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Originally published In Press as doi:10.1074/jbc.M511408200 on March 24, 2006

J. Biol. Chem., Vol. 281, Issue 22, 15215-15226, June 2, 2006
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Concordant Regulation of Gene Expression by Hypoxia and 2-Oxoglutarate-dependent Dioxygenase Inhibition

THE ROLE OF HIF-1{alpha}, HIF-2{alpha}, AND OTHER PATHWAYS*Formula

Gareth P. Elvidge{ddagger}, Louisa Glenny{ddagger}, Rebecca J. Appelhoff§, Peter J. Ratcliffe§, Jiannis Ragoussis{ddagger}1, and Jonathan M. Gleadle§2

From the §Oxygen Sensing Group, The Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford OX3 7BN, and the {ddagger}Genomics Group, Wellcome Trust Centre for Human Genetics, The Henry Wellcome Building for Genomic Medicine, University of Oxford, Oxford OX3 7BN, United Kingdom

Received for publication, October 20, 2005 , and in revised form, February 28, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies of gene regulation by oxygen have revealed novel signal pathways that regulate the hypoxia-inducible factor (HIF) transcriptional system through post-translational hydroxylation of specific prolyl and asparaginyl residues in HIF-{alpha} subunits. These oxygen-sensitive modifications are catalyzed by members of the 2-oxoglutarate (2-OG) dioxygenase family (PHD1, PHD2, PHD3, and FIH-1), raising an important question regarding the extent of involvement of these and other enzymes of the same family in directing the global changes in gene expression that are induced by hypoxia. To address this, we compared patterns of gene expression induced by hypoxia and by a nonspecific 2-OG-dependent dioxygenase inhibitor, dimethyloxalylglycine (DMOG), among a set of 22,000 transcripts, by microarray analysis of MCF7 cells. By using short interfering RNA-based suppression of HIF-{alpha} subunits, we also compared responses that were dependent on, or independent of, the HIF system. Results revealed striking concordance between patterns of gene expression induced by hypoxia and by DMOG, indicating the central involvement of 2-OG-dependent dioxygenases in oxygen-regulated gene expression. Many of these responses were suppressed by short interfering RNAs directed against HIF-1{alpha} and HIF-2{alpha}, with HIF-1{alpha} suppression manifesting substantially greater effects than HIF-2{alpha} suppression, supporting the importance of HIF pathways. Nevertheless, the definition of genes regulated by both hypoxia and DMOG, but not HIF, distinguished other pathways most likely involving the action of 2-OG-dependent dioxygenases on non-HIF substrates.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The response of cells to low oxygen (hypoxia) is characterized by coordinated regulation of the expression of a large number of genes whose products have widespread roles, including energy provision, vascular supply, and growth. Studies of the regulation of many such genes by oxygen have implicated a central role for the transcription factor hypoxia-inducible factor (HIF),3 which exists as a heterodimer of an {alpha} and a beta subunit (1). The mechanism of oxygen sensing, which controls this heterodimeric factor, has been elucidated recently (for reviews see Refs. 2 and 3).

In the presence of oxygen, HIF-{alpha} molecules undergo ubiquitination followed by rapid proteasomal degradation. The ubiquitination is facilitated by the product of the von Hippel-Lindau gene (VHL), which acts as an essential component of an E3 ubiquitin ligase (4). In the presence of oxygen, the VHL protein recognizes and binds to two specific hydroxyproline residues in HIF-1{alpha} and HIF-2{alpha} (5–7). Three homologous 2-oxoglutarate-dependent dioxygenases PHD1, PHD2, and PHD3 catalyze this prolyl hydroxylation (8, 9). Further oxygen-regulated control of the transcriptional potency of HIF-{alpha} is provided by another 2-oxoglutarate-dependent dioxygenase (FIH-1), which catalyzes the formation of a specific hydroxyasparagine in the C terminus of HIF-{alpha}, decreasing its binding to the transcriptional coactivator p300 (10, 11).

The identification of this mechanism of regulating HIF raises two important questions. First, to what extent are HIF and the HIF hydroxylases responsible for the global patterns of gene regulation by hypoxia? Second, are there other oxygen-regulated pathways controlled in a similar manner by the HIF hydroxylases or other 2-oxoglutarate-dependent dioxygenases? Although HIF appears to have a major role in the control of gene expression by oxygen, it is unclear to what extent other transcriptional mechanisms are also involved in the response to hypoxia. A role for HIF-independent regulation appears probable because there are examples of genes whose expression is regulated by hypoxia in cells lacking functional HIF-1{alpha} or HIF-1beta (12, 13). Furthermore, other transcription factors such as AP1, NF{kappa}B, and p53 have all been reported to show activation by hypoxia (14–16), and the stability of certain mRNA transcripts may also be regulated by hypoxia (17).

The understanding of the mechanism of oxygen sensing controlling HIF has also led to ways of pharmacologically manipulating the HIF response. Both the prolyl and asparaginyl hydroxylases require 2-oxoglutarate. The cell-permeant 2-oxoglutarate analogue dimethyloxalylglycine (DMOG) inhibits the HIF prolyl and asparaginyl hydroxylases (5, 8, 10, 11), collagen prolyl 4-hydroxylase (18), and is predicted to inhibit other members of this class of 2-oxoglutarate-dependent dioxygenases. DMOG can produce activation of the HIF system with enhanced transcription of target genes and might have a role in the therapy of ischemic disease (5, 19). Indeed, in a model of myocardial ischemia, 2-oxoglutarate-dependent dioxygenase inhibition appeared beneficial, but the precise mechanism of action is unclear (20). It remains to be established to what extent exposure of cells to 2-oxoglutarate-dependent dioxygenase inhibitors can mimic the hypoxic response because the extent to which they can inhibit all four HIF hydroxylases in cellsisunclear, and they also may inhibit the action of other 2-oxoglutarate-dependent dioxygenases or have other metabolic effects.

