Modulation of PKCδ signaling alters the shear stress-mediated increases in endothelial nitric oxide synthase transcription: role of STAT3

Neetu Sud, Sanjiv Kumar, Stephen Wedgwood, Stephen M. Black

Abstract

We have previously shown that the regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells isolated from fetal lamb under static conditions is positively regulated by PKCδ. In this study, we explore the role of PKCδ in regulating shear-induced upregulation of eNOS. We found that shear caused a decrease in PKCδ activation. Modulation of PKCδ before shear with a dominant negative mutant of PKCδ (DN PKCδ) or bryostatin (a known PKCδ activator) demonstrated that PKCδ inhibition potentiates the shear-mediated increases in eNOS expression and activity, while PKCδ activation inhibited these events. To gain insight into the mechanism by which PKCδ inhibits shear-induced eNOS expression, we examined activation of STAT3, a known target for PKCδ phosphorylation. We found that shear decreased the phosphorylation of STAT3. Further the transfection of cells with DN PKCδ reduced, while PKCδ activation enhanced, STAT3 phosphorylation in the presence of shear. Transfection of cells with a dominant negative mutant of STAT3 enhanced eNOS promoter activity and nitric oxide production in response to shear. Finally, we found that mutating the STAT3 binding site sequence within the eNOS promoter increased promoter activity in response to shear and that this was no longer inhibited by bryostatin. In conclusion, shear decreases PKCδ activity and, subsequently, reduces STAT3 binding to the eNOS promoter. This signaling pathway plays a previously unidentified role in the regulation of eNOS expression by shear stress.

  • cell signaling
  • phosphorylation
  • endothelial cell
  • biomechanical forces

it is well established that the nitric oxide (NO) required to regulate vascular tone is produced by the enzyme endothelial nitric oxide synthase (eNOS). eNOS is regulated by a number of factors such as shear, posttranslational modifications, and protein-protein interactions (8, 15, 47). Several studies (43, 52) report that shear stress stimulates NO synthesis and upregulates eNOS gene expression. Laminar shear stress increases eNOS transcription, while stimuli such as cell growth increase eNOS expression by prolonging the half-life of the eNOS mRNA (3738). In addition, factors such as intracellular location, protein-protein interactions (e.g., calmodulin, caveolin, and heat shock protein 90), phosphorylation, and substrate and cofactor availability may all dynamically regulate eNOS activity (4, 9, 12, 13, 16, 23, 28, 45, 48, 49, 55). The regulation of eNOS by mechanical forces is complex and incompletely understood. A variety of in vitro systems clearly demonstrate that fluid shear stress upregulates eNOS gene expression by activation of the 5′-promotor region (52). Similarly in vivo, increases in flow associated with exercise are associated with increased eNOS-derived NO signaling (21, 22, 37). Fluid shear stress has also been demonstrated to increase eNOS activity in vitro (32, 34, 46). This appears to be regulated, in part, by potassium channels and serine phosphorylation (2, 11, 17).

Previously, we identified PKC as one signal transduction molecule involved in regulating eNOS activity and gene expression by fluid shear stress. PKC is a family of phospholipid-dependent serine-threonine kinases (7, 38). We (50) have previously shown that the expression of eNOS is increased in response to increased 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 (50). Similar results were obtained with the other PKC inhibitors staurosporine, H-7, and bisindolylmaleimide, implicating PKC in the transduction of the shear stress signal transduction pathway, leading to an increase in eNOS gene expression (50). However, the PKC consists of 12 members and it is unclear the role of each isoform in regulating eNOS expression and/or activity. Indeed it is possible that there may be some iosforms that activate, while others inhibit, NO signaling. We (40) have recently shown that PKCδ is involved in maintaining basal eNOS expression through its ability to activate Akt and increase NO generation. In support of this premise, studies have shown that, depending on the tissue analyzed, PKC can either positively (25, 50) or negatively (29, 57) regulate eNOS expression and activity. Thus in this study we wished to determine the role, if any, played by PKCδ in regulating shear-mediated increases in eNOS expression. Here we find that PKCδ activity is decreased by shear stress in pulmonary arterial endothelial cells (PAECs) isolated from the fetal lamb. This, in turn, led to the identification of decreased STAT3 binding activity as a previously unidentified mechanism in the upregulation of eNOS promoter by shear stress.

