Mitochondrial Function in Enamel Development

Abstract

Enamel is the most calcified tissue in vertebrates. Enamel formation and mineralization is a two-step process that is mediated by ameloblast cells during their secretory and maturation stages. In these two stages, ameloblasts are characterized by different morphology and function, which is fundamental for proper mineral growth in the extracellular space. Ultrastructural studies have shown that the mitochondria in these cells localize to different subcellular regions in both stages. However, limited knowledge is available on the role/s of mitochondria in enamel formation. To address this issue, we analyzed mitochondrial biogenesis and respiration, as well as the redox status of rat primary enamel cells isolated from the secretory and maturation stages. We show that maturation stage cells have an increased expression of PGC1α, a marker of mitochondrial biogenesis, and of components of the electron transport chain. Oxygen consumption rate (OCR), a proxy for mitochondrial function, showed a significant increase in oxidative phosphorylation during the maturation stage, promoting ATP production. The GSH/GSSG ratio was lower in the maturation stage, indicative of increased oxidation. Because higher oxidative phosphorylation can lead to higher ROS production, we tested if ROS affected the expression of AmelX and Enam genes that are essential for enamel formation. The ameloblast cell line LS8 treated with H 2 O 2 to promote ROS elicited significant expression changes in AmelX and Enam. Our data highlight important metabolic and physiological differences across the two enamel stages, with higher ATP levels in the maturation stage indicative of a higher energy demand. Besides these metabolic shifts, it is likely that the enhanced ETC function results in ROS-mediated transcriptional changes.

Mitochondria modulate ameloblast Ca 2+ signaling

“…This task is likely important to help prevent toxic levels of c Ca 2+ . Therefore, results shown here and in our previous study 22 suggest that mitochondria play a dual and significant role in enamel mineralization by supplying high levels of ATP and differentially buffering the Ca 2+ fluxes via SOCE, advancing the notion that SOCE and m Ca 2+ uptake are complementary processes in biological mineralization.…”

“…The data shown here and in recent reports 22,50 provide a picture of the physiological role of mitochondria in enamel mineralization. Maturation ameloblasts are metabolically more active and produce more ATP than secretory cells 22 3,76,77 .…”

“…The purity of the dissection was validated by RT‐qPCR of specific enamel genes for the two stages ( Enam and Odam for the secretory and maturation stages, respectively; Figure S1A) and by Western blot of the enamel matrix protein amelogenin (Figure S1B), which is primarily translated in the secretory enamel stage. Fibroblasts were detected by either FITC fluorescent or PE anti‐rat CD90/mouse CD90.1 (Thy‐1.1) (1:500, 30 min at 37°C; BioLegend) and were excluded from our analysis, as we have reported previously 22 …”

“…It also underscores the possibility that mitochondria in ameloblasts may play a role in Ca 2+ signaling because mitochondria are known to shape Ca 2+ transients in cells by modulating SOCE 19‐21 . However, the function of mitochondria in ameloblasts is poorly defined 22 …”

“…The enamel studies have largely focused on morphological analysis and subcellular localization of the mitochondria in the ameloblasts, and the identification of mitochondrial enzymes, 47‐49 but there is an overwhelming dearth of functional studies. To gain a better understanding of the physiological role of mitochondria in enamel formation, we recently analyzed OXPHOS levels in ameloblasts and showed that this function was upregulated in maturation 22 . A recent report confirmed the important role of OXPHOS in mice with impaired mitochondrial DNA (mtDNA) replication which showed abnormal enamel 50 .…”