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a Department of Obstetrics and Gynecology, University of Tennessee Graduate School of Medicine, Knoxville, Tennessee 37922
b Methodist Research Institute, Clarian Health, Indianapolis, Indiana 46202
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and tumor necrosis factor 
. These results provide the first direct evidence of a biochemical and functional role for PlGF/Flt-1 in normal trophoblast and suggest that aberrant PlGF expression during pregnancy may impact upon trophoblast function as well as vascularity within the placental bed.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Receptors for the VEGF/PlGF family of growth factors include products of the fms-like tyrosine kinase (Flt-1) gene [9, 10] and the kinase-insert domain containing receptor (KDR) gene [11]. In spite of significant structural similarities between the receptors, Flt-1 receptors demonstrate high affinity binding for VEGF and PlGF, while KDR binds only VEGF [12]. Recently, we [7] and others [13, 14] have demonstrated Flt-1 receptors on trophoblast. Normal trophoblast does not appear to express KDR receptors, however [7, 14].
Prominent production of PlGF by trophoblast and the presence of Flt-1 receptors on trophoblast raises the possibility that PlGF may act in an autocrine manner to modulate normal trophoblast function [7]. However, the biochemical responses and physiological significance of Flt-1/PlGF interactions in normal trophoblast are not known. Consequently, we sought to characterize the signal transduction responses of PlGF-induced activation of Flt-1 and to delineate the possible cellular function of PlGF in normal primary human trophoblast.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant human (rh) PlGF was purchased from R & D Systems (Minneapolis, MN); recombinant human epidermal growth factor (EGF) and myelin basic protein (MBP) were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-c-Jun-N terminal kinase (JNK)-1, extracellular signal regulated kinase 2 (ERK-2), p38, goat anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies, and glutathione-S-transferase (GST)-c-Jun fusion protein were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-active mitogen-activated protein kinase (MAPK) and p38 kinase antibodies were from Promega (Madison, WI). Keratinocyte growth media (KGM) and keratinocyte basal media (KBM) were from Clonetics Corp (San Diego, CA); 3-4,5-dimethylthizol-2,5-diphenyl-tetrazolium bromide (MTT), Dulbecco's Modified Eagle's Medium (DMEM)-high glucose (HG), and heparin were from Sigma Chemical (St. Louis, MO). Chemiluminescence reagents were purchased from Pierce Chemical Co. (Rockford, IL).
Isolation of Trophoblast and Cell Culture
Human term trophoblast was isolated and cultured as previously described [7]. Briefly, ~50 mg of minced placental tissue from uncomplicated vaginal or Cesarean term deliveries was subjected to three successive trypsin/deoxyribonuclease (DNase) digestions. Tissue fragments were allowed to settle, and freed cells in the supernatant were layered over a discontinuous Percoll (Pharmacia and Upjohn, Kalamazoo, MI) gradient (25–50%). After centrifugation at 1200 x g for 20 min, cells banding between approximately 37% and 42% percoll were collected and cultured as described below. The purity of each trophoblast preparation was verified using antibodies to vimentin and cytokeratin [7], and cultures exhibiting 
90% pure trophoblast were studied.
Freshly isolated trophoblast was plated in KGM/10% fetal calf serum (FCS) for 48 h and rendered quiescent by incubation in KBM without FCS for 18–24 h. Endothelial cells were collected from human umbilical veins [15] and cultured in RPMI 1640 with 20% FCS as previously described [16]. Human umbilical vein endothelial (HUVE) cells were cultured to ~80% confluency and used before passage number 8. They were serum-starved in RPMI for 24 h before growth factor treatment.
Cell Stimulation and Preparation of Cell Extracts
Trophoblast cultures and HUVE cells were treated with 10 ng/ml of PlGF or 20 ng/ml EGF in the presence of 1 µg/ml heparin. EGF was used as a positive control for ERK-1/2 activity, and exposure to 80 joule/m2 of UV light for 2 min was used as a positive control for p38 and JNK activation [17]. Protein lysates from both trophoblast and HUVE cells were prepared in lysis buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 0.1% NP-40, 1 mM PMSF, 1 mM Na3VO4, and 10 mM NaF) at the indicated time points, and clarified by centrifugation (10 000 x g for 10 min). Lysates were aliquoted and stored at -80°C until used.
