Angiotensin II type 2 receptor-stimulated activation of plasma prekallikrein and bradykinin release: role of SHP-1

Abstract

ANG II type 2 receptors (AT₂R) elicit cardioprotective effects in part by stimulating the release of kinins; however, the mechanism(s) responsible have not been fully explored. We demonstrated previously that overexpression of AT₂R increased expression of prolylcarboxypeptidase (PRCP; a plasma prekallikrein activator) and release of bradykinin by mouse coronary artery endothelial cells (ECs). In the present study we hypothesized that the AT₂R-stimulated increase in PRCP is mediated by the tyrosine phosphatase SHP-1, which in turn activates the PRCP-dependent prekallikrein-kallikrein pathway and releases bradykinin. We found that activation of AT₂R using the specific agonist CGP42112A increased SHP-1 activity in ECs, which was blocked by the AT₂R antagonist PD123319. Activation of AT₂R also enhanced conversion of plasma prekallikrein to kallikrein, and this effect was blunted by a small interfering RNA (siRNA) to SHP-1 and abolished by the tyrosine phosphatase inhibitor sodium orthovanadate. Treating cells with a siRNA to PRCP also blunted AT₂R-stimulated prekallikrein activation and bradykinin release. Furthermore, blocking plasma kallikrein with soybean trypsin inhibitor (SBTI) abolished AT₂R-stimulated bradykinin release. These findings support our hypothesis that stimulation of AT₂R activates a PRCP-dependent plasma prekallikrein pathway, releasing bradykinin. Activation of SHP-1 may also play an important role in AT₂R-induced PRCP activation.

angiotensin II (ANG II) is classically considered a vasoconstrictor and pro-growth molecule acting via the ANG II type 1 receptor (AT₁R). Although the role of AT₂R remains controversial, they are generally thought to be mutually antagonistic, with AT₂R opposing the actions of AT₁R (6, 14, 21). Some of the beneficial effects of AT₂R are mediated by bradykinin/nitric oxide (NO) (5, 10), and overexpression of AT₂R in the vasculature reportedly stimulated the kinin system, causing dilatation (31). Likewise cardiac AT₂R overexpression attenuated fibrosis induced by ANG II or myocardial infarction, apparently acting via a kinin/NO-dependent mechanism (3, 7). We have demonstrated that the cardioprotective effects of AT₁R antagonists are mediated in part by activation of AT₂R and kinins, since they were diminished in AT₂R or kinin receptors knock out mice and kinin-deficient rats or by blocking the AT₂R or kinin receptors (11, 34–36). However, the precise mechanism(s) by which AT₂R mediates kinin release is not yet known.

Prolylcarboxypeptidase (PRCP) is a serine carboxypeptidase that cleaves a variety of COOH-terminal amino acids adjacent to proline and has been linked to hypertension and preeclampsia (1, 32) as well as obesity (23). Dysfunction of PRCP is associated with adverse cardiovascular events such as hypertension and inflammation (18). In recent years PRCP-dependent plasma prekallikrein activation on endothelial cells (ECs) has received much attention. When the complex of high-molecular-weight kininogen (HMWK) and plasma prekallikrein binds to the endothelial membrane, PRCP converts plasma prekallikrein to kallikrein, which then cleaves HMWK, generating bradykinin, which leads to NO and prostacyclin formation as well as vasodilatation, probably by activating constitutive kinin B₂ and inducible B₁ receptors (15, 24, 25). Overexpression of PRCP in cultured Chinese hamster ovary cells and ECs enhanced plasma prekallikrein activation and release of kinins and NO, and these effects can be blocked by a small interfering RNA (siRNA) to PRCP (18, 26, 37). These data confirm the role of PRCP-dependent plasma prekallikrein activation in bradykinin release. It is suggested that PRCP constitutively regulates bradykinin production, thereby maintaining the integrity of the endothelium (20). Because PRCP activates prekallikrein to kallikrein, leading to formation of bradykinin and NO, it might be considered a negative regulator of the pressor actions of the renin-angiotensin system (12) as well as a means of maintaining stores of NO (23).

