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BOR - Papers in Press, published online ahead of print November 19, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.022293
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BIOLOGY OF REPRODUCTION 70, 785–796 (2004)
DOI: 10.1095/biolreprod.103.022293
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Mechanisms of Shear Stress-Induced Endothelial Nitric-Oxide Synthase Phosphorylation and Expression in Ovine Fetoplacental Artery Endothelial Cells1

Yun Li3, Jing Zheng3, Ian M. Bird3,4, and Ronald R. Magness2,3,4,5

Perinatal Research Laboratories, Departments of Obstetrics and Gynecology,3 Pediatrics,4 Animal Sciences,5 University of Wisconsin-Madison, Wisconsin 53715


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Placental blood flow, nitric-oxide (NO) levels, and endothelial NO synthase (eNOS) expression increase during human and ovine pregnancy. Shear stress stimulates NO production and eNOS expression in ovine fetoplacental artery endothelial (OFPAE) cells. Because eNOS is the rate-limiting enzyme essential for NO synthesis, its activity and expression are both closely regulated. We investigated signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by OFPAE cells. The OFPAE cells were cultured at 3 dynes/cm2 shear stress, then exposed to 15 dynes/cm2 shear stress. Western blot analysis for phosphorylated ERK1/2, Akt, p38 mitogen activated protein kinase (MAPK), and eNOS showed that shear stress rapidly increased phosphorylation of ERK1/2 and Akt but not of p38 MAPK. Phosphorylation of eNOS Ser1177 under shear stress was elevated by 20 min, a response that was blocked by the phosphatidyl inositol-3-kinase (PI-3K)-inhibitors wortmannin and LY294002 but not by the mitogen activated protein kinase kinase (MEK)-inhibitor UO126. Basic fibroblast growth factor (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels otherwise induced by the 15 dynes/cm2 shear stress. Blockade of either signaling pathway changed the shear stress-induced increase in eNOS protein levels. In conclusion, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibition of MEK. Prolonged shear stress-stimulated increases in eNOS protein were not affected by inhibition of MEK- or PI-3K-mediated pathways.

growth factors, mechanisms of hormone action, nitric oxide, placenta, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The most striking cardiovascular adaptation observed during pregnancy is the dramatic elevation in placental and uterine blood flows that occurs to meet the increasing need for nutrients by the growing fetus [1–3]. Endothelium-derived nitric oxide (NO) plays a significant role in this pregnancy adaptation, as shown by evidence that NO is released in greater amounts by fetoplacental and uteroplacental endothelium [4] and that chronic inhibition of NO production induces fetal growth restriction [5, 6] via decreases in fetoplacental blood flow [7]. Moreover, NO produced by ovine placenta and endothelial NO synthase (eNOS) protein levels in placental artery endothelium are elevated gradually from Day 110 to Day 130 of pregnancy [8, 9].

Shear stress (i.e., frictional forces on the vessel wall from blood flow) modulates many physiological and pathological processes associated with endothelium, such as endothelial cell proliferation, vasodilation, vasoconstriction, and inflammatory responses [10, 11]. This mechanical force is a potent stimuli for endothelium-dependent NO production and vasodilation [12, 13]. Elevations in fetoplacental blood flow/shear stress also increase NO production by ovine fetoplacental endothelial (OFPAE) cells.

The only NOS family isoform identified in the endothelium of the ovine fetoplacental vascular bed is eNOS, the rate-limiting enzyme for NO synthesis [8, 9]. Activity of eNOS is regulated by Ca2+/calmodulin-dependent [14] and Ca2+-independent mechanisms. Phosphorylation of eNOS on Ser1177 and eNOS activity increased rapidly (1 min) at a steady shear stress of 25 dynes/cm2 [15, 16]. Shear stress-induced eNOS activity may be mediated through the phosphatidyl inositol-3-kinase (PI-3K) pathway, because wortmannin completely inhibited the shear stress-induced increase in cGMP and NO production in pig coronary artery segments, bovine aortic endothelial cells (BAEC), and human umbilical vein endothelial cells (HUVEC) [16–18]. Also, ERK1/2 was rapidly activated by shear stress in BAEC [19], and erbstatin A, a tyrosine kinase inhibitor, abrogated shear stress-induced phosphorylation of ERK1/2 [20]. It is unclear what interactive role PI-3K and mitogen activated protein kinase (MAPK) pathways play in eNOS Ser1177 phosphorylation.

Shear stress induces eNOS mRNA and protein expression in various endothelial cell types [21–23]. Little is known regarding which signaling cascade controls eNOS expression under shear stress. Upregulation of eNOS mRNA by shear stress in BAEC is inhibited by Ca2+ chelation or pertussis toxin [24, 25]. Tyrosine kinase c-Src may play a role in shear stress-stimulated eNOS mRNA transcription and stabilization with ERK1/2 being the downstream mediator to c-Src [26]. In contrast, PI-3K may have an inhibitory effect on shear stress-induced increases in eNOS mRNA level [25].

