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Carcinogenesis Advance Access originally published online on November 16, 2007
Carcinogenesis 2008 29(2):291-298; doi:10.1093/carcin/bgm262
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

CCAAT/enhancer-binding protein {alpha} antagonizes transcriptional activity of hypoxia-inducible factor 1 {alpha} with direct protein–protein interaction

L. Yang1, Y. Jiang2, S.F. Wu1, M.Y. Zhou1, Y.L. Wu2 and G.Q. Chen1,2,*

1 Institute of Health Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences–Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
2 Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

* To whom correspondence should be addressed. Tel: +86 21 63846590 ext. 776573; Fax: +86 21 64154900; Email: chengq{at}shsmu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Hypoxia-inducible factor 1 (HIF-1), a master heterodimeric transcriptional regulator consisting of HIF-1{alpha} and HIF-1β subunits for cellular response to hypoxia, plays an important role in carcinogenesis, while CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}) is proposed to act as a tumor suppressor in C/EBP{alpha}-expressing tissues. Previously, we reported that ectopically expressed HIF-1{alpha} protein interacts with and enhances transcriptional activity of C/EBP{alpha}, which favors leukemic cell differentiation. Here we further showed that such an interaction also occurred in their endogenously expressing state of leukemic U937 cells. Glutathione S-transferase pull-down assay proposed that the protein–protein interaction was direct, and transactivation domains of C/EBP{alpha} and the basic helix-loop-helix domain of HIF-1{alpha} were essential for such an interaction. More intriguingly, we provided the first demonstration that C/EBP{alpha} competed with HIF-1β for direct binding to HIF-1{alpha} protein. Correspondingly, C/EBP{alpha} overexpression significantly inhibited the DNA-binding ability of HIF-1 and expressions of hypoxia-responsive element-driven luciferase and HIF-1-targeted genes vascular endothelial growth factor, glucose transporter-1 and phosphoglycerate kinase 1. In parallel, suppression of C/EBP{alpha} expression by specific small hairpin RNA increased DNA-binding ability of HIF-1 and expression of these HIF-1-targeted genes in leukemic U937 cells. These results would provide new insights for antitumor potential of C/EBP{alpha} protein.

Abbreviations: bHLH, basic helix-loop-helix; ChIP, chromatin immunoprecipitation; Glut-1, glucose transporter-1; GST, glutathione S-transferase; HIF-1, hypoxia-inducible factor 1; PAS, Per-Arnt-Sim; PGK1, phosphoglycerate kinase 1; PCR, polymerase chain reaction; shRNA, short hairpin RNA; TAD, transactivation domain


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Hypoxia-inducible factor 1 (HIF-1), a master transcriptional regulator for cellular response to hypoxia, plays an important role in development, physiology and many pathological processes such as carcinogenesis (1–3). The transcription factor is a heterodimeric protein consisting of two basic helix-loop-helix (bHLH)–Per-Arnt-Sim (PAS) domain-containing subunits, constitutively expressed HIF-1β/ARNT (for aryl hydrocarbon receptor nuclear translocator) and highly regulated HIF-1{alpha} subunit, the latter determining overall transcriptional activity of HIF-1. Under normoxic condition, the hydroxylated HIF-1{alpha} protein at two highly conserved prolines (proline 402/564) binds to the von Hippel-Lindau (VHL) tumor suppressor, a part of an E3 ubiquitin ligase (4–6). Thus, the HIF-1{alpha} protein is degraded by the 26S proteasome system. By the way, lysine acetylation of HIF-1{alpha} enhances its binding to VHL and its subsequent degradation (7). Hypoxia or treatment of some hypoxia-mimetic agents such as cobalt chloride (CoCl2) results in stabilization of HIF-1{alpha} by inhibiting the hydroxylation of HIF-1{alpha} protein. In addition, Li et al. (8) reported recently that normoxic HIF-1 activity can be upregulated through nitric oxide-mediated S-nitrosylation and stabilization of HIF-1{alpha}. The stabilized HIF-1{alpha} protein is translocated to the nucleus, followed by heterodimerization with HIF-1β and recruitment of the transcription co-activator p300/CBP and SRC-1. Consequently, HIF-1 heterodimer binds to the hypoxia-responsive element (HRE) and regulates an impressive array of target genes such as vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1) and phosphoglycerate kinase 1 (PGK1), which involve in angiogenesis, metabolic adaptation, apoptosis induction/resistance and invasion/metastasis (3,9).