The role of PRCP in AT₂R-mediated bradykinin release is not fully understood. According to one study, the AT₁R antagonist losartan increased PRCP expression in hypertensive rats (22). Recently we demonstrated that overexpression of AT₂R in ECs increased expression of PRCP and release of bradykinin, whereas stimulating AT₂R with the agonist CGP42112A increased bradykinin release further (38). However, the signaling and molecular mechanism(s) by which AT₂R activates PRCP have not been explored to our knowledge. SHP-1, a Src homology region 2 domain-containing protein tyrosine phosphatase-1, has been implicated in the AT₂R intracellular pathway, which negatively interacts with AT₁R signaling (2, 27). We hypothesized that activation of AT₂R enhances tyrosine phosphatase SHP-1 activity, which stimulates PRCP and thereby increases conversion of plasma prekallikrein to kallikrein, liberating bradykinin. To test this possibility, we conducted studies in mouse coronary artery ECs transfected with the AT₂R gene and examined 1) the effect of AT₂R activation on SHP-1 activity, 2) whether inhibition of SHP-1 or protein tyrosine phosphatase diminishes or blocks AT₂R-induced plasma prekallikrein activation, 3) the effect of PRCP inhibition on AT₂R-stimulated plasma prekallikrein activation and bradykinin release, and 4) the effect of blocking plasma kallikrein with soybean trypsin inhibitor (SBTI) on AT₂R-stimulated bradykinin release.

MATERIALS AND METHODS

EC cultures.

Coronary artery ECs were isolated from 10-week-old male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) as described previously (30) and identified as ECs by uptake of acetylated low-density lipoprotein labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate and immunostaining with an antibody to von Willebrand factor (38). The study was approved by the Henry Ford Health System Institutional Animal Care and Use Committee (IACUC) in accord with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. ECs were maintained in medium 199-F-12 (1:1), 1% penicillin-streptomycin (Invitrogen), 100 μg/ml EC growth supplement (Sigma), and 10% FBS (HyClone). All experiments were conducted with cells between passages 3 and 5.

AT₂R gene transfer to ECs.

An adenoviral vector containing genomic AT₂R DNA and a green fluorescent protein gene driven by a cytomegalovirus promoter was used to induce overexpression of AT₂R in ECs (8). Cells were transfected with Ad-AT₂R at a multiplicity of infection of 80 for 24 h.

Immunoprecipitation and SHP-1 activity.

SHP-1 activity was determined by immunoprecipitation of SHP-1 followed by a colorimetric assay using a tyrosine phosphatase assay kit from Promega. Cells seeded in a 100-mm tissue culture dish were incubated overnight with medium 199-F-12 (1:1) containing 0.5% FBS, then washed with PBS and incubated for 1 h in serum free medium containing 20 nM HMWK. Prekallikrein (PK; 20 nM) was then added, and the mixture incubated for 1 h in the absence or presence of the AT₂R antagonist PD123319 (100 μM). Cells were then treated with or without (as control) the AT₂R agonist CGP42112A (0.1 μM) for 2 h. After the incubation, cells were solubilized for 30 min at 4°C in lysis buffer containing 25 mM Tris·HCl (pH 7.2), 2 mM EDTA, 0.4% Triton X-100, and a cocktail of protease inhibitors. After centrifugation at 16,000 g for 10 min at 4°C, the supernatant (200 μg proteins) was incubated with anti-SHP-1 antibodies (BD Transduction Laboratories) overnight at 4°C and then precipitated with protein A/G PLUS-agarose (Santa Cruz) according to the manufacturer's protocol. After extensive washing, the immunocomplexes were resuspended in 42 μl phosphatase buffer containing (in mM) 25 Tris·HCl (pH 7.2) and 2 EDTA, and phosphatase activity was measured using a tyrosine phosphatase assay kit as instructed by Promega. After 40 min incubation at 37°C, the reaction was terminated by adding molybdate dye, and color induced by free phosphate released from the phosphotyrosine peptide was measured in an enzyme-linked immunosorbent assay reader at 620 nm.

Small interfering RNA.