Endothelial cells are constantly exposed to pulsatile blood flow in vivo. We have reported successful culturing of OFPAE cells in a novel pulsatile perfusion system using artificial capillaries [27]. Pulsatile shear stress acutely elevated NO production before prolonged eNOS protein levels were increased. Thus, NO synthesis results initially from elevations in eNOS activity but subsequently from additional rises in eNOS expression. To understand vasodilation in pregnancy, it is important to evaluate the mechanisms underlying shear stress-induced increases in eNOS activity versus eNOS protein expression. To our knowledge, no reports have been published on signaling mechanisms controlling eNOS phosphorylation and eNOS levels in flow-adapted endothelial cells stimulated by increased pulsatile flow. We also tested the hypothesis that angiogenic factors (basic fibroblast growth factor [bFGF] and vascular endothelial growth factor [VEGF]) elevated in pregnancy [28, 29] would further augment shear stress-induced increases in eNOS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Antibodies

Dulbecco modified Eagle medium (DMEM), calf serum, fetal bovine serum, penicillin/streptomycin, and trypsin (0.025%)/EDTA (0.53 mM) were purchased from Life Technologies (Gaithersburg, MD). The UO126 was purchased from Promega (Madison, WI). 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), PD98059, SB203580-HClm, and wortmannin were purchased from Calbiochem (San Diego, CA). The LY294002 was purchased from Cell Signaling Technology (Beverly, MA). The bFGF and VEGF were obtained from R&D Systems, Inc. (Minneapolis, MN). Anti-phospho-Thr202Tyr204-ERK1/2 antibody, anti-ERK1/2 antibody, and horse radish peroxidase (HRP)-linked anti-rabbit immunoglobulin (Ig) G were purchased from New England Biolabs (Beverly, MA). Anti-phospho-Ser473-Akt antibody, anti-Akt antibody, anti-phospho-Thr180Tyr182-p38 MAPK antibody, anti-p38 MAPK antibody, anti-phospho-Thr183Tyr185-JNK antibody, anti-JNK antibody, and anti-phospho-Ser1177-eNOS antibody were purchased from Cell Signaling Technology. Anti-phospho-Thr495-eNOS and anti-phospho-Ser116-eNOS antibody were purchased from Upstate Biotechnology (Lake Placid, MA). Anti-eNOS antibody was purchased from Transduction Laboratories (Lexington, KY). The HRP-linked sheep-anti-mouse IgG was purchased from Amersham Biosciences (Arlington Heights, IL). The enhanced chemiluminescence (ECL) reagent detection system and ECL Plus Western blot detection system were obtained from Amersham Biosciences.

Cell Culture

The OFPAE cells were cultured in DMEM supplemented with 10% calf serum, 10% fetal bovine serum, and 1% penicillin/streptomycin in T75 flasks. When reaching confluence, the cells were passaged at 1:4. Cells of passage 13 were used in the experiments. For shear stress studies, the cells were inoculated into the lumens of Cellco CELLMAX artificial capillary modules (Spectrum Laboratories, Rancho Dominguez, CA). For static studies, the cells were plated in 60-mm tissue culture dishes. In both conditions, the culture media were DMEM supplemented with 10% calf serum, 10% fetal bovine serum, and 1% penicillin/streptomycin, and confluent cells were subjected to further treatment.

Cellco CELLMAX Artificial Capillary Module System

Passage 13 OFPAE cells (5 x 106) were inoculated into the Cellco CELLMAX artificial capillary modules and grown (i.e., adapted to flow) inside the capillary lumens with a pulsatile shear stress averaging 3 dynes/cm2 in each capillary as previously described [27]. Then, 24 hours before elevating shear stress to the endothelial cells, the entire flow path was gently flushed with serum-free DMEM media, and the culture media in the reservoir were replaced with DMEM without serum supplement. Further shear stress exposure studies were performed at Days 11–13 after inoculation, when the cells had reached confluence as indicated by a stable lactate production rate and morphology [27].

Acute Induction of ERK1/2, Akt, p38 MAPK, and eNOS Phosphorylation by Shear Stress

The confluent OFPAE cells were exposed to pulsatile shear stresses averaging 15 dynes/cm2 for 0, 5, 10, 20, or 30 min (n = 4–6 per time point) and solubilized in lysis buffer (10 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-100, 0.5% NP-40, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml of leupeptin, 10 µg/ml of aprotinin, and 1 mM phenylmethylsulfonylfluoride). Protein concentration was determined by BCA assay (Sigma Chemical Co., St. Louis, MO). To test the effects of signaling pathway blockade on eNOS phosphorylation, confluent OFPAE cells were pretreated at 3 dynes/cm2 with the mitogen activated protein kinase kinase (MEK)-inhibitor UO126 (10 µM) or the PI-3K-inhibitors wortmannin (100 nM) or LY294002 (10 and 50 µM) for 1 h before shear stress was elevated to 15 dynes/cm2. One experiment was stopped at 0 and 20 min and the other at 0 and 2 h. The dosages of these inhibitors were chosen based on the median inhibitory concentration (IC50) of UO126 (10–20 µM), wortmannin (2–4 nM), SB203580 (34 nM), and LY294002 (1.4–5 µM) as well as preliminary studies in static culture showing specific reductions in phosphorylated intermediates of both pathways or in OFPAE cell proliferation assays in which LY294002 had an IC50 of 2–5 µM.

