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Am J Physiol Lung Cell Mol Physiol 294: L582-L591, 2008. First published January 11, 2008; doi:10.1152/ajplung.00353.2007
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Protein kinase C{delta} regulates endothelial nitric oxide synthase expression via Akt activation and nitric oxide generation

Neetu Sud,1 Stephen Wedgwood,2 and Stephen M. Black1

1Vascular Biology Center, Medical College of Georgia, Augusta, Georgia; and 2Department of Pediatrics, Northwestern University, Chicago, Illinois

Submitted 28 August 2007 ; accepted in final form 1 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we explore the roles of the delta isoform of PKC (PKC{delta}) in the regulation of endothelial nitric oxide synthase (eNOS) activity in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). Pharmacological inhibition of PKC{delta} with either rottlerin or with the peptide, {delta}V1-1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177 (S1177). The chronic effects of PKC{delta} inhibition using either rottlerin or the overexpression of a dominant negative PKC{delta} mutant included the downregulation of eNOS gene expression that was manifested by a decrease in both eNOS promoter activity and protein expression after 24 h of treatment. We also found that PKC{delta} inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we conclude that PKC{delta} is actively involved in the activation of Akt. To determine the effect of Akt on eNOS signaling, we overexpressed a dominant negative mutant of Akt and determined its effect of NO generation, eNOS expression, and phosphorylation of eNOS at S1177. Our results demonstrated that Akt inhibition was associated with decreased NO production that correlated with reduced phosphorylation of eNOS at S1177, and decreased eNOS promoter activity. We next evaluated the effect of endogenously produced NO on eNOS expression by incubating FPAECs with the eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). ETU significantly inhibited NO production, eNOS promoter activity, and eNOS protein levels. Together, our data indicate involvement of PKC{delta}-mediated Akt activation and NO generation in maintaining eNOS expression.

cell signaling; gene expression; endothelial

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 (8, 13, 19, 39). This process is regulated by a complex and incompletely understood interplay between mechanic and metabolic factors (15). 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) (15, 17, 32, 52). NO is an endothelium-derived relaxing factor synthesized by the oxidation of L-arginine after activation of endothelial NO synthase (eNOS) (42). L-arginine increases pulmonary blood flow in fetal and newborn lambs and augments endothelium-dependent pulmonary vasodilation (12, 21). In addition, inhaled NO decreases pulmonary vascular resistance in fetal lambs and in newborn lambs with pulmonary hypertension (9, 12, 55). Conversely, inhibition of NO synthesis increases pulmonary vascular resistance in fetal lambs (12, 43). Basal NO production rises 2-fold from late gestation to 1 wk of life and another 1.6-fold from 1 wk to 4 wk of life in intrapulmonary arteries (16). Coinciding with these data, eNOS mRNA and protein increase in late gestation and then decrease postnatally in rat and sheep lung parenchyma (20, 28, 30). Together, these data strongly suggest 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. Thus, the regulation of eNOS gene expression likely plays an important role in controlling the successful transition to air-breathing life.

PKC represents a family of closely related serine/threonine kinases (29) that plays a key role in different cellular signal transduction pathways (29). Reports on the regulation of NOS activity by PKC are controversial. For example, PKC inhibitors have been shown to reduce purinoceptor-stimulated (6) and angiotensin II-stimulated (40) NO synthesis in bovine endothelial cells. In addition, PKC activation with phorbol esters has been shown to induce NO synthesis in isolated rat aorta (41) but inhibit endothelium-dependent vasodilator responses evoked by acetylcholine (38). In porcine endothelial cells, PKC activation reduced the bradykinin-stimulated release of NO, and calphostin C, a PKC inhibitor, augmented the NO release (18). Conversely, a study performed with bovine aortic endothelial cells suggested that downregulation or inhibition of PKC could increase endothelial NOS III expression (31).

