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Molecular Biology and Genetics |
Department of Molecular Biology, Pusan National University, Pusan 609-735 [S-K. B., M-H. B., M-Y. A., M. J. S., Y. M. L., M-K. B., O-H. L., K-W. K.], and Liver Clinic, Pusan 614-621 [B. C. P.], Korea
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies have shown that hypoxia detected in the central region of a tumor can be the leading cause of angiogenesis by activating the expression of angiogenic factors (2
, 6
, 7)
. Of the angiogenic factors, expression of the vascular endothelial growth factor (2
, 8)
, platelet-derived growth factor- 
(7)
, interleukin-6 (9)
, interleukin-8 (10)
, and acidic/basic fibroblast growth factor (11)
genes are known to be hypoxia-inducible.
Much interest in IGF-II4 has been generated by recent reports on the role of IGF-II protein as a potent mitogen (12) and inhibitor of apoptosis (13 , 14) in several normal and neoplastic cell types. IGF-II is also known to exhibit an angiogenic activity in rat cornea assay (15) and quantitative chorioallantoic membrane assay (16) . The human IGF-II gene is located on chromosome 11p15.5, downstream of the insulin gene, and spans 30 kb, which includes nine exons and four promoters (17) . Transcription of the human IGF-II gene yields six kinds of mRNA species, which are expressed on tissue-specific manners during the development phase (18) .
A dynamic control of the four promoters (P1-P4) of the IGF-II gene has been shown in hepatic development (19) . The P1 promoter is active exclusively in human adult liver, whereas the activity of the P2 promoter has been detected only in certain human tumor cell lines. In contrast, P3 and P4 are highly active in fetal hepatic tissues but down-regulated shortly after birth (20) . Enhanced levels of P3- and P4-driven IGF-II mRNA have been detected in many human tumors of different origins including hepatocellular carcinoma (21) . These data suggest a role for P3- and P4-derived transcripts in autocrine or paracrine growth stimulation during tumorigenesis. However, the molecular mechanisms underpinning the activation of P3 and P4 promoters during the hepatocarcinogenesis are still unclear.
In this study, we show that the expression of the IGF-II gene is induced by hypoxia-mediated P3 promoter activation. Furthermore, we present the first evidence that Egr-1, a zinc finger-containing transcription factor, is involved in the activation of IGF-II transcription by hypoxia.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Primers.
Oligonucleotide primers for PCR were designed as follows: -actin, 5'-GACTACCTCATGAAGATC-3' and 5'-GATCCACATCTGCTGGAA-3'; Egr-1, 5'-GAGCCGAGCGAACAACCCTACGAGCACCTG-3' and 5'-GCGCTGAGGATGAAGAGGTTGGAGGGTTGG-3'; Sp1, 5'-AATTTGCCTGCCCTGAGTGC-3' and 5'-TTGGACCCATGCTACCTTGC-3'; WT-1, 5'-ACGCCCTCGCACCATGCGGCGCAGTTCCCC-3' and 5'-CCTTTGGTGTCTTTTGAGCTGGTCTGAACG-3'. A double-stranded oligonucleotide (Ew) containing two Egr-1 consensus sequences (CGCCCCCGC) separated by one nucleotide and an oligonucleotide (Em) containing two mutated Egr-1 consensus sequences (CGCCCTAGC), and an oligonucleotide containing consensus sequences for transcriptional factor Sp1 (CCGCCC) were from Santa Cruz Biotechnology (Santa Cruz., CA).
Cell Culture and Hypoxic Condition.
The human hepatoma cell line, HepG2, was cultured in MEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml of penicillin, and 100 mg/ml of streptomycin. After 3 days, the cells were washed with serum-free MEM, replaced with complete medium, and incubated under normoxic (5% CO2, balanced with air) or hypoxic (1% O2, 5% CO2, balanced with N2) conditions in a humidified incubator with an interior temperature of 37°C.
RT-PCR.
Total RNA was isolated from cell cultures by a single-step procedure with TRI REAGENT (Life Technologies, Inc.). First-strand cDNAs were synthesized from 5 µ g of total RNA by incubating in 20 µ l of aliquots containing 200 units of superscriptase (Life Technologies, Inc.) and 500 ng of oligo-dT primer at 42°C for 1 h. PCR reaction was performed on the first-strand cDNA using the PCR reaction kit (Perkin-Elmer) and primer sets. Amplification by PCR was performed using an automated thermal cycler (Perkin-Elmer). A thermal cycler was set to the following cycle parameters: -actin, at 94°C for 4 min (1 cycle), 94°C for 30 s, 55°C for 30 s, 72°C for 1 min (35 cycles), 72°C for 5 min (1 cycle); Egr-1, at 94°C for 3 min (1 cycle), 94°C for 30 s, 55°C for 30 s, 72°C for 1 min (30 cycles), 72°C for 5 min (1 cycle); Sp1, at 94°C for 3 min (1 cycle), 94°C for 1 min, 60°C for 1 min, 72°C for 1 min (30 cycles), 72°C for 5 min (1 cycle); WT-1, at 94°C for 3 min (1 cycle), 94°C for 1 min, 55°C for 2 min, 72°C for 2 min (35 cycles), 72°C for 5 min (1 cycle).
