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BOR - Papers in Press, published online ahead of print February 11, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.025031
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BIOLOGY OF REPRODUCTION 70, 1822–1827 (2004)
DOI: 10.1095/biolreprod.103.025031
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Normoxic Induction of the Hypoxic-Inducible Factor-1{alpha} by Interleukin-1ß Involves the Extracellular Signal-Regulated Kinase 1/2 Pathway in Normal Human Cytotrophoblast Cells1

Dong Qian, Hai-Yan Lin, Hong-Mei Wang, Xuan Zhang, Dong-Lin Liu, Qing-Lei Li, and Cheng Zhu2

State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During early pregnancy, an environment of relative low oxygen tension is essential for normal embryonic and placental vasculature. In low-oxygen conditions, the hypoxic-inducible factor-1 (HIF-1), composed of {alpha} and ß subunits, controls the expression of a number of genes such as vascular endothelial growth factor (VEGF), a key angiogenic factor. The recent studies in some tumor cells have found that the labile component, HIF-1{alpha}, is not only activated by hypoxia but also by peptides such as interleukin-1 (IL-1) in normoxia. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to IL-1ß stimulated the expression of HIF-1{alpha} protein. Meanwhile, IL-1ß also induced the secretion of VEGF in normal human cytotrophoblast cells. Our data indicated that IL-1ß induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Moreover, treatment of cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited the stimulation of HIF-1{alpha} protein expression and VEGF secretion by IL-1ß. These data indicate that, in normal human cytotrophoblast cells, IL-1ß induces HIF- 1{alpha}-mediated VEGF secretion and that IL-1ß-stimulated ERK1/2 activation may be involved in this process.

cytokines, growth factors, pregnancy, signal transduction, trophoblast


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Successful pregnancy outcome depends on the proper development of the fetoplacental vasculature in the villous core, which provides structure for the delivery of oxygen and nutrients from the intervillous space to the fetus [1]. Thus, vasculogenesis and angiogenesis as well as the entire repertoire of associated growth factors are remarkably activated during placental development. It has been well known that these processes are critically regulated by oxygen [1–3]. Oxygen tension effects are mediated by the hypoxia inducible factor-1 (HIF-1) [4]. Moreover, studies have shown that HIF-1 is highly expressed in placenta and plays an important role during early pregnancy [5, 6].

HIF-1 is a transcription factor that binds DNA as a heteromeric complex composed of two subunits, the constitutively expressed HIF-1ß (ARNT) and the inducible HIF- 1{alpha}, both of which are members of the bHLH-PAS superfamily of proteins. These proteins have a region of homology called the PAS (PER-ARNT-SIM) domain, which is an interaction domain that acts as ligand binder environmental sensor, and signal transducer [7, 8]. In the presence of O2 and iron, proline residue 546 in HIF-1{alpha} is hydroxylated, which enables binding of the von Hippel-Lindau tumor suppressor E3 ligase complex, and then HIF-1{alpha} is degraded by the proteasome [9, 10]. In hypoxia, the {alpha} and ß subunits dimerize in the nucleus and bind to the consensus sequence (A) CGTG present in the hypoxia response element of O2- controlled target genes, such as those encoding vascular endothelial growth factor (VEGF), erythropoietin (EPO), and distinct glycolytic enzymes [10].

Recent studies have shown that HIF-1 is not only induced by hypoxia, but is activated in normoxic cells in response to various growth factors and cytokines, including insulin-like growth factors (IGF) [11], tumor necrosis factor {alpha} (TNF{alpha}) [12], and interleukin-1ß (IL-1ß) [10, 12]. IL-1ß, known as one of the multifunctional cytokines, can induce numerous physiological effects in a wide variety of cells. Soluble IL-1ß is the predominant form in biological fluids, and it binds to specific receptors in target tissues [13]. Several signaling systems have been reported to participate in IL-1-induced-HIF-1{alpha} expression. Stiehl and coworkers found that IL-1ß induced HIF-1{alpha} accumulation in nuclei and stimulated VEGF production in cultured normoxic HepG2 cells, and this process was mediated by phosphatidylinositol 3-kinase (PI-3K) pathway [10]. Furthermore, evidence that reactive oxygen species (ROS) signaling mediates IL-1ß-dependent regulation of HIF-1{alpha} was ascertained in alveolar epithelial cells [14]. In addition, evidence suggests that the transcriptional activity of HIF-1 is modulated by phosphorylation. The p42/44 (ERK1/2) mitogen- activated protein kinase (MAPKs) catalyzes HIF-1{alpha} phosphorylation [15, 16]. However, whether IL-1ß-regulated- HIF-1{alpha} expression in normoxic cells involves ERK1/2 signaling is still unknown.

