The International Journal of Biochemistry & Cell Biology
Cobalt stimulates HIF-1-dependent but inhibits HIF-2-dependent gene expression in liver cancer cells
Introduction
Hypoxia-inducible factors (HIFs) play a central role in hepatocellular carcinoma (HCC) (Mylonis and Simos, 2012). HIFs are frequently up-regulated in HCC and seem to control tumor progression and sensitivity to radiation therapy (Nath and Szabo, 2012). HIFs are heterodimers of the protein subunits HIF-α, which are induced by hypoxia and HIF-β or aryl hydrocarbon receptor nuclear translocator (ARNT), which is constitutively expressed (Keith et al., 2012). The HIF-α subunits are rapidly degraded in normoxia by a process involving prolyl hydroxylation, interaction with the von Hippel–Lindau protein (pVHL) E3 ubiquitin ligase complex and proteasomal degradation. Hydroxylation is catalyzed by a family of Fe(II) and 2-oxoglutarate-dependent prolyl hydroxylases (PHDs) whose absolute requirement for molecular oxygen confers sensitivity to hypoxia, under which HIF-α members rapidly accumulate, translocate inside the nucleus, heterodimerize with ARNT and bind to hypoxia-response elements (HREs) DNA elements in the promoters or enhancers of their target genes. The activity of PHDs and subsequent stabilization of HIF-1α is also affected by chemical agents (“hypoxia mimetics”) such as the iron chelator desferrioxamine (DFO) (Wang and Semenza, 1993), flavonoids such as quercetin (Triantafyllou et al., 2007), 2-oxoglutarate-dependent oxygenase inhibitors such as DMOG (Elvidge et al., 2006) and transition metals such as cobalt (Kaelin and Ratcliffe, 2008). Treatment with these agents (“chemical hypoxia”) is often used to simulate hypoxic conditions and induce HIF-1. However, the action of certain of these agents (e.g. cobalt and flavonoids) may be mediated by signaling pathways not necessarily shared by the “true” hypoxic response and may cause, therefore, different and oxygen-independent biological effects (Chachami et al., 2004, Triantafyllou et al., 2006, Triantafyllou et al., 2008).
HIF-1α was the first isoform of HIF-α to be identified by its capacity to bind to the hypoxia-responsive element (HRE) present in the erythropoietin (EPO) enhancer (Wang et al., 1995). HIF-1α is ubiquitously expressed under hypoxia and is responsible for the regulation of a wide range of cellular adaptation responses and more preferentially metabolic enzymes (Majmundar et al., 2010, Mylonis et al., 2012). A second isoform (HIF-2α), is encoded by the EPAS1 gene, its expression is restricted to specific cell types, including hepatocytes, it appears to be more involved in angiogenesis and erythropoiesis and its regulation is considerably less investigated (Keith et al., 2012). Like HIF-1α, HIF-2α is degraded under normal conditions via the same PHD-VHL-proteasome-dependent system and is stabilized under hypoxia due to PHD inhibition. However, it is not yet known whether oxygen-independent mechanisms that regulate HIF-1 activity, such as for example nuclear transport and phosphorylation (Mylonis et al., 2008, Kalousi et al., 2010), also apply to HIF-2. In the same line, the effects of “hypoxia mimetics” on different cell types is usually studied by monitoring HIF-1α expression and whether HIF-2α activity is also affected remains relatively unknown. This has important biological relevance given that HIF-α stabilizers are used both experimentally and clinically as erythropoiesis-stimulating agents.
HIF-1α and HIF-2α share certain overlapping functions by regulating common hypoxia-inducible genes. However, it has recently become clear that, through independent regulation of distinct target genes or unique protein-protein interactions, HIF-1α and HIF-2α can also mediate divergent functions when expressed in the same cell type and especially in the context of cancer (Keith et al., 2012). The predominant role of HIF-2 in erythropoiesis has been established by studies in HIF-2α-deficient (knock-out) mice documenting HIF-2α as the main regulator of hepatic EPO production and essential for the maintenance of systemic EPO and iron homeostasis (Scortegagna et al., 2005, Rankin et al., 2007, Kapitsinou et al., 2010). Other prominent HIF-2-specific targets are genes for major antioxidant enzymes such as superoxide dismutase (SOD2) (Scortegagna et al., 2003). However, these animal studies have not determined the exact degree to which individual HIF-α subunits contribute to EPO production in cancer cells. Many subtle differences on the regulation of the expression and transcriptional activity between HIF-1α and HIF-2α need to be elucidated, especially in HCC as inhibition of HIFs may have an important role in targeted cancer therapy (Nath and Szabo, 2012).
In this report, we investigate the expression and transcriptional activity of HIF-1α and HIF-2α as well as expression and secretion of EPO in response to hypoxia and the “hypoxia mimetic” cobalt in hepatic cancer cell lines expressing both HIF-α isoforms. Our findings show that cobalt has opposing effects on HIF-2α expression and activity, rendering it ineffective in EPO gene activation. This specific effect of cobalt on HIF-2 signaling was accompanied by reduced interaction of the HIF-2α with USF2. This suggests that HIF-2α, but not HIF-1α, requires the assistance of a cobalt-sensitive factor in order to activate transcription of its target genes in liver cancer cells.
Section snippets
Plasmids
pGL3-SOD2 promoter and pGL3-PGK promoter were kindly provided by Joseph A. Garcia (Department of Medicine, University of Texas) and M. Celeste Simon (Abramson Family Cancer Research Institute, University of Pennsylvania) respectively (Scortegagna et al., 2003, Hu et al., 2007). Plasmid pEGFP-HIF-2α was constructed by inserting the full length HIF-2α cDNA into the BamHI position of the multicloning site of the pEGFP-C1 plasmid (Clontech). pcDNA-HIF-2α, kindly provided from Dr. S. L. McKnight
Hypoxia or CoCl2 induce both HIF-2α and HIF-1α protein expression in Huh7 hepatoma cells
The expression of HIF-2α in Huh7 hepatoma cells under different “hypoxic” conditions, such as true hypoxia (1% O2) or in the presence 150 μM CoCl2 (chemical hypoxia) was compared to that of HIF-1α by western blotting analysis shown in Fig. 1A. When cells were exposed to 1% O2, HIF-2α as well as HIF-1α protein levels increased rapidly and remained elevated for up to 16 h. Then, both HIF-2α and HIF-1α expression gradually declined and returned to control levels by 48 h. When cells were treated with
Discussion
Many human cancer cell types exhibit increased expression of either one or both of the main HIF-α isoforms, HIF-1α and HIF-2α, and in many cases this overexpression is associated with poor prognosis (Keith et al., 2012). This has made HIFs major targets of anti-cancer therapy and many HIF inhibitors are currently under clinical evaluation (Semenza, 2010). However, there are certain cases of tumors in which one of the two HIF-α isoforms, but not the other, may actually confer favorable prognosis
Acknowledgments
We thank Drs. J. A. Garcia (University of Texas), M. C. Simon (University of Pennsylvania) and M. U. Muckenthaler (University of Heidelberg) for providing plasmids.
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