Northern Blot Analysis.
Total RNA was prepared as described above. RNAs (20 µg) were fractionated on 1.2% agarose-6.6% formaldehyde gels, transferred to Zeta-Probe nylon membrane (Bio-Rad, Hercules, CA), and were covalently linked by UV irradiation (UV Stratalinker 1800; Stratagene, La Jolla, CA). The membrane was prehybridized at 42°C for 1 h in 50% formamide, 0.12 M Na2HPO4 (pH 7.2), 0.25 M NaCl, 7% SDS, and 1 mM of EDTA. Hybridization was then performed with the addition of 20 ng of 
32P-labeled DNA probes at 42°C for 16 h. The membrane was washed twice with washing solution (0.5 x SSC/0.1% SDS) at 50°C for 30 min and exposed to X-ray film with intensifying screens at -70°C for 24 h. -32P-labeled DNA probes were prepared by using random primer oligonucleotides (redi prime DNA Labeling System, Amersham, United Kingdom).
mRNA Stability Assay.
The mRNA stability of Egr-1 was determined by treating HepG2 cells with the transcription inhibitor, actinomycin D, as follows. Actinomycin D was added into the growth medium (25 µg/ml) to block transcription. After 1 h, the cells were returned either to normoxic or to hypoxic conditions and incubated for 2 h. Total RNA was isolated at 3 h after treatment of actinomycin D, and RT-PCR was performed as described.
Plasmid Construction and Serial Deletions of IGF-II P3 Promoter.
The human IGF-II P3 construct, HUP3, was cloned into the pSLA3 vector, which contains the luciferase gene as a reporter, by the procedure reported previously (22)
. HUP3 was cut with Eco RI and Sal I, agarose gel-purified, and cloned between the Eco RI and Sal I sites in the multiple cloning site of the vector pBluescript (Stratagene, Heidelberg, Germany). This construct was then digested with Bam HI and Xho I and subcloned into pXP-2 luciferase vectors to construct N-terminal deletion mutants of the IGF-II P3 promoter, pXP-2-P3. p 
125 (from -169 to +110) and p255 (from -39 to +110) were made in pXP-2 luciferase vectors using the Sma I and Alu I site in the IGF-II P3 promoter sequence.
Transient Transfection.
The calcium phosphate-mediated cotransfection procedure (23)
for adherent cells was used with the following modifications. Briefly, 5 x 105 HepG2 cells were plated in 60-mm dishes and transfected after changing the medium the next day with the calcium phosphate method. The medium was removed after 24 h, and the cells were supplied with fresh medium and incubated under hypoxic conditions. Control cells were transfected in parallel and cultivated under normal oxygen pressure. After 6, 16, and 24 h, cells were harvested, lysed, and then assayed for luciferase activity. For studying involvement of Egr-1 in the induction of IGF-II P3 promoter activity under hypoxic conditions, HepG2 cells were cotransfected with the IGF-II P3 promoter-luciferase reporter plasmid and increasing amounts of Egr-1 expression vector pEgr-1. After the 24-h transfection, cells were washed with serum-free medium and supplied with fresh medium. After another 48 h, cells were harvested, lysed, and then assayed for luciferase activity.
Luciferase Assay.
Cells were harvested after transfection, and extracts were prepared with reporter lysis buffer (Promega, Madison, WI). Cell extracts were assayed for luciferase activity with the luciferase assay kit (Promega) and a luminometer (Turner Designs, Sunnyvale, CA). Extracts were also assayed for -galactosidase activity with the -galactosidase enzyme assay system (Promega) and assayed for protein concentration with the protein assay kit (Bio-Rad). Each extract was assayed three times, and the mean RLU was corrected by values obtained from an extract prepared from nontransfected cells. The relative luciferase activity was calculated as RLU/-galactosidase.
Preparation of Nuclear Extracts.
