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BOR - Papers in Press, published online ahead of print February 23, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.039172
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BIOLOGY OF REPRODUCTION 72, 1421–1428 (2005)
DOI: 10.1095/biolreprod.104.039172
© 2005 by the Society for the Study of Reproduction, Inc.

Angiotensin II Elevates Nitric Oxide Synthase 3 Expression and Nitric Oxide Production Via a Mitogen-Activated Protein Kinase Cascade in Ovine Fetoplacental Artery Endothelial Cells1

Jing Zheng 2 3, YunXia Wen 3, Dong-bao Chen 6, Ian M. Bird 3,4 , and Ronald R. Magness 3,4,5  

Departments of Obstetrics and Gynecology, Perinatal Research Laboratories,3 Pediatrics,4 Animal Sciences,5 University of Wisconsin, Madison, Wisconsin 53715 Department of Reproductive Medicine, Division of Maternal-Fetal Medicine,6 University of California, San Diego, California 92093


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Normal pregnancy is associated with high angiotensin II (ANG II) concentrations in the maternal and fetal circulation. These high levels of ANG II may promote production vasodilators such as nitric oxide (NO). ANG II receptors are expressed in ovine fetoplacental artery endothelial (OFPAE) cells and mediate ANG II-stimulated OFPAE cell proliferation. Herein, we tested whether ANG II stimulated NO synthase 3 (NOS3, also known as eNOS) expression and total NO (NOx) production via activation of mitogen-activated protein kinase 3/1 (MAPK3/1, also known as ERK1/2) in OFPAE cells. ANG II elevated (P < 0.05) eNOS protein, but not mRNA levels with a maximum effect at 10 nM. ANG II also dose dependently increased (P < 0.05) NOx production with a maximal effect at doses of 1–100 nM. Activation of ERK1/2 by ANG II was determined by immunocytochemistry and Western blot analysis. ANG II rapidly induced positive staining for phosphorylated ERK1/2, appearing in cytosol after 1–5 min of ANG II treatment, accumulating in nuclei after 10 min, and disappearing at 15 min. ANG II increased (P < 0.05) phosphorylated ERK1/2 protein levels. Activation of ERK1/2 was confirmed by an immunocomplex kinase assay using ELK1 as a substrate. PD98059 significantly inhibited ANG II-induced ERK1/2 activation, and the ANG II-elevated eNOS protein levels but only partially reduced ANG II-increased NOx production. Thus, in OFPAE cells, the ANG II increased NOx production is associated with elevated eNOS protein expression, which is mediated at least in part via activation of the mitogen-activated protein kinase kinase1 and kinase2 (MAP2K1 and MAP2K2, known also as MEK1/2)/ERK1/2 cascade. Together with our previous observation that ANG II stimulates OFPAE cell proliferation, these data suggest that ANG II is a key regulator for both vasodilation and angiogenesis in the ovine fetoplacenta.

growth factors, kinases, nitric oxide, placenta, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiotensin II (ANG II) regulates a variety of biological processes, including vascular tone, cellular growth, and electrolyte homeostasis [1, 2]. Of specific interest is that, during normal human and ovine pregnancy, dramatic increases in placental blood flows are associated with increased ANG II concentrations in the maternal circulation and high levels of ANG II in the fetal circulation [3–7]. For instance, it has been reported that the circulating levels of ANG II are elevated approximately 4.5-fold in pregnant ewes (30 ng/ml) as compared with those in nonpregnant ewes (6.7 ng/ml) [3]. More impressive is that fetal ANG II levels is another ~2.2-fold greater than the maternal levels [4, 5]. Thus, this association between ANG II and blood flow has led to the theory that ANG II, a well-described systemic vasoconstrictor, could also modulate vasodilation in the placental vasculature during pregnancy. Indeed, ANG II has been shown to enhance de novo synthesis of the endothelial-derived vasodilators, such as nitric oxide (NO) and prostaglandin I2 in uteroplacental endothelia during late ovine pregnancy [8–11]. The increases in these vasodilator production in turn may attenuate the ANG II-induced vasoconstriction in vascular smooth-muscle cells, so contributing to the refractoriness to vasoconstriction by infused ANG II in the uteroplacental unit [12–16].