More recently, hypoxia or mimetic hypoxia is also reported to induce or accelerate differentiation of acute myeloid leukemic (AML) cells and normal hematopoietic progenitors (10–14). During this event, HIF-1{alpha} exerts a critical role in its transcriptional activity-independent manner (15). Tiron (a non-toxic chelator to alleviate an acute metal overload) is shown to be a potent inducer of leukemic cell differentiation via increasing HIF-1{alpha} protein (16). By co-immunoprecipitation (Co-IP) assay, we reported previously that overexpressed HIF-1{alpha} interacts with and enhances transcriptional activity of CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}) (11), a member of the basic leucine zipper family of transcription factors, which plays a critical role in granulocytic hematopoiesis (17,18), and is proposed to act as a tumor suppressor in C/EBP{alpha}-expressing tissues beyond hematopoietic system (19). Furthermore, the inhibition of C/EBP{alpha} expression by specific short hairpin RNAs (shRNAs) or conditional expression of leukemogenic AML1–ETO fusion protein significantly eliminates HIF-1{alpha}/hypoxia-mediated myeloid leukemic cell differentiation (11,15). However, exact domains of HIF-1{alpha} and C/EBP{alpha} for their interaction and possible effects of such an interaction on transcriptional activity of HIF-1 remain unclear. After further confirming that HIF-1{alpha}–C/EBP{alpha} interaction also occurred in their endogenously expressing states of leukemic U937 cells, in this work, we map that transactivation domains (TADs) of C/EBP{alpha} and the bHLH domain of HIF-1{alpha} are essential for their interaction. More intriguingly, we provide the first demonstration that C/EBP{alpha} competes with HIF-1β for direct binding to HIF-1{alpha} protein. Especially, C/EBP{alpha} inhibits DNA-binding ability and transcriptional activity of HIF-1{alpha} protein. These results would shed new insights for understanding antitumor potential of C/EBP{alpha} protein.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell lines
Leukemic cell line U937 and the non-hematopoietic cell lines COS-7 and 293 were, respectively, cultured in RPMI-1640 and Dulbecco's Modified Eagle's Medium (Sigma, St Louis, MI) supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD) in a humidified incubator at 37°C and 5% CO2/95% air. Hypoxic treatment was carried out in a specially designed hypoxia incubator (Thermo Electron, Forma, MA) with 2% air, 5% CO2 and 93% N2 or cells were incubated with hypoxia-mimicking agent CoCl2 (Sigma). In all experiments, all cells remain to more than 95% of survival as determined by trypan-blue exclusion assay.

Plasmids and transfection
The plasmid pCMV-SPORT-C/EBP{alpha} was a gift of Dr A.F. Gombart (Cedars-Sinai Medical Center, Los Angeles, CA). The pEF-BOS, pEF-BOS-HIF-1{alpha}, pEF-BOS-HIF-1β and pGL3-EpoHRE-luciferase plasmids were kindly provided by Dr Y. Fujii-Kuriyama in University of Tsukuba, Japan (20). For in vitro translation, HIF-1{alpha} and HIF-1β cDNAs, which were amplified, respectively, from pEF-BOS-HIF-1{alpha} and pEF-BOS-HIF-1β plasmids, were subcloned into pcDNA3.0 vector (Invitrogen, Carlsbad, CA), and C/EBP{alpha} cDNA from pCMV-SPORT-C/EBP{alpha} was subcloned into pcDNA3.1 vector (Invitrogen). The fidelity of all inserted cDNAs was verified by sequencing (Invitrogen, Shanghai, China). All plasmids were transfected into the non-hematopoietic cell lines by using Polyfect Transfection Reagent according to the manufacturer's instruction (Qiagen, Hilden, Germany).

Co-immunoprecipitation
Cells were harvested and cell lysates were incubated with mouse monoclonal antibody against human HIF-1{alpha} (BD Transduction, San Jose, CA) and human C/EBP{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA) together with protein A plus-agarose (Santa Cruz) overnight at 4°C. Normal preimmune mouse and rabbit IgG (Santa Cruz) were also used as negative controls. The immunoprecipitates were eluted by the 2x SDS sample buffer and then detected by western blots.

Glutathione S-transferase pull-down assay
Full-length or truncated HIF-1{alpha} or C/EBP{alpha} cDNA was amplified, respectively, from pEF-BOS-HIF-1{alpha} and pCMV-SPORT-C/EBP{alpha} plasmids with specific primers and subcloned into pGEX-4T3 vector (Amersham Biosciences, Buckinghamshire, UK) as glutathione S-transferase (GST)-tagged expression vector. GST alone and GST-tagged full-length or truncated HIF-1{alpha} or C/EBP{alpha} fusion proteins were induced by 0.5mM of Isopropyl β-D-thiogalactoside (IPTG, Shanghai Chemical Agent Company, Shanghai, China) in E. coli BL21 and purified by the Bulk GST Purification Module according to the manufacturer's instructions (Amersham Biosciences). C/EBP{alpha}, HIF-1{alpha} and HIF-1β protein were translated, respectively, in vitro by using the TNT T7 Transcription/Translation System (Promega, Madison, WI). The purified GST and GST-tagged proteins were incubated with the in vitro translated protein for 2 h at room temperature. Then the precipitations were eluted by the 2x SDS sample buffer and followed by western blot or being stained by Commassie Brilliant Blue G250 (Shanghai Chemical Agent Company).

Nuclear extraction and electrophoretic mobility shift assay
One microgram of pEF-BOS-HIF-1{alpha} and/or 1 µg of pcDNA3.1-C/EBP{alpha} plasmids were transfected into 293 cells. Empty vectors were added to equal the amount of plasmids. Twenty-four hours later, 150 µM of CoCl2 was added to pEF-BOS-HIF-1{alpha}-transfected 293 cells for additional 8 h to stabilize HIF-1{alpha} protein. Thereafter, about 6 x 106 cells were collected and their nuclear proteins were extracted by NE-PER nuclear and cytoplasmic Extraction Reagents (Pierce, Rockford, IL) and were concentrated by MICROCON centrifugal Filter Device (Millipore, Bedford, MA). Protein was quantified by the DC Protein Assay kit (Bio-Rad, Hercules, CA). The wild-type probe 5'-CCACAGTGCATACGTGGGCTCCAACAGGTC-3' from human VEGF gene including the HRE (shown in underlined) and the parallel mutated probe 5'-CCACAGTGCATAAATGGGC TCCAACAGGTC-3' (mutated HRE shown in underlined) were labeled by Biotin 3' End DNA Labeling Kit (Pierce). Binding reactions were performed at 25°C for 15 min in the binding buffer (10 mM Tris–HCl, pH 7.5, 50 mM KCl, 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 5 mM DTT and 5% glycerol) with 5 µg of nuclear protein and labeled probe to total volume of 20 µl (21). For competitive assay, additional 500-fold molar excess of the unlabeled wild-type probe was added to binding reaction. The DNA–protein complex was separated in 4% non-denaturing polyacrylamide gel by electrophoresis in 0.5x TBE buffer at room temperature. After electrophoresis, the gel was transformed to Biodyne® B Pre-cut Modified Nylon Membranes (Pierce) and analyzed by Chemiluminescent Nucleic Acid Detection Module (Pierce).

