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Endocrinology Vol. 140, No. 3 1399-1407
Copyright © 1999 by The Endocrine Society

ARTICLES

Activation of the Mitogen-Activated Protein Kinase Cascade Is Necessary But Not Sufficient for Basic Fibroblast Growth Factor- and Epidermal Growth Factor-Stimulated Expression of Endothelial Nitric Oxide Synthase in Ovine Fetoplacental Artery Endothelial Cells1

Jing Zheng, Ian M. Bird, Amy N. Melsaether and Ronald R. Magness

Department of Obstetrics and Gynecology, Perinatal Research Laboratories (J.Z., I.M.B., A.M.M., R.R.M.), and the Department of Meat/Animal Science (R.R.M.), University of Wisconsin, Madison, Wisconsin 53715

Address all correspondence and requests for reprints to: Ronald R. Magness, Ph.D., Department of Obstetrics and Gynecology, Perinatal Research Laboratories, University of Wisconsin, 7E Meriter Hospital, 202 S Park Street, Madison, Wisconsin 53715. E-mail: rmagness{at}facstaff wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) may play important roles in the placental vasculature, not only by controlling cell growth and differentiation, but also by mediating production of local vasodilators such as nitric oxide. As the mitogen-activated protein kinase (MAPK) signal cascade has been widely associated with cell growth in response to growth factors, herein we investigate whether bFGF, EGF, and VEGF also stimulate expression of endothelial nitric oxide synthase (eNOS) via activation of the MAPK cascade in ovine fetoplacental artery endothelial cells. The presence of the receptors for all three growth factors was confirmed by both immunocytochemistry and a functional cell proliferation assay. All three growth factors at 10 ng/ml rapidly (<10 min) activated MAPK. This activation was inhibited by PD 98059, a specific MAPK kinase inhibitor. bFGF and EGF, but not VEGF, dose- and time-dependently increased eNOS protein levels. Maximal stimulatory effects of bFGF and EGF on eNOS protein expression were observed at 10 ng/ml for 24 h of treatment and were associated with elevated eNOS messenger RNA. PD 98059 also significantly inhibited bFGF- and EGF-induced increases in eNOS protein expression. Because treatment with all three growth factors resulted in activation of the MAPK cascade, while bFGF and EGF, but not VEGF, increased eNOS expression, we conclude that activation of the MAPK cascade is necessary, but not sufficient, for bFGF- and EGF-induced increases in eNOS protein expression in ovine fetoplacental artery endothelial cells. Thus, additional signaling pathways are implicated in the different controls of eNOS expression and mitogenesis by growth factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NORMAL human and ovine pregnancies are associated with increases in placental blood flow that are mediated by both placental angiogenesis and vasodilation (1, 2). The production of placental angiogenic (growth) factors increases during normal pregnancy in association with an elevation in placental vascular density (1, 2). Local endothelial production of vasodilators such as nitric oxide (NO) in uterine arteries also is increased during ovine pregnancy, and this increased NO production is associated with pregnancy-induced increases in the expression of endothelial NO synthase (eNOS) (3, 4, 5). Because inhibition of NOS activity causes increases in fetoplacental vascular resistance (6, 7, 8, 9), decreases in placental NO production could lead to a reduction of fetoplacental blood flow (10, 11). This reduced placental blood flow is associated with fetal growth disorders such as intrauterine growth retardation (10).

Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are widely regarded as angiogenic factors (1, 2). Epidermal growth factor (EGF) has also been shown to stimulate angiogenesis in vivo and endothelial cell proliferation in vitro (12). These three growth factors are expressed in placentas of many species including human and sheep (13, 14, 15, 16, 17, 18, 19). In addition to their roles in angiogenesis, these growth factors regulate local vasomotor tone by mediating the production of vasodilators such as NO. This is supported by the observation that the vasodilatory activity of bFGF and VEGF is mediated by the production of NO, which is controlled by activation of eNOS in aortic, basilar, uterine, and coronary arteries (20, 21, 22, 23). In addition, bFGF increases eNOS expression in bovine endothelial cell lines derived from adrenal cortex, retina, and aorta (24). Similarly, VEGF also stimulates NO production by endothelial cells of different origins (25, 26, 27); this action of VEGF may be mediated through the VEGF receptor-1, flt-1 (26).

