Developmental differences in the shear stress-induced expression of endothelial NO synthase: changing role of AP-1

Stephen Wedgwood1, Calista J. Mitchell1, Jeffrey R. Fineman2,3, and Stephen M. Black1,4

Departments of 1 Pediatrics and 4 Molecular Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611-3008; and 2 Department of Pediatrics and 3 Cardiovascular Research Institute, University of California, San Francisco, California 94143-0106


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial nitric oxide synthase (eNOS) mRNA and protein levels increase during late gestation and then decrease postnatally in sheep lung parenchyma. The increase in fluid shear stress at birth, resulting from increased pulmonary blood flow, is an important mediator of postnatal eNOS gene expression. Our objective was to identify factors stimulating eNOS expression in pulmonary arterial endothelial cells (PAEC) in response to shear stress and to determine if these factors are developmentally regulated. PAEC were isolated from fetal lambs and adult sheep. Transcriptional activity from a 1,600-bp eNOS promoter fragment increased in both fetal and adult PAEC exposed to 8 h of shear stress. Conversely, activity driven from an 840-bp promoter fragment containing a putative activator protein (AP)-1 binding site was increased only in fetal PAEC. This increase was completely abolished in an identical construct containing a mutant AP-1 sequence. The AP-1 protein c-Jun was localized to the cytosol in static adult PAEC and to the nucleus in static fetal PAEC. After shear, c-Jun was nuclear localized in both cell types. However, transcriptionally active phosphorylated c-Jun was elevated only in the nuclei of sheared fetal PAEC. Resting levels of eNOS and NO were 2- and 20-fold higher, respectively, in fetal cells. Shear increased eNOS and NO in both cell types: levels were ~2.5-fold higher in fetal PAEC. Phosphorylation of Akt and eNOS was evident in sheared fetal but not adult PAEC. We have therefore identified mechanisms of eNOS regulation at the transcriptional level and to be enzyme activation specific to the fetal pulmonary arterial circulation.

activator protein-1; nitric oxide; fetal sheep; pulmonary arterial endothelial cells; gene expression; maturational differences; protein translocation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AFTER BIRTH, WITH INITIATION of ventilation of the lungs, pulmonary vascular resistance decreases and pulmonary blood flow increases 8- to 10-fold to match systemic blood flow (9, 13, 28, 52). This process is regulated by a complex and incompletely understood interplay between mechanic and metabolic factors (18). Recent evidence suggests that normal pulmonary vascular tone is regulated by a complex interaction of vasoactive substances produced by the vascular endothelium including nitric oxide (NO) (18, 22, 27, 60). NO is an endothelium-derived relaxing factor synthesized by the oxidation of L-arginine after activation of endothelial NO synthase (eNOS) (46). L-Arginine increases pulmonary blood flow in fetal and newborn lambs and augments endothelium-dependent pulmonary vasodilation (1, 17). In addition, inhaled NO decreases pulmonary vascular resistance in fetal lambs and in newborn lambs with pulmonary hypertension (1, 32, 61). Conversely, inhibition of NO synthesis increases pulmonary vascular resistance in fetal lambs (1, 12).

Several studies have investigated the potential role of NO in the developing pulmonary circulation. Both basal and agonist-stimulated endothelial production of NO has been demonstrated in the fetal, newborn, and adult vasculature. Basal NO production rises twofold from late gestation to 1 wk of life and another 1.6-fold from 1 to 4 wk of life in intrapulmonary arteries (55). A physiological study of intrapulmonary arteries and isolated lung preparations from sheep revealed maturational increases in NO-mediated relaxation during the late fetal and early postnatal periods (55). Similarly, in the pig, there is an increase in NO-mediated relaxation during the first 2 wk of life, followed by a decrease (37, 48). Coinciding with these data, eNOS mRNA and protein increase in late gestation then decrease postnatally in rat and sheep lung parenchyma (26, 30, 45). In addition, lung mRNA levels for the alpha 1-and beta 1-subunits of soluble guanylate cyclase during late-gestation fetal and neonatal rats are sevenfold higher than those found in adult rats (7). This maturational increase in NO and cGMP production parallels the dramatic decrease in pulmonary vascular resistance that occurs at birth. Also, in the late-gestation fetal lamb, an infusion of the eNOS inhibitor Nomega -nitro-L-arginine markedly attenuates the increase in pulmonary blood flow associated with ventilation at birth (1, 17). Together, these data strongly suggests that NO activity mediates, in part, the fall in pulmonary vascular resistance during the transitional pulmonary circulation and maintains the normal low postnatal pulmonary vascular resistance. The developmental regulation of eNOS gene expression is likely to play a key role in controlling the fetal to newborn transition.

eNOS gene expression is influenced by several stimuli, with laminar shear stress making an important contribution. Fluid shear stress is defined as the tractive force produced by moving a viscous fluid (blood) on a solid body (vessel wall), constraining its motion (41). When endothelial cells (EC) are subjected to shear stress, diverse responses are initiated, some of which occur within minutes and others that develop over several hours or days (42). NOS activity, NO production, and eNOS mRNA and protein levels are increased in EC exposed to shear stress (33-35, 43, 44, 49, 57, 58). Furthermore, studies have shown that the increased pulmonary blood flow occurring at birth leads to an increase in eNOS expression in the pulmonary vascular endothelium of newborn lambs (5, 19).

