastern part of Norway from February 11-13, 2009. This conference was the first in a series of events to mark the 50’s anniversary of the Faculty of Dentistry at the University of Oslo. The Faculty of Dentistry was established at the University of Oslo in 1959. From 1928 and until 1959 dental education and research in Norway were carried out in the dental school, Tannlegehoyskolen, in Oslo only. By inviting young scientists in the beginning of their career, focus was directed against the future rather than the past. When the dental school became part of the University, more emphasis was put on science. Organised programs were established for scientists already in the 1960’s and dental education based more on a scientific background introduced. This has, without doubt, improved the quality of the dental education during these 50 years. The same development has, of course, occurred in all the Nordic countries. It was therefore not a surprise that we at this conference could gather so many young, talented scientists from almost all the Nordic countries. The site of the conference was the historic town Lillehammer at the gateway to the Gudbrandsdalen valley. The town is with its 26,000 inhabitants the commercial, administrative, culture and educational centre of the region. In addition the town is world famous for hosting the Olympic Winter Games in 1994 and a popular tourist destination – with more than 1 million visitors every year. Lillehammer is the home of the Norwegian Film School and Lillehammer University College with about 4,000 students, and the town is also well known for its architecture and its active cultural life hosting institutions as Lillehammer Art museum, Lillehammer Town Orchestra, the homes of the Nobel Prize winners of Literature; Sigrid Undset and Bjornstjerne Bjornson and the historic museum at Maihaugen. About 75 PhD students, post doctors and scientists participated. Therefore, the conference gave a fairly good view of the current research activity in dentistry in the Nordic countries. More than 20 oral presentations were given. Topics varied from craniofacial growth and development and radiology in juvenile idiopathic arthritis, periimplantitis, osseointegration of implants, squamous cell carcinoma, oral carcinoma-associated fibroblasts, oral health epidemiology and periodontal conditions in developing countries and Crohns disease, probiotics and oral health, ameloblastin expression and its effect in hard tissues and gene expression during tooth development. In addition a series of posters were open for discussion during a separate session. Four keynote speakers were invited to give talks about implants or microbiology. The first two speakers were established scientists and well known internationally. Dr. Daniel Thoma from Center for Dental and Oral Medicine and Cranio Maxillofacial Surgery, University of Zurich, Switzerland, talked about “Potential of growth factors for localised ridge augumentation”. Professor Jan Eirik Ellingsen from Institute of Dentistry at the Faculty of Dentistry, University of Oslo, gave a presentation on “The impact of surface topography and chemistry on osseintegration of dental implants”. The keynote lectures in microbiology were given by two young scientists from Institute of Oral Biology at The Faculty of Dentistry, University of Oslo. Dr. Nibras Abdul-Majeed had finished her PhD in 2008 and talked about “The hidden bacterial lifestyle: from signaling to biofilms and survival”. Dr. Dorita Preza will defend her PhD in May this year, and gave a presentation with the title: “Oral microbiota of the elderly assessed by molecular methods.” This conference gave also the PhD candidates and post doctors the opportunity to create research networks and share scientific knowledge and experience with each other. Many of these researchers will hold head positions in the Nordic countries in the years to come. Therefore a close knowledge of colleagues in the other Nordic countries will be of outmost importance for future collaboration and research across the countries. A popular brake in the conference was the outdoor activities in the winterlandscape with sunshine and minus 20 degrees Celsius and included tobogganing, bungy-run, snow volley and ice sculpturing. The conference was closed by the previous dean of research at the Faculty of Dentistry, professor Ingar Olsen who had taken the initiative to the conference. We hope that similar conferences could be arranged every second year among the Nordic countries and look forward to the next meeting in 2011 in either Sweden or Denmark.