MATERIALS AND METHODS

Cell culture.

Primary cultures of ovine PAECs were isolated as described previously (52). Cells were maintained in DMEM containing phenol red supplemented with 10% FCS (Hyclone, Logan, UT), antibiotics, and antimycotics (MediaTech, Herndon, VA) at 37°C in a humidified atmosphere with 5% CO2-95% O2. Cells were between passages 3 and 10, seeded at ∼50% confluence, and utilized when fully confluent.

Shear stress.

Laminar shear stress was applied using a cone-plate viscometer that accepts six-well tissue culture plates, as described previously (39, 42, 52). This method achieves laminar flow rates that represent physiological levels of laminar shear stress in the major human arteries, which is in the range of 5–20 dyn/cm2 (27) with localized increases to 30–100 dyn/cm2.

Western blotting.

Serum-starved PAECs (16 h) were sheared for 8 h and 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 then subjected to SDS-PAGE on 4–12% polyacrylamide gels and transferred to a nitrocellulose membrane (Bio-Rad). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween (TBST). The primary antibodies used for immunoblotting were anti-PKCδ (cat. no. 2056), anti-phospho Tyr 311 PKCδ (cat. no. 2055), anti-STAT3 (cat. no. 9139), anti-phospho Ser 727 STAT3 (cat. no. 9136), and anti-eNOS (cat. no. 9586; all from Cell Signaling Technology and used at 1:1,000 dilution). The membrane was then washed with TBST three times for 10 min, incubated with secondary antibodies coupled to horseradish peroxidase, and washed again with TBST as described above, and the protein bands were visualized with ECL reagent (Pierce).

Overexpression of dominant negative PKCδ and S727A STAT3.

Overexpression of dominant negative (DN) PKCδ was performed as described previously (40). To overexpress STAT3, pulmonary artery endothelial cell isolated from the fetal lamb were transfected with S727A STAT3 mutant construct (Addgene) in which Ser 727 is mutated to alanine. The overexpression of S727A STAT3 was analyzed by immunoblotting.

eNOS promoter analysis.

eNOS transcription was analyzed using a 1,600-bp promoter fragment fused to a luciferase reporter gene as described previously (52). Fetal endothelial cells were cotransfected with the 1.6-kb eNOS promoter-luciferase and β-galactosidase construct (to normalize for transfection efficiency). To analyze the effect of PKCδ or STAT3, the cells were cotransfected with either DN PKCδ (40) or a dominant negative mutant of STAT3, S727A STAT3. Transfected cells were serum starved overnight and then incubated with either bryostatin (10 ng/ml; 30 min) before shear. Luciferase activity was determined using the Luciferase assay kit (Promega) and a Fluoroskan Ascent FL luminometer (Thermo Electron). In addition, a mutant eNOS promoter construct was prepared in which the STAT3 binding site of eNOS (TTTCTTT) was mutated by 2 bp (to TTTCccT) to eliminate the binding of STAT3 (18). S727 STAT3.

Detection of nitrites/nitrates.

NO generated by PAECs in response to shear 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 (40).

DNA binding analysis.