To address these questions we have utilized microarray assays of mRNA abundance to examine the gene expression changes in response to hypoxia and to DMOG and following HIF-{alpha} siRNA. We demonstrate a large number of hypoxically regulated genes, both known and novel, and find a surprisingly high concordance between the hypoxic response and the response to the 2-oxoglutarate-dependent dioxygenase inhibitor, dimethyloxalylglycine, and a dominant role of HIF-1{alpha} for hypoxic regulation of gene expression. We also demonstrate pathways of hypoxic gene regulation that are HIF-independent but likely involve oxygen sensing 2-oxoglutarate-dependent dioxygenases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and RNA Preparation—MCF7 breast cancer and Hep3B hepatoblastoma cell lines were grown under conditions of either normoxia (21% oxygen) or hypoxia (1% oxygen) for 16 h in an In vivo2 Hypoxia Work Station (Ruskin Technologies, Kent, UK). All culture media included Dulbecco's modified Eagle's medium, 2 mM L-glutamine, and 10% fetal bovine serum (Sigma). RNA was extracted using the "Absolutely RNA" reverse transcription-PCR miniprep kit (Stratagene) and treated with DNase I. RNA quality and abundance were determined using an Agilent 2100 Bioanalyzer and Nanodrop ND-1000 spectrophotometer, respectively. All experiments were performed in triplicate from independent cell cultures, and in total, seven different types of samples were analyzed as follows: "normoxia," cells grown in normoxic (21% oxygen) conditions; "hypoxia," cells grown in hypoxic conditions (1% oxygen for 16 h); "DMOG," cells grown in normoxic conditions and exposed to DMOG (2 mM) for 16 h; "OF," cells grown in hypoxic conditions and exposed to oligofectamine transfection reagent (Invitrogen) alone; "HIF-1," cells grown in hypoxic conditions and transfected with HIF-1{alpha} siRNA; "HIF-2," cells grown in hypoxic conditions with HIF-2{alpha} siRNA; and "HIF-12," cells grown in hypoxic conditions with both HIF-1{alpha} and HIF-2{alpha} siRNAs. A summary of all the samples used in the study can be found in supplemental Table 1.

siRNA Treatment of Cells—MCF7 cells were seeded at 30% confluency and grown in normoxic conditions. Cells were transfected twice with 20 nM siRNA at 24 and 48 h using oligofectamine (Invitrogen) according to the manufacturers' instructions. At 55 h cells were exposed to hypoxic conditions, and after a further 16 h, RNA was extracted. The same protocol was used for the generation of protein extracts.

To achieve specific suppression of HIF-1{alpha} and/or HIF-2{alpha}, cells were transfected in triplicate with the siRNA oligonucleotides described by Sowter et al. (21). The HIF-1{alpha} siRNA duplex targeted nucleotides 1521–1541 of the HIF-1{alpha} mRNA sequence (GenBankTM accession number NM_001530 [GenBank] ) and included sense 5'-CUGAUGACCAGCAACUUGAdTdT-3' and antisense 5'-UCAAGUUGCUGGUCAUCAGdTdT-3'. The HIF-2{alpha} siRNA duplex targeted nucleotides 1260–1280 of the HIF-2{alpha} mRNA sequence (GenBankTM accession number NM_001430 [GenBank] ) and included sense 5'-CAGCAUCUUUGAUAGCAGUdTdT-3' and antisense 5'-ACUGCUAUCAAAGAUGCUGdTdT-3'. These sequences have been shown to achieve substantial suppression of their target mRNA and protein levels in several different cell types (21). To ensure specific and substantial knockdown was occurring in MCF7 cells under hypoxic conditions, we assayed HIF-{alpha} levels by immunoblotting (as described in Ref. 22) (Fig. 1a) following transfection with control and HIF-{alpha} siRNA sequences and additionally confirmed substantial suppression of the HIF-{alpha} mRNA transcripts with the Affymetrix GeneChip (Fig. 1b). To examine for the specificity of the siRNA effect, we additionally utilized alternative siRNA sequences for the Illumina microarray and real time PCR analyses targeting nucleotides 1378–1398 of HIF-1{alpha} 5'-GCCACUUCGAAGUAGUGCUdTdT-3' and targeting nucleotides 2274–2294 of HIF-2{alpha}, 5'-GCGACAGCUGGAGUAUGAAdTdT-3' as described by Warnecke et al. (23). The microarray expression results following each transfection were examined to ensure that the siRNA did not induce an interferon response. Genes that are known to be induced in the interferon response (24), such as OAS1 and MX1, were not found to be differentially expressed.


Figure 1
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  		FIGURE 1.
Protein and mRNA expression of HIF-1{alpha} and HIF-2{alpha} following siRNA transfection in MCF7 cells. a, immunoblot of cell extract obtained from MCF7 cells cultured at 1% oxygen and subjected to oligofectamine alone, transfection with control oligonucleotides, or with siRNA oligonucleotides targeting HIF-1{alpha} or HIF-2{alpha}. Substantial and specific reductions in protein levels are seen. b, Affymetrix results of mRNA expression levels for HIF-1{alpha} and HIF-2{alpha}, expressed as fold changes relative to oligofectamine alone, illustrating the efficacy and specificity of reduction in HIF-{alpha} mRNA. c, immunoblot detecting CAIX, BNIP3, PHD2, PHD3, ATF3, SOX9, LOXL2, ITPR1, SERPINE, and PRKCA in cell extracts from MCF7 cells cultured for 16 h in normoxia (N, 21% oxygen), hypoxia (H, 1% oxygen), DMOG (2 mM) or cultured at 1% oxygen and subjected to OF alone or transfection with siRNA oligonucleotides targeting either HIF-1{alpha}, HIF-2{alpha}, or HIF-1{alpha} and HIF-2{alpha}.

