F-actin microfilaments affect the LIPUS-promoted osteogenic differentiation of BMSCs through TRPM7

Abstract

The differentiation of bone marrow mesenchymal stem cells (BMSCs) toward osteogenesis can be induced by low-intensity pulsed ultrasound (LIPUS). However, the molecular mechanisms responsible for LIPUS stimulation are unclear. The possible molecular mechanisms by which LIPUS promotes osteogenic differentiation of BMSCs were investigated in this study. The quantification of alkaline phosphatase (ALP) activity, Alizarin Red S staining, ALP staining, and the establishment of a calvarial defect model were used to evaluate osteogenic effects. Immunofluorescence was performed to observe the expression of microfilaments and transient receptor potential melastatin 7 (TRPM7). The levels of F-actin/G-actin and osteogenesis-related proteins under LIPUS alone or LIPUS combined with cytoskeleton interfering drugs (Cytochalasin D [CytoD] or Jasplakinolide [JA]) were assayed by western blot. Quantitative real-time reverse transcription polymerase chain reaction was utilized to measure the expression of Trpm7 mRNA. Moreover, adenoviral Trpm7 knockdown was verified using western blot. The results demonstrated that LIPUS promoted bone formation in vivo. Under osteogenic induction in vitro, the osteogenesis of BMSCs induced by LIPUS was accompanied by the depolymerization and rearrangement of microfilaments and increased levels of TRPM7. By perturbing intracellular actin dynamics, CytoD enhanced the pro-osteogenicity of LIPUS and increased TRPM7 level, while JA inhibited the pro-osteogenicity of LIPUS and reduced TRPM7 level. Additionally, the knockdown of Trpm7 suppressed the osteogenic promotion of BMSCs induced by LIPUS. The transient depolymerization and rearrangement of the cytoskeleton microfilaments mediated by LIPUS can affect TRPM7 expression and subsequently promote the osteogenesis of BMSCs. This study provides further direction for exploring the molecular mechanism of LIPUS, as a mechanical stress, in facilitating the osteogenic differentiation of BMSCs.