It has been shown that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on Ser1177 and activate the enzyme increasing NO production (11, 14, 26). Also, various PKC isoenzymes have been shown to activate Akt (34). For example, PKC{alpha} and PKCβ are critical for phospholipase-modified LDL (PLA-LDL)-induced Akt phosphorylation and survival in THP-1 monocytic cells (36). PKC{alpha} increases Akt-1 activity via Ser473 phosphorylation in response to insulin growth factor-1 (34) via a direct phosphorylation of Akt-1 (34). In addition, exposure of mouse epidermal JB6 cells to vanadium has been shown to lead to activation of PKC{lambda} and {zeta}, and overexpression of a dominant negative mutant PKC{lambda} blocked Akt phosphorylation (22). However, the potential role of PKC{delta} in regulating Akt activation has not been determined. Also, as Akt has been shown to exert a positive effect on eNOS activity, we wished to determine the effect of PKC{delta} inhibition on eNOS expression and NO signaling.

Our data identify a signaling pathway involving PKC-mediated activation of Akt that plays a key role in maintaining eNOS expression and NO signaling under basal conditions and also reveal an important role for endogenous NO generation in regulating eNOS expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Primary cultures of ovine fetal pulmonary artery endothelial cells (PAECs) were isolated as described previously (50). Cells were maintained in DMEM containing phenol red supplemented with 10% fetal calf serum (Hyclone, Logan, UT), antibiotics, and antimycotics (MediaTech, Herndon, VA) at 37°C in a humidified atmosphere with 5% CO2-95% air. Cells were between passages 3 and 10, seeded at ~50% confluence, and utilized when fully confluent.

Infection of endothelial cells with adenovirus. Endothelial cells at 90% confluence were incubated with either an adenovirus containing a dominant negative mutant of Akt (Vector Biolabs) or green fluorescent protein (GFP) using a multiplicity of infection of 200:1.

Pharmacological inhibition of PKC{delta}. We utilized two different methodologies to inhibit PKC{delta}. We utilized the PKC{delta} specific inhibitor, rottlerin (10 µM), as well as the PKC{delta}-derived inhibitory peptide {delta}V1-1 (1 µM) or a control peptide (1 µM) conjugated to a Drosophila antennapedia peptide to allow transfer across the cell membrane (obtained from Dr. Daria Mochly-Rosen, Stanford Univ.).

Generation of a plasmid containing a dominant negative mutant of PKC{delta}. A cDNA containing amino acids 2-144 of PKC{delta} was subcloned into the mammalian expression plasmid pIRES (Clonetech). In addition, a FLAG epitope was introduced at the 5'-end of the cDNA to allow expression levels to be determined by Western blotting. The plasmid was designated pIRES-DNPKC{delta}.

Overexpression of a dominant negative PKC{delta}. PAECs were transfected with pIRES-DNPKC{delta} using Effectene Transfection Reagent (Qiagen) according to the manufacturer's directions. Overexpression was analyzed by immunoblotting using anti-Flag Tag MAb (GeneTex).

Western blotting. PAECs from fetal lambs were serum-starved for 16 h, treated with 10 µM rottlerin or 100 µM 2-ethyl-2-thiopseudourea (ETU), and then solubilized with a lysis buffer containing 1% Triton X-100, 20 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Pierce). Insoluble proteins were precipitated by centrifugation at 13,000 rpm for 10 min at 4°C, and the supernatants were boiled in SDS sample buffer, subjected to SDS-PAGE on 4–12% polyacrylamide gels, and transferred to a PVDF membrane. The blots were blocked with 2% BSA in Tris-buffered saline containing 0.1% Tween (TBST). The primary antibodies used for immunoblotting were anti-eNOS (BD Bioscience), anti-phospho Ser1177 eNOS, phospho Thr495 eNOS, Akt, anti-phospho Ser473 Akt, anti-PKC{delta}, and phospho Tyr311 PKC{delta} (1:1,000; cat. no. 9571, 9574, 9272, 4058, 2058, and 2055, respectively, from Cell Signaling Technology). The membrane was then washed with TBST three times for 10 min, incubated with secondary antibodies coupled to horseradish peroxidase, and washed with TBST. The protein bands were visualized with ECL reagent (Pierce).