 
Microarray Analysis Using Affymetrix GeneChip—Total RNA (10 µg) from each sample was amplified, labeled, hybridized to an Affymetrix GeneChip, and detected according to the Affymetrix user manual. The normoxia, hypoxia, and DMOG samples (replicates 1–3) were arrayed to HG-U133A GeneChips (~22,000 transcripts), and sample types OF, HIF-1, HIF-2, and HIF-12 (replicates 1–3) were arrayed to HG-U133 plus 2 GeneChips (~55,000 transcripts). The CEL files for each array were imported into GeneSpring version 7.2 (Agilent) and normalized using the GCRMA algorithm (25). Statistical significance for the difference in expression levels between different treatments was assessed using a Perl script implementation of the "Rank Products" algorithm (26). The method is particularly suitable for the data presented because it is applicable to small sample sizes, corrects for multiple testing using a permutation-based estimation procedure (we used 100 permutations), and restricts the number of significant genes to those that show a reproducible high fold change between the two treatments. To correct for multiple testing, we used an arbitrary false discovery rate (FDR) cutoff of 5% (q value <0.05) to identify probe sets that are statistically significantly up- or down-regulated between two treatments. Heat map plots and sample clustering were performed using functions within the "gplots" library of the R statistical programming language.

Microarray Analysis Using Illumina BeadChip—Microarray analysis of gene expression in response to an additional set of siRNA sequences (siRNA replicates 4–6, see supplemental Table 1) was performed using the Illumina BeadChip system. To examine for hypoxic regulation with this platform, replicates 1 and 2 (see supplemental Table 1) from the hypoxia, DMOG, and normoxia sample set were also hybridized to the Bead-Chips. 200 ng of total RNA was used to perform in vitro transcription amplification using the Illumina RNA amplification kit (Ambion). Amplified RNA (1.5 µg) was hybridized to the "whole genome" Sentrix Human-6 Expression BeadChips (Illumina). Data normalization was performed using quantile normalization, and fold changes and statistical significance were determined using the Limma package from the Bioconductor repository, implemented on the R platform.

Independent Assessment of Expression Changes by Real Time PCR—The expression of selected genes was assessed independently by real time PCR. Replicates 4–6 of all MCF7 sample types were used in these analyses (see supplemental Table 1). Hep3B cells were also studied to assess mRNA regulation in a different cell type. Total RNA was reverse-transcribed, and real time PCR amplification was performed using SYBR Green as described previously (27). Each assay was optimized to minimize primer-dimer and nonspecific product formation. The primer sequences and individual amplification conditions are shown in supplemental Table 2.

Fold changes between treatments were determined by the {Delta}Ct method (28), normalizing the results from MCF7 samples to the mean of two reference genes (60 S rRNA and cyclophilin), which have been used previously as normalization genes in hypoxia studies. Cyclophilin alone was used as a normalization gene for the Hep3B analyses as 60 S rRNA was found to show variable expression in this cell line. Statistical significance was assessed using a permutation-based procedure. The normalization and statistical procedures were performed as implemented in the REST Excel plug-in (29) and using t tests.

Protein Abundance Analysis by Immunoblotting—Cells were rinsed in phosphate-buffered saline and subsequently lysed in urea/SDS buffer (6.7 M urea, 10 mM Tris-Cl (pH 6.8), 1 mM dithiothreitol, 10% glycerol, and 1% SDS) supplemented with Complete protease inhibitor mixture (Roche Applied Science). Whole cell extracts were resolved by SDS-PAGE and electroblotted onto polyvinylidene difluoride membrane (Millipore). The membranes were probed overnight at 4 °C with mouse monoclonal anti-CAIX (30), mouse monoclonal anti-BNIP3 (ANa40; Sigma), mouse monoclonal anti-PHD3 (22), rabbit polyclonal anti-PHD2 (NB100-137; Novus Biologicals), rabbit polyclonal anti-ATF3 (sc-188; Santa Cruz Biotechnology), rabbit polyclonal anti-SOX9 (AB5535; Chemicon International), rabbit polyclonal anti-ITPR1 (ab5804; Abcam), rabbit polyclonal anti-LOXL2 (31), mouse monoclonal anti-SERPINE (sc-5297; Santa Cruz Biotechnology), and mouse monoclonal anti-PRKCA (sc-8393; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (DAKO) were used in conjunction with the ECL Plus system (Amersham Biosciences) to visualize immunoreactive bands.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of the Effect of DMOG with Those of Hypoxia on Gene Expression—The breast cancer cell line MCF7 was chosen for study as we have previously shown it to possess readily detectable levels of HIF-1{alpha}, HIF-2{alpha}, and all the HIF hydroxylase enzymes (22). To examine for the global transcriptional response to DMOG and to compare it with the effects of hypoxia, we undertook mRNA profiling of MCF7 cells exposed to hypoxia (1%), normoxia (21%), or to 2 mM DMOG using the Affymetrix U133A GeneChip array. The cells were exposed to precisely regulated oxygen tensions (1 and 21%) for 16 h.

A comparison of the normoxic and hypoxic exposures detected 246 transcripts that showed statistically significant up-regulation (FDR <0.05) (see Table 1 and supplemental Table 3), and 190 transcripts that showed statistically significant down-regulation (FDR <0.05) (see Table 2 and supplemental Table 4) in the hypoxia-treated cells when compared with the cells grown in normoxia. Furthermore, an additional 308 transcripts (a total of 554 transcripts; 2.5% of the total number of probe sets interrogated) exhibited greater than a 2-fold mean induction by hypoxia, whereas an additional 370 transcripts (a total of 560; 2.5% of the total number of probe sets interrogated) showed a greater than 2-fold mean repression in hypoxia but did not achieve statistical significance.


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  		TABLE 1
Transcripts induced by hypoxia in MCF7 cells

Mean fold changes in gene expression in MCF7 cells were assessed by Affymetrix microarray in response to hypoxia and DMOG compared with expression in cells cultured in parallel under normoxic conditions. The 20 gene transcripts that showed the greatest fold induction following hypoxic exposure are shown. The complete list of the 246 transcripts, which showed significant induction by hypoxia, can be found in supplemental Table 3. The fold changes in expression as a result of HIF-1{alpha} siRNA, HIF-2{alpha} siRNA, and HIF-1{alpha} + HIF-2{alpha} siRNA treatments are also shown; these fold changes are the expression in cells under hypoxic conditions following HIF-{alpha} siRNA transfection relative to the expression under hypoxic conditions with OF alone. A positive number indicates up-regulation by the indicated treatment, and a negative value indicates down-regulation.

