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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1547
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 3 1712-1719
Copyright © 2005 by The Endocrine Society

Hypoxia Up-Regulates Hypoxia-Inducible Factor-1{alpha} Expression through RhoA Activation in Trophoblast Cells

Masami Hayashi, Masahiro Sakata, Takashi Takeda, Masahiro Tahara, Toshiya Yamamoto, Ryoko Minekawa, Aki Isobe, Keiichi Tasaka and Yuji Murata

Department of Obstetrics and Gynecology (M.H., M.S., T.T., M.T., R.M., A.I., K.T., Y.M.), Osaka University Faculty of Medicine, Suita, Osaka 565-0871, Japan; and Osaka Medical Center for Cancer and Cardiovascular Diseases (T.Y.), Higashinari-ku, Osaka 537-0025, Japan

Address all correspondence and requests for reprints to: Masahiro Sakata, M.D., Ph.D., Department of Obstetrics and Gynecology, Osaka University, Faculty of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: msakata{at}gyne.med.osaka-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During early pregnancy, trophoblast cells are exposed to relatively low-oxygen tension. Recently, the Rho GTPase family has been shown to play a key role in hypoxia-inducible factor-1 (HIF-1) {alpha} induction in renal cell carcinoma. The present study was designed to investigate the effect of low-oxygen conditions on RhoA expression in trophoblast cells isolated from early stages of human placenta and in trophoblast-derived BeWo cells and JAR cells. Immunoblot and RT-PCR analyses showed that low-oxygen conditions (1% O2 or 250 µM CoCl2) stimulated expression of RhoA protein and mRNA. Pull-down assays demonstrated that these low-oxygen conditions increased RhoA activity. Preincubation of BeWo cells with Clostridium botulinum C3 exoenzyme, a specific inhibitor of Rho, inhibited hypoxia-induced HIF-1{alpha} expression. Under 1% O2 or 250 µM CoCl2, BeWo cells, transfected with a dominant-negative RhoA, exhibited decreased levels of HIF-1{alpha} protein and mRNA compared with the control vector transfectants. BeWo cells expressing constitutively active RhoA showed enhanced protein levels of not only HIF-1{alpha} but also vascular endothelial growth factor (VEGF) and glucose transporter 1, which are target gene products of HIF-1{alpha}. These findings suggest that up-regulation of RhoA induced by low-oxygen conditions may play an important role in regulation of HIF-1{alpha} expression in trophoblast cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE EARLY stage of pregnancy, the trophoblast cells invade into the decidua of the uterus, establishing a mixture of fetal and maternal cellular elements in the placental bed. During this period, maternal blood flow to the placenta is relatively limited, causing the trophoblast cells to be exposed to a low-oxygen environment (1). The proliferation of the trophoblast cells is stimulated in this low-oxygen environment (2, 3), which is essential for appropriate embryonic development. Several cellular responses, such as erythropoiesis and angiogenesis, are stimulated under hypoxia (4, 5). These enhanced responses are mediated by heterodimeric hypoxia-inducible factor-1 (HIF-1){alpha} (6). HIF-1 is a transcriptional activator that mediates expression of several genes in response to the cellular oxygen concentration (6).

HIF-1 possesses two subunits, HIF-1{alpha} and HIF-1ß, both of which belong to the basic-loop-helix-PAS protein family (7). The level of HIF-1{alpha} is dependent on the cellular oxygen concentration (7), whereas the level of HIF-1ß is not (8, 9). HIF-1{alpha} is maintained at a low level in normoxic cells by degradation of the protein through the ubiquitin-proteasome pathway (10). In the presence of normal levels of oxygen, the binding of von Hippel-Lindau protein to the conserved oxygen degradation domain of HIF-1{alpha} results in iron-dependent hydroxylation, whereas this hydroxylation is inhibited under hypoxic conditions. This mechanism accounts for HIF-1{alpha} stabilization in hypoxic cells, allowing HIF-1{alpha} to translocate into the nucleus and to dimerize with HIF-1ß (11, 12, 13). Previous reports have demonstrated that HIF-1{alpha} is highly expressed in human trophoblast cells in early stages of pregnancy (14, 15). However, little is known about the regulatory mechanisms of HIF-1{alpha} expression in placental cells.

