By performing cDNA microarray analyses of hypoxic hepa1c1c7 cells and BpRc1 cells, we identified both hypoxia-regulated genes and their Arnt dependence.. From the 259 genes, we selected h
Trang 1in an aryl hydrocarbon receptor nuclear translocator
(Arnt)-dependent manner
Su Mi Choi*, Hookeun Oh* and Hyunsung Park
Department of Life Science, University of Seoul, South Korea
Keywords
Arnt; gene expression; HIF; hypoxia;
microarray
Correspondence
H Park, Department of Life Science,
University of Seoul, 90 Jeonnong-dong,
Dongdaemun-gu, Seoul 130-743, South
Korea
Fax: +82 2 2210 2888
Tel: +82 2 2210 2622
E-mail: hspark@uos.ac.kr
*These authors made equal contributions to
this study
(Received 17 July 2008, revised
12 September 2008, accepted 17
September 2008)
doi:10.1111/j.1742-4658.2008.06686.x
We investigated hypoxia-inducible factor (HIF)-dependent changes in the expression of 5592 genes in response to hypoxia (0.1% O2, 16 h) by per-forming cDNA microarray analyses of mouse hepa1c1c7 and BpRc1 cells BpRc1 cells are a hepa1c1c7 variant defective in HIF-b⁄ aryl hydrocarbon receptor nuclear translocator (Arnt), and are therefore unable to induce HIF target genes in response to hypoxia By comparing hepa1c1c7 cells with BpRc1 cells, we were able to investigate hypoxia-regulated gene expression as well as the role played by HIF in regulating the hypoxic-dependent response of gene expression This study identified 50 hypoxia-induced genes and 36 hypoxia-repressed genes Quantitative PCR analysis
of nine genes confirmed our ability to accurately analyze changes in hypoxia-induced gene expression by microarray analysis By comparing quantitative PCR analyses of these nine genes in BpRc1 and hepa1c1c7 cells, we determined that eight of the nine hypoxia-induced genes are Arnt dependent Additional quantitative PCR analyses of eight hypoxia-repressed genes confirmed, with a 50% probability, that microarray analy-sis was able to predict hypoxia-repressed gene expression Only two of the four confirmed genes were found to be repressed in an Arnt-dependent manner Collectively, six of these 13 genes (46.2% probability) showed a pattern of expression consistent with the microarray analysis with regard to Arnt dependence Finally, we investigated the HIF-1a dependence of these
13 genes by quantitative PCR analysis in HIF-1a knockdown 3T3-L1 cells These analyses identified novel hypoxia-regulated genes and confirmed the role of Arnt and HIF-1a in regulating their expression These results identify additional HIF target genes and provide a more complete understanding of hypoxia signaling
Abbreviations
ABCC3, ATP-binding cassette, subfamily C (CFTR ⁄ MRP), member 3; Arnt, aryl hydrocarbon receptor (AhR) nuclear translocator; ATF-4, activating transcription factor-4; bHLH, basic helix–loop–helix; BNIP3, BCL-2 ⁄ adenovirus E1B 19 kDa-interacting protein 3; BSG, basigin; CCGN2, cyclin G2; DUSP12, dual specificity phosphatase 12; eIF1, eukaryotic translation initiation factor 1; ER, endoplasmic reticulum; FDR, false discovery rate; FKBP4, FK506 binding protein 4 (59 kDa); GNA11, guanine nucleotide binding protein, a 11; HIF, hypoxia-inducible factor; HSP60, heat shock protein, 60 kDa; IER3, immediate early response 3; MAD2L1, MAD2 (mitotic arrest deficient, homolog)-like 1 (yeast); MAPK, mitogen-activated protein kinase; MKP-1, mitogen-activated protein kinase phosphatase-1; MMP, matrix metalloproteinase; NDR1, N-myc downstream regulated 1; P4HA1, procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), a1 polypeptide; PAS, Per-Arnt-Sim; PERK, PKR-like ER kinase; PPAR, peroxisome proliferators-activated receptor; PSMA3, proteasome (prosome,
macropain) subunit, a type 3; PTPN16, protein tyrosine phosphatase, non-receptor type 16; SFRS3, splicing factor, arginine ⁄ serine-rich 3 (SRp20); shRNA, short hairpin RNA; SUI1-RS1, suppressor of initiator codon mutations, related sequence 1; VEGF, vascular endothelial growth factor.
