Ameloblasts require active RhoA to generate normal dental enamel.

RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up-regulated. Transgenic mice were generated that express a dominant-negative RhoA transgene in ameloblasts using amelogenin gene-regulatory sequences. Transgenic and wild-type (WT) molar tooth germs were incubated with sodium fluoride (NaF) or sodium chloride (NaCl) in organ culture. Filamentous actin (F-actin) stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared with WT ameloblasts treated with NaCl or with transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/Rho-associated protein kinase (ROCK) pathway in the transgenic mice. Little difference in quantitative fluorescence (an estimation of fluorosis) was observed between WT and transgenic incisors from mice provided with drinking water containing NaF. We subsequently found reduced transgene expression in incisors compared with molars. Transgenic molar teeth had reduced amelogenin, E-cadherin, and Ki67 compared with WT molar teeth. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein, amelogenin, and E-cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited.

Mouse genetic background influences the dental phenotype.

Dental enamel covers the crown of the vertebrate tooth and is considered to be the hardest tissue in the body. Enamel develops during secretion of an extracellular matrix by ameloblast cells in the tooth germ, prior to eruption of the tooth into the oral cavity. Secreted enamel proteins direct mineralization patterns during the maturation stage of amelogenesis as the tooth prepares to erupt. The amelogenins are the most abundant enamel proteins and are required for normal enamel development. Phenotypic differences were observed between incisors from individual Amelx (amelogenin) null mice that had a mixed 129xC57BL/6J genetic background and between inbred wild-type (WT) mice with different genetic backgrounds (C57BL/6J, C3H/HeJ, FVB/NJ). We hypothesized that this could be due to modifier genes, as human patients with a mutation in an enamel protein gene causing the enamel defect amelogenesis imperfecta (AI) can also have varied appearance of dentitions within a kindred. Enamel density measurements varied for all WT inbred strains midway during incisor development. Enamel thickness varied between some WT strains, and, unexpectedly, dentin density varied extensively between incisors and molars of all WT and Amelx null strains studied. WTFVB/NJ incisors were more similar to those of Amelx null mice than to those of the other WT strains in terms of incisor height/width ratio and pattern of enamel mineralization. Strain-specific differences led to the conclusion that modifier genes may be implicated in determining both normal development and severity of enamel appearance in AI mouse models and may in future studies be related to phenotypic heterogeneity within human AI kindreds reported in the literature.

Dental enamel structure is altered by expression of dominant negative RhoA in ameloblasts.

Using in vitrotooth germ cultures and analysis by confocal microscopy, ameloblasts treated with sodium fluoride were found to have elevated amounts of filamentous actin. Because this response is reduced by inhibitors of the Rho/ROCK signaling pathway, we generated mice that express dominant negative RhoA (RhoA(DN)) in ameloblasts for in vivo analysis. Expression of the EGFP-RhoA(DN) fusion protein was evaluated by RT-PCR and immunohistochemistry, and teeth were analyzed by scanning electron microscopy. The 3 strains expressed at either low (TgEGFP-RhoA(DN)-8), intermediate (TgEGFP-RhoA(DN)-2), or high (TgEGFP-RhoA(DN)-13) levels, and the molar teeth from the 3 strains had enamel hypoplasia and surface defects. We conclude that RhoA(DN) expressed in ameloblasts interferes with normal enamel development through the pathway that is induced by sodium fluoride.

Wnt-RhoA signaling is involved in dental enamel development.

Transgenic mice that express dominant-negative RhoA (RhoA(DN) ) in ameloblasts have hypoplastic enamel with defects in molar cusps. ß-catenin and Wnt5a were up-regulated in enamel organs of RhoA(DN) transgenic mice, which indicated that both canonical and non-canonical Wnt pathways are implicated in the process of enamel defect formation. It was hypothesized that expression of RhoA(DN) in ameloblasts interfered with normal enamel development through the pathways that were induced by fluoride. The Wnt and RhoA pathways were further investigated in an ameloblast-lineage cell line (ALC) by treatment with sodium fluoride (NaF). The activities of RhoA and Rho-associated protein kinase (ROCK) II decreased significantly by 8-12 hours, similar to decreased activity in RhoA(DN) transgenic mice. Both canonical and non-canonical Wnt pathways were activated by treatment with NaF, which was verified by western blotting and the ß-catenin-TCF/LEF (T cell factor lymphanoid/enhancer factor) reporter gene (TOPflash) assay. ß-catenin localization to both cytoplasm and nucleus was up-regulated in NaF-treated ALC, while Gsk-3ß, the negative regulator of the Wnt pathway, showed a decreased pattern of expression. The current results indicate that both Wnt and RhoA pathways are implicated in fluoride-induced signaling transductions in the ALC as well as in the development of enamel defects in RhoA(DN) transgenic mice.