Western Blot Analysis for ERK1/2, Akt, p38 MAPK, and eNOS Phosphorylation

Solubilized total cellular proteins (15 µg/lane) were separated on 7.5% SDS-PAGE and transferred onto Immobilon P membrane (Millipore, Bedford, MA). Phosphorylated and total ERK1/2, Akt (Ser473), p38 MAPK, and eNOS (Ser1177, Thr495, and Ser116) were detected by Western blot analysis using phospho-specific and nonphospho-specific antibodies. Anti-phospho-ERK1/2 and HRP-linked anti-rabbit IgG antibodies were both used at 1:2000 dilution. The anti-ERK1/2, anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-p38 MAPK, and anti-p38 MAPK antibodies were used at 1:1000 dilution, and their HRP-linked anti-rabbit IgG antibodies were used at 1:2000 dilution. The anti-phospho-Ser1177-eNOS, anti-phospho-Thr495-eNOS and anti-phospho-Ser116-eNOS were used at 1:1000, 1:750, and 1:500 dilution, respectively, followed by 1:2000 HRP-linked anti-rabbit IgG. The anti-eNOS and HRP-linked anti-mouse IgG were used at 1:750 and 1:3000 dilution, respectively. Signals were visualized by ECL Plus or ECL system, and intensities were quantified by transmission scanning densitometry (Bio-Rad 670 scanning densitometer; Bio-Rad Laboratories, Hercules, CA). Phosphorylation level of a particular protein was determined by the ratio of phosphorylated protein to total protein.

Prolonged Effects of bFGF and VEGF on eNOS Protein Levels in Static Culture

The OFPAE cells at passage 13 were cultured in 60-mm tissue culture dishes until confluence and then subjected to serum withdrawal for 24 h before further treatments. Cells were stimulated with bFGF (10 ng/ml) or VEGF (10 ng/ml) for 24 h in the absence or presence of UO126 (10 µM) or PD98059 (50 µM) for 1 h before treatment. Then, the cells were solubilized into lysis buffer (50 mM Tris, 0.15 M NaCl, 10 mM EDTA [pH 7.4], 0.1% Tween-20, 0.1% ß-mercaptoethanol, 5 µg/ml of leupeptin, 5 µg/ml of aprotinin, and 0.1 mM phenylmethylsulfonylfluoride), and the protein concentrations were quantified by modified Lowry assay (Bio-Rad Laboratories).

Prolonged Effects of Growth Factors and Signaling Pathway Inhibitors on Shear Stress-Induced eNOS Protein Levels

The OFPAE cells were cultured inside the Cellco CELLMAX capillary system at 3 dynes/cm2 shear stress until they reach confluence. After 24 h of serum withdrawal, bFGF or VEGF was added into the media reservoir, reaching the final concentration of 10 ng/ml 30 min before further shear stress treatment to let bFGF and VEGF circulate and distribute within the system evenly. Shear stress was then elevated to 15 dynes/cm2 or kept at 3 dynes/cm2 for up to 24 h. The cells were eluted from the capillaries using trypsin (0.025%)/EDTA (0.53 mM) treatment for 7 min at 0, 6, or 24 h of growth factor stimulation. Cell pellets were obtained by centrifugation at 1000 rpm, 4°C, for 10 min. The pellets were then solubilized in lysis buffer (50 mM Tris, 0.15 M NaCl, 10 mM EDTA [pH 7.4], 0.1% Tween-20, 0.1% ß-mercaptoethanol, 5 µg/ml of leupeptin, 5 µg/ml of aprotinin, and 0.1 mM phenylmethylsulfonylfluoride), and the protein concentrations were quantified by modified Lowry assay. To study the effects of signaling pathway blockade on shear stress-induced eNOS protein levels, confluent OFPAE cells within the artificial capillary modules were pretreated with UO126 (10 µM), wortmannin (100 nM), LY294002 (50 µM), SB203580 (20 µM), or BAPTA/AM (10 µM) for 1 h and then exposed to further shear stresses of 3 or 15 dynes/cm2 for 24 h. The cell lysates were obtained following the same procedure.

Statistics

Data are presented as the mean ± SEM. Data were analyzed by Student t-test or one-way ANOVA. A level of P < 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Acute Regulation of Shear Stress Signaling Mechanisms on eNOS Phosphorylation

Increasing shear stress from 3 to 15 dynes/cm2 produced a rapid (within 10 and 5 min, respectively) increase in phosphorylation of both ERK1/2 and Akt, but not of p38 MAPK, in OFPAE cells (Fig. 1). However, phospho-JNK could not be detected (data not shown). Phosphorylation of eNOS on Ser1177 was also elevated by the higher shear stress (Fig. 1), whereas the level of phospho-Thr495-eNOS was not changed (data not shown). Phospho-Ser116-eNOS was not detectable in OFPAE cells under these conditions (data not shown). The eNOS phosphorylation on Ser1177 induced by 15 dynes/cm2 shear stress reached significance at 20 min of treatment, which corresponds with the acute phase of the NOx production time course that we previously reported [27]. Therefore, all the following acute experiments studying the signaling pathways related to eNOS phosphorylation were performed for 20 min of stimulation of pulsatile shear stress to correspond to the maximal eNOS Ser1177 phosphorylation.