Detection of NOx production. Nitrite and nitrate (NOx) generated by PAECs was measured using an NO-sensitive electrode with a 2-mm-diameter tip (ISO-NOP sensor, WPI) connected to an NO meter (ISO-NO Mark II, WPI) as described previously (47).

eNOS promoter analysis. eNOS transcription was analyzed using a 1,600-bp promoter fragment fused to a luciferase reporter gene as described previously (53). Fetal endothelial cells were cotransfected with the 1.6-kb eNOS promoter-luciferase and β-galactosidase construct (to normalize for transfection efficiency). Transfected cells were serum-starved overnight and then incubated with either rottlerin or dominant negative Akt, ETU, or 1 mM sodium nitroprusside. Luciferase activity in protein extracts was determined using the Luciferase assay kit (Promega) and a Fluoroskan Ascent FL luminometer (Thermo Electron).

Statistical analysis. Statistical calculations were performed using the GraphPad Prism V. 4.01 software. The means ± SD or SE were calculated for all samples, and significance was determined by either the unpaired t-test or one-way ANOVA with Newman-Keuls post hoc test. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Acute effect of PKC{delta} inhibition on Akt activation and NO generation. Pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs) were exposed to the PKC{delta} specific inhibitor, rottlerin (10 µM, 30 min), the PKC{delta}-derived inhibitory peptide {delta}V1-1 (1 µM, 2h), or a control peptide (1 µM). Cell lysates were then prepared and subjected Western blot analysis to examine effects on the expression and activity of PKC{delta} and Akt. Our initial studies indicate that although total PKC{delta} levels were unchanged by either rottlerin (Fig. 1, A and B) or {delta}V1-1 (Fig. 1, C and D), there was a significant decrease in the activating (Tyr311) phosphorylation of PKC{delta} in cells exposed to either rottlerin (Fig. 1, A and B, P < 0.05 vs. control) or {delta}V1-1 (Fig. 1, C and D, P < 0.05 vs. control), indicating that both rottlerin and {delta}V1-1 are inhibitors of PKC{delta} activity. Our data also indicate that although total Akt levels were unchanged by either rottlerin (Fig. 1, E and F) or {delta}V1-1 (Fig. 1, G and H), there was a significant decrease in the activating (Ser473) phosphorylation of Akt in cells exposed to either rottlerin (Fig. 1, E and F, P < 0.05 vs. control) or {delta}V1-1 (Fig. 1, G and H, P < 0.05 vs. control), suggesting that PKC{delta} is a positive stimulator of Akt activity.


Figure 1
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  		Fig. 1. Acute effects of PKC{delta} inhibition on Akt activation in pulmonary arterial endothelial cells isolated from fetal lambs (FPAECs). FPAECs were acutely exposed to the PKC{delta} inhibitor, rottlerin (10 µM, 30 min), the PKC{delta}-derived inhibitory peptide {delta}V1-1 (1 µM, 2 h), or control peptide (1 µM, 2 h), and then whole cell lysates were subjected to Western blot analysis for total and phopsho-Tyr311 PKC{delta} (A–D) and Akt phospho-Ser473 Akt (E–H). Protein loading was also normalized for loading using β-actin. Representative images are shown for each. Although total PKC{delta} (A and C) and Akt (E and G) were unchanged by PKC{delta} inhibition, both phopsho-Tyr311 PKC{delta} (B and D) and phospho-Ser473 Akt (F and H) were significantly decreased. Data are presented as means ± SE, n = 3. *P < 0.05 vs. control cells.

 
Since Akt is known to be involved in activating eNOS through the phosphorylation at Ser1177 (11, 14, 26), we then determined the acute effect of rottlerin and the {delta}V1-1 peptide on eNOS expression, phosphorylation, and NO generation. Our data indicate that, as with Akt, acute inhibition of PKC{delta} with rottlerin or {delta}V1-1 did not alter total eNOS protein levels (Fig. 2, A–D). However, both eNOS phosphorylation at Ser1177 (Fig. 2, A–D) and NO generation (Fig. 2, E and F) were significantly reduced (P < 0.05 vs. control). However, phosphorylation of eNOS at Thr495 was unaffected by PKC{delta} inhibition (Fig. 2, A–D).