One of the cellular events that follow low-oxygen tension is ATP depletion, which results in the disruption of the actin cytoskeleton in diverse types of cells (16, 17, 18). Regulation of actin stress fibers is mediated mainly by the Rho GTPase family (19). Rho proteins have two conformations (19, 20): one is a GTP-bound state, and the other a GDP-bound state. Rho proteins function as molecular switches cycling between the inactive GDP-bound state and active GTP-bound state. The activity of the Rho GTPases is determined by the ratio of their GTP/GDP-bound forms in the cell (21). Previous reports have demonstrated that constitutively active RhoA prevented disruption of stress fibers and cortical F-actin during chemical ATP depletion caused by antimycin A (22), which suggests that recovery of stress fiber reassembly requires RhoA. A recent report demonstrated that Rho proteins are up-regulated in hypoxia and involved in HIF-1{alpha} induction in renal cell carcinoma (23). Therefore, the present study was designed to characterize the expression and activity of RhoA and to analyze the potential roles of RhoA during the low-oxygen condition in placental cells, using trophoblast cells isolated from first-trimester placentas. We also used trophoblast-derived BeWo cells and JAR cells as models for investigating the molecular regulatory mechanisms of placental gene and protein expression, using techniques such as transfection of DNAs. BeWo cells and JAR cells have been previously used as trophoblast models for studying several aspects of placental gene expression and trophoblast invasion (24, 25, 26, 27). In this report, we show, for the first time, that RhoA expression and activity are significantly increased under low-oxygen conditions in placental cells. Our findings demonstrated that hypoxia-induced RhoA regulates HIF-1{alpha} induction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Fetal bovine serum (FBS) was purchased from JRH BIOSCIENCES (Lenexa, KS). Percoll was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). CoCl2 was purchased from Sigma (St. Louis, MO). Clostridium botulinum C3 exoenzyme and Rhotekin Rho binding domain (Rhotekin RBD) were purchased from Upstate Biotechnology (Lake Placid, NY). Vector (pcDNA3.1) was obtained from Invitrogen Corp. (Carlsbad, CA). Constitutively active RhoA (pcDNA3.1-RhoA G14V-Myc) and dominant-negative RhoA (pcDNA3.1-RhoA T19N-Myc) vector plasmids were obtained from Guthrie cDNA Resource Center (Sayre, PA).

Tissue collection

Human placental tissues from the first trimester were collected from 12 normal pregnancies that were voluntarily terminated by dilation and curettage between 7 and 9 wk of gestation. Informed consent was obtained from each patient before obtaining the placental explants. The protocol was approved by the local ethics committee of the Department of Obstetrics and Gynecology, Faculty of Medicine, Osaka University. Placental tissues were collected in ice-cold PBS, transported to the laboratory on ice, and processed within 2 h as described previously (14).

Isolation and purification of trophoblast cells

Trophoblast cells were prepared as described by Kliman et al. (28), with modifications as follows. Placental tissue was washed in 0.9% (wt/vol) NaCl, dissected from the membranes, and cut into small pieces (2–3 mm) under the microscope. They were incubated in a shaking water bath for 20 min at 37 C in calcium- and magnesium-free Hanks’ solution containing 25 mM HEPES (pH 7.4), 0.125% trypsin (Sigma), and 0.2 mg/ml deoxyribonuclease I (1500 Kunitz units/mg protein) (Sigma). The tissue fragments were then allowed to settle for 1 min. The supernatant was layered over FBS and centrifuged at 1000 x g for 10 min. The resultant pellets were resuspended in 5 ml DMEM (Invitrogen Corp.) containing 25 mM HEPES and 25 mM glucose. The remaining placental tissue was subjected to the digestion procedure again, with addition of fresh solution containing trypsin and deoxyribonuclease. The resultant cell suspensions were pooled and centrifuged through a Percoll gradient as described by Kliman et al. (28). We obtained placental cells (3–4 x 107 cells) from placental tissue (approximately 18 g) and plated them at a density of 2 x 105 cells/cm2 in DMEM medium containing 10% FBS. After 18 h of incubation, the isolated cells were cultured in fresh medium every 24 h.

Purity of the cells isolated from first-trimester placental tissue was assessed concomitantly on the experiments by characterization using immunofluorescence analysis, as described previously (29), with modifications as follows. Cells were fixed with 3.7% paraformaldehyde in PBS for 20 min, incubated with 5% normal goat serum in PBS, and incubated with primary antibody at 4 C overnight. We used anticytokeratin 7 mouse monoclonal antibody (DaKoCytomation, Glostrup, Denmark), antivimentin mouse monoclonal antibody (DaKoCytomation), and anti-Von Willebrand Factor mouse monoclonal antibody (DaKoCytomation). After washing, samples were incubated with Alexa Fluor 488-labeled goat antimouse IgG. The images were recorded and analyzed using a Nikon confocal microscope TE2000-U (Nikon Corp., Tokyo, Japan). Over 94% of the cells were stained with anticytokeratin 7 antibody. Vimentin-positive cells and Von Willebrand Factor positive cells was less than 5% and less than 1%, respectively.