Infusion of ANG II into the ovine fetus has been shown to dose dependently increase mean arterial blood pressure and decrease fetoplacental blood flow [7, 17, 18]. Whether ANG II can upregulate NO synthase 3 (eNOS) expression and NO production in the fetoplacental endothelium still remained poorly understood. However, it is clear that NO, a potent vasodilator, plays a pivotal role in regulating fetoplacental vascular tone during pregnancy [19]. This is supported by the observation that inhibition of NO-mediated guanylate cyclase activation increases perfusion pressure of the human fetoplacental circulation [20]. In addition, inhibition of NO synthase (NOS) activity with analogues of arginine potentiates vasoconstriction of human stem villous fetoplacental arteries [21] and increases ovine umbilical vascular resistance, leading to reduction in umbilical blood flow [22]. We have also demonstrated that gestational-dependent NO production by the ovine fetoplacentomes parallels the changes in eNOS but not NOS2A (also known as iNOS) protein levels during late pregnancy, suggesting that eNOS is a predominant isoform for the NO production in ovine fetoplacentas [23].

Placental expression of ANG II receptors has been described in many species, including human [24–26] and ovine [27, 28]. Thus, it is highly likely that the similar NO responses to ANG II may also exist in the fetoplacental vasculature as in the uteroplacental unit. The recent studies from other non-fetoplacental-derived endothelial cells support this concept. For example, it has been shown that ANG II enhances in vitro the activity of eNOS and/or NO production in endothelial cells directly via activation of ANG II receptors [29–33]. Alternatively, ANG II may also stimulate endothelial eNOS expression and NO production indirectly via angiotensin IV (ANG IV), an ANG II metabolite, which executes these stimulatory effects by activating its own receptors [34, 35].

Activity of eNOS is regulated by a complex transcriptional and posttranscriptional mechanism [36–38]. At transcriptional levels, the eNOS promoter contains binding sites of numerous transcription factors, including activator protein 1 (AP1) and SV40 virus promoter-specific transcription protein 1 (Sp1), both of which could be activated by mitogen-activated protein kinase 3/1 (MAPK3/1, also known as ERK1/2) [39], so enhancing eNOS protein expression [36–38]. Thus, because ANG II is capable of activating ERK1/2 in endothelial cells [40], ANG II-activated ERK1/ 2 could also potentially mediate eNOS expression [36–38]. In addition to transcriptional regulation of eNOS, posttranscriptional modifications of eNOS (i.e., transient phosphorylation and dephosphorylation) are other critical mechanisms for eNOS activation [36–38]. Several protein kinases, including v-akt murine thymoma viral oncogene homolog 1 (AKT1) and ERK1/2, have been implicated to alter (either enhance or inhibit) eNOS activity via phosphorylating eNOS. Of these kinases, AKT1 is the best-studied one, whereas the role of ERK1/2 is less well defined. For example, activation of ERK1/2 has been shown to inhibit eNOS activity in endothelial cells [41, 42]. Conversely, we have observed that, under a static culture condition, vascular endothelial growth factor (VEGF)-increased acute NOx production by ovine fetoplacental artery endothelial (OFPAE) cells is greatly attenuated by inhibition of ERK1/ 2 activation (unpublished results), whereas shear stress-elevated NOx production in OFAPE cells is mediated primarily via phosphoinositide 3 kinase (PI3K)/AKT1, but not MEK1/2/ERK1/2, signaling pathways [43–45].

