Zinc promotes differentiation and proliferation of MC3T3-E1 cells through activation of Ca²⁺-activatide K⁺ channels

Abstract

Zinc, as the divalent cation (Zn²⁺) and the second most abundant trace metal in the human body, plays pivotal roles in the proliferation and differentiation of osteoblasts. It is well known that the Ca²⁺-activated K⁺ (K_Ca) channels regulate membrane potential and calcium signaling in the non-excitable cells. Although K_Ca channels are expressed in osteoblast-like cells including MC3T3-E1, their functions in osteoblasts have not been fully clarified. The molecular mechanisms of Zn²⁺ induced proliferation and differentiation of osteoblasts also remain unclear. In this research, the mechanisms of proliferation and differentiation through KCa channel activation by Zn²⁺ in MC3T3-E1 cells are investigated.

To clarify the physiological roles of Zn²⁺ in osteoblasts, we applied whole-cell mode of the patch-clamp technique as well as calcium and zinc imaging in MC3T3-E1 cells. One millimolar of Zn²⁺ hyperpolarized membrane potential under current-clamp mode and induced outward K⁺ currents and blockade of non-selective cation channel currents under voltage-clamp mode. Internal and external Ca²⁺ was necessary for the Zn²⁺-induced K⁺ currents. The K⁺ currents were inhibited by intermediate-conductance Ca²⁺-activated K⁺ (IK) channel blockers and a large-conductance Ca²⁺-activated K⁺ (BK) channel blocker, but not by a small-conductance Ca²⁺-activated K⁺ (SK) channel blocker. The phospholipase C (PLC) inhibitor and the non-hydrolysable GDP analog (GDPγS) blocked the increase in intracellular Ca²⁺ and Zn²⁺-induced K⁺ currents, respectively. TPEN (10 μM), a Zn²⁺-chelator, in the pipette solution abolished BK currents, but not IK currents, indicating that intracellular Zn²⁺ passed through TRP channels and/or Zrt-, Irt-related proteins (ZIPs) activates BK channel directly, but not IK and SK channels. Zn²⁺ promoted cell proliferation, and enhanced osteoprotegerin (OPG) mRNA expression. Interestingly, these biological effects of Zn²⁺ were diminished by TRAM-34, an IK inhibitor.

These results suggest that Zn²⁺ can activate IK and BK channels through two different pathways in MC3T3-E1 cells. External administration of Zn²⁺ increases intracellular free-Ca²⁺ and free-Zn²⁺ concentration, and hyperpolarizes MC3T3-E1 cells synergistically. Finally, Zn²⁺ promotes their proliferation and differentiation of MC3T3-E1 cells.