Shear stress stimulates nitric oxide signaling in pulmonary arterial endothelial cells via a reduction in catalase activity: role of protein kinase Cδ

Abstract

Previous studies have indicated that acute increases in shear stress can stimulate endothelial nitric oxide synthase (eNOS) activity through increased PI3 kinase/Akt signaling and phosphorylation of Ser1177. However, the mechanism by which shear stress activates this pathway has not been adequately resolved nor has the potential role of reactive oxygen species (ROS) been evaluated. Thus, the purpose of this study was to determine if shear-mediated increases in ROS play a role in stimulating Ser1177 phosphorylation and NO signaling in pulmonary arterial endothelial cells (PAEC) exposed to acute increases in shear stress. Our initial studies demonstrated that although shear stress did not increase superoxide levels in PAEC, there was an increase in H₂O₂ levels. The increases in H₂O₂ were associated with a decrease in catalase activity but not protein levels. In addition, we found that acute shear stress caused an increase in eNOS phosphorylation at Ser1177 phosphorylation and a decrease in phosphorylation at Thr495. We also found that the overexpression of catalase significantly attenuated the shear-mediated increases in H₂O₂, phospho-Ser1177 eNOS, and NO generation. Further investigation identified a decrease in PKCδ activity in response to shear stress, and the overexpression of PKCδ attenuated the shear-mediated decrease in Thr495 phosphorylation and the increase in NO generation, and this led to increased eNOS uncoupling. PKCδ overexpression also attenuated Ser1177 phosphorylation through a posttranslational increase in catalase activity, mediated via a serine phosphorylation event, reducing shear-mediated increases in H₂O₂. Together, our data indicate that shear stress decreases PKCδ activity, altering the phosphorylation pattern catalase, leading to decreased catalase activity and increased H₂O₂ signaling, and this in turn leads to increases in phosphorylation of eNOS at Ser1177 and NO generation.

nitric oxide (NO) is an endothelium-derived relaxing factor synthesized by the oxidation of the guanidino nitrogen moiety of l-arginine following activation of nitric oxide synthase (NOS) (35). Three isoforms of NOS are known. Constitutive forms are present in endothelial cells and neurons, and a third inducible isoform is present in macrophages (23, 26, 42). After certain stimuli, such as flow and the receptor binding of specific vasodilators (endothelium-dependent vasodilators), NO is synthesized and released from the endothelial cell by the activation of endothelial NOS (eNOS) (31, 40). Once released from endothelial cells, NO diffuses into vascular smooth muscle cells and activates soluble guanylate cyclase (sGC), a heterodimer with α₁- and β₁-subunits, which catalyzes the production of cGMP from GMP. cGMP induces vascular smooth muscle relaxation through activation of a cGMP-dependent protein kinase (18, 21, 32).

Although initially considered to be a constitutively expressed enzyme, an increasingly large body of literature demonstrates that eNOS is dynamically regulated at the transcriptional, posttranscriptional, and posttranslational levels (3, 11, 16, 36). Laminar shear stress increases eNOS transcription, whereas stimuli, such as cell growth, increase eNOS expression by prolonging the half-life of the eNOS mRNA (41, 59). In addition, factors such as intracellular location, protein-protein interactions (e.g., calmodulin, caveolin, and heat shock protein 90), phosphorylation, as well as substrate and cofactor availability, can all dynamically regulate eNOS activity (3, 11, 14, 16, 36, 39, 45). eNOS can also be regulated by mechanical forces through complex and incompletely understood mechanisms. Fluid shear stress has been demonstrated to increase eNOS activity in vitro (3, 11, 16, 36), whereas in vivo, increases in flow associated with exercise are associated with increased eNOS mRNA and protein expression (19, 43). This appears to be regulated, in part, by potassium channels and serine phosphorylation (8, 33). It appears that eNOS activity is also dependent on its phosphorylation status. Two key phosphorylation sites on eNOS are located at Ser1177 and Thr495. It has been demonstrated that, at least in part, the phosphorylation at Ser1177 is mediated by Akt (13, 15). Akt activation is important both for agonist and shear stress activation of eNOS (10). The major kinase that phosphorylates eNOS at Thr495 is protein kinase C (PKC) (12, 28, 29). Furthermore, data suggest there is a reciprocal regulation between the Thr495 and Ser1177 sites with Ser1177 being an activator site and Thr495 being a negative regulatory site, its phosphorylation being associated with a decrease in the enzyme activity. However, the mechanism by which this reciprocal regulation occurs has not been elucidated. Thus, the overall goal of this study was to determine, in pulmonary arterial endothelial cells (PAEC), if acute changes in shear stress altered phosphorylation of eNOS and to elucidate the mechanism by which this occurs.