Whether ANG II-regulated eNOS expression and NOx production in OFPAE cells as well as whether this regulation is mediated via activation of the MEK1/2/ERK1/2 cascades remain unknown. In the present study, we tested the hypothesis that ANG II stimulates eNOS expression and NOx production in association with activation of the MEK1/2/ERK1/2 cascade in OFPAE cells. Specifically, we evaluated in OFPAE cells 1) by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses, if ANG II elevated eNOS protein and mRNA expressions; 2) using a NO analyzer, if ANG II increased NOx production by cells; 3) if ANG II activated the MEK1/2/ERK1/2 cascade; and 4) if ANG II-regulated-eNOS expression and NOx production was mediated via the MEK1/2/ERK1/2 cascade.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Endothelial Cells

A primary OFPAE cell line, established and validated in our laboratory [28, 46, 47], was used at passages 8–10. The OFPAE cell isolation was approved by the University of Wisconsin-Madison Animal Care Committee. Cells were cultured and expanded in Dulbecco modified Eagle medium (DMEM; GIBCO-BRL, Gaithersburg, MD) containing 10% fetal bovine serum, 10% calf serum, and 1% penicillin-streptomycin (all from Life Technologies, Gaithersburg, MD).

Western Blot Analysis for eNOS Protein Expression

Western blot analysis was conducted as described previously [46]. After 24 h of serum starvation (to avoid/reduce serum interference on ANG II effect), cells cultured in DMEM were treated without or with ANG II at 0 (controls), 0.01, 0.1, 1, 10, or 100 nM for another 24 h. Media were collected for detecting NOx levels. Cells treated were harvested and lysed by sonication in 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, 5 µg/ml aprotinin). The lysates were centrifuged, and protein concentrations of the supernatant were determined by a modified Bradford method using BSA (Sigma, St. Louis, MO) as the standard. Proteins (15 µg/lane) were separated on 7.5% SDS-PAGE gels, electroblotted onto the membrane, immunoblotted with mouse monoclonal eNOS antibody (1:750; Transduction Laboratories, Lexington, KY), visualized by the enhanced chemiluminescence system (ECL; Amersham Life Science, Arlington Heights, IL), and quantified by scanning densitometry (Bio-Rad, Hercules, CA). After determining dose-dependent effects, an effective dose (10 nM, see below) of ANG II was used to evaluate the time dependency of ANG II on eNOS protein expression. To determine the role of ERK1/2 on eNOS protein expression and NOx production, additional cells were treated for 24 h with 10 nM of ANG II in the absence or presence of a highly selective MEK1/2 inhibitor, PD98059 (50 µM, 1 h pretreatment; Cal Biochem, La Jolla, CA).

RT-PCR eNOS mRNA Mass Assay

The eNOS mRNA was quantified by coupled RT-PCR amplification in a single-tube assay as described previously [46]. Cells were treated as described above. The forward and reverse primers, used for targeting amplification from the partial ovine eNOS coding region (GenBank accession # U76738) were 5'-TGTGGCTGTCTGCATGG-3' and 5'-TGGCTGGTAGCGGAAGG-3', respectively. The final PCR product was 300 bases. For eNOS mRNA quantification, cells cultured in DMEM were treated without or with ANG II (10 nM) for 0, 3, 6, 12, 24, or 36 h. Total RNA was extracted from cultured cells using a phenol/chloroform/isoamyl alcohol extraction procedure as described previously. The mRNA samples were incubated (0.1 µg per tube) in a 50-µl final volume containing 1x PCR buffer, 2 mM MgCl2, 10 nmoles of each dATP, dCTP, dTTP, and dGTP, and 30 pmoles of each forward and reverse temperature-matched primer. Amplification was performed in the presence of 1 µl AMV reverse transcriptase (2.5 U) and 1 µl of Taq Polymerase (5 U), except for RT(–) controls, which only contained Taq Polymerase. The program used was anneal 62°C, 5 min; reverse transcription, 50°C, 10 min; denature, 94°C, 2 min; amplify, 29 cycles using 94°C, 30 sec; 62°C, 30 sec; 72°C, 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 cDNA target sequence. At the end of the assay, 10 µl of products were separated on a 2% Tris-acetate-EDTA agarose gel and transferred to MagnaGraph hybridization membranes (Molecular Separations Inc., Westborough, MA) for Southern blotting against a probe (generated against partial ovine eNOS cDNA sequence using asymmetric PCR) encoding the same protein coding sequence. After hybridization, membranes were washed once in 2x saline-sodium citrate (SSC)/0.1% SDS for 15 min and twice in 0.1x SSC/0.1% SDS (2 x 30 min) before drying and direct exposure to a phosphorimager (Bio-Rad BI screen, 15 min) for direct quantification (Molecular Analysis v1.4, Bio-Rad). Data were obtained from six determinations of two experiments, and were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPD, also known as GAPDH) mRNA.

