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Department of Obstetrics and Gynecology (H.N., T.N., M.H., Y.O., O.I., K.I.), Tokyo Medical University, Tokyo 160-0023, Japan; Laboratory of Human Carcinogenesis (L.E.H.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255
Address all correspondence and requests for reprints to: Hirotaka Nishi, M.D., Ph.D., Department of Obstetrics and Gynecology, Tokyo Medical University, 6-7-1 Nishishinjuku Shinjuku, Tokyo 160-0023, Japan. E-mail: nishih{at}tokyo-med.ac.jp.
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Abstract |
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and TGF-ß3 are overexpressed in preeclamptic placenta. Hypoxia not only transactivates the TGF-ß3 promoter activity but also enhances endogenous TGF-ß3 expression. Using the TGF-ß3 promoter deletion mutants, we show that the region between –90 and –60, which contains a putative HIF-1 consensus motif, is crucial for HIF-1-mediated transactivation. Electrophoretic mobility shift assays show that HIF-1 binds to the oligonucleotide containing the HIF-1 motif. Also, introduction of an antisense oligonucleotide for HIF-1 diminishes TGF-ß3 expression during hypoxia, indicating that the up-regulation of TGF-ß3 by hypoxia is mediated through HIF-1. Our results provide evidence that regulation of TGF-ß3 promoter activity by HIF-1 represents a mechanism for trophoblast differentiation during hypoxia. |
Introduction |
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HIF-1 is a heterodimeric transcription factor composed of the basic helix loop helix-Per Arnt Sim-PAS proteins HIF-1 and the arylhydrocarbon receptor nuclear translocator (ARNT). HIF-1 mediates the transcriptional response to oxygen deprivation by binding to hypoxia response elements (HRE) within the promoters or enhancers of genes involved in glycolysis, glucose transport, erythropoiesis, and angiogenesis (9, 10). HIF-1 activity is critical for normal development. HIF-1 activity is essential for the proliferation, survival, and/or differentiation of multiple embryonic tissues (11).
Caniggia et al. (8) show that TGF-ß3 plays a vital role in trophoblast differentiation through hypoxia, and its overexpression can be found in preeclamptic placenta. They also show that antisense inhibition of HIF-1 down-regulates TGF-ß3 mRNA expression in villous explants (12). Although TGF-ß3 is an important factor in trophoblast differentiation, the molecular mechanisms by which hypoxia/HIF-1 activates TGF-ß3 are not fully understood. Interestingly, our computer-assisted homology searches have revealed potential binding sites for HIF-1 on the gene promoter of TGF-ß3.
Based on these observations, we support the hypothesis that early in the first trimester (<10 wk) the low oxygen tension environment maintains trophoblasts in a relatively immature, proliferative state, which is mediated by TGF-ß3 through HIF-1. However, the mechanism underlying HIF-1-induced TGF-ß3 expression is unclear. The present study was designed to test whether HIF-1 is involved in hypoxia-induced activation of the TGF-ß3 gene promoter. Our data provide evidence that the induction of TGF-ß3 promoter activity by hypoxia is mediated by HIF-1 through a HRE located at –84 to –77 in the proximal promoter. This study adds the TGF-ß3 gene to the list of hypoxia-inducible genes regulated by this transcription factor.
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Materials and Methods |
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and TGF-ß3 expression. The samples were taken from central cotyledons. The tissues were finely minced into small pieces with scissors, washed in 0.9% sterile saline to avoid contamination of red blood cells that probably interfere with PCR, snap frozen, and stored at –80 C until used for the telomeric repeat amplification protocol assay, Western blot, and real-time RT-PCR.
Cell culture
The human choriocarcinoma cell lines, JEG-3 and JAR, were maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum. For hypoxic exposure, cells were subjected to 1% oxygen in a water-jacketed CO2 incubator (NAPCO, Winchester, VA) at 37 C in a humidified atmosphere with 5% CO2 for indicated times.
RT-PCR analysis
Total RNA was isolated using Isogen (Nippon Gene, Tokyo, Japan). Five micrograms of total RNA were reverse transcribed into cDNA using an RT-PCR kit (Stratagene, La Jolla, CA) according to the manufacturer’s recommendations. The TGF-ß3 was amplified using the following primer pair (8): TGF-ß3 primers, 5'-CAAAGGGCTCTGGTGGTCCTG-3' (forward) and 5'-CTTGGAGGTAATTCCTTTAGGG-3' (reverse).
