| Previous studies have shown that in vitro and in vivo zinc (Zn) deficiency result ultimately in cell death, while pretreatment of cells with Zn blocks apoptosis. Using a Zn chelator, we prepared Zn deficient medium and further characterized the events preceding Zn deficiency-induced cell death. Zn deficient 3T3 fibroblasts showed signs of oxidative stress, alterations in mitochondrial distribution and potential, and displayed apoptotic and necrotic cell death.; Intracellular iron (Fe) levels were also found to be increased during Zn deficiency, a finding that is observed in most tissues in vivo as well. While investigators have suggested that Zn and Fe primarily interact at transport sites due to their chemical similarities, the increase in Fe levels observed in a Zn deficiency suggest an additional mechanism of accumulation. Therefore we hypothesized that Zn deficiency can trigger Fe accumulation via alterations in Iron Regulatory Proteins (IRPs), as these can be affected by increased reactive oxygen species, which is a consequence of Zn depletion. Our studies showed that during Zn deficiency, IRP-2 binding activity is increased, which was reflected in an increase in Transferrin Receptor (TfR) levels. Moreover, levels of Ferritin Light (FtL) Chain were increased while those of Ferritin Heavy (FtH) Chain remained unchanged, unexpectedly. In contrast, binding activity of IRP-1 was significantly decreased in the Zn deficient group.; Next, we tested whether Zn deficiency increases PKC activity, as all PKC isoforms contain cysteine-rich finger motifs that bind to two Zn atoms. In turn, PKC can phosphorylate IRPs, which has been shown to increase the RNA-binding activity of IRPs. This mimics an Fe deficient state in the cell, which responds by increasing Fe uptake. For these experiments, we focused on two PKC isoforms: -alpha and -delta, which are ubiquitously expressed and have a role in cell proliferation and apoptosis, respectively. Zn deficient 3T3 cells had decreased PKC-alpha activity, which may contribute to cell death in this group. PKC-delta was proteolytically cleaved into a kinase-active fragment with pro-apoptosic activity in the mitochondria. Moreover, inhibition of PKC-delta resulted in decreased PKC-delta fragmentation and translocation to the mitochondria, thereby rescuing Zn deficient cells from apoptosis. |