Measurement of NOx Levels in Media

The concentration of NOx (nitrite and nitrate) in media was determined by chemiluminesce using a NO Analyzer (Sievers Instruments, Boulder, CO) as described previously [23]. Cells were treated as described above. Media (100 µl) collected from the cells treated with or without ANG II was injected into the analyzer. Nitrite and nitrate were quickly converted to NOx with 0.8% (wt/vol) vanadium chloride in 1 N HCl at 95°C. The NOx was then propelled with nitrogen gas into another reaction chamber to react with ozone and so form NO2. The chemiluminescence signal generated by NOx was recorded and processed. NOx production was calculated by a standard curve generated with sodium nitrate (100 nM to 100 µM; Sigma) as the standard and normalized by the protein content of corresponding wells (see above).

ERK1/2 Activation

To determine if ANG II activates the MEK1/2/ERK1/2 cascade in OFPAE cells, immunocytochemistry, Western blot analysis, and immunocomplex kinase assay were performed for measuring the effects of ANG II on subcellular localization, phosphorylation, and enzymatic activity of activated ERK1/2, respectively, as described previously [46]. For all experiments, cells, after 16 h of serum deprivation, were treated with ANG II (10 nM) in the absence or presence of PD98059 (50 µM, 1 h pretreatment).

A) Immunolocalization of phosphorylated ERK1/2 Cells cultured in DMEM in the chamber slides were treated with ANG II for 0, 1, 5, 10, or 15 min. Cells in additional wells were treated with ANG II for 10 min in the presence of PD98059 or with PD98059 alone. After rinse in ice-cold PBS, cells were fixed and stained with the rabbit phospho-specific ERK1/2 antibody (1:250; New England BioLabs, Beverly, MA).

B) Western blot analysis After 10 min of ANG II treatment, total cell extracts were prepared as described above. Fibroblast growth factor 2 (FGF2, also known as bFGF) at 10 ng/ml was used as a positive control. Proteins (20 µg/lane) were electrophoresed, electroblotted onto Immobilon-P membrane, immunoblotted with either a rabbit phospho-specific (1: 2000) or total (1:1000) ERK1/2 antibody. The ERK1/2 on the membranes were visualized by ECL system and quantified by scanning densitometry.

C) Immunocomplex ERK1/2 kinase assay The assay was conducted using an ERK assay kit and followed the manufacturer's instructions (New England BioLabs). Cell lysates (200 µg) obtained were immunoprecipitated with a mouse monoclonal antibody (1:100) raised against phospho-specific ERK1/2 overnight at 4°C, followed by the incubation with protein A/G sepharose beads (Santa Cruz Biotechnology) for 2 h. After centrifugation, the immunocomplex 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 DTT, 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 addition of 5x 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.

Statistical Analysis

Data were analyzed using one-way ANOVA (SigmaStat; Jandel, Scientific Software, San Rafael, CA). When an F-test was significant, data were compared with their respective control by Bonfferoni multiple comparisons or Student t-test. Data are reported as means ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of ANG II on eNOS Protein and mRNA Expression

The OFPAE cell lysates on Western blots showed a single band at 140 kDa for eNOS (Fig. 1), corresponding to the molecular mass of ovine and human eNOS we previously reported [28, 46]. ANG II induced dose- and time-dependent increases in eNOS protein levels (Fig. 1). ANG II increased eNOS protein levels in OFPAE cells at all doses studied, and this stimulatory effect reached significant (P < 0.05) at doses of 1 and 10 nM (2.4- to 2.6-fold over control) and then declined at 100 nM (Fig. 1A). After determining the dose-dependent effects on eNOS protein expression, an appropriate dose (10 nM) of ANG II was chosen to determine the time dependency of ANG II on eNOS protein expression (Fig. 1B). The stimulatory effect of ANG II on eNOS protein expression was detectable (P < 0.05) at 12 h after treatment and remained elevated up to 36 h (Fig. 1B).



