Open AccessCav 1.3 damages the osteogenic differentiation in osteoporotic rats by negatively regulating Spred 2‐mediated autophagy‐induced cell senescence
Author(s) -
Fan Ping,
Pu Dan,
Lv Xiaohong,
Hu Nan,
Feng Xiuyuan,
Hao Zhiming,
Sun Yining,
He Lan
Publication year - 2020
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/jcmm.15978
Subject(s) - viability assay , microbiology and biotechnology , alkaline phosphatase , runx2 , chemistry , cell cycle , cell growth , cell , osteocalcin , flow cytometry , biology , biochemistry , enzyme
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.