Bioinformatic analysis and molecular modelling of human ameloblastin suggest a two‐domain intrinsically unstructured calcium‐binding protein

Ameloblastin (AMBN) was originally believed to be an enamel-specific extracellular matrix glycoprotein secreted by ameloblasts. Recently, AMBN expression was also detected in developing mesenchymal dental hard tissues, in trauma-induced reparative dentin, and during early craniofacial bone formation. The function and structure of AMBN still remain ambiguous, and there are no known proteins with similar primary sequences. We therefore performed a bio-informatic analysis of AMBN to model ab initio the three-dimensional structure of the molecule. The results suggest that AMBN is a two-domain, intrinsically unstructured protein (IUP). The analysis did not reveal any regions with structural similarity to known receptor-ligand systems, and did not identify any higher-order structures similar to functional regions in other known sequences. The AMBN model predicts 11 defined regions exposed on the surface, internalizing the rest of the molecule including a human-specific insert. Molecular dynamics analysis identified one specific and several non-specific calcium-binding regions, mostly at the C-terminal part of the molecule. The model is supported by previous observations that AMBN is a bipolar calcium-binding molecule and hints at a possible role in protein-protein interactions. The model provides information useful for further studies on the function of AMBN.

Ameloblastin expression during craniofacial bone formation in rats

Based on previous results showing the expression of ameloblastin (Ambn; amelin) in the formation of mesenchymal dental hard tissues, we investigated its presence during bone development. Immunohistochemistry (IHC), in situ hybridization (ISH), and reverse transcription-polymerase chain reaction (RT-PCR) were used to investigate the expression of ameloblastin protein and mRNA during craniofacial development in rats. Tissue samples were collected on embryonic day 18 and from days 2-28 postnatally. IHC revealed the expression of ameloblastin during bone formation at embryonic and early postnatal stages with different patterns of expression in intramembranous and endochondral ossification. In intramembranous ossification, ameloblastin expression was detected in the superficial layer of the condensed vascularized primitive connective tissue and in the cellular layer covering the surface of the newly formed woven bone. In endochondral ossification, ameloblastin was expressed within the extracellular matrix of the cartilage templates and in the perichondrium. Between days 2 and 28 the expression decreased markedly, concordant with the maturation of the bone, and disappeared after completion of bone remodeling. The results obtained by IHC were confirmed by ISH and RT-PCR, showing the expression of ameloblastin mRNA during craniofacial bone formation. This study indicates the expression of the putative dental protein ameloblastin during craniofacial bone development in rats.

Expression of ameloblastin during enamel formation in a crocodile

Ameloblastin is an enamel-specific protein that plays critical roles in enamel formation, as well as adhesion between ameloblasts and the enamel matrix, as shown by analyses of ameloblastin-null mice. In the present study, we produced two distinct antibodies that recognize the N-terminus and C-terminus regions of caiman ameloblastin, in order to elucidate the fate of ameloblastin peptides during tooth development. An immunohistochemical study using the antibodies showed that caiman ameloblastin was a tooth-specific matrix protein that may initially be cleaved into two groups, N- and C-terminal peptides, as shown in mammals. The distribution of the N-terminal peptides was much different from that of the C-terminal peptides during enamel formation; however, it was similar to that of mammalian ameloblastin. Although ameloblastin is thought to have a relationship with the enamel prismatic structure in mammals, in the caiman, which has non-prismatic enamel, functional ameloblastin has no relationship with any enamel structure. Consequently, it is suggested that ameloblastin has kept its original functions during the evolutionary transition from reptiles to mammals and that it has been conserved in both lineages during more than 200 million years of evolution. Our results support the notion that ameloblastin acts as a factor for ameloblast adhesion to enamel matrix, because distribution of the C-terminal peptides was consistently restricted on the surface layers of enamel matrix specimens ranging from immature to nearly completely mature. The principal molecules that provide the adhesive function are presumably C-terminal peptides.

Ameloblastin and amelogenin expression in posnatal developing mouse molars

Ameloblastin and amelogenin are structural proteins present in the enamel matrix of developing teeth. Here we report the results of in situ hybridization analyses with DNA probes of ameloblastin and amelogenin expression in the mandibular first molars of ICR/Jcl mice from postnatal day 1 to day 15. Ameloblastin mRNA expression was observed in ameloblasts at day 2 while amelogenin mRNA was detected in secretory ameloblasts at day 3. Significant expression of both molecules was observed at days 4, 5 and 6, after which the levels decreased. Amelogenin expression ended on day 10, while ameloblastin mRNA was only weakly detected on day 12. Neither amelogenin nor ameloblastin expression was observed in day 15 mouse molars. Amelogenin and ameloblastin mRNAs were restricted to ameloblasts. We conclude that amelogenin and ameloblastin expression is enamel-specific, and suggest that these genes might be involved in the mineralization of enamel. It is possible that ameloblastin could participate in the attachment of ameloblasts to the enamel surface. In this case, the downregulation of expression may indicate the beginning of the maturation stage in which the ameloblasts tend to detach from the enamel layer.