Nuclear extracts were prepared using the NE-PER kit (Pierce) as specified by the manufacturer. To quantify the STAT3-specific binding activity of nuclear extract, a Transfactor kit (Clonetech) was used according to the manufacturer's protocol. The wells of streptavidin-coated plates were blocked for 15 min with 1× TransFactor/blocking buffer, added, and incubated for 1 h at room temperature. Nuclear extract was diluted with 1× TransFactor/blocking buffer and mixed with poly(dI-dC) and either wild-type biotinylated double stranded oligonucleotide (5′-CTGAGCTTCCGTTTCTTTCTTAAACTTTCTCTCAGTC-3′) corresponding to the STAT3 binding site at −1,024 in eNOS gene or double-stranded mutant oligonucleotide (5′- CTGAGCTTCCGTTTCccTCTTAAACTTTCTCTCAGTC-3′) with a 2-bp mutation in STAT3 binding site at −1,024 and incubated on ice for 15 min. This was then added to the wells of the plate and incubated at room temperature for 60 min. Microtiter wells were then washed three times, and anti-STAT3 was added per well and incubated further at room temperature for an hour. After being washed extensively, diluted secondary antibody conjugated with horseradish peroxidase was added to each well and further incubated at room temperature for 30 min. After being washed repeatedly, 100 μl of tetramethylbenzidine substrate solution were added to each well in the dark for the color development at room temperature for 10 min and the binding intensity was measured as absorbance at 655 nm using a microtiter plate reader.

Statistical analysis.

Statistical calculations were performed using the GraphPad Prism V. 4.01 software. Means ± SD or SE were calculated for all samples, and significance was determined by either by the unpaired t-test or ANOVA. For ANOVA Neuman-Kuels, post hoc testing was also utilized. A value of P < 0.05 was considered significant.

RESULTS

Shear stress decreases PKCδ activity in PAECs.

Initially, we evaluated the effect of shear stress (8 h, 20 dyn/cm2) on PKCδ activity in PAECs isolated from fetal lambs (PAECs). We found that the membrane localization of PKCδ was significantly decreased by shear stress (Fig. 1, A and B). Similarly, shear stress significantly decreased the phosphorylation of PKCδ at amino acid position Tyr 311 (Fig. 1, C and D). Together these data indicate that shear stress decreases PKCδ activity.

Fig. 1.

Shear stress decreases PKCδ activation in ovine pulmonary arterial endothelial cells (PAECs). Ovine PAECs were exposed or not to shear stress (20 dyn/cm2, 8 h) and then whole cell extracts, membrane (M), and cytosolic (C) fractions were prepared by differential centrifugation. Extracts (20 μg) were then subjected to Western blot analysis using either a PKCδ antibody (A and C) or a phospho-specific antibody recognizing Tyr 311 (the activation site of PKCδ; C). C: whole cell extract data only. Shear stress decreases both PKCδ membrane translocation (B) and phopsho-Tyr 311 levels (D), indicating that PKCδ activity is diminished by shear stress. Data are means ± SD. *P < 0.05 vs. static; n = 3–4.

Effect of modulating PKCδ activity on shear stress-mediated increases in eNOS expression.

We next determined the effect of modulating PKCδ activity on shear stress-mediated increases on eNOS expression. To inhibit PKCδ activity, PAECs were transfected with DN PKCδ or treated with bryostatin (10 ng/ml; 30 min) to activate PKCδ before cells were exposed to shear stress (8 h, 20 dyn/cm2). Initially, we verified that our treatments were modulating PKCδ activity in the expected fashion. As expected, DN PKCδ further decreased the shear-mediated inhibition of PKCδ Tyr 311 phosphorylation (Fig. 2, A and B), while exposure of the cells to the PKCδ activator bryostatin before shear significantly increased PKCδ phosphorylation relative to shear alone (Fig. 2, A and B). Further, DN PKCδ abolished the bryostatin mediated increase in phosphorylation of PKCδ at Tyr 311 (Fig. 2, A and B). Further, we found that transfection with DN PKCδ potentiated the increase in eNOS promoter activity (measured using a 1.6-kb eNOS promoter construct linked to a luciferase reporter gene) induced by shear stress (Fig. 2C), while bryostatin significantly reduced the shear-mediated increase in eNOS promoter activity (Fig. 2C). DN PKCδ transfection restored the shear-mediated increase in eNOS promoter activity inhibited by bryostatin (Fig. 2C). In addition, we found that these changes in eNOS promoter activity were reflected at the level of eNOS protein levels. We found as expected that eNOS protein levels were significantly increased by shear but that pretreatment with bryostatin significantly inhibited the shear-induced increase in eNOS protein levels (Fig. 3, A and B). Moreover, DN PKCδ significantly enhanced the shear-mediated increase in eNOS protein levels (Fig. 3, A and B). These changes in eNOS protein correlated with alterations in NO generation. Thus bryostatin attenuated the shear-mediated increase in NO generation (Fig. 3C), while DN PKCδ significantly increased the increase in NO induced by shear (Fig. 3C).

