Distinct Roles of Protease-Activated Receptors in Signal Transduction Regulation of Endothelial Nitric Oxide Synthase
Protease-activated receptors (PARs), such as PAR1 and PAR2, have been implicated in the regulation of endothelial NO production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase through the selective phosphorylation of a positive regulatory site, Ser1179, and a negative regulatory site, Thr497, in bovine aortic endothelial cells. A selective PAR1 ligand, TFLLR, stimulated the phosphorylation of endothelial NO synthase at Thr497. It had a minimal effect on Ser1179 phosphorylation. In contrast, a selective PAR2 ligand, SLIGRL, stimulated the phosphorylation of Ser1179 with no noticeable effect on Thr497. Thrombin has been shown to transactivate PAR2 through PAR1. Thus, thrombin, as well as a peptide mimicking the PAR1 tethered ligand, TRAP, stimulated phosphorylation of both sites. Also, thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A Gq inhibitor blocked thrombin- and SLIGRL-induced Ser1179 phosphorylation, whereas it enhanced thrombin-induced Thr497 phosphorylation. In contrast, a G12/13 inhibitor blocked thrombin- and TFLLR-induced Thr497 phosphorylation, whereas it enhanced the Ser1179 phosphorylation. Although a Rho-kinase inhibitor, Y27632, blocked the Thr497 phosphorylation, other inhibitors that targeted Rho-kinase failed to block TFLLR-induced Thr497 phosphorylation. These data suggest that PAR1 and PAR2 distinctly regulate endothelial NO synthase phosphorylation and activity through G12/13 and Gq, respectively, delineating the novel signaling pathways by which the proteases act on protease-activated receptors to potentially modulate endothelial functions.
Thrombin is a multifunctional serine protease that not only mediates the coagulation cascade but also has a wide variety of actions within the endothelium and vascular smooth muscle. The signal transduction and functions of thrombin through the protease-activated receptors (PARs) are strongly implicated in vascular physiology and pathophysiology.1,2 PARs represent a unique class of G protein–coupled receptors activated by proteolytic cleavage of their extracellular N-terminal domains. The new N terminus acts as a “tethered ligand” and activates the receptor itself. Four members of this family have been cloned, of which PAR1 and PAR3 are essentially thrombin receptors, whereas PAR2 is activated by trypsin or mast cell tryptase and PAR4 is activated by both thrombin and trypsin.1 However, transactivation of PAR2 by thrombin through PAR1 (the tethered ligand of PAR1 acts as a PAR2 ligand to activate PAR2 if both receptors coexist) has been demonstrated in cultured human umbilical vein endothelial cells (HUVECs).3 Activation of PARs stimulate a myriad arrays of signal transduction pathways, which include the activation of distinct heterotrimeric G proteins and tyrosine and serine/threonine kinases.1,2
Importantly, multiple PARs seem to regulate the release of NO via endothelial NO synthesis (eNOS).4–6 Although these data suggest a significant link between eNOS and PARs, detailed mechanisms by which PARs regulate eNOS activity have been insufficiently characterized. Recent studies revealed that eNOS needs to be specifically phosphorylated to exert its full activity.7 There are multiple phosphorylation sites on eNOS; however, the most is known about the functional consequences of phosphorylation of Ser1179 (bovine)/1177 (human) and Thr497 (bovine)/495 (human). When the Ser1179 is phosphorylated, NO production is increased 2- to 3-fold above basal level. In contrast, Thr497 is a negative regulatory site of eNOS associated with decreased enzymatic activity. Several eNOS kinases, such as Akt, which phosphorylates Ser1179, or protein kinase C (PKC), which phosphorylates Thr497, have been identified.7
Here, we have hypothesized that PAR1 and PAR2 distinctly regulate the 2 phosphorylation sites on eNOS and NO production. We found several lines of evidence indicating the reciprocal regulation of the phosphorylation of these sites on eNOS by the PARs in bovine aortic endothelial cells (BAECs) through different G protein–dependent signal transduction pathways. These data demonstrate novel signal transduction characteristics of PAR1 and PAR2 in endothelial cells representing diverse physiological and pathophysiological roles of PARs in regulating endothelial functions.
