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Comparative Study
. 2005 May 24;102(21):7481-6.
doi: 10.1073/pnas.0502716102. Epub 2005 May 16.

The candidate tumor suppressor ING4 represses activation of the hypoxia inducible factor (HIF)

Abdullah Ozer  1 , Leeju C Wu, Richard K Bruick
Affiliations
Comparative Study

The candidate tumor suppressor ING4 represses activation of the hypoxia inducible factor (HIF)

Abdullah Ozer et al. Proc Natl Acad Sci U S A. .

Abstract

The hypoxia inducible factor (HIF) plays an important role in the progression of a number of pathophysiological processes including tumorigenesis. In addition to several well characterized oxygen-dependent modes of regulation, the function of the HIF transcription factor can also be influenced through the action of other regulatory pathways. Misregulation of these factors resulting in inappropriate HIF expression or activity can contribute to the progression of human cancers through the induction of genes promoting angiogenesis, glycolysis, cell survival, and metastasis, among other processes. The candidate tumor suppressor protein inhibitor of growth family member 4 (ING4) has recently been implicated as a repressor of angiogenesis and tumor growth through association with NF-kappaB. Here we demonstrate that suppression of ING4 further induces HIF transcriptional activity as well. ING4 directly associates with the HIF prolyl hydroxylase, an Fe(II)-dependent oxygenase previously shown to mediate HIF stability as a function of oxygen availability. However, rather than affecting HIF's stability, ING4 mediates HIF's activity. These data support a model in which, in addition to regulating HIF stability, HIF prolyl hydroxylases can modulate HIF function through the recruitment of ING4, a likely component of a chromatin-remodeling complex VSports手机版. .

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V体育2025版 - Figures

Fig. 1.
Fig. 1.
siRNA suppression of ING4 enhances HIF target gene expression under hypoxic conditions. (A) Western blot analysis indicating relative protein levels of HPH-2, ING4, and nuclear HIF-1α under normoxic (20% O2) or hypoxic (1% O2) conditions for 12 h after treatment of HeLa cells with siRNA duplexes specific for ING4 (ING4#1 or ING4#2) or GFP control. HIF-2α was undetectable in Western blots of HeLa cell extracts. (B) Northern blot analysis of the endogenous HIF target genes Nip3 and AK3 after siRNA-mediated suppression of ING4. Actin mRNA levels are shown to confirm equivalent RNA loading. All results are representative of multiple experiments.
Fig. 2.
Fig. 2.
ING4 interacts with HPH-2 through its C-terminal PHD (residues 191–249). (A Upper) Yeasts were transformed with vectors encoding HPH-2C fused to the LexA DNA-binding domain (DBD) and ING4 fused to the GAL4 activation domain. Positive protein–protein interactions are denoted by LacZ reporter expression. Transformations were performed in duplicate. (Lower) Yeast two-hybrid analysis of HPH-2C interactions with N- and C-terminal truncations of ING4 are shown. (B) 35S-labeled full-length ING4 (residues 1–249) or ING4 lacking the PHD (residues 1–198) were incubated with immobilized GST or the GST/HPH-2C fusion protein. Bound 35S-labeled ING4 was visualized after SDS/PAGE. (C) Lysates from HeLa cells transfected with N-terminal 3XFLAG-tagged ING4 were immunoprecipitated with anti-HPH-2 serum or preimmune serum as a control. ING4 that coimmunoprecipitated with HPH-2 was detected by Western blot analysis using an anti-FLAG M2 antibody. IP, immunoprecipitate.
Fig. 3.
Fig. 3.
Purified ING4 and HPH-2C directly associate in vitro. One hundred micrograms of purified recombinant ING4, HPH-2C, and/or FIH-1 proteins was incubated alone or together before resolution on a Superdex 200 10/30 column (Amersham Pharmacia Biotech). Eluted fractions were resolved by SDS/PAGE and visualized by Coomassie blue staining. The elution profile of protein standards is given at the top.
Fig. 4.
Fig. 4.
Both ING4 and HPH-2 are found among salt-extracted nuclear proteins. HeLa cells were incubated under normoxic (20% O2) or hypoxic (1% O2) conditions for 12 h. Soluble cytoplasmic (C) and salt-extracted nuclear (N) proteins were separated and analyzed by Western blot analysis with antibodies raised against ING4 or HPH-2. Antibodies to p-ATF-2 or annexin I were used to assess the integrity of the nuclear and cytoplasmic samples, respectively.
Fig. 5.
Fig. 5.
ING4 is not an HPH-2 substrate in vitro. Substrate utilization by HPH-2C was assessed by [14C]CO2 generation in the presence of either a control HIF-1α peptide substrate or a recombinant ING4 protein. Assays were performed in triplicate.
Fig. 6.
Fig. 6.
ING4 does not affect HPH-2C activity in vitro. Hydroxylase activity of recombinant HPH-2C was measured in a 35S-labeled VHL pull-down assay in the presence of a HIF-1α peptide substrate and increasing amounts of recombinant ING4 (the amount of ING4 protein relative to HPH-2C is indicated). Reaction conditions were chosen to provide approximately half-maximal substrate hydroxylation to observe either stimulation or inhibition of hydroxylase activity. 35S-labeled VHL binding to a fully hydroxylated peptide (+ control) is shown as a reference. Assays were performed in triplicate.
Fig. 7.
Fig. 7.
ING4 affects HIF activity in a chromatin-dependent manner. (A) Suppression of ING4 increases expression of a stably transfected HIF-driven luciferase reporter gene. A HeLa cell line stably expressing the HIF-responsive 3XHRE-tk-Luc reporter gene was transfected with siRNA duplexes specific for either ING4 (duplex ING4#1) or GFP followed by incubation under normoxic (20% O2) or hypoxic (1% O2) conditions for 15 h. Similar results were obtained with other independently isolated stably transfected HeLa cell lines (data not shown). (B) siRNA suppression of ING4 does not affect hypoxic induction of a transiently transfected HIF reporter gene. Wild-type HeLa cells were transfected with siRNA duplexes specific for either ING4 or a GFP control followed by the 3XHRE-tk-Luc HIF reporter construct and incubation under normoxic or hypoxic conditions. All assays were performed in triplicate, and the results are representative of multiple experiments.
Fig. 8.
Fig. 8.
ING4, HPH-2, and HIF associate with the HRE promoter. HeLa cells stably transfected with the 3XHRE-tk-Luc reporter construct were incubated under hypoxic (1% O2) conditions for 15 h followed by crosslinking, sonication, and immunoprecipitation using antibodies specific for ING4, HIF-β, or HPH-2. (Upper) Primers flanking the 3XHRE promoter element were used to amplify associated chromatin DNA by PCR. (Lower) Relative band intensities are indicated. No Ab, no antibody control; Pre Immune, preimmune serum control.
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