Quantitative reverse transcription–polymerase chain reaction
For quantitative real-time reverse transcription–polymerase chain reaction (RT–PCR) assay, total RNA was isolated by TRIzol kit (Invitrogen, Scotland, UK) with DNase (Promega) treatment, and cDNA was synthesized by the cDNA synthesis kit (Roche-Applied Biosystems, Foster, CA). Quantitative real-time PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) on the ABI PRISM 7900 system (Perkin-Elmer, Torrance, CA). Data were analyzed as described previously (22). The specific primers were 5'-CATCCTCACCCTGAAGTACCC-3' and 5'-AGCCTGGATAGCAACGTACATG-3' for β-actin, 5'-GCAGAATCATCACGAAGTGG-3' and 5'-GCATGGTGATGTTGGACTCC-3' for VEGF, 5'-CT GGATGGGCTTGGACTG-3' and 5'-GCAAGTGGCAGTGTCTCC-3' for PGK1 and 5'-GACTCCTGCCCTGTTGTG-3' and 5'-CGAAGTCTAAGCCGTTGC-3' for Glut-1.

Luciferase assay
COS-7 or 293 cells were seeded in a 12-well plate (Becton Dickinson, Franklin Lakes, NJ) and transfected with indicated amount of pcDNA3.1-C/EBP{alpha} only together with pGL3-EpoHRE-luciferase(Luc) (200 ng), a luciferase reporter construct containing four HREs of the promotor of erythropoietin (Epo) gene (20), pSV40-Renilla (10 ng) in the presence or absence of pEF-BOS-HIF-1{alpha} and pEF-BOS-HIF-1β (100 ng). The empty vectors were supplemented to 750 ng of the total plasmid concentration. Twenty-four hours later, 150 µM CoCl2 was added as described in the text, and cell lysates were analyzed by the Dual-Luciferase Assay system (Promega) according to the manufacturer's instruction. The relative Luc activity was normalized by renilla activity.

shRNA design and transfection
Pairs of complementary oligonucleotide shR-C2 against HIF-1{alpha} were synthesized by Invitrogen (Shanghai, China) and annealed and ligated into pSilencer 3.1-H1-neo vector (Ambion, Austin, TX). The shRNA vector and negative control pSilencer neo vector (Ambion) were, respectively, transfected into U937 cells using the Bio-Rad Gene-Pulser II with the square-wave electroporation of 2 pulses, 0.18 kV, 25 ms, 1 Hz. Twenty-four hours later, 1000 µg G418/ml (Calbiochem, La Jolla, CA) were added to the medium and the stable transformants were selected by testing targeted proteins.

Chromatin immunoprecipitation
After incubating with 1% formaldehyde at room temperature for 10 min, cells were pelleted and resuspended in 200 µl lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.0). Cell lysates were sonicated with Sonicator ultrasonic processor (Misonix, Inc, NY) until DNA was cleaved into 500–1000 bp in size. Then, these extracts were immunoprecipitated with mouse anti-human HIF-1{alpha} monoclonal antibody (BD Transduction) and normal preimmune mouse IgG (Santa Cruz), and chromatin immunoprecipitation (ChIP) experiments were performed using ChIP Assay Kit according to the manufacturer's instructions (Upstate, NY). Then, PCR for the HRE in the promotor of the VEGF was performed with primers 5'-GACGTTCCTTAGTGCTGGCGGGTAGGTTTGA-3' and 5'-GGCACCAAGTTTGTGGAGCTGAGAAC GGG-3' (23).

Western blot
Proteins were fractionated on 8% SDS–polyacrylamide gel and transferred to the HybondTM-C Extra nitrocellulose membrane (Amersham Biosciences). After blocked in 5% non-fat milk, the membrane was incubated with mouse monoclonal anti-HIF-1{alpha} (BD Transduction), anti-HIF-1β (BD Transduction), rabbit polyclonal anti-C/EBP{alpha} (Santa Cruz), followed by horseradish peroxidase (HRP)-linked secondary antibody (Cell Signaling, Beverly, MA). Chemiluminescence phototope-HRP kit (Cell Signaling) was used for detection. As necessary, blots were probed with anti-β-actin (Merck, Darmstadt, Germany) antibody as loading controls.