Cell culture

The human choriocarcinoma cell line BeWo was obtained from the Health Science Research Resources Bank (Osaka, Japan). The cells were cultured in Ham’s F12 medium (Sigma) containing 15% FBS. JAR cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in RPMI 1640 medium (Invitrogen Corp.) containing 10% FBS.

Low-oxygen experimental design

Before low-oxygen experiments, first-trimester trophoblast cells or trophoblast-derived cells (BeWo cells and JAR cells) were cultured at 37 C in 5% CO2 in air incubator. Then, the cells were incubated under the low-oxygen conditions. A low-oxygen environment from 1–10% O2 was attained by incubation of the cells in a controlled oxygen incubator. The oxygen was maintained at the above oxygen concentration by a compact gas oxygen controller APM-36 (ASTEC, Fukuoka, Japan) with a residual gas mixture composed of 94–85% N2-5% CO2. A low-oxygen environment of CoCl2 was obtained by incubation of the cells in a standard 5% CO2 in air incubator with medium containing 250 µM CoCl2.

Immunoblot analysis

Whole-cell proteins from primary cultured trophoblast cells, BeWo cells, and JAR cells were extracted as described previously (30). Samples were electrophoresed and transferred to a nitrocellulose filter (Bio-Rad, Hercules, CA) using standard procedures. For detection of RhoA, VEGF, or glucose transporter (GLUT) 1 protein, the filter was blocked with 5% (wt/vol) nonfat milk in 10 mM Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 2 h at room temperature followed by overnight incubation at 4 C with anti-RhoA rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology), or anti-GLUT1 rabbit polyclonal antibody (Chemicon International, Temecula, CA), respectively. After washing three times in TBS-T, the filter was incubated with secondary antirabbit antibody (Santa Cruz Biotechnology) in TBS-T for 1 h at room temperature and developed for the detection of specific protein bands using enhanced chemiluminescence reagents (Amersham Biosciences Corp., Piscataway, NJ).

Nuclear extracts were prepared from BeWo cells as described previously (31, 32). In brief, samples were electrophoresed and transferred to a nitrocellulose filter. The filter was presoaked and treated as described above, and then incubated with anti-HIF-1{alpha} mouse monoclonal antibody (Novus Biologicals, Littleton, CO), followed by incubation with secondary antimouse antibody (Santa Cruz Biotechnology). After washing, the filter was developed for the detection of specific protein bands using enhanced chemiluminescence reagents.

Both whole-cell extract and nuclear extract protein concentrations were measured with a DC protein assay kit (Bio-Rad) using the modified method of Lowry et al. (33).

The Rho pull-down assay

The Rho pull-down assay was performed as described previously (34). Briefly, BeWo cells were treated under normoxic conditions (20% O2) or low-oxygen conditions (1% O2 or 250 µM CoCl2) for 2 h. The cells were washed twice with PBS and lysed in magnesium lysis buffer (Upstate Biotechnology). After each cell lysate was centrifuged, half of the sample was treated with 30 µg Rhotekin RBD-agarose beads at 4 C for 60 min. The beads were washed three times with magnesium lysis buffer. Bound Rho proteins were detected by immunoblot analysis using anti-RhoA mouse monoclonal antibody (Santa Cruz Biotechnology). For the comparison of RhoA activity (level of GTP-bound Rho), immunoblot analysis of the total amount of RhoA in cell lysates was performed using the remainder of the samples.

RT-PCR

Total RNA was extracted from monolayers of cells with TRIzol reagent (Invitrogen) according to the manufacturer’s directions. Total RNA (1 µg) was reverse-transcribed using a first-strand cDNA synthesis kit (Invitrogen Corp.), following the manufacturer’s instructions. An aliquot (2 µl) of reverse transcription product was used for PCR amplification in a total vol of 100 µl. The PCR primer sets used for RhoA, HIF-1{alpha}, and ß-actin cDNA amplification were as follows: RhoA (35) sense 5'-CTG GTG ATT GTT GGT GAT GG-3', antisense 5'-GCG ATC ATA ATC TTC CTG CC-3'; HIF-1{alpha} (36) sense 5'-GTC GGA CAG CCT CAC CAA ACA GAG C-3'; antisense 5'-GTT AAC TTG ATC CAA AGC TCT GAG-3'; ß-actin (BD Biosciences Clontech, Palo Alto, CA) sense 5'-ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3', antisense 5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3'. The thermal cycle profile used for RhoA was 25 cycles of denaturation at 94 C for 30 sec, annealing at 58 C for 1 min, and extension at 72 C for 1 min (23). The profile for HIF-1{alpha} was 25 cycles of denaturation at 95 C for 30 sec, annealing at 55 C for 1 min, and extension at 72 C for 2.5 min (23). For ß-actin, 25 cycles of 45 sec at 94 C, 45 sec at 60 C, and 2 min at 72 C (37, 38) were carried out. PCR amplification for RhoA, HIF-1{alpha}, and ß-actin was performed in the range of the linear relationship between the cycle number and the intensity of RT-PCR product (data not shown). PCR fragments were analyzed by electrophoresis on 1.8% agarose gels and stained with ethidium bromide.

