Hypoxia-inducible factor ␣ proteins (HIF-␣s) are regulated oxygen dependently and transactivate numerous genes essential for cellular adaptation to hypoxia. NEDD8, a member of the ubiquitin-like family, covalently binds to its substrate proteins, and thus, regulates their stabilities and functions. In the present study, we examined the possibility that the HIF signaling is regulated by the neddylation. HIF-1␣ expression and activity were inhibited by knocking down APPBP1 E1 enzyme for NEDD8 conjugation but enhanced by ectopically expressing NEDD8. HIF-1␣ and HIF-2␣ were identified to be covalently modified by NEDD8. NEDD8 stabilized HIF-1␣ even in normoxia and further increased its level in hypoxia, which also occurred in von Hippel-Lindau (VHL) protein-or p53-null cell lines. The HIF-1␣-stabilizing effect of NEDD8 was diminished by antioxidants and mitochondrial respiratory chain blockers. This suggests that the NEDD8 effect is concerned with reactive oxygen species driven from mitochondria rather than with the prolyl hydroxylase (PHD)/VHL-dependent oxygen-sensing system. Based on these findings, we propose that NEDD8 is an ancillary player to regulate the stability of HIF-1␣. Furthermore, given the positive role played by HIF-␣s in cancer promotion, the NEDD8 conjugation process could be a potential target for cancer therapy. Hypoxia-inducible factors (HIFs)3 play crucial roles in tumor adaptation to hypoxia and angiogenesis by up-regulating numerous genes (1). HIF family members are composed of ␣ (HIF-1␣ and HIF-2␣) and ␤ (also known as aryl hydrocarbon receptor nuclear translocator (ARNT)) subunits. The HIF-␣s are tightly regulated by oxygen tension and function as prime transactivating factors, whereas aryl hydrocarbon receptor nuclear translocator is constitutively expressed and assists HIF-␣ binding to DNA. Under normoxic conditions, two proline residues (Pro-402 and Pro-564) in the oxygen-dependent degradation domain (ODDD) of HIF-1␣ are hydroxylated by prolyl hydroxylases (PHD1-3) (2); subsequently, HIF-1␣ is ubiquitinated by von Hippel-Lindau protein (pVHL) and finally degraded by 26 S proteasome (3). However, this hydroxylation is limited under hypoxic conditions, which stabilizes HIF-␣s. Given the essential roles played by HIFs in tumor promotion, the HIF inhibition has become a frontline topic in research on new cancer therapies (4).NEDD8 (neural precursor cell-expressed developmentally down-regulated 8) is conserved in eukaryotes and is ubiquitously expressed in most mammalian tissues. As NEDD8 is structurally similar to ubiquitin, it is classified as a member of the ubiquitin-like family (5). Furthermore, like ubiquitin, NEDD8 conjugates to its substrate proteins, which is named "neddylation," via a sequential process involving activation, conjugation, and ligation. Neddylation requires a unique set of conjugating enzymes, namely NEDD8-activating E1 complex, which is composed of APPBP1 and UBA3, NEDD8-conjugating E2 enzyme (UBC12), and various NEDD8-ligase E3 enzymes (6). Functionally, neddylation...

Amphotericin B (AmB) is widely used for treating severe systemic fungal infections. However, long-term AmB treatment is invariably associated with adverse effects such as anemia. The erythropoietin (EPO) suppression by AmB has been proposed to contribute to the development of anemia. However, the mechanism whereby EPO is suppressed remains obscure. In this study, we investigated the possibility that AmB inhibits the transcription of the EPO gene by inactivating HIF-1, which is a known key transcription factor and regulator of EPO expression. EPO mRNA levels were markedly attenuated by AmB treatment both in rat kidneys and in Hep3B cells. AmB inactivated the transcriptional activity of HIF-1␣, but did not affect the expression or localization of HIF-1 subunits. Moreover, AmB was found to specifically repress the C-terminal transactivation domain (CAD) of HIF-1␣, and this repression by AmB required Asn803, a target site of the factor-inhibiting HIF-1 (FIH); moreover, this repressive effect was reversed by FIH inhibitors. Furthermore, AmB stimulated CAD-FIH interaction and inhibited the p300 recruitment by CAD. We propose that this mechanism underlies the unexplained anemia associated with AmB therapy.

No abstract