Overexpression of the ZIP1 Zinc Transporter Induces an Osteogenic Phenotype in Mesenchymal Stem Cells

Zinc is an essential trace element that is involved in diverse metabolic and signaling pathways. Zinc deficiency is associated with retardation of bone growth. Previous in vitro studies have suggested a direct effect of zinc on both the proliferation and differentiation of osteoblast-like cells. However, the mechanisms for uptake of zinc into osteoblasts have not been examined in detail. Several families of zinc transporters have previously been characterized in mammalian cells; such transporters function in the uptake, intracellular sequestration or efflux of zinc. In the current study, we examined zinc transport in osteoprogenitor cells and have attempted to define a functional role for a zinc transport mechanism in osteogenic differentiation. We identified at least two zinc transporters in both human mesenchymal stem cells (MSCs) and in osteoblastic cells--the ubiquitous zinc transporter, ZIP1, and LIV-1, which was previously characterized as a protein that is expressed in breast cancer cells. The subcellular localization of both these zinc transporters suggested distribution in both the plasma membrane and also diffusely in the cytoplasm. During the differentiation process of pluripotent MSCs into osteoblast-like cells, both zinc uptake and expression of the ZIP1 protein were increased. An adenoviral-mediated overexpression of ZIP1 in MSCs resulted in Alizarin-red-positive mineralization and also increased expression of specific osteoblast-associated markers, such as alkaline phosphatase, and of several osteoblast differentiation genes, including osteopontin, Cbfa1/Runx2, promyelocytic leukemia zinc finger and bone sialoprotein. An siRNA-mediated reduction of ZIP1 protein expression in MSCs caused decreased zinc uptake and inhibition of osteoblastic differentiation under osteogenic culture conditions. Finally, following overexpression of ZIP1 in MSCs, cDNA microarray analysis revealed differential regulation of several genes associated with the proliferation of osteoprogenitor cells and osteoblast differentiation. In conclusion, these studies provide important insights into the role of a plasma membrane zinc transporter in the initiation of an osteogenic lineage from MSCs.