The SHP-1 and PRCP siRNAs were purchased from Santa Cruz, and the control scrambled siRNA was purchased from Qiagen. The siRNA was transfected into ECs in 6-well plates using 7.5-μl lipofectamine 2000 (Invitrogen). It was diluted in 250 μl Opti-MEM I, and the mixture was incubated for 5 min at room temperature. During the incubation period, 15 μl siRNA (10 μM) was mixed with 250 μl Opti-MEM I and incubated for 25 min at room temperature to allow a complex to form, after which 500 μl of the siRNA-lipofectamine mixture was added to each well.

Measurement of plasma prekallikrein activation on ECs.

For kallikrein activity, we followed the methods established by Ngo et al. (18) and Shariat-Madar et al. (24). Cells seeded in 96-well plates were washed three times with HEPES-NaHCO₃ buffer containing 137 mM NaCl, 3 mM KCl, 10 μM CaCl₂, 1 mM MgCl₂, 12 mM NaHCO₃, 5.5 mM dextrose, 0.1% gelatin, and 14.7 mM HEPES (pH 7.1). Human HMWK (20 nM; Research Enzyme Laboratory, South Bend, IN) was then added and incubated for 1 h at 37°C. Afterward, cells were washed and incubated with 20 nM human prekallikrein (PK) (28 PEU/mg; Research Enzyme Laboratory) in the absence or presence of 1) the AT₂R antagonist PD123319 (100 μM), 2) SBTI (1 μM), 3) the tyrosine phosphatase inhibitor sodium orthovanadate (100 μM), or 4) the AT₁R blocker valsartan (1 μM) for 1 h at 37°C. Then either the AT₂R agonist CGP42112A (0.1 μM) or ANG II (0.1 μM) was added and incubated for 2 h. Finally, cells were washed and 0.8 mM H-D-Pro-Phe-Arg-p-nitroanilide (S2302, a chemical substrate for plasma kallikrein; Dia-Pharma, Franklin, OH) was added in the same buffer and substrate hydrolysis was performed for 1 h at 37°C. The amount of kallikrein formed was determined by the rate of paranitroanilide liberation from S2302 at 405 nm absorbance compared with hydrolysis of S2302 by known amounts of human plasma kallikrein (0.1–10 nM; Research Enzyme Laboratory).

Measurement of bradykinin.

Generation of bradykinin was assessed by measuring the amount of bradykinin released into the incubation medium by ECs using an enzyme immunoassay kit as described previously (38). Cells were incubated overnight in 6-well plates with medium 199-F-12 (1:1) containing 0.5% FBS, then washed with PBS and incubated for 1 h in serum free medium containing 2 nM HMWK. Prekallikrein (PK; 2 nM) and 10 μM captopril were then added, and the mixture incubated for 1 h in the absence or presence of the AT₂R antagonist PD123319 (100 μM), SBTI (1 μM), or the tyrosine phosphatase inhibitor sodium orthovanadate (100 μM). The AT₂R agonist CGP42112A (0.1 μM) was then added, and the mixture incubated for 2 h. Finally, 1 ml of the medium was collected in a test tube containing the peptidase inhibitor cocktail and bradykinin measured as described previously (38). Bradykinin concentration was calculated according to a calibration curve, normalized to total protein, and expressed as picograms per microgram protein.

Statistical analysis.

Results are expressed as means ± SE. Student's two-sample t-test was used to compare differences between two groups. Bonferroni's adjustment was applied in multiple comparisons. A difference was considered significant if the adjusted P value was less than 0.05.

RESULTS

Effects of AT₂R activation on SHP-1 activity.

We first examined the effect of AT₂R activation on tyrosine phosphatase SHP-1 activity and found that activating AT₂R using its agonist CGP42112A significantly increased SHP-1 activity, and this effect was blocked by the AT₂R antagonist PD123319 (Fig. 1). We then examined the effect of AT₂R activation on prekallikrein activation by measuring kallikrein activity. Kallikrein activity is assayed by measuring liberation of paranitroanilide from S2302, a synthetic substrate for plasma kallikrein. We found that kallikrein activity was undetectable in the absence of HMWK and/or prekallikrein in AT₂R-transfected EC cells; however, when HMWK and prekallikrein were added, stimulating AT₂R with CGP42112A significantly increased conversion of plasma prekallikrein to kallikrein compared with AT₂R-transfected cells. This effect was diminished by the AT₂R antagonist PD123319 (Fig. 2). These data confirm that activating AT₂R increased plasma prekallikrein activation, and this effect was AT₂R specific.