Materials and Methods
Thrombin from bovine plasma was purchased from Sigma. A PAR1 selective agonist, TFLLR-NH2, a PAR4 selective agonist, AY-NH2, and a peptide mimicking the PAR1 tethered ligand, TRAP, were purchased from Tocris. A PAR2-selective agonist, SLIGRL-NH2, was purchased from Bachem. A Gq inhibitor, YM-254890, was a gift from Astellas Pharma Inc. Pertusis toxin, phorbol 12-myristrate 13-acetate, and Rho-kinase (ROCK) inhibitors, Y27632 and H-1152, and a PKC inhibitor, GF109203X, were purchased from Calbiochem. Antibodies against Ser1179-phosphorylated eNOS and Thr497-phosphorylated eNOS were purchased from Cell Signaling Technology. Total eNOS antibody was purchased from BD Transduction Laboratories. Antibody for Thr853-phosphorylated myosin phosphatase target subunit-1 (MYPT-1) was purchased from Upstate Biotechnology, and antibody against total myosin phosphatase target subunit-1 was purchased from Covance.
BAECs were purchased from Cambrex and grown in DMEM containing 10% FBS, penicillin, and streptomycin, as described previously.8 BAECs were subcultured using Versene (0.53 mmol/L of EDTA in PBS) to avoid trypsin exposure. HUVECs were a gift from Dr Yi Wu (Temple University School of Medicine).9 Cells from passage 4 to 12 were grown to ≈90% confluence and serum starved for 48 hours before the experiments.
Generation and characterization of replication-deficient adenoviruses encoding a dominant-negative mutant of Rho, myc-N19-RhoA, and myc-p115RGS were described in detail elsewhere.10,11 Adenovirus construct encoding GRK2/βARK-ct was generated by Dr Andrea Eckhart from Gene Transfer Vector Core (Thomas Jefferson University). An adenovirus vector encoding GFP was used as a control for other adenovirus vectors, which also encode GFP independent of their respective inserts. The adenovirus titers were determined by Adeno-X Rapid Titer kit (BD Biosciences). Subconfluent BAECs were infected with adenovirus for 2 days, as described previously.8
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 (Pierce).
Intracellular cGMP Assay
BAECs were incubated with agonists at 37°C for 20 minutes in the presence of 0.5 mmol/L of methylisobutylxanthine, and intracellular cGMP was determined by an enzyme immunoassay kit (Cayman Chemical).8
Intracellular Ca2+ Measurements
Intracellular Ca2+ was measured as described previously using fura 2 as an indicator.13 In brief, BAECs subcultured on cover slips were loaded with 3 μmol/L of fura 2-acetoxymethyl ester. The fura 2 fluorescence was measured at a frequency of 1 Hz, and intracellular Ca2+ values were then obtained as described.13
The data shown are either from 3 or 4 independent experiments (the total number is given in the figure legends) and presented as means±SEMs. The data were analyzed using 1-way ANOVA followed by a posthoc modified t test or a 2-way ANOVA. P<0.05 was considered significant.
eNOS Phosphorylation by Thrombin in BAECs
In cultured BAECs, we have examined whether thrombin stimulates phosphorylation of eNOS at Ser1179, a catalytically positive regulatory site, and at Thr497, a catalytically negative regulatory site.7 As shown in Figure 1A, thrombin (10 U/mL) stimulated eNOS phosphorylation at Ser1179, which began at 30 seconds, peaked at 1 minute, and declined thereafter. Also, thrombin stimulated eNOS phosphorylation at Thr497 from 30 seconds to 2 minutes. Figure 1B shows the concentration dependence of thrombin-induced phosphorylation of eNOS in BAECs. eNOS phosphorylation at Ser1179 was detectable with 1 U/mL of thrombin, and the maximum phosphorylation was observed at a concentration of 10 U/mL. In contrast, thrombin-stimulated phosphorylation at Thr497 occurred at an even lower concentration of thrombin (0.1 U/mL) in BAECs. Thus, these results suggest the possibility that thrombin mediates the phosphorylation of each eNOS site through distinct PARs or G proteins in BAECs.
eNOS Phosphorylation and Activation by Selective PAR Agonists in BAECs
To examine the participation of PARs in the regulation of NO production, we stimulated BAECs with a selective PAR1 agonist, TFLLR, or a selective PAR2 agonist, SLIGRL, and evaluated eNOS phosphorylation. As shown in Figure 2A, TFLLR stimulated eNOS phosphorylation at Thr497 at 1 to 2 minutes, whereas it had no effect on the Ser1179 phosphorylation. On the other hand, SLIGRL stimulated eNOS phosphorylation at Ser1179 but not Thr497 at 1 to 2 minutes. In line with a theory that thrombin activates PAR2 via the intermolecular transactivation of PAR2,3 a peptide mimicking the PAR1-tethered ligand, TRAP, stimulated both phosphorylation sites in a concentration-dependent manner (10 to 100 μmol/L). However, a PAR4 agonist, AY-NH2 (≤200 μmol/L), had no effect on either phosphorylation site (data not shown). The distinct responses by the PAR1 and PAR2 agonists were also confirmed in HUVECs and bovine pulmonary artery endothelial cells (data not shown). These results suggest that PAR1 and PAR2 have distinct roles in regulating eNOS phosphorylation at least in these types of endothelial cells.
