RESUMEN
OBJECTIVES: The aim of this study was to investigate the molecular mechanism underlying odontoblast damage repair in dentin hypersensitivity (DH) and the role of Yes-associated protein (YAP) in this process. METHODS: The DH model was constructed in Sprague-Dawley (SD) rats, and the in vivo expression of Piezo1, Integrin αvß3, YAP, and dentin sialophosphoprotein (DSPP) was detected by immunohistochemistry. COMSOL Multiphysics software was used to simulate the dentinal tubule fluid flow velocity and corresponding fluid shear stress (FSS) on the odontoblast processes. MDPC-23 cells were cultured in vitro and loaded with a peristaltic pump for 1 hour at FSS values of 0.1, 0.3, 0.5, and 0.7 dyne/cm2. The expression of Piezo1, Integrin αvß3, and YAP was detected by immunofluorescence. Verteporfin (a YAP-specific inhibitor) was utilised to confirm the effect of YAP on the expression of dentineogenesis-related protein under FSS. RESULTS: The level and duration of external mechanical stimuli have an effect on the functional expression of odontoblasts. In DH, the harder the food that is chewed, the faster the flow of the dentinal tubule fluid and the greater the FSS on the odontoblast processes. The expression of Piezo1, Integrin αvß3, and YAP can be promoted when the FSS is less than 0.3 dyne/cm2. After YAP inhibition, the DSPP protein expression level was reduced at 0.3 dyne/cm2 FSS. CONCLUSIONS: These results suggest that appropriate FSS can enhance the expression of odontoblast-related factors in odontoblasts via the Piezo1-Integrin αvß3-YAP mechanotransduction pathway and the YAP appears to play an essential role in the response of odontoblasts to external mechanical stimuli.
Asunto(s)
Sensibilidad de la Dentina , Odontoblastos , Proteínas Señalizadoras YAP , Animales , Ratas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sensibilidad de la Dentina/genética , Sensibilidad de la Dentina/metabolismo , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Inmunohistoquímica , Integrina alfaVbeta3/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana , Odontoblastos/metabolismo , Fosfoproteínas/metabolismo , Ratas Sprague-Dawley , Sialoglicoproteínas/metabolismo , Estrés Mecánico , Verteporfina/farmacología , Verteporfina/uso terapéuticoRESUMEN
OBJECTIVE: The aim of this study is to investigate the expression of pannexin3 (Panx3) in human odontoblast-like cells (hOBs) and its hemichannel function in mediating ATP release. METHODS: RT-PCR and immunofluorescence analysis were used to detect the expression of pannexins (Panxs) in human dental pulp tissue and cultured cells. To determine the role of Panx3 in ATP release, hOBs were infected with Panx3-overexpression lentivirus, Panx3-shRNA lentivirus or control lentivirus and then stimulated with cold buffer. Intracellular ATP was monitored using quinacrine, and then semi-quantitatively analyzed. In the meantime, the ATP release was quantitatively analyzed using the bioluminescence method when the cells were exposed to cold stimulus. RESULTS: Panx3 mRNA and protein were found in dental pulp tissue and cultured cells. Upon cold stimulus, intracellular ATP was released into the extracellular space. Overexpression of Panx3 accelerated ATP release, whereas inhibition of Panx3 suppressed this process. CONCLUSION: Panx3 hemichannel is expressed in human odontoblast-like cells and mediates ATP release into the extracellular space.
Asunto(s)
Adenosina Trifosfato/metabolismo , Conexinas/biosíntesis , Odontoblastos/metabolismo , Adolescente , Adulto , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Frío , Conexinas/genética , Pulpa Dental/citología , Pulpa Dental/metabolismo , Sensibilidad de la Dentina/genética , Sensibilidad de la Dentina/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Lentivirus/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Adulto JovenRESUMEN
Odontoblasts form the outermost cellular layer of the dental pulp where they have been proposed to act as sensory receptor cells. Despite this suggestion, evidence supporting their direct role in mediating thermo-sensation and nociception is lacking. Transient receptor potential (TRP) ion channels directly mediate nociceptive functions, but their functional expression in human odontoblasts has yet to be elucidated. In the present study, we have examined the molecular and functional expression of thermo-sensitive TRP channels in cultured odontoblast-like cells and in native human odontoblasts obtained from healthy wisdom teeth. PCR and western blotting confirmed gene and protein expression of TRPV1, TRPA1 and TRPM8 channels. Immunohistochemistry revealed that these channels were localised to odontoblast-like cells as determined by double staining with dentin sialoprotein (DSP) antibody. In functional assays, agonists of TRPV1, TRPA1 and TRPM8 channels elicited [Ca2+]i transients that could be blocked by relevant antagonists. Application of hot and cold stimuli to the cells also evoked rises in [Ca2+]i which could be blocked by TRP-channel antagonists. Using a gene silencing approached we further confirmed a role for TRPA1 in mediating noxious cold responses in odontoblasts. We conclude that human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth. Cultured and native human odontoblasts express functional TRP channels that may play a crucial role in mediating thermal sensation in teeth.
