Mechanism of Endothelial Nitric Oxide Synthase Phosphorylation and Activation by Thrombin

Reagents

Thrombin from bovine plasma and EGTA were purchased from Sigma Chemical Co. N^G-nitro-l-arginine methyl ester, phorbol 12-myristrate 13-acetate (PMA), LY294002, A23187, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxy-methyl ester, GF109203X, and rottlerin were purchased from Calbiochem. YM-254890 was a gift from Astellas Pharma Inc. Antibodies that selectively recognize Ser¹¹⁷⁹-phosphorylated eNOS, Thr⁴⁹⁷-phosphorylated eNOS, Ser⁴⁷³-phosphorylated Akt, and total Akt were purchased from Cell Signaling Technology, and antibody to detect Tyr311-phosphorylated PKCδ was purchased from Biosource International. Total eNOS antibody was purchased from BD Transduction Laboratories. Total PKCα, PKCδ, and extracellular signal-regulated kinase 2 antibodies were purchased from Santa Cruz Biotechnology, Inc.

Cell Culture

Bovine aortic endothelial cells (BAECs) were purchased from BioWhittaker and cultured in DMEM containing 10% FBS, penicillin, and streptomycin. Cells from passage 4 to 12 were grown to ≈90% confluence and serum depleted for 48 hours before the experiments. Cultured human umbilical vein endothelial cells were a gift from Dr Yi Wu (Temple University School of Medicine). Primary vascular smooth muscle cells (VSMCs) from rat aorta were obtained by the explant methods and subcultured for the experiment as described previously.¹²

Immunoblotting

Cell lysates were subjected to SDS-PAGE gel electrophoresis and electrophoretically transferred to a nitrocellulose membrane as described previously.¹² The membranes were then exposed to primary antibodies overnight at 4°C. After incubation with the peroxidase-linked secondary antibody for 1 hour at room temperature, immunoreactive proteins were visualized by enhanced chemiluminescence reagent.¹³ Results were expressed as percentage increase in which the maximum response to thrombin (10 U/mL) is defined as 100%, because the basal signals are more varied depending on film exposure than the stimulated signals.

Adenovirus Infection

The generation and characterization of adenovirus encoding wild-type and dominant-negative mutants of PKCα and PKCδ were described in detail elsewhere.¹⁴ Adenovirus encoding wild-type and the Ser1179 mutant, S1179A, were kindly provided by Dr William Sessa.¹⁵ BAECs or VSMCs were infected with the adenovirus for 2 days as described previously before treatment with thrombin.¹⁶

Intracellular cGMP Accumulation

BAECs were incubated with agonists at 37°C for 20 minutes in the presence of 0.5 mmol/L of methylisobutylxanthine,¹⁷ and intracellular cGMP was determined using an enzyme immunoassay kit (Cayman Chemical) under the manufacturer’s instruction. Results were expressed as percentage increase in which the maximum response to thrombin (10 U/mL) is defined as 100%.

Statistical Analysis of Data

Results shown in the blots are representative of ≥3 independent experiments. Densitometry data and cGMP data were analyzed using 1-way ANOVA with a significance level of P<0.05 (results shown are the mean±SEM of data from ≥3 separate experiments).

Thrombin Induces Ser¹¹⁷⁹ Phosphorylation and Activation of eNOS

First, we examined whether or not thrombin phosphorylates eNOS at Ser¹¹⁷⁹, a catalytically positive regulatory site, in BAECs. As shown in Figure 1A, thrombin stimulated eNOS phosphorylation in a concentration-dependent manner. Figure 1B demonstrates that thrombin stimulates a significant increase in cGMP production that was completely inhibited by an eNOS inhibitor, N^G-nitro-l-arginine methyl ester. To examine the functional relevance of Ser¹¹⁷⁹ phosphorylation of eNOS by thrombin, cultured rat VSMCs with no detectable eNOS expression were infected with adenovirus encoding wild-type or S1179A eNOS, and thrombin-induced cGMP production was evaluated. Despite comparable expression and induction of equivalent basal cGMP production, thrombin was able to further stimulate cGMP production in VSMCs expressing wild-type eNOS but not in VSMCs expressing S1179A, suggesting a positive contribution of Ser1179 phosphorylation in eNOS activation by thrombin (Figure 1C).