Figure 2
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  		Fig. 2. Acute effects of PKC{delta} inhibition on NO signaling in pulmonary arterial endothelial cells isolated from fetal lambs. FPAECs were acutely exposed to the PKC{delta} inhibitor, rottlerin (10 µM, 30 min), the PKC{delta}-derived inhibitory peptide {delta}V1-1 (1 µM, 2 h), or control peptide (1 µM, 2 h), and then whole cell lysates were subjected to Western blot analysis for eNOS as well as phospho-Ser1177 and phospho-Thr495 eNOS (A–D). Protein loading was also normalized for loading using β-actin. Representative images are shown for each. Although total eNOS levels were unchanged by PKC{delta} inhibition, phospho- phospho-Ser1177 eNOS (D) was significantly decreased. However, phospho-Thr495 eNOS was unchanged (D). The decrease in Ser1177 phosphorylation of eNOS was associated with a significant decrease in NO generation (E and F). Data are presented as means ± SE, n = 3. *P < 0.05 vs. control cells.

 
Generation of a PKC{delta} dominant negative mutant expression plasmid. A cDNA containing amino acids 2-144 of PKC{delta} and a FLAG epitope was subcloned into the mammalian expression plasmid pIRES to generate a PKC{delta} dominant negative mutant (pIRES-DNPKC{delta}). Initial studies were carried out to confirm the dominant negative effect. FPAECs were transfected or not with pIRES-DNPKC{delta}. After 24 h, cell extracts were prepared, and Western blot analysis was used to confirm expression of the mutant protein (Fig. 3A). In addition, we found that although PKC{delta} levels were unchanged by pIRES-DNPKC{delta} (Fig. 3, B and C), there was a significant decrease in the activating (Tyr311) phosphorylation of PKC{delta} (Fig. 3, B and C), confirming the dominant negative effect.


Figure 3
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  		Fig. 3. Generation of a PKC{delta} dominant negative mutant expression plasmid. FPAECs were transfected or not with the dominant negative PKC{delta} mutant plasmid, pIRES-DNPKC{delta}. After 24 h, whole cell lysates were subjected to Western blot analysis for the FLAG epitope (A) as well as total and phospho-Tyr311 PKC{delta} (B and C). Representative images are shown for each. Although total PKC{delta} (B and C) is unchanged by pIRES-DNPKC{delta} transfection phospho-Tyr311 PKC{delta} (C) levels were significantly decreased. Data are presented as means ± SE, n = 3. *P < 0.05 vs. control cells.

 
Prolonged effect of PKC{delta} inhibition on eNOS expression. FPAECs were exposed to rottlerin (10 µM) for 24 h and then analyzed for eNOS protein expression by Western blot analysis. eNOS protein expression was also compared in cells overexpressing a dominant negative PKC{delta} mutant, pIRES-DNPKC{delta}. Our data indicated that PKC{delta} inhibition significantly reduced eNOS protein levels (Fig. 4, P < 0.05 vs. control). To determine if this effect was at the level of transcription, we transfected FPAECs with a 1.6-kb eNOS promoter construct linked to a luciferase reporter gene (51) in the presence or absence of the dominant negative PKC{delta} mutant and then incubated with or without rottlerin. We then analyzed eNOS promoter activity after a further 24 h. Our data indicate that PKC{delta} inhibition with either rottlerin or dominant negative PKC{delta} mutant overexpression significantly reduces eNOS promoter activity (Fig. 5, P < 0.05 vs. control).


Figure 4
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  		Fig. 4. Prolonged PKC{delta} inhibition decreases eNOS expression in pulmonary arterial endothelial cells isolated from fetal lambs. FPAECs were exposed to the PKC{delta} inhibitor, rottlerin (10 µM), or transfected with the dominant negative PKC{delta} mutant, pIRES-DNPKC{delta}. After 24 h, whole cell lysates were subjected to Western blot analysis to determine the effect on eNOS protein levels. eNOS expression was also normalized for loading using β-actin. Representative images are shown for rottlerin (A) and pIRES-DNPKC{delta} (C). There is a significant decrease in eNOS expression after 24 h of PKC{delta} inhibition by both rottlerin (B) and pIRES-DNPKC{delta} (D). The data are expressed as means ± SE, n = 3. *P < 0.05 vs. control cells.