Statistical analysis
All experiments were repeated at least for three times with the same results. Student’s t-test was used to compare the difference between two groups. A value of P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
HIF-1{alpha} directly interacts with C/EBP{alpha} protein
To ascertain whether HIF-1{alpha}–C/EBP{alpha} interaction occurs between the endogenously expressed HIF-1{alpha} and C/EBP{alpha} proteins, leukemic U937 cells were treated with CoCl2 at 50 µM that rapidly stabilized HIF-1{alpha} protein (Figure 1a). The cell line also expressed endogenous C/EBP{alpha} protein, which was not affected by the stabilized HIF-1{alpha} protein (Figure 1a). Thus, extracts from U937 cells with CoCl2 treatment for 24 h were applied to Co-IP assay. As depicted in Figure 1b, preimmune rabbit or mouse IgG failed to precipitate C/EBP{alpha} or HIF-1{alpha} protein, while anti-HIF-1{alpha} or anti-C/EBP{alpha} antibody pulled its targeted protein down, confirming the specificity and effectiveness of the Co-IP assay. Like that seen in transfected cells (11), anti-C/EBP{alpha} antibody was capable of precipitating CoCl2-stabilized endogenous HIF-1{alpha} protein, while anti-HIF-1{alpha} antibody failed to precipitate C/EBP{alpha} protein. We extrapolated that interaction of C/EBP{alpha} with HIF-1{alpha} possibly covered the epitope of the anti-HIF-1{alpha} antibody. Thus, GST pull-down assay was used. The result showed that bacteria-expressed GST–HIF-1{alpha} but not GST itself effectively pulled the in vitro translated C/EBP{alpha} protein down. Vice versa, GST–C/EBP{alpha} also precipitated HIF-1{alpha} protein (Figure 1c), supporting a direct interaction of HIF-1{alpha} with C/EBP{alpha} protein.


Figure 1
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  		Fig. 1. Physical interaction of C/EBP{alpha} and HIF-1{alpha} protein by immunoprecipitation and GST pull-down assay. (a) U937 cells were incubated with 50 µM CoCl2 for hours as indicated. Cell lysate was blotted respectively for antibodies against HIF-1{alpha}, HIF-1β and C/EBP{alpha} with β-actin as loading control. (b) The nuclear extracts from U937 cells treated with 50 µM CoCl2 for 24 h were immunoprecipitated and blotted for antibodies against HIF-1{alpha} and C/EBP{alpha}. (c) E. coli BL21-expressing GST and GST–HIF-1{alpha} (left) or GST and GST–C/EBP{alpha} (right) proteins were purified, and these purified proteins at equal amount were incubated with the in vitro translated C/EBP{alpha} or HIF-1{alpha} protein. The GST pulled-down complexes were blotted with anti-C/EBP{alpha} or anti-HIF-1{alpha} antibody.

 
bHLH of HIF-1{alpha} and TADs of C/EBP{alpha} are required for their interaction
To map the minimum region of C/EBP{alpha} required for its interaction with HIF-1{alpha}, we tested the ability of various C/EBP{alpha} mutants to interact with HIF-1{alpha} by means of GST pull-down assay. For this experiment, various kinds of GST-tagged C/EBP{alpha} mutants were constructed (Figure 2a) and expressed in E. coli. Then, these mutants were incubated with the in vitro translated HIF-1{alpha} protein. The GST pulled-down complexes were collected, among which most were used for western blot with anti-HIF-1{alpha} antibody (top panel, Figure 2b), and the remnants were run on the SDS–polyacrylamide gel for Commassie Brilliant Blue staining to check loading amount of GST fusion proteins (bottom panel, Figure 2b). The results showed that the C-terminal basic zip domain of C/EBP{alpha} was not required for interaction with HIF-1{alpha} protein, since deletion of the C terminus (deletion of aa221 to 359) did not hinder protein interaction with HIF-1{alpha}. On the contrary, N-terminal fragment from aa1 to aa221, which includes three TADs (TA1–3), could pull HIF-1{alpha} protein down in the same ability as full-length C/EBP{alpha} protein, while their deletions made C/EBP{alpha} protein lose the ability to bind HIF-1{alpha} protein (top panel, Figure 2b). These results proposed that TA domains of C/EBP{alpha} are critical for its interaction with HIF-1{alpha} protein. Moreover, the fragment from aa1 to aa148 carrying TA1 and TA2 domains could pull HIF-1{alpha} down but with a lower ability than full-length C/EBP{alpha} protein and its N-terminal fragment including TA1–3, indicating that TA3 domain might affect this interaction although it did not directly bind to HIF-1{alpha} protein (top panel, Figure 2b).


Figure 2
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  		Fig. 2. Identification of structure requirement for C/EBP{alpha}–HIF-1{alpha} interaction. (a and c) Schematic diagrams of C/EBP{alpha} (a) or HIF-1{alpha} (c) and their deletion mutants assayed are shown. The numbers in the top represent amino acid sequences of C/EBP{alpha} or HIF-1{alpha} protein. (b and d) The purified GST-tagged C/EBP{alpha} (b) or HIF-1{alpha} (d) and their deletion mutants as shown in the top were incubated with the in vitro translated HIF-1{alpha} or C/EBP{alpha} protein. The in vitro translated proteins (input) and the GST pulled-down complexes were blotted with anti-HIF-1{alpha} or anti-C/EBP{alpha} antibody. Commassie Brilliant Blue staining of gels for the same GST pulled-down complexes was used to check loading amount of GST fusion proteins, of which the corresponding bands were pointed by white arrows according to their putative molecular weight.