Trang 2Cellular oxygen is an important regulatory stimulus
for many physiological and pathological processes
Mammalian cells adapt to hypoxia by inducing the
expression of genes involved in anaerobic metabolism,
oxygen delivery and cell survival These diverse target
genes are induced by a common heterodimeric
transcription factor: hypoxia-inducible factor-a⁄ b
(HIF-a⁄ b) [1–4] The HIF-a and HIF-b subunits belong
to the basic helix–loop–helix (bHLH)-Per-Arnt-Sim
(PAS) protein family HIF-a is rapidly degraded in
normoxic cells, whereas HIF-b, also known as Arnt
(aryl hydrocarbon receptor nuclear translocator), is
con-stitutively expressed Under hypoxic conditions, HIF-a
is stabilized and translocates into the nucleus, where it
forms a heterodimer with the nuclear protein Arnt
Structural analyses of bHLH-PAS proteins have
deter-mined that interaction between the HLH-PAS domains
of each subunit mediates dimerization between HIF-a
and HIF-b, and that individual basic regions from
each protein interact with their corresponding DNA
elements Therefore, dimerization of bHLH-PAS
proteins is required for DNA binding [5]
The stability and activity of the a subunit are
inhib-ited by post-translational modification, specifically by
hydroxylation HIF-a hydroxylation is catalyzed by
HIF-a-specific proly-4-hydroxylase 2 and
HIF-a-spe-cific asparaginyl-hydroxylase, which utilize molecular
oxygen and a-ketoglutarate as cosubstrates The
hydroxylated proline residues (human HIF-1a Pro402
and Pro564) are recognized by the E3 ubiquitin ligase,
a von Hippel–Lindau protein which mediates HIF-1a
polyubiquitination and degradation by the 26S
protea-some [6,7] The hydroxylation of the human HIF-1a
asparagine residue 803 prevents HIF-a from recruiting
the CBP⁄ p300 coactivator A lack of oxygen has been
shown to reduce the activities of these two
oxygen-dependent hydroxylases, resulting in the stabilization
of the transactive form of HIF-1a [8,9]
HIF-1a was the first HIF-a isoform identified by
affinity purification, and HIF-2a (endothelial PAS
domain-containing protein 1) was later identified
through an homology search [10] Both HIF-1a and
HIF-2a form functional heterodimers with Arnt
Although knockout mice experiments have shown that
HIF-1a and HIF-2a have unique functions and are
non-redundant [11], no HIF-2a-specific target genes have
been identified HIF-1a and HIF-2a share a number of
target genes, but HIF-1a appears to be the predominant
form responsible for the induction of target genes [12]
Arnt was originally identified as a partner protein of
aryl hydrocarbon receptor (AhR) Similar to Arnt,
AhR also contains a bHLH-PAS domain at its
N-ter-minal domain Dioxin, an environmental pollutant, is
the most potent ligand for AhR Once bound to ligand, cytosolic AhR translocates into the nucleus and forms a heterodimer with Arnt Therefore, Arnt is a binding partner for both HIF-a and AhR [13,14] Pre-vious studies by Miller and Whitlock [15] led to the isolation of variant mouse hepa1c1c7 cell lines that lose responsiveness to dioxin using benzo(a)pyrene selection and fluorescence-activated cell sorting One of the variant cell lines, BpRc1, has normal AhR, but is defective in the nuclear localization of AhR Arnt transfection can complement this defect in BpRc1 cells, indicating that these variant cells are defective in Arnt [16,17] As Arnt is also required for the hypoxic induc-tion of HIF target genes, BpRc1 cells are also unre-sponsive to hypoxia, even in the presence of HIF-a Several studies have shown the role of Arnt in the basal expression of genes [18–20] Here, we emphasize the role of Arnt, especially in the hypoxic responses of gene expression By performing cDNA microarray analyses of hypoxic hepa1c1c7 cells and BpRc1 cells,