Cells were washed once in PBS followed by preparation of nuclear extracts using modifications of the procedure of Dignam et al. (24)
. Briefly, the cells were lysed in buffer A [10 mM HEPES, 1.5 mM MgCl2, 10 mM NaCl, 0.25% NP40 (pH 7.5)] for 5 min at 4°C, followed by centrifugation at 4000 rpm for 2 min. The supernatant containing cytosol was removed, and the nuclei were extracted with buffer C[20 mm of HEPES, 25% glycerol, 0.42 M NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 0.25% NP40 (pH 7.5)]. The nuclei were vortexed vigorously several times over 20 min, followed by centrifugation at 14,000 rpm for 5 min. The supernatant (nuclear extract) was removed, diluted 1:2 with buffer D[20 mm of HEPES, 50 mM of KCl, 0.2 mM of EDTA, 20% glycerol (pH 7.5)], and frozen at -80°C until use.
EMSAs and Supershift Assays.
EMSAs were performed as previously described methods (25)
. Nuclear proteins from HepG2 cells were used in binding reactions. The double-stranded oligonucleotides for the Egr-1 were 5'-end-labeled with [
-32P]ATP (10 mCi/ml; Amersham, United Kingdom) using T4 polynucleotide kinase and purified by ethanol precipitation. The DNA binding reaction was performed by preincubating nuclear extract proteins (10 µg) in 20 mMof HEPES (pH 7.5), 50 mMof KCl, 1 mMof DTT, 1 mm of EDTA, 5% glycerol, 2 µg of poly(dI·dC) at 4°C for 15 min, followed by the addition of the double-strand 32P-labeled oligonucleotides (
0.2 ng) and a second incubation at 25°C for 30 min. Oligonucleotide competition experiments were performed with unlabeled wild-type or mutant Egr-1 or unlabeled Oct-1 double-stranded oligonucleotide. After preincubation for 15 min on ice, labeled wild-type Egr-1 probe was added and incubated at 25°C for 30 min.
In the supershift assays, the antibodies (Santa Cruz Biotechnology, Santa Cruz., CA) were added to the reaction mixture subsequent to the addition of the 32P-labeled oligonucleotide probe and incubated at 4°C for 1 h. Samples were subjected to electrophoresis on a native 5% polyacrylamide gel in 0.25 x Tris-borate EDTA for 2 h at 200 V. Gels were vacuum-dried before autoradiography.
Western Blot Analysis.
Nuclear proteins (10 µg) were separated on 12% nonreducing PAGE (26)
. After transfer, nitrocellulose membranes were washed in TBS-T solution [20 mM Tris-HCl buffer (pH 7.6) containing 137 mM NaCl, 1% Tween 20] and incubated in a blocking buffer (5% skim milk in TBS-T) at room temperature for 2 h. After washing with TBS-T three times, the filters were probed with first polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz., CA) against Egr-1 at room temperature for 5 h and then washed three times with TBS-T. The primary antibodies were detected by horseradish peroxidase-conjugated secondary antibody and visualized by the Amersham ECL system after intensive washing of the membranes.
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RESULTS |
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-gal plasmid constitutively expressing -galactosidase under the control of the SV40 promoter and enhancer. The transfected cells were exposed to hypoxia, harvested, and assayed for luciferase and -galactosidase activities. As shown in Fig. 1
, reporter gene expression by hypoxia was increased 1.4-fold at 6 h, 2.9-fold at 16 h, and 3.7-fold at 24 h. This result demonstrates that the activation of the P3 promoter was responsible for the induction of the IGF-II gene by hypoxia.
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, RT-PCR analysis demonstrated that the strong induction of Egr-1 transcripts was detected at 2 h of hypoxia, with declined expression up to 8 h. In contrast to Egr-1 mRNA, neither a significant increase nor decrease of Sp1 and -actin mRNA by hypoxia was observed at up to 8 h of hypoxia. Interestingly, the level of WT1 mRNA was dramatically decreased after 8 h of hypoxia. The induction of Egr-1 transcripts under hypoxia was confirmed by Northern blot analysis (Fig. 2B)
. Hypoxia could increase Egr-1 mRNA levels either by increasing its rate of transcription or by decreasing its destruction. To evaluate the effect of hypoxia on the steady-state level of Egr-1 mRNA, HepG2 cells were pretreated with actinomycin D for 1 h before hypoxic or normoxic treatment for 2 h. Total RNA was harvested, and the amount of Egr-1 mRNA was measured by RT-PCR. Hypoxia did not affect the stability of Egr-1 mRNA (Fig. 2C)
. Thus, the increase of Egr-1 mRNA levels during hypoxia was not due to the increased stability but to the transcriptional activation.