The cellular growth response to bFGF and EGF is mediated by binding and activating their specific receptors that have cytoplasmic tyrosine kinase domains (28, 29, 30). Upon activation, the receptor-tyrosine kinase initiates a cascade of cellular protein phosphorylation events by several protein kinases and leads to a variety of cellular responses (28, 29, 30). One family of these downstream kinases is mitogen-activated protein kinase (MAPK) p44 and p42. MAPK is phosphorylated and activated by MAPK kinase [MAPK kinase (MAPKK) or MEK] in the cytosol, translocates to the nucleus, and subsequently stimulates transcription of early response genes (28, 29, 30). It is also clear that activation of the MAPK cascade by bFGF and EGF regulates cell growth (30, 31, 32, 33). Stimulation of VEGF receptors also activates the MAPK cascade in endothelial cells and causes mitogenesis (27, 34).

Nothing is currently known about the role of growth factors in regulating placental eNOS protein expression or about the signaling mechanisms underlying this regulation. Recently, we reported that in the fetoplacental artery from late pregnant ewes, eNOS is localized in endothelium, and that its expression is maintained in a cultured fetoplacental artery endothelial cell model we recently developed (35). Thus, in this study, we tested the hypothesis that bFGF, EGF, and VEGF stimulate eNOS expression of fetoplacental artery endothelial cells via activation of the MAPK cascade. We demonstrate that bFGF and EGF, but not VEGF, increase eNOS protein expression, and that this could be blocked by a MAPK kinase inhibitor PD 98059. However, because all three growth factors, bFGF, EGF, and VEGF, activate the MAPK cascade, we conclude that activation of the MAPK cascade is necessary, but not sufficient, for bFGF- and EGF-induced increases in eNOS protein expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Endothelial cells
A primary ovine fetoplacental artery endothelial (OFPAE) cell line, recently established and validated in our laboratory (35), was used in this study. All cells used in this study were at passages 8–10.

Immunolocalization of receptors for FGF, EGF, and VEGF
Immunolocalization of receptors for FGF, EGF, and VEGF was performed as we described previously (5, 13, 35, 36). Subconfluent OFPAE cells cultured in chamber slides (Nunc, Inc., Naperville, IL) were fixed in 4% formaldehyde. Cells were stained with mouse antibovine FGF receptor-1 (FGFR; 2.5 µg/ml; Zymed Laboratories, South San Francisco, CA), mouse antihuman EGF receptor (EGFR; 2 µg/ml; NeoMarkers, Fremont, CA), or rabbit VEGF receptors (VEGFR; 0.5 µg/ml; antihuman flt-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) antibody for 1 h. Controls consisted of replacing the primary antibody with preimmune mouse (2.5 µg/ml for FGFR and EGFR) or rabbit (0.5 µg/ml for VEGFR) IgG (Vector Laboratories, Inc., Burlingame, CA). After immunostaining, cells were counterstained with hematoxylin.

Endothelial cell proliferation assays
To verify the presence of biologically functional receptors for bFGF, EGF, and VEGF in OFPAE cells, cell proliferation assays were performed as we described previously (13). Cells were precultured overnight in 96-well plates (5000 cells/well) in DMEM containing 10% FBS, 10% calf serum, and 1% penicillin-streptomycin (all from Life Technologies, Gaithersburg, MD). After preculture, media were changed to serum-free DMEM for 24 h. Cells were then treated with bovine bFGF, human recombinant EGF, or human recombinant VEGF165 (R & D Systems, Minneapolis, MN) at 0 (controls), 0.01, 0.1, 1, 10, or 100 ng/ml in serum-free DMEM (six wells per concentration). After an additional 72 h of culture, the number of cells was determined as described previously (13). Wells containing known cell numbers (0, 5,000, 10,000, 20,000, or 40,000 cells/well; 6 wells/cell density) were evaluated in a similar fashion to establish standard curves.