we identified both hypoxia-regulated genes and their Arnt dependence In addition, using HIF-1a knock-down cells, we investigated whether HIF-1a is required for the hypoxic responses of the identified genes
Results
Microarray analyses of hypoxia-regulated gene expression
We analyzed the changes in the expression of 5592 genes in response to hypoxic exposure (0.1% O2, 16 h) using Mouse 6K cDNA chips (TwinChip Mouse-6K) from Digital Genomics Inc (Seoul, South Korea) Mouse hepa1c1c7 and BpRc1 cells were exposed to hypoxia or normoxia (20% O2) for 16 h prior to RNA isolation and subsequent cDNA microarray analysis Four replicates were performed for each cell type using twin chips that incorporated dye-reversed hybridiza-tion Comparison of hepa1c1c7 and BpRc1 cells enabled us to investigate hypoxia-regulated gene expression, as well as the role played by HIF in the regulation of the hypoxic response Based on our anal-yses of 5592 genes, we selected statistically relevant genes with q values less than 0.1 for further analysis;
420 and 565 genes were selected from the analyses of hepa1c1c7 and BpRc1 cells, respectively Of these genes, 259 demonstrated q values of less than 0.1 in both analyses From the 259 genes, we selected hypoxia-induced genes that demonstrated a greater than 1.5-fold induction; 50 and 40 genes were selected from the analyses of hepa1c1c7 and BpRc1 cells, respectively (Tables 1 and S1) In addition, we selected
Trang 3Table 1 Hypoxia-induced genes identified by microarray analyses.
GenBank
accession
number
Gene
symbol Gene name
WT cells BpRc1 cells
Fold a q value b Fold a q value b Genes induced by hypoxia in both wild-type and BpRc1 cells
AI956848 BNIP3 c BCL2 ⁄ adenovirus E1B 19 kDa-interacting protein 3, NIP3 8.523 0.005 8.956 0.003 AI325917 PTPN16 c Protein tyrosine phosphatase, non-receptor type 16 6.409 0.005 2.264 0.003
AI323719 SUI1-RS1 c Suppressor of initiator codon mutations, related sequence 1
(Saccharomyces cerevisiae)
3.628 0.005 1.695 0.003
AI413228 – Mus musculus, clone MGC:18904 IMAGE:4240711, mRNA,
complete cds
2.168 0.005 1.637 0.003
AI504706 – ESTs, weakly similar to hair mouse hairless protein
(M musculus)
2.022 0.005 1.845 0.003
AI415729 – ESTs, moderately similar to sylm_human probable leucyl-tRNA
synthetase, mitochondrial precursor (Homo sapiens)
1.846 0.007 1.608 0.003 AW321053 AIRAP Arsenite inducible RNA-associated protein (Airap) 1.682 0.013 2.602 0.003
Genes induced by hypoxia in wild-type cells
AI323613 INPP5D Inositol polyphosphate-5-phosphatase, 145 kDa 3.271 0.005 1.357 0.072
AI323453 P4HA1 c Procollagen-proline, 2-oxoglutarate 4-dioxygenase
(proline 4-hydroxylase), a1 polypeptide
2.143 0.005 1.373 0.005
AI848411 BTG1 B-cell translocation gene 1, anti-proliferative 2.056 0.005 1.216 0.072 AI451895 RPGRIP1 Retinitis pigmentosa GTPase regulator interacting protein 1 1.838 0.005 1.269 0.051 AI452157 – ESTs, weakly similar to the KIAA0146 gene product is novel