Fig. 2.

Effect of modulating PKCδ activity on endothelial nitric oxide synthase (eNOS) promoter activity in response to shear stress. Ovine PAECs were transfected with dominant negative (DN) PKCδ and then treated or not with bryostatin (10 ng/ml for 30 min) followed by shear stress (20 dyn/cm2, 8 h). Extracts (20 μg) were then subjected to Western blot analysis using either a PKCδ antibody (A) or a phospho-specific antibody recognizing Tyr 311 (A). DN PKCδ significantly reduces the Tyr 311 phosphorylation of PKCδ relative to no shear condition, while bryostatin enhances PKCδ Tyr 311 phosphorylation relative to shear stress (B). To examine the effect of modulating PKCδ activity on eNOS promoter activity, cells were also transfected with 1.6-kb eNOS promoter-luciferase reporter construct (along with β-galactosidase as an internal control) along with DN PKCδ, treated or not with bryostatin (10 ng/ml, 30 min before shear), and then exposed to shear stress (20 dyn/cm2, 8 h). The activity of the eNOS promoter is significantly increased by shear (C); in addition, bryostatin inhibits, whereas overexpression of DN PKCδ potentiates, the shear-induced increase in eNOS promoter activity (C). The inhibitory effect of bryostatin is blocked by DN PKCδ (C). Values are means ± SD. *P < 0.05 vs. no shear; P < 0.05 vs. shear alone; n = 3.

Fig. 3.

Effect of modulating PKCδ activity on eNOS protein levels and nitric oxide (NO) production in response to shear stress. Ovine PAECs were transfected with DN PKCδ, then treated or not with bryostatin (10 ng/ml for 30 min), and then exposed to shear (20 dyn/cm2, 8 h). Extracts (20 μg) were then subjected to Western blot analysis using a specific antibody raised against eNOS. A: representative image. DN PKCδ significantly increases, while bryostatin attenuates, the shear-mediated increase in eNOS protein levels (B). DN PKCδ also reverses the effect of bryostatin on eNOS protein levels (B). Similarly, DN PKCδ significantly increases, while bryostatin attenuates, the shear-mediated increase in NO generation (C). DN PKCδ also reverses the effect of bryostatin on NO levels (C). Values are means ± SD. *P < 0.05 vs. no shear; P < 0.05 vs. shear alone; n = 3–6.

Shear stress decreases STAT3 activity via the inhibition of PKCδ.

It is well established that one of the downstream mediators of PKCδ is STAT3 (20). Thus we next investigated if shear stress alters STAT3 activity. We found that shear stress reduced STAT3 activity, as determined by a decrease in phosphorylation at amino acid Ser 727 (Fig. 4, A and B). Further, we found that treatment with bryostatin prevented the decrease in STAT3 Ser 727 phosphorylation mediated by shear stress (Fig. 4, A and B), whereas DN PKCδ further reduced the shear-mediated decrease in STAT3 Ser 727 phosphorylation (Fig. 4, A and B). DN PKCδ also abolished the effect of bryostatin, indicating that the bryostatin-induced effects are due to activation of PKCδ (Fig. 4, A and B). We next sought to determine the functional consequences of STAT3 serine phosphorylation on its DNA binding activity. We analyzed STAT3 binding in the presence of either biotinylated double-stranded oligonucleotide spanning the STAT3 binding site at the position −1,024 of eNOS gene or a mutant oligonucleotide in which this STAT3 binding site was disrupted by 2-bp mutation using site-directed mutagenesis. We found that DNA binding of STAT3 was significantly inhibited by shear (Fig. 4C). Pretreatment with bryostatin before shear enhanced STAT3 DNA binding activity (Fig. 4C), whereas overexpression of either DN PKCδ or a dominant negative mutant of STAT3 (S727 STAT3) further enhanced the shear-mediated decrease in DNA binding (Fig. 4C). In addition, we found that when the DNA binding assay was done in the presence of an oligonucleotide containing a mutation in the STAT3 sequence, the DNA binding activity was dramatically reduced in all cases (Fig. 4C). Together these data indicate that shear stress decreases STAT3 DNA binding activity through the inhibition of PKCδ activity.