 


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  		TABLE 2
Transcripts showing reduced level of expression in MCF7 cells under hypoxic conditions

Fold changes in gene expression in MCF7 cells were assessed by Affymetrix microarray in response to hypoxia and DMOG compared with expression in cells cultured in parallel under normoxic conditions. The 20 gene transcripts that showed the greatest reduction in expression following hypoxic exposure are shown. The complete list of the 190 transcripts, which showed significant repression by hypoxia, can be found in supplemental Table 4. The fold changes in expression as a result of HIF-1{alpha} siRNA, HIF-2{alpha} siRNA, and HIF-1{alpha} + HIF-2{alpha} siRNA treatments are also shown; these fold changes are the expression in cells under hypoxic conditions following HIF-{alpha} siRNA transfection relative to the expression under hypoxic conditions with OF alone. A positive number indicates up-regulation by the indicated treatment, and a negative value indicates down-regulation.

 
The difference in expression levels between normoxia and hypoxia ranged from an ~90-fold up-regulation to an ~10-fold down-regulation as determined from the GeneSpring GCRMA normalization algorithm. Many of the transcripts that were differentially expressed between the hypoxic and normoxic conditions have been reported previously to show hypoxic regulation, including genes encoding carbonic anhydrase IX, adrenomedullin, BNIP3, vascular endothelial growth factor, and the HIF prolyl hydroxylase PHD3 (1). However, we additionally detected substantial changes for gene transcripts that had not been reported previously as showing hypoxic regulation (see Table 3).


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  		TABLE 3
mRNA expression in MCF7 cells of a series of genes not previously reported to show hypoxic regulation assayed by real time PCR and by Affymetrix microarray

The mean fold regulation between hypoxic conditions or exposure to DMOG and normoxic conditions is shown. A positive number indicates up-regulation by the indicated condition, and a negative value indicates down-regulation.

 
By having established the changes in mRNA expression produced by hypoxia, we wished to examine the extent to which 2-OG-dependent dioxygenase inhibition was able to mimic the hypoxic response. The effects of DMOG on gene expression matched those of hypoxia both qualitatively and quantitatively to a very surprising extent (Fig. 2). This is also reflected in the sample clustering analysis (Fig. 3). A more focused analysis of the data showed that of the 246 transcripts that were significantly up-regulated in hypoxia, 190 transcripts showed a statistically significant (FDR <0.05) up-regulation with DMOG exposure. Furthermore, of the remaining 56 transcripts up-regulated by hypoxia, 42 transcripts showed a greater than 2-fold mean induction (but did not meet our stringent criteria for statistically significant up-regulation), and indeed all but one (PRKCBP1) showed some degree of induction by DMOG. Conversely, of the 266 transcripts that showed statistically significant induction by DMOG, 190 also showed significant induction by hypoxia. Of the remaining 76 genes induced by DMOG, a further 44 transcripts showed a greater than 2-fold mean induction, and all but one (CA8) showed some degree of induction by hypoxia.

For transcripts that were repressed in hypoxic conditions, there was somewhat less similarity to the effect of DMOG. Of the 191 transcripts that were significantly down-regulated by hypoxia, 69 transcripts were significantly down-regulated by DMOG. However, a further 39 transcripts showed a greater than 2-fold mean reduction in expression, and all but one of the hypoxically repressed transcripts showed some reduction in expression when exposed to DMOG. Of the 134 transcripts significantly down-regulated by DMOG, 69 showed significant down-regulation in hypoxia with a further 30 showing greater than 2-fold mean repression by hypoxia and all but 2 showing some reduction in expression.


Figure 2
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  		FIGURE 2.
Scatter plot of mean fold changes for each of the ~22,000 mRNA transcripts detected by Affymetrix microarray following exposure to 2 mM DMOG or hypoxia (1% oxygen) compared with normoxic levels. The data points are colored according to statistical significance (FDR, q < 0.05) (e.g. blue indicates transcripts that were significantly up-regulated by DMOG but not significantly affected by hypoxia exposure). The dashed lines indicate the position of a 2-fold difference between the fold change induced by hypoxia and the fold change induced by DMOG.

 
To verify the concordant regulation of these transcripts by DMOG and hypoxia, we sought independent validation by real time PCR assays. We selected nine transcripts that had not been reported previously to show hypoxic regulation with differing levels of induction by hypoxia (from 77-fold for FLJ10134 to 4-fold for PRRX1) and three transcripts that showed marked repression in hypoxia (RET, CXCL12, and NAPA) (see Table 3). Significant and substantial induction by hypoxia and DMOG was confirmed for eight of the nine induced transcripts when assessed by real time PCR assays, although for some transcripts the amplitude of regulation was less than had been indicated by the array (e.g. SOX9). Nevertheless, quantitative results were again concordant between the response to hypoxia and that to DMOG. Real time PCR assays for regulation of the repressed transcripts confirmed down-regulation by hypoxia and DMOG but did not achieve statistical significance for one transcript (NAPA).

Hypoxia-regulated, DMOG-independent Gene Regulation—To define pathways of hypoxic gene regulation that are independent of the HIF hydroxylases, we examined for transcripts that showed substantial regulation by hypoxic exposure but that were unaffected by DMOG exposure. As described above, of the 246 genes that showed significant induction by hypoxia, only 14 transcripts showed both nonsignificant regulation and less than 2-fold mean regulation. We chose to examine the regulation of three such transcripts (CYP1A1, CYP1B1, and PRKCBP1) that were significantly induced by hypoxia but were unaffected or minimally affected by DMOG treatment. As seen in Table 4, real time PCR analysis confirmed this pattern of regulation for all three of the transcripts studied. This suggests a pathway for hypoxic gene regulation that is independent of the HIF hydroxylases.