(H sapiens)
1.837 0.005 1.427 0.005
AI842276 B3GALT2 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 2 1.666 0.005 1.323 0.016
AI452202 – DNA segment, Chr 12, Wayne State University 95, expressed 1.577 0.005 1.273 0.012
Trang 4genes that were repressed in response to hypoxia and
that demonstrated a less than 0.6-fold induction; 36
and 40 genes were selected from the analysis of
hepa1c1c7 and BpRc1 cells, respectively (Tables S1
and 3) Compared with northern analyses or
quantita-tive real-time reverse transcription-polymerase chain
reaction (Q-PCR), we found that the cDNA
micro-array analyses underestimated the fold change of gene
expression In order to take more genes into
consider-ation, we arbitrarily chose the values of 1.5- and
0.6-fold induction
Genes that are induced by hypoxia
The 50 genes that demonstrated a greater than
1.5-fold induction in hepa1c1c7 cells were considered to
be induced by hypoxia (Table 1) Of these 50 genes,
26 demonstrated a less than 1.5-fold induction in
BpRc1 cells, suggesting that these genes were induced
by hypoxia in an Arnt-dependent manner The
remaining 24 genes demonstrated a greater than
1.5-fold induction in both cell lines, suggesting that their
expression was regulated independent of Arnt To confirm these findings, nine of the 50 up-regulated genes were selected for Q-PCR analysis The fold induction of each gene in both hepa1c1c7 and BpRc1 cells was analyzed by one-way analysis of variance (ANOVA) (Table 2) Q-PCR analysis confirmed that each of the nine genes [suppressor of initiator codon mutations, related sequence 1 (SUI1-RS1), protein tyrosine phosphatase, non-receptor type 16 (PTPN16), N-myc downstream regulated 1 (NDR1), cyclin G2 (CCNG2), vascular endothelial growth factor (VEGF), BCL-2⁄ adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), basigin (BSG), immediate early response 3 (IER3) and procollagen-proline, 2-oxoglut-arate 4-dioxygenase (proline 4-hydroxylase), a1 poly-peptide (P4HA1)] was induced by hypoxia with the indicated fold and P value (P < 0.05) in hepa1c1c7 cells (Fig 1 and Table 2) In contrast, Q-PCR analy-ses demonstrated that, in BpRc1 cells, the induction folds of eight genes (PTPN16, NDR1, CCNG2, VEGF, BNIP3, BSG, IER3 and P4HA1) were signifi-cantly lower, and their P values were greater than
Table 2 Hypoxia-induced gene expression analyzed by both microarray and Q-PCR up, hypoxia-induced expression pattern, fold ‡ 1.5; down, hypoxia-repressed expression pattern, fold < 0.6; nc, no change, 0.6 £ fold < 1.5; ns, not statistically significant, P value > 0.05; ), Arnt independent; +, Arnt dependent.
Gene
symbol
Arnt dependence
Arnt dependence
a Fold: a ratio of expression in hypoxia⁄ expression in normoxia b q value: significant difference, false discovery rate (FDR), selected q value < 0.1.
Table 1 Continued.
GenBank
accession
number
Gene
Fold a q value b Fold a q value b
AI324697 SNRPA Small nuclear ribonucleoprotein polypeptide A 1.519 0.025 1.449 0.005
a Fold: a ratio of expression in hypoxia⁄ expression in normoxia b q value: significant difference, false discovery rate (FDR), selected q value < 0.1 c Bold characters indicate genes that were further investigated by Q-PCR as reported in Fig 1.