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, EMSAs of nuclear extracts from hypoxia-exposed HepG2 cells showed enhanced binding activity of Egr-1 to its binding sequence (Lanes 3 and 4), compared with their counterparts in normoxia (Lane 2). Competition experiments showed that an excess of unlabeled Egr-1 oligonucleotides (Lanes 6 and 7) completely blocked the appearance of the hypoxia-induced gel-shift band. In contrast, excess unlabeled mutant Egr-1 (Lane 8) or Oct-1 oligonucleotides (Lane 9) had no effect, indicating that sequence-specific DNA binding activity of Egr-1 was involved. Moreover, the protein-DNA complex was supershifted by the anti-Egr-1 antibody (Lanes 11–13). But the DNA-binding activity of Sp1 was not changed by hypoxia (data not shown). Taken together, the results in Fig. 3
indicate that induction of the P3 promoter activity of the human IGF-II gene by hypoxia is, in part, mediated through the EBSs in the P3 promoter region and that hypoxia enhances the DNA binding activity of the Egr-1 protein.
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, which is consistent with observations from EMSA and supershift studies (Fig. 3)
. These results demonstrate that Egr-1 was constitutively induced in hypoxia-exposed HepG2 cells and that the enhanced level of Egr-1 by hypoxia contributes in part to the DNA binding activity.
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-gal control plasmid. As shown in Fig. 5A
, increasing Egr-1 expression vector relative to the reporter plasmid resulted in increasing activity of the P3 promoter, causing maximally 2.5-fold higher luciferase values than those when only the reporter vector is present. Furthermore, hypoxia synergistically induced the promoter activities observed in the Egr-1 cotransfectant (Fig. 5B)
. All these data support the possibility of Egr-1 as a critical factor in the activation of P3-directed IGF-II expression by hypoxia.
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. A construct containing the promoter sequence of the three EBSs, HUP3, was included as a positive control of the hypoxia-induced promoter. These constructs were transiently transfected in the HepG2 cells to assess a putative effect of EBSs on the transcription of IGF-II with the IGF-II P3 promoter. Luciferase activities were measured 24 h later and normalized to -galactosidase activities. The activity of the hypoxia-treated IGF-II P3 promoter compared to that of the control promoter was reproducibly 3.3-fold higher in the HepG2 cells (Fig. 6)
. Activities of the deletion constructs are shown as a relative activity of the longest HUP3 construct (Fig. 6A)
. The pXP-2-P3 construct (from -294 to +110 bp), which contains three EBSs, retains most of the promoter activity responding to hypoxic stress (Fig. 6B)
. In contrast, deletion of the region from -294 to -169 bp (p125) and -294 to -39 bp (p255) resulted in an almost complete loss of the promoter activity (Fig. 6B)
. Therefore, we propose that the region of -294 to -169 bp, the first EBS, is likely to contain the important regulatory elements in response to hypoxia.
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DISCUSSION |
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Reactivation of the P3 promoter of the IGF-II gene, resulting in continued expression of IGF-II, was found to be an important driving force of cell proliferation during hepatocarcinogenesis (21
, 28)
. However, little is known about the molecular mechanism of P3 promoter reactivation during tumorigenesis. In the present studies, we have demonstrated that hypoxia in the central region of the tumor cells may act as a triggering signal for reactivating the human IGF-II P3 promoter (Fig. 1)
. Our sequence analysis of the human IGF-II P3 promoter failed to find hypoxia-responsive element sequences that exist in the promoters of vascular endothelial growth factor and erythropoietin genes. This failure strongly implies the existence of other regulatory mechanisms for the IGF-II P3 promoter activation.
Transcription factors Egr-1 and Sp1 are known to play a role in the activation of the P3 promoter (27)
. Interestingly, WT1 that binds the same DNA sequences as Egr-1 displays opposite effects on the transcription of target genes of EBSs (29)
. In fact, it was previously reported that the human IGF-II P3 promoter was repressed by the WT1 product and a continued synthesis of large amounts of IGF-II was observed by functional loss of WT1 activity in Wilms’ tumor (30)
. Our data demonstrated for the first time that hypoxia up- and down-regulates the expression of the Egr-1 (Fig. 2, A and B)
and WT1 genes (Fig. 2A)
, respectively, and hypoxia also activates the DNA binding activity of the Egr-1 protein as detected by EMSA (Fig. 3)
. Moreover, our Western blot analysis reveals that the amount of Egr-1 is markedly elevated in the nuclei of HepG2 cells by hypoxia (Fig. 4)
. The increase of Egr-1 mRNA was not due to its increased stability because actinomycin D treatment did not change the Egr-1 mRNA level under hypoxic conditions (Fig. 2C)
. By deletion analysis of the IGF-II P3 promoter sequences, we showed that the sequences containing the upstream EBS1 is primarily responsible for transcriptional activation by hypoxia (Fig. 6B)
. From these results, we conclude that hypoxia increases the level and/or the activity of Egr-1, thus enhancing the transcription of the IGF-II gene through Egr-1-mediated P3 promoter activation. In addition, we further defined a critical role for the EBS1 region of the IGF-II P3 promoter in the elevated expression of the Egr-1 gene of HepG2 cells and identified that Egr-1 nuclear proteins bind to the IGF-II P3 element in a specific and a functional manner under hypoxic conditions.