Fig. 4.

Effect of modulating PKCδ activity on STAT3 Ser 727 phosphorylation in response to shear stress. Ovine PAECs were transfected with DN PKCδ, then treated or not with bryostatin (10 ng/ml for 30 min), and followed by shear stress (20 dyn/cm2, 8 h). Extracts (20 μg) were then subjected to Western blot analysis using an anti-phospho Ser 727 STAT3 antibody. A: representative image. DN PKCδ significantly decreases, while bryostatin increases, the shear-mediated attenuation in Ser 727 STAT3 phosphorylation (B). DN PKCδ also reverses the effect of bryostatin on Ser 727 STAT3 phosphorylation (B). Nuclear fractions were also prepared from PAECs transfected with DN PKCδ or S727A STAT3 and then treated or not with bryostatin (10 ng/ml for 30 min) followed by shear stress (20 dyn/cm2, 8 h). STAT3 DNA binding activity was then determined using both a STAT3 binding site at position −1,024 of eNOS promoter or a mutant oligonucleotide with a 2-bp mutation in this STAT3 binding site. With the use of the wild-type oligonucleotide, shear reduces STAT3 binding to the wild-type oligonucleotide (solid bars; C). DN PKCδ or S727A STAT3 significantly decreases, while bryostatin increases, the shear-mediated attenuation in STAT3 DNA binding to the wild-type oligonucleotide (C). With the use of the mutant oligonucleotide, bryostatin does not induce an increase in STAT3 binding activity upon shear (open bars; C). Values are means ± SD. *P < 0.05 vs. no shear; P < 0.05 vs. shear alone; n = 3–6.

Inhibiting STAT3 activity enhances the shear-induced increase in eNOS expression and activity.

To evaluate the effect of inhibiting STAT3 activity on shear-mediated increases in eNOS promoter activity, we again cotransfected PAECs with a 1.6-kb eNOS promoter construct linked to a luciferase reporter gene with the S727A STAT3 dominant negative mutant. After 24 h, PAECs were exposed to shear stress (8 h, 20 dyn/cm2). We found that transfection with the S727A STAT3 mutant enhanced shear-induced eNOS promoter activity (Fig. 5A). S727A STAT3 also abolished the negative effect of bryostatin (Fig. 5A). Further, the inhibition of STAT3 enhanced the shear-mediated increase in eNOS protein levels (Fig. 5, B and C) and NO generation (Fig. 5D). Again, S727A STAT3 also abolished the negative effect of bryostatin on eNOS protein levels (Fig. 5, B and C) and NO generation (Fig. 5D).

Fig. 5.

Effect of STAT3 inhibition on eNOS expression and activity in response to shear stress. To examine the effect of inhibiting STAT3 activity on eNOS promoter activity, cells were also transfected with a 1.6-kb eNOS promoter-luciferase reporter construct (along with β-galactosidase as an internal control) or a construct in which the STAT3 binding element at −1,024 was mutated along with a dominant mutant of STAT3, S727A STAT3. After 24 h, the cells were then treated or not with bryostatin (10 ng/ml, 30 min before shear) and then exposed to shear stress (20 dyn/cm2, 8 h). The increase in the activity of the eNOS promoter induced by shear stress is significantly increased by S727A STAT3 overexpression (A). The inhibitory effect of bryostatin is also blocked by S727A STAT3 overexpression (A). Similarly, S727A STAT3 overexpression significantly increases the shear-mediated increase in both eNOS protein levels (B and C) and NO generation (D). S727A STAT3 overexpression also reverses the effect of bryostatin on eNOS protein levels (B and C) and NO generation (D). In addition, shear stress significantly enhances the activity of the STAT3 mutant eNOS promoter compared with the wild-type promoter and this is not attenuated by bryostatin (E). Values are means ± SD. *P < 0.05 vs. no shear, P < 0.05 vs. shear alone, P < 0.05 vs. wild-type promoter; n = 6.

