Normoxic stabilization of hypoxia-inducible factor-1alpha by modulation of the labile iron pool in differentiating U937 macrophages: effect of natural resistance-associated macrophage protein 1
Authors: Helen J Knowles
Peter J Ratcliffe
David R Mole
Adrian L Harris
Journal: Cancer Research (2006)
Discipline: CLINICAL MEDICINE
Keywords: Hypoxia-inducible factor; HIF; normoxic conditions; macrophages; HIF-1alpha
Affiliations: Cancer Research UK Molecular Oncology Laboratory, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.

Abstract:  Hypoxia-inducible factor (HIF) is a transcription factor with major roles in many cellular and systemic responses to hypoxia. Activation of HIF pathways under hypoxia is mediated by suppression of the Fe(2+)- and O(2)-dependent HIF hydroxylase enzymes that normally inactivate HIFalpha subunits. Mechanisms underlying induction of HIF in normoxic conditions are less clearly understood. In human cancers, infiltrating macrophages show up-regulation of HIF and it has recently been shown that normoxic expression of HIF-1alpha is essential for macrophage function. Here, we report studies of HIF-1alpha induction following phorbol-12-myristate 13-acetate (PMA)-induced differentiation of monocytic U937 and THP1 cells. HIF-1alpha was markedly up-regulated under normoxia in this setting and this involved failure of HIF-1alpha prolyl hydroxylation despite the presence of O(2). Fluorescence measurements showed that differentiation was associated with marked reduction of the labile iron pool. Both the reduction in labile iron pool and the up-regulation of HIF-1alpha were suppressed by RNA interference-mediated down-regulation of the iron transporter natural resistance-associated macrophage protein 1. Up-regulation of HIF-1alpha following PMA-induced differentiation was also abolished by addition of Fe(2+) or ascorbate. These results indicate that physiologic changes in macrophage iron metabolism have an important effect on HIF hydroxylase pathways and suggest means by which the system could be manipulated for therapeutic benefit. 

Retracted: No

PubPeer comments:
 Figure 2: More similar than expected, could authors check, please? ![file] , 
 Thank you for your enquiries. I am going through each of these carefully both within my own laboratory and, in the case of collaborative work with any other laboratory that was involved. This will take a little time, but I will endeavour to get back with answers to your questions as soon as is possible. , 
 Same Actin in 2A and 2B. Different exposure ![file] , 
 Thank you for this comment. There does appear to be an error in this figure, which we think was due to mixing gels taken at multiple exposures. We cannot, so many years later ,find the originals, so we are repeating the experiments, which we will report to the journal when complete. , 
 Further to our earlier response we have now performed repeats of the experiments originally illustrated in Figure 2a and 2b. This is for THP1 cells in Figure 2a and for both THP1 cells and U937 cell in Figure 2b where the mistake made it impossible to know which of the two original panels was correct. Methods Methods were the same as in the original work except that a different antihydroxy-HIF-1 alpha 564 (anti HIF-OH) antibody was used, as currently available. As before, human monocytic THP1 and U937 were induced to differentiate by treatment with 20 nmol L PMA for 48 hours. Suspension cells and cells treated with PMA (P) were incubated under normoxia or hypoxia (0.1% O2, 5%CO2, balance N2 in a MiniGalaxy incubator; RS Biotech, Irvine, Scotland) for 16 hours. Cells were homogenized in lysis buffer (6.2 mol L urea, 10% glycerol, 5 mmol L DTT, and 1% SDS plus protease inhibitors). Whole cell extract was separated by 8% SDS-PAGE and transferred to PVDF membrane. Primary antibodies were monoclonal mouse anti-HIF-1 alpha (clone 54, BD Biosciences, Oxford, UK) and anti-beta-tubulin (clone TUB2.1, Sigma-Aldrich, Dorset, UK) and a hydroxy-HIF-1 alpha (Pro564) (D43B5) XP rabbit monoclonal antibody (Cell Signaling Technology, London, UK). Results Results are shown in the attached figures (2a, 2b) Figure 2A: THP-1 cells were normoxic (N) or made hypoxic and transiently reoxygenated (R, 5 minutes during lysate preparation). Total protein was assayed for HIF-1 alpha, HIF-OH, and beta-tubulin expression by Western blot. The repeat experiment confirms the original finding of reduced hydroxylated HIF-1 alpha in PMA-stimulated normoxic THP1 cells (lane 3). As previously, to validate the hydroxy-specific antibody (HIF-OH), cells were also treated with either MG132 (25 micromol L, 16 h, proteasomal inhibition) to cause accumulation of hydroxylated HIF-1 alpha (lanes 5 and 6) or MMOG (1 mmol L, 16 h) to cause accumulation of non-hydroxylated HIF-1 alpha (lanes 7 and 8). Figure 2B: Normoxic and hypoxic THP1 cells (left) and U937 cells (right) were additionally treated with either 25 micromol L ascorbate (Asc) or 40 micromol L FeCl2 (Fe) for 16 hours. Total protein was assayed for HIF-1 alpha and beta-tubulin expression by Western blot. The repeat experiments using both THP1 and U937 cells confirm the original findings that both ascorbate and FeCl2 reduce or abrogate PMA-mediated accumulation of HIF-1 alpha in these cells. We will inform the journal, as previously indicated. [file] ,