Effects of 12 metal ions on iron regulatory protein 1 (IRP-1) and hypoxia-inducible factor-1 alpha (HIF-1alpha) and HIF-regulated genes.

Several metal ions that are carcinogenic affect cellular iron homeostasis by competing with iron transporters or iron-regulated enzymes. Some metal ions can mimic a hypoxia response in cells under normal oxygen tension, and induce expression of HIF-1alpha-regulated genes. This study investigated whether 12 metal ions altered iron homeostasis in human lung carcinoma A549 cells as measured by an activation of IRP-1 and ferritin level. We also studied hypoxia signaling by measuring HIF-1alpha protein levels, hypoxia response element (HRE)-driven luciferase reporter activity, and Cap43 protein level (an HIF-1alpha responsive gene). Our results show the following: (i) Ni(II), Co(II), V(V), Mn(II), and to a lesser extent As(III) and Cu(II) activated the binding of IRP-1 to IRE after 24 h, while the other metal ions had no effect; (ii) 10 of 12 metal ions induced HIF-1alpha protein but to strikingly different degrees. Two of these metal ions, Al(III) and Cd(II), did not induce HIF-1alpha protein; however, as indicated below, only Ni(II), Co (II), and to lesser extent Mn(II) and V(V) activated HIF-1alpha-dependent transcription. The combined effects of both [Ni(II) + As(III)] and [Ni(II) + Cr(VI)] on HIF-1alpha protein were synergistic; (iii) Addition of Fe(II) with Ni(II), Co(II), and Cr(VI) attenuated the induction of HIF-1alpha after 4 h treatment; (iv) Ni(II), Co(II), and Mn(II) significantly decrease ferritin level after 24 h exposure; (v) Ni(II), Co(II), V(V), and Mn(II) activated HRE reporter gene after 20 h treatment; (vi) Ni(II), Co(II), V(V), and Mn(II) increased the HIF-1-dependent Cap43 protein level after 24 h treatment. In conclusion, only Ni (II), Co (II), and to a lesser extent Mn(II) and V(V) significantly stabilized HIF-1alpha protein, activated IRP, decreased the levels of ferritin, induced the transcription of HIF-dependent reporter, and increased the expression of Cap43 protein levels (HIF-dependent gene). The mechanism for the significant stabilization and elevation of HIF-1alpha protein which drives these other parameters was previously shown by us and others to involve a loss of cellular Fe as well as inhibition of HIF-1alpha-dependent prolyl hydroxylases which target the binding of VHL ubiquitin ligase and degrade HIF-1alpha. Even though there were small effects of some of the other metals on IRP and HIF-1alpha, downstream effects of HIF-1alpha activation and therefore robust hypoxia signaling were only observed with Ni(II), Co(II), and to much lesser extents with Mn(II) and V(V) in human A549 lung cells. It is of interest that the metal ions that were most effective in activating hypoxia signaling were the ones that were poor inducers of metallothionein protein and also decreased Ferritin levels, since both of these proteins can bind metal ions and protect the cell against toxicity in human lung cells. It is important to study effects of these metals in human lung cells since this represents a major route of human environmental and occupational exposure to these metal ions.