 

Figure 5
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  		Fig. 5. Transcriptional activity of the human eNOS promoter in response to PKC{delta} inhibition in pulmonary arterial endothelial cells isolated from fetal lambs. FPAECs were transfected with 1.6 kb of upstream sequence of the eNOS promoter fused to a luciferase reporter gene. Cells were also cotransfected with a construct expressing a β-galactosidase reporter gene as a transfection efficiency control. Cells were then either treated with rottlerin (10 µM) or transfected with the dominant negative PKC{delta} mutant, pIRES-DNPKC{delta}. After 24 h, luciferase activity was determined. PKC{delta} inhibition with either rottlerin (A) or pIRES-DNPKC{delta} (B) significantly decreases the activity of the 1.6-kb human eNOS promoter fragment. Values expressed are means ± SE, n = 6. *P < 0.05 vs. control cells.

 
Effect of Akt inhibition on eNOS expression and activity. To further elucidate the potential role of the PKC{delta}/Akt axis in regulating eNOS expression, we utilized an adenoviral construct to overexpress a dominant negative mutant of Akt in FPAECs. Initial Western blot analyses confirmed the overexpression of the dominant negative Akt mutant. We found that Akt expression was approximately twofold higher in dominant negative Akt mutant-transduced cells compared with GFP-transduced cells (Fig. 6, A and B, P < 0.05 vs. GFP control). We next determined if the dominant negative Akt mutant had effects on eNOS phosphorylation at Ser1177 or total eNOS expression. Our data indicate that the overexpression of the dominant negative Akt mutant significantly decreased both total eNOS protein levels (Fig. 6, C and D, P < 0.05 vs. control) and eNOS phosphorylation at Ser1177 (Fig. 6, C and D, P < 0.05 vs. control). These changes were associated with decreased NO generation (Fig. 6E, P < 0.05 vs. control). Furthermore, we found that overexpression of the dominant negative Akt mutant significantly decreased eNOS promoter activity (Fig. 7, P < 0.05 vs. control).


Figure 6
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  		Fig. 6. Effect of Akt inhibition on eNOS expression and activity in pulmonary arterial endothelial cells isolated from fetal lambs. FPAECs were transduced with adenoviruses expressing either a dominant negative mutant of Akt or green fluorescent protein (GFP) (as a transduction control). Western blot analysis in whole cell lysates was used to confirm Akt overexpression (A). There is a significant increase in Akt expression with the transduction of the dominant Akt adenovirus (B). The effect of Akt inhibition on phospho-Ser1177 eNOS was then determined by Western blot analysis (C). There is a significant decrease in phospho-Ser1177 with the transduction of the dominant Akt adenovirus (D). Representative images are shown, and in all cases, protein loading was normalized for loading using β-actin. The decrease in Ser1177 phosphorylation of eNOS was also associated with a significant decrease in NO generation (E). Data are presented as means ± SE, n = 3. *P < 0.05 vs. control cells.

 

Figure 7
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  		Fig. 7. Transcriptional activity of the human eNOS promoter in response to Akt inhibition in pulmonary arterial endothelial cells isolated from fetal lambs. FPAECs were transduced with adenoviruses expressing either a dominant negative mutant of Akt or GFP (as a transduction control). Then, after 24 h, there was further transfection with 1.6-kb of upstream sequence of the eNOS promoter fused to a luciferase reporter gene along with a construct expressing a β-galactosidase reporter gene (as a transfection efficiency control). Cells were harvested after a further 24 h, and the luciferase activity was determined. Akt inhibition significantly decreases the activity of the 1.6-kb human eNOS promoter fragment. Values expressed are means ± SE, n = 6. *P < 0.05 vs. control cells.

 
Effect of endogenous NO on eNOS expression. Our data indicated that the inhibition of PKC{delta}/Akt signaling leads to decreased NO generation and a subsequent decrease in NOS expression. Thus, we next determined whether the decrease in eNOS expression causally related to the reduced NO generation. Initially, we confirmed that the eNOS inhibition using ETU decreased NO generation. We found that NOx levels were significantly decreased after 24 h of ETU (100 µM) treatment (Fig. 8A, P < 0.05 vs. control). Furthermore, the reduction of NO generation caused significant decreases in both eNOS promoter activity (Fig. 8B, P < 0.05 vs. control) and eNOS protein levels (Fig. 8, C and D, P < 0.05 vs. control). To verify that NO signaling is involved in regulating eNOS promoter activity, cells were treated with both ETU and the NO donor, sodium nitroprusside (SNP). Our data indicate that the addition of SNP prevents the ETU-mediated reduction in eNOS promoter activity (Fig. 9A) and eNOS protein levels (Fig. 9, B and C).