Trang 50.05, suggesting that hypoxia induced these eight
genes in an Arnt-dependent manner (Table 2)
Fur-thermore, Q-PCR analysis confirmed that SUI1-RS1
expression was significantly induced in both
hepa1c1c7 and BpRc1 cells, suggesting that expression
of SUI1-RS1 was regulated in an Arnt-independent
manner in response to hypoxia
Based on these Q-PCR results, we were able to con-firm the validity of our microarray analysis of hypoxia-induced gene expression Microarray analysis demonstrated that six genes (SUI1-RS1, PTPN16, NDR1, CCNG2, VEGF and BNIP3) were induced by hypoxia in both wild-type and Arnt-defective cells, and that an additional three genes (BSG, IER3 and
A
B
Hypoxia – + – +
Cell WT BpRc1
5
4
3
2
1
0
BSG mRNA
Hypoxia – + – + Cell WT BpRc1
120
100
80
60
40
20
0
PTPN16 mRNA
Hypoxia – + – + Cell WT BpRc1
25
20
15
10
5
0
VEGF mRNA
+ Hypoxia – + – Hypoxia – + – Cell WT BpRc1
25
20
15
10
5
0
P4HA1 mRNA
Cell
Hypoxia – + – +
Cell WT BpRc1
6
5
4
3
2
1
0
SUI1 -RS1 mRNA
Hypoxia – + – + Cell WT BpRc1
80
60
40
20
0
NDR1 mRNA
20
15
10
5
0
CCNG2 mRNA
Hypoxia – + – +
Cell WT BpRc1
Hypoxia – + – + Cell WT BpRc1
20
15
10
5
0
BNIP3 mRNA
Hypoxia Cell
– + – +
WT BpRc1
18
15
12
9
6
3
0
IER3 mRNA
Fig 1 mRNA levels of hypoxia-induced genes analyzed by Q-PCR (A, B) Wild-type (WT) hepa1c1c7 cells and BpRc1 cells were incubated
in hypoxic conditions for 16 h Total RNA was isolated and quantified by Q-PCR 18S rRNA expression levels were used for normalization Values are presented as the average ± standard deviation of three independent experiments Statistical analysis of the Q-PCR data was evaluated using one-way ANOVA.
Trang 6P4HA1) were not induced in Arnt-deficient cells
How-ever, Q-PCR analysis indicated that, with the
excep-tion of SUI1-RS1, eight of the nine genes were found
to be induced by hypoxia in an Arnt-dependent
man-ner Therefore, only four of these genes (SUI1-RS1,
BSG, IER3 and P4HA1) showed a consistent
expres-sion pattern in both Q-PCR and microarray analyses,
indicating that our microarray analysis was able to
predict Arnt-dependent expression of each gene in
response to hypoxia with a 44.5% (4⁄ 9) probability
Genes that are repressed by hypoxia The 36 genes that demonstrated a fold induction of less than 0.6 were considered to be repressed by hypoxia (Table 3) Of these, nine demonstrated a fold induction between 0.6 and 1.5 in BpRc1 cells, suggest-ing that they were repressed in an Arnt-dependent manner An additional 27 genes that demonstrated a fold induction of less than 0.6 in both cell lines were believed to be repressed in an Arnt-independent
Table 3 Hypoxia-repressed genes identified by microarray analyses.
GenBank
accession
number
Gene
symbol Gene name
Fold a q value b Fold a q value b Genes repressed by hypoxia in wild-type cells
AI324252 MAD2L1 c MAD2 (mitotic arrest deficient, homolog)-like 1 (yeast) 0.593 0.010 0.710 0.003
AI843948 PSMA3c Proteasome (prosome, macropain) subunit, a type 3 0.537 0.014 0.744 0.012 AI528616 SFRS3 c Splicing factor, arginine ⁄ serine-rich 3 (SRp20) 0.532 0.058 0.670 0.003
Genes repressed by hypoxia in both wild-type and BpRc1 cells
AI504950 ABCC3 c ATP-binding cassette, sub-family C (CFTR ⁄ MRP), member 3 0.599 0.029 0.426 0.003
AI853888 HIRIP5 Histone cell cycle regulation defective interacting protein 5 0.577 0.005 0.590 0.003
AI507479 – ESTs, moderately similar to Z277_human zinc finger
protein 277 (Homo sapiens)
AI853883 SHD src homology 2 domain-containing transforming protein D 0.484 0.005 0.471 0.003
AI504558 – Mus musculus, similar to hypothetical protein FLJ20174,
clone IMAGE:3595651, mRNA, partial cds
a Fold: a ratio of expression in hypoxia⁄ expression in normoxia b
q value: significant difference, false discovery rate (FDR), selected q value < 0.1 c Bold characters indicate genes that were further investigated by Q-PCR as reported in Fig 2.
