Targeted p120-Catenin Ablation Disrupts Dental Enamel Development

Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows

The Acid Test of Fluoride: How pH Modulates Toxicity

Background: It is not known why the ameloblasts responsible for dental enamel formation are uniquely sensitive to fluoride ($F^−$). Herein, we present a novel theory with supporting data to show that the low pH environment of maturating stage ameloblasts enhances their sensitivity to a given dose of $F^−$. Enamel formation is initiated in a neutral pH environment (secretory stage); however, the pH can fall to below 6.0 as most of the mineral precipitates (maturation stage). Low pH can facilitate entry of $F^−$ into cells. Here, we asked if $F^−$ was more toxic at low pH, as measured by increased cell stress and decreased cell function. Methodology/Principal Findings: Treatment of ameloblast-derived LS8 cells with $F^−$ at low pH reduced the threshold dose of $F^−$ required to phosphorylate stress-related proteins, PERK, eIF2α, JNK and c-jun. To assess protein secretion, LS8 cells were stably transduced with a secreted reporter, Gaussia luciferase, and secretion was quantified as a function of $F^−$ dose and pH. Luciferase secretion significantly decreased within 2 hr of $F^−$ treatment at low pH versus neutral pH, indicating increased functional toxicity. Rats given 100 ppm $F^−$ in their drinking water exhibited increased stress-mediated phosphorylation of eIF2α in maturation stage ameloblasts (pH<6.0) as compared to secretory stage ameloblasts (pH∼7.2). Intriguingly, $F^−$-treated rats demonstrated a striking decrease in transcripts expressed during the maturation stage of enamel development (Klk4 and Amtn). In contrast, the expression of secretory stage genes, AmelX, Ambn, Enam and Mmp20, was unaffected. Conclusions: The low pH environment of maturation stage ameloblasts facilitates the uptake of $F^−$, causing increased cell stress that compromises ameloblast function, resulting in dental fluorosis

Mosaic phenotype of the K14-Cre p120-cKO mouse incisor.

<p>A section through a mosaic cKO enamel organ (A) showing normal ameloblasts (Am) in the middle and malformed ameloblasts to the left (arrow). Es, enamel space. SEM analysis of the same cKO mosaic incisor (B). The right side of the bracket touches normal enamel and the left side touches an abnormal, enamel-free area. Between the two sides of the bracket is the malformed dysplastic enamel. Note that the cKO mouse is capable of forming normal enamel. Therefore, the observed enamel dysplasia is not a secondary effect of conditional p120 ablation.</p

E-cadherin is only expressed in ameloblasts capable of expressing p120.

<p>In the same K14-Cre p120-cKO mosaic incisor shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012703#pone-0012703-g008" target="_blank">Figure 8</a>, E-cadherin (top) and p120 catenin (bottom) was immunolocalized in adjacent cross-sections. E-cadherin is expressed exclusively in normal appearing ameloblasts (brackets) that also express p120. However, in flattened, malformed ameloblasts where p120 was ablated, immunostaining for E-cadherin was not detectable. EO, enamel organ; PO, pulp organ.</p

N-cadherin is expressed in wild-type secretory stage ameloblasts, but not in p120 ablated ameloblasts.

<p>In the less mature second molar (M2), N-cadherin was not expressed (A) in the enamel organ (EO) or pulp organ (PO) of three day-old mice. In the more mature first molar (M1), N-cadherin was expressed (B, C). After dentin matrix deposition, odontoblasts (Od) and ameloblasts (Am) showed lateral membrane immunostaining for N-cadherin, and the apical and basal terminal web apparatus of ameloblasts were also stained positively (B). After enamel matrix deposition, the odontoblasts stain intensely (C). A developing cusp tip from the first molar of a P3 K14-Cre p120-cKO mouse stained for N-cadherin (D). N-cadherin expression was detected in odontoblasts, but not in the ameloblasts from this molar. Note that the dentin appears rough and mildly dysplastic.</p

Enamel from K14-Cre p120-cKO mice does not mineralize properly.

<p>Faxitron analysis of wild-type and cKO mouse skulls reveals either absence of enamel or a thin layer of poorly mineralized enamel that is indistinguishable from the underlying dentin (Top). This assessment was corroborated by micro-CT analysis of molar and incisor teeth (Bottom). The wild-type molars had clearly defined opaque enamel layers whereas molars from the cKO mice did not (bracket identifies molars lacking a highly mineralized enamel layer). Note that small spots of more highly mineralized enamel are present on the cKO mouse molars. These data indicate that the globular material observed on the tooth surface by SEM is poorly mineralized. The cKO incisor tooth (bottom) had a thin layer of mineralized enamel covering a small portion of the labial tooth surface whereas the wild-type incisor had thick enamel covering the entire labial surface of the dentin.</p