ERK-2 and JNK In Vitro Kinase Assays
Equal quantities of cell lysates (25 µg for ERK and 95 µg for JNK) were immunoprecipitated with 0.5 µg/sample ERK-2 or JNK-1 antibodies conjugated to protein A/G-coupled agarose beads for 16–18 h at 4°C. The beads were washed three times in lysis buffer and twice in kinase buffer (25 mM Hepes pH 7.5, 25 mM NaCl2, 25 mM glycerophosphate). The precipitates were resuspended in kinase buffer containing 1 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM ATP, 5 µCi/sample of [-32P]ATP, and 0.2 mg/sample MBP or GST-c-Jun protein as target substrates for ERK-2 and JNK-1, respectively. The kinase reactions were allowed to continue for 20 min at room temperature and were terminated by the addition of double-strength Laemmli sample buffer. Reaction products were resolved on 14% (ERK-2) or 12% (JNK) SDS polyacrylamide gel (SDS-PAGE), dried, and exposed to x-ray film. The radiolabeled substrate bands were quantitated by laser densitometry, and fold induction relative to control levels (Time 0) for each experiment was calculated.
Western Blot Analysis for ERK-2 and p38 Kinase Activity
Equal quantities of cell lysates (30 µg/lane for ERK-1/2, 90 µg/lane for p38) were separated by SDS-PAGE and were transferred to nitrocellulose membranes. The membranes were blocked with 0.5% casein in Tris-buffered saline containing 0.1% Tween (TBST) for 30 min, incubated with anti-active MAPK antibody (25 ng/ml) or anti-active p38 kinase antibody (1:2000) overnight at 4°C, and then washed with TBST several times. The immune complexes were probed with goat anti-rabbit HRP-conjugated antibody (1:2000) and washed well with TBST. The membrane was developed in chemiluminescent reagents as directed by the manufacturer (Pierce Chemical) for 5 min and exposed to x-ray film. The proteins bands were quantitated by laser densitometry.
To control for protein loading, the membranes were stripped in 0.2 M glycine pH 2.2, 0.1% SDS, and 10% Tween 20 for 20 min, washed three times in TBST, and reblocked with blocking buffer for 30 min. The membranes were probed with anti-ERK-2 antibody or p38 antibody (0.5 µg/ml) overnight and developed as above.
Apoptosis Assays
Cultured trophoblast was plated in KGM/10% FCS and allowed to adhere for 4 h at 37°C. Medium was replaced with DMEM-HG with or without 10% FCS, EGF (20 ng/ml), or PlGF (10 ng/ml). Cytokine-induced apoptosis was accomplished with 100 U/ml interferon (IFN)- and 10 ng/ml tumor necrosis factor (TNF) as previously described [18]. Apoptosis was determined via DNA fragmentation and MTT analyses after 72-h incubation. For DNA fragmentation analyses, both floating and attached cells were collected and placed in digestion buffer (10 mM EDTA, 50 mM Tris pH 8, 0.5% sodium lauryl sarcosine, 0.5 mg/ml proteinase K) for 3 h at 55°C. Digests were treated with DNase-free ribonuclease (RNase) for 1 h at 55°C, and DNA was extracted twice with phenol/chloroform and once with chloroform. Extracted DNA was precipitated with ethanol overnight at -80°C. Ten micrograms of DNA from each treatment group was resolved on a 1.6% agarose gel, stained with ethidium bromide, and photographed. For MTT analyses, spent media from quadruplicate trophoblast cultures were replaced with DMEM-HG containing 0.5 mg/ml MTT and incubated for 3.5 h at 37°C. Intracellular formazan was extracted with isopropanol, and absorbance of each well was measured at 570/650 nm. Percentage of apoptosis was calculated after normalizing to control cultures containing 10% FCS.
Statistical Analyses
Normalized data were compared by one-sample t-test against control values (100%), differences between multiple treatment groups were analyzed by ANOVA, and significance between two treatment groups determined by Tukey's honest significant difference post-hoc comparison. Statistics were calculated with STATISTICA (StatSoft, Inc., Tulsa, OK), and significance was indicated when p 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous studies documented that the Flt-1 receptor expressed in normal human trophoblast is functional and that VEGF induces JNK activity [7]. To extend these initial findings and to determine whether PlGF induces similar activation of the Flt-1 receptor, isolated normal syncytiotrophoblast was treated with rhPlGF, and time-course induction of JNK, p38, and ERK-1/2 activities was determined (Fig. 1). Exogenous rhPlGF rapidly induced transient JNK activity (Fig. 1A). Induction of JNK activity was evident at 5 min, and the response peaked (mean 6.1 ± 0.5 fold) at 10 min before diminishing at 20 min. Longer time-course experiments showed that JNK activity levels returned to background levels by 40 min (data not shown). Activation of JNK activity by PlGF was qualitatively and quantitatively similar to the increase induced within 10 min by UV treatment, a known activator of JNK [17]. Addition of exogenous rhPlGF to trophoblast also resulted in a time-dependent induction of p38 kinase activity (mean 7.5 ± 0.5 fold at 10 min; Fig. 1B). In contrast to these SAPK results, PlGF did not induce significant (p > 0.14) ERK-1 and -2 activity in syncytiotrophoblast (mean 2.2 ± 0.53 fold at 10 min; Fig. 1C). Treatment of the syncytiotrophoblast with EGF resulted in a large increase in ERK-1 and -2 activity, indicating that the ERK-1 and -2 pathway was functional in the cultured trophoblast.