Fig. 1.

Effect of ANG II type 2 receptor (AT₂R) stimulation with the agonist CGP42112A (CGP) on SHP-1 activity. AT₂R-transfected cells were activated with CGP (0.1 μM) in the presence or absence of the AT₂R antagonist PD123319 (PD; 100 μM; n = 4 to 5).

Fig. 2.

Effect of AT₂R stimulation on plasma kallikrein activity. AT₂R-transfected cells were incubated with HEPES-NaHCO₃ buffer in the presence or absence of 1) high-molecular-weight kininogen (HMWK) and/or plasma prekallikrein (PK), 2) the AT₂R agonist CGP42112A (CGP, 0.1 μM), or 3) the AT₂R antagonist PD123319 (PD; 100 μM; n = 12–15).

Effect of blocking SHP-1 or protein tyrosine phosphatase on AT₂R-stimulated prekallikrein activation.

We next studied whether knockdown of SHP-1 with a siRNA or inhibition of tyrosine phosphatase with sodium orthovanadate (vanadate) blocks AT₂R-mediated prekallikrein activation. We found that the SHP-1 siRNA reduced SHP-1 protein expression by 75% compared with the control, a scrambled siRNA (Fig. 3A); moreover, the increase in plasma kallikrein activity stimulated by the AT₂R agonist CGP42112A was significantly diminished by the SHP-1 siRNA compared with the scrambled control (Fig. 3B). In addition, the tyrosine phosphatase inhibitor sodium orthovanadate abolished both AT₂R-stimulated plasma kallikrein activity (Fig. 4A) and the AT₂R-stimulated increase in bradykinin release (Fig. 4B). These data suggest that SHP-1 could be an important upstream component in the AT₂R intracellular cascade and that tyrosine phosphatase activity could be a critical regulator of AT₂R-stimulated prekallikrein activation and bradykinin release.

Fig. 3.

Effect of SHP-1 blockade on AT₂R-stimulated plasma kallikrein activity. AT₂R-transfected cells were treated with a scrambled siRNA (Sc-siRNA) or SHP-1 small interfering RNA (SHP-1-siRNA). A: representative Western blot showing SHP-1 protein expression (top) and quantitative data (bottom). B: cells activated with the AT₂R agonist CGP42112A (CGP; 0.1 μM; n = 12–30).

Fig. 4.

Effect of tyrosine phosphatase inhibition on AT₂R-stimulated plasma kallikrein activity and bradykinin release. AT₂R-transfected cells were activated with the AT₂R agonist CGP42112A (CGP; 0.1 μM) in the presence or absence of the tyrosine phosphatase inhibitor sodium orthovanadate (vanadate; 100 μM). A: kallikrein activity (n = 23). B: bradykinin release (n = 6–10).

Effect of PRCP blockade on AT₂R-stimulated plasma prekallikrein activation.

We reported previously that overexpression of AT₂R increased PRCP mRNA and protein expression and blocking PRCP with a siRNA reduced basal and AT₂R-stimulated release of bradykinin, suggesting that PRCP plays an important role in AT₂R-mediated bradykinin release (38). We now tested whether the effect of PRCP on AT₂R-stimulated bradykinin release is mediated via activation of plasma prekallikrein. Because a specific PRCP inhibitor is not commercially available, we again used a PRCP siRNA to determine the effect of PRCP blockade on kallikrein activity and bradykinin release in AT₂R-transfected cells stimulated with the AT₂R agonist CGP42112A. We found that PRCP siRNA prevented CGP42112A-stimulated prekallikrein activation (Fig. 5A) and bradykinin release (Fig. 5B), which were not observed with the scrambled siRNA controls. These data confirm that AT₂R-stimulated prekallikrein activation is PRCP- dependent and contributes to AT₂R-stimulated bradykinin release.

Fig. 5.