Trang 7manner in response to hypoxia To validate these
results, eight of the 36 genes [MAD2 (mitotic arrest
deficient, homolog)-like 1 (yeast) (MAD2L1), heat
shock protein, 60 kDa (HSP60), FK506 binding
pro-tein 4 (59 kDa) (FKBP4), proteasome (prosome,
macropain) subunit, a type 3 (PSMA3), guanine
nucle-otide binding protein, a 11 (GNA11), splicing factor,
arginine⁄ serine-rich 3 (SRp20) (SFRS3), dual
specific-ity phosphatase 12 (DUSP12) and ATP-binding
cas-sette, subfamily C (CFTR⁄ MRP), member 3 (ABCC3)] were selected for Q-PCR (Fig 2) Our analysis deter-mined that four of the eight genes (MAD2L1, HSP60, FKBP4 and PSMA3) were significantly repressed in response to hypoxia as measured by Q-PCR analysis (P < 0.05) in hepa1c1c7 cells (Fig 2 and Table 4) Based on these results, we were able to demonstrate that our microarray analysis was able to predict hypoxia-repressed gene expression with a 50%
proba-A
B
2.0
1.5
1.0
0.5
0
DUSP12 mRNA
4
3
2
1
0
ABCC3 mRNA
2.5
2.0
1.5
1.0
0.5
0
MAD2L1 mRNA
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
PSMA3 mRNA
Hypoxia Cell
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0
GNA11 mRNA
1.2 1.0 0.8 0.6 0.4 0.2 0
HSP60 mRNA
1.2 1.0 0.8 0.6 0.4 0.2 0
FKBP4 mRNA
2.5
2.0
1.5
1.0
0.5
0
SFRS3 mRNA
Fig 2 mRNA levels of hypoxia-repressed genes analyzed by Q-PCR (A,B) Wild-type (WT) hepa1c1c7 cells and BpRc1 cells were incubated
in hypoxic conditions for 16 h The expression level of each gene was quantified by Q-PCR Values are presented as the average ± standard deviation of three independent experiments Statistical analysis of the Q-PCR data was evaluated using one-way ANOVA.