One-microliter aliquots of the cDNA and 10 pmol of each primer were subjected to PCR using Ready-to-Go PCR beads kit (Amersham Pharmacia Biotech, Uppsala, Sweden). The PCR conditions were 30 cycles of 94 C for 10 sec, 57 C for 10 sec, and 72 C for 30 sec for TGF-ß3. The amplified products were fractionated on a 1.5% agarose gel. The gel was stained with ethidium bromide. The efficiency of cDNA synthesis from each sample was estimated by PCR with glyceraldehyde-3-phosphate dehydrogenase primers.
Real-time RT-PCR analysis
Total RNA was isolated using Isogen reagent (Nippon Gene) and quantified by A260/A280 measurement using an Ultraspec 3000 (Amersham). Total RNAs (5 µg) were reverse transcribed into cDNAs. Real-time PCR was performed for the quantitative estimation. Twenty microliters of PCR reactions were set up with final concentrations of 5 mm MgCl2, 2 µl SYBR Green mastermix (Roche Molecular Biochemicals, Indianapolis, IN), 5 µl of 1:10 diluted cDNA, and 0.3 µM of both forward and reverse primers, which were the same as those used in RT-PCR analysis. The reactions were then cycled in the LightCycler (Roche) with the following parameters: denaturation for one cycle at 95 C for 10 sec, 45 cycles (temperature transition of 20 C/sec) of 95 C for 0 sec, 50 C for 10 sec, and 72 C for 15 sec and fluorescence reading taken at 72 C, and melting curve analysis with continuous fluorescence reading. If necessary, optimization of the protocol was achieved by changing MgCl2 concentrations and/or reading fluorescence at a higher temperature and/or using LightCycler-FastStart DNA master SYBR Green I (Roche). The LightCycler software generated a standard curve (measurements taken during the exponential phase of the amplification) that enabled the amount of each gene in each test sample to be determined.
Western blot analysis
Cells and tissues were lysed on ice for 30 min in lysis buffer (10 mM Tris, pH 8.0; 1 mM EDTA, 400 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 5 mM sodium fluoride, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol) containing complete protease inhibitor cocktail (Roche). The lysate was subjected to centrifugation at 14,000 rpm for 15 min, and the soluble fraction was collected. Protein concentrations were measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (40 µg) were loaded onto a 4–12% sodium dodecyl sulfate-polyacrylamide gel and subjected to electrophoresis at 200 V for 50 min. The protein was transferred onto a polyvinylidene difluoride membrane and probed with anti-TGF-ß3 antibodies (V; Santa Cruz Biotechnology, Santa Cruz, CA), anti-HIF-1 antibody (NB100-105; Novus Biologicals, Littleton, CO), anti-ARNT antibody (H-172; Santa Cruz Biotechnology), and antiactin antibody (C4; Roche). The same blot was probed after stripping the membrane with the different antibodies. Each protein was detected by horseradish peroxidase-conjugated secondary antibody coupled with enhanced chemiluminescence Western blotting detection reagents (Amersham). Each Western blot analysis was performed at least twice. Each band intensity was normalized by the intensity of the actin band.
DNA plasmids
The HIF-1 and ARNT expression vectors were described previously (10). pB3-1387-luc is a TGF-ß3 promoter-luciferase reporter plasmid in which a 1387-bp sequence upstream of the transcription start site of TGF-ß3 is cloned into pGL3-Basic (Promega, Madison, WI) from pB3-1387-CAT (13). Reporter plasmids pB3-499-luc, pB3-91-luc, and pB3-60-luc contain the 499-bp, 91-bp, and 60-bp promoter regions of TGF-ß3, respectively. pB3-1387-CAT, pB3-499-CAT, pB3-91-CAT, and pB3-60-CAT were kindly provided by Dr. Seong-Jin Kim (Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD).
Transfections and luciferase assays
Cells were seeded at 5 x 105 cells/35-mm dish and incubated overnight at 37 C in a 5% CO2 incubator. For each transfection, 1.0 µg of empty vector and/or expression vectors along with 0.3 µg of promoter-luciferase DNA were mixed in 0.2 ml of Opti-MEM (Life Technologies, Gaithersburg, MD), and a precipitate was formed using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s recommendations. Cells were washed with Opti-MEM, and complexes were applied to the cells. Cells were then exposed to hypoxia conditions (1% oxygen). Twenty-four hours after transfection, cells were harvested, and extracts were prepared with luciferase cell lysis buffer (PharMingen, San Diego, CA). Luciferase activity was measured in extracts from triplicate samples using the luciferase assay kit (PharMingen).
