Biomedical subjects

Mary MacDougall

Publications and source records attributed to Mary MacDougall.

18 recordsLinked to original sources

PubMed2006

Molecular basis of human dentin diseases.

In recent years, substantial progress has been made regarding the molecular etiology of human structural tooth diseases that alter dentin matrix formation. These diseases have been classified into two major groups with subtypes: dentin dysplasia (DD) types I and II and dentinogenesis imperfecta (DGI) types I-III. Genetic linkage studies have identified the critical loci for DD-II, DGI-II, and DGI-II to human chromosome 4q21. Located within the common disease loci for these diseases is cluster of dentin/bone genes that includes osteopontin (OPN), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein (MEPE), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP). To date, only mutations within dentin sialophosphoprotein have been associated with the pathogenesis of dentin diseases including DGI types-II and -III and DD-II. In this article, we overview the recent literature related to these dentin genetic diseases, their clinical features, and molecular pathogenesis.

Chromosome Mapping↗

PubMed2006

In vitro cytotoxicity of a low-shrinkage polymerizable liquid crystal resin monomer.

The objective of this study was to determine the in vitro cytotoxicity of novel, polymerizable liquid crystal resin monomers when placed in direct contact with dental and nondental cell lines. One common dimethacrylate and three liquid crystal compounds, Bis-glycidyl methacrylate (Bis-GMA), 2-(t-butyl)-1,4-bis-{4-(6-acryloxy-hexane-1-oxy)-benzoyloxy}-benzene (C6), 2-(t-butyl), 1-[6-(3-acryloxy-propionoxy)-hexane-1-oxy-benzoyloxy], 4-[4-(6-acryloxy-hexane-1-oxy)-benzoyloxy]-benxene (by-product), and a 3:2 mixture of C6 and by-product, respectively, were tested for relative cytotoxicity in vitro. Cultured dental and nondental cells were treated for 24 h with test compound dissolved in media over a fourfold range of concentration (10(-4) -10(-7) mol/L). Cytotoxicity was measured using the WST-1 reagent as an indicator of remaining cell numbers based on the reduction of WST-1 substrate by mitochondrial dehydrogenases in viable cells. Bis-GMA ID(50) was found to be consistent with ID(50) values reported in the literature. A small but significant difference in the sensitivity of the dental and nondental cells in regard to their response to this dimethacrylate was noted. The liquid crystal resin monomers were significantly less cytotoxic to all cell lines tested. ID(50) values of >1 x 10(-4) mol/L were registered for the C6 and by-product monomers alone. The 3:2 mixture of C6 and by-product had a slightly higher cytotoxicity (ID(50) = 1 x 10(-4) mol/L); however, this remained significantly less than that of Bis-GMA. The results demonstrate that the newly synthesized low-shrinkage, polymerizable liquid crystal resin monomers demonstrate a minimal cytotoxic effect on both dental and nondental cells. These data suggest that the low-shrinkage liquid crystal resin monomers will not elicit a response by oral tissues (pulp tissue) when used to repair carious lesions in posterior teeth.

Animals↗

PubMed2005

In vitro cytotoxicity of a remineralizing resin-based calcium phosphate cement.

UNLABELLED: Recently, a resin-based calcium phosphate cement (RCPC) has been reported as a remineralizing pulp-capping or lining cement. RCPC consists mainly of tetracalcium and dicalcium phosphates, ethoxylated bisphenol A dimethacrylate and pyromellitic glycerol dimethacrylate monomers and photo- and chemical initiators. OBJECTIVES: Here, the cytotoxic effects of RCPC were evaluated. The hypothesis was that RCPC induced only minor cytotoxic response in immortalized murine odontoblast and pulp cells, comparable to that produced by similar dimethacrylates due to unpolymerized dimethacrylate monomer present after curing. METHODS: Cytotoxicity was determined following the changes in cell succinate dehydrogenase activity after 24 h exposure to the cement components and after a 24 h recovery period. A fourfold range of concentrations was tested of the monomers, the eluate of cured RCPC leached in Dulbecco's modified Eagle's medium, and crushed cured cement in dimethyl sulfoxide. RESULTS: The monomers themselves had cytotoxicities similar to those reported for other dimethacrylates, although they are significantly less toxic than Bis-GMA. Differential cell sensitivity was demonstrated, with the pulp cells having greater sensitivity to the unpolymerized monomer than the odontoblast-like cells. The leached components have cytotoxicity similar to that of the free monomers. The crushed material demonstrated no apparent cytotoxicity at the dilutions tested. SIGNIFICANCE: These data demonstrate that RCPC has an in vitro cytotoxicity that is comparable to other materials containing dimethacrylate monomers and suggest that the material may be suitable for use in dental restorations. The data also indicate that the pulp cells appear more sensitive to dimethacrylates than the odontoblasts.

