Conditional Derepression of Ferritin Synthesis in Cells Expressing a Constitutive IRP1 Mutant

Abstract

Iron regulatory protein 1 (IRP1), a major posttranscriptional regulator of cellular iron and energy metabolism, is controlled by an iron-sulfur cluster switch. Cysteine-437 is critical for coordinating the cluster, and its replacement yields mutants that do not respond to iron perturbations and constitutively bind to cognate mRNA iron-responsive elements (IREs). The expression of IRP1_C437S in cells has been associated with aberrations in iron homeostasis and toxicity. We have established clones of human lung (H1299) and breast (MCF7) cancer cells that express high levels of IRP1_C437S in a tetracycline-inducible manner. As expected, IRP1_C437S stabilizes transferrin receptor mRNA and inhibits translation of ferritin mRNA in both cell types by binding to their respective IREs. However, H1299 transfectants grown at high densities are able to overcome the IRP1_C437S-mediated inhibition in ferritin synthesis. The mechanism involves neither alteration in ferritin mRNA levels nor utilization of alternative transcription start sites to eliminate the IRE or relocate it in less inhibitory downstream positions. The derepression of ferritin mRNA translation occurs under conditions where global protein synthesis appears to be impaired, as judged by a significant enrichment in the expression of the underphosphorylated form of the translational regulator 4E-BP1. Collectively, these data document an example where ferritin mRNA translation evades control of the IRE-IRP system. The physiological implications of this response are reflected in protection against iron-mediated toxicity, oxidative stress, and apoptosis.

We thank Xinbin Chen (University of Alabama—Birmingham, Birmingham) for providing us with the tTA-H1299 and tTA-MCF7 cells, Anne-Claude Gingras and Nahum Sonenberg for the eIF-4E and 4E-BP1 antibodies, Franca Sicilia for technical assistance with FACS, and Joan Buss, Prem Ponka, and Antonis Koromilas for critical readings of the manuscript.

This work was supported by a grant from the Canadian Institutes of Health Research (CIHR). J.W. is a recipient of a fellowship from CIHR; K.P. is a scholar of CIHR and a researcher of the Canada Foundation for Innovation.