Altered ion-responsive gene expression in Mmp20 null mice

Abstract

During enamel maturation, hydroxyapatite crystallites expand in volume, releasing protons that acidify the developing enamel. This acidity is neutralized by the buffering activity of carbonic anhydrases and ion transporters. Less hydroxyapatite forms in matrix metalloproteinase-20 null (Mmp20(-/-)) mouse incisors, because enamel thickness is reduced by approximately 50%. We therefore asked if ion regulation was altered in Mmp20(-/-) mouse enamel. Staining of wild-type and Mmp20(-/-) incisors with pH indicators demonstrated that wild-type mice had pronounced changes in enamel pH as development progressed. These pH changes were greatly attenuated in Mmp20(-/-) mice. Expression of 4 ion-regulatory genes (Atp2b4, Slc4a2, Car6, Cftr) was significantly decreased in enamel organs from Mmp20(-/-) mice. Notably, expression of secreted carbonic anhydrase (Car6) was reduced to almost undetectable levels in the null enamel organ. In contrast, Odam and Klk4 expression was unaffected. We concluded that a feedback mechanism regulates ion-responsive gene expression during enamel development.

Figure 1.

Expression of Odam and Klk4 in the maturation-stage mouse enamel organ. Expression of Klk4 (A) and Odam (B) was not significantly altered in the Mmp20 null mouse. Data are presented as mean ± SEM and represent measurements of 6 individual mice, with duplicate measurements for each mouse (n = 6). Results are presented as relative gene expression normalized to the geometric mean of Eef1α1, Gapdh, β-actin, and Casc3 mRNA expression. Statistical analysis was determined by t test.

Figure 2.

Staining of incisors with pH indicators. A comparison of wild-type rat and mouse incisor banding patterns was made by use of methyl red staining (top panels). Note that for mandibular incisors, the rat has at least one more band of acidity than does the mouse. The 4 bottom panels show incisors from wild-type and Mmp20 null mice. For each of these panels, the top incisor is stained with methyl red, the middle incisor is stained with bromophenol red, and the bottom incisor is stained with resazurin. The staining pattern of the Mmp20 null incisors is distinctly different from that of the wild-type control, and the areas of acidity are greatly reduced in the null mouse enamel.

Figure 3.

Ion transporter gene expression in maturation-stage mouse enamel organ. Atp2b4 (B), Car6 (D), Cftr (E), and Slc4a2 (F) all showed a significant decrease in mRNA expression in the absence of MMP20. Expression of Atp2b1 (A) and Car2 (C) was not significantly altered in the Mmp20 null mouse. Although a trend existed, the data for Slc4a4 (G) was not significant due to variability within the data sets. Data are presented as mean ± SEM and represent measurements of 6 individual mice, with duplicate measurements for each mouse (n = 6). Results are presented as relative gene expression normalized to the geometric mean of Eef1α1, Gapdh, β-actin, and Casc3 mRNA expression. Statistical analysis was determined by t test (*p < 0.05, **p < 0.01, ***p < 0.001).