Mechanical Stimulation-Induced Calcium Signaling by Piezo1 Channel Activation in Human Odontoblast Reduces Dentin Mineralization.

Mayumi Matsunaga,Toshifumi Azuma,Masayuki Ando,Maki Kimura,Takehito Ouchi,Takashi Nakamura,Sadao Ohyama,Yoshiyuki Shibukawa,Tatsuya Ichinohe,Sachie Nomura

doi:10.3389/fphys.2021.704518

Abstract

Odontoblasts play critical roles in dentin formation and sensory transduction following stimuli on the dentin surface. Exogenous stimuli to the dentin surface elicit dentinal sensitivity through the movement of fluids in dentinal tubules, resulting in cellular deformation. Recently, Piezo1 channels have been implicated in mechanosensitive processes, as well as Ca2+ signals in odontoblasts. However, in human odontoblasts, the cellular responses induced by mechanical stimulation, Piezo1 channel expression, and its pharmacological properties remain unclear. In the present study, we examined functional expression of the Piezo1 channel by recording direct mechanical stimulation-induced Ca2+ signaling in dentin matrix protein 1 (DMP-1)-, nestin-, and dentin sialophosphoprotein (DSPP)-immunopositive human odontoblasts. Mechanical stimulation of human odontoblasts transiently increased intracellular free calcium concentration ([Ca2+]i). Application of repeated mechanical stimulation to human odontoblasts resulted in repeated transient [Ca2+]i increases, but did not show any desensitizing effects on [Ca2+]i increases. We also observed a transient [Ca2+]i increase in the neighboring odontoblasts to the stimulated cells during mechanical stimulation, showing a decrease in [Ca2+]i with an increasing distance from the mechanically stimulated cells. Application of Yoda1 transiently increased [Ca2+]i. This increase was inhibited by application of Gd3+ and Dooku1, respectively. Mechanical stimulation-induced [Ca2+]i increase was also inhibited by application of Gd3+ or Dooku1. When Piezo1 channels in human odontoblasts were knocked down by gene silencing with short hairpin RNA (shRNA), mechanical stimulation-induced [Ca2+]i responses were almost completely abolished. Piezo1 channel knockdown attenuated the number of Piezo1-immunopositive cells in the immunofluorescence analysis, while no effects were observed in Piezo2-immunopositive cells. Alizarin red staining distinctly showed that pharmacological activation of Piezo1 channels by Yoda1 significantly suppressed mineralization, and shRNA-mediated knockdown of Piezo1 also significantly enhanced mineralization. These results suggest that mechanical stimulation predominantly activates intracellular Ca2+ signaling via Piezo1 channel opening, rather than Piezo2 channels, and the Ca2+ signal establishes intercellular odontoblast-odontoblast communication. In addition, Piezo1 channel activation participates in the reduction of dentinogenesis. Thus, the intracellular Ca2+ signaling pathway mediated by Piezo1 channels could contribute to cellular function in human odontoblasts in two ways: (1) generating dentinal sensitivity and (2) suppressing physiological/reactional dentinogenesis, following cellular deformation induced by hydrodynamic forces inside dentinal tubules.

Highlights

MATERIALS AND METHODSPiezo proteins are large membrane proteins and components of mechanosensitive non-selective cationic channels (Bagriantsev et al, 2014; Beech and Xiao, 2018)
The results showed that human odontoblasts, which showed dentin matrix protein 1 (DMP-1), nestin, and dentin sialophosphoprotein (DSPP)-immunoreactivity, functionally express Piezo1 channels activated by Yoda1 pharmacologically and by direct mechanical stimulation
Mechanical stimulation-induced-[Ca2+]i increases mediated intercellular signal networks among odontoblasts, as previously reported in rat studies (Sato et al, 2015; Shibukawa et al, 2015). These mechanical stimulation-induced [Ca2+]i responses were almost completely abolished in cells when Piezo1 channels were knocked down by short hairpin RNA (shRNA)

Summary

MATERIALS AND METHODS

Piezo proteins are large membrane proteins and components of mechanosensitive non-selective cationic channels (Bagriantsev et al, 2014; Beech and Xiao, 2018). Human odontoblasts were incubated for 60 min at 37°C and 5% CO2 in a standard solution containing 10 μM fura-2acetoxymethyl ester (Dojindo Laboratories, Kumamoto, Japan) and 0.1% (w/v) pluronic acid F-127 (Life Technologies). Standard ECS with or without pharmacological Piezo channel activator/inhibitors was applied by superfusion using a rapid gravity-fed perfusion system (ValveLink8.2 Controller; AutoMate Scientific, Berkeley, CA, United States; Figure 1E). Human odontoblasts, which were transfected with shRNA-Piezo and shRNA-Control, were transferred to eight-well glass chambers (AGC Techno Glass Co., Ltd.), maintained in culture conditions, and fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Co.). The odontoblasts transfected with shRNA, including vectors specific for human Piezo, or including an empty vector control (see above), were grown to full confluency in basal medium, and transferred to mineralization medium at 37°C in 5% CO2 for 28 days. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, United States)

RESULTS

DISCUSSION

DATA AVAILABILITY STATEMENT