Transient transfection

Lipofectamine (Life Technologies, Rockville, MD)-mediated transfection of BeWo cells was performed as described previously (31, 32), with modifications as follows. BeWo cells or JAR cells were plated into 6-well plates (2 x 105/well or 1 x 105/well, respectively) for 20 h. The cells were transfected with 2 µg pcDNA (control vector), pcDNA-RhoAG14V-2XMyc (constitutively active RhoA), or pcDNA-RhoAT19N-2XMyc (dominant-negative RhoA). Four hours after transfection, the medium was changed, and the cells were cultured for 48 h in complete medium.

Statistical analysis

Data were expressed as the mean ± SEM. Statistical analysis was performed by paired t test or ANOVA for multiple comparisons, followed by Scheffé’s F test. Differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Low-oxygen conditions increase RhoA protein and mRNA levels

To investigate the effects of low-oxygen concentration on the expression of RhoA protein in primary cultured trophoblast cells, we first examined the expression of RhoA protein, by immunoblot analysis, using protein samples prepared from first-trimester placentas. In preliminary experiments, the level of RhoA expression was examined by immunoblot analysis after various periods of time under 1% O2 or 250 µM CoCl2. Exposure of cells to the above conditions resulted in the greatest stimulation after 2 h (data not shown). As shown in Fig. 1AGo, RhoA protein expression was up-regulated by 2.6-fold under 1% O2 compared with that under 20% O2. In the presence of 250 µM CoCl2, an agent with effects that mimic those of low-oxygen, RhoA protein expression was also increased by 2.9-fold compared with that under 20% O2. We then examined the expression of RhoA protein in trophoblast-derived BeWo cells and JAR cells as trophoblast models. As shown in Fig. 1BGo, the level of RhoA protein in BeWo cells was up-regulated by 3.7-fold under 1% O2 and by 4.1-fold in the presence of 250 µM CoCl2 compared with that in BeWo cells cultured under 20% O2. RhoA protein expression in JAR cells was also increased under low-oxygen conditions compared with that under 20% O2 (3.2-fold under 1% O2 and 3.1-fold in the presence of 250 µM CoCl2) (Fig. 1CGo). Using the trophoblast model, we examined the effect of culture in a range of oxygen tensions (1% O2, 5% O2, 10% O2, and 20% O2) on RhoA protein expression. As shown in Fig. 1DGo, the level was significantly increased under both 5% O2 (3.8-fold) and 1% O2 (3.9-fold) compared with that under 20% O2, whereas the expression of RhoA under 10% O2 was not significantly increased compared with that under 20% O2. Furthermore, the expression under both 5% O2 (2.4-fold) and 1% O2 (2.5-fold) were significantly increased compared with that under 10% O2. To investigate whether the increases of RhoA protein levels under the conditions were due to enhanced expression at the transcriptional level, we compared the expression of RhoA mRNA in BeWo cells under 20% O2 to that in the cells under 1% O2 or in the presence of 250 µM CoCl2. As shown in Fig. 2Go, RhoA mRNA expression in BeWo cells was up-regulated by 2.2-fold under 1% O2 and by 2.1-fold in the presence of 250 µM CoCl2 compared with that under 20% O2.



