Store-operated Ca2+ Entry Modulates the Expression of Enamel Genes.

Dental enamel formation is an intricate process tightly regulated by ameloblast cells. The correct spatiotemporal patterning of enamel matrix protein (EMP) expression is fundamental to orchestrate the formation of enamel crystals, which depend on a robust supply of Ca2+. In the extracellular milieu, Ca2+ -EMP interactions occur at different levels. Despite its recognized role in enamel development, the molecular machinery involved in Ca2+ homeostasis in ameloblasts remains poorly understood. A common mechanism for Ca2+ influx is store-operated Ca2+ entry (SOCE). We evaluated the possibility that Ca2+ influx in enamel cells might be mediated by SOCE and the Ca2+ release-activated Ca2+ (CRAC) channel, the prototypical SOCE channel. Using ameloblast-like LS8 cells, we demonstrate that these cells express Ca2+ -handling molecules and mediate Ca2+ influx through SOCE. As a rise in the cytosolic Ca2+ concentration is a versatile signal that can modulate gene expression, we assessed whether SOCE in enamel cells had any effect on the expression of EMPs. Our results demonstrate that stimulating LS8 cells or murine primary enamel organ cells with thapsigargin to activate SOCE leads to increased expression of Amelx, Ambn, Enam, Mmp20. This effect is reversed when cells are treated with a CRAC channel inhibitor. These data indicate that Ca2+ influx in LS8 cells and enamel organ cells is mediated by CRAC channels and that Ca2+ signals enhance the expression of EMPs. Ca2+ plays an important role not only in mineralizing dental enamel but also in regulating the expression of EMPs.

The role of bioactive nanofibers in enamel regeneration mediated through integrin signals acting upon C/EBPα and c-Jun.

Enamel formation involves highly orchestrated intracellular and extracellular events; following development, the tissue is unable to regenerate, making it a challenging target for tissue engineering. We previously demonstrated the ability to trigger enamel differentiation and regeneration in the embryonic mouse incisor using a self-assembling matrix that displayed the integrin-binding epitope RGDS (Arg-Gly-Asp-Ser). To further elucidate the intracellular signaling pathways responsible for this phenomenon, we explore here the coupling response of integrin receptors to the biomaterial and subsequent downstream gene expression profiles. We demonstrate that the artificial matrix activates focal adhesion kinase (FAK) to increase phosphorylation of both c-Jun N-terminal kinase (JNK) and its downstream transcription factor c-Jun (c-Jun). Inhibition of FAK blocked activation of the identified matrix-mediated pathways, while independent inhibition of JNK nearly abolished phosphorylated-c-Jun (p-c-Jun) and attenuated the pathways identified to promote enamel regeneration. Cognate binding sites in the amelogenin promoter were identified to be transcriptionally up-regulated in response to p-c-Jun. Furthermore, the artificial matrix induced gene expression as evidenced by an increased abundance of amelogenin, the main protein expressed during enamel formation, and the CCAAT enhancer binding protein alpha (C/EBPα), which is the known activator of amelogenin expression. Elucidating these cues not only provides guidelines for the design of synthetic regenerative strategies and opportunities to manipulate pathways to regulate enamel regeneration, but can provide insight into the molecular mechanisms involved in tissue formation.

Biological response on a titanium implant-grade surface functionalized with modular peptides.

Titanium (Ti) and its alloys are among the most successful implantable materials for dental and orthopedic applications. The combination of excellent mechanical and corrosion resistance properties makes them highly desirable as endosseous implants that can withstand a demanding biomechanical environment. Yet, the success of the implant depends on its osteointegration, which is modulated by the biological reactions occurring at the interface of the implant. A recent development for improving biological responses on the Ti-implant surface has been the realization that bifunctional peptides can impart material binding specificity not only because of their molecular recognition of the inorganic material surface, but also through their self-assembly and ease of biological conjugation properties. To assess peptide-based functionalization on bioactivity, the present authors generated a set of peptides for implant-grade Ti, using cell surface display methods. Out of 60 unique peptides selected by this method, two of the strongest titanium binding peptides, TiBP1 and TiBP2, were further characterized for molecular structure and adsorption properties. These two peptides demonstrated unique, but similar molecular conformations different from that of a weak binder peptide, TiBP60. Adsorption measurements on a Ti surface revealed that their disassociation constants were 15-fold less than TiBP60. Their flexible and modular use in biological surface functionalization were demonstrated by conjugating them with an integrin recognizing peptide motif, RGDS. The functionalization of the Ti surface by the selected peptides significantly enhanced the bioactivity of osteoblast and fibroblast cells on implant-grade materials.