Animals↗

PubMed2005

Dentin sialoprotein and dentin phosphoprotein overexpression during amelogenesis.

The gene for dentin sialophosphoprotein produces a single protein that is post-translationally modified to generate two distinct extracellular proteins: dentin sialoprotein and dentin phosphoprotein. In teeth, dentin sialophosphoprotein is expressed primarily by odontoblast cells, but is also transiently expressed by presecretory ameloblasts. Because of this expression profile it appears that dentin sialophosphoprotein contributes to the early events of amelogenesis, and in particular to those events that result in the formation of the dentino-enamel junction and the adjacent "aprismatic" enamel. Using a transgenic animal approach we have extended dentin sialoprotein or dentin phosphoprotein expression throughout the developmental stages of amelogenesis. Overexpression of dentin sialoprotein results in an increased rate of enamel mineralization, however, the enamel morphology is not significantly altered. In wild-type animals, the inclusion of dentin sialoprotein in the forming aprismatic enamel may account for its increased hardness properties, when compared with bulk enamel. In contrast, the overexpression of dentin phosphoprotein creates "pitted" and "chalky" enamel of non-uniform thickness that is more prone to wear. Disruptions to the prismatic enamel structure are also a characteristic of the dentin phosphoprotein overexpressing animals. These data support the previous suggestion that dentin sialoprotein and dentin phosphoprotein have distinct functions related to tooth formation, and that the dentino-enamel junction should be viewed as a unique transition zone between enamel and the underlying dentin. These results support the notion that the dentin proteins expressed by presecretory ameloblasts contribute to the unique properties of the dentino-enamel junction.

Amelogenesis↗

PubMed2005

DLX3 mutation associated with autosomal dominant amelogenesis imperfecta with taurodontism.

Amelogenesis imperfecta hypoplastic-hypomaturation with taurodontism (AIHHT) is an autosomal dominant (AD) trait associated with enamel defects and enlarged pulp chambers. In this study, we mapped an AIHHT family to human chromosome 17 q21-q22 (lod score 3.3) and identify a two basepair deletion (CT) at nucleotide 560 in DLX3 associated with the disease. This mutation causes a frameshift altering the last two amino acids of the DNA-binding homeodomain introducing a premature stop codon truncating the protein by 88 amino acids. This is the first report of a mutation within the homeodomain of DLX3. Previous studies have shown a DLX3 mutation outside the homeodomain associated with tricho-dento-osseous syndrome (TDO) suggesting TDO and some forms of AIHHT are allelic.

Abnormalities, Multiple↗

PubMed2005

Effects of sodium fluoride on the actin cytoskeleton of murine ameloblasts.

Fluoride is associated with a decrease in the incidence of dental caries, but excess fluoride can lead to enamel fluorosis, a defect that occurs during tooth enamel formation. In fibroblasts, the Arhgap gene encodes a RhoGAP, which regulates the small G protein designated RhoA. Fluoride treatment of fibroblasts inactivates RhoGAP, thereby activating RhoA, which leads to elevation of filamentous actin (F-actin). Since RhoA is a molecular switch, our hypothesis is that in ameloblasts, fluoride may alter the cytoskeleton through interference with the Rho signaling pathway. Our objective was to measure the effects of sodium fluoride on F-actin using tooth organ culture and confocal microscopy. The results indicated that cellular responses to fluoride include elevation of F-actin in ameloblasts. It was concluded from immunohistochemistry, RT-PCR and confocal approaches that the components of the Rho pathway are present in ameloblasts, and that the response to fluoride involves the Rho/ROCK pathway.

Actins↗

PubMed2005

Dentin phosphoprotein compound mutation in dentin sialophosphoprotein causes dentinogenesis imperfecta type III.

A rare compound mutation involving a 36 bp deletion and 18 bp insertion within exon 5 of the dentin sialophosphoprotein (DSPP) gene has been identified in a family with dentinogenesis imperfecta type III (DGI-III). The DSPP gene encodes two major tooth matrix proteins dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). DSPP mutations associated with DGI-III results in an in frame truncation of the serine aspartic acid triplet repeat found in DPP near the highly conserved carboxyl terminal region shortening the protein by six amino acids. Clinically this family presents with discolored amber opalescent teeth and severe attrition of the tooth structure. This study is the first report of a mutation within DPP associated with a genetic dentin disease. Our study indicates that DGI-III is allelic with some forms of DGI-II with and without progressive hearing loss and dentin dysplasia type II that have been shown to be caused by mutations within the DSP coding or signal peptide regions.

