Uncoupling protein-2 is an antioxidant that is up-regulated in the enamel organ of fluoride-treated rats

Dental fluorosis is characterized by subsurface hypomineralization and retention of enamel matrix proteins. Fluoride (F−) exposure generates reactive oxygen species (ROS) that can cause ER-stress. We therefore screened oxidative stress arrays to identify genes regulated by F− exposure. Vitamin E is an antioxidant so we asked if a diet high in vitamin E would attenuate dental fluorosis. Maturation stage incisor enamel organs (EO) were harvested from F− treated rats and mice were assessed to determine if vitamin E ameliorates dental fluorosis. Uncoupling protein-2 (Ucp2) was significantly up-regulated by F− (~1.5 & 2.0 fold for the 50 or 100 ppm F− treatment groups respectively). Immunohistochemical results on maturation stage rat incisors demonstrated that UCP2 protein levels increased with F− treatment. UCP2 down-regulates mitochondrial production of ROS, which decreases ATP production. Thus, in addition to reduced protein translation caused by ER-stress, a reduction in ATP production by UCP2 may contribute to the inability of ameloblasts to remove protein from the hardening enamel. Fluoride treated mouse enamel had significantly higher quantitative fluorescence (QF) than the untreated controls. No significant QF difference was observed between control and vitamin E enriched diets within a given F− treatment group. Therefore, a diet rich in vitamin E did not attenuate dental fluorosis. We have identified a novel oxidative stress response gene that is up-regulated in vivo by F− and activation of this gene may adversely affect ameloblast function

Uncoupling protein-2 is an antioxidant that is up-regulated in the enamel organ of fluoride-treated rats

Dental fluorosis is characterized by subsurface hypomineralization and retention of enamel matrix proteins. Fluoride (F(−)) exposure generates reactive oxygen species (ROS) that can cause ER-stress. We therefore screened oxidative stress arrays to identify genes regulated by F(−) exposure. Vitamin E is an antioxidant so we asked if a diet high in vitamin E would attenuate dental fluorosis. Maturation stage incisor enamel organs (EO) were harvested from F(−) treated rats and mice were assessed to determine if vitamin E ameliorates dental fluorosis. Uncoupling protein-2 (Ucp2) was significantly up-regulated by F(−) (~1.5 & 2.0 fold for the 50 or 100 ppm F(−) treatment groups respectively). Immunohistochemical results on maturation stage rat incisors demonstrated that UCP2 protein levels increased with F(−) treatment. UCP2 down-regulates mitochondrial production of ROS, which decreases ATP production. Thus, in addition to reduced protein translation caused by ER-stress, a reduction in ATP production by UCP2 may contribute to the inability of ameloblasts to remove protein from the hardening enamel. Fluoride treated mouse enamel had significantly higher quantitative fluorescence (QF) than the untreated controls. No significant QF difference was observed between control and vitamin E enriched diets within a given F(−) treatment group. Therefore, a diet rich in vitamin E did not attenuate dental fluorosis. We have identified a novel oxidative stress response gene that is up-regulated in vivo by F(−) and activation of this gene may adversely affect ameloblast function

Matrix metalloproteinase-20 over-expression is detrimental to enamel development: a Mus musculus model.

