Cav 1.3 Damages the Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Osteoporotic Rats by Negatively Regulating Spred 2-Mediated Autophagy-Induced Cell Senescence

Background: Cav 1.3 can affect the classical osteoclast differentiation pathway through calcium signaling pathway. However, its effect and mechanism in osteoporosis (OS) are not clear. Methods: 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 analyze 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). Results: OS lead to the decreased of bone mineral density and differentiation ability of BMSCs in rats. Cav 1.3 was upregulated 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. 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. Funding Statement: National Natural Science Foundation of China (No.81900800); the Science and Technology Project of Shaanxi (2019JM-560). Declaration of Interests: All authors declare no conflict of interest. Ethics Approval Statement: The ethic approval was obtained from the Ethic Committee of the First Affiliated Hospital of Xi’an Jiaotong University School of Medicine.