Effect of prolylcarboxypeptidase (PRCP) blockade on AT₂R-stimulated plasma kallikrein activity and bradykinin release. AT₂R-transfected cells were treated with a Sc-siRNA or PRCP-siRNA and activated with the AT₂R agonist CGP42112A (CGP; 0.1 μM). A: kallikrein activity (n = 10–18). B: bradykinin release (n = 5).

Effect of plasma kallikrein inhibition on AT₂R-stimulated bradykinin release.

SBTI reportedly inhibits plasma kallikrein (16). To confirm the role of plasma kallikrein in AT₂R-stimulated bradykinin release, we blocked plasma kallikrein with SBTI and found that this abolished both endogenous kallikrein activity and AT₂R-stimulated prekallikrein activation or formed plasma kallikrein activity (Fig. 6A). Likewise bradykinin release by AT₂R-transfected cells or stimulated by the AT₂R agonist was also abolished by SBTI (Fig. 6B), confirming that the increased plasma kallikrein activity is responsible for AT₂R-stimulated bradykinin release.

Fig. 6.

Effect of soybean trypsin inhibitor (SBTI) on AT₂R-stimulated plasma kallikrein activity and bradykinin release. AT₂R-transfected cells were activated with the AT₂R agonist CGP42112A (CGP; 0.1 μM) in the presence or absence of SBTI. A: kallikrein activity (n = 10–18). B: bradykinin release (n = 5–11).

DISCUSSION

We found that activation of AT₂R in mouse coronary artery ECs increased both tyrosine phosphatase SHP-1 activity and plasma prekallikrein activation. Knockdown of SHP-1 or inhibition of tyrosine phosphatase reduced or prevented AT₂R-induced plasma prekallikrein activation. Blocking the plasma prekallikrein activator PRCP using a siRNA diminished AT₂R-stimulated plasma kallikrein activity and bradykinin release. We believe these results provide the first evidence that activation of AT₂R increases plasma kallikrein activity, which may involve SHP-1-dependent activation of PRCP. In turn PRCP transforms plasma prekallikrein to kallikrein, thereby liberating bradykinin from HMWK.

We and others have demonstrated a cardioprotective role of AT₂R, partially due to stimulation of kinins (5, 10, 11, 34–36). However, the mechanism by which AT₂R increases kinins is not fully understood. Tsutsumi et al. (31) reported that activation of AT₂R decreased cellular pH associated with increased kininogenase activity in the mouse aorta (31), suggesting the existence of an acid-optimal kininogenase or kininogenase-like acidic protease that is responsible for release of kinins from the vasculature. Interestingly, PRCP has an optimal acid pH (19) and has been described as a novel plasma prekallikrein activator. Shariat-Madar et al. (25) reported that when plasma prekallikrein binds to HMWK on ECs, PRCP rapidly converts prekallikrein to kallikrein, which cleaves HMWK and liberates bradykinin. We recently reported that overexpression of AT₂R in mouse coronary artery ECs increased PRCP mRNA and protein expression as well as bradykinin release (38). In the present study we further demonstrated that stimulation of AT₂R increased plasma kallikrein activity, whereas in the absence of HMWK and/or prekallikrein such kallikrein activity was undetectable. Pretreatment with SBTI completely abolished both kallikrein activity and bradykinin release. However, the SBTI data must be interpreted with caution, since this compound is not a specific inhibitor for plasma kallikrein. Importantly, blockade of PRCP using a siRNA blunted AT₂R-stimulated plasma prekallikrein activation and bradykinin release. Taken together, our data suggest that PRCP-dependent plasma prekallikrein activation is at least partially responsible for AT₂R-stimulated kinin release. We confirmed that activation of plasma prekallikrein by the AT₂R agonist CGP42112A or ANG II in the presence of the AT₁R blocker valsartan is AT₂R-specific, since it was blocked by the AT₂R antagonist PD123319 (data not shown).