 
Next, we also used GST pull-down assay to confirm the ability of various HIF-1{alpha} mutants (Figure 2c) to interact with C/EBP{alpha}, with Commassie Brilliant Blue staining to scale the amount of GST fusion proteins (bottom panel, Figure 2d). As depicted in the top panel of Figure 2d, the C-terminal (aa302-aa826) deletion of HIF-1{alpha}, including two TADs (aa531-575 and 813-826) and an oxygen-dependent degradation domain (ODD)(aa401-531) (24), did not damage HIF-1{alpha}–C/EBP{alpha} interaction, proposing that N-terminal fragment (aa1-302) containing the bHLH–PAS domain was required for this interaction. Furthermore, the bHLH domain itself (aa1-71) could pull C/EBP{alpha} protein down, while its deletion made HIF-1{alpha} protein fail to bind with the C/EBP{alpha} protein. These results propose that the bHLH domain of HIF-1{alpha} is essential for its interaction with HIF-1{alpha} protein, and PAS domain enhances HIF-1{alpha}–C/EBP{alpha} interaction.

C/EBP{alpha} competes with HIF-1β for direct binding to HIF-1{alpha} protein
Because HIF-1{alpha}–HIF-1β interaction also involves bHLH–PAS domain of HIF-1{alpha} protein (25), it was rationally speculated that the C/EBP{alpha} protein might impinge on HIF-1{alpha}–HIF-1β interaction. To confirm this comment, equal amount of GST–HIF-1{alpha} was incubated with the in vitro translated HIF-1β and/or C/EBP{alpha} protein. As shown in Figure 3a, GST alone did not but GST–HIF-1{alpha} could effectively pull HIF-1β protein down in the absence of C/EBP{alpha}, indicating the specificity of the GST pull-down assay. More intriguingly, GST–HIF-1{alpha}-bound HIF-1β protein was reduced when C/EBP{alpha} was added, which was dependent on the dose of C/EBP{alpha} protein (lanes 4–7, Figure 3a). Similarly, GST–HIF-1{alpha} could effectively pull C/EBP{alpha} protein down in the absence of HIF-1β (lane 3, Figure 3b), while HIF-1{alpha}-bound C/EBP{alpha} protein was decreased with the addition of increasing doses of HIF-1β protein (lanes 4–7, Figure 3b). All these results supported that C/EBP{alpha} inhibits binding of HIF-1β to HIF-1{alpha} protein.


Figure 3
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  		Fig. 3. Competitive effect of C/EBP{alpha} and HIF-1β for their interaction with HIF-1{alpha} protein. Bacterially expressed GST-tagged HIF-1{alpha} protein was incubated with equal volume (6 µl) of the in vitro translated HIF-1β protein and the increasing amount (4, 8, 16, 32 µl) of the in vitro translated C/EBP{alpha} protein (a) or with equal volume (16 µl) of the C/EBP{alpha} protein and the increasing (2, 4, 6, 8 µl) HIF-1β protein (b). The in vitro translated C/EBP{alpha} or HIF-1β protein (input) and GST pulled-down complexes were blotted, respectively, by anti-C/EBP{alpha} and anti-HIF-1β antibodies. Commassie Brilliant Blue stainings of gels for the same GST pulled-down complexes were shown in the bottom to check loading amount of GST fusion proteins.

 
C/EBP{alpha} inhibits the DNA-binding ability of HIF-1 protein
The fact that C/EBP{alpha} interferes with HIF-1{alpha}–HIF-1β interaction promoted us to ask for whether C/EBP{alpha} impacts on the transcriptional activity of HIF-1. Thus, we detected the DNA-binding activity of HIF-1 in the presence or absence of C/EBP{alpha} expression by electrophoretic mobility shift assay (EMSA). For this purpose, pEF-BOS-HIF-1{alpha}- and/or pcDNA3.1-C/EBP{alpha}-expressing plasmids were transfected into 293 cells, and their nuclear proteins were extracted and concentrated. As can be seen in Figure 4a, untransfected and CoCl2-free cells expressed a little HIF-1{alpha} protein without the detectable C/EBP{alpha} protein. C/EBP{alpha} transfection caused overexpression of C/EBP{alpha} and HIF-1{alpha} transfection led to higher HIF-1{alpha} protein level in the presence of CoCl2, indicating the effective transfections. Of note, overexpression of C/EBP{alpha} or HIF-1{alpha} protein did not alter expression of HIF-1{alpha} or C/EBP{alpha} as well as HIF-1β protein. These nuclear extracts were mixed with the biotin-labeled wild-type HRE oligonucleotide from the promotor of human VEGF gene, together with its mutant as negative control. Then, the DNA–protein complex was separated in 4% non-denaturing polyacrylamide gel. As demonstrated in Figure 4b, the nuclear extract of HIF-1{alpha}-overexpressing CoCl2-treated cells significantly bound to wild-type HRE but not mutated HRE, which was almost completely blocked by the addition of 500-fold molar excess of unlabeled wild-type HRE (cold probe). These results proposed the specificity for HRE–HIF-1 complex. Of great interest, when C/EBP{alpha} was co-transfected with HIF-1{alpha}, HIF-1–HRE complex was markedly reduced. By the way, ChIP assay also showed that overexpression of C/EBP{alpha} in 293 cells significantly reduced binding of transfected HIF-1 protein with the VEGF-HRE promoter under CoCl2 treatment (data not shown). All these experiments indicated that C/EBP{alpha} inhibits the DNA-binding ability of HIF-1 protein.


