17β-Estradiol Protects Rat Annulus Fibrosus Cells Against Apoptosis via α1 Integrin-Mediated Adhesion to Type I Collagen: An In-vitro Study
Chun-Ming Zhao, Qian Chen, Wen-Jie Zhang, Ai-Bing Huang, Wei Zhang, Hui-Lin Yang, Zhi-Ming Zhang
Department of Orthopedic Surgery, The Affiliated Taizhou People’s Hospital of Nantong University, Taizhou, Jiangsu, China (mainland)
Med Sci Monit 2016; 22:1375-1383
17β-Estradiol (E2) has been reported to protect annulus fibrosus (AF) cells in vitro against interleukin-1β (IL-1β)-induced apoptosis in a concentration-dependent manner. However, its time-response effect remains unexplored. In addition, integrin α2/collagen II interaction has been reported to influence the apoptosis of nucleus pulposus cells in vitro. Thus, we hypothesized that integrin α1/collagen II might play a role in exerting the anti-apoptosis effect by E2. The aim of the current study was to further investigate the anti-apoptotic effect of E2 and determine the role of integrin a1/collagen II interaction.
MATERIAL AND METHODS: Rat AF cells were primary cultured and used for the following experiments. AF cells were identified by immunocytochemistry of type I collagen. Cell apoptosis was detected by fluorescence-activated cell sorter (FACS) analysis. The activity of active caspase-3 was determined by use of a caspase-3 detection kit. AF cell adhesion to type I collagen was determined by cell adhesion assay. Protein level of integrin subunit α1 was quantified by Western blot and mRNA expression was determined by real-time qPCR.
RESULTS: The immunocytochemistry of type I collagen revealed that cell purity was eligible for the following experiments with 98% of purity. FACS analysis indicated time-dependent anti-apoptosis effect of E2 at time points of 6 h, 12 h, and 24 h, which was confirmed by Caspase-3 activity. Furthermore, cell adhesion assay showed that E2 significantly increased cell binding to 95% of control, and qPCR and Western blot analysis showed that E2 effectively upregulated integrin α1. However, estrogen receptor antagonist ICI182780 prohibited the effect of E2.
CONCLUSIONS: This study shows that E2 protects against apoptosis in a time-dependent manner, and α1 integrin-mediated adhesion to collagen II is essential for estrogen-dependent anti-apoptosis in rat annulus fibrosus cells in vitro.
Keywords: Apoptosis - drug effects, Annulus Fibrosus - cytology, Animals, Blotting, Western, Caspase 3 - metabolism, Cell Adhesion - drug effects, Cell Shape - drug effects, Collagen Type I - metabolism, Cytoprotection - drug effects, Estradiol - pharmacology, Flow Cytometry, Integrin alpha1 - metabolism, Rats, Wistar, Real-Time Polymerase Chain Reaction