Previous studies have demonstrated that IL-1 is expressed in placental tissues and is implicated as an important mediator of angiogenesis [17, 18]. But whether IL-1 is involved in the expression of HIF-1 in normal cytotrophoblast cells is not clear. Herein, the possible involvement of ERK1/2 pathway in IL-1ß-regulated-HIF-1{alpha} expression and VEGF secretion in normal human cytotrophoblast cells was studied.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Recombinant human IL-1ß was provided by PEPROTECH EC Ltd. (London, England). PD98059 was purchased from Sigma (Oakville, ON, Canada). The oligonucleotides primers were synthesized from SBS Genetech (Beijing, China). Reagents and all general supplies not indicated otherwise were obtained from Sigma.

Isolation and Cultivation of Human Cytotrophoblast Cells

The normal cytotrophoblast cells were isolated and maintained as previously described, with some modifications [19–22]. Human chorionic villi tissues were obtained from patients who underwent therapeutic termination of pregnancy after 6–8 wk of gestation. Informed consent was provided by the patients, and the project was approved by the local ethics committee at the State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences. The time of gestation was defined according to the first day of the last menstrual period and further morphological examination was conducted by means of stereomicroscope. The experiments were repeated more than three times. Each tissue sample was minced separately and digested with 0.25% trypsin at 4°C for 45 min and 37°C for 10 min, then with 15 IU/ml DNase I at 37°C for 15 min. Trypsinization was stopped by addition of double volumes of FD medium (Ham F-12:Dulbecco modified Eagle medium: 1:1; Gibco-BRL, Gaithersburg, MD). After washing, the dispersed cells were filtered through a nylon sieve to remove the gross villous core residues. The filtered cell suspension (1–2 ml) was then added slowly to the top of a BSA gradient (prepared by sequential addition of 3 ml of 5%, 3%, 2%, and 1% BSA in FD medium to a 15-ml centrifuge tube). The cells were sedimented for 1 h at unit gravity, and cytotrophoblast cells were collected from the bottom of the tube. The purified cytotrophoblast cells were plated at 0.5–1 x 105 cells with 1 ml of FD medium supplemented with 10% FBS (Invitrogen, Gaithersburg, MD), 1 mM sodium pyruvate, and 2 mM glutamine in fibronectin-coated 24-well dishes. The cells were cultured in 95% air and 5% CO2 at 37°C and began to attach within 2 h after plating. The cells spread and showed monolayer epithelial cell morphology after 24 h. Immunocytochemical studies revealed that more than 99% of the cells exhibited positive staining for cytokeratin and GnRH and were vimentin negative, consistent with their identification as cytotrophoblast cells.

RNA Isolation and Semiquantitative Reverse Transcription-Polymerase Chain Reaction

Isolation of total cellular RNA was performed using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. Semiquantitative polymerase chain reaction (PCR) was based on the Esteve protocol [23] with some modifications. After removal of contaminating chromosomal DNA with DNase I treatment, aliquots of 1 µg of total cellular RNA were used for first-strand cDNA synthesis in 20 µl of reaction volume using 100 units of Superscript II reverse transcriptase (Invitrogen). Semiquantitative PCR was performed in a final volume of 25 µl containing 0.25 mM primers, 2.5 U of Taq polymerase (Takara, Dalian, China), 0.25 mM of each dNTP (Takara) and 1x reaction buffer (Takara). In order to compare the PCR products in a semiquantitative way, we i) determined the exponential phase of amplification by performing 25-30-35-40 cycles and, ii) amplified the gene for ß-actin (15-20-25 cycles) as internal control for cDNA quantity and quality [24]. The cDNA template was denatured for 5 min at 94°C and amplified as follows: HIF-1{alpha} (94°C 45 sec, 56°C 45 sec, 72°C 1 min, 30 cycles), ß-actin (94°C 45 sec, 55°C 45 sec, 72°C 45 sec, 25 cycles). Sequences of the primers were HIF-1{alpha}, forward 5'-TGG ACT CTG ATC ATC TGA CC-3' and reverse 5'-CTC AAG TTG CTG GTC ATC AG-3'; ß-actin, forward 5'-GTG GGG CCC CCC AGG CAC CA-3' and reverse 5'-CTC CTT AAT GTC ACG CAC GAT TTC-1;3'. The lengths of the HIF-1{alpha} and ß-actin amplicons were 433 and 548 base pairs, respectively. As internal controls for the reverse transcription (RT), samples without RNA or without reverse transcriptase were prepared in parallel, and these yielded no amplification products (data not shown). As negative controls for the PCR, samples without reverse-transcribed cDNA or without Taq enzyme were used (data not shown). PCR amplifications were performed on Perkin-Elmer 2400 GeneAmp PCR System (Perkin- Elmor, Wellesley, MA). PCR products were visualized on 1.5% agarose gels stained by ethidium bromide and ultraviolet transillumination. The bands were analyzed using MetaView image analyzing system (Version 4.50, Universal Imaging Corp., Downington, PA), and the intensities of HIF-1{alpha} bands were corrected by comparison of corresponding ß-actin mRNA levels. The cDNA was cloned into pGEM-T Easy vector (Promega, Madison, WI). The sequencing was performed commercially (Sangon Corp, Shanghai, China) by using an Applied Biosystems Automated sequencer, ABI PRISM 377-96 (Perkin-Elmer; data not shown).

