Loss of novel mechanosensitive ion channel TMEM63A impairs osteogenic differentiation and proliferation via disruption of cellular mechanotransduction.

Skeletal homeostasis depends critically on the local mechanobiological microenvironment. While well-known mechanosensitive ion channels like Piezo1 and Piezo2 translate physical forces into immediate membrane ion fluxes, the proximal sensors regulating medium- and long-term osteogenic pathways remain poorly understood. Low-intensity pulsed ultrasound (LIPUS) is widely used clinically to accelerate fresh fracture repair, yet its underlying upstream mechanotransduction machinery is not fully characterized. Here, we identify TMEM63A - a stretch-activated cation channel - as a crucial mechanosensor required for mechanical stimuli-driven osteogenic differentiation and anabolic signaling for sustained response to the force. Thus, the objective of this study was to elucidate the function of TMEM63A in driving osteogenic differentiation and modulating ultrasound-driven anabolic signaling during mechanotransduction with both short- and long-term effects. In MC3T3-E1 pre-osteoblasts, stable shRNA-mediated knockdown (KD) of TMEM63A (∼63% mRNA and ∼60% protein reduction) significantly impaired osteogenic capacity. KD cells exhibited reduced alkaline phosphatase activity at Days 7 and 14, suppressed proliferation, attenuated early marker expression (Runx2, Alp, and Col1a1), and a marked reduction of LIPUS-evoked Ca2+ oscillations and nuclear β-catenin translocation. Genome-wide RNA sequencing revealed coordinated downregulation of bone formation, mineralization, and ossification networks in KD cells across Days 7 and 14. Gene set enrichment analysis (GSEA) demonstrated negative enrichment for Hallmark Wnt/ β -catenin and PI3K-AKT-mTOR signaling pathways at Day 14, paired with positive enrichment for matrix-degradation and remodeling programs, consistent with Mmp13 upregulation. Crucially, LIPUS treatment failed to rescue or engage these osteogenic programs in TMEM63A-deficient cells. Under Day 14 LIPUS, knockdown cells maintained negative enrichment of Hallmark Wnt/β-catenin signaling and Reactome Axin degradation, a positive readout of canonical Wnt activation, indicating that the β-catenin destruction complex remains active and continues to target β-catenin for degradation. Taken together, these findings establish TMEM63A as an essential mechanosensor that couples acoustic force stimulation via LIPUS to a Ca2+ → AKT → GSK3 β → β-catenin signaling axis to drive osteogenic transcription and bone formation through cellular mechanotransduction.