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The ability of PlGF to induce JNK and p38 kinase, but not ERK-1/2 activity, in syncytiotrophoblast is in sharp contrast to the effects elicited in HUVE cells, in which exogenous PlGF induced a large transient increase in ERK-1 and -2 activity (Fig. 2A). PlGF induced a peak activation of both ERK-1 (p44) and ERK-2 (p42) in the HUVE cells by ~5 min which returned to background levels by 20 min. This response was similar to that invoked by the known endothelial cell mitogen EGF. PlGF did not induce JNK activity (Fig. 2B) nor p38 kinase activity (data not shown) in HUVE cells.
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PlGF Protected Trophoblast from Apoptosis Induced by Growth Factor Withdrawal
Activation of JNK and p38 kinase pathways has been shown to induce or inhibit apoptosis depending on cell type and culture conditions [19]. We were unable to induce apoptosis in trophoblast cultures by exogenous PlGF (data not shown). Thus, we examined the ability of PlGF to protect trophoblast from apoptosis induced by growth factor withdrawal as well as apoptosis induced by the pro-inflammatory cytokines TNF and IFN-. Under our culture conditions, syncytiotrophoblast maintained for 72 h in 10% FCS showed little DNA fragmentation (Fig. 3). However, serum withdrawal induced marked apoptosis in the trophoblast cultures as evidenced by significant DNA fragmentation. The addition of exogenous PlGF to the serum-free media significantly reduced DNA fragmentation at 72 h and protected the cells from apoptosis. However, supplementing the serum-free media with 20 ng/ml of EGF, a known inducer of ERK-1 and -2 in trophoblast (see Fig. 1), did not prevent apoptosis.
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The DNA fragmentation results were confirmed, and relative apoptosis was quantitated by measuring loss of trophoblast viability as measured by the metabolic capacity of the trophoblast to reduce MTT [18]. As shown in Figure 4, serum deprivation significantly reduced MTT activity as compared to results in control trophoblast maintained in 10% FCS (p < 0.001). The addition of rhPlGF significantly increased MTT reduction compared to levels under serum-free culture conditions (p < 0.02). Indeed, inclusion of rhPlGF in serum-free culture media significantly (p = 0.05) increased MTT activity to 146% of normal control cultures containing 10% FCS. These data quantitatively support the DNA fragmentation data and confirm that PlGF can function to protect cultured term trophoblast from growth factor withdrawal-induced apoptosis.
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PlGF Did Not Protect Trophoblast from Cytokine-Induced Apoptosis
To determine whether PlGF functions broadly to protect trophoblast from apoptosis, we tried to inhibit IFN-/TNF-induced apoptosis with exogenous PlGF (Fig. 5). As previously shown [18], IFN- and TNF induced significant (p < 0.005) apoptosis in primary trophoblast cultures by 48 h. The cytokine-induced apoptosis was prevented by the addition of 20 ng/ml exogenous EGF (p < 0.01). However, we were not able to significantly (p > 0.80) inhibit cytokine-induced apoptosis with 10 ng/ml exogenous PlGF (Fig. 5). Higher concentrations of PlGF (50 and 100 ng/ml) also failed to protect the trophoblast from IFN-/TNF-induced apoptosis, and qualitatively similar results were obtained when apoptosis was monitored by DNA fragmentation analyses (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The biological function of Flt-1 receptor activation in endothelial cells is unclear. Activation of Flt-1 receptors by PlGF has been reported to have little or no cellular effect in some studies [12, 20–22], while other studies have shown significant responses [1–3, 23, 24]. The reasons for these discrepant findings are not clear, but they may be due to differences in sources of PlGF, target cell heterogeneity, and/or assay end points [25]. Cellular function of PlGF/Flt-1 in cells other than endothelial cells is limited to monocytes [26]. Here we show that PlGF induces rapid activation of JNK and p38 kinase activities in primary trophoblast. In contrast, PlGF induces strong ERK-1 and -2 activation, but no JNK or p38 kinase responses, in endothelial cells. These findings in endothelial cells are consistent with a recent study showing that PlGF induces ERK activity in porcine aortic endothelial cells overexpressing Flt-1 [23]. Collectively, these results provide further molecular support for PlGF as a mitogenic and chemotactic factor for endothelial cells and provide novel evidence for a unique function in trophoblast via activation of the SAPK pathways.