Trang 8bility (4⁄ 8) in hepa1c1c7 cells We next compared the
fold induction of these eight genes in Arnt-defective
BpRc1 cells Microarray analyses showed that two
genes (DUSP12 and ABCC3) were repressed by
hypoxia in both hepa1c1c7 and BpRc1 cells, whereas
the remaining six genes (MAD2L1, HSP60, FKBP4,
PSMA3, GNA11 and SFRS3) were only repressed in
wild-type cells However, Q-PCR analyses indicated
that only two (MAD2L1 and HSP60) of the
four (MAD2L1, HSP60, FKBP4, and PSMA3)
confirmed genes were believed to be repressed in an
Arnt-dependent manner These results suggested that
our microarray analysis was able to predict
Arnt-dependent repression of each gene with a 50%
probability
Genes that are regulated by hypoxia in
a HIF-1a⁄ b-dependent manner
Q-PCR analyses confirmed that nine genes were
induced and four were repressed in response to
hypoxia (Tables 2 and 4), and that 10 of the 13
con-firmed genes were regulated by hypoxia in an
Arnt-dependent manner The remaining three genes
(SUI1-RS1, FKBP4 and PSMA3) were found to be
regulated in an Arnt-independent manner To
substan-tiate the specific role of Arnt, we used BpRc1 cells
infected with retrovirus expression full-length Arnt
[17] BpRc1 cells reconstituted with full-length Arnt
restored the hypoxia-induced (IER3, BSG, BNIP3,
VEGF, CCNG2, NDR1, P4HA1, PTPN16) or
hypoxia-repressed (HSP60, MAD2L1) gene expression
(Table 5), confirming that these 10 genes are regulated
by hypoxia in an Arnt-dependent manner The fold
induction of the genes was often greater in BpRc1 cells reconstituted with Arnt, reflecting that overexpression
of Arnt endowed the cells with increased responsibili-ties to hypoxia In addition, the Arnt-dependent induc-tion of five genes (IER3, BSG, BNIP3, VEGF and NDR1) and Arnt-dependent repression of two genes (MAD2L1 and HSP60) were validated by northern blot analysis (Fig 3)
Finally, we investigated the role of HIF-1a in regu-lating the expression of these genes in response to hypoxia by analyzing murine preadipoctyes 3T3-L1,
in which HIF-1a was knocked down by infection of retrovirus encoding a short hairpin RNA (shRNA) against HIF-1a Western blot analyses confirmed a specific reduction of HIF-1a protein by the cognate shRNA in 3T3-L1 cells (Fig 4A) Q-PCR analysis determined that IER3, BSG, BNIP3, VEGF, CCNG2 and NDR1 were induced in response to hypoxia in both an Arnt- and HIF-1a-dependent manner, indicat-ing that they are the target genes for HIF-1a⁄ b (Table 5 and Fig 4B,C)
Interestingly, it was determined that hypoxia induced both P4HA1 and PTPN16 in an Arnt-depen-dent, but HIF-1a-independent manner shRNA knockdown of HIF-1a failed to completely abolish hypoxia-induced expression of both P4HA1 and PTPN16, but instead reduced the fold induction of these genes by approximately one-half, thereby sug-gesting that HIF-1a plays a role in regulating hypoxia-mediated induction of these genes, at least in part (Table 5) As Arnt is a common binding partner for both HIF-1a and HIF-2a, HIF-2a may also play a partial role in regulating the hypoxia-dependent induc-tion of these genes
Table 4 Hypoxia-repressed gene expression analyzed by both microarray and Q-PCR up, hypoxia-induced expression pattern, fold ‡ 1.5; down, hypoxia-repressed expression pattern, fold < 0.6; nc, no change, 0.6 £ fold < 1.5; ns, not statistically significant, P value > 0.05; ), Arnt independent; +, Arnt dependent.
Gene
symbol
Arnt dependence
Arnt dependence
a Fold: a ratio of expression in hypoxia⁄ expression in normoxia b q value: significant difference, false discovery rate (FDR), selected q value < 0.1 c na, not applied; Arnt dependence was not applied when gene expression pattern of Q-PCR was different from microarray in hepa1c1c7 cells.