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression.

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.

A Transgenic Animal Model Resembling Amelogenesis Imperfecta Related to Ameloblastin Overexpression

Genetic diseases that affect tooth enamel are grouped under the classification amelogenesis imperfecta. Human pedigrees and experiments on transgenic and null mice have all demonstrated that mutations to the secreted proteins amelogenin, enamelin, and enamelysin result in visibly, structurally, or mechanically defective enamel. In an attempt to better define a physiologic function for ameloblastin during enamel formation, we have produced transgenic mice that misexpress the ameloblastin gene. These transgenic animals exhibit imperfections in their enamel that is evident at the nanoscale level. Specifically, ameloblastin overexpression influences enamel crystallite habit and enamel rod morphology. These findings suggest enamel crystallite habit and rod morphology are influenced by the temporal and spatial expression of ameloblastin and may implicate the role of the ameloblastin gene locus in the etiology of a number of undiagnosed autosomally dominant cases of amelogenesis imperfecta.

Differential Epithelial and Mesenchymal Regulation of Tooth-Specific Matrix Proteins Expression by 1,25-Dihydroxyvitamin D 3 In Vivo

Enamel defects have been reported in rickets and related to disturbed expression of amelogenin in ameloblasts. The present study is devoted to amelogenin, enamelin, ameloblastin, and dentin sialo-phosphoprotein (DSPP) expression in both the epithelium and mesenchyme of vitamin D-deficient rat incisors. Quantitative Northern blotting analysis (relatively to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA steady-state levels) was performed on microdissected cells of rachitic ( m D) and control (+D) 56 day-old rats. Steady-state levels of amelogenin and enamelin mRNA were significantly reduced in the m D epithelium versus the +D epithelium ones. In contrast, ameloblastin expression was slightly increased in m D epithelium. In the same samples, DSPP mRNA levels remained unchanged in m D dental mesenchyme. Comparative electron microscopy studies between +D and m D animals showed a dramatic decrease of intraprismatic enamel (amelogenin and enamelin immunoreactive) consistent with our molecular results. In conclusion, tooth formation results from the coordinated expression of several matrix proteins that may be controlled by 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ].

Cloning, characterization and immunolocalization of human ameloblastin.

Amelogenesis imperfecta is a broad classification of hereditary enamel defects, exhibiting both genetic and clinical diversity. Most amelogenesis imperfecta cases are autosomal dominant disorders, yet only the local hypoplastic form has been mapped to human chromosome 4q between D4S242 1 and the albumin gene. An enamel protein cDNA, termed ameloblastin (also known as amelin and sheathlin), has been isolated from rat, mouse and pig. Its human homolog has been mapped to chromosome 4q21 between markers D4S409 and D4S400, flanking the local hypoplastic amelogenesis imperfecta critical region. Therefore, ameloblastin is a strong candidate gene for this form of amelogenesis imperfecta. To facilitate genetic studies related to this dental disease, we isolated and characterized a human ameloblastin cDNA. A human third molar cDNA library was screened and two ameloblastin clones identified. Nucleotide sequencing of these cDNAs indicated alternative splicing of the putative open reading frame, use of different polyadenylation signals, and a high degree of similarity to reported rat, mouse and porcine cDNAs. Immunohistochemistry studies on embryonic human teeth using an antibody to recombinant ameloblastin indicated ameloblastin expression by ameloblasts with localization in the enamel matrix associated with the sheath structures.

Sheath proteins: synthesis, secretion, degradation and fate in forming enamel.