To assess eNOS activation by the PAR agonists, intracellular cGMP production was measured as a marker of NO production after stimulation of BAECs by thrombin or PAR agonists. Although thrombin stimulated intracellular cGMP production (basal 3.55±0.36 pmol per well versus thrombin 17.50±2.53 pmol per well), a PAR1 agonist, TFLLR, at concentrations from 50 to 200 μmol/L did not stimulate cGMP production (Figure 2B). In contrast, a PAR2 agonist, SLIGRL, stimulated intracellular cGMP production in a concentration-dependent manner from 2 to 50 μmol/L (Figure 2C). Also, AY-NH2, a PAR4 agonist, did not increase in cGMP production (data not shown). We confirmed that PAR2 stimulated cGMP through NO production, because NG-nitro-l-arginine methyl ester treatment completely blocked PAR2-induced cGMP production (data not shown). We have shown previously that NG-nitro-l-arginine methyl ester inhibited cGMP production by thrombin.9 These data indicate a preferential role of PAR2 in eNOS activation in BAECs.
PAR Activation of Gq Is Required for Phosphorylation of eNOS at Ser1179 but not Thr497
PARs are known to couple to multiple G proteins, including Gq, Gi, and G12/13.1 Thus, the difference in eNOS phosphorylation profile by PARs may be because of distinct G proteins coupling to PAR1 and PAR2 in endothelial cells. We have confirmed our recent observation that a selective Gq inhibitor, YM-254890,14 markedly inhibited thrombin-induced eNOS Ser1179 phosphorylation,9 whereas it enhanced the thrombin-induced phosphorylation of eNOS at Thr497 (Figure 3A). The maximum inhibition was observed with 100 to 1000 nmol/L (Figure 3A and Figure S1A, please see http://hyper.ahajournals.org). However, neither pertussis toxin (100 ng/mL; 24 hours), a Gi inhibitor, nor infection of adenovirus (100 multiplicities of infection; 48 hours) encoding GRK2-ct, a Gβγ inhibitor, blocked thrombin-induced eNOS phosphorylation at Ser1179 or Thr497 (data not shown). YM-254890 also markedly inhibited PAR2 (SLIGRL)-induced eNOS Ser1179 phosphorylation, whereas it had no inhibitory effect on PAR1 (TFLLR)-induced eNOS Thr497 phosphorylation (Figure 3B and 3C). YM-254890 also inhibited PAR2-induced cGMP production (Figure S1B), as it did in BAECs stimulated with thrombin.9
Gq activation by PARs leads to phospholipase C–dependent rapid and transient intracellular Ca2+ elevation.1 We have also demonstrated that thrombin-induced eNOS Ser1179 phosphorylation requires intracellular Ca2+ elevation.9 To test whether Gq coupling of PARs is distinct in BAECs, the effects of the PAR agonists on intracellular Ca2+ concentrations were examined. Both TFLLR and SLIGRL significantly elevated intracellular Ca2+ concentrations, which were completely inhibited by 1 μmol/L of YM-254890 (data not shown). These data suggest that Gq coupling and subsequent intracellular Ca2+ elevation are required but not sufficient enough for eNOS Ser1179 phosphorylation, as well as its activation.
G12/13 and a Y27632-Sensitive Kinase but not ROCK Are Involved in eNOS Phosphorylation at Thr497 by PAR1
PAR1 has been shown to couple to G12/13 leading to its downstream Rho and ROCK activation in endothelial cells.15 As shown in Figure 4, a specific G12/13 inhibitor, p115RGS, markedly inhibited thrombin- or TFLLR (PAR1)-induced eNOS Thr497 phosphorylation. It also inhibited thrombin- or TFLLR-induced ROCK activation, as judged by phosphorylation of a ROCK substrate, myosin phosphatase target subunit-1, at Thr853. Inhibition of G12/13 leads to stimulation of Ser1179 phosphorylation by TFLLR or thrombin. p115RGS by itself had no obvious effect on either site of eNOS phosphorylation (Figure S2A). A ROCK inhibitor, Y27632, markedly inhibited thrombin- or PAR1 (TFLLR)-induced eNOS Thr497 phosphorylation, whereas this inhibitor minimally affected eNOS Ser1179 phosphorylation stimulated by thrombin (Figure 5A) or the PAR2 agonist (data not shown). Y27632 also inhibited TFLLR (PAR1)-induced eNOS Thr497 phosphorylation (Figure 5B). However, Y27632 did not modulate cGMP production induced by thrombin in BAECs (Figure S2B).
