Functional Expression of Mechanosensitive Piezo1/TRPV4 Channels in Mouse Osteoblasts

Abstract

Mechanical stress is an important regulatory factor in bone homeostasis. Mechanical stimulation of osteoblasts has been shown to elicit an increase in the concentration of intracellular free Ca²⁺ ([Ca²⁺]_i). The pattern of functional expression of mechanosensitive ion channels remains unclear, however. Therefore, the purpose of this study was to investigate the pharmacological characteristics of [Ca²⁺]_i in response to direct mechanical stimulation in osteoblasts. The morphological expression of mechanosensitive ion channels was also examined. Mouse osteoblast-like cells (MC3T3-E1 cells) were loaded with fura-2-acetoxymethyl ester, after which [Ca²⁺]_i was measured. Increased levels of [Ca²⁺]_i were observed in MC3T3-E1 cells in response to direct mechanical stimulation by means of a glass micropipette, but no desensitization. Application of a hypotonic solution also induced an increase in [Ca²⁺]_i but was accompanied by a desensitizing effect. Extracellular Gd³⁺, GsMTx4, or RN-1734 reversibly inhibited this mechanical stimulation-induced increase in [Ca²⁺]_i, whereas no inhibitory effect was observed with HC030031 or clemizole. When osteoblasts were stimulated with Yoda1, an increase was observed in [Ca²⁺]_i together with a significant desensitizing effect. Immunoreactivity against Piezo1 and TRPV4 channel antibodies was detected in MC3T3-E1 cells. These results suggest that osteoblasts express Piezo1 and TRPV4 channels, which are involved in mechanosensitive processes during mechanical stress.