Overall our data indicate that acute increases in shear stress stimulate NO generation through the inhibition of PKCδ signaling. The decrease in PKCδ in turn leads to a decrease in phosphorylation of eNOS at Thr495 and an increase in eNOS phosphorylation at Ser1177. The increase in Ser1177 phosphorylation of eNOS appears to be due to an increase in H₂O₂ levels secondary to a decrease in catalase activity and the stimulation of Akt signaling.

MATERIALS AND METHODS

Cell culture.

Primary cultures of ovine fetal PAEC were isolated as described previously (56) and under the approval of the AUC of the Medical College of Georgia. 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 utilized 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 (48, 51, 56). 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 (25) with localized increases to 30–100 dyn/cm².

Western blotting.

Serum-starved PAEC (16 h) were sheared for 4 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 (Biorad). 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, anti-phospho Tyr311 PKCδ, anti-catalase, Akt, anti-phospho Ser473 Akt, anti-phospho Thr495 eNOS, anti-phospho-Ser1177 eNOS, and anti-eNOS (1:1,000; Cell Signaling Technology). The membrane was then washed with TBST three times for 10 min, incubated with the appropriate secondary antibody coupled to horseradish peroxidase, washed again with TBST as described above, and the protein bands visualized with ECL reagent (Pierce) using a Kodak 440CF image station.

Transient transfection of wild-type and dominant negative mutant-PKCδ.

Overexpression of wild-type or a dominant negative mutant-PKCδ was performed using a transient transfection technique as described previously (49). Parental vectors served as controls.

Adenoviral-mediated overexpression of catalase.

Catalase was overexpressed using an adenoviral construct as we have previously described (6). For these studies, an MOI of 200:1 was used. An adenoviral construct expressing green fluorescent protein (GFP) was used as a transduction control.

Preparation of recombinant human catalase.

A full-length cDNA of the human catalase was cloned into pET28b⁺ vector and overexpressed in the protease-deficient Escherichia coli strain BL21 (DE3) pLysS. For protein purification, cells were grown in 1 l of terrific broth. Expression was induced by the addition of 2 mM IPTG. Cells were then grown at 22°C for a further 18 h. Cells were harvested by centrifugation (20 min at 13,000 rpm at 4°C) and resuspended in lysis buffer, then broken by sonication, followed by freezing and thawing. Cell debris was then removed by ultracentrifugation, and the supernatant was brought to 25 mM imidazole and loaded into 2 × 5 ml HisTrap HP columns, using an AKTA FPLC Purifier System equipped with a 150-ml superloop with a flow of 0.05 ml/min. The bound protein was then isocratic eluted with elution buffer (20 mM Tris-OH, 300 mM NaCl, 300 mM imidazole, and 10% glycerol, pH 7.5).

Assay for catalase activity.

Catalase activity was measured as described (1). This method is based on the rate of degradation of H₂O₂ to form water and oxygen over time. For the cell studies, total protein extracts (40 μg) were diluted to 1,000 μl in 50 mM phosphate buffer (pH 7.0). One milliliter of 10 mM H₂O₂ solution was added, and the decomposition of the substrate was recorded by the decrease in absorbance at 240 nm over a 30-s period. For the in vitro assays, recombinant catalase (20 μg) was incubated with recombinant PKCδ (50 ng, Panvera cat. no. P-2348) and 20 μM of ATP in the presence of kinase buffer (150 mM NaCl, 4 mM MnCl₂, 10% glycerol, 1 mM DTT, 100 μM sodium orthovanadate, 50 mM HEPES, 20 μm ATP) (54) at 30°C for 20 min. As a control, denatured PKCδ (boiled for 2 min) was incubated with recombinant catalase and ATP at 30°C for 30 min. Catalase activity was expressed as degradation of H₂O₂·μg protein⁻¹·min⁻¹.

Detection of NO_x.

NO generated by PAEC in response to shear was measured using a NO-sensitive electrode with a 2-mm diameter tip (ISO-NOP sensor, WPI) connected to a NO meter (ISO-NO Mark II, WPI) as described previously (49).

Electron paramagnetic resonance spectroscopy and spin trapping.