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  		FIG. 1. Effect of ANG II on eNOS protein expression in OFPAE cells. A) Dose response. Cells were treated with ANG II (0–100 nM) for 24 h. B) Time course. Cells were treated with ANG II at 10 nM for 0, 6, 12, 24, or 36 h. Proteins (15 µg) were separated and detected as described in Materials and Methods. A representative blot is shown. Quantitative data are expressed as the mean + SEM percentage of the control value (in the absence of ANG II) from three independent experiments. Means with asterisks differ (P < 0.05) from the control

Coupled RT-PCR analysis of eNOS mRNA levels revealed that the eNOS mRNA levels, normalized to GAPDH mRNA in OFPAE cells, were not altered by treating cells with 10 nM ANG II, up to 24 h (Fig. 2). These data showed that ANG II-induced eNOS protein expression did not require an elevation of eNOS mRNA.



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  		FIG. 2. Effect of ANG II on eNOS mRNA levels in OFPAE cells. Cells were treated with ANG II (10 nM) for 0, 3, 6, 12, or 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 number/tube). The sensitivity limit was <1 x 103 copies of eNOS mRNA/ µg total RNA. Data are from two independent experiments

Effects of ANG II on NOx Production

NOx (nitrate and nitrite) levels in media obtained from the OFPAE cells treated with ANG II are shown in Figure 3. Corresponding to its effects on eNOS protein expression, ANG II also elevated NOx levels in OFPAE cells at all doses studied, and this stimulatory effect reached statistical significance (P < 0.05) at doses of 1, 10, and 100 nM (5.1-, 4.8-, and 5.5-fold over control, respectively) (Fig. 3).



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  		FIG. 3. Effect of ANG II on total NOx production by OFPAE cells. Cells were treated with ANG II for 24 h. Media were collected and NOx levels in media were determined using a NO analyzer (NOA 280, Silvers Instruments) based on a reaction of conversion of nitrite and nitrate to NOx. NOx production was calculated by a standard curve generated with sodium nitrate as the standard and normalized by the protein content of corresponding wells. Data have been subtracted from those in unconditioned media controls and are expressed as means ± SEM from three independent experiments. *, Means with asterisks differ (P < 0.05) from the control

Activation of ERK1/2 by ANG II

To define ERK1/2 activation by ANG II, immunocytochemistry was first used to examine intracellular translocation of phosphorylated ERK1/2, which is a critical step for the ERK1/2 cascade in the ERK1/2-mediated cell growth and differentiation. ANG II treatment rapidly induced positive phosphorylated ERK1/2 staining in a time-dependent manner (Fig. 4). Following ANG II treatment, positive phosphorylated ERK1/2 staining was not observed in any cell compartments at time zero, first appeared in cytosol after 1 min, accumulated heavily in nuclei after 10 min, and disappeared from cells after 15 min (Fig. 4). Positive phosphorylated ERK1/2 staining induced by ANG II was blocked in all cells pretreated with PD98059, a highly selective MEK1/2 inhibitor. PD98059 alone had no effect on phosphorylation of ERK1/2.