Enamel matrix proteins; old molecules for new applications.

Emdogain (enamel matrix derivative, EMD) is well recognized in periodontology, where it is used as a local adjunct to periodontal surgery to stimulate regeneration of periodontal tissues lost to periodontal disease. The biological effect of EMD is through stimulation of local growth factor secretion and cytokine expression in the treated tissues, inducing a regenerative process that mimics odontogenesis. The major (>95%) component of EMD is Amelogenins (Amel). No other active components have so far been isolated from EMD, and several studies have shown that purified amelogenins can induce the same effect as the complete EMD. Amelogenins comprise a family of highly conserved extracellular matrix proteins derived from one gene. Amelogenin structure and function is evolutionary well conserved, suggesting a profound role in biomineralization and hard tissue formation. A special feature of amelogenins is that under physiological conditions the proteins self-assembles into nanospheres that constitute an extracellular matrix. In the body, this matrix is slowly digested by specific extracellular proteolytic enzymes (matrix metalloproteinase) in a controlled process, releasing bioactive peptides to the surrounding tissues for weeks after application. Based on clinical and experimental observations in periodontology indicating that amelogenins can have a significant positive influence on wound healing, bone formation and root resorption, several new applications for amelogenins have been suggested. New experiments now confirm that amelogenins have potential for being used also in the fields of endodontics, bone regeneration, implantology, traumatology, and wound care.

Role of NBCe1 and AE2 in secretory ameloblasts.

The H(+)/base transport processes that control the pH of the microenvironment adjacent to ameloblasts are not currently well-understood. Mice null for the AE2 anion exchanger have abnormal enamel. In addition, persons with mutations in the electrogenic sodium bicarbonate co-transporter NBCe1 and mice lacking NBCe1 have enamel abnormalities. These observations suggest that AE2 and NBCe1 play important roles in amelogenesis. In the present study, we aimed to understand the roles of AE2 and NBCe1 in ameloblasts. Analysis of the data showed that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical membrane. Transcripts for AE2a and NBCe1-B were detected in RNA isolated from cultured ameloblast-like LS8 cells. Our data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts in vivo in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation.

Cellular uptake of amelogenin, and its localization to CD63, and Lamp1-positive vesicles.

Proteins of the developing enamel matrix include amelogenin, ameloblastin and enamelin. Of these three proteins amelogenin predominates. Protein-protein interactions are likely to occur at the ameloblast Tomes' processes between membrane-bound proteins and secreted enamel matrix proteins. Such protein-protein interactions could be associated with cell signaling or endocytosis. CD63 and Lamp1 are ubiquitously expressed, are lysosomal integral membrane proteins, and localize to the plasma membrane. CD63 and Lamp1 interact with amelogenin in vitro. In this study our objective was to study the molecular events of intercellular trafficking of an exogenous source of amelogenin, and related this movement to the spatiotemporal expression of CD63 and Lamp1 using various cell lineages. Exogenously added amelogenin moves rapidly into the cell into established Lamp1-positive vesicles that subsequently localize to the perinuclear region. These data indicate a possible mechanism by which amelogenin, or degraded amelogenin peptides, are removed from the extracellular matrix during enamel formation and maturation.

Controlled failure mechanisms toughen the dentino-enamel junction zone.

