Hypoxia-associated factor, a novel E3-ubiquitin ligase, binds and ubiquitinates hypoxia-inducible factor 1alpha, leading to its oxygen-independent degradation

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

The polypyrimidine tract-binding protein stimulates HIF-1alpha IRES-mediated translation during hypoxia

When oxygen supply is restricted, protein synthesis is rapidly abrogated owing to inhibition of global translation. However, HIF-1α protein expression can persist during hypoxia, owing to an internal ribosome entry site (IRES) in the 5′-untranslated region of its mRNA. Here, we report on the molecular mechanism of HIF-1α IRES-mediated translation during oxygen deprivation. Using RNA affinity chromatography and UV-crosslinking experiments, we show that the polypyrimidine tract binding protein (PTB) can specifically interact with the HIF-1α IRES, and that this interaction is enhanced in hypoxic conditions. Overexpression of PTB enhanced HIF-1α IRES activity, whereas RNA interference-mediated downregula-tion of PTB protein expression inhibited HIF-1α IRES activity. Furthermore, hypoxia-induced stimulation of the HIF-1α IRES was reduced in cells in which PTB function was downregulated. In agreement with these results, the IRES activity of HIF-1α IRES deletion mutants that are deficient in PTB-binding could not be stimulated by oxygen deprivation. All together, our data suggest that PTB plays a stimulatory role in the IRES-mediated translation of HIF-1α when oxygen supply is limited.     

Adenomatous Polyposis Coli and Hypoxia-inducible Factor-1α Have an Antagonistic Connection

The tumor suppressor adenomatous polyposis coli (APC) is mutated in the majority of colorectal cancers and is best known for its role as a scaffold in a Wnt-regulated protein complex that determines the availability of β-catenin. Another common feature of solid tumors is the presence of hypoxia as indicated by the up-regulation of hypoxia-inducible factors (HIFs) such as HIF-1α. Here, we demonstrate a novel link between APC and hypoxia and show that APC and HIF-1α antagonize each other. Hypoxia results in reduced levels of APC mRNA and protein via a HIF-1α–dependent mechanism. HIF-1α represses the APC gene via a functional hypoxia-responsive element on the APC promoter. In contrast, APC-mediated repression of HIF-1α requires wild-type APC, low levels of β-catenin, and nuclear factor-κB activity. These results reveal down-regulation of APC as a new mechanism that contributes to the survival advantage induced by hypoxia and also show that loss of APC mutations produces a survival advantage by mimicking hypoxic conditions.     

EAF2 Suppresses Hypoxia-Induced Factor 1α Transcriptional Activity by Disrupting Its Interaction with Coactivator CBP/p300

Previous studies revealed that the potential tumor suppressor EAF2 binds to and stabilizes pVHL, suggesting that EAF2 may function by disturbing the hypoxia signaling pathway. However, the extent to which EAF2 affects hypoxia and the mechanisms underlying this activity remain largely unknown. In this study, we found that EAF2 is a hypoxia response gene harboring the hypoxia response element (HRE) in its promoter. By taking advantage of the pVHL-null cell lines RCC4 and 786-O, we demonstrated that hypoxia-induced factor 1α (HIF-1α), but not HIF-2α, induced EAF2 under hypoxia. Subsequent experiments showed that EAF2 bound to and suppressed HIF-1α but not HIF-2α transactivity. In addition, we observed that EAF2 inhibition of HIF-1α activity resulted from the disruption of p300 recruitment and that this occurred independently of FIH-1 (factor inhibiting HIF-1) and Sirt1. Furthermore, we found that EAF2 protected cells against hypoxia-induced cell death and inhibited cellular uptake of glucose under hypoxic conditions, suggesting that EAF2 indeed may act by modulating the hypoxia-signaling pathway. Our findings not only uncover a unique feedback regulation loop between EAF2 and HIF-1α but also provide a novel insight into the mechanism of EAF2 tumor suppression.     

Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes

This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O₂) and severe hypoxia (0.1% O₂). We found that chenodeoxy cholic acid (CDCA) reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR), a CDCA receptor and its target gene, Small heterodimer partner (SHP) are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein.     

Hypoxia-inducible factor-1α and poly [ADP ribose] polymerase 1 cooperatively regulate Notch3 expression under hypoxia via a noncanonical mechanism

Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxia-inducible factor-1α (HIF-1α) in the Notch3 response are still unclear. In this study, we found that HIF-1α and poly [ADP-ribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1α knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1α and also another molecule that binds the Notch3 promoter’s guanine-rich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1α associates with PARP-1 on the Notch3 promoter in a hypoxia response element–independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1α, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies.