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Open Access Publications from the University of California

Scholarly Works (11 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2011)

Ameloblasts continuously differentiate from epithelial stem cells to maturation stage ameloblasts, modulating and directing the formation of a mineralized enamel matrix. Calcium is required for enamel mineralization, however, as in other epithelial tissues; calcium may also be a modulator for ameloblast differentiation. Several studies have suggested a role for the calcium sensing receptor (CaSR) in ameloblast differentiation. Hypothesis: Calcium regulates ameloblast differentiation through the activity of the calcium sensing receptor. Methods: A primary ameloblast cell culture model was used to determine whether calcium could direct ameloblast differentiation, cellular morphology, and gene expression. Gene expression in response to calcium was assessed via microarray analysis. In vivo studies using K14-Cre-CaSR-/- mice treated with a normal diet, low calcium diet or high fluoride diet were completed to determine the role of the CaSR on ameloblast differentiation. Enamel formation, mineralization, and ameloblast differentiation were imaged by microCT, histochemistry, and immunohistochemistry. Gene expression was compared by qPCR. Results: In vitro primary cell culture data showed that elevated levels of calcium could induce mineralization, changes in cell morphology and gene expression related to dose and time of exposure. Primary ameloblast lineage cells is very heterogeneous, with a proportion of stellate reticulum cells, which accounts for the high levels of collagen Type I levels found in the microarray study. In vivo K14-Cre-CaSR-/- mouse model data showed earlier enamel formation compared to wild type mice, with no effects on ameloblast morphology and gene expression. The early enamel formation phenotype in K14-Cre-CaSR-/- mice was enhanced in the presence of fluoride and in the absence of calcium in the diet. Conclusion: Calcium can modulate expression of extracellular matrix proteins in a mixed primary cell culture. CaSR activity can influence the timing of enamel formation, but has a limited role in regulating ameloblast differentiation. Calcium mediated changes in gene expression and ameloblast cellular function appears to be controlled through mechanisms other than CaSR.

    Cover page: Calcium mediated differentiation of ameloblasts via the calcium sensing receptor
    • Thesis
    • Peer Reviewed
    UC San Francisco Electronic Theses and Dissertations (2007)

    Amelogenins make up over 90% of the secretory enamel proteins and play a critical role in tooth enamel formation. There are several amelogenin isoforms identified within the enamel extracellular matrix as a result of proteolysis and alternative splicing of the primary amelogenin mRNA transcript. Leucine-rich amelogenin peptide (LRAP) is one of the major alternatively spliced amelogenins, whose function in tooth formation remains unclear. The purpose of this thesis is to explore the role and mechanism of LRAP and its MMP-20-mediated proteolytic products in biomineralization, and their biological effects on odontogenic cell (i.e. dental pulp cell and ameloblast-lineage cell) proliferation and/or differentiation. The purified recombinant LRAP was hydrolyzed by active MMP-20. LRAP proteolytic products were analyzed by mass spectrometry to identify specific cleavage sites. Subsequently, LRAP and its proteolytic products were allowed to interact with synthetic carbonate hydroxyapatites (CAP) in vitro to determine the binding mechanism. After binding onto the apatite substrates, these peptides were incubated in supersaturated calcium phosphate solution, similar to secretory stage enamel-like fluid, to study their ability to promote enamel-like crystal growth. In addition, LRAP and its proteolytic fragments were also exogenously added to human dental pulp cell and primary ameloblast-lineage cell cultures to detect the evidence of cell proliferation and/or differentiation. Expressions of regulatory genes and cell-surface receptors associated with these specific pathways were also identified by gene arrays, reverse-transcription PCR and immunohistochemistry. LRAP is a specific substrate for MMP-20 hydrolysis to cleave its C-terminal domain, which was responsible for the binding of LRAP directly onto the surface of CAP by ionic interactions. Unlike its parent amelogenin, LRAP did not function as a structural protein to promote crystal formation in vitro; however, it acted as a cell-signaling molecule to affect odontoblast cell proliferation and ameloblast-lineage cell differentiation by up-regulating and interacting with its cell membrane receptor lysosome-associated membrane glycoprotein 1 (LAMP-1). Therefore, LRAP functions in the early stages of cell development to control odontogenesis and amelogenesis. These are novel findings that allow us for the first time to understand how the alternatively spliced amelogenin protein, LRAP and its MMP-20 hydrolytic products, interact with cells and mineral in the developing tooth. This knowledge is critical for further studies of tooth regeneration and the use of amelogenins in biomineralization.