Figure 4
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  		Fig. 4. Inhibition of the DNA-binding ability of HIF-1 by C/EBP{alpha} protein. (a) 293 cells were transfected with HIF-1{alpha} and/or C/EBP{alpha}-expressing vectors. Twenty-four hours later, 150 µM CoCl2 was added to HIF-1{alpha} transfected cells for additional 8 h to stabilize HIF-1{alpha} protein. Thereafter, nuclear extracts were concentrated as described in Materials and methods, and were blotted respectively for proteins as indicated. (b) Nuclear extracts from 293 cells with the indicated transfection were incubated with biotin-labeled HRE probe from the promotor of VEGF, containing either normal or mutated binding site of HIF-1{alpha}. Cold probe was 500 folds of unlabeled wild-type probe. Then electrophoresis was performed and a non-radioactive LightShift Chemiluminescent EMSA Kit was employed for analysis.

 
C/EBP{alpha} overexpression inhibits the transcriptional activity of HIF-1 protein
We investigated whether C/EBP{alpha} influences the transcriptional activity of HIF-1 protein by detecting HRE-driven Luc activity and mRNA levels of HIF-1-targeted genes. To detect HRE-driven Luc activity, COS-7 cells were co-transfected with HIF-1{alpha} and/or C/EBP{alpha}-expressing vectors together with pEF-BOS-HIF-1β and pGL3-EpoHRE-Luc plasmids. Although COS-7 cells displayed detectable HIF-1{alpha} protein with HIF-1{alpha} transfection in the air, and only HIF-1{alpha} protein significantly induced HRE-Luc expression in the absence of CoCl2 treatment (data not shown), we still added 150 µM CoCl2 to cells for additional 8 h at 24 h after HIF-1{alpha} transfection so as to avoid possible effects of N803 hydroxylation by factor inhibiting HIF-1 (FIH) on HIF-1 activity (26). Results showed that in the presence of HIF-1{alpha} expression, as shown in Figure 5a, C/EBP{alpha} expression significantly antagonized HRE-Luc expression in a manner dependent upon doses of C/EBP{alpha}. The similar results were also seen in 293 cells (data not shown). Furthermore, 293 cells were transfected with HIF-1{alpha} together with HIF-1β and/or C/EBP{alpha}-expression vectors. Twenty-four hours later, CoCl2 at 150 µM was added for additional 8 h to stabilize HIF-1{alpha} protein. Western blot clearly showed the effective transfection (top panel, Figure 5b). Consistent with that seen by detecting HRE-driven Luc activity (Figure 5a), HIF-1{alpha} but not C/EBP{alpha} transfection alone significantly increased expression of three known HIF-1-targeted genes VEGF, Glut-1 and PGK1, as evidenced by quantitative real-time RT–PCR assay (bottom panel, Figure 5b). Compared with HIF-1{alpha} transfection alone, C/EBP{alpha}–HIF-1{alpha} co-transfection significantly inhibited expression of VEGF and Glut-1. PGK1 expression was also decreased by the co-transfection, although it was not significant by the statistic analysis (bottom panel, Figure 5b). In addition, we also tested possible effects of C/EBP{alpha} overexpression on transcriptional activity of endogenous HIF-1{alpha} that was stabilized by 150 µM CoCl2 treatment for additional 24 h. As shown in Figure 5c and d, C/EBP{alpha} expression also significantly antagonized HRE-Luc activity and expression of VEGF. All these observations proposed that C/EBP{alpha} expression effectively antagonizes the transcriptional activity of HIF-1 protein.


Figure 5
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  		Fig. 5. Inhibition of the transcriptional activity of HIF-1 protein by C/EBP{alpha} overexpression. (a and c) COS-7 cells were transfected with the indicated doses of pcDNA3.1-C/EBP{alpha} plasmids with or without 100 ng of pEF-BOS-HIF-1{alpha} plasmid in the presence of pEF-BOS-HIF-1β (a) or transfected with the indicated doses of pcDNA3.1-C/EBP{alpha} plasmids (c), together with pGL3-EpoHRE-Luc and pSV40-Renilla. Twenty-four hours later, 150 µM CoCl2 was added for additional 8 h (a) or 24 h (c) as shown. Western blots were performed to confirm the effective transfections, and the relative HRE-Luc activity was normalized by pSV40-Renilla, and folds of increase against lane 1 were calculated. Symbols # and * represent P values, respectively, compared with lane 1 and lane 3. (b and d) 293 cells were transfected with plasmids as indicated (b) or with empty vector (EV) or C/EBP{alpha}-expressing plasmids with untransfected cells (vehicle) as control (d). Twenty-four hours later, 150 µM CoCl2 was added for additional 8 h (b) or 24 h (d) as indicated to stabilize HIF-1{alpha} protein. Then, cells were harvested for western blots and quantitative real-time RT–PCR assay for mRNAs as indicated. Folds of increase of mRNAs were calculated against vehicle cells. Symbol * represents P values compared with HIF-1-transfected cells (b) or with CoCl2-treated EV-transfected cells (d). All data represent means + SDs of triplicates in an independent experiment, which was repeated for three times with the same results.

