PIEZO1 promotes ATP release from periodontal ligament cells following compression force

Abstract

Objectives: Orthodontic mechanical force on the periodontal ligament induces extracellular adenosine triphosphate (ATP) release. However, mechanosensitive molecules have not been confirmed functionally in periodontal ligament cells. In the present study, we examined the roles of mechanosensitive PIEZO channels in the mechanically stimulated release of ATP in human periodontal ligament fibroblasts (HPdLFs).

Materials and methods: To examine PIEZO expression in HPdLFs, we performed reverse transcription-quantitative polymerase chain reaction, fluorescent immunostaining, and Ca2+ imaging. ATP concentrations were measured in culture medium after applications of the PIEZO1 agonist Yoda1 and compression force in a newly developed in vitro weight-loaded cell model (IVWLC) using balance weights and a 48-well plate. The mechanosensitive channel inhibitor GsMTx4 and the ATP-releasing route inhibitors clodronic acid, meclofenamic acid, and probenecid were used. To suppress PIEZO1 expression, short interference RNA (siRNA) treatment of the PIEZO1 gene was performed.

Results: PIEZO1 mRNA was expressed more abundantly than PIEZO2 mRNA in HPdLFs. HPdLF cell bodies were immunoreactive to anti-PIEZO1 antibody. Yoda1 increased intracellular Ca2+ and extracellular ATP concentrations in a dose-dependent manner. ATP release was inhibited by GsMTx4 and inhibitors of ATP release routes. In the IVWLC, HPdLFs released ATP in response to compression force but not in response to hypoxic stimulation that was simultaneously applied to cells. Mechanically stimulated ATP release was inhibited by GsMTx4, inhibitors of ATP-releasing routes and siRNA treatment of PIEZO1.

Conclusions: PIEZO1 on the cell membranes of HPdLFs is activated by compression force and then induces ATP release via intracellular Ca2+-dependent exocytosis and ATP-permeable channels.

Keywords: PIEZO1; TRPA1; adenosine triphosphate; bone remodelling; orthodontics; pain.