The upstream regulatory factors controlling the disparate signal transduction responses of PlGF/Flt-1 in HUVE cells and trophoblast are not known, but they may reflect differences in proliferation potentials between the cell types or possible signal facilitation by KDR in HUVE cells; or the responses may reflect the presence or absence of yet-to-be-defined signaling protein(s) in trophoblast. The limited molecular and cellular responses of fibroblasts that overexpress ectopic Flt-1 [27] support the hypothesis that endogenous signal transduction molecules, perhaps unique to trophoblast, regulate Flt-1-mediated responses. These possibilities are currently under investigation.
Activation of SAPK responses has been shown in other cell types to regulate apoptosis [19]. We showed that incubation of primary trophoblast under serum-free conditions, which induces apoptosis in many cell types [28], resulted in significant apoptosis. The stress-induced apoptosis could be inhibited with exogenous PlGF. However, EGF, an inducer of ERK-1 and -2 kinase activity, failed to protect trophoblast from growth factor withdrawal-induced apoptosis. Thus, PlGF may have a unique role in regulating placental function.
Studies have shown that TNF and IFN- can induce apoptosis in primary trophoblast in vitro [18, 29, 30]. We and others [29] are able to inhibit the pro-inflammatory cytokine-induced trophoblast apoptosis by exogenous EGF. However, even relatively high levels of exogenous PlGF do not provide significant protection of the trophoblast against cytokine-induced apoptosis. These results further suggest that the mechanism by which PlGF protects trophoblast from apoptosis is distinct from those mediated by EGF. This functional difference is probably due to the different signal transduction pathways that each growth factor activates. Although the mechanisms of action are not understood, one potential target may be Bcl-2. VEGF has recently been shown to induce the expression of Bcl-2 and A1 anti-apoptotic proteins in endothelial cells, and these molecules functioned to protect the cells from growth factor withdrawal-induced apoptosis [31]. Furthermore, activation of JNK, but not ERK-1 and -2, in other cells has been shown to regulate phosphorylation and functional activity of Bcl-2 [32]. Irrespective of the molecular mechanism(s), our findings suggest that PlGF/Flt-1-mediated SAPK activation provides protection against stress-induced apoptosis whereas EGF/EGF receptor-mediated ERK activation may not affect trophoblast survival in the absence of growth factors.
The ability of PlGF to modulate apoptotic events may be clinically important because trophoblast apoptosis occurs in both normal pregnancies and pregnancies complicated by infections or other pathologies. Discontinuities in the integrity of the syncytiotrophoblast may be due to apoptosis [33], and apoptosis is responsible for progressive disappearance of trophoblast in the chorion laeve as pregnancy approaches term in normal pregnancies [34]. Apoptosis has been demonstrated in normal placentae from both the first and the third trimester, and the rate of apoptosis appears to increase significantly as pregnancy progresses [35–37]. These observations suggest that apoptosis plays an important role in the normal development and aging of placentae.
Although the mechanisms regulating trophoblast apoptosis during gestation are not known, the incidence of placental apoptosis during the first and third trimesters of normal pregnancies [37] is associated with low serum PlGF levels at these gestational time points [38]. Thus, it may be that low levels of PlGF increase trophoblast susceptibility to apoptosis in early and late stages of pregnancy.
In addition to its potential role in normal pregnancy, trophoblast apoptosis has been implicated in several obstetrical complications. Apoptosis in the placenta leads to fetal growth retardation in rats [39], and the incidence of placental apoptosis is significantly greater in human pregnancies complicated with intrauterine growth restriction (IUGR) [40]. Placental bed hypoxia is generally thought to occur in preeclampsia and IUGR, and it is known to induce apoptosis in many cell types [41] as well as to decrease PlGF expression in primary trophoblast [7]. Hence, relative placental bed hypoxia may lead to decreased production of PlGF by trophoblast, which may function to increase trophoblast susceptibility to stress-induced apoptosis. Conceivably, the significantly decreased serum levels of PlGF observed during preeclampsia [38] contribute to increased trophoblast apoptosis, which in turn would have a significant role in the pathophysiology of the disease.
Based on our results, we propose that trophoblast expression of PlGF could influence the maternal-fetal interface via two mechanisms. PlGF could control decidua and villus vascular function via paracrine mechanisms and influence trophoblast function directly via autocrine mechanisms. Consequently, aberrant trophoblast expression of PlGF may contribute significantly to both the vascular and the placental pathologies commonly noted in perfusion compromised pregnancies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Donald S. Torry, Department of OB/GYN, University of Tennessee Graduate School of Medicine, Box U-27, 1924 Alcoa Highway, Knoxville, TN 37922. FAX: 423 544 6822; dtorry{at}utk.edu
Accepted: November 13, 1998.
Received: August 20, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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