Western immunoblot analysis for MAPK
Western immunoblot analysis was performed as described previously (5, 13, 35, 36). After 16 h of serum deprivation, cells were treated for 10 min with bFGF, EGF, and VEGF (10 ng/ml) in the absence or presence of PD 98059 (50 µM; 1-h pretreatment), a specific MAPKK inhibitor (Calbiochem, La Jolla, CA) (37, 38). Controls consisted of cells cultured with DMEM or PD 98059 alone. Cells were washed with ice-cold PBS, harvested, and lysed by sonication in buffer [4 mM sodium pyrophosphate, 50 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM EDTA, 10 mM sodium fluoride, 2 mM sodium orthovanadate (Na3VO4), 1 mM phenylmethylsulfonylfluoride, 1% Triton X-100, 5 µg/ml leupeptin, and 5 µg/ml aprotinin]. The lysates were centrifuged, and protein concentrations of the supernatant were determined. Proteins (20 µg/lane) were separated on 12% SDS-PAGE gels, electroblotted onto Immobilon-P membrane (Millipore Corp., Bedford, MA), and immunoblotted with either rabbit p44/p42 MAPK (1:1000) or phospho-specific p44/p42 MAPK (1:2000) antibody (New England Biolabs, Inc., Beverly, MA). The former recognizes total p44/p42 MAPK, whereas the latter recognizes only phosphorylated forms of p44/p42 MAPK (39, 40). The p44/p42 MAPK on the membranes were visualized by an enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL) and quantified by scanning densitometry (model GS 670, Bio-Rad Laboratories, Inc., Hercules, CA). Standard phosphorylated and nonphosphorylated p42 MAPK (New England Biolabs, Inc., Beverley, MA) were included as positive controls. In preliminary time-course studies, we found that bFGF, EGF, and VEGF all rapidly increased phosphorylation of MAPK. Phosphorylation of MAPK appeared after 5 min of treatment, reached maximum levels after 10 min, and decreased after 20 min (data not shown).

MAPK activity assay
MAPK activity assays were performed using a p44/p42 MAPK assay kit and followed the instructions of the manufacturer (New England Biolabs, Inc.). Cell lysates (200 µg), obtained as described above, were immunoprecipitated with a mouse monoclonal antibody (1:100) raised against phospho-specific p44/p42 MAPK overnight at 4 C and incubated with protein A/G Sepharose (Santa Cruz Biotechnology, Inc.) for 2 h. After centrifuge, the pellet was washed in ice-cold lysis buffer, followed by a kinase buffer (25 mM Tris, 10 mM MgCl2, 5 mM ß-glycerophosphate, 2 mM dithiothreitol, and 0.1 mM Na3VO4, pH 7.5). The pellet was then incubated in the kinase buffer containing 200 µM ATP and 40 µg/ml Elk1 fusion protein, which served as the substrate. The reaction was terminated after 30 min at 30 C by the addition of 5 x Laemmli buffer. Samples (15 µl) were electrophoresed and transferred to the membrane as described above. The membrane was immunoblotted with a phospho-specific Elk1 antibody (1:2000). Phosphorylated Elk1 on the membrane was visualized by ECL system. Activated MAPK standards (20 ng; New England Biolabs, Inc.) run parallel to OFPAE cell samples served as positive controls.

Immunolocalization of phosphorylated MAPK
Immunolocalization of phosphorylated MAPK was performed using a method similar to that described above. After 16 h of serum starvation, cells cultured in the chamber slides were treated with 10 ng/ml bFGF, EGF, or VEGF for 0, 1, 5, 10, or 15 min. Cells in additional wells were treated with each growth factor for 10 min in the presence of PD 98059 (50 µM; 1-h pretreatment) or with PD 98059 alone. Cells were rinsed in ice-cold PBS, fixed, and stained with the rabbit phospho-specific p44/p42 MAPK antibody (1:250; New England Biolabs, Inc.). After immunostaining, cells were counterstained with hematoxylin.

Western immunoblot analysis for eNOS protein expression
Cells were cultured in DMEM containing 20% serum in culture dishes (60 x 15 mm; Becton Dickinson and Co., Mountain View, CA; ~1 x 106 cells/dish). After 24 h of serum starvation, cells were treated without or with bFGF, EGF, or VEGF at 0 (controls), 0.01, 0.1, 1, 10, or 100 ng/ml for 24 h. After determining dose-dependent effects, an effective (based upon both cell proliferation and eNOS protein expression assays) dose (10 ng/ml) of bFGF, EGF, and VEGF was used to determine the time dependency and synergy among bFGF, EGF, and VEGF on levels of eNOS protein. To determine the role of activated MAPKK/MAPK cascade on eNOS protein expression induced by bFGF and EGF, additional cells were treated for 24 h with 10 ng/ml bFGF and EGF in the absence or presence of PD 98059 (20 µM; 1-h pretreatment).