Antisense oligonucleotide and transfections
To inhibit the expression of endogenous HIF-1, we prepared the HPLC-purified antisense phosphorothioate oligonucleotide (AS) and, as a control, the sense oligonucleotide (SE) according to the sequence of the HIF-1 gene (12). The sequences of the HIF-1 AS and SE were 5'-GCCGGCGCCCTCCAT-3' and 5'-ATGGAGGGCGCCGGC-3', respectively. All nucleotides were phosphorothioated. The AS or SE (0.6 mM) was complexed with Lipofectamine 2000 reagent (Life Technologies) and applied to cells. Cells were then exposed to hypoxia conditions (1% oxygen). Twenty-hour hours after transfection, cells were harvested, and lysates were prepared for Western blot analysis.
In vitro transcription/translation
HIF-1 protein was synthesized in vitro in the presence of unlabeled amino acids using the pcDNA3-HIF-1 and pcDNA3-ARNT expression constructs with the coupled transcription/translation system (TNT) from Promega. Translated products were analyzed by Western blotting.
Electrophoretic mobility shift assays
Oligonucleotides containing the HIF-1consensus DNA binding site were purchased as single-stranded DNAs from Genosys Biotechnologies (Woodlands, TX). Double-stranded oligonucleotides were prepared by annealing complementary oligonucleotides in a buffer containing 10 mM Tris (pH 8.0), 500 mM NaCl, and 1 mM EDTA. The sequences of the complementary pairs are as follows: HRE/TGF-ß3, 5'-AAGAGGCTGCGTGCGCTGGTCC-3' and 5'-GGACCAGCGCACGCAGCCTCTT-3'; and HRE, 5'-TCTGTACGTGACCACACTCACCTC-3' and 5'-GAGGTGAGTGTGGTCACGTACAGA-3'.
Equimolar amounts of the complementary oligonucleotides were mixed in a 1.5-ml microcentrifuge tube and placed in a heat block at 95 C. The heat block was allowed to cool to room temperature, and the samples were desalted on a G-25 microspin column (Amersham). The double-stranded oligonucleotides were end-labeled with 32P using T4 polynucleotide kinase and [
-32P]ATP. For EMSA, end-labeled double-stranded oligonucleotides (5000 cpm) were incubated with 2 µl of HIF-1 protein prepared by in vitro transcription/translation at room temperature (22 C) for 30 min in the presence of a binding buffer containing 25% glycerol, 250 mM NaCl, 50 mM Tris (pH 8.0), 2.5 mM dithiothreitol, 5 mM EDTA (pH 7.5), and 150 ng polydeoxyinosinic deoxycytidylic acid. When competition assays were performed, an unlabeled HRE or TGF-ß3/HRE was incubated with protein and buffer for 5 min before the addition of each labeled oligonucleotide. When supershift assays were performed, 0.5 µg of anti-HIF-1 antibody (OZ15; Lab Vision Corporation, Fremont, CA) was incubated with protein and buffer for 5 min before the addition of each labeled oligonucleotide. Samples (20 µl) were loaded onto a 5% nondenaturing polyacrylamide gel and subjected to electrophoresis at 100 V for 1.5 h using 0.5x Tris-borate EDTA (1x Tris-borate EDTA: 89 mM Tris, 8 mM boric acid, and 2 mM EDTA, pH 8.3) as running buffer. After electrophoresis, gels were exposed to Kodak XAR film (Kodak, Rochester, NY) with intensifying screens at –80 C.
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Results |
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and TGF-ß3 expression in human placentas
). Quantitative real-time RT-PCR analysis revealed that preeclamptic placentas expressed higher levels of TGF-ß3 mRNA than the placenta tissues from normal pregnancy (Fig. 1B). We next examined HIF-1 and TGF-ß3 protein expression in placentas by Western blot analysis. We found that all eight preeclampsia placentas expressed both HIF-1 and TGF-ß3 protein (Fig. 1C and data not shown). Neither HIF-1 nor TGF-ß3 protein expression was detected in placenta samples from normal pregnancy. Therefore, TGF-ß3 mRNA expression was correlated with HIF-1 protein levels.