To identify the factors involved in this pathway leading from increased pulmonary blood flow to increased eNOS expression, we have previously exposed fetal pulmonary arterial endothelial cells (FPAEC) to laminar shear stress. NO production was found to be biphasic with an increase in NO up to 1 h, a plateau phase until 4 h, then a second release between 4 and 8 h (59). The initial release may have been due to the activation of preformed enzyme. In contrast, the second NO release coincided with an increase in eNOS mRNA and protein and may have been stimulated by factors that regulate eNOS gene transcription. PKC isoforms appear to be important in both stages of NO release, although our data suggest that the two pathways are distinct (59).

The purpose of this study was to identify additional components of the shear stress-induced signaling pathway. To identify which of these components are potentially developmentally regulated, we also determined whether the same mechanisms activate eNOS expression in adult PAEC (AdPAEC). Here we demonstrate the involvement of activator protein (AP)-1 in the activation of eNOS transcription in FPAEC, but not in AdPAEC, exposed to laminar shear. In addition, shear stress-induced pathways that mediate the phosphorylation of Akt, eNOS, and c-Jun are present only in FPAEC.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Animals. All procedures and protocols were approved by the Committee on Animal Research of the University of California at San Francisco. Five mixed-breed Western ewes at each maturational age [118-120 days (d), 138-140 d of gestation, newborn (<2 d), and adult] were euthanized with an intravenous injection of pentobarbital sodium (Euthanasia CII; Central City Medical, Union City, CA) and were subjected to bilateral thoracotomy. These animals had not undergone previous surgery or study.

Tissue preparation. The heart and lungs were removed en bloc. To prepare total RNA, lung tissue was removed, snap-frozen in liquid nitrogen, and stored at -70°C until used. Total RNA was prepared from snap-frozen lung tissue (after pulverization) or from cultured ovine pulmonary arterial EC (see below) by brief homogenization in 4 M guanidinium isothiocyanate, followed by extraction with acid phenol and precipitation in isopropanol (11). To prepare protein, lung tissue was rinsed in cold (4°C) normal saline to remove blood, then minced, and finally homogenized using a Tissuemizer (2 × 15 s at 80% power) at 4 vol/wet wt of Triton lysis buffer (20 mM Tris · HCl, pH 7.6, 0.5% Triton X-100, and 20% glycerol) supplemented with protease inhibitors; cultured ovine pulmonary arterial EC were sonicated in 3 vol/wet wt. Extracts were then centrifuged at 15,000 g for 15 min. The supernatant was then removed for protein determination and Western blot analysis (29, 30).

Cell culture techniques. The heart and lungs were obtained from fetal lambs (138- to 140-d gestation), newborn lambs (<2 d), or adult sheep after death. These animals had not undergone previous surgery or study. The main and branch pulmonary arteries were removed and dissected free; the adventitia was removed. The exterior of the vessel was rinsed with 70% ethanol. The vessel was then opened longitudinally, and the interior was rinsed with PBS to remove any blood. Using a cell scraper, we lightly scraped away the endothelium, placed it in medium DMEM-H16 (with 10% fetal bovine serum and antibiotics), and incubated it at 37°C in 21% O2-5% CO2 balanced with N2. After 5 days, islands of EC were cloned to ensure purity. Basic fibroblast growth factor (1 ng/ml, a gift from Dr. Denis Gospodarowicz, Chiron, Emeryville, CA) was added to the media every other day. When confluent, the cells were passaged to maintain them in culture or frozen in liquid nitrogen. EC identity was confirmed by their typical cobblestone appearance, contact inhibition, specific uptake of 1,1'-dioctadecyl-1,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (Molecular Probes, Eugene, OR), and positive staining for von Willebrand factor (Dako, Carpinteria, CA). Ovine pulmonary arterial EC were studied between passages 3 and 10.

Generation and transfection of promoter constructs. We generated promoter constructs by PCR using human genomic DNA as a template. The reverse primer common to all three constructs binds immediately upstream of the ATG initiation codon of eNOS (5'-GTTACTGTGCGTCCACTCTGCTGCC). Forward primers were: 5'-ATCTGATGCTGCCTGTCACCTTGAC (1,600-bp fragment), 5'-TGT-AGTTTCCCTAGTCCCCC (840-bp fragment), and 5'-GGTGTGGGGGTTCCA-GGAAA (650-bp fragment). A 2-bp mutation was introduced into the AP-1 consensus sequence within the 840-bp construct. The sequence TGAGTCA at -661 in the human eNOS promoter (39) was changed to TGAGTtg by site-directed mutagenesis (Retrogen, San Diego, CA) based on previous studies (8, 29). All promoter constructs were sequenced to confirm their identity. Fetal and adult EC were transfected with 10 µg of plasmid DNA on a 10-cm2 tissue culture plate at 90% confluence with Lipofectamine (GIBCO-BRL) according to the manufacturer's instructions. After 48 h, the two cell types were split onto three wells of two six-well plates and allowed to adhere for a further 24 h. Medium was replaced with serum-free DMEM, and cells were left static or were subjected to laminar shear stress (for 8 h) as described below, such that sheared cells and static controls came from the same transfection reaction. We determined luciferase activity of 10 µl of protein extracts using the Dual-Luciferase Reporter Assay System (Promega) and a Femtomaster FB12 luminometer (Zylux).