Matrix metalloproteinase-20 (Mmp20) ablated mice have enamel that is thin and soft with an abnormal rod pattern that abrades from the underlying dentin. We asked if introduction of transgenes expressing Mmp20 would revert this Mmp20 null phenotype back to normal. Unexpectedly, for transgenes expressing medium or high levels of Mmp20, we found opposite enamel phenotypes depending on the genetic background (Mmp20(-/-) or Mmp20(+/+) ) in which the transgenes were expressed.Amelx-promoter-Mmp20 transgenic founder mouse lines were assessed for transgene expression and those expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Regardless of expression level, each transgene brought the null enamel back to full thickness. However, the high and medium expressing Mmp20 transgenes in the Mmp20 null background had significantly harder more mineralized enamel than did the low transgene expresser. Strikingly, when the high and medium expressing Mmp20 transgenes were present in the wild-type background, the enamel was significantly less well mineralized than normal. Protein gel analysis of enamel matrix proteins from the high and medium expressing transgenes present in the wild-type background demonstrated that greater than normal amounts of cleavage products and smaller quantities of higher molecular weight proteins were present within their enamel matrices.Mmp20 expression levels must be within a specific range for normal enamel development to occur. Creation of a normally thick enamel layer may occur over a wider range of Mmp20 expression levels, but acquisition of normal enamel hardness has a narrower range. Since over-expression of Mmp20 results in decreased enamel hardness, this suggests that a balance exists between cleaved and full-length enamel matrix proteins that are essential for formation of a properly hardened enamel layer. It also suggests that few feedback controls are present in the enamel matrix to prevent excessive MMP20 activity

Assessment of incisor microhardness on incisor longitudinal sections.

<p>Approximately 20 indentations throughout the enamel layer were obtained per incisor and the results were average to generate one data point for the graph. Measurements from at least 4 incisors from each genotype were used to generate a bar on the graph. Whiskers denote the data range and the horizontal line within the box represents the median microhardness value. When present in the <i>Mmp20</i> null background (A), the Tg6i (M) <i>Mmp20^−/−</i> (N = 4) enamel was slightly softer (P<0.05) than wild-type mouse enamel (Tg– <i>Mmp20^+/+</i>, N = 8), but the Tg24i (H) <i>Mmp20^−/−</i> (N = 4) enamel had a wider range of microhardness values and was therefore not significantly different from wild-type. The Tg42i (L) <i>Mmp20^−/−</i> (N = 7) enamel was much softer than enamel from wild-type mice and this difference was highly significant (P<0.0001). In contrast, each of the transgenic mice had enamel that was harder than the enamel from the <i>Mmp20</i> null mouse incisors (Tg– <i>Mmp20^−/−</i>, N = 11) and these differences were all highly significant (P<0.0001). Enamel hardness values positively correlated to the level of transgene expression in mouse incisors when the transgenes were in the <i>Mmp20</i> null background. When present in the <i>Mmp20</i> wild-type background (B), the Tg6i (M) <i>Mmp20^+/+</i> (N = 4) incisor enamel was softer (P<0.01) than enamel from wild-type mice (Tg– <i>Mmp20^+/+</i>, N = 8) and the Tg24i (H) <i>Mmp20^+/+</i> (N = 4) enamel was much softer than enamel from wild-type mice (P<0.0001). However, no difference in enamel hardness was observed between wild-type and Tg42i (L) <i>Mmp20^+/+</i> (N = 4) enamel. Enamel hardness values negatively correlated to the level of transgene expression in mouse incisors when the transgenes were in the wild-type background.</p

Assessment of enamel thickness in incisor cross-sections.

<p>Thickness was measured in the widest portion of the enamel layer of each incisor cross section as illustrated by the line in the top right panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086774#pone-0086774-g005" target="_blank">Figure 5</a>. Each bar (genotype) on this graph represents enamel measurements from three different mouse incisors. No significant difference in enamel thickness was observed among wild-type and transgenes present in the <i>Mmp20</i> null background (A). Each of the three transgenes brought the null enamel back to its normal thickness. When the transgenes were present in the wild-type background (B), the most highly expressed transgene [Tg24i (H)] had an enamel layer that was significantly thinner (P<0.001) than wild-type enamel.</p

Assessment of control and <i>Mmp20</i> transgenic enamel by light microscopy.