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and TGF-ß3 expression, ARNT, and TGF-ß3 protein expression in choriocarcinoma cancer cells, JAR and JEG-3. To analyze whether there is a correlation between HIF-1 and TGF-ß3, we examined the expression of the two genes in response to 1% oxygen. Whole-cell extracts prepared from control and cells exposed to 1% oxygen for 24 h were subjected to Western blot analysis. As shown in Fig. 2, both HIF-1 and TGF-ß3 expression were increased significantly by this condition, whereas ARNT protein expression was not altered.
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, and ARNT. HIF-1 overexpression transactivated the TGF-ß3 promoter by more than 20-fold (Fig. 3B). We prepared two reporter constructs (pB3-499-luc and pB3-90-luc) that lack the distal putative HRE and another reporter construct (pB3-61-luc) that lacks both the distal and proximal putative HREs (Fig. 3A). With these deletion mutants, HIF-1 overexpression was able to increase the activity of pB3-499-luc and pB3-90-luc but not of pB3-61-luc, suggesting that only proximal HRE is involved in TGF-ß3 transactivation by HIF-1 (Fig. 3B). We obtained similar results by luciferase assays using JAR cells (data not shown).
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and ARNT prepared by in vitro transcription/translation and subjected to electrophoresis. A DNA-protein complex was formed when TGF-ß3/HRE was incubated with HIF-1programmed rabbit reticulocyte lysate (Fig. 4A) but not with unprogrammed lysate (data not shown). This complex was specifically retarded by anti-HIF-1 antibody (Fig. 4A). The addition of a 50-fold molar excess of unlabeled HIF-1 consensus oligonucleotide (Santa Cruz Biotechnology) markedly reduced the binding, and a 200-fold molar excess completely diminished it (Fig. 4A). To further substantiate an HIF-1 binding to these oligonucleotides, we performed competition assays using end-labeled HIF-1 consensus oligonucleotide and cold TGF-ß3/HRE as a competitor. The unlabeled TGF-ß3/HRE competed with the HIF-1 binding to the wild-type probe when present in the reaction at 50-fold and 200-fold molar excess (Fig. 4B). The top band in Fig. 4 implies DNA-HIF-1 complex, whereas the others imply the constitutive binding of ATF-like proteins (14). These results confirm that HIF-1 binds to the TGF-ß3/HRE and indicate that the affinity of HIF-1 binding to the TGF-ß3/HRE is almost the same as that to the consensus HIF-1 sequence. The data indicate that hypoxia-induced TGF-ß3 expression is due to the enhanced activity of HIF-1 on the TGF-ß3 promoter. Taken together, these result indicate that the putative HIF-1 binding site in the proximal region of TGF-ß3 promoter is essential for the regulation of TGF-ß3 by HIF-1.
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expression (12). Thus, we used the AS to confirm whether hypoxia-induced TGF-ß3 expression is mediated by HIF-1 activation. When JAR and JEG-3 cells were transfected with AS and subsequently exposed to hypoxia (1% oxygen), AS down-regulated TGF-ß3 expression by more than 50% compared with the result from the SE-transfected cells (Fig. 5A). As a control, the level of actin was monitored and found to be unaltered by AS (Fig. 5A).
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). These results indicate that HIF-1 is required for hypoxic induction of TGF-ß3 expression. |
Discussion |
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protein in the human placenta significantly decreased with gestational age (12, 15), as shown in the present study. The finding of increased HIF-1 protein expression in the first trimester is consistent with the low intervillous blood flow and physiological hypoxia that has been reported during the early stages of placental development (7, 16). HIF-1 has been shown to be a major regulator of cell function and differentiation in a number of in vivo animal models and cell systems (10, 17, 18, 19). Our data support the observation that HIF-1 mediates the effects of oxygen on human trophoblast development. Thus, expression of HIF-1 protein in placental trophoblast is high early on between 5 and 8 wk and then falls precipitously around 10–12 wk, precisely at the time when the intervillous space is perfused by maternal blood and pressure of oxygen levels are believed to increase (7). This observation is consistent with a recent report showing that hypoxic exposure of isolated trophoblasts induces changes in the expression of proteins that control the cell cycle (20). Moreover, HIF-1 has recently been reported to control proliferation and apoptosis of embryonic stem cells by regulating genes such as p53, p21, and Bcl-2 (21). Together, these data suggest a key role for HIF-1 in regulating trophoblast proliferation under conditions of low oxygen tension. TGF-ß3 plays an important role in trophoblasts (12). The previous results showed that TGF-ß3 is associated with invasion of trophoblasts, and exposure of human trophoblasts to hypoxic conditions results in the induction of TGF-ß3 expression (8, 12). A failure to down-regulate expression of TGF-ß3 at around 9 wk of gestation results in shallow trophoblast invasion and predisposes the pregnancy to preeclampsia (12). However, it is unclear what mechanisms are involved in hypoxia-induced TGF-ß3 expression. To delineate the molecular mechanism for the activation of the TGF-ß3 gene during hypoxia, we examined the induction of TGF-ß3 promoter activity by hypoxia and HIF-1 in a transient expression system.