Fluid shear stress. A cone-plate viscometer similar to that described by Sdogous et al. (54) was designed and built such that it accepts six-well tissue culture plates. This apparatus consists of a cone of shallow angle (alpha ) rotating at angular velocity (omega ) on top of a tissue culture plate containing medium of a known viscosity (µ) on which EC were present as a monolayer. This allowed the monolayer to be subjected to a radially constant fluid-shear stress (tau , dyn/cm2) as calculated with the formula tau  = alpha omega /µ. A cone angle of 0.5° is used to achieve laminar flow rates representing levels of shear stress within physiological parameters. Typical physiological shear stress in the major human arteries is in the range of 5-20 dyn/cm2 (38) with localized increases to 30-100 dyn/cm2 (15). Thus we imparted a shear stress of 20 dyn/cm2 to mimic the upper limit of the physiological range. Also, since previous studies have shown that pulsatile and laminar flow appears to induce similar effects on NO production, laminar shear stress was employed (44).

RNase protection assay. The plasmid containing the ovine eNOS cDNA fragment was linearized with the appropriate restriction enzyme (GIBCO-BRL, Grand Island, NY). We synthesized antisense single-stranded ovine eNOS riboprobes by in vitro transcription using T3 RNA polymerase (Boehringer-Mannheim, Indianapolis, IN) in the presence of cold rCTP, rGTP, and rATP. RNA probes labeled with [32P]UTP (New England Nuclear, Boston, MA) were used for RNase protection assays (4-6). RNase protection assays were performed as previously described (4-6). We did studies on total RNA prepared from peripheral lung tissue, using the antisense radiolabeled cRNA eNOS probe. Antisense radiolabeled single-stranded ovine RNA probes were hybridized overnight at 42°C with total RNA isolated peripheral lung (50 µg) in 80% formamide, 50 mM PIPES (pH 6.4), 0.4 M NaCl, and 1 mM EDTA. Single-stranded RNA was digested with an RNase A-T1 mixture (Ambion, Austin, TX) for 1 h at 37°C. After phenol-CHCl3 extraction and ethanol precipitation, the protected fragments were analyzed by electrophoresis on a 6% denaturing polyacrylamide gel. Also included was a probe for 18S to serve as a control for the amount of input total RNA and the recovery of protected probe fragments. In all experiments, sufficient counts were added such that the eNOS and 18S riboprobes were always in molar excess.

Western blot analysis. Western blot analysis was performed as previously described (4, 5, 59). Briefly, protein extracts (20 µg) were separated on 7.5% denaturing polyacrylamide gels. All gels were electrophoretically transferred to Hybond-polyvinylidene difluoride membranes (Amersham, Arlington Heights, IL). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween. After being blocked, the membranes were incubated at room temperature with a specific monoclonal antiserum raised against eNOS (1:2,500; Transduction Laboratories) then incubated with a goat anti-mouse IgG-horseradish peroxidase conjugate. After washing, chemiluminescence was used to detect the protein bands.

Immunocytochemistry and fluorescence detection. Sheared and static cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Washington, PA) for 20 min at 4°C. After three washes in phosphate-buffered saline (PBS; Cellgro), cells were permeabilized in 0.1% Igepal CA-630 (Sigma) for 1 min at room temperature and washed three more times in PBS. Cells were blocked in 5% (wt/vol) nonfat dry milk (Challenge Dairy Products) and 0.05% Tween (Fisher Biotech) in PBS for 60 min at room temperature. Cells were washed three times in PBS and incubated with primary antibodies against c-Jun (1:100; Oncogene Research Products), phosphorylated c-Jun (serine 73, 1:1,000; Cell Signaling), eNOS (1:1,000, Transduction Laboratories), phosphorylated eNOS (serine 1,177, 1:1,000; Cell Signaling), Akt (1:100, Cell Signaling), and phosphorylated Akt (serine 473, 1:100; Cell Signaling) in 5% BSA (Sigma), 0.05% Tween in PBS at 4°C for 16 h. Cells were washed three times in PBS and incubated with goat anti-rabbit (c-Jun, phosphorylated c-Jun, phosphorylated eNOS, and Akt) or goat anti-mouse (eNOS and phosphorylated Akt) IgG conjugated to rhodamine red or Oregon green (all from Molecular Probes) in 5% nonfat dry milk, 0.05% Tween in PBS for 60 min in the dark at room temperature. Cells were washed three times in PBS and visualized by fluorescence microscopy with excitation at 518 nm and emission at 605 nm for rhodamine red and with excitation at 485 nm and emission at 530 nm for Oregon green. Cells were imaged under a Nikon Eclipse TE-300 fluorescent microscope. Fluorescent images were captured with a CoolSnap digital camera, and the average fluorescent intensities (to correct for differences in cell number) were quantified with Metamorph imaging software (Fryer). Statistical analyses between treatments were carried out as detailed below (see Statistical analysis).

Diaminofluorescein fluorescence. Cells were loaded with 10 µM 4,5-diaminofluorescein diacetate (DAF2-DA) and incubated at 37°C for 30 min. Cells were washed three times in PBS and visualized by fluorescence microscopy at 485 nm for excitation and 530 nm for emission. Images were captured and quantified as described above.

Statistical analysis. Relative luminescent intensity was determined for the four promoter-luciferase constructs in sheared AdPAEC and FPAEC and expressed as fold change (shear/static) ± SD. The relative fluorescent intensity was calculated for secondary antibodies and DAF2-DA, then expressed as fold change (vs. AdPAEC static) ± SD. We made comparisons between treatment groups by the unpaired t-test using the GB-STAT software program. A P < 0.05 was considered statistically significant.