Figure 8
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  		Fig. 8. Effect of endogenous NO generation on eNOS protein levels in pulmonary arterial endothelial cells isolated from fetal lambs. FPAECs were exposed to the NOS inhibitor 2-ethyl-2-thiopseudourea (ETU) (100 µM, 24 h). NOS inhibition was confirmed by a significant decrease in NO generation (A). FPAECs were then transfected with 1.6 kb of upstream sequence of the eNOS promoter fused to a luciferase reporter gene. Cells were also cotransfected with a construct expressing a β-galactosidase reporter gene as a transfection efficiency control. Cells were then treated with ETU (100 µM, 24 h), and the luciferase activity was determined. NOS inhibition significantly decreases the activity of the 1.6-kb human eNOS promoter fragment (B). Values expressed are means ± SE, n = 6. *P < 0.05 vs. control cells. Furthermore, to examine effects of NOS inhibition on eNOS protein levels, whole cell lysates were subjected to Western blot analysis. eNOS expression was also normalized for loading using β-actin. A representative image is shown (C). There is a significant decrease in eNOS expression after 24 h of NOS inhibition (D). The data are expressed as means ± SE, n = 3. *P < 0.05 vs. control cells.

 

Figure 9
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  		Fig. 9. Sodium nitroprusside (SNP) prevents the decrease in eNOS expression in response to NOS inhibition in pulmonary arterial endothelial cells isolated from fetal lambs. FPAECs were transfected with 1.6 kb of upstream sequence of the eNOS promoter fused to a luciferase reporter gene. Cells were also cotransfected with a construct expressing a β-galactosidase reporter gene as a transfection efficiency control. Cells were then treated with ETU (100 µM) in the presence or absence of the NO donor SNP (1 mM). After 24 h, luciferase activity was determined. The significant decrease in eNOS expression induced by NOS inhibition is attenuated by SNP (A). Values expressed are means ± SE, n = 6. *P < 0.05 vs. control cells. Whole cell lysates were also subjected to Western blot analysis to examine eNOS protein levels. Expression was also normalized for loading using β-actin. A representative image is shown (B). The significant decrease in eNOS expression associated with NOS inhibition is attenuated by SNP (C). The data are expressed as means ± SE, n = 3. *P < 0.05 vs. control cells; {dagger}P < 0.05 vs. ETU alone.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 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. Increasing evidence suggests that changes in pulmonary vascular tone are mediated by NO, possibly in response to increased shear stress on the pulmonary vascular endothelium (20, 28, 30). eNOS plays an important role in providing the NO necessary to allow these birth-related changes. 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) (27). Thus, a better understanding of the mechanisms that regulate the pulmonary vascular changes at birth may lead to new prevention and treatment strategies for patients with PPHN. In particular, identification of factors that govern basal expression of eNOS is likely to be of great benefit.

Regulation of eNOS by PKC been extensively investigated by many groups, and the issue of eNOS regulation by PKC is somewhat controversial with some studies indicating that PKC activates eNOS (18, 41), whereas others demonstrate that it inhibits eNOS (6, 38, 40). However, most of the studies completed so far have investigated PKC signaling as a whole rather than determining the effects of individual PKC isoforms. However, previous studies have suggested that specific activation of the PKC{alpha} isoform can increase NO production in endothelial cells and plays a role in regulation of blood flow in vivo (35). Similarly, platelet-activating factor stimulates NO release via PKC{alpha} in epithelial cells (10). Data suggest that when PKC signaling in endothelial cells inhibits eNOS activity, this occurs via the phosphorylation of Thr495 coupled with a dephosphorylation at Ser1177. Interestingly, we found that phosphorylation of eNOS at Thr495 was not affected by PKC{delta} inhibition, suggesting that PKC signaling through Thr495 may not be involved in basal NO generation or eNOS expression in FPAECs. However, the role of PKC{delta} in conditions of eNOS stimulation (such as shear stress) needs to be investigated.