Amino Acid Sequence↗

PubMed2005

Altered gene expression in human cleidocranial dysplasia dental pulp cells.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder characterised by defects of bone and tooth development. The dental manifestations in CCD patients include supernumerary teeth, delayed tooth eruption, tooth hypoplasia and absence of cellular cementum formation. This disorder is associated with mutations in the osteoblast-specific transcription factor Runx2. To identify morphological and molecular alterations associated with CCD dental tissues, human primary dental pulp cell cultures were established from age- and sex-matched CCD and normal patients. Dental pulp cells were compared for general morphology, proliferation rates, and gene expression profiles using cDNA microarray technology. CCD pulp cells were about four-fold larger than normal cells, however the normal pulp proliferation rates were two- and three-fold greater at time points tested than the CCD cells. Of the 226 genes analysed by blot microarray, 18.6% displayed significant differences at least two-fold in expression levels. This includes 25 genes (11.1%) that were up-regulated, while 17 (7.5%) that were down-regulated in the CCD cells as compared to the normal cells. Expression of selected genes was further verified by quantitative real-time polymerase chain reaction (qRT-PCR). Comparison between the CDD and normal cells revealed that gene expression of cytokines and growth factors, such as leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and transforming growth factor beta receptor II (TGF-betaRII) and vascular endothelial growth factor B (VEGFB) were higher while bone morphogenetic protein 2 (BMP2) was lower in the CCD cells. Furthermore, potential Runx2 binding sites were found in all putative target gene promoters. This study suggests that in addition to bone and tooth cell differentiation, Runx2 may be involved in controlling cell growth during tooth development.

Case-Control Studies↗

PubMed2004

Regulation of the Cell Type-specific dentin sialophosphoprotein gene expression in mouse odontoblasts by a novel transcription repressor and an activator CCAAT-binding factor.

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) with a highly restricted expression pattern in tooth and bone. Mutations of the DSPP gene are associated with dentin genetic diseases. Regulation of tissue-specific DSPP expression has not been described. To define the molecular basis of this cell-specific expression, we characterized the promoter responsible for the cell-specific expression of the DSPP gene in odontoblasts. Within this region, DNase I footprinting and electrophoretic mobility shift assays delineated one element that contains an inverted CCAAT-binding factor site and a protein-DNA binding site using nuclear extracts from odontoblasts. A series of competitive electrophoretic mobility shift assay analyses showed that the protein-DNA binding core sequence, ACCCCCA, is a novel site sufficient for protein binding. These two protein-DNA binding sequences are conserved at the same proximal position in the mouse, rat, and human DSPP gene promoters and are ubiquitously present in the promoters of other tooth/bone genes. Mutations of the CCAAT-binding factor binding site resulted in a 5-fold decrease in promoter activity, whereas abolishment of the novel protein-DNA binding site increased promoter activity by about 4.6-fold. In contrast to DSPP, expression levels of the novel protein were significantly reduced during odontoblastic differentiation and dentin mineralization. The novel protein was shown to have a molecular mass of 72 kDa. This study shows that expression of the cell type-specific DSPP gene is mediated by the combination of inhibitory and activating mechanisms.

Animals↗

PubMed2004

Binding of two nuclear factors to a novel silencer element in human dentin matrix protein 1 (DMP1) promoter regulates the cell type-specific DMP1 gene expression.

DMP1 is an acidic phosphorylated protein with the spatial and temporal expression that is largely restricted to bone and tooth tissues. The biological function of DMP1 is associated with biomineralization of bone, cartilage and tooth development. To study the cell-specific expression of DMP1, a 2,512 bp upstream segment of the human gene was isolated and characterized. A series of progressive deletions of the human DMP1 5' flanking sequence were ligated to the luciferase reporter gene, and their promoter activities examined in transfected human osteoblast-like (MG-63) and dental pulp (HDP-D) cells that express DMP1 and hepatic (HepG2) and uterine (HeLa) cells lacking DMP1 expression. A critical cis-regulatory element located between nt -150 and -63 was found to act as a specific silencer responsible for the negative regulation of DMP1 in HepG2 and HeLa cells. The transcriptional activity of this element in MG-63 and HDP-D cells had a 5-7-fold increase than that observed in HepG2 and HeLa cells. Electrophoretic mobility shift assays (EMSAs) showed that a 6-bp DNA sequence in this element was bound by two nuclear factors that are expressed at high levels in HepG2 and HeLa versus MG-63 and HDP-D cells. Competitive assays by EMSAs suggest that the 6-bp core DNA sequence, AG(T/C)C(A/G)C, is a novel DNA-protein binding site and conserved with high identity in reported DMP1 promoters for all species. Furthermore, point mutations of the core sequence caused a marked increase of DMP1 promoter activity in HepG2 and HeLa cells. We speculate that this silencing cis-element may play a critical role in the regulation of DMP1 cell-specific expression.