Western analysis was conducted as described above. Cells were harvested and lysed by sonication in 50–100 µl buffer [50 mM Tris, 0.15 M NaCl, 10 mM EDTA (pH 7.4); 0.1 M phenylmethylsulfonylfluoride, 0.1% ß-mercaptoethanol, 0.1% Tween-20, 5 µg/ml leupeptin, and 5 µg/ml aprotinin]. The lysates were centrifuged, and protein concentrations of the supernatant were determined. Proteins (2 µg/lane) were separated on 7.5% SDS-PAGE gels, electroblotted onto the membrane, immunoblotted with a mouse monoclonal eNOS antibody (1:750; Transduction Laboratories, Inc., Lexington, KY), and visualized by the ECL system. Levels of eNOS protein were then quantified by scanning densitometry. Protein from human umbilical vein endothelial cells (2.5 µg; Transduction Laboratories, Inc.) (5, 37) was included on each gel as a positive control. Linearity for eNOS quantification was confirmed throughout the working range (r2 = 0.99; P < 0.0001).

RT/PCR eNOS messenger RNA (mRNA) mass assay
eNOS mRNA was quantified by coupled RT-PCR amplification in a single tube assay as described previously (41). The forward and reverse primers, used for targeting amplification from part of the ovine eNOS protein-coding region (GenBank accession no. U76738) were 5'-TGTGGCTGTCTGCATGG-3' and 5'-TGGCTGGTAGCGGAAGG-3', respectively. The final product was 300 bases. For eNOS mRNA quantification, cells were treated without or with three growth factors. After 24 h, total RNA was extracted from cultured cells using a phenol/chloroform/isoamyl alcohol extraction procedure, as described previously (36), and incubated (0.1 µg per tube) in a 50-µl final volume containing 1 x PCR buffer; 2 mM MgCl2; 10 nmol of each deoxy (d)-ATP, dCTP, dTTP, and dGTP; and 30 pmol of each forward and reverse temperature-matched primers. Amplification was performed in the presence of 1 µl AMV reverse transcriptase (2.5 U) and 1 µl Taq polymerase (5 U), except for RT- controls, which only contained Taq polymerase. The program used was annealing at 62 C for 5 min; RT at 50 C for 10 min; denaturing at 94 C for 2 min; and amplification for 29 cycles at 94 C for 30 sec, at 62 C for 30 sec, and at 72 C for 30 sec. Final products were extended to full length by incubation at 72 C for 30 sec. Controls for each assay included pooled RNA extracted from ovine uterine artery endothelial cells, and a standard curve containing known copy numbers of eNOS complementary DNA (cDNA) target sequence. At the end of the assay 10 µl of products were separated on a 2% Tris acetate-EDTA gel and transferred to MagnaGraph hybridization membranes (Molecular Separations, Inc., Westborough MA) for Southern blotting against a probe (generated against pOeNOS using asymmetric PCR) (42) encoding the same protein-coding sequence. After hybridization, membranes were washed once in 2 x SSC (standard saline citrate-0.1% SDS for 15 min and twice in 0.1 x SSC-0.1% SDS (twice, 30 min each time) before drying and direct exposure to a phosphorimager (Bio-Rad BI screen; 15 min) for direct quantification (Molecular Analysis version 1.4, Bio-Rad Laboratories, Inc.). Data were normalized to 28S ribosomal RNA and expressed as a percentage of the control value.

Statistical procedures
For growth factor dose, time, and synergy responses, data were analyzed using one-way ANOVA (SigmaStat, Jandel Scientific, San Rafael, CA). When an F test was significant, data were compared with their respective control by Bonferroni’s multiple comparisons or Student’s t test. Data are reported as the mean ± SEM


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Presence of FGFR, EGFR, and VEGFR
Immunocytochemistry and cell proliferation assays were performed on OFPAE cells to determine whether bFGF, EGF, and VEGF receptors are both present and functionally coupled. Positive immunolocalization demonstrated the presence of receptors for FGF, EGF, and VEGF on OFPAE cells (Fig. 1AGo). Moreover, all three growth factors stimulated proliferation of OFPAE cells across all doses studied (Fig. 1BGo). Both bFGF and EGF stimulated (P < 0.05) cell proliferation in a dose-dependent manner. bFGF stimulated cell proliferation with a 50% increase over the control value at 0.01 ng/ml, reached its maximum effect (~210%) at 10 ng/ml, and then declined slightly (~170%). For EGF, the threshold stimulatory effect (P < 0.05) was similar to that of bFGF at 0.01 ng/ml (~50%), but its maximum effect (~120% at 100 ng/ml) appeared lower than that of bFGF. For VEGF, the threshold stimulatory effect on cell proliferation (P < 0.05) also occurred at 0.01 ng/ml (~110%) and was elevated progressively up to 10 ng/ml (~160%), but then declined slightly at 100 ng/ml (~110%). Thus, these observations indicated that OFPAE cells in culture express functionally coupled receptors for bFGF, EGF, and VEGF.