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  		FIG. 4. Translocalization of phosphorylated ERK1/2 induced by ANG II in OFPAE cells. After 16 h of serum starvation, cells were treated with ANG II (10 nM) for 0, 1, 10, or 15 min or 10 min after 1 h of pretreatment with PD98059 (50 µM). Cells were fixed and stained with a phospho-specific ERK1/2 antibody. PD: PD98059. Note: Brown staining indicates positive phosphorylated ERK1/2. Blue nuclear staining is hematoxylin counterstaining. No positive staining was observed in control wells in the absence of the primary antibody (not shown) or in wells as stained with preimmune rabbit IgG. For all images, the original magnification is x20

Western blot analysis confirmed the ANG II-induced ERK1/2 phosphorylation (Fig. 5). ANG II and bFGF served as a positive control [46], and treatments for 10 min caused phosphorylation of ERK1/2 but did not alter the total ERK1/2 protein levels (Fig. 5A). ANG II increased (P < 0.05) phosphorylation of ERK1 and ERK2 isoforms by approximately 1.9- and 1.5-fold over the control, respectively (Fig. 5B). The ANG II-induced phosphorylation of ERK1/ 2 was effectively inhibited (P < 0.05) by pretreatment with PD98059. Parallel to its action on ERK1/2 phosphorylation, ANG II stimulated ERK1/2 activity, as indicated by the significant elevation in phosphorylation of ELK1 fusion protein, an ERK1/2 substrate (Fig. 6). Moreover, the ANG II-increased ELK1 phosphorylation was inhibited by PD98059, whereas phosphorylation of ELK1 in controls and with PD 98059 alone was undetectable (Fig. 6).



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  		FIG. 5. Phosphorylation of ERK1/2 induced by ANG II in OFPAE cells. Cells were treated with ANG II (10 nM) for 10 min alone or with PD98059 (50 µM, 1 h pretreatment). Proteins (20 µg/lane) were separated, and ERK1/2 were detected using a phospho-specific ERK1/2 or total ERK1/2 antibody. Data are expressed as means ± SEM (percentage of the control from three independent experiments). Means with asterisks differ (P < 0.05) from control. Within each isoform of ERK, means with different letters differ (P < 0.05). pERK1/2, Phosphorylated ERK1/2; tERK1/2, total ERK1/2. Standard, phosphorylated (2 ng protein) or total (10 ng protein) ERK1/2 (New England BioLabs)



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  		FIG. 6. Activity of ERK1/2 activated by ANG II in OFPAE cells. Proteins (200 µg) were immunoprecipitated with a phospho-specific pERK1/2 antibody and subsequently incubated with its substrate ELK1 fusion protein. Phosphorylated ELK1 was detected using a phospho-specific ELK1 antibody. pERK. Activated ERK (New England BioLabs; positive control); PD, PD98059; pELK1, phosphorylated ELK1 fusion protein. Molecular weight (MW) markers are indicated

Role of ERK1/2 Activation in ANG II Increased eNOS Protein Expression and NOx Production

PD98059 significantly inhibited (P < 0.05) the ANG II-increased eNOS protein levels (Fig. 7) and partially attenuated (~31% reduction; P < 0.05) the ANG II-elevated NOx production (Fig. 8), suggesting that ERK1/2 activation is critical for ANG II-induced increases in eNOS protein expression and, to some extent, for NOx production in OFPAE cells. Treatment of OFPAE cells with PD98059 alone did not alter eNOS protein and NOx levels, as shown in Figures 7 and 8, respectively.



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  		FIG. 7. Effect of PD98059 on ANG II-increased eNOS protein expression. Cells were treated with ANG II (10 nM) for 24 h in the absence or presence of PD98059 (50 µM, 1 h of pretreatment). Proteins (15 µg) were separated and detected as described above. A represent Western blot is shown. Quantitative data are expressed as means ± SEM percentage of the control from three independent experiments. Means with different letters differ significantly (P < 0.05)



