The C-terminus of amelogenin enhances osteogenic differentiation of human cementoblast lineage cells.

BACKGROUND AND OBJECTIVES: Amelogenin proteins are the major constituent of developing extracellular enamel matrix and are believed to have an exclusively epithelial origin. Recent studies have suggested that amelogenins might induce the differentiation and maturation of various cells, including cementoblast lineage cells. However, the residues comprising the active site of amelogenin remain unclear. The purpose of this study was to identify the active site region of amelogenin by studying the effects of amelogenin fragments on the osteogenic differentiation of cementoblasts. MATERIAL AND METHODS: Amelogenin fragments lacking the C-terminus (rh163) and N-terminus (rh128) and a fragment consisting of the C-terminal region of rh174 (C11 peptide) were synthesized and purified. Human cementoblast lineage cells were cultured in osteogenic differentiation medium and treated with 0, 10, 100 or 1000 ng/mL of rh163, rh128 or C11 peptide. The mRNA levels of bone markers were examined by real-time polymerase chain reaction analysis. Alkaline phosphatase activity and calcium deposition were also determined. Mineralization was evaluated by alizarin red staining. RESULTS: The osteogenic differentiation of human cementoblast lineage cells was significantly enhanced by treatment with rh128 or C11 peptide, whereas rh163 had no significant effect as compared with untreated controls. CONCLUSIONS: The C-terminus of amelogenin promotes the osteogenic differentiation of human cementoblast lineage cells, indicating the possible utility of C11 peptide in periodontal tissue regeneration.

NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse.

During the formation of dental enamel, maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by cotransporting HCO3 (-) with Na(+). Mutation in SLC4A4 (coding for the sodium-bicarbonate cotransporter NBCe1) induces developmental defects in human and murine enamel. We have hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in wild-type dental epithelium and examined the effect of the NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice, weak to moderate immunostaining for NBCe1 with antibodies that recognized isoforms A/B/D/E and isoform C was seen in ameloblasts at the secretory stage, with no or low staining in the early maturation stage but moderate to high staining in the late maturation stage. The papillary layer showed the opposite pattern being immunostained prominently at the early maturation stage but with gradually less staining at the mid- and late maturation stages. In NBCe1 (-/-) mice, the ameloblasts were disorganized, the enamel being thin and severely hypomineralized. Enamel organs of CFTR (-/-) and AE2a,b (-/-) mice (CFTR and AE2 are believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Thus, the expression of NBCe1 in ameloblasts and the papillary layer cell depends on the developmental stage and possibly responds to pH changes.

Enamel pits in hamster molars, formed by a single high fluoride dose, are associated with a perturbation of transitional stage ameloblasts.

Excessive intake of fluoride (F) by young children results in the formation of enamel subsurface porosities and pits, called enamel fluorosis. In this study, we used a single high dose of F administered to hamster pups to determine the stage of ameloblasts most affected by F and whether pit formation was related to F-related sub-ameloblastic cyst formation. Hamster pups received a single subcutaneous injection of either 20 mg or 40 mg NaF/kg body weight, were sacrificed 24 h later, and the number of cysts formed in the first molars were counted. Other pups were sacrificed 8 days after F injection, when the first molars had just erupted, to score for enamel defects. All F-injected pups formed enamel defects in the upper half of the cusps in a dose-dependent way. After injection of 20 mg NaF/kg, an average of 2.5 white spots per molar was found but no pits. At 40 mg NaF/kg, almost 4.5 spots per molar were counted as well as 2 pits per molar. The defects in erupted enamel were located in the upper half of the cusps, sites where cysts had formed at the transition stage of ameloblast differentiation. These results suggest that transitional ameloblasts, located between secretory- and maturation-stage ameloblasts, are most sensitive to the effects of a single high dose of F. F-induced cysts formed earlier at the pre-secretory stage were not correlated to either white spots or enamel pits, suggesting that damaged ameloblasts overlying a F-induced cyst regenerate and continue to form enamel.

Micromolar fluoride alters ameloblast lineage cells in vitro.