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  		FIG. 1. Phosphorylation of ERK1/2, Akt, p38 MAP kinase, and eNOS (Ser1177) by pulsatile shear stress. OFPAE cells were cultured (adapted) inside the lumens of the Cellco CELLMAX capillary modules with a flow of 3 dynes/cm2 shear stress until confluent. Shear stress was then elevated to 15 dynes/cm2, and the cells were solubilized at 0, 5, 10, 20, and 30 min of stimulation. Western blot analysis for phosphorylated and total ERK1/2, Akt, p38 MAPK, and eNOS (Ser1177) was performed with specific antibodies, respectively. Phosphorylation levels were determined by the ratio of the phosphorylated form to the total amount of each molecule. Data are expressed as the mean ± SEM of the fold-value of the phosphorylation levels at 0 min (n = 4–6 per time point). *P < 0.05 compared to 0 min

Effects of UO126 on Shear Stress-Induced Phosphorylation of ERK1/2 and eNOS

Because ERK1/2 was activated by pulsatile shear stress before eNOS phosphorylation on Ser1177, the question arose whether it might also be involved in shear stress-induced phosphorylation of eNOS in these cells. Therefore, OFPAE cells were pretreated with the MEK-inhibitor UO126 (10 µM) for 1 h before shear stress was elevated to 15 dynes/cm2 for 20 min. We observed that although UO126 completely blocked the phosphorylation of ERK1/2 both at basal and stimulated pulsatile shear stresses, it had no effects on the Ser1177 phosphorylation levels of eNOS (Fig. 2). These data suggested that a UO126-sensitive (MEK-ERK1/2) pathway is not involved in shear stress-stimulated eNOS Ser1177 phosphorylation.



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  		FIG. 2. Effects of UO126 (10 µM) on pulsatile shear stress-induced phosphorylation of ERK 1/2 and eNOS. A) Confluent, adapted OFPAE cells were pretreated with the MEK-inhibitor UO126 (10 µM) for 1 h before shear stress was elevated to 15 dynes/cm2. The cells were solubilized at 0 and 20 min of shear stress elevation. The ERK1/2 phosphorylation and eNOS phosphorylation on the Ser1177 residue were detected by Western blot analysis. B) Intensities of phosphorylated ERK1/2 or eNOS were normalized by total ERK1/2 or eNOS, respectively, and expressed as the fold-value of the 0-min control. Data are expressed as the mean ± SEM (n = 4 per group). *P < 0.05 compared to 0 min, #P < 0.05 compared to control

Effects of Wortmannin on Shear Stress-Induced Akt and eNOS Phosphorylation

One of the downstream signaling molecules of PI-3K, Akt was shown to be transiently activated before eNOS phosphorylation under pulsatile shear stress (Fig. 1). The PI-3K-inhibitor wortmannin (100 nM) was introduced into the artificial capillary module 1 h before shear stress was elevated to 15 dynes/cm2 for 20 min. We observed that wortmannin substantially decreased Akt phosphorylation at basal shear stress of 3 dynes/cm2 after pretreatment as well as blocked the 15 dynes/cm2 shear stress-induced increase in Akt activation (Fig. 3). Moreover, the treatment of wortmannin at this dose significantly inhibited eNOS phosphorylation on Ser1177 induced by shear stress of 15 dynes/cm2 (Fig. 3). Because wortmannin may also inhibit signaling pathways other than PI-3K, similar experiments were performed with a more specific PI-3K-inhibitor, LY294002. At 10 µM, LY294002 reduced Akt phosphorylation at basal shear stress but could only partially blocked its increase at 15 dynes/cm2, and it did not inhibit the induction of eNOS phosphorylation on Ser1177 (Fig. 4). When the concentration of LY294002 was elevated to 50 µM, it caused the similar inhibitions on Akt and eNOS Ser1177 phosphorylation (Fig. 5), as observed with wortmannin. Therefore, shear stress-induced increase in OFPAE cell eNOS phosphorylation on Ser1177 residue detected at 20 min of stimulation appears to be mediated by the PI-3K pathway.



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  		FIG. 3. Effects of wortmannin (100 nM) on shear stress-induced Akt and eNOS phosphorylation. A) Confluent, adapted OFPAE cells were pretreated with the PI-3K-inhibitor wortmannin (100 nM) for 1 h before shear stress was elevated to 15 dynes/cm2. The cells were solubilized at 0 and 20 min of shear stress elevation. Phosphorylation of Akt and eNOS phosphorylation on the Ser1177 residue were detected by Western blot analysis. B) Intensities of phosphorylated Akt and eNOS were normalized by total Akt and eNOS, respectively, and expressed as the fold-value of the 0-min control. Data are expressed as the mean ± SEM (n = 3–4 per group). *P < 0.05 compared to 0 min, #P < 0.05 compared to control



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  		FIG. 4. Effects of LY294002 (10 µM) on pulsatile shear stress-induced Akt and eNOS phosphorylation. A) Confluent, adapted OFPAE cells were pretreated with the PI-3K-inhibitor LY294002 (10 µM) for 1 h before shear stress was elevated to 15 dynes/cm2. The cells were solubilized at 0 and 20 min of shear stress stimulation. Phosphorylation of Akt and eNOS phosphorylation on the Ser1177 residue were detected by Western blot analysis. B) Intensities of phosphorylated Akt and eNOS were normalized by total Akt and eNOS, respectively, and expressed as the fold-value of the 0-min control. Data are expressed as the mean ± SEM (n = 4 per group). *P < 0.05 compared to 0 min, #P < 0.05 compared to control