<p>Wild-type control (Tg– <i>Mmp20^+/+</i>) incisors had a sharp incisal tip and the characteristic yellow-brown coloration. The molars contained a fully thick enamel layer and the cusp tips were well defined. In contrast, <i>Mmp20</i> null (Tg– <i>Mmp20^−/−</i>) incisors had a blunted tip and showed little or no enamel and no yellow-brown color. The molars were worn and the remaining cusp tips appeared thin from abrasion and absence of a full thickness enamel layer. For the transgenes in the <i>Mmp20</i> null background (Tg+ <i>Mmp20^−/−</i>): The Tg6 transgenic enamel had a sharp incisal tip, but the yellow-brown color was mostly missing while the molars appeared fully recovered from the null phenotype. The Tg24 transgenic animals had incisors that appeared sharp and somewhat yellow-brown in color, but the enamel surface was rough and appeared slightly chalky rather than translucent. The molar cusp tips were worn and the enamel layer appeared hypoplastic. The Tg42 transgenic mice had incisors that were blunted with a rough enamel surface that was mildly yellow-brown color with a chalky appearance like the Tg24 transgenic incisors. The molars appeared well formed and fully recovered from the null phenotype. For the transgenes in the <i>Mmp20</i> wild-type background (Tg+ <i>Mmp20^+/+</i>): The Tg6 transgenic mice had well pigmented but blunted incisors with a chalky-white appearance and rough enamel surface. The molars were severely compromised having abraded cusp tips with pitted enamel surfaces. The Tg24 transgenic enamel appeared almost identical to the Tg6 enamel in the wild-type background. In contrast, Tg42 transgenic incisors had a sharp tip and smooth enamel, but with almost no yellow-brown color. The Tg42 transgenic molars appeared no different than the wild-type molars. The observed Tg6 transgenic enamel was the best example of an overall recovery (both incisors and molars) from the <i>Mmp20</i> null phenotype.</p

Assessment of MMP20 protein content from extracted enamel.

<p>Immunoblots performed on proteins extracted from 5 day-old molars (A) or adult incisors (C) assessed MMP20 quantity. Zymography of extracted enamel from 5 day-old molars assessed MMP20 proteolytic activity (B). MMP20 was not detected in <i>Mmp20</i> null (–/–) mouse enamel, low levels were observed in the heterozygous (+/–) enamel and wild-type (+/+) enamel had more MMP20 protein than did the heterozygotes. In molar (m) enamel from <i>Mmp20</i> null mice, the Tg6 transgene [Tg6m (H)] expressed the highest quantities of MMP20 followed by the Tg24m (M) transgene at mid-levels with the Tg42m (L) transgene expressing the lowest levels of MMP20. The Tg42m (L) transgene expressed lower MMP20 amounts than were present in enamel from wild-type mice (A). Zymography results for MMP20 activity (B) were consistent with the immunoblot results. In contrast to the molar results, immunoblots performed on extracted incisor (i) enamel showed that enamel from Tg24i (H) transgenic mice contained the highest amount of MMP20 protein followed by Tg6i (M) enamel and then Tg42i (L) transgenic enamel which, like the molars, also contained less MMP20 than wild-type enamel (C). As expected, enamel from transgenes expressed in the <i>Mmp20</i> wild-type background had total MMP20 quantities that were higher for each transgene than were observed in the null background. Arrows point to the MMP20 doublet bands that each represents an active form of MMP20.</p

Enamel matrix protein profile and immunoblotting of amelogenin splice and cleavage products.