    RESULTS
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RESULTS
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RNase protection assays were performed to determine eNOS gene expression in peripheral lung tissue from fetal lambs [120-d gestational age (F120d) and F140d], newborns (<2 d old), and adult sheep. The expression of eNOS mRNA was found to be maximally expressed in the newborn (P < 0.05 vs. F120d) and then to be significantly decreased in the adult (P < 0.05 vs. F120d; Fig. 1, A and B). To determine whether these maturational differences in eNOS expression could be maintained in culture, we prepared EC from F140d or newborn lambs or adult sheep (Fig. 1, C and D). These cells were then passaged 10 times (1:4 split when cultures obtained confluency). At passage 10, RNA was prepared, and eNOS expression determined by RNase protection assay (Fig. 1C). The results obtained indicate that the eNOS developmental phenotype can be maintained during 10 passages such that eNOS expression is highest in newborn PAEC and is less in PAEC obtained from adult sheep (Fig. 1D). Similar results were obtained when eNOS protein levels were examined (Fig. 5).


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Fig. 1.   Maturational differences in endothelial nitric oxide synthase (eNOS) mRNA expression in peripheral lung are maintained in cultured pulmonary arterial endothelial cells (PAEC). A: a cRNA probe for ovine eNOS was hybridized overnight to 50 µg of total lung RNA prepared from 120- and 140-day fetal (F120d and F140d, respectively) lambs, newborns (NB, <2 days old), and adult sheep (Ad). No protected fragments were detected in the lanes where the probe was hybridized without RNA (PD) or in the presence of tRNA. eNOS is undigested probe. A cRNA for ovine 18S was also hybridized to serve as a control for RNA loading. A representative RNase protection assay is shown. B: the densitometric values for relative eNOS mRNA (normalized to 18S mRNA and to F120d) from 5 sets of animals at each maturational age. Values are means ± SE. *P < 0.05 vs. F120d. C: a cRNA probe for ovine eNOS was hybridized overnight to 20 µg of total lung RNA prepared from PAEC prepared from F140d lambs, newborns (<2 days old), and adult sheep and subjected to culturing for 10 passages. No protected fragments were detected in the lanes where the probe was hybridized without RNA (PA) or in the presence of tRNA. eNOS is undigested probe. A cRNA for ovine 18S was also hybridized to serve as a control for RNA loading. A representative RNase protection assay is shown. Maturational differences in eNOS expression are maintained in culture. D: the densitometric values for relative eNOS mRNA (normalized to 18S mRNA and to F140d) from PAEC isolated from 3 sets of animals at each age. Values are means ± SE. *P < 0.05 vs. F140d; dagger P < 0.05 vs. adult lung.

We then wished to determine whether there are maturational differences in the cis-elements used to regulate the eNOS promoter. Because we have shown previously that eNOS mRNA levels increase in FPAEC exposed to 8 h of laminar shear (59), we used this to examine differences between PAEC isolated from either fetal (FPAEC) or adult sheep (AdPAEC). Thus we transfected both FPAEC and AdPAEC with eNOS promoter fragments fused to a luciferase reporter gene. Using a promoter fragment containing 1.6 kb of DNA 5' of the eNOS transcription start site, we found luciferase activity increased 1.64-fold in FPAEC and 1.77-fold in AdPAEC after 8 h of laminar shear (P < 0.05 vs. static controls, Fig. 2). A construct containing 840 bp of the eNOS 5'-flanking region gave a 1.95-fold increase in luciferase activity in FPAEC (P < 0.05 vs. static controls) but no significant increase in AdPAEC after 8 h of shear (Fig. 2). Conversely, a third construct containing 650 bp of the eNOS 5'-flanking region was unresponsive to shear in both cell types (Fig. 2). Inspection of the eNOS promoter sequence (39) between -840 and -650 identified a potential binding site for AP-1 at -661. Mutation of two base pairs within the consensus AP-1 binding sequence specifically disrupts DNA binding (8, 29). Introduction of the same mutations within the AP-1 sequence of the 840-bp construct completely abolished the shear-induced increase in luciferase activity in the fetal cells (Fig. 2).


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Fig. 2.   Transcriptional activity of the eNOS promoter in response to laminar shear stress. Adult and fetal pulmonary arterial endothelial cells (AdPAEC and FPAEC, respectively) were transfected with eNOS promoter constructs fused to a luciferase reporter gene. The three constructs used contained 1,600, 840, and 650 bp of eNOS promoter DNA 5' of the translational initiator ATG. Activator protein (AP)-1mut is identical to the 840-bp construct except for a 2-bp mutation in the AP-1 consensus sequence. After 8 h of shear, protein extracts were made, and luciferase assays were performed. Activities are expressed as fold change relative to static controls. Promoter activity increased in response to shear in AdPAEC and FPAEC transfected with the 1,600-bp promoter fragment. Activity was increased from the 840-bp fragment only in sheared FPAEC; this was abolished in the mutant promoter construct. No increases were detected with the 650-bp fragment in either cell type. Values expressed are means ± SD of triplicate readings from 3 individual experiments. *P < 0.05 vs. static; dagger P < 0.05 vs. sheared 840-bp construct.

To confirm that the endogenous eNOS gene in FPAEC requires the AP-1 element to respond to shear, we carried out an experiment in which FPAEC were exposed to laminar shear stress for 8 h in the presence or absence of the AP-1 inhibitor curcumin (10 µM). In the absence of curcumin, eNOS mRNA levels increased by 4.9-fold from baseline; however, in the presence of curcumin this increase was abrogated (Fig. 3), indicating the importance of AP-1-mediated signaling in regulating eNOS gene expression by laminar shear stress in FPAEC.


