Decreased Zn2+ Influx Underlies the Protective Role of Hypoxia in Rat Nucleus Pulposus Cells

• Published in: Biological trace element research 2015-11, Vol.168 (1), p.196-205
• Main Authors: Xiao-Fan, Yin, Li-Bo, Jiang, Yi-Qun, Ma, Jun, Xu, Hui-Jie, Gu, Xu-Hua, Wu, Xi-Lei, Li, Jian, Dong
• Format: Article
• Language: English
• Subjects: Biochemistry; Biomedical and Life Sciences; Biotechnology; Life Sciences; Nutrition; Oncology
• ISSN: 0163-4984; 1559-0720
• DOI: 10.1007/s12011-015-0335-2

Zn 2+ is an essential component of metalloproteinases, and is required for their activity in cartilage; however, the effect of Zn 2+ on nucleus pulposus (NP) cells has not been widely investigated. The aim of this paper was to investigate the effect of intracellular Zn 2+ concentration ([Zn 2+ ]i) in hypoxia-induced regulation of metalloproteinases (MMPs) and extracellular matrix (ECM) production in NP cells. NP cells from Sprague-Dawley (SD) rats were cultured as monolayers or in alginate beads. [Zn 2+ ]i was assayed by FluoZin-3 AM staining. Alcian Blue staining, immunochemistry, 1,9-dimethylmethylene blue (DMMB) assay, and real-time PCR were used to assay collagen II, proteoglycan, and COL2A1 , MMP - 13 , and ADAMTS - 5 mRNA expression. ZIP8, a main Zn 2+ transporter in chondrocytes, was assayed by immunochemistry and in Western blotting. Interleukin (IL)-1β- and ZnCl 2 -induced increases of [Zn 2+ ]i were significantly inhibited by hypoxia. Hypoxia did not reverse a decline of ECM expression caused by IL-1β and ZnCl 2 in monolayer cultures, but did significantly attenuate the decreases of proteoglycan, glycosaminoglycan (GAG), and COL2A1 mRNA expression following IL-1β and ZnCl 2 treatment in alginate bead cultures. However, ZnCl 2 inhibited the protective effect of hypoxia. Both an intracellular Zn 2+ chelator and hypoxia prevented the increase in MMP - 13 mRNA expression. IL-1β and ZnCl 2 treatment increased ZIP8 expression in NP cells, and hypoxia inhibited ZIP8 expression. In conclusion, decrease of Zn 2+ influx mediates the protective role of hypoxia on ECM and MMP-13 expression. Consequently, changes in intracellular Zn 2+ concentration maybe involved in intervertebral disc degeneration.