 
The suppression of C/EBP{alpha} expression by specific shRNA increases DNA-binding ability of HIF-1 protein and expression of HIF-1-targeted genes in leukemic U937 cells
Finally, we evaluated the potential inhibitory effect of C/EBP{alpha} on the transcriptional activity of HIF-1 protein in the physiologic condition. For this purpose, C/EBP{alpha} expression in leukemic U937 cells was knocked down by stable transfection of shRNA specifically against C/EBP{alpha} (shR-C2) with empty vector as negative control (NC). Then, these cells were incubated in the air and 2% O2 for 24 h, the latter stabilizing HIF-1{alpha} protein (Figure 6a). As reported previously (15), C/EBP{alpha} protein was significantly reduced in shR-C2-transfected U937 cells, compared with NC-transfected cells (Figure 6a). It appeared that siRNA presented the better effect on C/EBP{alpha} expression under hypoxia than in the air, of which mechanisms remain to be investigated. Of note, the knock-down of C/EBP{alpha} had no influence on HIF-1{alpha} and HIF-1β protein levels (Figure 6a). Then, we also used ChIP assay to investigate the DNA-binding ability of HIF-1 protein. As shown in Figure 6b, anti-HIF-1{alpha} antibody but not normal preimmune mouse IgG could effectively immunoprecipitate HRE of the promoter of VEGF gene, supporting the specificity of the ChIP assay. Our results revealed that the suppression of C/EBP{alpha} expression in shR-C2 cells significantly increased binding of HIF-1 with the VEGF-HRE promoter under 2% O2 (Figure 6b), supporting that the suppression of C/EBP{alpha} expression increases DNA-binding ability of HIF-1 protein in leukemic U937 cells. Further, we also tested expressions of HIF-1-targeted genes in the cell context. In the air, all these cells were absent from the detectable HIF-1{alpha} protein (Figure 6a), and thus, expressions of all three HIF-1-targeted genes examined had no difference between them (Figure 6c). When NC-transfected cells were put into hypoxia, HIF-1-targeted genes Glut-1 and PGK1 only presented a little of increase with no alteration of VEGF mRNA compared with NC-transfected cells in the air (Figure 6c), although HIF-1{alpha} was detected clearly (Figure 6a). More intriguingly, expressions of these HIF-1-targeted genes were significantly increased in the suppression of C/EBP{alpha} expression by shRNA-C2 (Figure 6c).


Figure 6
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  		Fig. 6. Increased expressions of HIF-1-targeted genes and DNA-binding ability of HIF-1 protein by specific shRNA against C/EBP{alpha} expression in leukemic U937 cells. U937 cells stably transfected with negative control (NC) and shRNA against C/EBP{alpha} (shR-C2) were incubated in the air or 2% O2 for 24 h. (a) The indicated proteins were detected by western blot with β-actin as loading control. (b) ChIP experiments were performed as described in Materials and method section, and HIF-1-bound oligonucleotide in the promoter of VEGF was amplified. (c) The relative VEGF, Glut-1 and PGK1 mRNA levels were measured by quantitative real-time RT–PCR. Folds of increase of mRNA were calculated against the NC-transfected cells in the air. Symbol # represents P value compared between NC- and shR-C2-transfected cells in 2% O2. All data represent means + SDs of triplicates in an independent experiment. All experiments were repeated for three times with the same results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
HIF-1{alpha} protein has been widely identified to interact with many proteins such as β-catenin and heat shock protein 90 (27,28). These interactions regulate stability and/or transcriptional activity of HIF-1{alpha} protein or modulate functions of HIF-1{alpha}-interacting proteins, as reviewed by Wenger et al. (29). As an example, HIF-1{alpha} interacts with and antagonizes c-Myc activity via its N-terminal region-dependent and transcriptional activity-independent manner (30). Furthermore, HIF-1 also negatively regulates mitochondrial biogenesis and O2 consumption in renal carcinoma cells by inhibiting c-Myc activity via binding to and activating transcription of the MXI1 gene, which encodes a repressor of c-Myc transcriptional activity, and promoting MXI1-independent, proteasome-dependent degradation of c-Myc (31). Previously we also showed that ectopically expressed C/EBP{alpha} protein interacts with HIF-1{alpha} protein (11). In this work, we further confirmed that CoCl2-stabilized endogenously expressed HIF-1{alpha} protein can also interact with physiologically expressed C/EBP{alpha} protein in leukemic U937 cells. GST pull-down assay proposed a direct interaction of HIF-1{alpha} with C/EBP{alpha} protein. Thereafter, the minimum structure requirement for HIF-1{alpha} and C/EBP{alpha} to form a complex with each other was searched by GST pull-down assay based on various kinds of domain-deleted mutants of HIF-1{alpha} and C/EBP{alpha}. Results demonstrated that N-terminal TA domains of C/EBP{alpha}, especially TA1 and TA2, were required for HIF-1{alpha} interaction. For C/EBP{alpha} interaction, the N-terminal bHLH domain but not the C-terminal TADs and ODD domain of HIF-1{alpha} was essential, which was similar to the minimum requirement for HIF-1{alpha} to form a complex with c-Myc (30).