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Fig. 3.   eNOS mRNA expression in FPAEC in the presence or absence of AP-1 inhibition. A: a cRNA probe for ovine eNOS was hybridized overnight to 20 µg of total RNA prepared from FPAEC exposed to laminar shear stress for 8 h in the presence or absence of the AP-1 inhibitor curcumin (10 µM). From baseline, eNOS mRNA expression was increased by shear stress, but this was blocked in the presence of curcumin. No protected fragments were detected in the lanes where the probe was hybridized without RNA (PD) or in the presence of tRNA. eNOS is undigested probe. A cRNA probe for ovine 18S was also hybridized to serve as a control for RNA loading. B: the densitometric values for relative eNOS mRNA from 3 different experiments with different primary cultures. From baseline, shear stress increased relative eNOS mRNA by 4.9-fold after 8 h of shear; in the presence of curcumin, this was reduced to 1.13-fold. Values are means ± SD. *P < 0.05 vs. baseline.

AP-1 complexes comprise c-Jun homodimers or c-Jun/c-Fos heterodimers (50, 53). Immunocytochemistry using an anti-c-Jun antibody revealed a nuclear localization in resting FPAEC and a cytosolic localization in resting AdPAEC, with 2.6-fold higher fluorescence in FPAEC (P < 0.05 FPAEC vs. AdPAEC; Fig. 4, A and C). NO release by sheared FPAEC is biphasic, with an increase up to 1 h, a plateau, then a second release between 4 and 8 h (59). We were therefore interested in determining differences between fetal and adult cells after 1 and 8 h of shear. After 1 h of shear, c-Jun became nuclearly localized in adult cells with a 2.6-fold and a 1.5-fold increase in fluorescence in adult and fetal cells, respectively (P < 0.05; Fig. 4, A and C). By 8 h, fluorescence was increased by eightfold relative to resting AdPAEC in both cell types (P < 0.05; Fig. 4, A and C). The same localization and similar increases in fluorescence in response to shear were seen using an anti-c-Fos antibody (data not shown). Once bound at its cognate promoter sequence, c-Jun is phosphorylated at serine 63 and serine 73 by c-Jun NH2-terminal kinase (JNK), rendering it transcriptionally active (3, 14, 36, 56). Immunocytochemistry indicated a twofold increase in phosphorylated c-Jun at serine 73 in FPAEC exposed to 8 h of laminar shear (P < 0.05 vs. FPAEC static controls; Fig. 4, B and D). However, no increase in phosphorylated c-Jun was detected in sheared AdPAEC (Fig. 4, B and D). Although we found a 1.5-fold increase in FPAEC sheared for only 1 h, it was not statistically significant (Fig. 4, B and D).


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Fig. 4.   Immunocytochemistry to detect c-Jun and phosphorylated c-Jun in static and sheared AdPAEC and FPAEC. A: representative images of c-Jun localization. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Igepal, and probed with an anti-c-Jun antibody. Cells were visualized by fluorescence microscopy and images captured. Under identical imaging conditions, cytosolic c-Jun became nuclear localized in AdPAEC after 1 h of shear, whereas c-Jun was nuclearly localized both in static and sheared FPAEC. B: representative images of phosphorylated (phospho)-c-Jun localization. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Igepal, and probed with an anti-phospho-c-Jun antibody. Cells were visualized by fluorescence microscopy, and images were captured. Under identical imaging conditions, levels of phospho-c-Jun increased only in sheared FPAEC. C: changes in indirect c-Jun fluorescence in response to shear. After 8-h shear, fluorescent intensity increased eightfold in AdPAEC and threefold in FPAEC to give similar levels in both cell types. Values are means ± SD; n = 6. *P < 0.05 vs. AdPAEC static; dagger P < 0.05 vs. FPAEC static. D: changes in indirect phospho-c-Jun fluorescence in response to shear. Fluorescent intensity increased twofold in FPAEC sheared for 8 h. Values are means ± SD; n = 6. *P < 0.05 vs. static.

Western blots were then performed to identify differences in eNOS protein levels in peripheral lung tissue from fetal lambs (F120d and F140d), newborns (<2 d old), and adult sheep. The expression of eNOS protein was found to be maximally expressed in the newborn (P < 0.05 vs. F120d) and then to be significantly decreased in the adult (P < 0.05 vs. F120d; Fig. 5, A and B). We then looked for differences in eNOS protein levels in cells in culture. Resting FPAEC had twofold greater eNOS protein than resting AdPAEC, as determined by Western blot analysis (P < 0.05 vs. FPAEC vs. AdPAEC static; Fig. 5, C and D). After 1 h of shear, eNOS levels were unchanged, but by 8 h they had increased by 2.2-fold in FPAEC and 2.1-fold in AdPAEC (P < 0.05; Fig. 5, E and F). We also used immunocytochemistry to detect changes in eNOS protein levels. Quantification of fluorescent images was in good agreement with the Western data, indicating twofold greater eNOS protein in resting FPAEC (P < 0.05; Fig. 6, A and C). No changes were detected after 1 h of shear, but by 8 h there was a 2.7-fold and 1.7-fold increase in FPAEC and in AdPAEC, respectively (P < 0.05; Fig. 6, A and C). eNOS activity is increased following AKT/PKB-mediated phosphorylation at serine 1,177 (16, 20). Immunocytochemistry identified a 2.9-fold increase in phosphorylated eNOS in FPAEC exposed to 8 h of laminar shear (P < 0.05 vs. static controls; Fig. 6, B and D). However, no increase in eNOS phosphorylation at serine 1,177 was detected in sheared AdPAEC (Fig. 6, B and D).