      Cover page: FUNCTIONAL ANALYSIS OF LEUCINE-RICH AMELOGENIN PEPTIDE AND ITS MMP-20-MEDIATED PROTEOLYTIC PRODUCTS IN TOOTH FORMATION
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (1999)
      Enamelysin is a matrix metalloproteinase (MMP-20) secreted by ameloblasts, previously shown to hydrolyze recombinant amelogenin. The purpose of this study was to use recombinant MMP-20 to further investigate the specific hydrolysis of peptide fragments containing cleavage sites for tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide (LRAP). MMP-20 cDNA was isolated from a subtracted bovine cDNA library, reconstructed into pRSET A vector, and overexpressed in BL21 Escherichia coli. The recombinant MMP-20 was purified using Mono-S ion exchange and nickel affinity chromatography. The proteinase was renatured by dialysis against buffer containing 50 microM zinc and 5 mM calcium and autolysed to form several active fragments. The varying sizes and activities of the activated enzyme fragments appeared to be due to sequential autolysis at different location of the carboxyl terminus of the intact enzyme. Two synthetic peptides corresponding to amelogenin amino acid sequences 36-49 and 181-188 were hydrolyzed by the activated rMMP-20. Mass spectrometry and amino acid composition analysis showed that the cleavage sites were between the tryptophan and leucine (45 and 46) for TRAP and between proline and alanine (186-187) for LRAP. These results indicate that MMP-20 can be autoactivated, and activated MMP-20 has a functional role in the initial cleavage of amelogenin.
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2011)
        Enamel fluorosis has been related to an increase in the amount of amelogenin in fluorosed enamel compared with normal enamel in the maturation stage. In this study we tested the hypothesis that fluoride incorporated into carbonated apatite alters amelogenin hydrolysis. Recombinant human amelogenin (rh174) was allowed to bind to 0.15 mg of carbonated hydroxyapatite (CAP) or to fluoride-containing carbonated hydroxyapatite (F-CAP) synthesized to contain 100, 1,000, or 4,000 ppm F(-). After 3 h of digestion with recombinant human matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4), bound protein was characterized by reverse-phase high-performance liquid chromatography (HPLC). Proteolytic fragments of amelogenin formed after 24h of digestion with MMP20 of KLK 4 were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The hydrolysis, by both MMP20 and KLK4, of amelogenin bound to F100-CAP was significantly reduced in a dose-dependent manner compared with the hydrolysis of amelogenin bound to CAP. After 24 h of hydrolysis, a similar number of MMP20 cleavage sites was found for amelogenin bound to CAP and amelogenin bound to F100-CAP; however, 24 fewer KLK4 cleavage sites were identified for amelogenin bound to F100-CAP than for amelogenin bound to CAP. These results suggest that the reduced hydrolysis of amelogenins in fluorosed enamel may be partially caused by the increased fluoride content in fluoride-containing apatite, contributing to the hypomineralized enamel matrix phenotype observed in fluorosed enamel.
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2009)
          Amelogenins, the chief proteins in enamel matrix, undergo progressive degradation by matrix metalloproteinase-20 (MMP-20) to facilitate crystal growth. Proline is the most abundant residue in amelogenin and is located upstream to all MMP-20 cleavage sites in the amelogenin sequence. Pro(41) is critical for amelogenin N-terminal processing, while the role of prolines at the amelogenin C terminus have not been determined. This study sought to elucidate the effect of the C-terminal prolines on apatite binding and MMP-20 hydrolysis. To compare apatite affinity, recombinant full-length human amelogenin (rh174) and mutated variants (P156T and P164T) were incubated with hydroxyapatite (HAP), and the unbound proteins were quantified by the Bradford assay. rh174 and mutants, as well as 3 oligopeptides, including wild-type peptide and peptides containing 2 mutations, were incubated with MMP-20 both in solution and on HAP. The digested products were analyzed by SDS-PAGE, reverse-phase high-performance liquid chromatography and mass spectrometry. Mutated amelogenins displayed a significantly lower affinity to HAP than the wild type (P156T < P164T < rH174). The proline mutation at amino acid location 164 significantly reduced the initial hydrolysis of either the amelogenins in solution or the proteins bound on HAP, which was confirmed by amelogenin oligopeptide assays. It was concluded that prolines at the amelogenin C terminus are essential for the initial processing of amelogenin and amelogenin-mineral interactions.