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  		FIG. 1. Effect of low levels of oxygen on RhoA protein expression in first-trimester human trophoblast cells, BeWo cells, and JAR cells. Whole-cell extract proteins (50 µg) were subjected to immunoblot analysis using anti-RhoA and -ß-actin antibodies. A, Trophoblast cells isolated from human first-trimester placental tissue were cultured under 20% O2 (lane 1), 1% O2 (lane 2), or 250 µM CoCl2 (lane 3) for 2 h. Histograms show densitometric analyses of relative protein expression levels. The density of the control bands (20% O2) was set at 100. Values shown represent the mean ± SEM from three separate experiments with different placenta. Significant differences from the control band (20% O2) density are indicated by asterisks (*, P < 0.05). B and C, Parallel experiments and quantitative analyses were performed using BeWo cells and JAR cells, respectively. The density of the control band (20% O2) was set at 100. Values shown represent the mean ± SEM from three separate experiments. Significant differences from the control band (20% O2) density are indicated by asterisks (*, P < 0.05; **, P < 0.01). D, BeWo cells were cultured under 20% O2, 10% O2, 5% O2, or 1% O2 for 2 h. Histograms show densitometric analyses of relative protein expression levels. The density of the control band (20% O2) was set at 100. Values shown represent the mean ± SEM from three separate experiments. Significant differences from the control band (20% O2) density are indicated by asterisks (**, P < 0.01).

 


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  		FIG. 2. Effect of low levels of oxygen on RhoA mRNA expression in BeWo cells. Total RNA was isolated from BeWo cells cultured under 20% O2 (lane 1), 1% O2 (lane 2), or 250 µM CoCl2 (lane 3) for 1 h. RT-PCR amplification (25 cycles) was performed using primers for RhoA or ß-actin. Histograms show densitometric analyses of relative mRNA expression levels. The density of the control bands (20% O2) was set at 100. Values shown represent the mean ± SEM from three separate experiments. Significant differences from the control band (20% O2) density are indicated by asterisks (*, P < 0.05).

 
Low-oxygen conditions activate RhoA

To understand whether the increased level of RhoA under low-oxygen tension depends on the increase of the activated state of Rho, we measured the intracellular level of the GTP-bound Rho, which is the active form, using the pull-down assay system. As shown in Fig. 3Go, the level of the active form of RhoA (Rhotekin RBD-bound RhoA) was elevated in BeWo cells by 3.5-fold under 1% O2 and by 3.7-fold in the presence of 250 µM CoCl2 compared with that under 20% O2. These findings demonstrate that the increase of RhoA protein expression under low-oxygen tension was accompanied by an increase of the active form of RhoA.



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  		FIG. 3. RhoA activation in BeWo cells under low-oxygen conditions. The activation state of RhoA protein was evaluated by affinity precipitation with Rhotekin RBD-agarose. Cells were exposed to 20% O2 (lane 1), 1% O2 (lane 2), or 250 µM CoCl2 (lane 3) for 2 h, lysed, and incubated with fusion proteins. After pull-down precipitation, the Rhotekin RBD-agarose-bound form of RhoA protein was analyzed by SDS-PAGE and immunoblotting. Parallel experiments were performed to assess the total amounts of RhoA protein under these conditions. The density of the control band (20% O2) was set at 100. Values shown represent the mean ± SEM from three separate experiments. Significant differences from the control band (20% O2) density are indicated by asterisks (**, P < 0.01).

 
HIF-1{alpha} induction under low-oxygen conditions is inhibited by C3 exoenzyme

The level of HIF-1{alpha} protein has been reported to increase in response to hypoxia in various tissues (7, 8) and BeWo cells (32). To determine whether low-oxygen conditions affect HIF-1{alpha} protein expression in our experimental system, we analyzed the expression of HIF-1{alpha} protein in both trophoblast cells and BeWo cells using immunoblot analysis (Fig. 4Go, A and B). As shown in Fig. 4Go, A and B, the cells cultured under both 1% O2 and 250 µM CoCl2 showed increased expression of HIF-1{alpha} protein compared with those cultured under 20% O2.



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  		FIG. 4. Effect of low levels of oxygen on HIF-1{alpha} protein expression. Nuclear extract proteins (15 µg) were subjected to immunoblot analysis using anti-HIF-1{alpha} and -ß-actin antibodies. A, Trophoblast cells isolated from human first-trimester placental tissue were cultured under 20% O2 (lane 1), 1% O2 (lane 2), or 250 µM CoCl2 (lane 3) for 4 h. B, Parallel experiments were performed using BeWo cells. Molecular mass markers are shown on the right, and the locations of the 120-kDa HIF-1{alpha} bands are shown by arrows. The experiment was repeated three times with similar results.