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Fig. 5.   eNOS protein expression in peripheral lung and static and sheared AdPAEC and FPAEC. A: protein extracts (100 µg) from peripheral lung tissue prepared from F120d and F140d lambs, newborns (<2 days old), and adult sheep were separated on a 6% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against eNOS. A representative Western blot is shown. B: the densitometric values for relative eNOS protein normalized to F120d from 5 sets of animals at each maturational age. Values are means ± SE. *P < 0.05 vs. F120d. C: protein extracts (20 µg) prepared from ovine FPAEC or AdPAEC were separated on 7.5% denaturing polyacrylamide gels, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against eNOS. D: eNOS protein expression is increased in FPAEC compared with AdPAEC. eNOS protein expression is 2.1-fold higher in FPAEC compared with AdPAEC. Values are means ± SD; n = 3. *P < 0.05 vs. static. E: protein extracts (20 µg) prepared from ovine FPAEC or AdPAEC either under static conditions or after 8 h of laminar shear stress (20 dyn/cm2) were separated on 7.5% denaturing polyacrylamide gels, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against eNOS. F: changes in eNOS protein expression in response to shear. eNOS protein expression increased 2.1-fold in sheared AdPAEC and 2.2-fold in sheared FPAEC. Values are means ± SD; n = 6. *P < 0.05 vs. static.



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Fig. 6.   Immunocytochemistry to detect eNOS and phospho-eNOS in static and sheared AdPAEC and FPAEC. A: representative images of eNOS localization utilizing immunocytochemistry. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Igepal, and probed with an anti-eNOS antibody. Cells were visualized by fluorescence microscopy, and images were captured. Under identical imaging conditions, eNOS levels increased in sheared AdPAEC and FPAEC. B: representative images of phospho-eNOS localization in sheared PAEC. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Igepal, and probed with an anti-phospho-eNOS antibody. Cells were visualized by fluorescence microscopy, and images were captured. Under identical imaging conditions, levels of phospho-eNOS are increased only in FPAEC sheared for 8 h. C: changes in indirect eNOS fluorescence in response to shear. After 8 h of shear, fluorescent intensity increased 1.7-fold in AdPAEC and 2.7-fold in FPAEC. Values are means ± SD; n = 6. *P < 0.05 vs. static. D: changes in indirect phospho-eNOS fluorescence in response to shear. Fluorescent intensity increased 2.9-fold in FPAEC sheared for 8 h. Values are means ± SD; n = 6. *P < 0.05 vs. FPAEC static.

We then used immunocytochemistry to confirm a role for Akt in the shear-induced phosphorylation of eNOS in FPAEC. Akt protein levels were similar in static fetal and adult cells, with no changes detected after 1 h of shear (Fig. 7, A and C). After 8 h, there was a 2.4-fold increase in sheared fetal cells (P < 0.05 vs. static; Fig. 7, A and C) but no significant increase in the sheared adult cells (Fig. 7, A and C). Phosphorylated Akt levels were similar in static fetal and adult cells, with no changes detected after 1 h of shear (Fig. 7, B and D). After 8 h of shear, there was an 8.1-fold increase in the fetal cells (P < 0.05 vs. static; Fig. 7, B and D) but no significant increase in the sheared adult cells (Fig. 7, B and D).


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Fig. 7.   Immunocytochemistry to detect Akt and phosphorylated Akt in static and sheared AdPAEC and FPAEC. A: representative images of Akt localization by immunocytochemistry. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Igepal, and probed with an anti-Akt antibody. Cells were visualized by fluorescence microscopy, and images were captured. Under identical imaging conditions, Akt levels increased in only in FPAEC sheared for 8 h. B: representative images of phospho-Akt localization in sheared PAEC. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Igepal, and probed with an anti-phospho-Akt antibody. Cells were visualized by fluorescence microscopy, and images were captured. Under identical imaging conditions, levels of phosphorylated Akt are increased only in FPAEC sheared for 8 h. C: changes in indirect Akt fluorescence in response to shear. After 8 h of shear, fluorescent intensity increased by 2.4-fold in FPAEC. Values are means ± SD; n = 6. *P < 0.05 vs. static. D: changes in indirect phospho-Akt fluorescence in response to shear. After 8 h of shear, fluorescent intensity increased by 8.1-fold in FPAEC. Values are means ± SD; n = 6. *P < 0.05 vs. static.

Using the NO-sensitive fluorescent dye DAF2-DA, we determined that resting NO production was 20-fold higher in FPAEC relative to AdPAEC (P < 0.05 FPAEC vs. AdPAEC; Fig. 8, A and B). After 1 h of shear, DAF2-DA fluorescence increased 2.4-fold and 8.0-fold in FPAEC and AdPAEC, respectively (P < 0.05 vs. static; Fig. 8, A and B). By 8 h of shear, DAF2-DA fluorescence had increased 2.7-fold and 20.4-fold in FPAEC and AdPAEC, respectively (P < 0.05 vs. static; Fig. 8, A and B). Thus the levels of both eNOS protein and NO in AdPAEC sheared for 8 h were similar to those found in resting FPAEC.