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  		FIG. 5. Effects of LY294002 (50 µM) on pulsatile shear stress-induced Akt and eNOS phosphorylation. A) Confluent, adapted OFPAE cells were pretreated with the PI-3K-inhibitor LY294002 (50 µM) for 1 h before shear stress was elevated to 15 dynes/cm2. The cells were solubilized at 0 and 20 min of shear stress stimulation. Phosphorylation of Akt and eNOS phosphorylation on the Ser1177 residue were detected by Western blot analysis. B) Intensities of phosphorylated Akt and eNOS were normalized by total Akt and eNOS, respectively, and expressed as the fold-value of the 0-min control. Data are expressed as the mean ± SEM (n = 4 per group). *P < 0.05 compared to 0 min, #P < 0.05 compared to control

Effects of MEK and PI-3K Inhibition on Shear Stress-Induced eNOS Ser1177 Phosphorylation at 2 h

Based on our previously reported time course for NOx production by OFPAE cells [27], phosphorylation of eNOS, ERK1/2, and Akt were also tested after 2-h shear stress treatment (Fig. 6). The phosphorylation of eNOS on Ser1177 was still significantly higher than the basal level, as was Akt activation, whereas ERK1/2 phosphorylation had returned to baseline. Inhibition of PI-3K with LY294002 (50 µM) blocked both Akt and eNOS phosphorylation on this particular residue. The UO126 (10 µM), however, greatly inhibited ERK1/2 activation without affecting Akt phosphorylation. The U0126 also tended to decrease eNOS phosphorylation to the control 0-h level, but this did not reach statistical significance compared to the control 2-h level (0.05 < P < 0.1).



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  		FIG. 6. Effects of MEK and PI-3K inhibition on pulsatile shear stress-induced eNOS Ser1177 phosphorylation at 2 h. The OFPAE cells were cultured in the CELLMAX artificial capillary modules until confluence. After 24 h of serum withdrawal, the cells were pretreated with LY294002 (50 µM) or UO126 (10 µM) for 1 h, followed by elevating shear stress to 15 dynes/cm2. The cells were solubilized at 0 and 2 h of shear stress stimulation. The ERK1/2 phosphorylation, Akt phosphorylation, and eNOS phosphorylation on Ser1177 residue were detected by Western blot analysis. Intensities of phosphorylated ERK1/2, Akt, and eNOS were normalized by total ERK1/2, Akt, and eNOS, respectively, and expressed as the fold-value of the 0-h control. Data are expressed as the mean ± SEM (n = 3–4 per group). *P < 0.05 compared to 0-h control, #P < 0.05 compared to 2-h shear stress control

bFGF-Induced Increase in eNOS Protein Levels in Static Culture Are Blocked by MEK Inhibitors

Previously, it has been reported that bFGF, but not VEGF, elevated eNOS protein levels in OFPAE cells in static culture and that the effect of bFGF was inhibited by the MEK-inhibitor PD98059 [30]. In that study, the OFPAE cells tested were of passages 8–10. Additional static culture experiments were performed to test if OFPAE cells of passage 13, which were used in shear stress studies, maintain the same responsiveness to bFGF, VEGF, and MEK inhibitors. As shown in Figure 7, 24-h treatment with bFGF (10 ng/ml) promoted greater eNOS protein levels versus control, and the increase was blocked by PD98059 (50 µM) and another MEK inhibitor, UO126 (10 µM), which was not tested in our previous study. On the other hand, VEGF (10 ng/ml) had no effects on eNOS protein levels in these cells.



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  		FIG. 7. The bFGF-induced increase in eNOS protein levels in static culture are blocked by MEK inhibitors. A) The OFPAE cells at passage 13 were cultured in 60-mm tissue culture dishes until confluent. After serum withdrawal for 24 h, they were pretreated with or without UO126 (10 µM) or PD98059 (50 µM) for 1 h, followed by treatment with bFGF (10 ng/ml) or VEGF (10 ng/ml) for 24 h. The solubilized proteins were subjected to Western blot analysis for eNOS. B) The eNOS intensities in each treatment group are shown as the fold-value of the control. Data are expressed as the mean ± SEM (n = 4 per group). *P < 0.05 compared to control

Effects of bFGF and VEGF on Shear Stress-Induced eNOS Protein Levels in OFPAE Cells

The effects of bFGF or VEGF on eNOS protein in the OFPAE cells adapted inside the artificial capillary modules were tested by treating the cells with bFGF (10 ng/ml) or VEGF (10 ng/ml) at 3 or 15 dynes/cm2 for up to 24 h (Fig. 8). Shear stress of 15 dynes/cm2 alone caused a time-dependent elevation in eNOS protein. The addition of VEGF had no effects on eNOS protein at all time points tested at both basal and stimulatory levels of shear stresses. When the OFPAE cells were treated with bFGF while exposed to the basal 3 dynes/cm2 shear stress, eNOS protein levels at 24 h showed a small, but not statistically significant (0.05 < P < 0.1), rise compared to control. When the OFPAE cells were exposed to 15 dynes/cm2 shear stress, bFGF did not elevate eNOS protein any further than shear stress alone up to 24 h.



