Cav 1.3 damages the osteogenic differentiation in osteoporotic rats by negatively regulating Spred 2‐mediated autophagy‐induced cell senescence

Abstract

Cav 1.3 can affect the classical osteoclast differentiation pathway through calcium signalling pathway. Here, we performed cell transfection, real-time fluorescence quantitative PCR (qPCR), flow cytometry, SA-β-Gal staining, Alizarin Red S staining, ALP activity test, immunofluorescence, Western blot and cell viability assay to analyse cell viability, cell cycle, osteogenesis differentiation and autophagy activities in vitro. Meanwhile, GST-pull down and CHIP experiments were conducted to explore the influence of Cav 1.3 and Sprouty-related EVH1 domain 2 (Spred 2) on bone marrow-derived mesenchymal stem cells (BMSCs). The results showed that OS lead to the decreased of bone mineral density and differentiation ability of BMSCs in rats. Cav 1.3 was up-regulated in OS rats. Overexpression of Cav 1.3 inhibited the activity of BMSCs, the expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN), as well as promoted the cell cycle arrest and senescence. Furthermore, the negative correlation between Cav 1.3 and Spred 2 was found through GST-pull down and CHIP. Overexpression of Spred 2 increased the expressions of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1 of BMSCs, which ultimately promoted the cell activity of BMSCs and ALP, RUNX2, OCN expression. In conclusion, Cav 1.3 negatively regulates Spred 2-mediated autophagy and cell senescence, and damages the activity and osteogenic differentiation of BMSCs in OS rats.