Angiotensin-Converting Enzyme Is Involved in Outside-In Signaling in Endothelial Cells

From the Institut für Kardiovaskuläre Physiologie (K.K, R.P.B., R.B., I.F.), and Institut für Biochemie II (W.M.-E.), Klinikum der J.W.Goethe-Universität, Frankfurt am Main, Germany.

　 　

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Not all of the cardiovascular effects of angiotensin-converting enzyme (ACE) inhibitors can be attributed to changes in angiotensin II and bradykinin levels. Because the cytoplasmic tail of ACE is phosphorylated, we determined whether ACE inhibitors affect the phosphorylation of ACE and whether ACE possesses the characteristics of a signal transduction molecule. The ACE inhibitors ramiprilat and perindoprilat, and the substrate bradykinin (but not angiotensin I), enhanced the activity of ACE-associated CK2 and the phosphorylation of ACE Ser¹²⁷⁰ in cultured endothelial cells. Mitogen-activated protein kinase kinase 7 and c-Jun N-terminal kinase (JNK) coprecipitated with ACE, and stimulation of endothelial cells with ACE inhibitors increased the activity of ACE-associated JNK and elicited the accumulation of phosphorylated c-Jun in the nucleus. Ramiprilat was however unable to activate JNK or to stimulate the nuclear accumulation of c-Jun in endothelial cells expressing a S1270A ACE mutant or in ACE-deficient cells. Because the ACE inhibitor–induced increase in ACE expression has been linked to the formation of c-Jun homodimers, we investigated whether ACE signaling via JNK contributes to this response in vitro and in vivo. Prolonged ramiprilat treatment increased ACE expression in primary cultures of human endothelial cells and in vivo (mouse lung), a response that was prevented by pretreatment with the JNK inhibitor SP600125. Thus, ACE is involved in outside-in signaling in endothelial cells and "ACE signaling" may be an important cellular mechanism contributing to the beneficial effects of ACE inhibitors.

Key Words: angiotensin-converting enzyme c-Jun N-terminal kinase  CK2  bradykinin  angiotensin I

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The angiotensin-converting enzyme (ACE) is an ectoenzyme that catalyzes the conversion of angiotensin I to the vasoconstrictor angiotensin II as well as the degradation of the potent vasodilator bradykinin (see review¹). Two distinct forms of ACE are expressed in humans, a somatic form that is particularly abundant on the endothelial surface of the lungs, and a smaller isoenzyme that is found exclusively in testis. The activity of somatic ACE is thought to play a crucial role in blood pressure regulation and in processes involved in vascular remodeling, effects best highlighted by the fact that the in vivo gene transfer of ACE into the uninjured rat carotid artery results in the development of vascular hypertrophy independent of systemic factors and hemodynamic effects. The inhibition of ACE activity is reported to improve endothelial function² and to stimulate vascular remodeling,³ as well as attenuate the progression of arteriosclerosis⁴ and the occurrence of cardiovascular events in humans.^4–6 The deleterious effects of ACE on the cardiovascular system were initially thought to be a consequence of the formation of angiotensin II, which initiates a cascade of events involving increased free radical production and vascular smooth muscle cell proliferation. However, as bradykinin is much more readily hydrolyzed by ACE than angiotensin I,^1,7 the hydrolysis of bradykinin may also contribute to this phenomenon. Indeed, the cardioprotective effects of the ACE inhibitor ramipril are diminished in mice lacking the B₂ kinin receptor gene.⁸ Over the last 10 years however, there have been a number of reports showing that ACE inhibitors can amplify responses to bradykinin in continuously perfused systems in which the accumulation of the peptide cannot be assumed to occur fast enough to account for the effects observed.^9,10 Moreover, ACE inhibitors also potentiate the effects of ACE-resistant analogues of bradykinin,^9,11 prevent the sequestration of the B₂ receptor via caveolae,¹⁰ and reactivate the desensitized B₂ kinin receptor.^10,12 As ACE inhibitors do not directly bind to the B₂ receptor,¹³ we and others have proposed that some form of cross-talk may occur between ACE and the B₂ kinin receptor.^10,13 Because we recently demonstrated that the cytoplasmic tail of ACE is phosphorylated in endothelial cells,¹⁴ the aim of the present study was to determine whether ACE is involved in endothelial cell signaling and whether or not the binding of either an ACE inhibitor or an ACE substrate to the enzyme can affect such signaling processes. For such an outside-in signaling to take place, we also hypothesized that the cytoplasmic tail of ACE should be able to bind to signal molecules and/or adaptor proteins that initiate a chain of discrete signaling events.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
Human umbilical vein endothelial cells or porcine aortic endothelial cells were isolated and cultured as described.¹⁵ The human somatic ACE cDNA used¹⁶ was provided by François Alhenc-Gelas (Hôpital Lariboisière, Paris, France) and the point mutants were generated using a PCR-based mutagenesis kit (Stratagene). The mutation of Ser¹²⁵³, Ser¹²⁶³, and Ser¹²⁷⁰ to alanine was verified by sequencing. Porcine aortic endothelial cells were stably transfected with ACE, S1253A, S1263A, or S1270A using electroporation as described.¹⁴