The specificity of Y27632 as a ROCK inhibitor has been questioned.16 H-1152, which has a better selectivity for ROCK than Y27632,17 inhibited TFLLR-induced myosin phosphatase target subunit-1 Thr853 phosphorylation but not eNOS Thr497 phosphorylation (Figure S3A). Also, dominant-negative mutant of Rho markedly inhibited TFLLR-induced MYPT Thr853 phosphorylation but not eNOS Thr497 phosphorylation (Figure S3B). In addition, PKC did not contribute to the PAR1-induced eNOS Thr497 phosphorylation, because it was insensitive to the PKC inhibitor GF109203X, whereas this inhibitor blocked the phorbol ester–induced eNOS Thr497 phosphorylation in BAECs (Figure S3C). Taken together, these data suggest that PAR1-induced phosphorylation of eNOS at Thr497 requires G12/13 and subsequent activation of a Y27632-sensitive eNOS Thr497 kinase that is distinct from ROCK or PKC.
The major findings of the present study are as follows: (1) stimulation of PAR2 in BAECs results in eNOS Ser1179 phosphorylation and subsequent NO production; (2) whereas stimulation of PAR1 solely stimulates eNOS Thr497 phosphorylation and does not lead to NO production; and (3) the downstream mechanisms used involve Gq for Ser1179 phosphorylation by PAR2 and G12/13 and a previously unidentified Y27632-sensitive eNOS kinase for Thr497 phosphorylation by PAR1 (Figure 6). These data suggest distinct physiological and pathophysiological roles of PAR1 and PAR2 in regulating eNOS activity, representing novel mechanisms of PAR signal transduction in endothelial cells.
In line with a theory of PAR2 transactivation by PAR1 proposed in HUVECs,3 our data suggest that both PAR1 and PAR2 mediate eNOS regulation by thrombin in BAECs. It is likely that thrombin at a low concentration induces Thr497 phosphorylation through its high-affinity receptor, PAR1, whereas at a higher concentration, thrombin further transactivates PAR2 through the PAR1-tethered ligand leading to Ser1179 phosphorylation and subsequent eNOS activation. This is further supported by the findings with a PAR1-tethered peptide used in the present study. Although a marked difference of G protein–coupling affinity of PAR1 on stimulation with thrombin or an agonistic peptide (the peptide prefers Gq coupling more than G12/13) has been demonstrated,15 it appears not to be applicable to our findings. This is because no difference was observed between thrombin- or TFLLR-induced elevation of intracellular Ca2+ (Gq-dependent signal) or stimulation of MYPT phosphorylation (G12/13-dependent signal) in BAECs.
We have observed that the Gq inhibitor YM-254890 enhanced thrombin-induced eNOS phosphorylation at Thr497. This is most likely because of competition of PAR1 coupling between Gq and G12/13, which leads to the enhanced G12/13 signal transduction on inhibition of Gq. The presence of such competition is further supported by the enhanced Ser1179 phosphorylation by TFLLR or thrombin when G12/13 activities were inhibited with p115RGS. Thus, G12/13 inhibition might unmask the PAR1 coupling to eNOS Ser1179 phosphorylation via Gq, as illustrated in Figure 6. However, it remains unclear why the phenomenon was not observed on TFLLR stimulation with the Gq inhibitor. It is possible that TFLLR binding to the PAR1 may not be sufficient enough to change the competition, which favors G12/13 activation on inhibition of Gq. In addition, TFLLR may not fully mimic the conformational change of the receptor induced by thrombin, which causes cleavage of the receptor.
In the present study, we found that Gq is critical in PAR2-induced eNOS Ser1179 phosphorylation and subsequent enzymatic activation, as was shown recently in BAECs stimulated with thrombin or angiotensin II.8,9 Although thrombin-induced NO production has been shown to be markedly inhibited by Ca2+ chelators,9 the Gq-mediated Ca2+/calmodulin–dependent eNOS activation mechanism alone may be insufficient to stimulate NO production in BAECs. In this regard, we have demonstrated previously that thrombin-induced eNOS Ser1179 phosphorylation is mediated through a non-Akt kinase acting downstream of Ca2+ and that the Ser1179 phosphorylation is functionally indispensable for NO production stimulated by thrombin.9
We have demonstrated the specificity of the Gq inhibitor, YM-254890, at the concentrations of 1 to 10 μmol/L in COS7 cells and vascular smooth muscle cells.13,14 Recently, the specificity was also verified in BAECs pretreated for 30 minutes with 30 nmol/L of YM-254890. This concentration appeared to be sufficient to inhibit intracellular Ca2+ elevation by bradykinin. It also inhibited NO production induced by thrombin but not by ionomycin in BAECs.18 However, the concentrations >50 nmol/L were required for the inhibition of eNOS Ser1179 phosphorylation by thrombin (Figure S1A) or extracellular signal regulated kinase phosphorylation by angiotensin II,14 which is most likely because of the shorter treatment time of 10 minutes.