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  		FIG. 8. Effects of bFGF and VEGF on pulsatile shear stress-induced eNOS protein levels in OFPAE cells. The OFPAE cells were cultured inside the CELLMAX capillary system at 3 dynes/cm2 shear stress until confluent. After 24 h of serum withdrawal, bFGF (10 ng/ml) or VEGF (10 ng/ml) was added into the reservoir 30 min before further shear stress treatment, at which time the shear stress was either elevated to 15 dynes/cm2 or kept at 3 dynes/cm2 for 24 h. The cells were recovered from the capillaries by trypsin (0.025%)/EDTA (0.53 mM) at 0, 6, or 24 h. The cell pellets were solubilized in lysis buffer. Western blot analysis for eNOS was performed on solubilized proteins, and eNOS intensities are expressed as the fold-value of the control at 0 h. A) 3 dynes/cm2. B) 15 dynes/cm2. Data are expressed as the mean ± SEM (n = 3–6 per group). *P < 0.05 compared to 0 h within treatment, +P < 0.05 compared to 6 h within treatment, #0.05 < P < 0.1 compared to control between treatment

MEK Inhibition Does Not Block Shear Stress-Induced Increase in eNOS Protein Levels

The fact that bFGF significantly increased eNOS protein in OFPAE cells in static culture but had much less or no effect in the flow system suggested that bFGF and shear stress might share the same signaling pathways leading to eNOS protein expression. To determine whether ERK1/2 also mediates shear stress-induced eNOS protein expression, OFPAE cells were pretreated with UO126 (10 µM) for 1 h and exposed to either 3 or 15 dynes/cm2 shear stress for 24 h. Figure 9 shows that shear stress of 15 dynes/cm2 increased eNOS protein levels and that this increase was not inhibited by UO126. Because UO126 was able to completely block shear stress-induced ERK1/2 activation, this suggests that in adapted OFPAE cells, MEK-ERK1/2 does not play a crucial role in determining shear stress-stimulated eNOS protein levels.



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  		FIG. 9. Inhibition by MEK does not block pulsatile shear stress-induced increase in eNOS protein levels. A) The OFPAE cells were cultured inside the artificial capillary modules at 3 dynes/cm2 shear stress until confluent. The cells were then subjected to 24 h of serum withdrawal, pretreated with UO126 (10 µM) for 1 h (still with the shear stress of 3 dynes/cm2), and then underwent to 24 h of either 3 or 15 dynes/cm2 shear stress. The cells were recovered from the capillaries by trypsin (0.025%)/EDTA (0.53 mM) and solubilized in lysis buffer for eNOS Western blot analysis. B) The eNOS intensities were expressed as the fold-value of the control at 3 dynes/cm2. Data are expressed as the mean ± SEM (n = 4 per group). *P < 0.05 compared to 3 dynes/cm2 control

PI-3K Inhibition Does Not Block Shear Stress-Induced eNOS Protein Elevation

We have shown that in our model, Akt, one of the downstream signaling molecules of PI-3K, was stimulated by shear stress (Figs. 1 3, and 5). The possible involvement of PI-3K on shear stress-induced eNOS protein elevation was tested by using the PI-3K-inhibitors wortmannin (100 nM) and LY294002 (50 µM). Both these inhibitors had no effect on basal eNOS expression and failed to modulate the upregulation of eNOS protein after 24-h treatment with 15 dynes/cm2 (Fig. 10), suggesting that PI-3K-mediated pathways are unlikely to be involved in shear force-stimulated eNOS protein upregulation in OFPAE cells.



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  		FIG. 10. Inhibition by PI-3K does not block shear stress-induced eNOS protein elevation. The OFPAE cells were cultured inside the artificial capillary modules at 3 dynes/cm2 shear stress until confluent. The cells were then subjected to 24 h of serum withdrawal, pretreated with (A) wortmannin (100 nM) or (B) LY294002 (50 µM) for 1 h, and then underwent 24 h of either 3 or 15 dynes/cm2 shear stress. The cells were recovered from the capillaries by trypsin (0.025%)/EDTA (0.53 mM) and solubilized in lysis buffer for eNOS Western blot analysis. The eNOS intensities were expressed as the fold-value of the control at 3 dynes/cm2. Data are expressed as the mean ± SEM (n = 3–4 per group). *P < 0.05 compared to 3 dynes/cm2 control

p38 MAPK and Ca2+ Chelation Studies on Shear Stress-Induced eNOS Protein Elevation

Although p38 MAPK was not activated acutely by shear stress, it might still play a role in mediating the prolonged eNOS protein expression. Western blot analysis indicated that p38 MAPK-inhibitor SB203580 (20 µM) did not modulate eNOS protein levels at shear stresses of 3 and 15 dynes/cm2 (data not shown). Our efforts to assess the effects of intracellular Ca2+ chelation on shear stress-induced eNOS protein were not successful, because 24 h of treatment with BAPTA/AM (10 µM) caused complete cell loss in the CELLMAX system.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Development-dependent cell signaling mechanisms controlling endothelial NO production appear to underlie the normal gestational regulation of uterine and placental blood flow [3, 4, 8, 9, 30, 31]. This is particularly important, because fetal growth disorders, such as intrauterine growth retardation, are attributed partly to decreases in fetoplacental blood flow and placental/vascular NO production [32, 33].

