The hypoxia-inducible factor 1 (HIF-1) is the master regulator of the cellular response to hypoxia. A key regulator of HIF-1 is von Hippel-Lindau protein (pVHL), which mediates the oxygen-dependent, proteasomal degradation of HIF-1 in normoxia. Here, we describe a new regulator of HIF-1, the hypoxia-associated factor (HAF), a novel E3-ubiquitin ligase that binds HIF-1 leading to its proteasome-dependent degradation irrespective of cellular oxygen tension. HAF, a protein expressed in proliferating cells, binds and ubiquitinates HIF-1 in vitro, and both binding and E3 ligase activity are mediated by HAF amino acids 654 to 800. Furthermore, HAF overexpression decreases HIF-1 levels in normoxia and hypoxia in both pVHL-competent and-deficient cells, whereas HAF knockdown increases HIF-1 levels in normoxia, hypoxia, and under epidermal growth factor stimulation. In contrast, HIF-2 is not regulated by HAF. In vivo, tumor xenografts from cells overexpressing HAF show decreased levels of HIF-1 accompanied by decreased tumor growth and angiogenesis. Therefore, HAF is the key mediator of a new HIF-1-specific degradation pathway that degrades HIF-1 through a new, oxygen-independent mechanism.

The hypoxia-inducible factor 1 (HIF-1) regulates the cellular response to oxygen deprivation or hypoxia. HIF-1 is composed of an oxygen-regulated HIF-subunit and a constitutive HIF-1 subunit (45). To date, three HIF-isoforms have been described, of which HIF-1 and HIF-2 are the best characterized. HIF-1 is expressed ubiquitously, while HIF-2 displays more tissue-specific expression (51). The HIF-1 heterodimer binds to a conserved HIF binding sequence within the hypoxia-responsive element (HRE) in the promoter or enhancer regions of target genes, causing their transactivation and an adaptive response of the tissue to hypoxia (44). HIF-1 activation is important in development and in normal adult tissues such as in skin during wound healing or in the kidney during hematopoiesis (17, 19). HIF-1 is also upregulated in many solid tumors which contain hypoxic regions because of the inability of the local vasculature to supply sufficient oxygen to the growing tumor (45). HIF-1 is a positive factor in tumor growth, and its increased expression has been correlated with poor patient prognosis (43).