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Fig. 8.   Increased NO production in response to shear. Cells were sheared for 1 and 8 h. Thirty minutes before, the end cells were loaded with 10 µM 4,5-diaminofluorescein diacetate (DAF2-DA). Cells were visualized by fluorescence microscopy, and images were captured. A: representative images of NO production. B: DAF2-DA fluorescence levels were 20-fold higher in static FPAEC relative to static AdPAEC. After 1 h of shear, DAF2-DA fluorescence increased 2.4- and 8.0-fold in FPAEC and AdPAEC, respectively. After 8 h of shear, DAF2-DA fluorescence had increased 2.7- and 20.4-fold in FPAEC and AdPAEC, respectively. Values are means ± SD; n = 6. *P < 0.05 vs. adult static; dagger P < 0.05 vs. fetal static.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the fetus, pulmonary vascular resistance is high and pulmonary blood flow is low. With the initiation of ventilation and oxygenation at birth, pulmonary vascular resistance decreases and pulmonary blood flow increases. These rapid changes in pulmonary blood flow and resistance are then followed by more gradual changes that occur over the next several hours. Increasing evidence suggests that the changes in pulmonary vascular tone are mediated by NO, possibly in response to increased shear stress on the pulmonary vascular endothelium (26, 30, 45). In a number of clinical conditions, the pulmonary circulation fails to undergo the normal transition to postnatal life, resulting in persistent pulmonary hypertension of the newborn (PPHN) (2, 6, 40). A better understanding of the mechanisms that regulate the pulmonary vascular changes at birth may lead to new prevention of and improved treatment strategies for PPHN. However, the factors that mediate these changes remain unclear. Our initial studies indicated that there are maturational differences in expression of eNOS in the sheep lung. Our data indicate that eNOS mRNA and protein expression peaked in the perinatal period and was significantly lower in the adult. This data confirm that previously published by Parker et al. (47). However, of more interest was our finding that these developmental differences in eNOS mRNA and protein expression could be maintained for at least 10 passages in culture, such that eNOS expression was highest in PAEC isolated from newborn lambs followed by those obtained from late-gestation lambs (F136d-F140d), whereas expression in PAEC isolated from adult sheep was ~30% of that of the late-gestation animals. This maintenance of maturational phenotype then enabled us to determine whether there are differences in the response of eNOS to relevant physiological stimuli. To examine this, we chose to determine the effect of increased pulmonary blood flow, as we have previously shown that this can increase eNOS gene expression in both fetal lambs and in PAEC isolated from these same lambs (5, 59). To mimic the effects of increased blood flow, we used a cone-plate viscometer that allowed us to expose the PAEC monolayer to a controlled level of shear stress. We have demonstrated an increase in NO production and eNOS mRNA in FPAEC using this technique (59). Using a 1.6-kb eNOS promoter construct, we found an increase in eNOS transcription in both AdPAEC and FPAEC exposed to 8 h of shear. Data from promoter deletion constructs indicate that the shear-induced increase in eNOS transcription is mediated via cis-elements located between -840 and -650 bp in fetal, but not in adult, PAEC. This suggests that additional upstream sequences are required for transcriptional activity in the adult cells. Interestingly, the eNOS promoter contains a cis-acting regulatory element identical to the previously identified shear stress-responsive element (SSRE) (51) located at -994 bp, raising the possibility that shear stress-induced factors regulate eNOS transcription in AdPAEC via this sequence. Indeed, studies have indicated that the SSRE is involved in the shear stress-induced upregulation of the PDGF-B gene via the binding of the NF-kappa B trans-acting factor (31). Further studies will be required to determine whether this same pathway is activated by shear stress in AdPAEC to increase eNOS expression.

A potential AP-1 binding site is located at -667 bp in the eNOS promoter (39). Mutation of two base pairs in the consensus AP-1 binding sequence, predicted to specifically disrupt AP-1 binding (8, 29) completely abolished the shear-induced increase in eNOS promoter activity in fetal cells. Immunocytochemistry revealed developmental differences in the localization of the AP-1 protein c-Jun, which was anuclear in static AdPAEC but nuclear in static FPAEC. After 1 h of laminar shear, c-Jun was nuclearly localized in AdPAEC. After 8 h of shear, levels of c-Jun were identical in fetal and adult cells. Furthermore, AdPAEC sheared for 8 h had levels of eNOS protein and NO comparable with levels in static FPAEC. Thus the sheared adult cells adopt a static fetal cell phenotype with respect to NO production and c-Jun localization. The nuclear localization signal (NLS) of c-Jun has been identified and has been shown to direct nuclear translocation of an unrelated protein (10). Although c-Jun is thought to be constitutively nuclear localized, the nuclear localization of its viral homolog v-Jun is cell cycle dependent. Regulation of this translocation was mapped to a single amino acid substitution in the NLS of v-Jun (10). The mechanisms that determine c-Jun localization in FPAEC and AdPAEC are unclear. It is possible that a c-Jun-binding protein masks the NLS in static AdPAEC rendering it cytosolic, in the same way that NF-kappa B is maintained within the cytosol by Ikappa B binding (23). It seems likely that this putative protein would be downregulated in sheared AdPAEC and in FPAEC. Alternatively, NO-mediated pathways may stimulate nuclear translocation of c-Jun, since sheared AdPAEC and static FPAEC have similar NO levels. However, further studies will be required to determine the mechanisms involved.