Protein Extraction and Western Blotting

The ERK1/2 activity assay was performed by Western blotting of the whole cell extracts using antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), which specifically recognized phosphyorylated ERK1/2. Total ERK1/2 was detected as the control. Briefly, the normal human cytotrophoblast cells were washed with phosphate-buffered saline before lysis in buffer containing 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1% Triton X- 100, 1 µg/ml leupeptin, 1 µg/ml pepstatin 50 mM NaF, 2.5 mM Na3VO4, 1 µM aprotinin, and 1 mM PMSF. The cytoplasmic extracts were collected after centrifugation at 12 000 x g for 10 min. The analysis of HIF-1{alpha} expression was performed using the total proteins extracted by Trizol reagent according to the manufacturer's instructions. The proteins extracted by Trizol were lysed in 1% SDS, and the total amount of protein present in solutions was then detected by UV spectrophotometer (Beckman DU530; Fullerton, CA). All protein samples were adjusted to the same concentration.

Proteins obtained from cell extraction were boiled in SDS/ß-mercaptoethanol sample buffer and then loaded onto each lane of the 12% PAGE gels. The proteins were separated by electrophoresis and were then transferred onto microporous polyvinylidene fluoride membranes. Blots were blocked in 3% BSA in 37°C for 30 min and incubated overnight at 4°C with phosphyorylated ERK1/2-directed antibodies diluted 1:400, total ERK1/2-directed antibodies diluted 1:1000, and HIF-1{alpha}-primary antibodies in a dilution of 1:200, in succession. Then the corresponding secondary antibodies conjugated with AP were added at 37°C for 30 min. Color development was performed by nitro blue tetrazolium/5-bromo-4chloro- 3indolyphosphate. The bands were analyzed using a MetaView image analyzing system. The intensities of HIF-1{alpha} bands were corrected by comparison with their corresponding actin levels.

Analysis of VEGF Protein Production by ELISA

VEGF protein production was analyzed by ELISA using the Quantikine VEGF ELISA kit (R&D Systems, Oxford, U.K.) according to the manufacturer's instructions.

Statistics

Statistical analysis was performed using the Statistical Package for Social Science (SPSS for Windows package release 11.0; SPSS Inc., Chicago, IL). Results are presented as the average ± SEM of at least three separate experiments. Statistical comparisons among groups were analyzed by analysis of variance. A value of P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of HIF-1{alpha} Protein but Not HIF-1{alpha} mRNA Expression by IL-1ß

Exposure of serum-starved normal human cytotrophoblast cells to IL-1ß for 9 h resulted in a concentration- dependent induction of HIF-1{alpha} protein expression with a maximal effect observed in the presence of 10 ng/ml of IL- 1ß (Fig. 1A). HIF-1{alpha} protein expression was also induced by exposure of cells to CoCl2 (50 µM) (Fig. 1A), which blocks HIF-1{alpha} degradation. In the presence of IL-1ß, HIF- 1{alpha} protein levels peaked at 9 h and declined thereafter (Fig. 1B). In contrast, neither IL-1ß nor CoCl2 induced HIF-1{alpha} mRNA expression (Fig. 1, A and B), demonstrating specific effects of these agents on HIF-1{alpha} protein expression.