Fluorosed enamel is caused by exposure to fluoride during tooth formation. The objective of this study was to determine whether epithelial ameloblast-lineage cells, derived from the human enamel organ, are directly affected by micromolar concentrations of fluoride. Cells were cultured in the presence of fluoride, and proliferation was measured by BrdU incorporation. The effect of 0, 10, or 20 microM fluoride on apoptosis was determined by the flow cytometry apoptotic index. The effects of fluoride on gene expression were investigated by SuperArray microarray analysis and real-time PCR. Fluoride had a biphasic effect on cell proliferation, with enhanced proliferation at 16 microM, and reduced proliferation at greater than 1 mM F. Flow cytometry showed that both 10 microM and 20 microM NaF significantly increased the apoptotic index of ameloblast-lineage cells. There was no general effect of fluoride on gene expression. These results indicate multiple effects of micromolar fluoride on ameloblast-lineage cells.

The effect of LRAP on enamel organ epithelial cell differentiation.

Leucine-rich amelogenin peptide (LRAP) is an alternatively spliced amelogenin found in the developing enamel organ. LRAP functions to regulate the development of mesenchymal-derived cells; however, its effect on cells of the enamel organ remains unclear. The hypothesis tested in this study is that LRAP also regulates human enamel organ epithelial cells. Recombinant human LRAP (rH58) was synthesized in E. coli, purified, and exogenously added to cultures of human primary enamel epithelial cells, which were analyzed for changes in cell proliferation and differentiation. rH58 had no effect on cell proliferation, but altered enamel epithelial cell morphology, resulting in larger, more rounded cells. Immunofluorescence showed that rH58 treatment increased amelogenin synthesis, but down-regulated Notch1 expression in enamel epithelial cells. LAMP-1, a membrane receptor for LRAP in mesenchymal cells, was identified and was up-regulated in the presence of rH58. These results suggest that rH58 promotes differentiation of human enamel organ epithelial cells.

Characterization of human primary enamel organ epithelial cells in vitro.

UNLABELLED: Tooth enamel is formed by ameloblasts, which are derived from the epithelial cells of the enamel organ. OBJECTIVE: The purpose of this study was to grow human ameloblast-like epithelial cells in culture. DESIGN: Human fetal tooth organs were isolated, and the cells were separated by digestion in collagenase/dispase. The cells were cultured in KGM-2 media with and without serum and at different calcium concentrations. The expression of enamel matrix proteins was analyzed by RT-PCR and cytokeratin 14 was detected by immunohistochemistry. The cells were further characterized by osteogenesis/odontogenesis-related DNA array. RESULTS: Cells isolated from the tooth organs grown in KGM-2 media containing 2-10% serum, were mixture of cobblestone and spindle shaped cells. Culturing these cells in KGM-2 with 0.05 mM calcium was selective for cobblestone ameloblasts-like cells (CAB), which were immunopositive for cytokeratin 14. Amelogenin, ameloblastin, enamelin, MMP-20 and KLK-4 were detected in CAB cells by RT-PCR. Osteogenesis SuperArray analyses could not detect the presence of typical molecules related to mesenchymal odontoblast or osteoblast lineage cells in these cultures. CONCLUSIONS: These studies showed that cobblestone-shaped ameloblast-like cells are selected from the tooth organ cells, by culture in KGM-2 media with 0.05 mM calcium.

Slc26a3/Dra and Slc26a6 in Murine Ameloblasts.

Formation of apatite crystals during enamel development generates protons. To sustain mineral accretion, maturation ameloblasts need to buffer these protons. The presence of cytosolic carbonic anhydrases, the basolateral Na(+) bicarbonate cotransporter Nbce1, and the basolateral anion exchanger Ae2a,b in maturation ameloblasts suggests that these cells secrete bicarbonates into the forming enamel, but it is unknown by which mechanism. Solute carrier (Slc) family 26A encodes different anion exchangers that exchange Cl(-)/HCO3 (-), including Slc26a3/Dra, Slc26a6/Pat-1, and Slc26a4/pendrin. Previously, we showed that pendrin is expressed in ameloblasts but is not critical for enamel formation. In this study, we tested the hypothesis that maturation ameloblasts express Dra and Slc26a6 to secrete bicarbonate into the enamel space in exchange for Cl(-). Real-time polymerase chain reaction detected mRNA transcripts for Dra and Slc26a6 in mouse incisor enamel organs, and Western blotting confirmed their translation into protein. Both isoforms were immunolocalized in ameloblasts, principally at maturation stage. Mice with null mutation of either Dra or Slc26a6 had a normal dental or skeletal phenotype without changes in mineral density, as measured by micro-computed tomography. In enamel organs of Slc26a6-null mice, Dra and pendrin protein levels were both elevated by 52% and 55%, respectively. The amount of Slc26a6 protein was unchanged in enamel organs of Ae2a,b- and Cftr-null mice but reduced in Dra-null mice by 36%. Our data show that ameloblasts express Dra, pendrin, or Slc26a6 but each of these separately is not critical for formation of dental enamel. The data suggest that in ameloblasts, Slc26a isoforms can functionally compensate for one another.