Thors et al19 proposed that Ser1179 phosphorylation of eNOS stimulated by thrombin is mediated through the Ca2+-dependent activation of an eNOS Ser1179 kinase, AMPK, by using a nonselective AMPK inhibitor in HUVECs. However, adenovirus transduction of dnAMPK did not prevent thrombin-induced eNOS Ser1179 phosphorylation in HUVECs.20 In addition, the species differences in eNOS regulation by PARs18 and the involvement of thrombomodulin in thrombin-induced eNOS phosphorylation have been reported.21 Therefore, further investigation is needed to identify the Ser1179 kinase, as well as its exact upstream signal transduction used by PAR2.
Little was known about the regulation of eNOS Thr497 by G protein–coupled receptors. We found that a Rho-kinase/ROCK inhibitor, Y27632, inhibited PAR1-induced eNOS Thr497 phosphorylation. Rho has been reported to inhibit NO production in arteries22 and to inhibit eNOS activation through inhibition of Akt in endothelial cells.23 In this regard, a recent study proposed ROCK as the eNOS Thr497 kinase activated by thrombin.24 Although this study demonstrated that ROCK was able to phosphorylate eNOS in vitro, only Y27632 was used to block the phosphorylation in vivo. Our data using a dominant-negative mutant of Rho and a more selective ROCK inhibitor rather suggest the presence of a Y27632-sensitive novel eNOS Thr497 kinase distinct from the Rho/ROCK.
A negative regulatory role of the eNOS Thr497 phosphorylation has been reported25; however, we could not observe enhancement of cGMP production on inhibition of the phosphorylation with Y27632 in the present study. A mutational experiment of eNOS Thr497 to mimic constitutive phosphorylation (Asp495 in human eNOS) with in vitro measurement of the enzymatic activity suggests that eNOS Thr497 phosphorylation reduces Ca2+ sensitivity of the enzyme.25 However, expression and stimulation of an eNOS Thr497 phosphorylation mutant to mimic dephosphorylation (Ala497) expressed in COS7 cells did not enhance NO production over that of the wild type, whereas a mutation in Asp497 inhibited NO production.26 Taken together with our Ca2+ stimulation data by PAR agonists, it is likely that eNOS phosphorylation at Thr497 blocks eNOS activity against Ca2+/calmodulin but is insufficient to block the activity with a concurrent phosphorylation of Ser1179.
Although we have observed similar regulation of eNOS by PARs in BAECs and HUVECs in the present study, the expression ratio of PARs may be different in endothelial cells from distinct species and/or vascular beds, as may be that of G proteins as well. Moreover, expression of PARs in endothelial cells is under the regulation of distinct extracellular conditions, such as inflammation.27 PAR1 mRNA was upregulated in rat aorta associated with angiotensin II–induced hypertension.28 However, exact roles of PARs in vascular tonus regulation still remain unclear. Therefore, additional experiments in various in vivo settings will be necessary to better generalize our findings in certain vascular pathophysiology, such as in hypertension.
Selective manipulation of Gq or G12/13 in vascular smooth muscle cells revealed the importance of these G proteins in the etiology of high blood pressure.29 The close link between endothelial G13 and PAR1 has been demonstrated.30 Moreover, positive regulation of eNOS expression by G12 has been reported.31 Additional detailed research specifically on eNOS phosphorylation regulation by PARs will shed light on critical mechanisms by which multiple G protein–coupled receptors expressed in the endothelium potentially regulate endothelial dysfunction associated with cardiovascular diseases.
We thank Kyoko Hinoki for her technical assistance.
Sources of Funding
This work was supported by National Institutes of Health grant HL076770 (to S.E.), by American Heart Association Established Investigator Award 0740042N (to S.E.), and by W.W. Smith Charitable Trust grant H0605 (to S.E.).
- Received October 15, 2008.
- Revision received October 23, 2008.
- Accepted November 13, 2008.
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