To detect superoxide generation in intact cells, EPR measurements were performed as described previously (58). Following overnight serum starvation of the cells, 20 μl of spin-trap stock solution consisting of 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine·HCl (20 μM in DPBS CMH; Alexis Biochemicals, San Diego, CA), desferrioxamine (25 μM; Calbiochem, La Jolla, CA), diethyldithiocarbamate (5 μM; Alexis Biochemicals, Lausen, Switzerland), and DMSO (2 μl of 100% stock solution) was added to each well before shear stimulation. Adherent cells were trypsinized and pelleted at 500 g after a 45-min incubation at 37°C postshear to allow entrapment of superoxide by the spin trap. The cell pellet was washed and suspended in a final volume of 35 μl DPBS (including desferrioxamine and diethyldithiocarbamate), loaded into a 50-μl capillary tube, and analyzed with a MiniScope MS200 EPR (Magnettech, Berlin, Germany) at a microwave power of 40 mW, modulation amplitude of 3,000 mG, and modulation frequency of 100 kHz. EPR spectra were analyzed and measured for amplitude using ANALYSIS software (version 2.02; Magnettech).

Measurement of cellular H₂O₂ levels.

PAEC were sheared for 4 h, and the media was collected at 15-, 30-, 60-, and 240 min. A modified H₂DCFDA oxidation method was used to detect H₂O₂ levels (57). Briefly, 50 μl of media was incubated with 25 μM H₂DCFDA (Calbiochem) for 30 min in the dark with or without 100 U/ml catalase (Sigma, St. Louis, MO). Purified catalase was used to confirm that the oxidation of H₂DCFDA was H₂O₂ dependent. The samples were measured with excitation at 485 nm and emission at 530 nm in Fluoroskan Ascent FL (Thermo Electron, Waltham, MA).

Immunoprecipitation analysis to detect phosphocatalase.

For these experiments, PAEC were transiently transfected with either wild-type or a dominant negative mutant-PKCδ. After 24 h, the cells were exposed to shear stress (20 dyn/cm²) for 4 h. The cells were then lysed in ice-cold lysis buffer. For each immunoprecipitation, cell lysates were incubated with anti-catalase antibody for 2 h at 4°C and then with protein G Plus/Protein A agarose suspension (Calbiochem) for 1 h at 4°C. The immune complexes were washed three times with the lysis buffer and boiled in SDS-PAGE sample buffer for 5 min. Agarose beads were pelleted by centrifugation, and the protein supernatants were loaded and run on 4–20% polyacrylamide gels, followed by transfer of the proteins to nitrocellulose membranes. The membranes were blocked with 2% BSA in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 2 h at room temperature, incubated with anti-phosphoserine or anti-phospho-threonine antibody (Calbiochem) for 2 h at room temperature, washed three times with TBST (room temperature, 10 min), and then incubated with a horseradish peroxidase-conjugated secondary antibody (Pierce). The reactive bands were visualized with the SuperSignal West Femto maximum sensitivity substrate kit (Pierce) using a Kodak 440CF image station. The same blot was reprobed with anti-catalase antibody to normalize for the levels of catalase immunoprecipitated in each sample.

Statistical analysis.

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

RESULTS

Acute shear stress increases H₂O₂, but not superoxide, levels in PAEC exposed to shear stress.

To evaluate NO signaling in the absence of changes in protein expression, PAEC were exposed to shear stress (20 dyn/cm²) for 4 h. We have previously shown that after 4 h of this magnitude of shear stress, NO signaling is increased, but eNOS protein levels are not (5, 55). Initially, we evaluated the effect of this acute shear stress on ROS levels in PAEC. Using EPR, we found that shear stress did not increase either total (Fig. 1A) or NOS-derived superoxide levels (Fig. 1A), although as expected, NO levels increased in a NOS-dependent manner (Fig. 1B). However, our data indicate that H₂O₂ levels are significantly increased in shear-exposed PAEC (from ∼18 μM to 50 μM, Fig. 1D), suggesting that increases in H₂O₂ are independent of increases in superoxide. One of the major antioxidant enzyme systems responsible for metabolizing H₂O₂ is catalase. Thus, we determined if shear stress altered catalase expression and/or activity. Our data indicate that after 4 h of shear stress (20 dyn/cm²), there was no change in catalase protein levels (Fig. 2, A and B), but catalase activity was decreased (Fig. 2 C), suggesting that shear stress can alter catalase activity through a posttranslational mechanism.

Fig. 1.