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  		FIG. 8. Effects of PD98059 on ANG II-increased NOx production. Cells were treated with ANG II (10 nM) for 24 h in the absence or presence of PD98059 (50 µM, 1 h of pretreatment). Media were collected for NOx detection as described above. Data have been subtracted from those in unconditioned media controls, normalized by the protein content of corresponding wells, and are expressed as means ± SEM from five independent experiments. *, Means with asterisks differ (P < 0.05) from the control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Herein we have demonstrated, for the first time, that ANG II increases eNOS protein expression and NOx production in the ovine fetoplacental artery endothelial cells and have further shown the MEK1/2 inhibitor, PD98059, significantly inhibits the ANG II-increased eNOS protein levels and attenuates the ANG II-elevated NOx production in OFPAE cells. Thus, we conclude that activation of the MEK1/2/ERK1/2 cascade is a key intracellular signal pathway, which is required for the ANG II-increased eNOS protein expression and is partially involved in the ANG II-elevated NOx production in OFPAE cells. These data also suggest that, similar to the uteroplacental vasculature [8, 9], the high levels of ANG II in the fetal circulation during late pregnancy [5–7] may be capable of upregulating local eNOS protein expression and endothelial NOx production.

Our findings that ANG II increased eNOS protein expression and NOx production in OFPAE confirm previous studies by many other investigators using several other types of endothelial cells derived from bovine and porcine pulmonary arteries, rat aortic arteries, and human and bovine coronary microvasculture [29–35]. Our current observation that ANG II increases eNOS protein expression is also similar to the bFGF's effect on eNOS protein in OFPAE cells we previously reported [46]. However, unlike our previous observation that bFGF increases both eNOS protein and mRNA expressions, ANG II-induced increase in eNOS protein expression is not associated with elevation of eNOS mRNA. These data suggest that ANG II stimulation of eNOS protein expression possibly occurs primarily at the levels of translation but not at transcription and posttranscription regulation (i.e., message stability). Moreover, although both ANG II and bFGF elevate eNOS protein expression and activate the MEK1/2/ERK1/2, the maximal stimulatory effect of ANG II (~2-fold over control) is much weaker than that of bFGF (~5- to 6-fold over control). This difference may be, in part, explained by the difference in the magnitudes of ERK1/2 activation induced by bFGF and ANG II because, compared with an approximately 2-fold increase in ERK1/2 activation in ANG II-treated cells, the maximal stimulatory effects of bFGF on ERK1/2 activation was greater than 20-fold over the control [46].

It is noteworthy that blockade of the ERK1/2 activation by pretreatment with PD98059 is able to greatly inhibit the ANG II-induced increase in eNOS protein expression, but only partially attenuate ANG II-elevated NOx production (~31% reduction) in OFPAE cells. These data suggest that activation of the MEK1/2/ERK1/2 cascade differentially modulates ANG II-increased eNOS protein expression and NOx production in OFPAE cells. This phenomenon is not surprising because endothelial NOx production could be enhanced by either upregulating eNOS protein levels or increasing eNOS enzymatic activity via acute phosphorylation or dephosphorylation of eNOS [36–38] or with shear stress [43, 44]. In OFPAE cells, we recently observed that NOx production does not correlate to changes in eNOS protein expression in response to bFGF and VEGF treatments (unpublished results). Thus, we postulate that the ANG II-activated ERK1/2 is required for upregulation of eNOS protein expression, whereas additional distinct signaling events must be involve in ANG II-elevated NOx production in OFPAE cells. Such a premise is consistent with a growing body of evidence from studies of vascular smooth-muscle cells, showing that ANG II is capable of activating multiple signaling pathways, including ERK1/2, protein kinase C, and Janus kinase/signal transducer and activator of transcription [47–49].

The mechanisms underlying the ANG II-induced and ERK1/2-mediated increase in eNOS expression in OFPAE cells is currently unknown. This upregulation of eNOS expression may be mediated via activation of eNOS transcription factors such as AP1 and Sp1 because both of these two transcription factors have been shown to be activated by ERK1/2 [39] and are involved in enhancing eNOS expression in other endothelial cells [36–38]. Moreover, whether this ANG II-increased NOx production in OFPAE cells is mediated via phophosphrylation of eNOS, especially at Ser1179 and Thr479, two of the best studied phosphorylation sites for regulation of eNOS activity [36–38], is also unclear. We recently reported that shear-stress-increased NOx production by OFPAE cells is associated with increases in Ser1179 phosphorylation, which is largely mediated via the PI3K/AKT1, but not MEK1/2/ERK1/2, pathway [43, 44]. Thus, a similar mechanism may also exist for modulating the ANG II-increased NOx production by OFPAE cells observed in the current study, especially because PD98059 only partially attenuated this ANG II action.