Base Sequence↗

PubMed2004

Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization, and expanded cavities of pulp and root canal during postnatal tooth development.

The dentin matrix protein-1 (DMP-1) gene is identified in odontoblasts during both embryonic and postnatal development. In vitro study suggests that this noncollagen acidic phosphoprotein plays a role in mineralization. However, deletion of the Dmp-1 gene has little effect on tooth development during embryogenesis. To address the role of DMP-1 in tooth during postnatal development, we analyzed changes of dentinogenesis in Dmp-1 null mice from 3 days after birth to 1 year. Here we show that Dmp-1 null mice postnatally develop a profound tooth phenotype characterized by a partial failure of maturation of predentin into dentin, enlarged pulp chambers, increased width of predentin zone with reduced dentin wall, and hypomineralization. The tooth phenotype of these mice is strikingly similar to that in dentin sialophosphoprotein (Dspp) null mice and shares some features of the human disease dentinogenesis imperfecta III. We have also demonstrated that DSPP levels are reduced in Dmp-1 null mice, suggesting that DSPP is probably regulated by DMP-1 during dentinogenesis. Finally, we show the absence or delayed development of the third molar in Dmp-1 null mice, which is probably secondary to defects in Dmp-1 null bone. Taken together, these studies suggest that DMP-1 is essential for later dentinogenesis during postnatal development.

Animals↗

PubMed2004

In vivo overexpression of tuftelin in the enamel organic matrix.

The primary sequences of human and mouse tuftelin are 89% identical. Both proteins comprise 390 amino acids and produce an acidic protein with an isoelectric point of 5.7, and an unmodified molecular weight of 44 kD. Using fluorescent-tagged tuftelin and amelogenin plasmid constructs we saw little evidence that these two enamel proteins colocalize in ameloblast-like LS-8 cells. Tuftelin is primarily localized to distinct 'speckled' domains within the cell cytoplasm. In an attempt to better define a physiological function for tuftelin during amelogenesis, we have produced transgenic mice that overexpress tuftelin in ameloblasts and subsequently the enamel matrix. Tuftelin overexpression impacts dramatically upon the enamel crystallite habit and the enamel prismatic structure. Overexpressing tuftelin results in gross imperfections in enamel that is evident both at the nanoscale and the mesoscale. The most notable difference observed in the transgenic animals, when compared to wild-type animals, is an apparent loss of restricted growth of enamel crystallites along their a-axis and b-axis. This equates to a change in the crystallite aspect ratio. In the transgenic animals the crystallite structures appear more 'plate'-like in contrast to the symmetric, 'ribbon'-like crystallite morphology that is a characteristic feature of mammalian enamel.

Ameloblasts↗

PubMed2003

Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III.

Dentin sialophosphoprotein (Dspp) is mainly expressed in teeth by the odontoblasts and preameloblasts. The Dspp mRNA is translated into a single protein, Dspp, and cleaved into two peptides, dentin sialoprotein and dentin phosphoprotein, that are localized within the dentin matrix. Recently, mutations in this gene were identified in human dentinogenesis imperfecta II (Online Mendelian Inheritance in Man (OMIM) accession number 125490) and in dentin dysplasia II (OMIM accession number 125420) syndromes. Herein, we report the generation of Dspp-null mice that develop tooth defects similar to human dentinogenesis imperfecta III with enlarged pulp chambers, increased width of predentin zone, hypomineralization, and pulp exposure. Electron microscopy revealed an irregular mineralization front and a lack of calcospherites coalescence in the dentin. Interestingly, the levels of biglycan and decorin, small leucine-rich proteoglycans, were increased in the widened predentin zone and in void spaces among the calcospherites in the dentin of null teeth. These enhanced levels correlate well with the defective regions in mineralization and further indicate that these molecules may adversely affect the dentin mineralization process by interfering with coalescence of calcospherites. Overall, our results identify a crucial role for Dspp in orchestrating the events essential during dentin mineralization, including potential regulation of proteoglycan levels.