The precise signaling and molecular mechanism(s) by which AT₂R activates PRCP has not been explored to our knowledge. AT₂R reportedly enhanced activity of protein tyrosine phosphatase SHP-1 (a tyrosine phosphatase containing Src homology 2 domains) in various cells and tissues, including rat fetal vascular smooth muscle cells (VSMC), rat neurons, and fetal rat tissues (2, 4, 9, 29, 33). Sohn et al. (28) reported that treating human umbilical vein ECs with ANG II for 15 min markedly increased SHP-1 activity, whereas Min et al. (13) reported that ANG II significantly increased SHP-1 activity in vascular smooth muscle cells, reaching a maximum at 4 h. Because we found that AT₂R activated plasma prekallikrein and increased bradykinin within 2 h, we wanted to know whether SHP-1 activity was altered at the same time. We observed that AT₂R stimulation activated SHP-1 in mouse coronary artery ECs, and this effect was blocked by the AT₂R antagonist PD123319, confirming that the role of AT₂R in activating protein tyrosine phosphatases. Activation of SHP-1 has been known to negatively regulate cell proliferation and growth via dephosphorylation of critical molecules involved in proliferative pathways (2, 4, 17), which may contribute to the antihypertrophic and antigrowth effects of AT₂R. However, we know of no study addressing whether AT₂R-induced activation of PRCP and/or kinin release involves SHP-1 or any other tyrosine phosphatases. Using a siRNA to knock down SHP-1, we found that SHP-1 siRNA partly but significantly reduced the AT₂R-induced increase in plasma kallikrein activity. Moreover, inhibition of tyrosine phosphatase with sodium orthovanadate abolished AT₂R-stimulated plasma prekallikrein activation and bradykinin release. Because plasma prekallikrein activation is an indirect measure of PRCP activity (18, 25) and PRCP is known to have several tyrosine phosphorylation sites, our data suggest that AT₂R-stimulated PRCP-dependent plasma prekallikrein activation is mediated at least in part via SHP-1.

In summary, we have shown that activation of AT₂R by a specific agonist increases plasma prekallikrein activation and bradykinin release from mouse coronary artery ECs. These effects were blocked by the AT₂R antagonist PD123319 or knockdown of PRCP. Furthermore, knockdown of SHP-1 or inhibition of the tyrosine phosphatase significantly reduced PRCP activity as indicated by decreased plasma prekallikrein activation. Therefore, we conclude that AT₂R-stimulated bradykinin release is mediated in part by a PRCP-dependent plasma prekallikrein activation pathway in mouse coronary artery ECs, while SHP-1 may play an important role in AT₂R-stimulated PRCP activation. (The schematic flow chart of the possible signaling mechanism in ECs is shown in Fig. 7.)

Fig. 7.

Schematic diagram of the pathways involved in AT₂R-stimulated bradykinin release in endothelial cells. Stimulation of AT₂R with an AT₂ agonist or ANG II in the presence of an AT₁R blocker (ARB) activates the protein tyrosine phosphatase SHP-1, which may positively regulate activation of PRCP; in turn, PRCP activates PK to plasma kallikrein, which liberates bradykinin from HMWK. Arrows (↑) indicate an increase in factors.

Perspectives

A large body of evidence has suggested that AT₂R expressed in the vasculature may have vasodilator properties via release of kinins and NO, counteracting AT₁R and leading to cardiovascular protection. However, the precise mechanism(s) by which AT₂R stimulates kinins and NO is not fully understood. The present study demonstrates the signaling cascade involved in AT₂R-mediated bradykinin release in ECs, which could help to clarify the mechanisms responsible for the cardioprotective role of AT₂R. Because AT₂R stimulation could be a potential therapeutic option in the treatment of cardiovascular disease, a study of this signaling cascade is of great clinical relevance.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-28982, PPG Project II (to X.-P. Yang), and Henry Ford Hospital Institutional Funds (to X.-P. Yang).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: L.Z., O.A.C., and X.-P.Y. conception and design of research; L.Z., J.X., and L.W. performed experiments; L.Z., J.X., L.W., J.J.Y., and X.-P.Y. analyzed data; L.Z., O.A.C., P.H., N.-E.R., C.S., and X.-P.Y. interpreted results of experiments; L.Z., J.X., L.W., and X.-P.Y. prepared figures; L.Z. drafted manuscript; L.Z., O.A.C., P.H., N.-E.R., C.S., and X.-P.Y. edited and revised manuscript; X.-P.Y. approved final version of manuscript.