Iron-regulatory proteins 1 and 2 (IRP1 and IRP2) are RNA-binding proteins that post-transcriptionally regulate the expression of mRNAs that code for proteins involved in the maintenance of iron and energy homeostasis. Here we show that hypoxia differentially regulates the RNA binding activities of IRP1 and IRP2 in human 293 and in mouse Hepa-1 cells. In contrast to IRP1, where hypoxic exposure decreases IRP1 RNA binding activity, hypoxia increases IRP2 RNA binding activity. The hypoxic increase in IRP2 RNA binding activity results from increased IRP2 protein levels. Cobalt, which mimics hypoxia by activation of hypoxia-inducible factor 1 (HIF-1), also increases IRP2 protein levels; however, cobalt-induced IRP2 lacks RNA binding activity. Addition of a reductant to cobalt-treated extracts restored IRP2 RNA binding activity. Hypoxic activation of IRP2 is not because of an increase in transcriptional activation by HIF-1, because IRP2 accumulates in Hepa-1 cells lacking a functional HIF-1 subunit, nor is it because of an increase in IRP2 mRNA stability. Rather, our data indicate that hypoxia increases IRP2 levels by a post-translational mechanism involving protein stability. Differential regulation of IRP1 and IRP2 during hypoxia may regulate specific IRP target mRNAs whose expression is required for hypoxic adaptation. Furthermore, these data imply mechanistic parallels between the hypoxia-induced post-transcriptional regulation of IRP2 and HIF-1␣.Cellular hypoxia is an important component of several pathophysiological conditions, including tumorigenesis and ischemia-related disorders. In these and other hypoxic situations mammalian cells alter gene expression to counter the effects of limited O 2 . Hypoxia induces the transcriptional activation of a variety of genes, including erthyropoietin, vascular endothelial growth factor, transferrin, tyrosine hydroxylase, and various glycolytic enzymes, all of whose products are involved in cellular adaptation to decreased O 2 (reviewed in Ref. 1). Increased expression of these genes is mediated primarily by the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), 1 which is composed of HIF-1␣ and HIF-1 subunits (1-4). Decreased O 2 tension stimulates HIF-1␣ protein accumulation by decreasing its proteasomal degradation (5, 6). Hypoxic stabilization of HIF-1␣ is blocked in the presence of H 2 O 2 , suggesting that hypoxia-induced changes in the level of this reactive oxygen species may be involved in HIF-1 activation (7). Stabilization of HIF-1␣ allows for heterodimerization with constitutive levels of HIF-1, also known as aryl hydrocarbon nuclear translocator or ARNT. In turn HIF-1 binds to specific enhancer elements resulting in transcriptional activation.The current understanding of the regulation of gene expression during hypoxia is primarily at the level of transcriptional activation by HIF-1. However, it is clear that post-transcriptional mechanisms are also employed. These mechanisms involve the induction of mRNA-binding proteins that interact wi...
BACKGROUND Amplification of the N‐myc oncogene is associated with adverse outcomes in the common childhood tumor, neuroblastoma. Because the transforming properties of Myc are related to its ability to modulate gene expression, the authors used cDNA microarrays to identify potential Myc target genes. METHODS Expression levels of 4608 genes were analyzed in a series of neuroblastoma cell lines. Identical analyses were performed in a panel of medulloblastoma cell lines to identify c‐Myc targets and to determine the extent to which N‐Myc targets and c‐Myc targets were shared. Comparisons were made between cell lines with high levels versus low levels of Myc protein expression. RESULTS Array analyses yielded 121 genes with increased expression levels (≥ 1.65‐fold) and 9 genes with decreased expression levels in N‐Myc‐expressing versus nonexpressing cell lines. Many of these were newly identified targets of biologic interest. Fifty percent of the N‐Myc targets (60 of 121) were mutual c‐Myc targets. A significant correlation between the level of N‐myc and selected target gene expression was demonstrated independently in 27 neuroblastoma tumor samples and in an N‐myc‐inducible cell line system. CONCLUSIONS A number of diverse pathways are modulated by N‐Myc in neuroblastoma. Although, overall, there was significant correlation between myc and target transcript expression among cohorts of tumors, great variability in levels of target expression was seen among individual tumor samples, and this biologic heterogeneity in the levels of target gene expression may offer insight into differences in the clinical behavior of neuroblastoma and may prove to be of prognostic significance in the future. Cancer 2003;98:841–53. © 2003 American Cancer Society. DOI 10.1002/cncr.11584
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