In this study, we present evidence that the basal regulation of eNOS in PAECs isolated from fetal lambs is dependent on an autocrine signaling pathway that is NO dependent and regulated by PKC{delta}-dependent phosphorylation, and activation of Akt. We utilized a number of modalities to inhibit PKC{delta}: rottlerin, a PKC{delta} inhibitory peptide, and the overexpression of a PKC{delta} dominant negative mutant protein. We utilized these multiple modalities since the specificity of pharmacological agents can always be questioned. Indeed, the use of rottlerin as a specific PKC{delta} inhibitor is controversial (1, 24, 33, 45, 46). Thus, we utilized alternative agents to modulate PKC{delta} signaling. We have utilized a dominant negative mutant peptide of PKC{delta} ({delta}V1-1) developed by Dr. Mochly-Rosen at Stanford (4, 5). In addition, we generated a dominant negative mutant plasmid in which a FLAG-tagged V1 domain of PKC{delta} was cloned into a pIRES vector under the control of the CMV early promoter (pIRES-DNPKC{delta}). We demonstrate that when FPAECs are treated with rottlerin or the PKC{delta}-derived inhibitory peptide {delta}V1-1, there is an acute inhibition of NO generation followed by a subsequent reduction in eNOS protein levels secondary to a decrease in eNOS promoter activity. In accordance with this, we observed reduced NO generation, eNOS protein levels, and promoter activity in cells overexpressing pIRES-DNPKC{delta}.

We have previously identified PKC as one signal transduction molecule involved in regulating eNOS activation by fluid shear stress (51). In this study, we found that both eNOS mRNA and eNOS protein were induced by fluid shear stress. However, when PKC activity was attenuated with the specific PKC inhibitor calphostin C, the shear stress-induced increase in eNOS gene expression was attenuated. When other PKC inhibitors were used (staurosporine, H-7, and bisindolylmaleimide), similar results were obtained. Together, our data implicated PKC in the transduction of the shear stress signal transduction pathway responsible for increased eNOS gene expression. This was further corroborated by the fact that the exposure of FPAECs to the PKC activator phorbol 12-myristate 13-acetate led to an increase in eNOS mRNA levels. However, our data also demonstrated that PKC inhibition alone could lead to a decrease in eNOS mRNA expression (51), suggesting that PKC signaling is required to maintain basal eNOS expression. Thus, the data presented here expand our earlier study and identify PKC{delta} signaling as being a key component in maintaining basal eNOS expression.

Rather than being a constitutive enzyme as was first suggested, eNOS is dynamically regulated at both the transcriptional, posttranscriptional, and posttranslational levels. Protein kinases, other than PKC, have been shown to regulate eNOS activity through phosphorylation at multiple sites. Because several consensus sites for phosphorylation by protein kinases such as PKA, PKB (Akt), and CaM kinase II are found on eNOS, the key role of these kinases in the regulation of NOS through phosphorylation at a specific site of this enzyme becomes of critical interest. Ser1177 on eNOS can be phosphorylated by a variety of protein kinases, including Akt (11, 14, 26) and PKA (2, 3, 7). Akt activity is believed to be important for both agonist and shear stress activation of eNOS (11). Furthermore, Akt is known to be regulated by different isoenzymes of PKC. For example, PKC{alpha} is known to stimulate Akt activation and suppress apoptosis induced by interleukin-3 withdrawal (23). Previously, it has been shown that PKC{alpha} increases Ser473 phosphorylation and Akt-1 activity, whereas inhibition of its activity or expression decreases IGF-I-dependent activation of Akt-1 (34). PKC{alpha} has been shown to directly phosphorylate Akt-1 at the Ser473 site in vitro (34). Another study shows that PKC{alpha} and PKCβ are critical for PLA-LDL-induced Akt phosphorylation and survival in THP-1 monocytic cells (36). Enzastaurin (LY-317615), a PKCβ inhibitor, inhibits the Akt pathway and induces apoptosis in multiple myeloma cell lines (37). Our data show that inhibition of PKC{delta} with rottlerin attenuates Akt activation, which indicates that Akt activation is dependent on PKC{delta}. Furthermore, we found that Akt activity is important in maintaining basal eNOS expression. To our knowledge, this is the first study in endothelial cells describing that PKC{delta}-mediated activation of Akt regulates basal eNOS expression and activity. However, a limitation of our study is that it fails to elucidate the NO-mediated signaling pathway responsible for modulating eNOS transcription. It is interesting to speculate on the possible mechanism of action. A recent study has investigated the effect of NOS inhibition on the activity of a number of transcription factors. The data obtained indicated that L-NAME exposure decreased the activities of CREB, STAT, Sp-1, and c-Jun (57). Previous studies have demonstrated a key role for Sp-1 binding in regulating basal eNOS expression (48, 49, 56), whereas our data have shown that c-Jun plays a key role regulating eNOS expression both through development and by increased flow (51). Thus, we speculate that perhaps NO regulates eNOS transcriptional activity through its ability to increase Sp-1 and/or AP-1 binding activity. However, further studies will be required, using mutant eNOS promoter fragments, to validate this possibility.