<p>Total extracted enamel matrix proteins (mostly amelogenins) from 5-day old molars were run on an SDS PAGE gel (A). Comparison of the first two lanes containing extracted wild-type or <i>Mmp20</i> null enamel matrix proteins demonstrate that the null enamel has a prominent band at approximately 27 kDa that is only weakly present in the wild-type lane indicating that this band is cleaved by MMP20. Also a doublet just above 20 kDa was present in the wild-type lane but was a single band in the null lane indicating that one of the wild-type bands is an MMP20 cleavage product. The protein profile for the Tg6m (H) transgene in the <i>Mmp20^−/−</i> background was similar to that of the protein extracted from wild-type enamel. However, the protein profile for Tg6m (H) and Tg24m (M) in the <i>Mmp20^+/+</i> background had a strong band below the 20 kDa marker and a weaker band beneath that indicating that more cleavage products than normal were present. Both transgenes in the wild-type background had bands above 20 kDa that were less prominent than the bands observed from wild-type enamel with no transgene. In contrast, regardless of the <i>Mmp20</i> background, the Tg42m (L) positive mice had protein banding patterns that looked substantially like the wild-type results. Amelogenin immunoblot results from the various geneotypes (B) were similar to the protein gel results. An approximate 27-kDa amelogenin band was present in the <i>Mmp20</i> null but not wild-type lanes. In the wild-type background, both Tg6m (H) and Tg24m (M) transgenes had prominent amelogenin bands that located below 20 kDa and the bands above 20 kDa appeared less prominent than those observed in the Tg– wild-type lane. Also the amelogenin profile of Tg42m (L) in the wild-type background looked similar to that of wild-type mice.</p

Assessment of incisor enamel mineralization by backscatter SEM and pseudo-color mapping.

<p>Backscatter images of incisor cross sections were sectioned at a site located 8-color mapping of the image above. Color mapping allows easier visualization of differences in enamel mineralization within the enamel layer. Blue and white colors indicate decreased mineralization relative to the highly mineralized red color. For wild-type mice (Tg– Mmp20+/+), color mapping shows that enamel on the mesial (left) side is not quite as mineralized as is the rest of the enamel layer at this level of sectioning just prior to where the incisor erupts into the mouth. The <i>Mmp20</i> null mouse (Tg– Mmp20–/–) enamel illustrates the variations in appearance of the enamel layer that is typical for these teeth. For transgenes in the <i>Mmp20</i> null background, Tg6i (M) transgenic incisors had less mineralized enamel especially at the lateral (right) side and the surface. Tg24i (H) transgenic incisors showed similar results as well as an irregular enamel surface. Enamel from mice transgenic for Tg42i (L) in the <i>Mmp20</i> null background had rougher surfaces and large areas of poor mineralization near the dentin-enamel junction. For the transgenes present in the <i>Mmp20</i> wild-type background (Tg+ Mmp20+/+), the situation was reversed from what occurred in the <i>Mmp20</i> null background. Mice transgenic for Tg6i (M) had poorly mineralized enamel throughout while Tg24i (H) transgenic mice had disorganized and clearly disrupted enamel formation. Conversely, the Tg42i (L) transgene in the wild-type background appeared relatively normal. In the top right panel, the black line extending from the dentin enamel junction to the outer edge of the enamel shows where enamel thickness measurements were obtained.</p

µCT analyses of incisor enamel from <i>Mmp20</i> transgenics and controls.

<p>Enamel on rodent incisors is present only on the labial side (arrows). The presented longitudinally oriented incisors were reconstructed from µCT images. The incisors protrude from bone (arrowheads) and are arranged so that the labial side is to the left. The wild-type incisors (Tg– MMP20^+/+, top right panel) have a bright line of mineralized enamel that extends from the apical region (bottom arrow) to the labial incisal tip (top arrow). This mineralized enamel was mostly missing from the <i>Mmp20</i> null incisors (Tg– <i>Mmp20^−/−</i>, top left panel). The transgenic incisors in the null background (Tg+ <i>Mmp20^−/−</i>) recovered some or most of the enamel layer along the labial surface. When the transgenes were present in the wild-type background (Tg+ Mmp20^+/+), the enamel layer seemed relatively normal on incisors from mice transgenic for Tg6i (M) or Tg42i (L), but it was severely disrupted on the Tg24i (H) <i>Mmp20^+/+</i> incisors. For incisors, Tg24 was the highest, Tg6 the middle and Tg42 the lowest expressing transgene.</p