Trang 9Gene symbol
Arnt dependence
Trang 10In contrast, our results demonstrated that SUI1-RS1
expression was induced in response to hypoxia, even in
the absence of both Arnt and HIF-1a, suggesting that
neither HIF-1a nor HIF-2a was required for
hypoxia-induced expression of this gene Therefore, our results
demonstrated that HIF was not required for the
induc-tion of SUI1-RS1 in response to hypoxia In addiinduc-tion,
our results demonstrated that PSMA3 was repressed
by hypoxia in the absence of both Arnt and HIF-1a,
and that HSP60 was repressed by hypoxia in an
Arnt-and HIF-1a-dependent manner Finally, we determined
that MAD2L1 and FKBP4 were repressed in
hepa1c1c7 cells, but not in 3T3-L1 cells, suggesting
that these genes were repressed in response to hypoxia
in a cell type-specific manner
Discussion
In this study, we identified 259 hypoxia-regulated genes
by microarray analysis and confirmed the expression
profiles of 17 of these genes by Q-PCR analysis Col-lectively, we determined that 13 of the 17 genes (76.5%) were regulated, as predicted, by microarray analysis By comparing the results of our microarray analysis between wild-type cells and BpRc1 cells, we predicted the Arnt dependence of the confirmed 13 genes (Tables 2 and 4) Q-PCR analyses determined that only six of the 13 genes (46.2%) showed a consis-tent pattern of expression when compared with our microarray analysis for Arnt-dependent regulation in response to hypoxia
BNIP3, VEGF, CCNG2 and NDR1 have been iden-tified as HIF-1 target genes [21,22] However, the results of microarray analyses indicated that they were also induced in BpRc1 cells in response to hypoxia Compared with microarray analysis, it was determined that Q-PCR analysis of wild-type hepa1c1c7 cells resulted in a greater fold induction of these genes, sug-gesting that our microarray analysis quantitatively underestimated the changes in gene expression in wild-type cells In contrast, both microarray and Q-PCR analyses resulted in comparable levels of fold induction
in BpRc1 cells (Table 2) [19] However, the P value of fold induction measured by Q-PCR was determined to
be too large to accept the difference between the norm-oxic and hypnorm-oxic mRNA level of the gene in BpRc1 cells (P > 0.05) Therefore, Q-PCR analysis demon-strates that Arnt is important for the induction, but less necessary for the repression, of hypoxia-mediated gene expression when compared with predictions gen-erated by microarray analysis We next investigated whether the expression of these 13 genes was also regu-lated by HIF-1a (Table 5) It was determined that seven of the 13 genes (IER3, BSG, BNIP3, VEGF, CCNG2, NDR1 and HSP60) were regulated by hypoxia in both an Arnt- and HIF-1a-dependent manner Two genes (P4HA1 and PTPN16) were found
to be induced in response to hypoxia in an Arnt-dependent manner, but only partially regulated in a HIF-1a-dependent manner An additional two genes (SUI1-RS1 and PSMA3) were determined to be regu-lated under hypoxic conditions in both an Arnt- and HIF-1a-independent manner For the final two genes (MAD2L1 and FKBP4), it was determined that they were not repressed in 3T3-L1 cells, indicating that these genes are regulated in a cell type-specific manner
In addition to identifying previously known HIF-1 target genes, including BNIP3, VEGF, CCNG2, NDR1 and P4HA1 [21,22], and other known hypoxia-induc-ible genes, including IER3, BSG and PTPN16 [21–23], this report is the first to identify a number of novel hypoxia-regulated genes, including SUI1-RS1, HSP60 and PSMA3 (Table 5)
Hypoxia – + – + – +
BNIP3
NDR1
BSG IER3
VEGF
28S/18S
NB
α-Arnt
WB
MAD2L1 HSP60
28S/18S
Hypoxia – + – + – +
NB
A
B
Fig 3 Northern analyses of hypoxia-regulated genes (A,B)
Wild-type (WT) mouse hepa1c1c7 cells, the variant BpRc1 cells and
BpRc1 cells reconstituted with Arnt were exposed to hypoxic
condi-tions (0.1% O2) for 6 h Western blot analysis was performed using
30 lg of the cell lysates and HIF-1b (ARNT) antibody (top panel:
WB) For northern blot (NB) analysis, wild-type mouse hepa1c1c7
cells, the BpRc1 variant cell line and BpRc1 cells reconstituted with
Arnt were exposed to hypoxic conditions for 16 h; 20 lg of total
RNA from the treated cells was transferred onto a nitrocellulose
membrane Each blot was hybridized with the indicated [a- 32
P]-labeled probes Information on the probes is presented in Table S3.