In a previous study, we investigated the effects of the NO donor SNP in cultured ovine FPAECs. SNP was found to be a potent inhibitor of eNOS activity and could reduce eNOS activity in these cells to a significant level within 2 h of treatment (44). A 24-h treatment with SNP did not alter eNOS mRNA and protein levels. Thus, our results demonstrated that NO acts to reduce the catalytic activity of the eNOS protein without altering the transcription or translation of the eNOS gene. However, a previous study from Yuhanna et al. (54) indicated that exogenous NO increased eNOS protein and mRNA levels, resulting in a parallel increase in NOS enzymatic activity in fetal intrapulmonary artery endothelium. These two studies were difficult to reconcile. However, contrary to the effect of exogenous NO observed in our earlier study, we observed that endogenous NO has a positive effect on eNOS activity and abundance such that when FPAECs were treated with eNOS inhibitor ETU, there was significant attenuation of eNOS protein expression and eNOS promoter activity. Furthermore, we found that the addition of SNP prevented the ETU-mediated decrease in eNOS expression. These data imply that endogenous NO has positive feedback effect on eNOS abundance and activity. Thus, our data now suggest the reason for the apparent opposite effects of the two prior studies. The FPAECs used in our studies appear to be capable of producing the amount of NO required to maintain eNOS expression, whereas the FPAECs used by Yuhanna and colleagues (54) do not appear to do so. As our cells are isolated from 136-day-old fetal lambs, whereas the studies from Yuhanna et al. (54) used cells isolated from 125-day-old lambs, this suggests that there are developmental differences in basal NO regulation that are maintained in culture. Indeed, we have previously shown this to be true for PAECs isolated from 136-day-old fetal lambs and from juvenile sheep (25).

In summary, data in the current study indicate that 1) under basal conditions, PKC{delta} triggers a pathway leading to NO generation via the activation of Akt; 2) PKC{delta} acts upstream of Akt as the inhibition of PKC{delta} is able to inhibit Akt activity in endothelial cells; and 3) endogenous NO upregulates eNOS in an autocrine fashion. In conclusion, our data indicate that PKC{delta}, Akt, and NO are crucial components of the signaling cascade that controls eNOS in ovine FPAECs under basal conditions. Furthermore, this study suggests that pharmacological modulation of PKC{delta} activity in the endothelium may have potential for the treatment of vascular disorders associated with vascular dysfunction and impaired blood flow.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported in part by National Heart, Lung, and Blood Institute Grants HL-60190, HL-67841, HL-72123, and HL-70061 (all to S. M. Black), and by a grant from the Fondation Leducq (to S. M. Black).


   ACKNOWLEDGMENTS
 
We thank Dr. Jeffrey R. Fineman (Univ. of California, San Francisco) for critical reading of this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. M. Black, Vascular Biology Center, Medical College of Georgia, 1459 Laney Walker Blvd., CB-3210B, Augusta, GA 30912 (e-mail: sblack{at}mcg.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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