 
C3 exoenzyme has been found to inhibit selectively RhoA, RhoB, and RhoC via ADP ribosylation (39). To evaluate the role of Rho GTPase in HIF-1{alpha} induction under low-oxygen tension in placental cells, we next examined whether C3 exoenzyme affects hypoxia-induced HIF-1{alpha} protein expression, using BeWo cells as a trophoblast model. As shown in Fig. 5AGo, HIF-1{alpha} expression was increased under 1% O2 or 250 µM CoCl2, and incubation with C3 exoenzyme inhibited the hypoxia-induced increase of HIF-1{alpha} protein expression, respectively. Under 20% O2, however, pretreatment with C3 exoenzyme had no significant effect on HIF-1{alpha} expression. We also examined the effect of C3 exoenzyme on HIF-1{alpha} mRNA expression (Fig. 5BGo). The increased level of HIF-1{alpha} mRNA under 1% O2 or 250 µM CoCl2 was inhibited by preincubation with C3 exoenzyme. These results suggest that the hypoxia-induced increase of HIF-1{alpha} expression is mediated by Rho.



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  		FIG. 5. Effect of C3 exoenzyme on low levels of oxygen-induced HIF-1{alpha} protein and mRNA expression. BeWo cells were pretreated with 5 µg/ml C3 exoenzyme for 24 h under 20% O2. A, Then the cells were placed under 20% O2 (lanes 1 and 2), 1% O2 (lanes 3 and 4), or 250 µM CoCl2 (lanes 5 and 6) for 4 h and lysed. Nuclear extract proteins (15 µg) from the cells were subjected to immunoblot analysis using anti-HIF-1{alpha} and -ß-actin antibodies. The experiment was repeated three times with similar results. B, After incubation under 20% O2 (lanes 1 and 2), 1% O2 (lanes 3 and 4), or 250 µM CoCl2 (lanes 5 and 6) for 2 h, total RNA was isolated. HIF-1{alpha} or ß-actin mRNA expression was evaluated by RT-PCR amplification. Histograms show densitometric analyses of relative mRNA expression levels. The density of the control bands (20% O2, without C3) was set at 100. Values shown represent the mean ± SEM from three separate experiments. Significant differences from the control band density are indicated by asterisks (**, P < 0.01).

 
Dominant-negative RhoA inhibits HIF-1{alpha} protein and mRNA induction

To further understand the relation between Rho GTPase and HIF-1{alpha} induction, we transfected BeWo cells with a control vector (pcDNA) or vector encoding dominant-negative RhoA (RhoA T19N). Because this mutated protein was tagged with Myc, the expression of RhoA T19N was confirmed with an anti-Myc antibody (data not shown). As shown in Fig. 6AGo, the level of HIF-1{alpha} protein under 20% O2 did not show any difference between the control vector transfectants and the dominant-negative RhoA transfectants. In contrast, the induction of HIF-1{alpha} protein observed under 1% O2 or 250 µM CoCl2 in the control vector transfectants was inhibited in the dominant-negative RhoA transfectants, respectively. These data indicate that Rho GTPase, especially RhoA, is a regulatory factor in the induction of HIF-1{alpha} protein under low-oxygen conditions. To investigate whether this effect was exerted at the transcriptional level, we next examined the expression of HIF-1{alpha} mRNA in BeWo cells transfected with the dominant-negative RhoA and those transfected with the control vector (Fig. 6BGo). The level of HIF-1{alpha} mRNA after transfection with the control vector showed significant increases (P < 0.01) in the presence of 1% O2 or 250 µM CoCl2 compared with 20% O2. In contrast, after transfection of the dominant-negative RhoA, the up-regulation of HIF-1{alpha} expression induced by the above conditions was inhibited. These findings demonstrate that the up-regulation and activation of RhoA induced by low-oxygen tension contribute to both HIF-1{alpha} mRNA and protein induction in placental cells.



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  		FIG. 6. Effect of dominant-negative RhoA on low levels of oxygen-induced HIF-1{alpha} protein and mRNA expression. BeWo cells were transfected with a control vector (pcDNA3.1) or a vector encoding dominant-negative RhoA (pcDNA3.1-RhoA T19N-Myc) for 48 h using Lipofectamine reagent. A, After incubation under 20% O2 (lanes 1 and 2), 1% O2 (lanes 3 and 4), or 250 µM CoCl2 (lanes 5 and 6) for 4 h, nuclear extract proteins (15 µg) were subjected to immunoblot analysis using anti-HIF-1{alpha} and -ß-actin antibodies. The experiment was repeated three times with similar results. B, After incubation under 20% O2 (lines 1 and 2), 1% O2 (lines 3 and 4), or 250 µM CoCl2 (lines 5 and 6) for 2 h, total RNA was isolated. HIF-1{alpha} or ß-actin mRNA expression was evaluated by RT-PCR amplification. The lower panel shows densitometric analysis to assess relative mRNA expression levels. The density of the control band (lane 1) was set at 100. Values shown represent the mean ± SEM from three separate experiments. Significant differences from the control band (lane 1) are indicated by asterisks (*, P < 0.05; **, P < 0.01).