Immunoprecipitation and Immunoblotting
Cells or lung homogenates were lysed in Nonidet lysis buffer, left on ice for 10 minutes, and centrifuged at 10 000g for 10 minutes. Either ACE or JNK was immunoprecipitated from whole cell lysates with a mixture of protein A/G Sepharose or anti-mouse IgG–covered Dynabeads M-450 (DYNAL) as described.¹⁷ Proteins in the immunoprecipitate were heated with SDS-PAGE sample buffer and separated by SDS-PAGE. Immunoprecipitated proteins were detected using their respective antibodies. In some experiments, endothelial cells were labeled with ³²P for 12 hours, ACE was then immunoprecipitated, and phosphorylation determined by autoradiography as described.¹⁴

The ACE monoclonal antibody (clone 9B9) used for immunoprecipitation was from Chemicon International, and the monoclonal antibody used for Western blotting was provided by Peter Bünning (Aventis, Frankfurt, Germany). The anti-mitogen–activated protein kinase kinase 7 (MKK7) and anti-JNK 1 antibodies were from Santa Cruz Biotechnology, the antibodies recognizing phosphorylated (Ser⁶³) c-Jun and the GST-c-Jun were from Cell Signaling. The monoclonal antibody against the -subunit of protein kinase CK2 as well as the specific CK2 substrate were from Calbiochem-Novabiochem. The phosphospecific Ser¹²⁷⁰ ACE antibody was generated from the peptide sequence HGPQFGpSEVELR (position 1263 to 1275 in human somatic ACE protein) by Eurogentec.

In Vitro Kinase Assays
ACE-associated CK2 activity was determined using ACE immunoprecipitated from endothelial cells as described.¹⁴ To assess JNK activity, JNK was immunoprecipitated as described above and in vitro kinase activity measured using 2 µg GST-c-Jun as substrate, as described.¹⁷ In some experiments, JNK activity was measured in ACE immunoprecipitates. The reactions were stopped and the products were resolved by SDS-PAGE (12%). The incorporation of ³²P was visualized by autoradiography and quantified by scanning densitometry.

Immunofluorescence
Endothelial cells were grown on glass coverslips and stimulated as described. After fixation with formaldehyde (2% in phosphate buffered saline) and washing with glycine (2% in PBS) and PBS, cells were permeabilized with Triton X-100 (0.2% v/v). After overnight incubation with the phospho-Jun antibody, followed by a fluorescein-conjugated anti-rabbit IgG (Dako Diagnostika GmbH) for 1 hour, preparations were mounted with ProLong Antifade kit (Molecular Probes) and viewed using a confocal microscope.