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Figure 1. The effect of thrombin on eNOS phosphorylation and cGMP formation. BAECs were stimulated with the indicated concentrations of thrombin for 1 minute, and (A) phosphorylation of eNOS at Ser¹¹⁷⁹ was determined using immunoblot analysis with a phosphospecific antibody, or (B) cGMP was measured as described in the Methods section. BAECs were also pretreated with the eNOS inhibitor N^G-nitro-l-arginine methyl ester (100 μmol/L) for 30 minutes and stimulated with thrombin (10 U/mL) for 20 minutes. C, VSMCs infected with adenovirus encoding eNOS (WT), eNOS-S1179A (SA), or control vector (Vec) were stimulated with thrombin (10 U/mL) for 20 minutes, and cGMP concentrations were measured. Also, expression of eNOS in the infected VSMCs was evaluated. The data shown are mean±SEM (*P<0.05).

Thrombin-Induced Phosphorylation and Activation of eNOS Is Independent From the PI3-K/Akt Pathway

Because eNOS phosphorylation at Ser¹¹⁷⁹ and subsequent eNOS activation are mediated by PI3-K/Akt activation in response to several distinct eNOS activators, such as vascular endothelial growth factor, in cultured endothelial cells,⁶ we investigated whether thrombin activates Akt and whether the activation precedes eNOS phosphorylation. Activation of Akt was assessed by its phosphorylation at Ser⁴⁷³. As shown in Figure 2A and 2B, thrombin-induced eNOS phosphorylation could be observed as early as 0.5 minutes with maximum phosphorylation occurring at 1 minute. In contrast, we observed that thrombin induces the phosphorylation of Akt as early as 5 minutes (Figure 2A and 2B), which was much later than the thrombin-induced phosphorylation of eNOS (Figure 2C). These data indicate that Akt may not participate in the phosphorylation of eNOS by thrombin in BAECs.

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Figure 2. Time-dependent effect of thrombin on eNOS and Akt phosphorylation. BAECs were stimulated with 10 U/mL of thrombin for the indicated time points, and phosphorylation of (A) eNOS at Ser¹¹⁷⁹ and (B) Akt at Ser⁴⁷³ was determined using immunoblot analysis with phosphospecific antibodies. C, The densitometric analysis of the phosphorylation of eNOS and Akt. The data shown are mean±SEM (*P<0.05).

To further explore our interpretation that the PI3-K/Akt signaling pathway is not required for thrombin-induced eNOS phosphorylation, we pretreated the BAECs with LY294002, an inhibitor of PI3-K before exposure to thrombin. Figure 3A and 3B show that LY294002 did not inhibit thrombin-induced eNOS phosphorylation at Ser¹¹⁷⁹ but that thrombin-induced Akt phosphorylation was completely inhibited by the agent. In addition, LY294002 had no inhibitory effect on cGMP production stimulated by thrombin in BAECs (Figure 3B). These studies demonstrate that thrombin activation of the PI3-K/Akt pathway is not essential for thrombin-induced phosphorylation and activation of eNOS in BAECs.

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Figure 3. Effect of LY294002, a PI3-K inhibitor, on the thrombin-induced phosphorylation of eNOS and Akt. BAECs were pretreated with LY294002 (10 μmol/L) for 30 minutes and stimulated with 10 U/mL thrombin for the indicated time points. A, Phosphorylation of eNOS at Ser¹¹⁷⁹ and Akt at Ser⁴⁷³ was determined using immunoblot analysis with phosphospecific antibodies. B, Percent phosphorylation of eNOS at 1 minute is shown in the first panel by densitometric analysis, and the percent cGMP production for 20 minutes is shown in the second panel using an assay kit. The data shown are mean±SEM (*P<0.05).