Hematopoiesis is achieved by a finely tuned cooperation of hematopoietic genes in progenitor cells (32). Multiple important hematopoiesis-related transcription factors such as C/EBP{alpha}, Runt-related protein 1 (Runx1, also called as acute myeloid leukemia 1, AML1) and PU.1 (33) synergistically regulate the differentiation of immature progenitors in bone marrow to terminally differentiated/mature cells in peripheral blood (34–36). Genetic or/and epigenetic abnormalities of these transcription factors have been widely identified in various kinds of leukemia, proposing that they play a role in the pathogenesis of human leukemia (37,38). As described above, hypoxia also induces differentiation of myeloid leukemic cells and normal hematopoietic progenitors, in which HIF-1{alpha} exerts a critical role in its transcriptional activity-independent mechanism (15,39). Recently, HIF-1{alpha} is reported to increase DNA-binding ability and transcriptional activity of Runx1 protein by their interaction (40). Previously we also showed that HIF-1{alpha} increases transcriptional activity of C/EBP{alpha} protein. As such, we suggested that HIF-1{alpha} induces differentiation of AML cells through enhancing transcriptional activity of C/EBP{alpha} and Runx1 (11). Recently, Kaidi et al. (28) reported that HIF-1{alpha} competes with T-cell factor-4 (TCF-4) for direct binding to β-catenin, thus inhibiting β-catenin–TCF-4 complex formation and transcriptional activity. Considering that bHLH–PAS domain of HIF-1{alpha} protein is also required for its dimerization with HIF-1β (25), we hypothesized that C/EBP{alpha} competes with HIF-1β for direct binding to HIF-1{alpha} protein. As expected, HIF-1{alpha}–HIF-1β interaction was inhibited by C/EBP{alpha}, while HIF-1{alpha}–C/EBP{alpha} interaction was also antagonized by the addition of HIF-1β protein. Of great importance, C/EBP{alpha} overexpression significantly inhibited DNA-binding ability of HIF-1 protein, as evidenced by EMSA and ChIP assays. In parallel, C/EBP{alpha} transfection also suppressed expressions of HIF-1-bound HRE-driven Luc and HIF-1-targeted genes including VEGF, Glut-1 and PGK1. Moreover, the suppression of C/EBP{alpha} expression by specific shRNA increased DNA-binding ability of HIF-1 in the ChIP assay, and it also elevated expressions of HIF-1-targeted genes in leukemic U937 cells. All these results support that C/EBP{alpha} inhibits the transcriptional activity of HIF-1. Moreover, the promyelocytic leukemia (PML) tumor suppressor, which also contributes to leukemic cell differentiation (41), is identified as a negative regulator of the synthesis rate of HIF-1{alpha} by repressing mammalian target of rapamycin. Thus, PML is also a critical inhibitor of neoangiogenesis (42). All these results suggest that inhibition of HIF-1 function is possibly a common feature of hematopoiesis-related transcription factors. The inhibiton of HIF-1 function leads to the reduced expression of VEGF, and thus inhibiting angiogenesis, which favors to form a hypoxic microenvironment and thus for leukemic cell differentiation.

On the other hand, HIF-1 is an important regulator of the growing tumor's response to hypoxia (43,44). In tumors, HIF-1 activity depends on the availability of the HIF-1{alpha} subunit. Substantial reports showed that no HIF-1{alpha} expression was detected in normal tissue and in benign tumors such as breast fibroadenoma and uterine leiomyoma (45), whereas overexpression of HIF-1{alpha} can be detected in the majority of solid tumors (46). Clinically, increased tumor HIF-1{alpha} is correlated with increased angiogenesis, aggressive tumor growth, poor patient prognosis and treatment failure in a number of cancers. All these observations lead to the current interest in HIF-1{alpha} as a cancer drug target, as widely reviewed (44,47). On the contrary, C/EBP{alpha} acts as a classical transcription factor in a range of cell types including lung, liver, mammary gland and skin besides the hematopoietic system. Therefore, C/EBP{alpha} is absent in most solid tumors, and C/EBP{alpha} also acts as a tumor suppressor in C/EBP{alpha}-expressing tissues beyond hematopoietic system (19). Increasing lines of evidence showed that C/EBP{alpha} is down-regulated in a large proportion of some kinds of cancers such as lung cancer (48), hepatocellular carcinoma (49), skin squamous cell carcinoma (50) and primary breast cancer (51). Recently, Tada et al. (52) reported that DNA hypermethylation of the upstream C/EBP{alpha} promoter region is critical in the regulation of C/EBP{alpha} expression in human lung cancer. Furthermore, C/EBP{alpha} has been shown to interact with and alter the activities of several cell cycle-related proteins (19), and antitumor function of C/EBP{alpha} mainly contributes to its anti-mitotic potential. According to our results that C/EBP{alpha} protein significantly inhibited transcriptional activity-dependent function of HIF-1 through their direct interaction, we propose that inhibition of HIF-1 activity is also involved in antitumor feature of C/EBP{alpha} protein in C/EBP{alpha}-expressing tissues. By the way, Runx1 also inhibits the DNA-binding and transcriptional activity of HIF-1 protein, as reported previously (40). Thus, HIF-1 presents higher transcriptional activity in solid tumors due to the absence or decreased expression of C/EBP{alpha} and Runx1 protein, which will favor angiogenesis formation and pathogenesis/metastasis of solid tumors.

Collectively, this work provides the first demonstration that C/EBP{alpha} significantly inhibits the DNA-binding ability and transcriptional activity of HIF-1 through their interaction, which disturbs formation of HIF-1{alpha} and HIF-1β dimerization. These important discoveries explore mechanisms of C/EBP{alpha} to interfere microenvironment of tissues, which favors leukemic cell differentiation, and provide new insights for antitumor potential of C/EBP{alpha} protein.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Ministry of Science and Technology (NO2002CB512806, No.2006CB910104); National Natural Science Foundation of China (30630034, 30500265); Science and Technology Commission of Shanghai (05JC14032). Ms L. Yang is a PhD candidate at Shanghai Institutes for Biological Sciences, and this work is submitted in partial fulfillment of the requirement for the PhD. Dr G.Q. Chen is a Chang Jiang Scholar of Ministry of Education of People's Republic of China, and is supported by Shanghai Ling-Jun Talent Program.


   Acknowledgments
 
We thank Drs A.F. Gombart and Y. Fujii-Kuriyama for generously providing plasmids. We are grateful to Mr L.P. Song and Ms J. Zhang for their assistance in shRNA-related work.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received August 2, 2007; revised November 5, 2007; accepted November 10, 2007.


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