Faculty Bibliography

Transcellular calcium transport is an essential activity in mineralized tissue formation, including dental hard tissues. In many organ systems, this activity is regulated by membrane-bound sodium/calcium (Na(+)/Ca(2+)) exchangers, which include the NCX and NCKX [sodium/calcium-potassium (Na(+)/Ca(2+)-K(+)) exchanger] proteins. During enamel maturation, when crystals expand in thickness, Ca(2+) requirements vastly increase but exactly how Ca(2+) traffics through ameloblasts remains uncertain. Previous studies have shown that several NCX proteins are expressed in ameloblasts, although no significant shifts in expression were observed during maturation which pointed to the possible identification of other Ca(2+) membrane transporters. NCKX proteins are encoded by members of the solute carrier gene family, Slc24a, which include 6 different proteins (NCKX1-6). NCKX are bidirectional electrogenic transporters regulating Ca(2+) transport in and out of cells dependent on the transmembrane ion gradient. In this study we show that all NCKX mRNAs are expressed in dental tissues. Real-time PCR indicates that of all the members of the NCKX group, NCKX4 is the most highly expressed gene transcript during the late stages of amelogenesis. In situ hybridization and immunolocalization analyses clearly establish that in the enamel organ, NCKX4 is expressed primarily by ameloblasts during the maturation stage. Further, during the mid-late maturation stages of amelogenesis, the expression of NCKX4 in ameloblasts is most prominent at the apical poles and at the lateral membranes proximal to the apical ends. These data suggest that NCKX4 might be an important regulator of Ca(2+) transport during amelogenesis.

In rodent incisors two distinct stages of enamel formation can be identified visually based on cell morphology: the secretory stage and the maturation stage. The expression profiles of many genes characterize both stages, including the bicarbonate transport protein NBCe1. Bicarbonate is a requirement for the mineralizing enamel matrix to buffer excessive protons that form as a consequence of hydroxyapatite formation. NBCe1-B mRNA is up-regulated during the maturation stage of amelogenesis, where hydroxyapatite formation predominates. In this study, a presumed 572-bp NBCe1-B promoter region was subcloned into a reporter construct, and within this 572-bp region of DNA we characterized a 285-bp segment that shows an increase of approximately 2.3-fold in gene-transcription activity when transfected into ameloblast-like cells and cultured in medium maintained at pH 6.8 (vs. pH 7.4). A presumed pH-responsive transcriptional factor-binding domain(s) thus resides in the 285-bp NBCe1-B promoter region where candidate domains include the nuclear factor of kappa light polypeptide gene enhancer in B-cells1(NFKB1), jun proto-oncogene (JUN), and tumor protein p53(TP53)-binding sites. Mutagenesis studies identify that both the NFKB1- and TP53-binding sites are responsive to changes in the extracellular pH. These data help to explain how ameloblasts respond to the altered extracellular milieu of protons by changing their gene-expression profile throughout the stages of amelogenesis.

Enamel maturation is a dynamic process that involves high rates of mineral acquisition, associated fluctuations in extracellular pH, and resorption of extracellular enamel proteins. During maturation, ameloblasts change from having a tall, thin, and highly polarized organization, characteristic of the secretory stage, to having a low columnar and widened morphology in the maturation stage. To identify potential differences in gene expression throughout maturation, we obtained enamel organ epithelial cells derived from the early- and late-maturation stages of rat incisor and analyzed the global gene-expression profiles at each stage. Sixty-three candidate genes were identified as having potential roles in the maturation process. Quantitative PCR was used to confirm the results of this genome-wide analysis in a subset of genes. Transcripts enriched during late maturation (n = 38) included those associated with lysosomal activity, solute carrier transport, and calcium signaling. Also up-regulated were transcripts involved in cellular responses to oxidative stress, proton transport, cell death, and the immune system. Transcripts down-regulated during the late maturation stage (n =25) included those with functions related to cell adhesion, cell signaling, and T-cell activation. These results indicate that ameloblasts undergo widespread molecular changes during the maturation stage of amelogenesis and hence provide a basis for future functional investigations into the mechanistic basis of enamel mineralization.

Huang Z, Kim J, Lacruz RS, Bringas P Jr, Glogauer M, Bromage TG, Kaartinen VM, Snead ML. Epithelial-specific knockout of the Rac1 gene leads to enamel defects. Eur J Oral Sci 2011; 119 (Suppl. 1): 168-176. (c) 2011 Eur J Oral Sci The Ras-related C3 botulinum toxin substrate 1 (Rac1) gene encodes a 21-kDa GTP-binding protein belonging to the RAS superfamily. RAS members play important roles in controlling focal adhesion complex formation and cytoskeleton contraction, activities with consequences for cell growth, adhesion, migration, and differentiation. To examine the role(s) played by RAC1 protein in cell-matrix interactions and enamel matrix biomineralization, we used the Cre/loxP binary recombination system to characterize the expression of enamel matrix proteins and enamel formation in Rac1 knockout mice (Rac1(-/-) ). Mating between mice bearing the floxed Rac1 allele and mice bearing a cytokeratin 14-Cre transgene generated mice in which Rac1 was absent from epithelial organs. Enamel of the Rac1 conditional knockout mouse was characterized by light microscopy, backscattered electron imaging in the scanning electron microscope, microcomputed tomography, and histochemistry. Enamel matrix protein expression was analyzed by western blotting. Major findings showed that the Tomes' processes of Rac1(-/-) ameloblasts lose contact with the forming enamel matrix in unerupted teeth, the amounts of amelogenin and ameloblastin are reduced in Rac1(-/-) ameloblasts, and after eruption, the enamel from Rac1(-/-) mice displays severe structural defects with a complete loss of enamel. These results support an essential role for RAC1 in the dental epithelium involving cell-matrix interactions and matrix biomineralization