Amelogenins as potential buffers during secretory-stage amelogenesis.

Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX(-/-)) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX(-/-) mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX(-/-) mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX(-/-) mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX(-/-) mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation.

Ameloblast Modulation and Transport of Cl⁻, Na⁺, and K⁺ during Amelogenesis.

Ameloblasts express transmembrane proteins for transport of mineral ions and regulation of pH in the enamel space. Two major transporters recently identified in ameloblasts are the Na(+)K(+)-dependent calcium transporter NCKX4 and the Na(+)-dependent HPO4 (2-) (Pi) cotransporter NaPi-2b. To regulate pH, ameloblasts express anion exchanger 2 (Ae2a,b), chloride channel Cftr, and amelogenins that can bind protons. Exposure to fluoride or null mutation of Cftr, Ae2a,b, or Amelx each results in formation of hypomineralized enamel. We hypothesized that enamel hypomineralization associated with disturbed pH regulation results from reduced ion transport by NCKX4 and NaPi-2b. This was tested by correlation analyses among the levels of Ca, Pi, Cl, Na, and K in forming enamel of mice with null mutation of Cftr, Ae2a,b, and Amelx, according to quantitative x-ray electron probe microanalysis. Immunohistochemistry, polymerase chain reaction analysis, and Western blotting confirmed the presence of apical NaPi-2b and Nckx4 in maturation-stage ameloblasts. In wild-type mice, K levels in enamel were negatively correlated with Ca and Cl but less negatively or even positively in fluorotic enamel. Na did not correlate with P or Ca in enamel of wild-type mice but showed strong positive correlation in fluorotic and nonfluorotic Ae2a,b- and Cftr-null enamel. In hypomineralizing enamel of all models tested, 1) Cl(-) was strongly reduced; 2) K(+) and Na(+) accumulated (Na(+) not in Amelx-null enamel); and 3) modulation was delayed or blocked. These results suggest that a Na(+)K(+)-dependent calcium transporter (likely NCKX4) and a Na(+)-dependent Pi transporter (potentially NaPi-2b) located in ruffle-ended ameloblasts operate in a coordinated way with the pH-regulating machinery to transport Ca(2+), Pi, and bicarbonate into maturation-stage enamel. Acidification and/or associated physicochemical/electrochemical changes in ion levels in enamel fluid near the apical ameloblast membrane may reduce the transport activity of mineral transporters, which results in hypomineralization.

Identification of the calcium-sensing receptor in the developing tooth organ.

Calcium (Ca2+) is a critical component of tooth enamel, dentin, and the surrounding extracellular matrix. Ca2+ also may regulate tooth formation, although the mechanisms for such action are poorly understood. The Ca2+-sensing receptor (CaR) that is expressed in the parathyroid gland, kidney, bone, and cartilage has provided a mechanism by which extracellular Ca2+ can regulate cell function. Because these tissues play an important role in maintaining mineral homeostasis and because Ca2+ is hypothesized to play a crucial role in tooth formation, we determined whether the CaR was present in teeth. In this study, using immunohistochemistry, CaR protein was detected in developing porcine molars localized in the predentin (pD), early secretory-stage ameloblasts, maturation-stage smooth-ended ameloblasts (SA), and certain cells in the stratum intermedium. CaR protein and messenger RNA (mRNA) were detected also in an immortalized ameloblast-like cell line (PABSo-E) using immunofluorescence, reverse-transcription polymerase chain reaction (RT-PCR), and Northern analysis. Based on the observation that the CaR is expressed in cultured ameloblasts, we determined whether increments in medium Ca2+ concentration could activate the intracellular Ca2+ signal transduction pathway. In PABSo-E cells, increasing extracellular Ca2+ in the medium from 0 (baseline) to 2.5mM or 5.0 mM resulted in an increase in intracellular Ca2+ above baseline to 534 +/- 69 nM and 838 +/- 86 nM, respectively. Taken together, these results suggest that the CaR is expressed in developing teeth and may provide a mechanism by which these cells can respond to alterations in extracellular Ca2+ to regulate cell function and, ultimately, tooth formation.

Reduced hydrolysis of amelogenin may result in X-linked amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.