In addition, the level of TGF-ß3 was also increased under hypoxic conditions as determined by real-time RT-PCR analysis. In choriocarcinoma cell lines, cotransfection with both HIF-1 and ARNT expression vectors activated the TGF-ß3 promoter. When AS was used to reduce HIF-1 expression, TGF-ß3 expression was significantly diminished during hypoxia. According to electrophoretic mobility shift assays, we identified the active HRE in the TGF-ß3 core promoter. The HRE (5'-RCGTG-3') overlaps the E-box (CACGTG), which is known to bind several nuclear factors, such as c-Myc, Max, and Mad (11, 22, 23, 24). It is unclear whether HIF-1 competes with these factors for binding to these sites, whereas recent findings show that hypoxia down-regulates the c-Myc expression (25). Under hypoxic conditions, HIF-1 may play a predominant role in regulating promoter activity of TGF-ß3. Taken together, these data strongly indicate that hypoxia-induced TGF-ß3 expression is due to the enhanced activity of HIF-1 on the TGF-ß3 promoter. Furthermore, the proximal HRE in the TGF-ß3 promoter are essential for the up-regulation of TGF-ß3 during hypoxia because the TGF-ß3 promoter was not transactivated by hypoxia when the HRE-deleted TGF-ß3 promoter-luciferase construct was used.
There have been several descriptive reports of various markers associated with preeclampsia, including TNF-, vascular endothelial growth factor, and IGFs (26, 27, 28). The precise role, if any, of these proteins in preeclampsia is unclear. The data presented here demonstrate not only that abnormalities in HIF-1 expression are associated with preeclampsia but also that TGF-ß3 expression may restore the proliferating capability of preeclamptic trophoblasts. In placenta predisposed to preeclampsia, HIF-1 expression may be abnormally elevated through hypoxic conditions, and trophoblasts remain in a relatively immature state of differentiation. Consequently, trophoblast invasion into the uterus is limited, and uteroplacental perfusion is reduced. This conclusion is consistent with the clinical manifestations of preeclampsia, including shallow trophoblast invasion into the uterus and abnormally high trophoblast proliferation. Nonetheless, a large number of factors in preeclamptic pregnancies suggests that the pathogenesis of this disease is complex and likely involves many regulatory systems. The present study suggests that the monitoring of HIF-1 and TGF-ß3 expression may be a useful diagnostic marker of preeclampsia. These molecules may be novel targets for therapeutic intervention for preeclampsia.
In conclusion, we demonstrate that TGF-ß3 expression is up-regulated after induction of HIF-1 during exposure of hypoxia and that HIF-1 directly induces the TGF-ß3 transcription. Given that low oxygen level and TGF-ß3 activation are crucial for placental development, this function of HIF-1 may represent an important mechanism of placental growth mediated by TGF-ß3 during hypoxia.
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Acknowledgments |
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Footnotes |
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Abbreviations: ARNT, Arylhydrocarbon receptor nuclear translocator; AS, antisense oligonucleotide; HIF, hypoxia-inducible factor; HRE, hypoxia response element; SE, sense oligonucleotide.
Received December 2, 2003.
Accepted for publication May 14, 2004.
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References |
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subunit. J Biol Chem 271:32253–32259 in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394:485–490[CrossRef][Medline]
-fetoprotein gene expression under hypoxic conditions in human hepatoma cells: characterization of a negative hypoxia response element that mediates opposite effects of hypoxia inducible factor-1 and c-Myc. Cancer Res 62:1158–1165 concentrations and mRNA expression are increased in preeclamptic placentas. J Reprod Immunol 32:157–169[CrossRef][Medline]
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