Involvement of Intracellular Calcium in eNOS Phosphorylation and Activation

Thrombin is known to cause the intracellular release of Ca²⁺ stores and the influx of extracellular Ca²⁺ in endothelial cells.^18,19 This may stimulate eNOS activity through a Ca²⁺/calmodulin-dependent mechanism, whereas the possible contribution of intracellular Ca²⁺ to the phosphorylation of eNOS remains uncertain. We observed that A23187, a Ca²⁺ ionophore, stimulated eNOS phosphorylation at Ser¹¹⁷⁹ in BAECs (Figure 4A). In Figure 4B, we observed a comparable increase in the production of cGMP by thrombin and A23187. Moreover, we found a critical role for Ca²⁺ in the thrombin-induced phosphorylation of eNOS. Pretreatment with an intracellular Ca²⁺ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxy-methyl ester (Figure 4C), or an extracellular Ca²⁺ chelator, EGTA (Figure 4D), inhibits eNOS phosphorylation. We confirmed that the thrombin-induced production of cGMP was inhibited by these chelators as well (Figure 4E and 4F). These data suggest that both intracellular Ca²⁺ stores and influx of extracellular Ca²⁺ are required for Ser¹¹⁷⁹ phosphorylation and activation of eNOS by thrombin.

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Figure 4. Involvement of Ca²⁺ in thrombin-induced eNOS phosphorylation. BAECs were stimulated with (A) 10 μmol/L of A23187, a Ca²⁺ ionophore, for 1 minute, and eNOS phosphoprylation at Ser¹¹⁷⁹ was observed. B, A23187 was also used to stimulate cGMP production. BAECs were pretreated with 10 μmol/L 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxy-methyl ester, an intracellular Ca²⁺ chelator, for 30 minutes (C and E) or 5 mmol/L EGTA, an extra Ca²⁺ chelator, for 3 minutes (D and F) and stimulated with or without 10 U/mL thrombin for 1 minute (C and D) or for 20 minutes (E and F). eNOS phosphorylation at Ser¹¹⁷⁹ was observed by immunoblot analysis with a phosphospecific antibody (C and D), as well as cGMP production using an assay kit (E and F). The data shown are mean±SEM (*P<0.05 vs basal; †P<0.05 vs stimulated control).

The Role of PKCδ in the Thrombin-Induced Phosphorylation and Activation of eNOS

Thrombin activates several PKC isoforms, such as PKCα and PKCδ, in endothelial cells, and each isoform appears to mediate distinct downstream functions.¹¹ We observed that rottlerin, a PKCδ inhibitor, attenuates the phosphorylation of eNOS. Rottlerin, but not LY294002, also blocked the phosphorylation of eNOS by thrombin in human umbilical vein endothelial cells (Figure IA, available online at http://hyper.ahajournals.org). In contrast, nonsubtype selective PKC inhibitor GF10932X had only a small inhibitory effect on the phosphorylation of Ser¹¹⁷⁹ eNOS (Figure 5A). Thrombin-induced cGMP production was almost completely inhibited by rottlerin (Figure 5B), consistent with the importance of PKCδ in eNOS activation by thrombin. In contrast to A23187, a nonsubtype selective PKC activator, PMA, dephosphorylated eNOS at Ser¹¹⁷⁹ (Figure 5C). When both agonists are combined, A23187 seems to have the dominate effect on the Ser¹¹⁷⁹ phosphorylation of eNOS. In addition, PMA did not stimulate cGMP production in BAECs (Figure 5D). We also observed that thrombin rapidly induced phosphorylation of PKCδ Tyr³¹¹, the site linked to its activity²⁰ (Figure IB). To study the relationship between Ca²⁺ and PKCδ in eNOS phosphorylation, the effect of rottlerin on A23187-induced eNOS phosphorylation was examined. Rottlerin markedly inhibited A23187-induced eNOS phosphorylation in BAECs (Figure IC).

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Figure 5. The effect of PKC inhibitors on thrombin-induced phosphorylation of eNOS. BAECs were pretreated with 10 μmol/L rottlerin, a PKCδ inhibitor, or 2 μmol/L GF109203X, a general PKC inhibitor, for 30 minutes and then stimulated with 10 U/mL thrombin for 1 minute (A) or 20 minutes (B). A, Phosphorylation of eNOS at Ser¹¹⁷⁹ was observed using immunoblot analysis, and (B) cGMP was measured using an assay kit. The data shown are mean±SEM (*P<0.05 vs basal; †P<0.05 vs stimulated control). C, BAECs were stimulated with 10 μmol/L A23187, 1 μmol/L PMA, or combination of both for 1 minute. Phosphorylation of eNOS at Ser¹¹⁷⁹ was observed using immunoblot analysis. D, BAECs were stimulated with 1 μmol/L of PMA or 10 U/mL of thrombin for 20 minutes. cGMP was measured using an assay kit. The data shown are mean±SEM (*P<0.05 vs basal).