We investigated expression of ameloblastin and sheathlin, recently cloned enamel matrix proteins from the rat and pig, in forming enamel immunocytochemically and immunochemically, using region-specific antibodies. The results obtained from the rat and pig were essentially the same. Antibodies which recognize the N-terminal region stained the secretory machinery of the secretory ameloblast and the entire thickness of the enamel matrix, especially the peripheral region of the enamel rod. Immunostained protein bands were observed near 65 or 70 kDa and below 20 kDa. C-terminal-specific antibodies stained the secretory machinery of the ameloblast and the immature enamel adjacent to the secretion sites. Immunostained protein bands were found ranging from 25 to 70 kDa. Antibodies which recognize a region in the protein just prior to the C-terminal region stained the cis-side of the Golgi apparatus but not the enamel matrix. Immunostained protein bands were observed of about 55 kDa. These results suggest that post-translational and post-secretory modifications of ameloblastin and sheathlin are similar to each other, and further showed that their cleaved N-terminal polypeptides concentrate in the prism sheath. We propose that sheathlin and ameloblastin share the same role in amelogenesis and should be classified as sheath proteins.

Identification and Characterization of a cDNA for Mouse Ameloblastin

Ameloblastin was first identified as one of the most abundant novel transcripts from a random screening of a rat incisor cDNA library. In situ hybridization experiments have shown ameloblastin expression to be specific to ameloblasts, with highest levels in secretory and maturation stage ameloblasts and cells of the epithelial root sheath. Ameloblastin has been identified as a candidate gene for the local hypoplastic form of autosomal dominant amelogenesis imperfecta, by virtue of it's location within the critical disease locus. The purpose of this study was to isolate a full length mouse ameloblastin cDNA and determine its temporal expression pattern during odontogenesis. A newborn mouse molar cDNA library was screened using a rat ameloblastin cDNA probe. Positive clones were confirmed by PCR analysis with ameloblastin-specific primers, and their size determined with vector-specific primers. Phage clones were rescued to phagemid using Exassist helper phage and the nucleotide sequence determined. We report here the identification of two clones, exhibiting alternative splicing of the putative open reading frame, and use of multiple polyadenylation signals. Nucleotide sequence analysis indicated a high degree of similarity to rat ameloblastin, rat amelin 1 and 2 and porcine sheathlin. Reverse transcriptase-PCR analysis using mouse first and second mandibular molar mRNA indicated initial expression at E-14. This is one day after the initial expression of tuftelin (E-13) and one day prior to that of amelogenin (E-15).

Ameloblastin expression in rat incisors and human tooth germs.

We recently identified ameloblastin as an ameloblast-specific gene product from a rat incisor cDNA library (Krebsbach et al., J. Biol. Chem. 271: 4431-4435, 1996). Here we report the developmental pattern of expression of ameloblastin in rat incisors and human tooth germs as visualized by in situ hybridization and immunochemistry. Compared to the expression of amelogenin, the major ameloblast product, ameloblastin mRNA was more widely expressed in ameloblasts from the presecretory to the late maturation stage of development. Ameloblastin mRNA was first observed in the juxtanuclear cytoplasm or presecretory stage ameloblasts, gradually increased in the distal cytoplasm of secretory stage ameloblasts and was found throughout the cytoplasm of early to late maturation stage ameloblasts. The immunostaining of ameloblastin, using a monospecific antibody raised against a recombinant protein, showed intense reactivity in Tomes' processes of secretory stage ameloblasts and surrounding enamel. The immunoreaction was concentrated in the juxtanuclear cytoplasm of late maturation stage ameloblasts. High-resolution colloidal gold immunocytochemistry established the presence of ameloblastin antigenicity in the Golgi apparatus, secretory granules in Tomes' process and enamel. Human tooth germs in early to late bell stage also expressed ameloblastin mRNA and ameloblastin antigenicity in the ameloblasts. Western blot analysis of protein extracts from rat incisor tissues indicated that ameloblastin can be found in the enamel epithelial tissue and in mineralized enamel, as well as in the EDTA decalcification solution. These data indicate that ameloblastin is an ameloblast secretory product which is sequentially expressed from the presecretory to the late maturation stage in rat and human teeth. This unique developmental pattern suggests that ameloblastin may have a broader role in amelogenesis than amelogenin and tuftelin.