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  		TABLE 4
Transcripts showing significant induction in response to hypoxia but whose expression was unaffected by exposure to DMOG in MCF7 cells

Genes were selected from the Affymetrix data, which showed significant induction by hypoxia (FDR, q<0.05) but a fold change in expression of <1.4 when exposed to DMOG. The results of gene expression assays performed by real time PCR and in response to HIF siRNA are also shown. Affymetrix siRNA data were generated using the first set of siRNA sequences (replicates 1–3) and the real time data were generated using the second set of siRNA sequences (replicates 4-6). Hypoxia and DMOG results are given as fold changes compared to normoxic conditions. The fold changes in expression as a result of HIF-1{alpha} siRNA, HIF-2{alpha} siRNA, and HIF-1{alpha} + HIF-2{alpha} siRNA treatments are also shown; these fold changes are the expression in cells under hypoxic conditions following HIF-{alpha} siRNA transfection relative to the expression under hypoxic conditions with OF alone. A positive number indicates up-regulation by the indicated treatment, and a negative value indicates down-regulation. The differences in magnitude of CYP1A1 induction by hypoxia when assayed by the different techniques may reflect differential sensitivities of detection of the very low levels of normoxic expression of CYP1A1.

 
The Influence of HIF-1{alpha} and HIF-2{alpha} siRNA on Hypoxic Gene Regulation—Given the very high concordance of regulation between transcripts showing induction by hypoxia and by HIF hydroxylase inhibition, we wished to examine the extent to which HIF-1{alpha} and HIF-2{alpha} were responsible for such regulation. We undertook siRNA suppression of HIF-1{alpha}, HIF-2{alpha}, and combined HIF-1{alpha} and HIF-2{alpha} suppression under conditions of 1% hypoxia for 16 h. A further control sample of cells was transfected with oligofectamine only and grown under identical conditions. Protein extracts were prepared in parallel from identically treated cells and immunoblotted for HIF-1{alpha} and HIF-2{alpha}, confirming a very effective suppression of the target proteins (Fig. 1a).


Figure 3
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  		FIGURE 3.
Heat map plot of mean changes in mRNA expression in response to hypoxia, DMOG, or HIF-{alpha} siRNA. The left-hand panel represents the changes in expression for all the transcripts studied. Hypoxia and DMOG results are given as fold changes compared with normoxic conditions, and the HIF-{alpha} siRNA results are given as fold changes under hypoxic conditions relative to the oligofectamine control. Sample clustering according to Euclidean distance is shown by the dendrogram above the heat map. The right-hand panel is a magnified version of those transcripts that showed significant changes in expression following hypoxic exposure. Transcripts are ordered from top to bottom according to the fold change in hypoxia. Yellow indicates little change in expression; blue indicates an increase in expression, and red indicates a decrease in expression. The cyan-colored lines also indicate the magnitude of the fold change for a particular transcript, and the dotted line indicates no change in expression.

 
Three independent RNA samples for each condition were studied with Affymetrix arrays, and the expression for each siRNA treatment was compared with the oligofectamine control. To examine the extent to which each HIF-{alpha} isoform was responsible for the hypoxic response, we specifically analyzed the changes in expression of those 246 transcripts that had shown significant induction by hypoxia (supplemental Table 3). HIF-1{alpha} siRNA treatment resulted in significant (FDR <0.05) down-regulation of a substantial proportion (127 of 246) of the hypoxia-induced transcripts. In marked contrast, HIF-2{alpha} siRNA, despite showing an equivalent or greater knockdown by siRNA than HIF-1{alpha} (see Fig. 1a), was associated with a statistically significant reduction in the expression of only 5 of 246 transcripts under hypoxic conditions. Combining HIF-1{alpha} and HIF-2{alpha} siRNA treatments increased further the number of transcripts that showed significantly reduced induction by hypoxia to 141 and reduced the hypoxic expression of 167 transcripts by greater than 2-fold. Furthermore, many of the transcripts showed a greater reduction in hypoxic expression when exposed to both HIF-1{alpha} and HIF-2{alpha} siRNA than with either siRNA alone. These data can be seen in supplemental Tables 3 and 4 and are also displayed graphically in a sample clustering heat map (Fig. 3).

To examine the role of the HIF pathway in the regulation of the three genes for which we had seen induction by hypoxia but not DMOG, we also examined the effects of HIF-{alpha} siRNA on these transcripts. The hypoxic induction of PRKCBP1 and CYP1B1 was not significantly affected by HIF-1{alpha} and HIF-2{alpha} siRNA when assessed by the Affymetrix array and real time PCR (Table 4), although some influence of the second set of HIF-{alpha} siRNA sequences was seen on the hypoxic induction of CYP1A1 as determined by real time PCR.

Given this major effect of HIF-1{alpha} in controlling hypoxic gene regulation, we also wished to verify the existence of a smaller group of genes that were solely regulated by HIF-2{alpha}. Five genes (PRKCA, SERPINE, KIAA1199, AKAP12, and ITPR1) fulfilled the criteria of showing hypoxic expression that was unaffected by HIF-1{alpha} siRNA but were affected by HIF-2{alpha} siRNA (Table 5). To help exclude "off-target" effects of the siRNA sequences used, we undertook suppression of HIF-{alpha} expression with different siRNA sequences and determined expression independently with the Illumina BeadChip and real time PCR. For three of the transcripts (PRKCA, AKAP12, and ITPR1), we confirmed dependence on HIF-2{alpha} for hypoxic induction and independence of HIF-1{alpha}. For SERPINE, we found dependence on the presence of both HIF-1{alpha} and HIF-2{alpha}, although a greater influence of HIF-2{alpha} siRNA was seen (fold regulation with HIF-1{alpha} siRNA –1.8 and HIF-2{alpha} siRNA –3.6 when assayed by real time PCR). For KIAA1199, a more modest effect of HIF-2{alpha} siRNA suppression was seen with the second set of siRNA sequences (Table 5).