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2001)
            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.
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2002)
              In addition to seven known exons of the amelogenin gene, recent studies have identified two exons downstream of amelogenin exon 7 in genomic DNA of mouse and rat. Here the spatial and temporal expression of mRNAs and of the translated proteins derived from alternative splicing of the amelogenin gene ending with exon 8 and exon 9 were examined by in situ hybridization (ISH) and immunohistochemistry (IHC). RNA signals for exons 8 and 9 were expressed in the ameloblast layer extending from early presecretory to postsecretory transitional stages of amelogenesis. IHC of amelogenin proteins that include sequences encoded by these exons demonstrated identical localization of these proteins in the ameloblast layer corresponding to RNA signals identified by ISH. There was intense immunostaining of the enamel matrix secreted by these cells. Western blotting analysis of rat enamel proteins revealed three distinct protein bands with sequences encoded by the new exons. These data confirmed the existence of the transcripts of alternatively spliced mRNAs coding for exons 8 and 9 of the amelogenin gene in rat tooth germs and suggest that the translated proteins contribute to the heterogeneity of amelogenins and have some significant roles in enamel formation and mineralization.
                • Article
                • Peer Reviewed
                UCSF Open Access Publishing Fund (2020)
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2006)
                  Leucine-rich amelogenin peptide (LRAP) is an alternately spliced amelogenin. LRAP is known to bind to hydroxyapatite, and has been shown to signal mesenchymal cells to proliferate, but its function in enamel formation is unclear. The purpose of this study was to determine the calcium-binding properties and structure of recombinant human LRAP (rLRAP) compared with full-length amelogenin (rH174). rLRAP and rH174 were synthesized in Escherichia coli and purified by affinity chromatography and reverse-phase high-performance liquid chromatography. Calcium binding was measured by isothermal titration calorimetry (ITC) at pH 7.5 and 25 degrees C, and raw data were analyzed by origin 7.0 software. The structure of rLRAP was analyzed by nuclear magnetic resonance (NMR) and circular dichroism (CD) in the absence or presence of Ca2+, pH 7.5 and 4.0, at 25 degrees C. Thermodynamic values showed that rLRAP had a Ca2+-binding affinity approximately 6.4-times greater than rH174. NMR and CD data revealed that rLRAP was randomly coiled, and that this structure was not altered by Ca2+, which bound to rLRAP and rH174 via ionic interactions. Unlike r174 (beta-spiral), rLRAP had a random-coiled structure. The calcium binding and structural differences between rLRAP and rH174 suggest that these proteins have different functions in enamel biomineralization.
                    • Article
                    • Peer Reviewed
                    UC San Francisco Previously Published Works (2006)
                    Amelogenins are a unique group of alternatively spliced proteins. While the full-length amelogenin is known to assemble into nanospheres and alter apatite crystal growth and alignment, the function of the leucine-rich amelogenin peptide (LRAP) in biomineralization is not understood. This study tested the hypothesis that LRAP self-assembles into a supramolecular structure and guides crystal growth similarly to the full-length protein. Synthetic LRAP and recombinant full-length amelogenin (rH175) were used at different concentrations and either immobilized onto fluoroapatite substrates (FAP) or immersed into saturated calcium-phosphate solutions. The structure of the assembled protein and the height of apatite crystals formed on the FAP template were determined using atomic force microscopy. Both LRAP and rH175 assembled into nanospheres. LRAP self-assembly, however, was only observed at concentrations of >0.5 mg ml-1 and limited to sizes between 5 and 30 nm. Apatite crystal growth was not significantly affected by LRAP, while rH175 accelerated crystal growth by up to 50-fold. The increased growth rate was only observed when rH175 precipitated at concentrations of >0.8 mg ml-1. It was concluded that the ability of amelogenins to self-assemble into nanospheres and to bind to apatite in vitro is not inevitably an indication for the ability to control apatite crystal growth.
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