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  		FIG. 1. The effect of IL-1ß and CoCl2 on HIF-1{alpha} protein and mRNA expression in normal human cytotrophoblast cells. A) Analysis of HIF-1{alpha} protein and mRNA expression as a function of IL-1ß concentration. The normal human cytotrophoblast cells were cultured in the absence of serum for 12 h, then exposed to vehicle, 0.1–10 ng/ml IL-1ß, or 50 µM CoCl2 for 9 h. Then each whole cellular protein was subject to Western blotting assay for expression of HIF-1{alpha} protein and actin (top). Bar graphs show the levels of HIF-1{alpha} quantity corrected for actin. Bars represent the mean ± SEM of values from three independent experiments. Bars with different letters on the top are significantly different (P < 0.05). Total RNA was extracted from the serum-starved normal human cytotrophoblast cells, which were treated by different concentrations of IL-1ß (0, 0.1, 1, 5, 10 ng/ml) and CoCl2 (50 µM) for 9 h. Semiquantitative RT-PCR experiments were performed as described in the Materials and Methods (middle). The optic densities of the DNA bands were analyzed by optical scanning densitometry. Bar graphs show the intensity of the bands from three independent experiments, which was corrected by comparison of ß-actin mRNA levels. M, Marker. B) Kinetics of HIF-1{alpha} protein and mRNA induction. Serum-starved cells were exposed to vehicle or 10 ng/ml IL-1ß for 3–12 h prior to Western blotting analysis of whole cellular protein. Bar graphs show the levels of HIF-1{alpha} quantity corrected for actin (top). Bars represent the mean ± SEM of values from three independent experiments. Bars carrying different letters differ within each panel (P < 0.05). Serum-starved cells were exposed to vehicle or 10 ng/ml IL-1ß for 3–12 h prior to semiquantitative RT-PCR analysis of total RNA (middle). The optic densities of the DNA bands were analyzed by optical scanning densitometry. Bar graphs show the intensity of the bands from three independent experiments, which was corrected by comparison of ß-actin mRNA levels. M, Marker

Stimulation of VEGF Secretion by IL-1ß

To determine whether IL-1ß treatment affected VEGF secretion, the serum-starved normal human cytotrophoblast cells were exposed to IL-1ß. The quantity of VEGF protein was examined by ELISA. When treated with exogenous IL- 1ß at different concentrations (0, 0.1, 1, 5, 10 ng/ml) for 9 h, VEGF protein secretion in normal human cytotrophoblast cells was stimulated in a dose-dependent fashion (Fig. 2). IL-1ß at 1 ng/ml was able to increase the VEGF protein level in normal human cytotrophoblast cells and 10 ng/ml of IL-1ß caused the maximal stimulation. In addition, CoCl2 also increased VEGF protein levels, but to a lesser extent than that of 1 ng/ml IL-1ß (Fig. 2).



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  		FIG. 2. VEGF protein secretion induced by IL-1ß in normal human cytotrophoblast cells. Dose-dependent VEGF secretion stimulated by IL-1ß. The serum-starved normal human cytotrophoblast cells were treated with IL-1ß (0, 0.1, 1, 5, 10 ng/ml) for 9 h. VEGF protein secretion in the culture medium was determined by ELISA. Data are expressed as the mean of triplicate ± SEM. Bars with different letters on the top are significantly different (P < 0.05)

Activation of ERK1/2 by IL-1ß

The signaling intermediates activated by IL-1ß were also investigated. Previous reports have suggested that some of the biological actions of IL-1ß are mediated by activation of the MAPK signaling pathway [25–27]. To investigate the potential intermediate role of ERK1/2 on IL-1ß-induced accumulation of VEGF, the phosphorylation status of ERK1/ 2 was analyzed by using antibodies that specifically recognize the phosphorylated form of ERK1/2. As shown in Figure 3, exogenous IL-1ß (10 ng/ml) increased the phosphorylation of ERK1/2 at 10 min of stimulation and the phosphorylation reached a maximum at 60 min. Moreover, phosphorylated ERK1/2 remained at a higher level for at least 12 h. The amount of the total ERK1/2 remained unchanged during the experiment, as indicated by Western blotting of the lysates using antibodies that detect total (phosphorylation state-independent) ERK1/2 protein (Fig. 3).



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  		FIG. 3. ERK1/2 pathway activation by IL-1ß. Normal human cytotrophoblast cells were incubated with exogenous IL-1ß (10 ng/ml) for the times indicated. The figure shows a representative of three experiments in the same conditions. Phosphorylation of ERK1/2 MAPK from cellular extracts was determined using Western blotting with phospho-p44/42 MAPK antibody (phospho-ERK1/2). Western blotting of total ERK1/2 protein (total ERK1/2) in cellular extracts is also shown. Bar graphs show the levels of activated ERK1/2 quantity corrected for total ERK1/2. Bars represent the mean ± SEM of values from three independent experiments. Bars with different letters on the top are significantly different (P < 0.05)

Inhibition of IL-1ß-Stimulated HIF-1{alpha} Protein Expression and VEGF Secretion by ERK1/2 Inhibition

In order to determine the signal transduction pathways mediating the effects of IL-1ß on HIF-1{alpha} protein and VEGF protein expression, the normal human cytotrophoblast cells were pretreated with PD98059, a selective pharmacologic inhibitor of the ERK1/2 route, and then prevented the activation of the ERK1/2 pathway (Fig. 4). The results showed that PD98059 inhibited the induction of HIF-1{alpha} protein expression in IL-1ß-treated cells in a dose- dependent manner (Fig. 5). Meanwhile, the induction of VEGF protein secretion was inhibited by PD98059 (Fig. 6). In contrast to its effects on the accumulation of HIF-1{alpha} protein induced by IL-1ß treatment, PD98059 failed to inhibit the accumulation of HIF-1{alpha} in CoCl2-treated, normal human cytotrophoblast cells.