However, it remains unclear by what precise mechanisms hypoxia induces the expression of Egr-1. Recently, it was shown that a posttranslational modification of the transacting factors, such as a phosphorylation event (31)
or redox shift (32)
, might play a role in the hypoxia signal transduction pathway. Previous reports (33)
indicate that the possible phosphorylation event is required for the activation of the Egr-1 protein and also that the induction of Egr-1 gene expression mediates through its own binding site because the Egr-1 promoter contains a high-affinity binding site for the Egr-1 protein. From these reports, it seems that hypoxia causes at first the phosphorylation of the Egr-1 protein or other proteins that regulate the activity of Egr-1. Then the active form of the Egr-1 protein increases the expression of the Egr-1 gene itself and other target genes containing EBSs in their promoter regions such as the IGF-II gene. As shown in Fig. 4B
, a synergistic response was observed when cells were cotransfected with Egr-1 and exposed to hypoxia. If the effect of hypoxia per se was to increase the synthesis of Egr-1 and such synthesis was sufficient for a maximal transcriptional response to hypoxia, the effects of Egr-1 and hypoxia should be additive at best.
Therefore, the observed synergism indicates that additional events, including the posttranslational modification (e.g., phosphorylation) of Egr-1, should occur under hypoxia. Another possible explanation for this synergism relates to the level of other proteins affecting the activity of Egr-1. This possibility is supported by our present result that WT1, functioning as a repressor of Egr-1, is reduced in its expression under hypoxia. This decreased level may be necessary for a maximal activation of the IGF-II P3 promoter in hypoxic cells.
It has been previously reported that Egr-1 and c-fos have a similar signaling pathway (34) . Therefore, it might be that the mitogen-activated protein kinase-dependent pathway is involved in the induction of Egr-1 by hypoxia. In fact, a recent report (35) shows that p38 and Jun kinase 1, not extracellular signal-regulated kinase 2 that is activated by various stresses such as heat shock, sodium arsenite, UV radiation, and anisomycin are involved in the induction of Egr-1 in NIH 3T3 cells. However, our data do not exclude these possibilities or any other unknown pathways of signal transduction in hypoxia-induced Egr-1 expression. Further delineations are needed to define underlying mechanisms.
In conclusion, our studies demonstrate that Egr-1 activated by hypoxia can positively regulate the activity of the human IGF-II P3 promoter. Egr-1 induction and the role of IGF-II as a autocrine/paracrine growth factor as well as an angiogenic factor can be significant in pathological conditions such as hepatoma where cells are exposed to hypoxic environments. Thus, it is likely that the Egr-1 protein is an important nuclear intermediate in signal transduction processes under hypoxia and one of the crucial targets in hepatocellular carcinogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant of the 1998 Korean Cancer Control Program, the Ministry of Health and Welfare, and the Molecular Medicine Research Group Program in the Ministry of Science and Technology, Korea. 
2 Present address: Institute for Virus Research, Kyoto University, Shogoin-Kawahara, Sakyo-ku, Kyoto 606-8507, Japan.
3 To whom requests for reprints should be addressed, at Department of Molecular Biology, Pusan National University, Pusan 609–735, Korea. Phone: 82-51-510-2277; Fax: 82-51-513-9258; E-mail: kimkw{at}hyowon.pusan.ac.kr
4 The abbreviations used are: IGF-II, insulin-like growth factor II; WT1, Wilms’ tumor suppressor gene; MEM, minimal essential medium; RT-PCR, reverse transcription-PCR; RLU, relative light units; EMSA, electrophoretic mobility shift assay; EBS, Egr-1 binding site; TBS, Tris-buffered saline.
Received 5/28/99. Accepted 10/ 4/99.
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, indicates EBS s (GCGGGGGCG), and each EBS is numbered. B, the plasmids (9 µg) were transfected into HepG2 cells in the presence of 1 µg of the pSV-