Acute increases in shear stress does not alter nitric oxide synthase (NOS)-dependent superoxide generation but stimulates NO and H₂O₂ levels in pulmonary arterial endothelial cells (PAEC). PAEC were exposed or not to shear stress (20 dyn/cm², 4 h). Then, the levels of superoxide were determined by EPR (A). NO (B) and H₂O₂ (C and D) were also determined. To determine the μM of H₂O₂ produced by shear stress a standard curve was generated using 50, 25, 12.5, 6.2, 3.1, 1.5, and 0.75 μM of H₂O₂ (C). For the EPR and NO_x studies, PAEC were also examined in the presence or absence of the NOS inhibitor ETU (100 μM) to determine changes in NOS-derived superoxide and NO, respectively. To confirm the specificity of the H₂DCFDA oxidation for H₂O₂, duplicate wells were run in the presence of PEG-catalase (100 U/ml). Shear stress increases H₂O₂ levels independently of increases in either total or NOS-derived superoxide levels. Data are means ± SD; n = 6. *P < 0.05 vs. no shear, †P < 0.05 vs. shear no ETU.

Fig. 2.

Acute increases in shear stress decrease catalase activity but not protein levels in PAEC. PAEC were exposed or not to shear stress (20 dyn/cm², 4 h), and then whole cell extracts (20 μg) were subjected to Western blot analysis to determine changes in catalase protein levels (A and B) or catalase activity (C). A representative image is shown for the Western blot analysis (A). Densitomeric values are means ± SE normalized to β-actin (B). Although catalase protein levels are unchanged by acute shear stress, catalase activity is significantly reduced. Data are means ± SE; n = 3. *P < 0.05 vs. no shear.

Effect of H₂O₂ treatment on Akt activation and eNOS Ser1177 phosphorylation.

To determine if the increase in H₂O₂ observed after shear stress was able to alter cellular signaling, we exposed PAEC for 1 h to a bolus dose of 50 μM H₂O₂ (equivalent to that produced by cells exposed to shear stress for 4 h, Fig. 1D) and then analyzed the effect on both Akt activation as determined by changes in phospho-Ser473 levels (Fig. 3A) and phospho-Ser1177 eNOS (Fig. 3B). Our data indicate that H₂O₂ significantly increases phospho-Ser473 Akt levels while total Akt levels are unchanged (Fig. 3A). Similarly, H₂O₂ significantly increases phospho-Ser1177 eNOS levels with total eNOS levels again unchanged (Fig. 3B).

Fig. 3.

Modulating cellular H₂O₂ levels alters Akt activity and NO generation in PAEC. The exposure of PAEC to a bolus dose of H₂O₂ (50 μM, equivalent to that obtained with 4 h of shear stress) significantly increased Akt activity, as measured by phospho-Ser473- (A) and phospho-Ser1177-eNOS levels (B). Data are means ± SE; n = 4. *P < 0.05 vs. no H₂O₂. PAEC were transduced with adenoviruses expressing either GFP (as a control) or catalase. After 24 h, cells were exposed or not to shear stress (20 dyn/cm², 4 h). Whole cell extracts (20 μg) were then subjected to Western blot analysis to determine changes in catalase protein levels (C and D), catalase activity (E), phospho Akt levels (F and G), phospho-Ser1177- and phospho-Thr495-eNOS levels (H–J). Representative images are shown (C, F, H). Modest increases in catalase expression and activity (∼2.5- and 3-fold respectively, C–E) lead to a significant attenuation of the shear-induced increase in both phospho Akt (F and G) and phospho Ser1177 eNOS (H and I). The overexpression of catalase also significantly attenuates the shear-mediated increase in NO (K). Data are means ± SE; n = 4–6. *P < 0.05 vs. unsheared AdGFP; †P < 0.05 vs. AdGFP + shear.

Catalase overexpression limits shear-mediated increases in NO generation in PAEC.

Previous studies from Keaney et al. (52) have shown that H₂O₂ can stimulate eNOS activity though its ability to activate a pp60^Src/PI3 kinase/Akt signaling pathway resulting in eNOS phosphorylation at Ser1177. As our data indicated that H₂O₂ levels were increased by shear stress (Fig. 1D), we next investigated the effect of increasing H₂O₂ scavenging activity on the shear-mediated increases in NO signaling in PAEC. To accomplish this, we used an adenoviral system as we have previously described (6). Our data indicate that modest overexpression of catalase (∼2.5-fold, Fig. 3, C–E) reduced the shear-mediated increase in both Akt activation (Fig. 3, F and G) and Ser1177 phosphorylation of eNOS (Fig. 3, H and I) as well as NO generation (Fig. 3K) and attenuated the shear-mediated dephosphorylation of eNOS at Thr495 (Fig. 3, H and J).

Role of PKCδ in the NO signaling induced by shear stress in PAEC.