The role of amelogenin during enamel-crystallite growth and organization in vivo.

Amelogenin is critical for enamel formation, and human amelogenin gene (AMELX) mutations cause hypoplastic and/or hypomaturation enamel phenotypes. The Amelx null (AKO) mouse has a severe hypoplastic phenotype. This study evaluated the effect of amelogenin loss on enamel formation and crystallite morphology. Enamel from AKO and wild-type (WT) mice was used. The AKO mice were mated with transgenic mice expressing the most abundant known amelogenin isoform, TgM180-87, to rescue (KOM180-87) the enamel crystallite phenotype. Molar enamel was embedded, sectioned with a diamond microtome, and images were obtained by transmission electron microscopy. The crystallite sizes from multiple sections were measured using Image J. The mean thicknesses (WT = 26 nm, AKO = 16 nm, and KOM180-87 = 25 nm) and the mean widths (WT = 96 nm, AKO = 59 nm, KOM180-87 = 85 nm) of crystallites were measured. Despite a complete loss of amelogenin in AKO mice, a mineralized enamel layer with well-defined and organized crystallites is formed. In the absence of amelogenin, enamel crystallites were reduced in thickness and width. For the first time we show that introduction of the m180 amelogenin isoform into the AKO mouse through cross-breeding rescues the crystallite phenotype. We conclude that amelogenin is essential for the development of normal crystallite size.

Rescue of the murine amelogenin null phenotype with two amelogenin transgenes.

The amelogenin proteins are required for normal enamel development, and the most abundant amelogenins expressed from alternatively spliced mRNAs are M180 and leucine-rich amelogenin protein (LRAP). The X-Chromosomal Amelogenin (Amelx) null [knockout (KO)] mouse has an enamel defect similar to human X-linked amelogenesis imperfecta. The disorganized enamel layer in KO mice is 10-20% of the thickness of wild-type (WT) enamel and lacks prismatic structures. When the KO mice were mated with mice that express the transgene M180-87, (TgM180-87) partial rescue of the phenotype was observed such that enamel thickness, volume, and density increased. A second transgene was introduced by mating TgM180 KO mice with TgLRAP mice, and male offspring were characterized for genotype and tooth phenotype was evaluated by scanning electron microscopy. The molar enamel thickness of TgM180-LRAP KO mice was further increased, and the structure was improved, with a more defined decussation pattern compared with singly rescued mice. We conclude that TgM180 provides significant rescue of the KO phenotype. Although the effectiveness of the LRAP transgene, alone, to rescue is less obvious, the addition of the LRAP transgene to the M180 transgene in KO enamel leads to an added improvement in both amount and structure and thus these transgenes function in a complementary manner. Together, the two most abundant amelogenins lead to the formation of obvious enamel decussation patterns.

Leucine-rich amelogenin peptide: a candidate signaling molecule during cementogenesis.

BACKGROUND: Cementum is a critical mineralized tissue; however, control of its formation remains undefined. One hypothesis is that enamel matrix proteins/peptides secreted by ameloblasts and/or epithelial rest cells contribute to the control of cementum formation via epithelial-mesenchymal interactions. Here, we focused on determining whether or not leucine-rich amelogenin peptide (LRAP), translated from an alternatively spliced amelogenin RNA, altered cementoblast behavior. METHODS: Immortalized murine cementoblasts (OCCM-30) were exposed to LRAP and evaluated for: 1) proliferative activity; 2) gene expression using Northern blot for Cbfal (core binding factor alpha-1); OCN (osteocalcin), OPN (osteopontin), and real-time reverse transcription-polymerase chain reaction (RT-PCR) for OPG (osteoprotegerin); and RANKL (receptor activator of NF-kappaB ligand); 3) signaling pathway using inhibitors of PKA (THFA), PKC (GF109203X), and MAPK (UO126); and 4) mineralization evaluated by von Kossa and Alizarin-red. RESULTS: LRAP had no effect on cell proliferation up to 6 days, with a decrease in cell growth observed at the highest dose by 9 days versus untreated cells. LRAP down regulated OCN and up regulated OPN in a dose- and time-response fashion, and inhibited the capacity of mineral nodule formation. Transcripts for OPG were increased in LRAP-treated cells compared to control, but RANKL mRNA levels were not affected. Core binding factor alpha (Cbfa) mRNA, expressed constitutively, was not affected by LRAP. Signaling pathway assays suggested involvement of the MAPK pathway, since the addition of the MAPK inhibitor suppressed OPN expression in LRAP-treated cells. CONCLUSION: Leucine-rich amelogenin peptide appears to have a direct effect on cementoblast activity that may prove significant during development as well as in regeneration of periodontal tissues.