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  		TABLE 5
Transcripts showing induction by hypoxia and DMOG and dependence on the presence of HIF-2{alpha} in MCF7 cells

Gene expression was determined with the Affymetrix microarray, and transcripts selected, which showed significant induction by DMOG and hypoxia (FDR, q < 0.05), were not down-regulated by exposure to HIF-1{alpha} siRNA (the maximum down-regulation allowed was –1.1-fold) but showed significant reduction in expression under hypoxic conditions when exposed to HIF-2{alpha} siRNA (down-regulation more than –1.4-fold). The results of gene expression determined by the Illumina system and real time PCR in response to hypoxia, DMOG, and to a second set of HIF-targeted siRNAs (siRNA replicates 4-6) are also given. Hypoxia and DMOG results are given as fold changes compared with normoxic conditions. The fold changes in expression as a result of HIF-1{alpha} siRNA, HIF-2{alpha} siRNA, and HIF-1{alpha} + HIF-2{alpha} siRNA treatments are also shown; these fold changes are the expression in cells under hypoxic conditions following HIF-{alpha} siRNA transfection relative to the expression under hypoxic conditions with OF alone. A positive number indicates up-regulation by the indicated treatment, and a negative value indicates down-regulation.

 
HIF-independent, DMOG-regulated Gene Expression—To examine for HIF-independent mechanisms of hypoxic gene regulation that might be mediated via 2-OG-dependent hydroxylases, we sought transcripts that showed high levels of regulation by hypoxia and DMOG but whose expression was unaffected by the individual or combined HIF-{alpha} siRNA suppression. In selecting such genes for further analysis, we used the stringent criteria that they should show significant regulation by hypoxia and DMOG but hypoxic induction reduced by less than 1.1-fold by either or both HIF-{alpha} siRNA treatments. Seven genes induced by hypoxia fulfilled these criteria (PIM1, PHLDA1, GDF15, IGSF4, ATF3, MET, and ASPH) (see Table 6). We examined for a similar pattern of regulation utilizing the Illumina platform and different siRNA sequences. The regulation by hypoxia and DMOG but lack of effect of HIF{alpha} siRNA was confirmed for PIM1, PHLDA1, GDF15, and ASPH. ATF3 was not detected by the Illumina platform but did show a similar pattern of regulation at the protein level, whereas the hypoxic induction of IGSF4 was reduced by the different HIF-1{alpha} siRNA sequence when assayed by the Illumina platform. The expression of two transcripts (PIM1 and MET) was additionally validated by independent examination with real time PCR following treatment with the different HIF-targeted siRNAs. PIM1 again showed induction by hypoxia and DMOG and hypoxic induction that was unaffected by either or both HIF siRNAs. Surprisingly, with real time PCR and the Illumina platform, we were unable to demonstrate hypoxic induction of MET, perhaps reflecting exon-specific regulation within this gene.


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  		TABLE 6
Transcripts selected from the Affymetrix microarray expression data, which showed significant up-regulation in response to hypoxia and DMOG but whose levels were unaffected by HIF-{alpha} siRNA under hypoxic conditions

The gene transcripts were selected using the criteria that there was statistically significant induction by both hypoxia and DMOG (FDR, q < 0.05) and that all HIF-{alpha} siRNA treatments (HIF-1{alpha} siRNA, HIF-2{alpha} siRNA, and HIF-1{alpha} + HIF-2{alpha} siRNA) were not down-regulated by greater than –1.1-fold. The results of gene expression determined by the Illumina system and real time PCR in response to hypoxia, DMOG, and to a second set of HIF-targeted siRNAs (siRNA replicates 4-6) are also given. Hypoxia and DMOG results are given as fold changes compared with normoxic conditions. The fold changes in expression as a result of HIF-1{alpha} siRNA, HIF-2{alpha} siRNA, and HIF-1{alpha} + HIF-2{alpha} siRNA treatments are also shown; these fold changes are the expression in cells under hypoxic conditions following HIF-{alpha} siRNA transfection relative to the expression under hypoxic conditions with OF alone. A positive number indicates up-regulation by the indicated treatment, and a negative value indicates down-regulation.

 
Gene Expression Changes in Hep3B Cells—To examine to what extent these patterns of regulation were operative in a different cell type, we undertook reverse transcription-PCR assays of mRNA expression in Hep3B cells of 22 genes that were representative of the different pathways of regulation that we had observed in the breast cancer cell line, MCF7. Of the 19 genes assayed that were newly recognized to show hypoxic induction of mRNA expression in MCF7 cells, 13 also showed significant and often substantial induction in the hepatoblastoma cell line Hep3B (see supplemental Table 5). Of the three genes assayed that had shown suppression of mRNA levels in hypoxia, two were also repressed in Hep3B cells, although one was not detected. The three genes that had shown induction by hypoxia but not DMOG in MCF7 cells were not induced by hypoxia in Hep3B cells (see supplemental Table 5). Of the genes that had shown a greater dependence on HIF-2{alpha} for hypoxic regulation in MCF7 cells, AKAP12 again showed a much greater dependence on HIF-2{alpha} in Hep3B cells, whereas the hypoxic induction of SERPINE was reduced by both HIF-1{alpha}- and HIF-2{alpha}-targeted siRNAs. GDF15 did show HIF-independent induction by hypoxia and induction by DMOG in the Hep3B cells, suggesting a more widespread operation of such a HIF-independent pathway (see supplemental Table 6).