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  		Figure 1. Expression and functional coupling of receptors for bFGF, EGF, and VEGF on OFPAE cells. A, Immunolocalization of FGFR-1, EGFR, and VEGFR on OFPAE cells. In a and b, cells were stained with a mouse monoclonal FGFR-1 or EGFR antibody (IgG fraction), respectively. In c, cells were stained with a rabbit VEGFR (flt-1) antibody (IgG fraction). In d and e, the primary antibody was replaced with preimmune mouse and rabbit IgG (controls), respectively. Note that dark cytoplasmic staining indicates positive FGFR-1, EGFR, and VEGF staining. Dark nuclear staining is hematoxylin counterstaining. No positive cytoplasmic staining is observed in control wells in the absence of the primary antibody (not shown) or in wells stained with preimmune mouse and rabbit IgG. B, Effects of bFGF, EGF, and VEGF on proliferation of OFPAE cells. Cells were counted after 72 h of treatment. Data for each point are averaged from 18 wells from three experiments and expressed as the mean ± SEM percentage of the control value. The number of cells per well in controls was 6388.8 ± 369.0. Within each growth factor treatment, means with different letters differ significantly (P < 0.05).

 
Activation of MAPK by bFGF, EGF, and VEGF
Phosphorylation and activation of MAPK by bFGF, EGF, and VEGF were determined (Fig. 2Go). All three growth factors rapidly caused phosphorylation of MAPK, but did not alter protein levels of total p44/p42 MAPK isoforms (overall averages, 97 ± 7% and 87 ± 7% of control for p44 and p42, respectively; Fig. 2AGo). After 10 min of treatment, bFGF increased (P < 0.05) phosphorylation of both p44 and p42 isoforms of MAPK by about 2000% over the control value. VEGF increased (P < 0.05) phosphorylation of both p44 and p42 isoforms by about 800%. EGF treatment phosphorylated p44, but not p42, isoform, with a slight increase (~160% over the control value; P < 0.05) compared with bFGF and VEGF. The phosphorylation of MAPK induced by all three growth factors was effectively inhibited (P < 0.05) by pretreatment with PD 98059. Parallel to their actions on MAPK phosphorylation, bFGF, EGF, and VEGF stimulated MAPK activity, as indicated by the significant elevation in phosphorylation of its substrate, Elk1 fusion protein (Fig. 2BGo). Moreover, these growth factor-induced increases in MAPK activity were inhibited by PD 98059 treatment. Phosphorylation of Elk1 in controls and with PD 98059 treatment alone was undetectable. To further define MAPK activation by these growth factors, immunocytochemistry was used to examine intracellular translocation of phosphorylated MAPK, which is a critical step for the MAPK cascade in growth factor-induced cell growth and differentiation (29, 30). All three growth factor treatments showed similar temporal changes in positive phosphorylated MAPK staining, and staining intensity paralleled those changes observed in Western analysis (i.e. positive staining was observed in many more cells in bFGF and VEGF treatments than in EGF treatments; Fig. 3Go). Positive phosphorylated MAPK staining was not observed in any cell compartment at time zero of treatment, first appeared in cytosol after 1 min of treatment, appeared in nuclei after 5 min of treatment, accumulated heavily in nuclei after 10 min of treatment, and started to disappear from nuclei after 15 min (not shown). Positive nuclear phosphorylated MAPK staining induced by all three growth factors was blocked in all cells when pretreated with PD 98059, and no positive phosphorylated MAPK staining was observed in cells treated with PD 98059 alone.