Our current observations that ANG II increases NOx production by OFPAE cells contrasts with previous findings that ANG II decreases NOx production by ovine fetoplacentomes (COT) explants from late pregnant ewes [27]. We have shown that eNOS is a predominant NOS isoform in the COT and its protein levels are correlated with NOx production by the COT [23]. It is unlikely that this discrepancy in ANG II-induced NOx production is due to differential responses of another NOS isoform (i.e., iNOS) to ANG II. Because eNOS and ANG II receptors are also colocalized in trophoblast cells [23, 27], endothelial and trophoblast cells may respond differently to ANG II to generate NOx in the ovine placenta. However, this is also unlikely because it has been shown that ANG II can increase NOx production by trophoblast cells from human term placentas [26]. Alternatively, because the COT vasculature mainly consists of microvessels (i.e., arterioles, venules, and capillaries), one reasonable explanation for this discrepancy is that opposite mechanisms may exist to regulate NOx production in relatively large versus small blood vessels.

The actions of ANG II are mostly mediated via activation of at least two specific subtypes of ANG II receptor, type 1 (AGTR1, also known as AT1) and type 2 (AGTR2, also known as AT2) [1, 2]. The AT1 is thought to be responsible for most of the known ANG II-induced physiological effects, whereas the functions of the AT2 remain unclear, but the evidence implies that it may suppress AT1-mediated actions on blood pressure and cell proliferation [1, 2]. Thus, an important question raised from the current study is whether, in OFPAE cells, ANG II mediates eNOS protein expression and NOx production directly via activating AT1 and/or AT2 or indirectly via its metabolite, ANG IV [29]. We have shown that, in OFPAE cells, AT1 is the predominant (~75% of total bindings) subtype of ANG II receptor and is largely responsible for mediating ANG II-stimulated cell proliferation [47]. Additionally, we recently also observed that bFGF- and VEGF-stimulated OFPAE cell proliferation is mediated via the MEK1/2/ ERK1/2-NO pathway (unpublished results). These data suggest that ANG II-stimulated eNOS protein expression and NOx production may also be mediated primarily via activation of AT1. This is supported by the previous observations that ANG II enhances the eNOS activity and NOx production in endothelial cells directly via activation of AT1 [29, 33]. However, AT2 alone [30, 31], combined AT1 and AT2 [32], and ANG IV [34, 35] have all been implied to mediate these ANG II-induced effects on eNOS protein expression and NOx production in nonplacental endothelial cells. Thus, we cannot exclude the possibility that, in OFPAE cells, AT2 and/or ANG IV could also modulate ANG II-induced increases in eNOS protein expression and NOx production. Future studies are needed to solve these puzzles.

In conclusion, we have shown that ANG II increases eNOS protein expression and NOx production in OFPAE cells and that the MEK1/2/ERK1/2 mediated the ANG II-elevated eNOS protein expression and NOx production. Thus, the current study supports our hypothesis that ANG II can elevate eNOS expression and NOx production via activation of the MEK1/2/ERK1/2 cascade in fetoplacental endothelial cells in culture. Moreover, together with the evidence that AT1 is colocalized with eNOS in endothelium of ovine fetoplacental vasculature [27], our data further suggest that fetoplacental endothelium in vivo could also respond to ANG II to increase NOx production via upregulating eNOS protein expression, so attenuating ANG II vasoconstriction action on the fetoplacental vascular smooth muscle.


   FOOTNOTES
 
1 Supported, in part, by NIH HL64703 and HD38843S to J.Z., HL74947 and HL70562 to D.B.C., and HL49210, HL57653, and HD33255 to R.R.M. Back

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

Received: 15 December 2004.

First decision: 7 January 2005.

Accepted: 16 February 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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