Animals↗

PubMed2003

Mechanical loading stimulates dentin matrix protein 1 (DMP1) expression in osteocytes in vivo.

Dentin matrix protein 1 (DMP1) was originally postulated to be dentin specific. Further analysis showed that DMP1 is also expressed in mature cartilage and bone. In bone tissue, DMP1 is expressed predominantly in late osteoblasts and osteocytes. DMP1 belongs to the SIBLING (Small Integrin Binding Ligand N-linked Glycoprotein) family of cellular matrix proteins that also includes osteopontin, bone sialoprotein, dentin sialophosphoprotein, and others. In this study, we examined the effect of mechanical loading on expression of DMP1 mRNA and DMP1 protein in alveolar bone in the mouse tooth movement model by in situ hybridization and immunocytochemistry. The expression of DMP1 mRNA was determined quantitatively in mechanically loaded and control sites of dento-alveolar tissue at several time points from 6 h to 7 days after loading. The tooth movement model allows simultaneous evaluation of bone resorption and bone formation sites. Expression of DMP1 mRNA in osteocytes increased 2-fold as early as 6 h after treatment in both the bone formation and bone resorption sites. After 4 days, DMP1 expression in osteocytes increased to a maximum of 3.7-fold in the bone formation sites and 3.5-fold in the resorption sites. Osteoblasts responded in the opposite manner and showed a transient 45% decrease of DMP1 mRNA in bone formation sites and a constant decrease of DMP1 mRNA during the entire course of treatment in the bone resorption sites, with a peak inhibition of 67% at day 2. By immunocytochemistry using a C-terminal region peptide antibody to DMP1, we found that there was a transient decrease in immunoreactivity at 3 days after treatment on both the formation side and the resorption side compared with the matched contralateral control tissue. However by 7 days of loading, there was a dramatic increase in DMP1 protein immunoreactivity on both the formation side and the resorption side. These results represent changes in epitope availability using this antibody or true changes in protein levels. The observations imply that the DMP1 protein is undergoing dynamic changes in either synthesis or other protein/matrix interaction after mechanical loading of alveolar bone. The findings indicate that DMP1 is involved in the responses of osteocytes and osteoblasts to mechanical loading of bone. These results support the hypothesis that osteocytes alter their matrix microenvironment in response to mechanical loading.

Animals↗

PubMed2003

Dental structural diseases mapping to human chromosome 4q21.

Genetic diseases affecting tooth structure have been classified by the tissue affected enamel versus dentin, and their pattern of inheritance autosomal dominant, autosomal recessive, or X-linked. Advances in molecular genetics and the Human Genome Project have provided substantial progress regarding the identification of genes involved in the pathogenesis of human diseases. These include dental diseases affecting enamel and dentin formation: amelogenesis imperfecta (AI), dentinogenesis imperfecta (DGI) types II and III, and dentin dysplasia (DD) type II. Linkage studies using large informative families have provided insight identifying two proximal gene clusters on human chromosome 4q21 that contain the critical loci for five dental structural diseases. Studies related to the autosomal dominant forms of AI, representing approximately 85% of all cases, have established linkage to 4q21 for two forms: local hypoplastic and smooth hypoplastic AI. Two enamel matrix proteins, ameloblastin and enamelin, have been mapped within the critical regions for these diseases. Located more toward the telomere is another cluster containing loci for three dentin diseases: DGI type II, type III, and DD type II. Located within an overlapping segment of these diseases is a dentin/bone gene cluster that contains osteopontin, bone sialoprotein, matrix extracellular phosphoglycoprotein also known as osteoblast/osteocyte factor 45 or osteoregulin, dentin matrix protein 1, and dentin sialophosphoprotein. Continuing molecular genetic studies will facilitate the identification of novel tooth matrix proteins within these two tooth matrix gene clusters as well as the identification of additional autosomal dominant AI loci.

Amelogenesis Imperfecta↗

PubMed2002

MEPE/OF45, a new dentin/bone matrix protein and candidate gene for dentin diseases mapping to chromosome 4q21.