As our prior studies had indicated that decreased PKCδ signaling played a key role in increasing eNOS expression in response to chronic shear stress (50), we next investigated the potential role of PKCδ in regulating NO signaling in response to acute shear stress. To estimate PKCδ activity, we examined the levels of phospho Tyr311 PKCδ as we have described (50). We found that phospho Tyr311 PKCδ levels are significantly decreased by shear stress (Fig. 4, A and B). These data suggest that shear stress decreases PKCδ activity in PAEC. Based on these data, we next overexpressed PKCδ in PAEC, exposed the cells to shear stress, and evaluated the effect on eNOS phosphorylation and activity. Our data indicate that PKCδ overexpression (Fig. 4, C and D) did not change the ratio of total to phospho to total PKCδ (Fig. 4, E and F). However, phospho-PKCδ levels themselves were enhanced by PKCδ overexpression. Our data indicate that PKCδ overexpression prevented the shear-mediated decrease in eNOS phosphorylation at Thr495 and the shear-mediated increase in eNOS phosphorylation at Ser1177 (Fig. 4, G–I) and attenuated the increase in NO generation (Fig. 4J). In addition, NOS-dependent cellular superoxide levels were enhanced (Fig. 4K). Our data also indicate that the overexpression of catalase has no affect on the levels of either total or phospho-PKCδ in the presence or absence of shear (Fig. 5, A and B).

Fig. 4.

Acute increases in shear stress decreases PKCδ activity in PAEC. PAEC were exposed or not to shear stress (20 dyn/cm², 4 h), and then whole cell extracts (20 μg) were subjected to Western blot analysis using either a PKCδ antibody or a phospho-specific antibody recognizing Tyr311 (the activation site of PKCδ, A–F). A representative image is shown for the Western blot analysis (A, C, E). Densitomeric values are means ± SE normalized to total PKCδ. Shear stress significantly decreases phospho-Tyr311 levels indicating that PKCδ activity is diminished by shear stress. In addition, PAEC were transfected with an expression plasmid for PKCδ (pCMS-PKCδ) or the parental plasmid pCMS and then exposed or not to shear stress (20 dyn/cm², 4 h). Phopsho-Tyr311 (E), phospho-Ser1177-, and phospho-Thr495-eNOS levels (G–I), NO (J), and superoxide (K) levels were then determined. The overexpression of PKCδ increased NO production (J) and does not alter superoxide generation under basal conditions (K), whereas the overexpression of PKCδ significantly attenuates the shear-mediated increase in NO (J) and significantly increases NOS-derived superoxide (K). Data are means ± SE; n = 4–6. *P < 0.05 vs. unsheared; †P < 0.05 vs. pCMS + shear; ‡P < 0.05 vs. shear + PKCδ.

Fig. 5.

Effect of increased catalase expression on PKCδ protein and activity. PAEC were transduced with either AdGFP or Adcatalase. After 24 h, the cells were harvested, and Western blot analyses used to determine the effect on the levels of total and phospho-Tyr311 PKCδ were determined. The overexpression of catalase has no effect on either total or phospho-Tyr311 PKCδ levels in the presence or absence of shear stress (20 dyn/cm², 4 h). Data are means ± SE; n = 4. *P < 0.05 vs. no shear.

Effect of PKC inhibition on shear-mediated increases in NO signaling.

PAEC were transiently transfected with pIRES-DN PKCδ or the parental plasmid, pIRES. After 24 h, cell extracts were prepared, and Western blot analysis was performed to confirm expression of the mutant protein (Fig. 6A). We also determined the effect of the DN PKCδ on catalase expression and activity, H₂O₂ levels, NO generation, and Ser1177 eNOS levels after exposure to acute shear stress. Our data indicate that DN PKCδ overexpression has no effect on catalase expression (Fig. 6B), but there is a further significant reduction in catalase activity in response to shear (Fig. 6C), and this enhances the shear-mediated increase in H₂O₂ levels (Fig. 6D). Furthermore, DN PKCδ overexpression potentiates both the shear-mediated increase in phopsho-Ser1177 eNOS levels (Fig. 6E) and NO (Fig. 6F).

Fig. 6.

PKCδ inhibition decreases catalase activity and increases NO production in PAEC. PAEC were transfected with an expression plasmid for either Flag-DNPKCδ or the parental plasmid pIRES. After 24 h, cells were exposed or not to shear stress (20 dyn/cm², 4 h), and then whole cell extracts (20 μg) were subjected to Western blot analysis to determine levels of Flag-DNPKCδ (A), catalase protein (B), and Ser1177-eNOS (E). The cell lysates were also used to measure catalase activity (C). H₂O₂ levels (D) and NO generation (F) were also measured. Data are means ± SE; n = 3–6. *P < 0.05 vs. pIRES no shear; †P < 0.05 vs. shear + pIRES.