STATEMENT OF PROBLEM: The dentino-enamel junction (DEJ) durably unites dissimilar hard brittle enamel and tough flexible dentin. In contrast to artificial bonds between restorations and dentin, the DEJ rarely fails except when it is affected by inherited disorders. Knowledge of DEJ toughening mechanisms is important in understanding inherited disorders, in biomimetic engineering of junctions between artificial restorations and teeth, and in tissue-engineering a DEJ. PURPOSE: The purpose of this study was to identify specific DEJ-zone failure mechanisms and to survey the fracture toughness of the human DEJ zone. MATERIAL AND METHODS: Fracture toughness indentations were made at 3 sites across the DEJ zone of 10 human incisor teeth. Failure modes identified using optical microscopy and fracture toughness (MPa.m(1/2)) were calculated following Vickers microindentation. Site mean values were then calculated and compared using 1-way analysis of variance (alpha=.05). RESULTS: The DEJ did not undergo catastrophic interfacial delamination; instead, damage was distributed over a broad zone. The primary damage mode involved cracking and damage dispersion in the specialized first-formed enamel close to the DEJ. Multiple, somewhat convoluted and sometimes branching, cracks spread and diffused damage over a wide area of adjacent enamel rather than producing catastrophic interfacial failure. Other secondary mechanisms included short microcracks in the DEJ adjacent dentin with possible cracked bridging, as well as plastic deformation of the DEJ without delamination. A DEJ-zone fracture toughness of approximately 0.8 to 0.9 MPa.m(1/2) was calculated. CONCLUSION: DEJ-zone damage occurred primarily within the adjacent layer of specialized first-formed enamel, and the optical DEJ interface resisted delamination.

Tooth developmental biology: disruptions to enamel-matrix assembly and its impact on biomineralization.

Dental enamel is a composite bioceramic material that is the hardest tissue in the vertebrate body, containing long, thin crystallites of substituted hydroxyapatite (HAP). Over a lifetime of an organism, enamel functions under repeated and immense loads, generally without catastrophic failure. Enamel is a product of ectoderm-derived cells called ameloblasts. Recent investigations on the formation of enamel using cell and molecular approaches are now being coupled to biomechanical investigations at the nanoscale and mesoscale levels. For amelogenin, the principal structural protein for forming enamel, we have identified two domains that are required for its proper self-assembly into supramolecular structures referred to as nanospheres. Nanospheres are believed to control HAP crystal habit. Other structural proteins of the enamel matrix include ameloblastin and enamelin, but little is known about their biological importance. Transgenic animals have been prepared to investigate the effect of overexpression of wild-type or mutated enamel proteins on the developing enamel matrix. Amelogenin transgenes were engineered to contain deletions to either of the two self-assembly domains and these alterations produced significant defects in the enamel. Additional transgenic animal lines have been prepared and studied and each gives additional insights into the mechanisms for enamel biofabrication. This study summarizes the observed enamel phenotypes of recently derived transgenic animals. These data are being used to help define the role of each of the enamel structural proteins in enamel and study how each of these proteins impact on enamel biomineralization.

Structural organization and cellular localization of tuftelin-interacting protein 11 (TFIP11).

Tuftelin-interacting protein (TFIP11) was first identified in a yeast two-hybrid screening as a protein interacting with tuftelin. The ubiquitous expression of TFIP11 suggested that it might have other functions in non-dental tissues. TFIP11 contains a G-patch, a protein domain believed to be involved in RNA binding. Using a green fluorescence protein tag, TFIP11 was found to locate in a novel subnuclear structure that we refer to as the TFIP body. An in vivo splicing assay demonstrated that TFIP11 is a novel splicing factor. TFIP11 diffuses from the TFIP body following RNase A treatment, suggesting that the retention of TFIP11 is RNA dependent. RNA polymerase II inhibitor (-amanitin and actinomycin D) treatment causes enlargement in size and decrease in number of TFIP bodies, suggesting that TFIP bodies perform a storage function rather than an active splicing function. The TFIP body may therefore represent a new subnuclear storage compartment for splicing components.

Ultrastructure of forming enamel in mouse bearing a transgene that disrupts the amelogenin self-assembly domains.

The mouse X-chromosomal amelogenin gene promoter was used to drive the expression of mutated amelogenin proteins in vivo. Two different transgenic mouse lines based on deletions to either the amino-terminal (A-domain deletions) or to the carboxyl-region (B-domain deletions) were bred. In the molars of newborn A-domain deleted transgenic mice the formation of the initial layer of aprismatic enamel was delayed. There were severe structural alterations in the enamel of incisors of newborn mice bearing the A-domain deletion which were not apparent in animals bearing the B-domain deletion. In the A-domain-deleted animals, stippled material accumulated throughout the entire thickness of the forming enamel apparently causing a disruption of the normal rod-to-inter-rod relationship. This stippled material was likened to and interpreted as being groupings of amelogenin nanospheres. In the B-domain-deleted animals the stippled material was detected only in minute defects of the forming enamel. These data suggest significant differences in nanosphere assembly properties for animals bearing either the A-domain or the B-domain-deleted transgene. The present in vivo experimental approach suggests that at early stages of enamel formation, the A-domain plays a greater role than does the B-domain in amelogenin self-assembly, and consequently in enamel architecture and structure.