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  		Figure 2. Activation of MAPK induced by bFGF, EGF, and VEGF in OFPAE cells. Cells were treated with bFGF, EGF, or VEGF (10 ng/ml) for 10 min alone or for 10 min 1 h after pretreatment with PD 98059 (50 µM). A, Phosphorylation of p44/p42 MAPK. Proteins (20 µg/lane) were separated, and p44/p42 MAPK were detected using a phospho-specific MAPK or MAPK antibody, as described in Materials and Methods. Representative blots from three experiments are shown. Data from three blots of three experiments are expressed as the mean ± SEM. Means with asterisks differ significantly (P < 0.05) from the control values. Within each growth factor treatment, means with different letters differ significantly (P < 0.05). B, Activation of MAPK. Proteins (200 µg) were immunoprecipitated with a phospho-specific p44/42 MAPK antibody and subsequently incubated with its substrate Elk1 fusion protein. Phosphorylated Elk1 was detected using a phospho-specific Elk1 antibody as described above. A representative blot from three experiments is shown. pp44/42, Phosphorylated p44/42 MAPK; PD, PD 98059; standard, phosphorylated (2 ng protein) or unphosphorylated (10 ng protein) p44/p42 MAPK (New England Biolabs, Inc.; positive control); pElk1, phosphorylated Elk1 fusion protein; ppMAPK, activated MAPK (New England Biolabs, Inc.; positive control). Mol wt (MW) markers are indicated.

 


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  		Figure 3. Immunocytochemical detection of phosphorylated MAPK translocation induced by bFGF, EGF, and VEGF in OFPAE cells. After 16 h of serum starvation, cells were treated with bFGF, EGF, or VEGF (10 ng/ml) for 0, 1, 5, 10 or 15 min or for 10 min after 1 h of pretreatment with PD 98059 (50 µM). Cells were fixed and stained with a phospho-specific p44/p42 MAPK antibody. Representative pictures from each growth factor treatment are shown. Note that brown staining indicates positive phosphorylated MAPK. Blue nuclear staining is hematoxylin counterstaining. No positive staining in any cellular compartment is observed in control wells in the absence of the primary antibody or in wells stained with preimmune rabbit IgG (not shown). PD, PD 98059; GF, growth factor.

 
Effects of bFGF, EGF, and VEGF on protein expression of eNOS
The OFPAE cell lysates on Western immunoblots showed a signal major band at 140 kDa for eNOS (Fig. 4Go), which corresponded to its respective positive control. Both bFGF and EGF induced a dose-dependent increase in eNOS protein levels (Fig. 4AGo). bFGF increased (P < 0.05) eNOS protein levels in OFPAE cells at all doses studied, starting at 0.01 ng/ml (~140% over the control value), reaching a maximum at 10 ng/ml (~850%) and then declining at 100 ng/ml (~490%). The stimulatory (P < 0.05) effect of EGF started at 0.1 ng/ml, yielding a maximum response of approximately 160% over the control value, which remained relatively constant through 100 ng/ml (~100%). Compared with bFGF, this EGF-induced response was quite small, especially when considering these effects based upon molar concentrations (molecular masses for bFGF and EGF are 18 and 6 kDa, respectively). Thus, bFGF is more efficacious than EGF in stimulating eNOS protein expression in OFPAE cells. VEGF did not significantly alter eNOS protein expression with any of the doses used.



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  		Figure 4. Effects of bFGF, EGF, and VEGF on eNOS protein levels in OFPAE cells. A, Dose response. Cells were treated with the growth factors (0.01–100 ng/ml) for 24 h. Data are from three blots of three experiments for each growth factor. B, Time course. Cells were treated with each growth factor at 10 ng/ml for 0, 6, 12, 24, or 36 h. Data are from three blots of three or four experiments for each growth factor. C, Synergistic effects of bFGF, EGF, and VEGF. Cells were treated with each growth factor (10 ng/ml) or combinations of each growth factor for 24 h. Data are from four blots of two to four experiments for each growth factor treatment. For all growth factor treatments, proteins (2 µg) were separated and detected as described in Materials and Methods. Representative blots are shown. Quantitative data are expressed as the mean ± SEM percentage of the control value (in the absence of growth factor). Within each growth factor treatment, means with different letters differ significantly (P < 0.05). HUVEC, Human umbilical vein endothelial cells (2 µg protein; positive control for eNOS).

 
After determining the dose-dependent effects of each growth factor on protein expression of eNOS, an appropriate dose (10 ng/ml) of bFGF, EGF, and VEGF was used to determine the time dependency of eNOS protein expression (Fig. 4BGo). The stimulatory effect of bFGF on eNOS protein expression was detectable (P < 0.05) 6 h after treatment, continued increasing up to 12 h, and remained elevated up to 36 h. The EGF-stimulated increase in eNOS protein expression was noted at 12 h (P < 0.05) of treatment, and the effect remained relatively unchanged thereafter up to 36 h. Consistent with our dose-response data, VEGF had no significant effect on eNOS protein levels throughout the time course studied.