Substantial progress has been made regarding the molecular etiology of the dentin diseases dentinogenesis imperfecta types II and III, and dentin dysplasia type II. Genetic linkage studies have identified the critical loci for these diseases on human chromosome 4q21. Located within an overlapping segment of these disease loci is a dentin/bone gene cluster that includes osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein, and a new gene MEPE also known as OF45, renamed osteoregulin. In this paper, we report the location of MEPE/OF45 in relationship to the other members of the dentin/bone gene cluster as well as the genomic organization of the human gene. For the first time, MEPE/OF45 expression was shown in dental tissue, in particular odontoblasts, by reverse-transcription polymerase chain reaction (RT-PCR) amplification and characterization of a partial mouse cDNA. Our data provide the first evidence that MEPE/OF45 is expressed during odontogenesis and should be considered as a candidate gene for dentin structural diseases mapping to human chromosome 4q21.

Animals↗

PubMed2002

Spatial expression of Cbfa1/Runx2 isoforms in teeth and characterization of binding sites in the DSPP gene.

Cbfa1/Runx2 is an essential transcription factor for osteoblast and odontoblast differentiation. Heterogeneous mutations of Cbfa1 gene result in cleidocranial dysplasia, an autosomal dominant syndrome, characterized by abnormal skeletal genesis and dental disorders. Recently three Cbfa1/Runx isoforms (Pebp2 alpha A/type I, til-1/type II, and Osf2/type III) have been identified that differ in their amino-terminal sequences. The precise roles of Cbfa1/Runx2 isoforms in odontoblast development are not known. The purpose of this study was to determine and compare expression patterns of the three Cbfa1/Runx2 isoforms in newborn tooth organs. Toward this aim, we developed three probes: type I and type II, which specifically hybridize with Pebp2 alpha A and til-1, respectively, and type II/III, which hybridizes with osf2 and partially with til-1. In addition, Cbfa1/Runx2 binding sites were identified in the regulatory elements of mouse dentin sialophosphoprotein (mDSPP) gene, which encodes a matrix protein expressed during odontogenesis. In situ hybridization performed with the specific Cbfa1/Runx2 isoform probes demonstrated that all isoforms are expressed in teeth and bone. The type I isoform was expressed at higher levels than isoforms type II and type II/III in developing newborn mouse incisors. Genomic mDSPP clones were isolated and characterized containing approximately 2.6 kb of the promoter region. Computer analysis of the promoter segment and intron 1 revealed a number of potential transcriptional factor binding sites including five Cbfa1/Runx2 binding sites, three in the promoter region and two within intron 1. DNA-protein assay and antibody supershift experiments showed that these binding sites interact with nuclear extracts isolated from the mouse odontoblast cell line MO6-G3. Further characterization of the functional role of Cbfa1/Runx2 in the regulation of the mDSPP gene expression is being investigated.

Animals↗

PubMed2002

Dentin matrix protein 1, a target molecule for Cbfa1 in bone, is a unique bone marker gene.

Dentin matrix protein 1 (Dmp1), a phosphoprotein highly linked to dentin formation, has also been reported to be expressed in the skeleton. However, the role of Dmp1 in skeletal tissues remains unclear. To clarify the role of Dmp1 in bone formation, we characterized the expression profile of Dmp1 in bone and cartilage and examined whether Dmp1 expression was regulated by core-binding factor a1 (Cbfa1). Studies of fetal rat calvarial (FRC) cell cultures showed that the expression of Dmp1 was associated closely with "bone nodule" formation and mineralization in vitro. In situ hybridization studies were performed to examine the spatial and temporal expression patterns of Dmp1 during development in mouse embryos from 12.5 day postcoitus (dpc) to 8 weeks postnatal; these studies showed that Dmp1 first appeared in hypertrophic cartilage cells, followed by osteoblasts, and later was expressed strongly in osteocytes. The expression profiles of Cbfa1 and Dmp1 overlapped in both cartilage and bone during development, with Cbfa1 preceding Dmp1. Examination of Dmp1 expression in Cbfa1-/- mice revealed that Dmp1 was absent in the developing bones of Cbfa1-null mice, whereas there was essentially no change in Dmp1 expression in the arrested tooth bud. Transient transfection studies showed forced expression of Dmp1 under the control of Cbfa1 and gel shift data indicated the presence of a functional osteocalcin-specific element (OSE)-2 response element in the Dmp1 proximal promoter region. However, in vitro promoter studies suggested that regulation of Dmp1 by Cbfa1 was not mediated by direct binding of Cbfa1 to this site and may be through indirect mechanisms. These studies highlight Dmp1 as a unique marker gene for osteoblastic differentiation. The close association of Dmp1 and Cbfa1 in the developing skeleton suggests that Dmp1 may play an important role in bone formation.

Animals↗