Two main proteases cleave enamel extracellular matrix proteins during amelogenesis. Matrix metalloprotease-20 (Mmp20) is the predominant enzyme expressed during the secretory stage, while kallikrein-related peptidase-4 (Klk4) is predominantly expressed during maturation. Mutations to both Mmp20 and Klk4 result in abnormal enamel phenotypes. During a recent whole-genome microarray analysis of rat incisor enamel organ cells derived from the secretory and maturation stages of amelogenesis, the serine protease chymotrypsin C (caldecrin, Ctrc) was identified as significantly up-regulated (> 11-fold) during enamel maturation. Prior reports indicate that Ctrc expression is pancreas-specific, albeit low levels were also noted in brain. We here report on the expression of Ctrc in the enamel organ. Quantitative PCR (qPCR) and Western blot analysis were used to confirm the expression of Ctrc in the developing enamel organ. The expression profile of Ctrc is similar to that of Klk4, increasing markedly during the maturation stage relative to the secretory stage, although levels of Ctrc mRNA are lower than for Klk4. The discovery of a new serine protease possibly involved in enamel development has important implications for our understanding of the factors that regulate enamel biomineralization.

Strepsirrhines are members of a primate suborder that has a distinctive set of features associated with the development of the dentition. Amelogenin (AMEL), the better known of the enamel matrix proteins, forms 90% of the secreted organic matrix during amelogenesis. Although AMEL has been sequenced in numerous mammalian lineages, the only reported strepsirrhine AMEL sequences are those of the ring-tailed lemur and galago, which contain a set of additional proline-rich tandem repeats absent in all other primates species analyzed to date, but present in some non-primate mammals. Here, we first determined that these repeats are present in AMEL from three additional lemur species and thus are likely to be widespread throughout this group. To evaluate the functional relevance of these repeats in strepsirrhines, we engineered a mutated murine amelogenin sequence containing a similar proline-rich sequence to that of Lemur catta. In the monomeric form, the MQP insertions had no influence on the secondary structure or refolding properties, whereas in the assembled form, the insertions increased the hydrodynamic radii. We speculate that increased AMEL nanosphere size may influence enamel formation in strepsirrhine primates

Recent discoveries at the new hominin-bearing deposits of Malapa, South Africa, have yielded a rich faunal assemblage associated with the newly described hominin taxon Australopithecus sediba. Dating of this deposit using U-Pb and palaeomagnetic methods has provided an age of 1.977 Ma, being one of the most accurately dated, time constrained deposits in the Plio-Pleistocene of southern Africa. To date, 81 carnivoran specimens have been identified at this site including members of the families Canidae, Viverridae, Herpestidae, Hyaenidae and Felidae. Of note is the presence of the extinct taxon Dinofelis cf. D. barlowi that may represent the last appearance date for this species. Extant large carnivores are represented by specimens of leopard (Panthera pardus) and brown hyaena (Parahyaena brunnea). Smaller carnivores are also represented, and include the genera Atilax and Genetta, as well as Vulpes cf. V. chama. Malapa may also represent the first appearance date for Felis nigripes (Black-footed cat). The geochronological age of Malapa and the associated hominin taxa and carnivoran remains provide a window of research into mammalian evolution during a relatively unknown period in South Africa and elsewhere. In particular, the fauna represented at Malapa has the potential to elucidate aspects of the evolution of Dinofelis and may help resolve competing hypotheses about faunal exchange between East and Southern Africa during the late Pliocene or early Pleistocene.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR), a phosphorylation- and ATP-regulated anion channel. CFTR expression and activity is frequently associated with an anion exchanger (AE) such as AE2 coded by the Slc4a2 gene. Mice null for Cftr and mice null for Slc4a2 have enamel defects, and there are some case reports of enamel anomalies in patients with CF. In this study we demonstrate that both Cftr and AE2 expression increased significantly during the rat enamel maturation stage versus the earlier secretory stage (5.6- and 2.9-fold, respectively). These qPCR data im- ply that there is a greater demand for Cl(-) and bicarbonate (HCO(3)(-)) transport during the maturation stage of enamel formation, and that this is, at least in part, provided by changes in Cftr and AE2 expression. In addition, the enamel phenotypes of 2 porcine models of CF, CFTR-null, and CFTR-DeltaF508 have been examined using backscattered electron microscopy in a scanning electron microscope. The enamel of newborn CFTR-null and CFTR-DeltaF508 animals is hypomineralized. Together, these data provide a molecular basis for interpreting enamel disease associated with disruptions to CFTR and AE2 expression

Proximal renal tubular acidosis (pRTA) is a syndrome caused by abnormal proximal tubule reabsorption of bicarbonate resulting in metabolic acidosis. Patients with mutations to the SLC4A4 gene (coding for the sodium bicarbonate cotransporter NBCe1), have pRTA, growth delay, ocular defects, and enamel abnormalities. In an earlier report, we provided the first evidence that enamel cells, the ameloblasts, express NBCe1 in a polarized fashion, thereby contributing to trans-cellular bicarbonate transport. To determine whether NBCe1 plays a critical role in enamel development, we studied the expression of NBCe1 at various stages of enamel formation in wild-type mice and characterized the biophysical properties of enamel in NBCe1(-/-) animals. The enamel of NBCe1(-/-) animals was extremely hypomineralized and weak with an abnormal prismatic architecture. The expression profile of amelogenin, a known enamel-specific gene, was not altered in NBCe1(-/-) animals. Our results show for the first time that NBCe1 expression is required for the development of normal enamel. This study provides a mechanistic model to account for enamel abnormalities in certain patients with pRTA.