 
Constitutively active RhoA stimulates HIF-1{alpha} expression under 20% O2

To determine whether overexpression of RhoA directly mediates an increase of HIF-1{alpha} mRNA and protein expression in placental cells under 20% O2, we transfected BeWo cells and JAR cells with a control vector (pcDNA) or a mutant vector that encodes constitutively active RhoA (RhoA G14V). Because this protein was tagged with Myc, the expression of RhoA G14V was confirmed with an anti-Myc antibody (data not shown). Enhanced HIF-1{alpha} protein expression under 20% O2 was shown in BeWo cells and JAR cells transfected with RhoA G14V compared with that in cells transfected with the control vector (P < 0.01) (Fig. 7Go, A and B, respectively). We also investigated RhoA-mediated regulation of HIF-1{alpha} expression at the transcriptional level. As shown in Fig. 7CGo, BeWo cells expressing constitutively active RhoA showed an increased level of HIF-1{alpha} mRNA, compared with control vector transfectants, under 20% O2 (P < 0.05). These data demonstrate that active RhoA directly affects the induction of HIF-1{alpha}.



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  		FIG. 7. Effect of a constitutively active form of RhoA on HIF-1{alpha} protein and mRNA expression under 20% O2. BeWo cells and JAR cells were transfected with a control vector (pcDNA3.1) (lane 1) or a vector encoding constitutively active RhoA (pcDNA3.1-RhoA G14V-Myc) (lane 2) using Lipofectamine reagent. A, Nuclear extract proteins (15 µg) from BeWo cells cultured under 20% O2 for 48 h were subjected to immunoblot analysis using anti-HIF-1{alpha} and -ß-actin antibodies. B, Nuclear extract proteins (30 µg) from JAR cells cultured under 20% O2 for 48 h were subjected to immunoblot analysis using anti-HIF-1{alpha} and -ß-actin antibodies. C, Total RNA was isolated from BeWo cells cultured under 20% O2 for 48 h. RT-PCR amplification was performed for 25 cycles using primers for HIF-1{alpha} or ß-actin. Histograms show densitometric analyses of relative protein or mRNA levels. The density of the control band (control vector) was set at 100. Values shown represent the mean ± SEM from three separate experiments. Significant differences from the control band (control vector) are indicated by asterisks (*, P < 0.05; **, P < 0.01).

 
Constitutively active RhoA stimulates VEGF and GLUT1 protein expression under 20% O2

To investigate the functional effect of HIF-1{alpha} induced by constitutively active RhoA, we examined the expression of VEGF (Fig. 8AGo) and GLUT1 protein (Fig. 8BGo) in BeWo cells. Cells expressing constitutively active RhoA showed significantly enhanced protein level of VEGF, compared with that in cells transfected with the control vector, under 20% O2 (P < 0.01). The level of GLUT1 protein was also significantly increased by constitutively active RhoA (P < 0.05).



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  		FIG. 8. Effect of a constitutively active form of RhoA on VEGF and GLUT1 protein expression under 20% O2. BeWo cells were transfected with a control vector (pcDNA3.1) (lane 1) or a vector encoding constitutively active RhoA (pcDNA3.1-RhoA G14V-Myc) (lane 2) for 48 h using Lipofectamine reagent. Whole-cell extract proteins (50 µg) were subjected to immunoblot analysis using anti-VEGF (A), -GLUT1 (B), and -ß-actin antibodies. Histograms show densitometric analyses of relative VEGF or GLUT1 protein levels. The density of the control band (control vector) was set at 100. Values shown represent the mean ± SEM from three separate experiments. Significant differences from the control band (control vector) are indicated by asterisks (*, P < 0.05; **, P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HIF-1 is a transcriptional activator that mediates changes in expression of several genes in cellular homeostatic responses to the oxygen concentration (6). HIF-1 is regulated mainly through oxygen-dependent degradation of its {alpha}-subunit. In the presence of oxygen, the binding of von Hippel-Lindau protein to oxygen degradation domain results in enzymatic hydroxylation and subsequent proteasomal degradation. However, stabilization of HIF-1{alpha} protein per se is not the only regulatory mechanism of HIF. Recently, it has been reported that HIF-1{alpha} expression could also be regulated at the transcriptional level (40, 41).