PKCδ stimulates catalase activity through a posttranslational mechanism.

To further explore the relationship between PKCδ and catalase, we evaluated the effect on catalase expression and activity when PKCδ was overexpressed. Our data indicate that PKCδ overexpression had no effect on catalase protein levels (Fig. 7, A and B) but significantly increased catalase activity (Fig. 7C) and blocked the shear-mediated increases in cellular H₂O₂ levels (Fig. 7D). In addition, we found that recombinant PKCδ alone can stimulate the activity of purified human catalase in vitro (Fig. 8).

Fig. 7.

PKCδ overexpression increases catalase activity in PAEC. PAEC were transfected with an expression plasmid for PKCδ (pCMS-PKCδ) or the parental plasmid pCMS. After 24 h, cells were exposed or not to shear stress (20 dyn/cm², 4 h), and then whole cell extracts (20 μg) were subjected to Western blot analysis to determine changes in catalase protein levels (A and B) and catalase activity (C). A representative image is shown for the Western blot analysis (A). Densitomeric values are means ± SE normalized to β-actin (B). H₂O₂ levels were also determined using H₂DCFDA oxidation (D). Data are means ± SE; n = 3–6. *P < 0.05 vs. pCMS no shear; †P < 0.05 vs. pCMS + shear.

Fig. 8.

PKCδ stimulates catalase activity in vitro. Recombinant human catalase (100 ng) was incubated with purified or heat-inactivated (boiled for 2 min) PKCδ (50 ng) for 20 min at 30°C, and then the catalase activity was determined. The presence of PKCδ significantly increases catalase activity, and this is attenuated when PKCδ is heat inactivated. Data are means ± SE; n = 4. *P < 0.05 vs. no PKCδ; †P < 0.05 vs. heat-inactivated PKCδ.

Effect of wild-type and dominant negative mutant-PKCδ overexpression on catalase phosphorylation.

Our data indicate that PKCδ stimulates catalase activity both in PAEC (Fig. 7C) and in vitro using recombinant protein (Fig. 8), suggesting that PKCδ can stimulate catalase activity posttranslationally through a direct phosphorylation event. PKCδ is a serine/threonine kinase. To further explore the effect of the PKCδ on catalase phosphorylation, we overexpressed wild-type or dominant negative mutant-PKCδ and then determined the effect on phospho-catalase serine and phospho-catalase threonine. Our data demonstrate an increase in phospho-catalase serine levels in PAEC overexpressing wild-type PKCδ, which was attenuated with shear (Fig. 9, A and B). However, the overexpression of wild-type PKCδ had no effect on phospho-catalase threonine (Fig. 9, A and C). Conversely, the dominant negative mutant PKCδ decreased phospho-catalase serine levels under basal conditions, and shear stress had no additive effect (Fig. 9, D–F). Again, the overexpression of dominant negative PKCδ had no effect on phospho-catalase threonine levels (Fig. 9, D–F).

Fig. 9.

Shear stress decreases phospho-serine but not phospho-threonine levels in catalase. Phospho-serine and phospho-threonine catalase levels were determined by immunoprecipitation (IP) using a specific antiserum raised against catalase in PAEC overexpressing or not PKCδ (A–C) or DN-PKCδ (D–F). Immunoprecipitated extracts were then separated on 4–20% SDS-polyacrylamide gradient gels, electrophoretically transferred to Hybond membranes, and analyzed using antisera against phospho-serine or phospho-threonine. Levels were normalized by reprobing with the antibody to catalase. Shear stress alone causes a decrease in phospho-serine catalase levels (A and B), but phospho-threonine catalase levels are not changed (A and C). An increase in phospho-serine catalase levels was observed in PAEC overexpressing wild-type PKCδ that is attenuated by shear (A and B). Dominant negative PKCδ decreased phospho-serine catalase levels (D and E) and again left phospho-threonine catalase levels unchanged (D and F). Data are means ± SE; n = 4. *P < 0.05 vs. vector no shear; †P < 0.05 vs. PKCδ no shear.