Biological mechanisms of fluorosis and level and timing of systemic exposure to fluoride with respect to fluorosis.

Enamel fluorosis can occur following either an acute or chronic exposure to fluoride during tooth formation. Fluorosed enamel is characterized by a retention of amelogenins in the early-maturation stage, and by the formation of a more porous enamel with a subsurface hypomineralization. The mechanisms by which fluoride affects enamel development include specific effects on both the ameloblasts and on the developing enamel matrix. Maturation-stage ameloblast modulation is more rapid in fluorosed enamel as compared with control enamel, and proteolytic activity in fluorosed early-maturation enamel is reduced as compared with controls. Secretory enamel appears to be more susceptible to the effects of fluoride following acute fluoride exposure, such as may occur with the use of fluoride supplements. However, both human and animal studies show that the transition/early-maturation stage of enamel formation is most susceptible to the effects of chronic fluoride ingestion at above-optimal levels of fluoride in drinking water.

Alternative splicing of amelogenin mRNA from rat incisor ameloblasts.

Amelogenin proteins are a major component of the developing enamel matrix, and are likely to have a key role in the control of enamel biomineralization. The heterogeneity of amelogenin is in part due to alternative splicing of the amelogenin RNA transcripts. Several patterns of alternative splicing have been described in mouse, bovine, porcine, and human enamel, with all alternatively spliced products having homologous 5' and 3' sequences within the coding regions. In these studies, we have used anchored PCR to identify alternatively spliced amelogenin cDNA sequences in the rat. We found amelogenin cDNAs that could be divided into two groups based on their 3' sequence. Group 1 cDNAs had a novel terminal sequence that has not been previously identified, while group 2 cDNAs were similar to those previously identified in other animal species. We identified a sequence, identical to the novel 3' amelogenin cDNA sequence, in rat genomic DNA downstream from the previously identified exon 7. The putative amelogenin proteins predicted for the two groups differed in their predicted isoelectric points, with the novel group 1 proteins having a more basic isoelectric point.

Changes in the fluoride-induced modulation of maturation stage ameloblasts of rats.

The maturation stage of enamel development is characterized by a cyclic modulation of the ameloblasts between bands of smooth-ended cells and longer bands of ruffle-ended cells. There are cyclic patterns of calcein staining of and 45Ca uptake in the enamel associated with this cellular modulation. Rats were given 0, 75, 100, or 150 ppm fluoride in their drinking water. Fluoride disrupted the cyclic patterns of the maturation stage, resulting in fewer bands of smooth-ended ameloblasts, fewer calcein-stained stripes, and fewer cycles of 45Ca uptake. When animals were given water containing 0 ppm fluoride following ingestion of water containing 100 ppm fluoride, the pattern of calcein staining returned to that of the control enamel. The disruption of the cyclic patterns in the maturation stage and the increased protein content of maturation enamel seem to be among the early events in the development of fluorosis.

Dentonin, a fragment of MEPE, enhanced dental pulp stem cell proliferation.

Matrix extracellular phosphoglycoprotein (MEPE) is a SIBLING protein, found in bone and dental tissues. The purpose of this study was to determine whether a 23-amino-acid peptide derived from MEPE (Dentonin or AC-100) could stimulate dental pulp stem cell (DPSC) proliferation and/or differentiation. DPSCs were isolated from erupted human molars, and the mitogenic potential of Dentonin in DPSCs was measured by BrdU immunoassay and cell-cycle gene SuperArray. Differentiation of DPSCs with Dentonin was characterized by Western blot and by osteogenesis gene SuperArray. Dentonin enhanced DPSC proliferation by down-regulating P16, accompanied by up-regulation of ubiquitin protein ligase E3A and human ubiquitin-related protein SUMO-1. Enhanced cell proliferation required intact RGD and SGDG motifs in the peptide. This study shows that Dentonin can promote DPSC proliferation, with a potential role in pulp repair. Further studies are required to determine the usefulness of this material in vivo.

Amelogenin-guided crystal growth on fluoroapatite glass-ceramics.