Endothelium is continuously exposed to mechanical forces exerted by pulsatile flowing blood, which modulates vasodilator production via shear stress-activated signaling pathways. In contrast to the irreversible pulsatile shear stress system described herein, nearly all previous data were obtained using devices that generate elevated, steady laminar shear stress from static conditions. This is substantially different from in vivo situations, in which the endothelial cells are adapted to pulsatile blood flows. The major observations of the present studies using confluent flow-adapted OFPAE cells are that pulsatile shear stress elevates Ser1177 eNOS phosphorylation through a PI-3K-dependent pathway, not by an ERK1/2 or p38 MAPK pathway. We also show, to our knowledge for the first time, that bFGF, which enhanced eNOS protein expression in static culture but to a lesser extent at basal shear stress, could not augment higher levels of shear stress-stimulated elevation in eNOS protein. Furthermore, inhibition of PI-3K, MEK, and p38 MAPK did not significantly affect shear stress-upregulated eNOS protein levels.

Production of NO by OFPAE cells (i.e., acute eNOS activation) is rapidly induced by pulsatile shear stress many hours before the eNOS protein levels are upregulated [27]. Posttranslational modification, such as Ser1177 phosphorylation, is related to increased eNOS activity [16]. Steady laminar shear stress stimulated eNOS phosphorylation on Ser1177 in unadapted BAEC as early as 2 min after shear onset and reached a maximum by 30 min [17]. We observed a rapid pulsatile shear stress-induced elevation of eNOS phosphorylation on Ser1177 (not Thr495) that peaked at 20 min, coinciding with the acute phase of NO production by adapted OFPAE cells with elevated shear stress [27]. Thus, phosphorylation of this specific eNOS residue and eNOS activity induced by elevating pulsatile flow are correlated. The NOx production rate at 2 h also appeared to be higher [27]; however, because eNOS Ser1177 phosphorylation was still elevated but declining (compare Figs. 1 and 6), other mechanisms must contribute to the regulation of eNOS activity, such as phosphorylation or dephosphorylation (via protein phosphatases; e.g., PP1, PP2A, and PP2B) of other residues. Phosphorylation of eNOS on Thr495 or Ser116 residues may decrease eNOS catalytic activity [34, 35]. In agreement with a study on steady shear stress-treated BAEC [36], no change was observed in eNOS Thr495 phosphorylation level by 30 min of pulsatile 15 dynes/cm2 shear stress. Phosphorylation on eNOS Ser116 was unaltered by steady shear stress [36], but it was undetectable in OFPAE cells. The list of other phosphorylation sites identified on eNOS, which may regulate eNOS activity, is continually expanding [36, 37]. Phosphorylation/dephosphorylation is an important, but not the only, mechanism to regulate eNOS activity. Shear stress indeed induces rapid increases in intracellular Ca2+ release at the onset of the flow [38], and eNOS relies somewhat on its interactions with Ca2+/calmodulin [14]. Moreover, association of heat shock protein 90 to eNOS with shear stress exposure elevates eNOS activity [39]. Shear stress may also induce NO production by affecting L-arginine transport, availability of cofactors, or eNOS recruitment onto the membrane.

Steady flow stimulates ERK1/2 phosphorylation and PI-3K-dependent Akt phosphorylation. Steady shear stress-induced ERK1/2 activation reached a peak by 5 min and declined to control levels by 30 min, and Akt activation was sustained for several hours [18, 40]. We report herein, to our knowledge for the first time, that elevating pulsatile flow to 15 dynes/cm2 in low shear level-adapted OFPAE cells acutely increases both Akt and ERK1/2 phosphorylation at 5 and 10 min, respectively. Increased ERK1/2 phosphorylation returned to basal level by 20 min and stayed low for up to 2 h, whereas Akt activation was still observed after 2 h of exposure to pulsatile flow, suggesting that both ERK1/2 and PI-3K pathways could be involved. The PI-3K-inhibitor wortmannin completely inhibited steady shear stress-induced increase in eNOS Ser1177 phosphorylation, cGMP, and NO production in HUVEC and BAEC, although these conclusions were recently questioned in BAEC by the same group [16, 18]. We observed that the PI-3K-inhibitors LY294002 or wortmannin blocked the pulsatile shear stress-activated Akt phosphorylation but also strongly blocked eNOS Ser1177 phosphorylation at 20 min and 2 h of treatment of shear stress, suggesting that PI-3K is the key player in Ser1177 eNOS phosphorylation level. Our attempt to directly demonstrate the inhibition of NO production by these inhibitors failed because of the lack of sensitivity of the NO analyzer [27]. Whether PI-3K is the only player remains an open question, because the doses of inhibitors necessary to block this response are higher than their IC50 doses. However, the longer time course may have been associated with substantial degradation of the inhibitors; a more likely explanation is that the signaling pathway phosphorylating a given eNOS residue may alter with time, especially under dynamic pulsatile shear stress conditions. To that end, we also demonstrated that although the MEK-inhibitor UO126 completely inhibited ERK1/2 activation induced by 15 dynes/cm2 shear stress, it did not affect eNOS Ser 1177 phosphorylation after 20 min of stimulation. A novel observation is that UO126 appeared to decrease eNOS Ser1177 phosphorylation at 2 h of shear stress treatment without affecting Akt phosphorylation. Others have reported that besides PI-3K-mediated pathways, AMP-activated protein kinase and cGMP-activated protein kinase II also phosphorylated eNOS on Ser1177 [34, 41]. It is possible that MEK-ERK1/2 may be involved in regulating these kinases to modulate eNOS Ser1177 phosphorylation after 2 h of pulsatile shear stress stimulation. We cannot, however, rule out the possibility that MEK-ERK1/2 pathways may play a role in the dephosphorylation of Ser1177 by activating serine/threonine protein phosphatase at 2 h.