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  		FIG. 4. Inhibition of the ERK1/2 pathway with PD98059 blocks IL-1ß- induced ERK activation. Representative blot phosphorylation of ERK from cellular extracts pretreated for 1 h with PD98059 (50 µM) and then exposed to IL-1ß (10 ng/ml) for 9 h, as determined using Western blotting with anti-phospho-ERK antibody (phospho ERK1/2). Western blotting of total ERK1/2 protein (total ERK1/2) in cellular extracts is also shown. Bar graphs show the levels of activated ERK1/2 quantity corrected for total ERK1/2. Bars represent the mean ± SEM of values from three independent experiments. Bars with different letters on the top are significantly different (P < 0.05)



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  		FIG. 5. Inhibition of the ERK1/2 pathway with PD98059 blocks IL-1ß- induced HIF-1{alpha} protein expression. Serum-starved normal human cytotrophoblast cells were pretreated for 1 h with PD98059 (0, 1, 10, 50, 100 µM) and then incubated in the presence (+) or absence (–) of exogenous IL-1ß (10 ng/ml) and CoCl2 for 9 h. Cells were harvested for analysis of HIF-1{alpha} protein by Western blotting. Bar graphs show the levels of HIF- 1{alpha} quantity corrected for actin. Bars represent the mean ± SEM of values from three independent experiments. Bars with different letters on the top are significantly different (P < 0.05)



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  		FIG. 6. The effect of PD98059 on IL-1ß-induced VEGF secretion. Serum- starved normal human cytotrophoblast cells were pretreated for 1 h with PD98059 (50 µM) and then incubated in the presence (+) or absence (–) of exogenous IL-1ß (10 ng/ml) for 9 h. The secretion of VEGF protein in the culture medium was quantified using ELISA. Each value represents the mean ± SEM of three experiments, each in triplicate. Bars with different letters on the top are significantly different (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The human placenta is a highly invasive tumor-like structure in which a subpopulation of placental trophoblast cells known as the cytotrophoblast invades the uterine decidua and its vasculature to established adequate fetal-maternal exchange of molecules [28]. A principal mediator of angiogenesis is VEGF, and the major transcriptional activator of the VEGF gene is HIF-1 [29]. Previous studies have shown that IL-1ß regulated the expression of HIF-1{alpha} protein [10, 12]. However, little is known about the molecular mechanism of IL-1ß-regulated HIF-1{alpha} expression in normal human cytotrophoblast cells. In the current study, we addressed for the first time a possible route that IL-1ß induced HIF-1{alpha} expression and VEGF secretion in normal human cytotrophoblast cells.

Our data showed that exposure of serum-starved normal human cytotrophoblast cells to IL-1ß for 9 h resulted in a concentration-dependent induction of HIF-1{alpha} protein expression, with a maximal effect observed in the presence of 10 ng/ml. HIF-1{alpha} protein expression was also induced by exposure of cells to CoCl2 (50 µM), which blocks HIF- 1{alpha} degradation. In addition, in the presence of IL-1ß, HIF- 1{alpha} protein levels peaked at 9 h and declined thereafter. These results were similar to those were obtained in HepG2 cells treated by IL-1ß under normoxic conditions [10, 12]. But unlike the results in human gingival and synovial fibroblasts in which IL-1ß induced HIF-1{alpha} mRNA expression [30], our observations showed that neither IL-1ß nor CoCl2 induced HIF-1{alpha} mRNA expression. These data demonstrate the specific effects of these agents on HIF-1{alpha} protein expression in normal human cytotrophoblast cells. This difference is likely due to the fact that we examined normal human cytotrophoblast cells in contrast with the study of Thornton et al. [30], where human gingival and synovial fibroblasts were studied. Our data provided the direct evidence that IL-1ß stimulated HIF-1{alpha} protein expression in normal human cytotrophoblast cells.