DISCUSSION

Laminar shear stress increases NO signaling, which exerts a beneficial effect on endothelial cell function: inhibition of platelet aggregation, low density lipoprotein uptake, adhesion molecule expression, and vascular smooth muscle cell proliferation. Shear stress stimulates production of NO from eNOS both in EC and in intact vessels. We and others have shown shear stress stimulates phosphorylation of eNOS at Ser1177 and that this stimulates enzyme activity (10, 13, 27). This Ser1177 phosphorylation in response to shear stress is mediated by PI3K and the downstream serine/threonine protein kinase, Akt (protein kinase B) (10, 13). Furthermore, studies have suggested that there is a reciprocal regulation between the Thr495 and Ser1177 sites with Ser1177 being an activator site and Thr495 being a negative regulatory site. However, the mechanism by which the reciprocal regulation between Thr495 and Ser1177 occurs is incompletely understood. Our data have identified PKCδ and catalase as being two previously unidentified signaling molecules that are important in regulating shear-mediated NO signaling in PAEC. Furthermore, the shear-mediated decrease in the activities we have observed could explain how the reciprocal regulation of eNOS phosphorylation at Thr495 and Ser1177 is regulated under conditions of increased shear stress.

The major kinase that phosphorylates eNOS at Thr495 is protein kinase C (PKC) (12, 28, 29). However, PKC is a multi-gene family with at least 12 members that can be classified by their activation criteria, and the distinct role of each PKC isoform in cell signaling has not been elucidated. There are three families of PKC: conventional (α, βI, βII, γ), which are Ca²⁺ and lipid activated, whereas the novel (δ, ε, η, θ) and atypical (ι, ζ, ν, μ), which are Ca²⁺ independent but activated by distinct lipid moieties. Although the majority of studies suggest that PKC signaling inhibits eNOS activity in EC (7, 29), there are exceptions to this. For example, we found that blockade of PKC activity did not prevent the NO-mediated inhibition of eNOS activity (46), whereas a recent study demonstrated the overexpression of PKCα in EC increased Ser1177 eNOS phosphorylation and increased NO production while inhibiting PKCα activity, either by siRNA transfection or by overexpression of a dominant negative mutant, and decreased FGF2-induced Ser1177 eNOS phosphorylation and NO production (38). Thus, the role of PKC in regulating eNOS activity and expression is complex. Like other PKC isoforms, PKCδ is activated by its translocation from cytosol to membrane. However, recent studies have also demonstrated that the activation of PKC isoforms can be associated with phosphorylation of certain tyrosine residue. PKCδ can be phosphorylated at Tyr311. The significance of this is controversial since studies have correlated this phosphorylation event with both increasing and decreasing PKC activity. It is also postulated that the phosphorylation at Tyr311 may be a necessary precursor for activation of kinase activity. However, we have recently shown that Tyr311 phosphorylation matches well with PKCδ activation in PAEC (50). Our data indicate that under conditions of acute shear stress, there is a decrease in phospho Tyr311 PKCδ levels corresponding to a decrease in PKCδ activity, and this correlates with enhanced NO signaling. This stands in contradiction to our recent study in which we found that PKCδ signaling is required to maintain basal eNOS expression through an Akt-dependent increase in NO signaling (50). In our prior study we found that pharmacological inhibition of PKCδ with either rottlerin or with the peptide, deltaV1–1, acutely attenuated NO production, and this was associated with a decrease in phosphorylation of eNOS at Ser1177. We also found that PKCδ inhibition blunted Akt activation as observed by a reduction in phosphorylated Akt at position Ser473. Thus, we concluded that PKCδ is involved in the activation of Akt. These two apparently contradictory findings can, however, be resolved. It is well established that the activation of Akt plays an important signaling role through the stimulation of eNOS phosphorylation at Ser117 (10, 13). Thus, it is likely in the basal state Akt is activated by PKCδ-mediated phosphorylation as we have previously shown (50). But under shear conditions, the decrease in PKCδ activity is replaced by H₂O₂-mediated activation of PI3 kinase/Akt to maintain Ser1177 phosphorylation of eNOS and enhanced NO signaling. See Fig. 10 for a more detailed scheme of how we propose that Akt is activated under basal and flow conditions to maintain NO signaling. It is also worth noting that we have recently shown that increasing ET-1 levels in PAEC through the transient overexpression of a preproET-1 cDNA, led to a decrease in eNOS expression mediated by increased activation of the STAT3 transcription factor and enhanced PKCδ signaling (47). As we have also previously shown that acute increases in blood flow leads to a decrease in NO signaling through an ET-1-mediated mechanism (34), it is interesting to speculate that ET-1 can reverse the flow-mediated decrease in PKCδ activity we have observed here. Thus, enhanced ET-1 signaling could prevent the increase in NO normally induced by increases in flow, by stimulating catalase activity, preventing H₂O₂ levels from rising, and so preventing eNOS activation via PI3 kinase/Akt/Ser1177 signaling. However, further in vivo studies will be required to test this possibility. These studies could be of high clinical significance as acute changes in pulmonary blood flow and the resulting increases in shear stress can occur during thoracic surgery, lung and/or heart transplantation, corrections of congenital heart defects, and exercise (13, 14, 15, 16).

