Mitochondria modulate ameloblast Ca ²⁺ signaling

Abstract

The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation‐stage ameloblasts focusing on their role in Ca2+ signaling. Quantification of the Ca2+ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca2+ pools by adenosine triphosphate in rhod2AM‐loaded cells showed similar mitochondrial Ca2+ (mCa2+) uptake. However, mCa2+ extrusion via Na+‐Li+‐Ca2+ exchanger was more prominent in maturation. To address if mCa2+ uptake via the mitochondrial Ca2+ uniporter (MCU) played a role in cytosolic Ca2+ (cCa2+) buffering, we stimulated Ca2+ influx via the store‐operated Ca2+ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented cCa2+ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher mCa2+ uptake in maturation ameloblasts. The uptake of Ca2+ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca2+ handling in maturation but only upon stimulation of Ca2+ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca2+ physiology, but also advance the concept that SOCE and mCa2+ uptake are complementary processes in biological mineralization.

Dinuclear nitrido-bridged osmium complexes inhibit the mitochondrial calcium uniporter and protect cortical neurons against lethal oxygen–glucose deprivation

“…[18][19][20][21][27][28][29] This work led to the discovery of the nitrido-bridged diruthenium complex Ru265 (Scheme 1), which inhibits MCU-mediated mt-Ca 2+ uptake in intact cells 19,20 and protects against mt-Ca 2+ overload in an in vivo model of ischemic stroke. 27 Further studies have also used this complex to investigate the role of the MCU in cancer cell death, 30 ameloblast Ca 2+ signaling, 31 and astrocyte function. 32 In continuing our study of Ru265, we sought to understand the influence of the metal center on the biological properties of this compound.…”

Overexpression of RCAN1, a Gene on Human Chromosome 21, Alters Cell Redox and Mitochondrial Function in Enamel Cells

“…Taken together, this suggests that the reduction in mitochondrial respiration we observed in LS8 +RCAN1 cells could have been the result of elevated m Ca 2+ uptake. In support of this notion is the observation that, although mitochondria of secretory- and maturation-stage ameloblasts show similar levels of m Ca 2+ uptake in response to ATP stimulation [ 11 ], m Ca 2+ uptake was higher in LS8 +RCAN1 cells compared with controls. LS8 cells are most similar to secretory ameloblasts, which we postulate may be more sensitive to changes in ROS levels; however, increased RCAN1 levels may increase the capacity for m Ca 2+ uptake in all stages of ameloblast development, with the metabolic outcome depending upon the physiological state and cell type in question.…”

“…However, the precise cause of the corresponding decrease in mitochondrial respiration is unclear. Mitochondria can uptake cytosolic Ca 2+ following its elevation in response to the opening of Ca 2+ channels either in the plasma membrane, such as ORAI1 [ 11 , 30 ], or in the ER, such as IP 3 R [ 31 ]. The latter source is considered a more efficient mechanism for stimulating mitochondrial function [ 32 ].…”

“…Our recent studies demonstrated that mitochondria participate in the maintenance of Ca 2+ homeostasis in ameloblasts [ 11 ] and that there is an increase in reactive oxygen species (ROS) levels in maturation-stage ameloblasts [ 12 ]. Because overexpression of RCAN1 can cause mitochondrial dysfunction and increase the susceptibility of cells to oxidative stress [ 13 ], we postulated that ameloblasts of DS patients may experience a disruption in mitochondrial function, or enhanced ROS, altering enamel formation.…”

PMCA Ca 2+ clearance in dental enamel cells depends on the magnitude of cytosolic Ca 2+

“…9, 10 The Ca 2+ buffering and extrusion toolkit in this context is less known but plasma membrane Ca 2+ pumps and Ca 2+ exchangers, Ca 2+ -binding proteins and buffering by intracellular organelles have been reported. 3,[11][12][13][14][15] Enamel is the most calcified vertebrate tissue, requiring a steady supply of Ca 2+ during development (amelogenesis), a process commonly classified as the formative (secretory) and mineralization (maturation) stages. 16 In ameloblasts, the general consensus is that Ca 2+ is transported via a transcellular route before its extrusion into the enamel space at the apical pole of the cells.…”