HIF-1 is considered as the main trans-acting factor in controlling the rate of transcription of the VEGF gene. We have proven that HIF-1{alpha} protein expression was induced by IL-1ß in normal human cytotrophoblast cells. Moreover, IL-1ß has been found to increase VEGF mRNA levels in rat and human first trimester trophoblast cells [18], aortic smooth muscle cells [31], and human synovial fibroblasts [32, 33]. In order to view proteins encoded by HIF-1 target genes in normal human cytotrophoblast cells, effects of IL- 1ß on the rates of the production of VEGF protein were studied. Our data showed that culture of the primary normal human cytotrophoblast cells in the presence of IL-1ß resulted in the secretion of significant levels of VEGF in culture medium in a dose-dependent fashion. In addition, under the exposure of normal human cytotrophoblast cells to CoCl2 (50 µM), VEGF secretion was increased to a lesser extent but has a negligible effect on the VEGF mRNA expression (data not shown). The above results led us to hypothesize that the stimulation of HIF-1{alpha} protein expression by IL-1ß may be one of the reasons for the increasing of VEGF secretion in the culture medium when the normal human cytotrophoblast cells were administrated with IL-1ß.

Recently, much effort has been made to define the signal transduction pathways induced by IL-1ß. Cellular responses to IL-1ß stimulation trigger a cascade of protein kinases that transmit signals that ultimately regulate gene expression. In mammalian cells, three subgroups of the MAPKs have been detected: the ERK1/2, the JNKs, and the P38. The mammalian ERK1/2 is activated by multiple stimuli, such as growth factors, neurotrophic factors, neurotransmitters, and cytokines. [34–36]. It has also been shown that the MAPKs are activated by IL-1ß [37–39]. In this study, the ERK1/2 pathway was regarded as an important route to be examined. Some evidence has proven that MAPKs play important roles in the expression of HIF-1{alpha} protein [40]. Our results further support these findings in normal human cytotrophoblast cells. Treatment of the normal cytotrophoblast cells with exogenous IL-1ß resulted in ERK1/2 phosphorylation. A rapid activation of the ERK1/2 pathway in normal human cytotrophoblast cells occurred as early as 10 min and reached a maximum at 60 min. ERK1/2 remained phosphorylated for at least 12 h. However, in this study, we have found that CoCl2 had a negligible effect on the ERK1/2 activation (data not shown). These data indicate that activation of the ERK1/2 signal cascade is an early event in normal human cytotrophoblast cells in response to IL-1ß stimulation. We also evaluated the effect of inhibiting the ERK1/2 pathway on this process. We found that PD98059, a specific inhibitor of ERK1/2 signaling, decreases the expression of HIF-1{alpha} stimulated by the exogenous IL-1ß in normal cytotrophoblast cells in a dose-dependent fashion. In contrast with its effect on the expression of HIF- 1{alpha} induced by IL-1ß treatment, PD98059 had little inhibitory effect on the expression of HIF-1{alpha} in CoCl2-treated normal human cytotrophoblast cells, providing direct evidence that IL-1ß and CoCl2 act by distinct molecular mechanisms. In addition, the increased VEGF secretion in the culture medium of normal human cytotrophoblast cells treated with exogenous IL-1ß was reduced because of the presence of PD98059. It does indicate that PD98059 prevents not only the ERK1/2 activity, but also the expression of HIF-1{alpha} protein and the secretion of VEGF induced by exogenous IL-1ß in normal human cytotrophoblast cells. These novel findings raise the possibility that ERK1/2 pathway may participate in the IL-1ß-induced HIF-1{alpha}-mediated-VEGF secretion in normal human cytotrophoblast cells.

In summary, our data demonstrate that activation of the ERK1/2 pathway by IL-1ß may result in the accumulation of HIF-1{alpha} protein, which initiates VEGF secretion in normal human cytotrophoblast cells. What emerges from our current findings presents a further clarification of the molecular mechanism that IL-1ß regulates the expression of angiogenesis-related molecules in human placenta during embryo implantation.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Wenping Guo (Peking University Third Hospital, Beijing, China) for her assistance with tissue collection. We also appreciate Dr. Qingyuan Sun's (Institute of Zoology, Chinese Academy of Sciences, Beijing, China) helpful discussions and careful reading of this manuscript.


   FOOTNOTES
 
1 Supported by the Special Funds for Major State Basic Research Project (G1999055903), the Knowledge Innovation Project of the Chinese Academy of Sciences (KSCX3-IOZ-07), and the National Natural Science Foundation of China (30200133). Back

2 Correspondence: Cheng Zhu, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 25, Bei Si Huan Xi Lu, Beijing 100080, China. FAX: 86 10 62529248; zhuc{at}panda.ioz.ac.cn Back

Received: 31 October 2003.

First decision: 6 December 2003.