Next, we examined the effect of mutating the STAT3 binding site located at −1,024 of the human eNOS promoter, which has previously been shown to mediate STAT3-induced inhibition of eNOS expression (35). Thus we transfected PAECs with an eNOS-luciferase construct bearing a 2-bp mutation in the STAT3 binding site at position −1,024. Our data indicate the shear-induced eNOS promoter activity is significantly higher in cells transfected with a mutant promoter compared with cells transfected with wild-type eNOS promoter (Fig. 5E). Further, bryostatin fails to inhibit eNOS promoter activity in cells transfected with a mutant eNOS promoter (Fig. 5E). Together these data demonstrate that PKCδ exerts its negative effect on eNOS promoter activity through the stimulation of STAT3 activity.

Finally, we investigated the effect of inhibiting PKCδ or STAT3 activities on NO signaling in resting PAECs. Our data indicate that inhibiting the activity of PKCδ or STAT3 in PAECs has opposing effects on NO signaling. We found that PKCδ inhibition significantly decreases eNOS promoter activity (Fig. 6A) and attenuates NO generation (Fig. 6B), while inhibiting STAT3 activity significantly increases eNOS promoter activity (Fig. 6A) and stimulates NO generation (Fig. 6B).

Fig. 6.

Effect of PKCδ or STAT3 inhibition on NO signaling in resting PAECs. To examine the effect of inhibiting PKCδ or STAT3 activities on eNOS promoter activity in resting PAECs, cells were trasnfected with DN PKCδ or S727A STAT3 along with the 1.6-kb eNOS promoter-luciferase reporter construct (along with β-galactosidase as an internal control). After 24 h, the luciferase activities were determined. PKCδ inhibition significantly attenuates, while STAT3 inhibition significantly enhances, eNOS promoter activity (A). Similarly, PKCδ inhibition significantly attenuates, while STAT3 inhibition significantly enhances, NO generation (B). Values are means ± SD. *P < 0.05 vs. control; n = 3.

DISCUSSION

We (40) have previously demonstrated that the novel PKC (PKCδ) positively regulates eNOS under basal conditions in endothelial cells isolated from fetal lamb. Herein we focus on its role in regulating shear-mediated increase in eNOS gene expression. Our data indicate that shear stress increases eNOS expression at least in part by decreasing PKCδ activity, which, in turn, decreases STAT3 phosphorylation at Ser 727 leading to a decrease in STAT3 binding to the eNOS promoter and reduced inhibition of eNOS transcription. Our results elucidating the key role played by PKCδ-STAT3 signaling in regulating eNOS expression are noteworthy for two major reasons. First, they elucidate a new mechanism by which shear stress increases eNOS transcription, and second, they suggest new mechanisms that may be involved in the decrease in eNOS expression and activity that occur in conditions of endothelial dysfunction such as pulmonary hypertension.