The formation of aligned fibrous apatite crystals in enamel is predominantly attributed to the involvement of amelogenin proteins. We developed a model to study interactions of matrix proteins with apatite mineral in vitro and tested the hypothesis that amelogenin solubility affects the ability to induce protein-guided mineralization. Crystal growth experiments were performed on fluoroapatite (FAP) glass-ceramics in mineralizing solutions containing recombinant full-length amelogenin (rH174) at different concentrations. Using atomic force microscopy, we observed that mineral precipitated randomly on the substrate, but also formed thin layers (height, 10 nm) on FAP within 24 hrs. This growth pattern was unaffected when 0.4 mg/mL of rH174 was added. In contrast, crystals grew on FAP at a rate up to 20 times higher, at 1.6 mg/mL protein. Furthermore, nanospheres and mineral bound specifically to FAP and aligned in strings approximately parallel to the c-axis of FAP, leading us to the conclusion that amelogenin proteins indeed control direction and rate of growth of apatite in enamel.

Neurotoxicity of sodium fluoride in rats.

Fluoride (F) is known to affect mineralizing tissues, but effects upon the developing brain have not been previously considered. This study in Sprague-Dawley rats compares behavior, body weight, plasma and brain F levels after sodium fluoride (NaF) exposures during late gestation, at weaning or in adults. For prenatal exposures, dams received injections (SC) of 0.13 mg/kg NaF or saline on gestational days 14-18 or 17-19. Weanlings received drinking water containing 0, 75, 100, or 125 ppm F for 6 or 20 weeks, and 3 month-old adults received water containing 100 ppm F for 6 weeks. Behavior was tested in a computer pattern recognition system that classified acts in a novel environment and quantified act initiations, total times and time structures. Fluoride exposures caused sex- and dose-specific behavioral deficits with a common pattern. Males were most sensitive to prenatal day 17-19 exposure, whereas females were more sensitive to weanling and adult exposures. After fluoride ingestion, the severity of the effect on behavior increased directly with plasma F levels and F concentrations in specific brain regions. Such association is important considering that plasma levels in this rat model (0.059 to 0.640 ppm F) are similar to those reported in humans exposed to high levels of fluoride.

Separation by polyacrylamide gel electrophoresis of multiple proteases in rat and bovine enamel.

At least seven protein bands of protease activity were found in rat secretory enamel and six bands in bovine secretory enamel. The bovine proteases had molecular weights similar to those found in rat enamel. Two bands were found to be active in gels containing enamel proteins. All proteases were acid-resistant and calcium-dependent. In vitro digestion of secretory enamel resulted in a changing profile of proteases. Rat maturation enamel contained a 23,000 and a 33,000 molecular-weight protease which were not present in the secretory enamel. The 33,000 molecular weight protease was also active against enamel proteins. Thus multiple proteases are present in rat and bovine secretory enamel and in rat maturation enamel; some of these may be important in hydrolysis of enamel proteins.

Characterization of amelogenin mRNA from secretory- and maturation-stage rat incisor enamel.

Amelogenins are extracellular matrix proteins expressed at the secretory and the maturation stages of enamel formation. To characterize amelogenin transcripts from these two stages, rat incisor enamel-organ mRNA was hybridized to a mouse amelogenin cDNA. Northern analysis of the mRNA showed four amelogenin transcripts at the secretory stage. Two of these were larger than previously described, and would code for high molecular-weight amelogenins (> 40 kDa). These transcripts were similar in size to at least three bovine amelogenin mRNAs. At the maturation stage, one transcript slightly smaller than the major amelogenin transcript from the secretory stage was apparent. These differences may indicate a selective splicing of amelogenin mRNA in the maturation stage as compared to the secretory stage of enamel development.

The effects of chronic high fluoride levels on forming enamel in the rat.

Sixty-gramme rats were given either 0, 75, 100 or 150 parts/10(6) fluoride in their drinking water. After five weeks, the fluoride, the phosphorus and the protein contents of the enamel were compared in control and experimental animals at three stages of enamel development. The mineral content was reduced in pigmented enamel from animals given 75 parts/10(6) or more fluoride in their drinking water. The fluoride content was elevated in all stages of fluorosed enamel development. At the lowest fluoride level (75 parts/10(6], a larger proline content was found in the proteins of the maturing, fluorosed enamel but there was no increase in the protein content. In animals given 100 parts/10(6) fluoride in their drinking water, the proline content of the protein was greater in maturing, fluorosed enamel, and the total protein content of the post-secretory enamel (maturing and pigmented) was greater than in the controls. These observations indicate that, with increasing levels of fluoride in drinking water, there was an initial delay in the loss of the amelogenin proteins followed by a decreased removal of total protein from the enamel. These results indicate that fluoride interfered with the normal post-secretory, pre-eruptive development of enamel.