Regulated gene expression dictates enamel structure and tooth function.

Enamel is a complex bioceramic tissue. In its final form, enamel is a reflection of the unique molecular and cellular activities occurring during organogenesis. From the ectodermal origins of ameloblasts, their gene activity and protein expression profiles exist for the sole purpose of producing a mineralized shell, almost entirely devoid of protein, deposited over the 'bone-like' dentine. The interface between enamel and dentine is referred to as the dentine enamel junction and it is also unique in its biology. This review article is narrow in its scope. We restrict our review to selected advances in our understanding of the genetic, molecular and structural aspects of enamel biology. We present a model of enamel formation that relates gene expression to the assembly of an extracellular protein matrix that in turn controls the structural hierarchy and mechanical aspects of enamel and the tooth organ.

Biological organization of hydroxyapatite crystallites into a fibrous continuum toughens and controls anisotropy in human enamel.

Enamel forms the outer surface of teeth, which are of complex shape and are loaded in a multitude of ways during function. Enamel has previously been assumed to be formed from discrete rods and to be markedly aniostropic, but marked anisotropy might be expected to lead to frequent fracture. Since frequent fracture is not observed, we measured enamel organization using histology, imaging, and fracture mechanics modalities, and compared enamel with crystalline hydroxyapatite (Hap), its major component. Enamel was approximately three times tougher than geologic Hap, demonstrating the critical importance of biological manufacturing. Only modest levels of enamel anisotropy were discerned; rather, our measurements suggest that enamel is a composite ceramic with the crystallites oriented in a complex three-dimensional continuum. Geologic apatite crystals are much harder than enamel, suggesting that inclusion of biological contaminants, such as protein, influences the properties of enamel. Based on our findings, we propose a new structural model.

Antagonistic regulation of Dlx2 expression by PITX2 and Msx2: implications for tooth development.

The transcriptional mechanisms underlying tooth development are only beginning to be understood. Pitx2, a bicoid-like homeodomain transcription factor, is the first transcriptional marker observed during tooth development. Because Pitx2, Msx2, and Dlx2 are expressed in the dental epithelium, we examined the transcriptional activity of PITX2 in concert with Msx2 and the Dlx2 promoter. PITX2 activated while Msx2 unexpectedly repressed transcription of a TK-Bicoid luciferase reporter in a tooth epithelial cell line (LS-8) and CHO cell line. Surprisingly, Msx2 binds to the bicoid element (5'-TAATCC-3') with a high specificity and competes with PITX2 for binding to this element. PITX2 binds to bicoid and bicoid-like elements in the Dlx2 promoter and activates this promoter 45-fold in CHO cells. However, it is only modestly activated in the LS-8 tooth epithelial cell line that endogenously expresses Msx2 and Pitx2. RT-PCR and Western blot assays reveal that two Pitx2 isoforms are expressed in the LS-8 cells. We further demonstrate that PITX2 dimerization can occur through the C-terminus of PITX2. Msx2 represses the Dlx2 promoter in CHO cells and coexpression of both PITX2 and Msx2 resulted in transcriptional antagonism of the Dlx2 promoter. Electrophoretic mobility shift assays demonstrate that factors in the LS-8 cell line specifically interact with PITX2. Thus, Dlx2 gene transcription is regulated by antagonistic effects between PITX2, Msx2, and factors expressed in the tooth epithelia.

Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy.

Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in amelogenin to protein-protein interactions during the self-assembly process. Mouse amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and amelogenins having single or double amino acid mutations identical to those found in cases of amelogenesis imperfecta were analyzed. The polyhistidine-containingfull-length recombinant amelogenin protein [rp(H)M180] generated nanospheres with monodisperse size distribution (hydrodynamic radius of 20.7 +/- 2.9 nm estimated from DLS and 16.1 +/- 3.4 nm estimated from AFM images), comparable to nanospheres formed by full-length amelogenin rM179 without the polyhistidine domain, indicating that this histidine modification did not interfere with the self-assembly process. Deletion of the N-terminal self-assembly domain from amelogenin and their substitution by a FLAG epitope ("A"-domain deletion) resulted in the formation of assemblies with a heterogeneous size distribution with the hydrodynamic radii of particles ranging from 3 to 38 nm. A time-dependent dynamic light scattering analysis of amelogenin molecules lacking amino acids 157 through 173 and containing a hemagglutinin epitope ("B"-domain deletion) resulted in the formation of particles (21.5 +/- 6.8 nm) that fused to form larger particles of 49.3 +/- 4.3 nm within an hour. Single and double point mutations in the N-terminal region resulted in the formation of larger and more heterogeneous nanospheres. The above data suggest that while the N-terminal A-domain is involved in the molecular interactions for the formation of nanospheres, the carboxy-terminal B-domain contributes to the stability and homogeneity of the nanospheres, preventing their fusion to larger assemblies. These in vitro findings support the notion that the proteolytic cleavage of amelogenin at amino- and carboxy-terminii occurring during enamel formation influences amelogenin to amelogenin interactions during self-assembly and hence alters the structural organization of the developing enamel extracellular matrix, thus affecting enamel biomineralization.

Functional antagonism between Msx2 and CCAAT/enhancer-binding protein alpha in regulating the mouse amelogenin gene expression is mediated by protein-protein interaction.

Ameloblast-specific amelogenin gene expression is spatiotemporally regulated during tooth development. In a previous study, the CCAAT/enhancer-binding protein alpha (C/EBPalpha) was identified as a transcriptional activator of the mouse amelogenin gene in a cell type-specific manner. Here, Msx2 is shown to repress the promoter activity of amelogenin-promoter reporter constructs independent of its intrinsic DNA binding activity. In transient cotransfection assays, Msx2 and C/EBPalpha antagonize each other in regulating the expression of the mouse amelogenin gene. Electrophoresis mobility shift assays demonstrate that Msx2 interferes with the binding of C/EBPalpha to its cognate site in the mouse amelogenin minimal promoter, although Msx2 itself does not bind to the same promoter fragment. Protein-protein interaction between Msx2 and C/EBPalpha is identified with co-immunoprecipitation analyses. Functional antagonism between Msx2 and C/EBPalpha is also observed on the stably transfected 2.2-kilobase mouse amelogenin promoter in ameloblast-like LS8 cells. Furthermore, the carboxyl-terminal residues 183-267 of Msx2 are required for protein-protein interaction, whereas the amino-terminal residues 2-97 of Msx2 play a less critical role. Among three family members tested (C/EBPalpha, -beta, and -gamma), Msx2 preferentially interacts with C/EBPalpha. Taken together, these data indicate that protein-protein interaction rather than competition for overlapping binding sites results in the functional antagonism between Msx2 and C/EBPalpha in regulating the mouse amelogenin gene expression.

A tuftelin-interacting protein (TIP39) localizes to the apical secretory pole of mouse ameloblasts.

Enamel biomineralization is a complex process that involves interactions between extracellular matrix proteins. To identify proteins interacting with tuftelin, a potential nucleator of enamel crystallites, the yeast two-hybrid system was applied to a mouse tooth expression library and a tuftelin-interacting protein (TIP) was isolated for further characterization. Polyclonal antibodies were prepared against two recombinant variants of this protein. Both antibodies identified a major protein product in tooth organs at 39 kDa, and this protein has been called TIP39. Northern analysis showed TIP39 messenger RNA in multiple organs, a pattern similar to that of tuftelin messenger RNA. In situ hybridization of mandibles of 1-day-old mice detected TIP39 RNA in secretory ameloblasts and odontoblasts. Immunolocalization of TIP39 and tuftelin in cultured ameloblast-like cells showed that these two proteins colocalize. Within the developing tooth organ, TIP39 and tuftelin immunolocalized to the apical pole of secretory ameloblasts (Tomes' processes) and to the newly secreted extracellular enamel matrix. TIP39 amino acid sequence appears to be highly conserved with similarities to proteins in species as diverse as yeast and primates. Available sequence data and the findings reported here suggest a role for TIP39 in the secretory pathway of extracellular proteins.

Identification of CCAAT/enhancer-binding protein alpha as a transactivator of the mouse amelogenin gene.