PKCδ translocates from cytosol to membrane like other PKCs. We therefore examined the effect of shear on PKCδ translocation. Our data show that under basal conditions PKCδ resides in both the cytosolic and membrane fractions, while shear stress significantly reduced the fraction of PKCδ localized to the membrane. In addition, recent studies (24) have demonstrated that the activation of PKC isoforms, in addition to the cytosol to membrane translocation, involves the phosphorylation of key tyrosine residues. These include α, β, γ, ε, δ, and ζ (24). PKCδ also has been shown to be phosphorylated at tyrosines, but the significance of this phosphorylation is controversial, since some studies report this phosphorylation can either increase (25, 26, 33, 44) or decrease PKCδ activity (58) or may not be involved in the activation of its kinase activity. Our results show that shear stress decreases phosphor-Tyr 311 PKCδ levels, while the overexpression of a dominant negative mutant of PKCδ potentiated this decrease in response to shear. Conversely, pretreatment with bryostatin before shear stimulation resulted in higher phospho-Tyr 311 PKCδ compared with shear alone. As bryostatin is known to activate both PKCδ and PKCε (5), we confirmed the bryostatin-induced effects were due to PKCδ activation, as the overexpression of dominant negative mutant of PKCδ before bryostatin treatment abolished bryostatin-mediated inhibition of eNOS transcription. Analyzing the effect of dominant negative mutant PKCδ overexpression as well as bryostatin on eNOS promoter activity, eNOS protein levels, and NO generation in response to shear, we found that the overexpression of dominant negative mutant PKCδ potentiated shear-induced eNOS promoter activity, eNOS protein levels, and NO generation, whereas bryostatin treatment before shear decreased these events. The effect of bryostatin was abolished by dominant negative mutant PKCδ, thus establishing the negative role of PKCδ in regulating shear-induced eNOS expression and NO generation. These data indicate that PKCδ inhibits shear-induced upregulation of eNOS promoter activity, protein levels, and NO generation. Further, we also confirmed our earlier data (40) in which we found that inhibition of PKCδ activity in resting PAECs led to a decrease in eNOS expression and NO signaling, indicating that the role of PKCδ signaling in regulating eNOS is complex and may depend on the stimulus applied. This is shown by the fact that our data indicate that even in the presence of a mutant STAT3 binding site at −1,024 the presence of bryostatin still reduced the shear-dependent increase in eNOS promoter activity. The reasons for this are unclear. It is possible that there are other PKCε-dependent effects on eNOS that we have not been resolved or that that the other STAT3 binding sites in the eNOS promoter (35) may still be functional and exerting an effect. However, further studies will be required to resolve this issue.

We next investigated the mechanism by which PKCδ was exerting its effect on eNOS transcription. To accomplish this we evaluated the potential role of STAT3. STAT3 is a transcription factor that can be activated by several cytokines and growth factors (1), and recent evidence suggests that it can be phosphorylated by PKCδ in vitro as well as in vivo (14, 20). Several studies have established that tyrosine phosphorylation of STATs is a prerequisite for their DNA binding and transactivation (3), although growth factors and cytokines induce phosphorylation of STATs on both tyrosine and serine (6, 53, 59). The key activation phosphorylation appears to be localized on Ser 727 (14, 20), and it has been shown that the phosphorylation of Ser 727 induced by IL-6 was enhanced by transfection of the wild-type PKCδ but inhibited by the dominant negative mutant of PKCδ (20). Thus we determined whether PKCδ phosphorylates STAT3 in PAECs and whether STAT3 phosphorylation is reduced by shear in the same manner as PKCδ phosphorylation. Interestingly, we observed that the shear-induced decrease in STAT3 Ser 727 phosphorylation paralleled the decrease in phosphorylation of PKCδ at Tyr 311. Moreover, we found that in DN PKCδ overexpressing cells there was a further inhibition in the shear-mediated decrease in STAT3 phosphorylation, while bryostatin treatment before shear enhanced STAT3 phosphorylation. Together our data suggest that PKCδ is involved in mediating Ser 727 phosphorylation of STAT3. These results are in agreement with other studies that reveal PKCδ activation as a primary regulator of STAT3 serine phosphorylation in keratinocytes (14, 20) and that shear stress can reduce STAT3 activity in endothelial cells (31), while STAT3 has been shown to inhibit eNOS transcription through its binding to a core sequence localized to −1,024 (35).