In vivo and in vitro studies have shown that shear stress upregulates eNOS mRNA and protein expression [22, 23, 42]. We reported that pulsatile shear stress elevates eNOS protein levels in a time- and shear stress-dependent manner in flow-adapted OFPAE cells [27]. Bovine eNOS promoter sequence indicates the presence of activator protein-1 (AP-1) and other transcription factor-binding sites [43]. Shear stress induces c-Fos expression and promotes DNA-binding activity of AP-1 [44, 45], whereas ERK activation triggers induction of c-Fos and subsequent stimulation of AP-1 activity [46, 47]. The current lack of synergy of shear stress and bFGF for eNOS expression suggests that they might share part of the same signaling pathways, possibly MEK-ERK1/2. However, UO126 did not change eNOS levels after 24 h of 15 dynes/cm2, suggesting that MEK-ERK1/2 is not crucial for shear stress-stimulated eNOS protein levels. However, involvement of MEK-ERK1/2 in eNOS gene expression cannot be totally ruled out, because gene transcription, mRNA degradation, translational efficiency, and enzyme stability all contribute to final eNOS protein levels. It is possible that interfering with one regulatory mechanism may not be enough to affect the amount of final product, especially with redundant stimulated pathways. Davis et al. [26] reported that in BAEC, early upregulation of eNOS mRNA (within 6 h) in response to steady shear stress was Src and ERK1/2 dependent. During long-term shear stress stimulation, mRNA stability may become the regulatory point in determining eNOS mRNA/protein levels. Indeed, shear stress greatly prolonged eNOS mRNA half-life via a non-ERK1/2 pathway. In agreement to our observations, neither PD98059 nor UO126 was able to inhibit the increase of eNOS mRNA level after 18 h of steady shear stress stimulation [26].

We also observed that shear stress-induced eNOS protein levels in adapted OFPAE cells were not altered by PI-3K inhibition. Davis et al. [26] reported that the PI-3K inhibitor did not affect steady shear stress eNOS mRNA expression in BAEC. Induction of eNOS mRNA, however, was greatly reduced by buffering intracellular calcium in BAEC with BAPTA/AM [24, 25] or by blockade of calcium entry with SKF96535 [25]. In our model, the long-term effects of calcium chelation were assessed, but 24 h of treatment with BAPTA/AM caused complete cell loss.

Flow/shear stress is the most potent stimuli for NO production by normal endothelium in vivo, leading to further vasodilation and higher placental blood flow, which is critical for successful gestation [3, 27]. Endothelial dysfunction in fetoplacental vasculature has been indicated in preeclampsia [4]. Insufficient endothelial-derived NO production may be one mechanism for elevated vascular resistance and arterial pressure in preeclampsia. Bradykinin-stimulated NO release by umbilical artery and vein from preelamptic women showed an 80%–90% reduction [48]. Moreover, HUVEC from preeclamptic deliveries had altered intracellular Ca2+ and NO regulation [49]. Flow/shear stress-induced, endothelium-dependent vasodilation is also impaired in women with preeclampsia [50], suggesting that malfunctioning endothelial cells in preeclampsia have changes in their signal transduction mechanisms, including those related to pulsatile shear stress.


   ACKNOWLEDGMENTS
 
The authors wish to thank Terrance M. Phernetton and Gladys Lopez for technical assistance and Cindy Goss for help in preparing this manuscript for submission. We also are grateful to Peter I. Lelkis, PhD, Thaddeus G. Golos, PhD, and Paul J. Bertics, PhD, for their valuable scientific input into these studies.


   FOOTNOTES
 
1 Supported in part by National Institutes of Health Grants HL49210, HL57653, HL64703, HD33255, and HD38843. The present study is in partial fulfillment of a PhD degree for Y.L. in the Endocrinology and Reproductive Physiology Training Program. Back

2 Correspondence: Ronald R. Magness, Department of Obstetrics and Gynecology, University of Wisconsin, Perinatal Research Laboratories, 7E Meriter Hospital, 202 S. Park St., Madison, WI 53715. FAX: 608 257 1304; rmagness{at}facstaff.wisc.edu Back

Received: 13 August 2003.

First decision: 3 September 2003.

Accepted: 22 October 2003.


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