Amelogenin expression is ameloblast-specific and developmentally regulated at the temporal and spatial levels. In a previous transgenic mouse analysis, the expression pattern of the endogenous amelogenin gene was recapitulated by a reporter gene driven by a 2. 2-kilobase mouse amelogenin proximal promoter. To understand the molecular mechanisms underlying the spatiotemporal expression of the amelogenin gene during odontogenesis, the mouse amelogenin promoter was systematically analyzed in mouse ameloblast-like LS8 cells. Deletion analysis identified a minimal promoter (-70/+52) containing a CCAAT/enhancer-binding protein (C/EBP)-binding site upstream of the TATA box. In transient transfection assays, C/EBPalpha up-regulated the promoter activity in a dose-dependent manner. The C/EBP-binding site was necessary for both C/EBPalpha-mediated transactivation and basal promoter activity. Electrophoresis mobility shift assays demonstrated that C/EBPalpha bound to its cognate site in the amelogenin promoter and that the binding was specific. Endogenous C/EBPalpha was detected in LS8 cells, and overexpression of exogenous C/EBPalpha in LS8 cells was able to increase the expression level of the endogenous amelogenin protein. The activity of the amelogenin promoter in rat parotid Pa-4 cells and Madin-Darby canine kidney cells was minimal, ranging from 20 to 30% of the activity in ameloblast-like cells. Transient transfection experiments showed that C/EBPalpha transactivated the mouse amelogenin reporter gene in Pa-4 cells, but not in Madin-Darby canine kidney cells. Taken together, these data indicate that C/EBPalpha is a bona fide transcriptional activator of the mouse amelogenin gene in a cell type-specific manner.

Enamel biomineralization defects result from alterations to amelogenin self-assembly.

Enamel formation is a powerful model for the study of biomineralization. A key feature common to all biomineralizing systems is their dependency upon the biosynthesis of an extracellular organic matrix that is competent to direct the formation of the subsequent mineral phase. The major organic component of forming mouse enamel is the 180-amino-acid amelogenin protein (M180), whose ability to undergo self-assembly is believed to contribute to biomineralization of vertebrate enamel. Two recently defined domains (A and B) within amelogenin appear essential for this self-assembly. The significance of these two domains has been demonstrated previously by the yeast two-hybrid system, atomic force microscopy, and dynamic light scattering. Transgenic animals were used to test the hypothesis that the self-assembly domains identified with in vitro model systems also operate in vivo. Transgenic animals bearing either a domain-A-deleted or domain-B-deleted amelogenin transgene expressed the altered amelogenin exclusively in ameloblasts. This altered amelogenin participates in the formation an organic enamel extracellular matrix and, in turn, this matrix is defective in its ability to direct enamel mineralization. At the nanoscale level, the forming matrix adjacent to the secretory face of the ameloblast shows alteration in the size of the amelogenin nanospheres for either transgenic animal line. At the mesoscale level of enamel structural hierarchy, 6-week-old enamel exhibits defects in enamel rod organization due to perturbed organization of the precursor organic matrix. These studies reflect the critical dependency of amelogenin self-assembly in forming a competent enamel organic matrix and that alterations to the matrix are reflected as defects in the structural organization of enamel.

Cloning and characterization of the murine ameloblastin promoter.

The molecular mechanisms directing the highly restricted expression pattern of murine ameloblastin were characterized by cloning and functional analysis of the ameloblastin promoter. The transcription start site, mapped by primer extension, was located 19 base pairs (bp) 5' of the published cDNA. The promoter was analyzed in a mouse ameloblast-like cell line (LS8) and was compared with promoter activity in primary gingival fibroblasts and pulp fibroblasts. Sequential 5'-deletion mutants encompassing DNA sequences from -1616 to -781 bp exhibited high promoter activity in LS8 cells, whereas the promoter activity decreased to 50% of the full-length construct in the -781- and -477-bp regions. The -217-bp promoter region regained promoter activity that approached the activity of the full-length promoter construct, suggesting that both positive and negative cis-acting regions may be involved in ameloblastin transcriptional regulation. Activity of the ameloblastin promoter in gingival and pulp fibroblasts was minimal and ranged from 8 to 30% of the activity in ameloblast-like cells. Several DNA-protein complexes were formed between functionally important promoter fragments and nuclear extracts from LS8 cells. The inactivity of promoter constructs in pulp and gingival fibroblasts as well as the absence of similar DNA-protein complexes from these cells suggest that regulatory regions of the murine ameloblastin promoter may function in a cell-specific manner.