Although c-Jun becomes nuclearly localized in sheared AdPAEC, it does not appear to be phosphorylated and is therefore unlikely to be transcriptionally active. This may explain why the AP-1 promoter construct was insufficient to drive shear-stress-dependent luciferase transcription in the adult cells. Phosphorylation and activation of c-Jun are mediated by JNK (3, 14, 36). The reactive oxygen species peroxynitrite has been implicated in the activation of JNK in response to laminar shear stress in EC (25). Further investigation identified Akt-mediated eNOS phosphorylation as an important mediator of this pathway (24). Our finding that eNOS and c-Jun are phosphorylated in sheared FPAEC suggests that a similar pathway may be active in these cells. Because we were using the cloned human eNOS promoter in ovine PAEC, we confirmed the requirement for AP-1 in regulating the endogenous eNOS gene in FPAEC. Using the AP-1 antagonist curcumin, we were able to demonstrate, with RNase protection assays, that the shear-induced increase in eNOS mRNA expression was inhibited. This further strengthens our conclusion of the importance of the AP-1 cis-element in regulating shear-induced expression of eNOS in FPAECs.

Western blot analysis identified higher levels of eNOS in peripheral lung tissue from fetal lambs relative to adult sheep. Furthermore, eNOS levels were twofold higher in static FPAEC relative to static AdPAEC. Because equivalent levels of phosphorylated c-Jun were detected in static fetal and adult cells, it appears that AP-1 may not play a significant role in regulating basal eNOS transcription. Additional experiments are required to identify cis-elements within the eNOS promoter that are necessary for gene expression under static conditions. These studies may identify additional transcription factors that are developmentally regulated. NO levels were almost 20-fold higher in static FPAEC relative to static AdPAEC. These data suggest that eNOS is more active in the fetal cells, although the mechanisms that give rise to these basal differences are as yet unknown. eNOS enzyme activity is regulated by several proteins, including calmodulin and heat shock protein (HSP) 90 (21). The availability of substrate and cofactors may also account for differences in eNOS activity. Additional studies are therefore required to determine whether these molecules are also developmentally regulated in PAEC.

NO production was elevated after 1 h in both cell types with no corresponding change in eNOS protein, suggesting an increase in enzyme activity. We did not detect increases in phosphorylated Akt or phosphorylated eNOS after 1 h of shear, indicating that this pathway is not involved. The positive regulation of eNOS by calmodulin and HSP90 is enhanced by shear stress (21), raising the possibility that these proteins activate eNOS in the acute response to shear in PAEC. NO production increased eightfold in adult cells with only a 2.5-fold increase in fetal cells exposed to 1 h of shear. This suggests a greater increase in eNOS activity in the adult cells; since eNOS appears to be more active in resting fetal cells, the potential for further activation may be less. However, eNOS protein levels were twofold higher in fetal cells, but NO levels were about fivefold higher after 1 h of shear, indicating that eNOS enzyme activity is still higher in the fetal cells. The mechanisms that generate these developmental differences are unclear, although we have shown previously that NO production in FPAEC in response to acute shear stress involves PKC (59). Whether the same PKC pathway is involved in adult cells remains to be elucidated.

After 8 h of shear, eNOS and NO increased further, with the levels in sheared adult cells being similar to those in resting fetal cells. Akt-mediated phosphorylation of eNOS provides an additional means of increased NO production in fetal cells, although this pathway does not appear to be active in the adult cells. Surprisingly, NO levels were not significantly higher in fetal cells sheared for 8 h relative to cells sheared for only 1 h. This may be due to limited availability of the fluorophore at higher concentrations of NO and/or saturation of the images captured such that the software does not detect the true increase in fluorescence intensity. In a previous study we found a sevenfold increase and a 22-fold increase in NOx release in FPAEC sheared for 1 and 8 h, respectively (59). This suggests that we may be underestimating the amount of NO in FPAEC sheared for 8 h in this study.

We have demonstrated maturational changes in eNOS gene expression in ovine peripheral lung tissue, although the mechanisms involved are complex and poorly understood. Thus the objectives of this study were to identify developmental differences within PAEC and to characterize the shear stress response pathways in these cells. Factors that increase eNOS protein levels and enzyme activity are in effect in static fetal cells. Furthermore, by having nuclearly localized c-Jun, EC within the fetal pulmonary circulation appear to be primed to respond immediately to shear stress. Shear stress-induced activation of signaling pathways leading to the phosphorylation of c-Jun, Akt, and eNOS occurs only in FPAEC. These events are predicted to increase eNOS transcription and enzyme activity. This is likely to be of great importance considering that sufficient NO must be synthesized to decrease pulmonary vascular resistance and increase pulmonary blood flow at birth. By identifying the molecular events involved, we hope that better treatment strategies can be developed for conditions arising from an abnormal fetal-to-newborn transition.


    ACKNOWLEDGEMENTS

This research was supported in part by National Institutes of Health Grants HL-60190, HL-67841, and HD-398110; March of Dimes Grant FY00-98; and American Heart Association, Midwest Affiliates, Grant 0051409Z (all to S. M. Black). S. M. Black is a member of the Feinberg Cardiovascular Research Institute.


    FOOTNOTES

Address for reprint requests and other correspondence: S. M. Black, Northwestern Univ. Medical School, Ward 12-191, 303 E. Chicago Ave., Chicago, IL 60611-3008 (E-mail: steveblack{at}northwestern.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 17, 2003;10.1152/ajplung.00252.2002

Received 29 July 2002; accepted in final form 4 December 2002.


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