Protein Abundance Assays in MCF7 Cells—We examined the response in MCF7 cells of proteins encoded by a selection of genes in response to hypoxia, DMOG, and HIF siRNAs. Several genes previously shown to be induced by hypoxia in a HIF-dependent manner (CAIX, BNIP3, PHD2, and PHD3) were also examined and found to be regulated in a concordant manner at the protein level (see Fig. 1c). Several other proteins were also regulated by hypoxia and DMOG in a HIF-1{alpha}-dependent manner (e.g. LOXL2 and SOX9). In addition, a gene that was regulated by hypoxia in a HIF-independent manner and to an even greater extent by DMOG (ATF3) showed a similar pattern of regulation of protein levels to the mRNA regulation. A gene that appears to show a greater dependence on HIF-2{alpha} for hypoxic induction (ITPR1) showed a similar pattern of regulation at the protein level. However, for several genes (e.g. PRKCA) we did not observe hypoxic regulation of protein abundance (see Fig. 1c).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Global alterations in gene expression, surveyed in this study among 22,000 human transcripts, revealed very strong concordance between responses of cells exposed to hypoxia and to the 2-OG analogue DMOG. Striking similarities were observed both in the direction and magnitude of the response, which extended across several hundred genes and included both those up-regulated and those down-regulated by the stimuli. Given other recent reports, these results were somewhat unexpected. For instance, substantial differences have been reported recently between genes induced by hypoxia and genes induced by exposure of cells to desferrioxamine, the transitional element ions cobalt (II) and nickel(II) (32), all of which inhibit 2-OG-dependent dioxygenases, and presumably induce hypoxia-regulated genes by this mechanism. The much greater similarity between responses to hypoxia and DMOG observed in the current study most probably reflects more specific and more complete inhibition of 2-OG-dependent dioxygenases than that achieved by desferrioxamine and metal ions. Overall our findings would suggest that the large majority of changes in gene expression observed following hypoxic exposure at this duration and severity is because of interference with 2-OG-dependent dioxygenase functions that affect gene expression pathways directly or indirectly. It must be noted, however, that under other hypoxic conditions results might be different. For instance, exposure of MCF-7 cells to 1% oxygen for 16 h does not result in substantial cell damage, and activation of cellular responses to injury might be anticipated to induce different patterns of gene expression.

This is one of the most extensive surveys of genes induced by hypoxia to date, and in keeping with this we observed hypoxic regulation of a large number of genes not reported previously to manifest this property. The use of multiple repetitions, and stringent criteria for assessment of differential expression, provided a high level of accuracy. Independent validation of differential expression, with both real time PCR assays and Illumina BeadChip assays, indicated that for the majority of transcripts array results were qualitatively and quantitatively robust. Newly recognized hypoxia and DMOG-inducible genes included examples with diverse roles in sex determination (SOX9) (33), collagen cross-linking (LOXL2) (34), Wnt signaling (WISP2) (35), antigen presentation (HLA DRB3), oncogenes (FOS), and responses to estrogen (E2IG5) (36), extending the known functions of the hypoxia pathways. Other genes identified in the arrays as showing substantial regulation by hypoxia and DMOG have been identified previously as HIF target genes and included functional groups whose protein products have roles in glycolysis (e.g. enolase, aldolase c, and phosphoglycerate kinase), in angiogenesis (e.g. vascular endothelial growth factor), in apoptosis (e.g. BNIP3), in the regulation of the HIF pathway itself (e.g. CITED2 and PHD3), and other 2-oxoglutarate-dependent dioxygenases (e.g. collagen prolyl 4-hydroxylase) (for review see Ref. 1).

Although DMOG may act relatively specifically on the family of 2-OG-dependent dioxygenases, it is likely to inhibit many other members of this family of enzymes. Thus, in addition to inhibiting the HIF prolyl hydroxylases (PHD1, -2, and -3) and the HIF asparaginyl hydroxylase (FIH), DMOG inhibits procollagen prolyl hydroxylases (18) and most probably other members of this family that have as yet unknown functions (37, 38). Thus the concordance between responses to hypoxia and DMOG may reflect inhibition of a number of different 2-OG-dependent dioxygenases by both these conditions. To determine the role of HIF hydroxylase pathways in the observed effects of hypoxia and DMOG exposure, we used siRNA to examine the effect of near complete knock-down of HIF-1{alpha}, HIF-2{alpha}, and both HIF-1{alpha} and HIF-2{alpha}. Suppression of both HIF-1{alpha} and HIF-2{alpha} greatly reduced the responses of a large number of genes that were induced by hypoxia and DMOG, affirming the importance of the HIF system as the central mediator of transcriptional response to hypoxia. Nevertheless, a number of genes were identified that were strongly induced by hypoxia and DMOG but unaffected by suppression of both HIF-1{alpha} and HIF-2{alpha}. These genes include examples encoding proteins with diverse functions that include a serine-threonine protein kinase oncogene (PIM1) (39), an aspartate beta-hydroxylase (ASPH) (40), and a growth differentiation factor (GDF15) (41). Interestingly, GDF15 (also known as MIC-1, PLAB, and NAG-1) has been identified previously as a gene induced by anoxia in a HIF-independent manner (42), and we also found it to be regulated in a HIF-independent manner in Hep3B cells. Our findings were validated using real time PCR and independent siRNAs directed against HIF-1{alpha} and HIF-2{alpha} transcripts, suggesting that the genes respond to novel oxygen-sensitive pathways controlled by 2-OG-dependent dioxygenases. Although a role for HIF-3{alpha} is not excluded, given the proposed role of HIF-3{alpha} in opposing HIF signaling (43, 44), it appears more likely that the responses are mediated by the action of the HIF hydroxylases on non-HIF targets or by the action of other 2-OG-dependent oxygenases. Interestingly, among the hypoxia- and DMOG-inducible genes that were suppressed by the combination of HIF-1{alpha}- and HIF-2{alpha}-directed siRNAs, a range of responses was observed from modest levels of suppression to essentially total abrogation. This variation may reflect requirements for different levels of HIF-{alpha} in the transactivation of different target genes, or may indicate that a proportion of HIF target genes are subject to additional 2-OG-dependent dioxygenase-dependent but HIF-independent controls on transcript abundance such as by the regulation of mRNA stability.