The functional consequences of Ser 727 phosphorylation of STAT3 are still controversial, as some studies (56) have shown it to be required for the DNA binding of STAT3 in certain cell types, while others (19) show that it inhibits DNA binding. Thus to further evaluate the role of STAT3 signaling in regulating eNOS expression, we focused on the eNOS promoter. Of the four consensus sequences for STAT3 binding in the eNOS promoter at positions −1,520, −1,024, −840, and −540, the one at −1,024 has been reported to inhibit eNOS promoter activity (35). Thus we next analyzed if there was a correlation between STAT3 Ser 727 phosphorylation and its binding ability to the eNOS promoter. To test this, we evaluated STAT3 DNA binding activity using a DNA binding assay in which either a biotinylated double stranded oligonucleotide encompassing the STAT3-binding element of the eNOS gene at position −1,024 or a mutant oligonucleotide with a 2-bp mutation at −1,024 was used. Our data demonstrate that shear induced a decrease in STAT3 DNA binding. Activating PKCδ before shear resulted in a significant increase in STAT3 activity, while attenuating PKCδ activity by overexpressing our dominant negative mutant further attenuated STAT3 DNA binding activity in response to shear. These data suggest that PKCδ is involved in regulating STAT3 activity and that the increase in eNOS transcription induced by shear stress involves a decrease in STA18 last line and T3 activity mediated through the inhibition of PKCδ signaling.

To further confirm the significance of PKCδ-mediated serine phosphorylation of STAT3 in reducing the binding to the eNOS promoter, we overexpressed a mutant of STAT3, S727A STAT3, deficient in its ability to be phosphorylated at Ser 727. Our results show that overexpression of S727A STAT3 before shear stimulation resulted in a potentiated decrease in DNA binding activity, suggesting that Ser 727 phosphorylation does indeed promote STAT3 binding to the eNOS promoter. This is in accordance with a previous study that shows that STAT3 phosphorylation at Ser 727 stimulates its DNA binding activity (30, 36, 56). However, it is again worth noting that the role of Ser 727 phosphorylation of STAT3 is controversial, as it has also been reported that this phosphorylation caused an attenuation of its DNA binding ability (19, 20, 54). Together our data in combination with previously published studies suggest that the DNA binding activity of Ser 727 phosphorylated STAT3 may depend on the cell type evaluated. We also analyzed the significance of Ser 727 phosphorylation of STAT3 on eNOS promoter activity, eNOS protein levels, and NO generation. Our data demonstrate that the overexpression of S727A STAT3 potentiated the shear-induced increase in eNOS promoter activity as well as the increases in eNOS protein levels and NO generation. In addition, we evaluated wild-type eNOS promoter, and STAT3 mutant eNOS promoter, luciferase constructs transfected into PAECs and exposed to shear. Our data indicate that the increase in eNOS promoter activity in response to shear is significantly increased when the STAT3 binding sequence located at −1,024 is mutated. Together these data highlight the important role of STAT3 inhibition in the upregulation of eNOS expression induced by shear stress. Further, our data suggest that STAT3 inhibits eNOS promoter activity in resting PAECs, suggesting that there is a basal level of STAT3 activity in the cell that reduces eNOS expression and NO signaling.

In conclusion, our results establish a key role for decreases in PKCδ signaling in the upregulation of eNOS in response to shear stress. Further, we found that the mechanism for this effect was mediated by a decrease in STAT3 binding to the eNOS promoter located at −1,024. Further, we found that increasing STAT3 phosphorylation at Ser 727 by stimulating PKCδ signaling promotes its binding to the eNOS promoter and inhibits the shear-induced increase in eNOS promoter activity, eNOS protein, and NO generation. To our knowledge, this study provides the first description of the role of decreased PKCδ-STAT3 signaling in the increase in eNOS expression induced by shear stress. We also speculate that PKCδ inhibition may be an attractive target for the development of strategies to restore eNOS expression and activity, with consequent improvement of endothelial function in conditions where eNOS expression and activity are attenuated and STAT3 activity is stimulated.

Acknowledgments

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).

Footnotes

  • 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.

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View Abstract