Fig. 10.

Models of Akt activation and NO signaling in PAEC under basal and acute shear stress conditions. In the basal state (A), PKCδ is active, and this leads to Akt activation and subsequent phosphorylation of eNOS at Ser1177. This maintains basal NO generation. In addition, catalase is in a phosphorylated, “high-activity” state keeping H₂O₂ levels depressed and attenuating Akt activation through pp60^Src/PI3 kinase (PI3K). Under conditions of acute shear stress (B), PKCδ is inhibited, and catalase phosphorylation decreases, returning it to a “low-activity” state. This leads to a rise in H₂O₂ levels and the activation of Akt via pp60^Src/PI3K signaling. This replaces the loss of PKCδ activation of Akt and enhances Ser1177 phosphorylation and NO generation.

The other major finding in this study is the apparent shear-mediated increases in H₂O₂ levels occurring independently of increases in superoxide. The response of EC to shear stress with respect to superoxide generation is complex. Although flow has been shown to stimulate the generation of superoxide in intact isolated vessels (24) and in cultured EC (9), the source of ROS remains controversial. This may involve NADPH oxidase, xanthine oxidase, mitochondrial enzymes, or uncoupled NOS (2, 9, 27, 46, 48). There may also be differences in response based on the type of shear employed. For example, oscillatory shear stress of human umbilical endothelial vein endothelial cells increases NADPH oxidase activity through increased expression of p22^phox, gp91^phox, and Nox4 in endothelial cells (9). However, pulsatile shear stress downregulates these same subunits (17, 53). Similarly, we have previously shown that there is a developmental phenotype with respect to superoxide generation in PAEC exposed to laminar shear stress that corresponds to the presence or absence of uncoupled NOS (27).

Previous studies have shown that pp60^Src activation by H₂O₂ mediates the stimulation of eNOS activity in ECs in vitro (52), while acutely, the generation of H₂O₂ has been implicated as a mediator of flow-induced vasodilatation in vivo in coronary and cerebral vessels (4, 30, 37) as well as juvenile sheep (22, 27). Together, these studies show that H₂O₂ can play a positive role in regulating NO signaling. H₂O₂ is normally generated through the dismutation of superoxide by the action of superoxide dismutase. However, cellular levels of H₂O₂ can be increased in the face of constant superoxide generation if the levels of scavenging are attenuated. Indeed, we have previously shown that increases in shear stress in vivo can result in decreased catalase expression and activity (44). Furthermore, it has also been previously reported that laminar shear stress (10 dyn/cm², 16 h) decreases catalase activity as well as the activities of other phase 2 detoxifying enzymes (glutathione reductase, glutathione peroxidase, and glutathione S-transferase) in human umbilical vein ECs (20). In this study, we have identified a new mechanism by which flow can inhibit catalase activity through a decrease in PKCδ activity. Our data indicate that PKCδ can stimulate catalase activity posttranslationally through a direct serine phosphorylation event. An initial bioinformatics approach using the NetPhos 2.0 predictive software with multiple mammalian catalase sequences (rat, mouse, cow, human) to identify likely serine phosphorylation sites identifies multiple serine residues (S114, S120, S167, S248, and S254) within the catalase protein sequence with high predictive values for phosphorylation (P > 0.9). Thus, a mass spectroscopy approach will likely be required to identify the important phospho-serine residue(s) sensitive to PKCδ phosphorylation.

In conclusion, our data indicate that acute increases in shear stress stimulate NO generation through the inhibition of PKCδ signaling. This leads to a decrease in phosphorylation of eNOS at Thr495 and an increase in eNOS phosphorylation at Ser177. The increase in Ser1177 phosphorylation of eNOS appears to be due to an increase in H₂O₂ levels secondary to a decrease in catalase activity and the stimulation of Akt signaling. We speculate that therapies targeted at PKCδ could be used to enhance NO signaling in conditions of endothelial dysfunction.

GRANTS

This research was supported in part by National Institutes of Health Grants HL-60190, HL-67841, HL-72123, HL-70061, HL-084739, and R21-HD-057406 (all to S. M. Black) and by a grant from the Fondation Leducq (to S. M. Black). N. Sud was supported in part by a postdoctoral fellowship award from the American Heart Association Southwest Affiliates and by Grant K99-HL-097153. F. V. Fonseca was supported in part by National Institutes of Health Training Grant 5T32-HL-06699. This work was also supported in part by a Seed award (to S. Kumar) from the Cardiovascular Discovery Institute of the Medical College of Georgia.

DISCLOSURES

No conflicts of interest are declared by the author(s).