Unveiling the Role of Plasma Membrane Calcium ATPases in Enamel Formation: Implications for Dental Health and Therapeutic Potential

Dental enamel, the hardest and most mineralized tissue in the human body, plays a vital role in protecting and ensuring the durability of teeth. The process of enamel formation, known as amelogenesis, is a complex undertaking consisting of two main stages: secretory and maturation. During the secretory stage, specialized epithelial cells called ameloblasts secrete a proteinaceous matrix that serves as a foundation for crystal growth. One crucial aspect of amelogenesis is the regulation of cytosolic calcium ion (Ca²⁺) concentration in ameloblasts. This regulation is essential for maintaining the pH gradient, facilitating crystal growth, supporting cell survival, and enabling gene expression during enamel formation.

Ameloblasts are tasked with managing the influx and extrusion of Ca²⁺ ions across their plasma membrane, ensuring that an optimal balance is maintained to avoid overload or depletion. One potential mechanism for Ca²⁺ extrusion in ameloblasts involves the activity of plasma membrane Ca²⁺ ATPases (PMCA1-4). These ATP-dependent pumps transport Ca²⁺ ions from the cytosol to the extracellular space. PMCAs exhibit high Ca²⁺ affinity and low clearance rates, implying their ability to bind Ca²⁺ ions at low concentrations but transport them at a slower pace. Although PMCAs are expressed in ameloblasts at different stages of amelogenesis, their functional role and regulation in enamel formation are not yet well understood.

In a recent study published in The FASEB Journal, Dr. Guilherme Bomfim and Professor Rodrigo Lacruz from the New York University College of Dentistry in collaboration with Assistant Professor Marta Giacomello from the University of Padova, aimed to investigate the involvement of plasma membrane PMCA1-4 in cytosolic Ca²⁺ extrusion in ameloblasts. They also sought to explore how the activity of PMCAs changes across the process of enamel formation, known as amelogenesis. The researchers hypothesized that PMCAs possess high Ca²⁺ affinity and low clearance rates, which may limit their contribution to enamel formation, particularly during the maturation stage when ameloblasts handle high Ca²⁺ loads.

To test their hypotheses, the research team utilized rat secretory and maturation ameloblasts as a model system. They measured cytosolic Ca²⁺ levels in these cells using a Ca²⁺ probe called Fura-2-AM and a fluorescence microscope. The study also involved manipulating PMCA activity by introducing an external alkaline solution with a pH of 9.0, using PMCA blockers such as vanadate and caloxin 1b1, or employing PMCA potentiators such as forskolin and 8- Br- cAMP. Additionally, they activated the ORAI1 Ca²⁺ channel using thapsigargin to induce higher cytosolic Ca²⁺ transients. These experimental approaches allowed the researchers to observe the behavior and function of PMCAs in ameloblasts.

The results of the study revealed that secretory ameloblasts exhibited faster Ca²⁺ clearance than maturation ameloblasts through the activity of PMCAs under conditions of low to moderate cytosolic Ca²⁺ elevations. This clearance process was completely inhibited by the alkaline solution and significantly delayed by the PMCA blockers in both secretory and maturation ameloblasts. However, when higher cytosolic Ca²⁺ transients were induced by ORAI1 activation, maturation ameloblasts cleared Ca²⁺ more efficiently through PMCAs compared to secretory ameloblasts. Moreover, inhibiting PMCAs decreased the rate of Ca²⁺ influx via ORAI1, while potentiating PMCAs had no effect on Ca²⁺ influx in both secretory and maturation ameloblasts.

The research team proposed that PMCAs function as active Ca²⁺ ATPase pumps during amelogenesis and regulate cytosolic Ca²⁺ levels in response to low and/or moderate Ca²⁺ stimuli in the secretory stage. The discussion also suggested that PMCAs may have different isoforms or regulatory mechanisms accounting for their varying activity levels throughout amelogenesis. Furthermore, the researchers speculated that PMCAs might collaborate with other Ca²⁺ transporters, such as sodium/calcium exchangers (NCX) or calcium/calmodulin-dependent protein kinase II (CaMKII), to modulate cytosolic Ca²⁺ clearance and influx.

Additionally, the authors reported that PMCAs likely play a role in enamel mineralization since proper cytosolic Ca²⁺ clearance is crucial for maintaining the pH gradient and facilitating crystal growth across the enamel matrix. According to the authors, PMCAs may also contribute to enamel cell survival, as maintaining cytosolic Ca²⁺ homeostasis is critical for preventing apoptosis and necrosis induced by Ca²⁺ overload.

In conclusion, the new study by Professor Rodrigo Lacruz and colleagues provides evidence that PMCAs are involved in cytosolic Ca²⁺ extrusion in ameloblasts and that their activity changes throughout the process of amelogenesis. The study highlights the high Ca²⁺ affinity and low Ca²⁺ clearance rates of PMCAs, which may limit their contribution to enamel formation. The researchers suggest that PMCAs likely interact with other Ca²⁺ transporters and signaling pathways to regulate enamel mineralization. The findings of this study also indicate that PMCAs have the potential to be novel targets for enhancing enamel formation and preventing enamel defects, offering new avenues for therapeutic interventions in the field of dentistry.