HIF-1{alpha} is highly expressed in human trophoblast cells during early pregnancy (14, 15). However, the precise mechanism of HIF-1{alpha} induction in placental cells has not been fully clarified. The results of the present study show that low-oxygen conditions induce expression and activation of RhoA in placental cells, which, in turn, is responsible for HIF-1{alpha} mRNA and protein induction. This RhoA pathway may play a major role in inducing these changes of HIF-1{alpha} expression because they are prevented by C3 exoenzyme or a dominant-negative form of RhoA. These observations are in agreement with a recent study reporting the relation between Rho GTPase and HIF-1{alpha} induction in renal cell carcinoma (23).

One of the cellular events that occur during hypoxia is ATP depletion (16), which disrupts the actin cytoskeleton in many cell types (17, 18, 42), followed by disruptions of cell polarity and intercellular junctions. It is well known that regulation of the actin cytoskeleton is mediated by Rho proteins (19, 20). Previous studies have demonstrated that transfection of a vector encoding constitutively active Rho prevented disruption of stress fibers and cortical F-actin during ATP depletion (22), suggesting that the activation of Rho signaling protects cells from damage by ATP depletion. These results lead us to speculate that Rho-proteins may play an important role in preventing ATP depletion and cytoskeleton disruption during low-oxygen conditions in placental cells. Thus, we characterized RhoA expression to investigate its potential roles in pathways involved in responses under low-oxygen conditions. Our results demonstrate, for the first time, that RhoA up-regulation and activation occurring under low-oxygen conditions are early events that contribute to the induction of HIF-1{alpha} mRNA and protein in placental cells.

Early placentation begins with the invasion of trophoblast cells into the maternal decidua. This is a critical and closely regulated process for placental development. Therefore, a better understanding of the mechanisms controlling trophoblast migration is required. During this period, the trophoblast cells are exposed to a hypoxic physiological environment (1). However, little is known about the mechanisms underlying cellular adaptations to changes in oxygen tension in normal embryonic development and placentation. In the present study, low-oxygen tension-mediated up-regulation of RhoA expression and activation in placental cells is responsible for the induction of HIF-1{alpha}. What is the physiological significance of the up-regulation and activation of RhoA during placental development? HIF-1 binds to specific DNA sequences and activates transcription of various genes encoding cytokines, growth factors, and enzymes such as VEGF (43), platelet-derived growth factor (44), aldolase A (45), lactate dehydrogenase A (46), and GLUT1 (47). We showed that BeWo cells, transfected with a mutant vector encoding constitutively active RhoA, stimulated VEGF and GLUT1 protein expression under 20% O2. These results suggest that RhoA induction is one of cellular adaptations to changes in oxygen tension.

Production of reactive oxygen species (ROS) is a possible sensor mechanism by which cells could detect the decrease in oxygen tension (48). In renal cell carcinoma cells, Turcotte et al. (23) reported that hypoxia stimulates ROS production, which is required to increase RhoA expression. Further research will be required to clarify whether ROS are involved in the up-regulation of RhoA under low-oxygen conditions in trophoblast cells.

The small GTPase RhoA mediates a variety of cellular responses, including activation of the contractile apparatus, growth, gene transcription, and differentiation (19, 49). RhoA also represents a key regulatory molecule during cell migration that is associated with a reorganization triggering changes in cell morphology (50, 51, 52). Previous reports have demonstrated that the pathway between RhoA and its effector, Rho-associated coiled coil-forming protein kinase, is one of the signals regulating the migration of cytotrophoblast cells under the physiologically low-oxygen environment during early placentation (53). Consequently, in addition to HIF-1{alpha} induction, hypoxia-mediated RhoA up-regulation itself in placental cells may lead to phenotype modulation in response to low-oxygen conditions and may play an important role in the migration of trophoblast cells under the low-oxygen environment that occurs physiologically during early placentation.

In summary, the data presented herein indicate that low-oxygen tension induces RhoA expression and activation, which leads to HIF-1{alpha} induction in placental cells. Data from this study may provide clues for future directions in clarifying the molecular mechanisms of placental HIF-1{alpha} regulation during early placentation. Additional studies will be required to understand the consequences of RhoA activation and HIF-1{alpha} expression in placental cells and their roles in the development of normal placentation or abnormal placentation, such as that in preeclampsia.


   Footnotes
 
First Published Online December 14, 2004

Abbreviations: FBS, Fetal bovine serum; GLUT1, glucose transporter 1; HIF-1, hypoxia-inducible factor-1; RBD, Rhotekin Rho binding domain; ROS, reactive oxygen species; TBS, Tris-buffered saline; TBS-T, TBS containing 0.1% Tween 20; VEGF, vascular endothelial growth factor.

Received August 4, 2004.

Accepted December 2, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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