Research Article: Real-Time Imaging of HIF-1α Stabilization and Degradation

Date Published: April 4, 2009

Publisher: Public Library of Science

Author(s): Ekaterina Moroz, Sean Carlin, Katerina Dyomina, Sean Burke, Howard T. Thaler, Ronald Blasberg, Inna Serganova, Joanna Mary Bridger. http://doi.org/10.1371/journal.pone.0005077

Abstract: HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α.

Partial Text: The HIF-1 (hypoxia inducible transcriptional factor 1) controls the expression of genes involved in critical aspects of cancer biology, such as angiogenesis, glucose metabolism, cell survival, invasion and tumor progression [1], [2], [3], [4], [5], [6], [7]. HIF-1 is a heterodimeric protein complex composed of two subunits: a stable and constitutively expressed HIF-1β, and an inducible, O2- and growth factor-regulated HIF-1α-subunit [8], [9]. HIF-1α protein is constantly modified posttranslationally by prolyl hydroxylases at Pro402 and/or Pro564 within the oxygen-dependent degradation (ODD) domain which promotes binding with pVHL (von Hippel-Lindau protein) and subsequent targeting for rapid proteasomal degradation. The half-life of HIF-1α protein, as determined by standard immunoblotting method, is about 5–8 min under normal oxygenated conditions [10], [11], [12]. It has been proposed, that interaction between pVHL and HIF-1α occurs in the nucleus, where HIF-1α protein is ubiquitinated and then exported to the cytoplasm for further proteasomal degradation [13]. Under hypoxic conditions, the prolyl hydroxylation reaction is inhibited and pVHL-HIF-1α interaction is abrogated, resulting in HIF-1α accumulation in the nucleus and dimerization with HIF-1β [14], [15]. The degradation of HIF-1α is also regulated in an O2-independent manner by the competitive binding to either heat shock protein 90 (HSP90), which stabilizes the protein [16], [17], or to the anchoring protein (RACK1), which leads to HIF-1α degradation by an oxygen-independent process [18].

The two HIF-1α chimeric reporter systems that were developed in this study provide an opportunity to investigate HIF-1α stabilization/degradation process in different cell lines, both in culture and in xenografts. Several groups have studied and employed the fusion between the HIF-1α-ODD domain and Firefly Luciferase. The resulting fusion protein (ODD-Luc) is responsive to hypoxia and hypoxia mimetics in live cells and can be used for imaging HIF-1 oxygen/VHL-regulated activity in real-time under different conditions [32], [33], [34]. A mouse that ubiquitously expresses the ODD-Luc reporter has been successfully used to study the action of small molecule inhibitors of HIF prolyl hydroxylase activity [35]. However, the ODD-Luc reporter is useful only for studying the O2-ODD-VHL-dependent mechanism of regulating HIF-1α expression. HIF-1α protein levels are also regulated by oxygen-independent mechanisms that reflect genetic alterations in signaling pathways or regulatory factors, and result in constitutive high levels of HIF-1α and HIF-1 transcriptional activity [36], [37]. Two PAS domains, A and B, of the HIF-1α subunit in the N-terminal region frequently mediate protein-protein interactions. One or both of the HIF-1α PAS domains have been functionally implicated in heterodimer formation, nuclear translocation and HIF-1α stabilization via HSP90 association [38]. The C-terminal part of the HIF-1 α is involved in protein transactivation. That is why the presence of the entire length of HIF-1α in reporter protein is essential for understanding HIF-1α biology and regulation of HIF-1α stability, since this occurs at multiple levels and involves more than the ODD domain.

Source:

http://doi.org/10.1371/journal.pone.0005077