To determine possible synergy among the effects of bFGF, EGF, and VEGF on eNOS protein expression, OFPAE cells were treated with each growth factor alone or in combination at 10 ng/ml for 24 h. The effects of these three growth factors alone showed results similar to those reported above. There were no significant synergistic effect of any combination of these growth factors on eNOS levels (Fig. 4CGo). Instead, the addition of VEGF, but not both EGF plus VEGF, to bFGF-treated cells tended to slightly attenuate the bFGF-induced increase in eNOS protein expression. VEGF did not alter the EGF-induced increase in eNOS protein expression.

RT-PCR mass assay for eNOS mRNA
Coupled RT-PCR analysis of eNOS mRNA levels confirmed that the eNOS mRNA levels, normalized to 28S ribosomal RNA in OFPAE cells, were increased by bFGF (233 ± 29% of the control value; P < 0.05) and EGF (118 ± 7%), but not VEGF (92 ± 25%; Fig. 5Go). These data correspond to the elevation of eNOS protein observed above. Results were obtained from six determinations of two experiments.



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  		Figure 5. Effects of bFGF, EGF, and VEGF on eNOS mRNA levels in OFPAE cells. Cells were treated with bFGF, EGF, and VEGF (10 ng/ml) for 24 h. Total RNA was extracted, and eNOS mRNA levels were quantified by RT-PCR as described in Materials and Methods. The standard curve using known copy numbers (103–107) of eNOS cDNA templates showed excellent correlation (r2 = 0.957; P < 0.001) between log(counts) and log(copy number per tube). The sensitivity limit was less than 1 x 103 copies of eNOS mRNA/µg total RNA. A representative blot from two experiments is shown. No signals was observed in RT- pooled RNA extracted from ovine uterine artery endothelial cells. The number of copies of eNOS mRNA per µg total RNA in controls after 24 h of treatment was 1.81 ± 0.75 x 109.

 
Requirement of activation of MAPK for bFGF- and EGF-induced increases in eNOS protein levels
Treatment of cells with PD 98059 alone did not alter eNOS protein levels; however, PD 98059 significantly (P < 0.05) inhibited the effect of bFGF by 93% and completely blocked the effect of EGF on stimulating eNOS protein expression (Fig. 6Go).



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  		Figure 6. Effects of PD 98059 on bFGF- and EGF-induced increases in eNOS protein levels in OFPAE cells. Cells were treated with bFGF and EGF (10 ng/ml) for 24 h in the absence or presence of PD 98059 (50 µM; 1 h of pretreatment). Proteins (2 µg) were separated and detected as described in Materials and Methods. A representative blot from three experiments is shown. HUVEC, Human umbilical vein endothelial cells (2 µg protein; positive control for eNOS). Quantitative data from three blots of three experiments are expressed as the mean ± SEM. Means with asterisks differ significantly (P < 0.05) from the control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Herein we demonstrated that the previously characterized OFPAE cells express receptors to bFGF, EGF, and VEGF (flt-1), all of which are functionally coupled, as demonstrated by robust mitogenic responses. In addition, all three growth factors activated the MAPK cascade, but in a manner that did not correlate directly with the magnitude of the subsequent mitogenic response. More importantly, for the first time, we have also shown that bFGF and EGF can induce the expression of eNOS, as seen at the level of protein and mRNA, and further, that this response is blocked by the MAPK inhibitor PD 98059. In contrast VEGF had no effect on eNOS expression, even though we confirmed MAPK activation and the presence of the VEGF receptor flt-1 in OFPAE cells, and VEGF is known to have an acute effect on the release/production of NO through flt-1 in human umbilical vein endothelial cells (26). Thus, we conclude that activation of the MAPK cascade is necessary, but not sufficient, for the induction of eNOS expression in OFPAE cells.

The lack of a close correlation between the dose-dependent magnitude of MAPK activation and the magnitude of both the mitogenic response and the expression of eNOS in OFPAE in response to each agonist suggests the involvement of additional distinct signaling events. Such a premise is consistent with a growing body of evidence from studies of mitosis in other systems, including NIH-3T3 cells (43) and vascular smooth muscle cells (44), and is of physiological importance because this would provide additional means for the differential regulation of these two important cell functions. This concept is also supported by the reports of Ziche and colleagues, who demonstrated that the activation of MAPK is required for VEGF-induced, but not for bFGF-induced, mitogenesis of coronary venule endothelium (27, 45).

An important question that follows is whether these additional signaling events involve activation of distinct signaling pathways or, alternatively, may reflect the translocation of MAPK to the nucleus upon activation. The translocation of activated MAPK into the nuclei is a key step for cellular responses to growth factors (29, 30), and the current data also show that translocation does indeed occur in OFPAE cells in response to all three of the growth factors studied. However, as all three of these growth factors stimulate mitogenesis, but VEGF fails to induce eNOS expression even in the face of potent MAPK activation and translocation, it seems that the activation and translocation are less important for eNOS expression than for mitogenesis. We conclude, therefore, that although mitogenesis may simply require that a threshold level of MAPK activation/translocation be achieved, distinct signaling pathways must be activated in addition to the MAPK cascade to control eNOS expression.

Another interesting question is why VEGF, which is a known growth factor and activator of MAPK, does not increase eNOS expression in OFPAE cells. One possibility is given by the studies of Ziche et al. (27, 45), who demonstrated that VEGF may be unusual in using NO as an intermediate in the activation of MAPK in coronary venule endothelium. It remains to be seen whether this is the case in OFPAE cells, but activation of eNOS to promote MAPK activity followed by further induction of eNOS expression would represent a feed-forward loop that may run out of control if left unchecked. One solution would be to require additional pathways to be activated independently to control eNOS expression, as seems to be the case in OFPAE cells.

Our findings that bFGF induced increases in eNOS expression in OFPAE cells confirm previous studies in bovine adrenal capillary endothelial cells by Kostyk et al. (24). Based upon our dose- and time-course studies, we also demonstrated that bFGF is a more efficacious stimulator of eNOS expression than either EGF or VEGF. We also showed that bFGF significantly increased eNOS mRNA levels, whereas the effects of EGF did not reach significance. This apparent lack of effect of EGF should be interpreted cautiously, however, because we observed that along with their effects on eNOS mRNA, bFGF (157.9% of the control value) and EGF (213.5%), but not VEGF (102.8%), also elevated levels of ribosomal RNA. Thus, our data, normalized to ribosomal RNA, represent a conservative estimate of changes in eNOS message. Without such normalization, the stimulatory effects of bFGF (374.9%) and EGF (229.0%) reached significance (P < 0.05), whereas those of VEGF did not (133.6%). It is not clear from these data alone whether the bFGF- and EGF-increased steady state mRNA levels are due to increased transcription or elevations in message stability. Furthermore, the relative magnitudes of these changes in mRNA levels for each growth factor are not entirely consistent with the magnitude of the corresponding changes in protein levels. Further studies will be necessary to determine the extent to which bFGF- and EGF-increased eNOS expressions are regulated at the level of transcription, message stability, or translation.

In conclusion, we have shown that bFGF and EGF, but not VEGF, increase eNOS expression (protein and mRNA) in OFPAE cells, and that MAPK activation is necessary, but not sufficient, for this response. Together with the evidence that eNOS (35) and bFGF and EGF receptors are present in fetoplacental artery endothelium, the current study supports our hypothesis that bFGF and EGF can directly elevate eNOS levels in fetoplacental endothelial cells in culture. Our data further suggest that fetoplacental endothelium in vivo may also respond to these growth factors, which are known to be produced locally (13, 18, 19), to increase eNOS expression and NO production. This proposed action in vivo seems all the more likely in view of the finding that a decrease in fetal plasma bFGF and EGF is associated with intrauterine growth retardation (46, 47), which could be interpreted in the light of our findings as a failure to maintain eNOS expression and a concomitant fall in circulating NO, thus resulting in decreased fetoplacental blood flow.


   Acknowledgments
 
The authors thank Dr. C. P. Weiner, Department of Obstetrics and Gynecology, University of Maryland (Baltimore, MD), for kindly providing partial ovine eNOS cDNA sequence, and Drs. Lewis. G. Sheffield and Paul. J. Bertics, University of Wisconsin (Madison, WI), for their critical reading of this manuscript. We also thank J. M. Cale and T. M. Phernetton for their expert technical assistance.


   Footnotes
 
1 This work was supported in part by NIH Grants HL-57653, HL-49210, HD-33255 (to R.R.M.), HL-56702 (to I.M.B.), USDA Grant 9601773 (to I.M.B.), and American Heart Association, Wisconsin Affiliate, Fellowship Award 62936 (to J.Z.) and Grant 95-GS-74 (to R.R.M.). Back

Received July 21, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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