Accepted: 7 February 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rajakumar A, Conrad KP. Expression, ontogeny, and regulation of hypoxia-inducible transcription factors in the human placenta. Biol Reprod 2000 63:559-569[Abstract/Free Full Text]
  2. Conrad KP, Benyo DF. Placental cytokines and the pathogenesis of preeclampsia. Am J Reprod Immunol 1997 37:240-249
  3. Lim K-H, Zhou Y, Janatpour M, McMaster M, Bass K, Chun S-H, Fisher SJ. Human cytotrophoblast differentiation/invasion in abnormal pre-eclampsia. Am J Pathol 1997 151:1809-1818[Abstract]
  4. Caniggia I, Winter J, Lye SJ, Post M. Oxygen and placental development during the first trimester: implications for the pathophysiology of pre-eclampsia. Placenta 2000 21:suppl AS25-S30
  5. Caniggia I, Mostachfi H, Winter J, Gassmann M, Lye SJ, Kuliszewski M, Post M. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFbeta(3). J Clin Invest 2000 105:577-587[Medline]
  6. Adelman DM, Gertsenstein M, Nagy A, Simon MC, Maltepe E. Placental cell fates are regulated in vivo by HIF-mediated hypoxia responses. Genes Dev 2000 14:3191-3203[Abstract/Free Full Text]
  7. Semenza GL. Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochem. Pharmacol 2000 59:47-53[CrossRef][Medline]
  8. Caniggia I, Winter JL. Adriana and Luisa Castellucci Award lecture 2001. Hypoxia inducible factor-1: oxygen regulation of trophoblast differentiation in normal and pre-eclamptic pregnancies—a review. Placenta 2002 23:suppl AS47-S57
  9. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, Kriegsheim AV, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, Ratcliffe PJ. Targeting of HIF-alpha to the von Hippel- Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001 292:468-472[Abstract/Free Full Text]
  10. Stiehl DP, Jelkmann W, Wenger RH, Hellwig-Burgel T. Normoxic induction of the hypoxia-inducible factor 1alpha by insulin and interleukin-1beta involves the phosphatidylinositol 3-kinase pathway. FEBS Lett 2002 512:157-162[CrossRef][Medline]
  11. Feldser D, Agani F, Iyer NV, Pak B, Ferreira G, Semenza GL. Reciprocal positive regulation of hypoxia-inducible factor 1alpha and insulin-like growth factor 2. Cancer Res 1999 59:3915-3918[Abstract/Free Full Text]
  12. Hellwig-Burgel T, Rutkowski K, Metzen E, Fandrey J, Jelkmann W. Interleukin-1beta and tumor necrosis factor-alpha stimulate DNA binding of hypoxia-inducible factor-1. Blood 1999 94:1561-1567[Abstract/Free Full Text]
  13. Franzen R, Pautz A, Brautigam L, Geisslinger G, Pfeilschifter J, Huwiler A. Interleukin-1beta induces chronic activation and de novo synthesis of neutral ceramidase in renal mesangial cells. J Biol Chem 2001 276:35382-35389[Abstract/Free Full Text]
  14. Haddad JJ. Recombinant human interleukin (IL)-1 beta-mediated regulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) stabilization, nuclear translocation and activation requires an antioxidant/reactive oxygen species (ROS)-sensitive mechanism. Eur Cytokine Netw 2002 13:250-260[Medline]
  15. Berra E, Pages G, Pouyssegur J. MAP kinases and hypoxia in the control of VEGF expression. Cancer. Metastasis Rev 2000 19:139-145[CrossRef][Medline]
  16. Michiels C, Minet E, Michel G, Mottet D, Piret JP, Raes M. HIF-1 and AP-1 cooperate to increase gene expression in hypoxia: role of MAP kinases. IUBMB Life 2001 52:49-53[CrossRef][Medline]
  17. Baergen R, Benirschke K, Ulich TR. Cytokine expression in the placenta. The role of interleukin 1 and interleukin 1 receptor antagonist expression in chorioamnionitis and parturition. Arch Pathol Lab Med 1994 118:52-55[Medline]
  18. Choi SJ, Park JY, Lee YK, Choi HI, Lee YS, Koh CM, Chung IB. Effects of cytokines on VEGF expression and secretion by human first trimester trophoblast cell line. Am J Reprod Immunol 2002 48:70-76
  19. Zhang J, Cao YJ, Zhao YG, Sang QX, Duan EK. Expression of matrix metalloproteinase-26 and tissue inhibitor of metalloproteinase-4 in human normal cytotrophoblast cells and a choriocarcinoma cell line, JEG-3. Mol Hum Reprod 2002 8:659-666[Abstract/Free Full Text]
  20. Knofler M, Mosl B, Bauer S, Griesinger G, Husslein P. TNF-alpha/ TNFRI in primary and immortalized first trimester cytotrophoblasts. Placenta 2000 21:525-535[CrossRef][Medline]