To complement these studies with rottlerin, which may have nonspecific effects, the role of PKCδ was further studied by infection of BAECs with adenovirus encoding dominant-negative (dn) mutants of PKCδ or PKCα. As shown in Figure 6A, infection of BAECs with dnPKCα adenovirus enhanced both basal- and thrombin-induced Ser¹¹⁷⁹ phosphorylation of eNOS, whereas infection of BAECs with dnPKCδ adenovirus inhibited thrombin-induced Ser¹¹⁷⁹ phosphorylation of eNOS. We also observe that dnPKCδ, but not dnPKCα, causes a significant reduction in cGMP production induced by thrombin in BAECs (Figure 6B). These findings suggest that PKC isoforms have distinct regulatory roles in eNOS phosphorylation and activation, and the positive regulatory role of PKCδ on Ser¹¹⁷⁹ is overridden if other negative regulatory PKCs, such as PKCα, are simultaneously activated by PMA in BAECs. Because reciprocal negative eNOS regulation through eNOS Thr⁴⁹⁷ phosphorylation has been reported,²¹ we have further examined phosphorylation of this site by thrombin in BAECs pretreated with or without PMA. Consistent with a recent report,²² thrombin also stimulated eNOS Thr⁴⁹⁷ phosphorylation. Pretreatment with PMA further enhanced Thr⁴⁹⁷ phosphorylation but partially inhibited Ser¹¹⁷⁹ phosphorylation induced by thrombin (Figure ID).

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Figure 6. The effects of dominant negative (dn) PKC and a G_q inhibitor on thrombin-induced phosphorylation and activation of eNOS. BAECs were infected with adenovirus encoding dn adenoviruses for PKCα or PKCδ (100 multiplicities of infection) for 48 hours and then stimulated with 10 U/mL of thrombin for 1 minute (A) or for 20 minutes (B). BAECs were pretreated with or without 10 μmol/L YM-254890 for 10 minutes and stimulated with or without 10 U/mL thrombin for 1 minute (C) or 20 minutes (D). eNOS phosphorylation at Ser¹¹⁷⁹ was observed by immunoblot analysis with a phosphospecific antibody (A and C). These data are representative of 3 separate experiments giving similar results. cGMP production was measured using an assay kit (B and D). The data shown are mean±SEM (*P<0.05 vs basal; †P<0.05 vs stimulated control).

The requirement of Ca²⁺ and a PKC isoform for thrombin-induced Ser¹¹⁷⁹ eNOS phosphorylation suggests that G_q may be required for the phosphorylation of eNOS at Ser¹¹⁷⁹. In fact, a selective G_q inhibitor, YM-254890,²³ inhibited thrombin-induced Ser¹¹⁷⁹ phosphorylation of eNOS (Figure 6C). YM-254890 also blocked thrombin-induced cGMP production in BAECs (Figure 6D). Taken together, these data indicate that, in addition to a previously known Ca²⁺/calmodulin-dependent mechanism, phosphorylation of Ser¹¹⁷⁹ eNOS is mediated through the activation of G_q and subsequent elevation of intracellular Ca²⁺ combined with the activation of PKCδ, which are indispensable for activation of eNOS by thrombin in endothelial cells (Figure 7).

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Figure 7. Proposed signal transduction cascade by which thrombin mediates eNOS activation. CaM indicates calmodulin.

Perspectives

Thrombin induces Ser¹¹⁷⁹ eNOS phosphorylation and activation through a Ca²⁺-dependent, PKCδ-sensitive, but PI3-K/Akt-independent pathway. Because dysfunction of endothelial NO production is one of the major predictors of cardiovascular events, these findings will contribute to a better understanding of the signaling mechanisms of thrombin in regulating the endothelial function. Therefore, further in vivo studies focusing on human pathophysiology in this area will contribute to the development of specific-acting drugs that can be used to treat and/or prevent endothelial dysfunctions.