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.

Keywords: BMSCs; Cav 1.3; Spred 2; osteogenic differentiation.