  21. Xu P, Wang YL, Zhu SJ, Luo SY, Piao YS, Zhuang LZ. Expression of matrix metalloproteinase-2, -9, and -14, tissue inhibitors of metalloproteinase-1, and matrix proteins in human placenta during the first trimester. Biol Reprod 2000 62:988-994[Abstract/Free Full Text]
  22. Wu D, Luo S, Wang Y, Zhuang L, Chen Y, Peng C. Smads in human trophoblast cells: expression, regulation and role in TGF-beta-induced transcriptional activity. Mol Cell Endocrinol 2001 175:111-121[CrossRef][Medline]
  23. Esteve PO, Chicoine E, Robledo O, Aoudjit F, Descoteaux A, Potworowski EF, St-Pierre Y. Protein kinase C-zeta regulates transcription of the matrix metalloproteinase-9 gene induced by IL-1 and TNF- alpha in glioma cells via NF-kappa B. J Biol Chem 2002 277:35150-35155[Abstract/Free Full Text]
  24. Gros J, Gerhardt CC, Strosberg AD. Expression of human (beta) 3- adrenergic receptor induces adipocyte-like features in CHO/K1 fibroblasts. J Cell Sci 1999 112:3791-3797[Abstract]
  25. Laporte JD, Moore PE, Abraham JH, Maksym GN, Fabry B, Panettieri RA Jr, Shore SA. Role of ERK MAP kinases in responses of cultured human airway smooth muscle cells to IL-1beta. Am J Physiol 1999 277:L943-L951
  26. Wang YZ, Zhang P, Rice AB, Bonner JC. Regulation of interleukin- 1beta-induced platelet-derived growth factor receptor-alpha expression in rat pulmonary myofibroblasts by p38 mitogen-activated protein kinase. J Biol Chem 2000 275:22550-22557[Abstract/Free Full Text]
  27. Miwa M, Kozawa O, Tokuda H, Uematsu T. Mitogen-activated protein (MAP) kinases are involved in interleukin-1 (IL-1)-induced IL-6 synthesis in osteoblasts: modulation not of p38 MAP kinase, but of p42/ p44 MAP kinase by IL-1-activated protein kinase C. Endocrinology 1999 140:5120-5125[Abstract/Free Full Text]
  28. Lala PK, Lee BP, Xu G, Chakraborty C. Human placental trophoblast as an in vitro model for tumor progression. Can J Physiol Pharmacol 2002 80:142-149[CrossRef][Medline]
  29. Semenza GL. HIF-1 and tumor progression: pathophysiology and therapeutics. Trends Mol Med 2002 8:S62-S67[CrossRef][Medline]
  30. Thornton RD, Lane P, Borghaei RC, Pease EA, Caro J, Mochan E. Interleukin 1 induces hypoxia-inducible factor 1 in human gingival and synovial fibroblasts. Biochem J 2000 350:307-312
  31. Li J, Perrella MA, Tsai JC, Yet SF, Hsieh CM, Yoshizumi M, Patterson C, Endege WO, Zhou F, Lee ME. Induction of vascular endothelial growth factor gene expression by interleukin-1 beta in rat aortic smooth muscle cells. J Biol Chem 1995 270:308-312[Abstract/Free Full Text]
  32. Ben-Av P, Crofford LJ, Wilder RL, Hla T. Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS Lett 1995 372:83-87[CrossRef][Medline]
  33. Jackson JR, Minton JA, Ho ML, Wei N, Winkler JD. Expression of vascular endothelial growth factor in synovial fibroblasts is induced by hypoxia and interleukin 1beta. J Rheumatol 1997 24:1253-1259[Medline]
  34. Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995 80:179-185[CrossRef][Medline]
  35. Reddy KB, Nabha SM, Atanaskova N. Role of MAP kinase in tumor progression and invasion. Cancer Metastasis Rev 2003 22:395-403[CrossRef][Medline]
  36. Cobb MH, Goldsmith EJ. How MAP kinases are regulated. J Biol Chem 1995 270:14843-14846[Free Full Text]
  37. Kyriakis JM, Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays 1996 18:567-577[CrossRef][Medline]
  38. LaPointe MC, Isenovic E. Interleukin-1beta regulation of inducible nitric oxide synthase and cyclooxygenase-2 involves the p42/44 and p38 MAPK signaling pathways in cardiac myocytes. Hypertension 1999 33:276-282[Abstract/Free Full Text]
  39. Yang CM, Chien CS, Wang CC, Hsu YM, Chiu CT, Lin CC, Luo SF, Hsiao LD. Interleukin-1beta enhances bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine tracheal smooth- muscle cells: involvement of the Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway. Biochem J 2001 354:439-446[CrossRef][Medline]
  40. Fukuda R, Hirota K, Fan F, Jung YD, Ellis LM, Semenza GL. Insulin- like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. J Biol Chem 2002 277:38205-38211[Abstract/Free Full Text]



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