Zinc (Zn) supplementation stimulates bone growth in Zn-deficient humans and animals. A biphasic pattern of mineralization has been observed in cultured osteoblasts; an initiation phase and a progression phase. We used MC3T3-E1, a murine osteoblastic cell line, to elucidate the physiological role of Zn in osteoblast mineralization and cellular Zn trafficking during the mineralization event. Cells were cultured in media containing Chelex-treated fetal bovine serum and 1, 4, 10 and 20 μM Zn as ZnSO(4) for 14 days (early phase of mineralization) or 21 days (mid-to-late phase of mineralization). During the early phase of mineralization, Alizarin Red staining indicated that mineralization was increased by Zn in a dose-dependent manner. Although Zn exposure did not affect monolayer Zn concentration, metallothionein (MT) mRNA expression increased dose-dependently as assessed by real-time PCR. During the late phase of mineralization, mineralization was maximal at 1 μM Zn and monolayer Zn concentration reflected Zn exposure. The increase in MT mRNA expression during the late phase was similar to that during the early phase, but the difference in expression between culture Zn concentrations tended to be smaller. ZnT-2 mRNA expression decreased significantly with increasing zinc concentrations in the culture medium during the early phase, but increased significantly during the late phase. Osteocalcin mRNA levels were positively correlated to Zn exposure at both time points. Taken together, we propose that Zn may play an important role in osteoblast mineralization through Zn trafficking involving Zn storage proteins and Zn transporters.
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