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

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Abstract

Zn2+ is an essential component of metalloproteinases, and is required for their activity in cartilage; however, the effect of Zn2+ on nucleus pulposus (NP) cells has not been widely investigated. The aim of this paper was to investigate the effect of intracellular Zn2+ concentration ([Zn2+]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. [Zn2+]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 Zn2+ transporter in chondrocytes, was assayed by immunochemistry and in Western blotting. Interleukin (IL)-1β- and ZnCl2-induced increases of [Zn2+]i were significantly inhibited by hypoxia. Hypoxia did not reverse a decline of ECM expression caused by IL-1β and ZnCl2 in monolayer cultures, but did significantly attenuate the decreases of proteoglycan, glycosaminoglycan (GAG), and COL2A1 mRNA expression following IL-1β and ZnCl2 treatment in alginate bead cultures. However, ZnCl2 inhibited the protective effect of hypoxia. Both an intracellular Zn2+ chelator and hypoxia prevented the increase in MMP-13 mRNA expression. IL-1β and ZnCl2 treatment increased ZIP8 expression in NP cells, and hypoxia inhibited ZIP8 expression. In conclusion, decrease of Zn2+ influx mediates the protective role of hypoxia on ECM and MMP-13 expression. Consequently, changes in intracellular Zn2+ concentration maybe involved in intervertebral disc degeneration.

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Acknowledgments

The study was supported by the Natural Science Foundation of China (81372002), (31170925), (81301335) and the “Technology Innovation Action Plan” Key Project of Shanghai Science and Technology Commission (12411951300), (12JC1402600).

Conflicts of Interest

The authors declare no conflict of interest.

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    Authors and Affiliations

    1. Department of Orthopedic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China

      Yin Xiao-Fan, Jiang Li-Bo, Ma Yi-Qun, Li Xi-Lei & Dong Jian

    2. Department of Orthopedic Surgery, Minhang Hospital, Fudan University, Shanghai, China

      Yin Xiao-Fan, Xu Jun, Gu Hui-Jie & Wu Xu-Hua

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    1. Yin Xiao-Fan
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    Correspondence to Li Xi-Lei or Dong Jian.

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    Yin Xiao-Fan and Jiang Li-Bo contributed equally to this paper.

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    Xiao-Fan, Y., Li-Bo, J., Yi-Qun, M. et al. Decreased Zn2+ Influx Underlies the Protective Role of Hypoxia in Rat Nucleus Pulposus Cells. Biol Trace Elem Res 168, 196–205 (2015). https://doi.org/10.1007/s12011-015-0335-2

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