Comparison of responses to siRNA-directed suppression of HIF-1{alpha} versus HIF-2{alpha} indicated that the majority of genes induced by hypoxia and DMOG was strikingly more dependent on HIF-1{alpha} than HIF-2{alpha}. The results are in keeping with previous studies that have reported strong dependence on expression of the HIF-1{alpha} isoform among a limited range of HIF target genes (12, 21, 45, 46). For instance, the current results for genes encoding glycolytic enzymes, and carbonic anhydrase IX, were consistent with previous data demonstrating specific responsiveness to HIF-1{alpha} and not HIF-2{alpha} in other cell types (47, 48). The new data indicate that absolute or relative dependence on HIF-1{alpha} as opposed to HIF-2{alpha} is also observed for many newly identified hypoxia-inducible HIF target genes and indeed extends across the large majority of hypoxia-inducible transcripts. This result is in apparent contrast with those of a recent report of the effects of inducible expression of either HIF-1{alpha} or HIF-2{alpha} in normoxic cells in which a substantial number of genes were induced by HIF-2{alpha} overexpression (49). Interestingly, the authors commented that most of the genes induced by HIF-2{alpha} overexpression were not themselves responsive to hypoxia (49). Taken together with the current results, this suggests that despite the considerable potential of HIF-2{alpha} overexpression to induce gene expression, it contributes relatively little to the overall transcriptional response to hypoxia.

Nevertheless among those genes targeted primarily by HIF-1{alpha}, a spectrum of responses was observed from groups of genes that appeared completely dependent on HIF-1{alpha}, and unaffected by HIF-2{alpha}, to others for which some suppression by HIF-2{alpha}-directed siRNA was observed, and suppression by HIF-2{alpha} plus HIF-1{alpha}-directed siRNA was clearly greater than with HIF-1{alpha}-directed siRNA. A much smaller number of genes were solely responsive to HIF-2{alpha} but included ITPR1, PRKCA (the target of tumor promoting phorbol esters (50)), and AKAP12, which are linked in intracellular calcium signaling pathways (AKAP12 is involved in the localization of PRKCA) (51), although ITPR1 is a substrate of PRKCA (52) and can affect tumor growth and apoptosis. HIF-2{alpha} dependence was further confirmed at the mRNA level for AKAP12 in Hep3B cells.

Interestingly several recent studies of the HIF pathway have suggested that (at least in certain settings) HIF-2{alpha} is more strongly pro-tumorigenic than HIF-1{alpha}. For example, a recent study of mouse ES cell teratoma xenografts demonstrated enhanced growth of cells bearing a HIF-2{alpha} knock-in allele at the HIF-1{alpha} locus (53). Studies of retrovirally mediated overexpression or siRNA-based suppression of HIF-2{alpha} have indicated a positive role for HIF-2{alpha} activation that is not shared by HIF-1{alpha} in promoting experimental renal carcinoma growth (47, 54–56). Thus, further analysis of pathways that are specifically regulated by HIF-2{alpha} may be illuminating in understanding these effects. Although mechanistic links to the current findings are unclear, it is interesting that a hyperphosphorylated form of atypical protein kinase C has been identified (in addition to HIF-{alpha}) as a target of the VHL tumor suppressor (57, 58).

Although the results utilizing HIF hydroxylase inhibition and HIF-{alpha} siRNA indicated a dominant role for the HIF system in hypoxic gene regulation, we were also able to define mechanisms of hypoxic gene regulation that were independent of the HIF hydroxylases. We found several genes whose regulation by hypoxia was not mimicked by DMOG, and we confirmed the pattern of regulation by real time PCR. The significant induction by hypoxia of CYP1A1, CYP1B1, and PRKCBP1 but the lack of effect of DMOG suggests a HIF hydroxylase-independent pathway of oxygen sensing underlies their regulation in MCF7 cells, although this pattern of regulation was not preserved for these genes in Hep3B cells.

An examination of the regulation of protein levels encoded by these genes revealed that similar patterns of regulation were operative on a protein level with examples of genes and their protein products that were regulated by hypoxia and DMOG in a HIF-1{alpha}-dependent manner (e.g. LOXL2 and SOX9), by hypoxia and DMOG in a HIF-independent manner (e.g. ATF3), and an example showing greater dependence on HIF-2{alpha} for hypoxic induction (ITPR1). However, for several genes we did not observe hypoxic regulation at the protein level (e.g. PRKCA) or observed slightly different patterns of regulation (e.g. SERPINE). This likely reflects differences in the time course of regulation of mRNA and protein levels and the operation of other regulatory mechanisms such as the well described hypoxic suppression of translation (59).

However, overall the study underlines the importance of HIF hydroxylase, HIF-1{alpha}-mediated pathways in directing the global transcriptional response to hypoxia. The definition of HIF-2{alpha}-specific pathways and identification of genes responding to hypoxia- and 2-OG-dependent dioxygenase inhibition in a HIF-independent manner should provide new entry points into mechanistically and physiologically distinct hypoxia pathways.


   FOOTNOTES
 
* This work was supported by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1–6.

The microarray data can be accessed through Gene Expression Repository under GEO accession number GSE3188 [NCBI GEO] . Back

1 To whom correspondence may be addressed. Tel.: 44-1865-287526; Fax: 44-1865-287501; E-mail: ioannisr{at}well.ox.ac.uk. 2 To whom correspondence may be addressed. Tel.: 44-1865-287788; Fax: 44-1865-287787; E-mail: jgleadle{at}well.ox.ac.uk.

3 The abbreviations used are: HIF, hypoxia-inducible factor; VHL, von Hippel-Lindau; PHD, prolyl hydroxylase domain; DMOG, dimethyloxalylglycine; siRNA, short interfering RNA; OF, oligofectamine; FDR, false discovery rate; 2-OG, 2-oxoglutarate. Back


   ACKNOWLEDGMENTS
 
We thank Christopher Pugh for helpful discussions and other contributions, Christopher Schofield for the gift of dimethyloxalylglycine, Katalin Csiszar for the gift of the anti-LOXL2 antibody, and Michael Wiesener for the gift of the HIF-{alpha} siRNA oligonucleotides described in Ref. 23.



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