Animals
To study the acute effects of ACE inhibition, male mice (C57 black 6, 6 weeks, Charles River, Sulzfeld, Germany) were anesthetized (isoflurane 1.5%) and ramipril (250 µg/mouse dissolved in HEPES-modified Tyrode’s solution) or solvent were administered intraperitoneally (i.p., 100 µL bolus). After 15 minutes, animals were euthanized by a transverse cut through the large abdominal vessels and the lungs perfused rapidly with cold phosphate-buffered saline and snap-frozen.

For prolonged administration, ramipril (5 mg/kg per day) was given with the drinking water for 5 days. The JNK inhibitor SP600125 (Tocris, Bristol, UK) was dissolved in polyethylene glycol (PEG400) as described¹⁸ and applied daily as subcutaneous injection (30 mg/kg per day, 40% in water, 100 µL) for 7 days (ie, beginning 2 days before ACE inhibitor treatment). Experiments conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23) and were approved by the local government (II25.3-19c20/15-F28/02).

Statistical Analysis
Data are expressed as mean±SEM and statistical evaluation was performed using Student’s t test for unpaired data or one-way analysis of variance (ANOVA) followed by a Bonferroni t test where appropriate. Values of P<0.05 were considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of Ramiprilat on the Phosphorylation of ACE in Endothelial Cells
To assess the effects of ramiprilat on the phosphorylation of ACE, the enzyme was immunoprecipitated from ³²P-labeled primary cultures of human umbilical vein endothelial cells (A), which also express the angiotensin AT₁ receptor and the B₂ kinin receptor, or porcine aortic endothelial cells overexpressing human somatic ACE (data not shown) but lacking both G protein–coupled receptors. In both cell types, the ACE inhibitor (100 nmol/L) transiently increased ACE phosphorylation with a maximal effect (a 1.3±0.1-fold increase in ³²P incorporation versus control, n=9, P<0.05) evident after 2 minutes. Phosphorylation then decreased over the next 5 minutes (A).

fig.ommitted Figure 1. ACE phosphorylation on Ser1270 is enhanced by ramiprilat. A, Autoradiograph and Western blot showing the effect of solvent (CTL) and ramiprilat (100 nmol/L, 2 to 7 minutes) on the phosphorylation of ACE immunoprecipitated from 32P-labeled human umbilical vein endothelial cells. Similar results were obtained in 3 additional experiments. B, Autoradiograph and Western blot showing the effect of solvent (S) and ramiprilat (R, 100 nmol/L, 2 minutes) on the phosphorylation of ACE immunoprecipitated from porcine aortic endothelial cells stably transfected with ACE S1253A, S1263A, or S1270A. Identical results were obtained in 3 additional experiments. C, Representative Western blots showing the time course (2 to 7 minutes) of ramiprilat-induced changes in Ser1270 phosphorylation in human endothelial cells assessed with a phosphospecific antibody. D, Prolonged time course (6 to 48 hours) of the effects of ramiprilat (100 nmol/L) on the phosphorylation of ACE Ser1270 in porcine endothelial cells stably overexpressing human somatic ACE. E, Concentration-dependent effect of ramiprilat (1 to 100 nmol/L, 2 minutes) on the phosphorylation of ACE Ser1270 in porcine endothelial cells stably overexpressing human somatic ACE. Bar graph summarizes results obtained in 12 to 16 different experiments. **P<0.01 vs CTL.

The cytoplasmic tail of ACE contains five potentially phosphorylatable serine residues, three of which (Ser¹²⁵³, Ser¹²⁶³, and Ser¹²⁷⁰) are within recognition sequences for known protein kinases (PKC, PKA, and CK2, respectively). Mutation of either Ser¹²⁵³ or Ser¹²⁶³ to alanine failed to affect the basal or ramiprilat-induced phosphorylation of ACE overexpressed in porcine endothelial cells, whereas the S1270A mutant was not phosphorylated in either the absence or presence of ramiprilat (100 nmol/L, 2 minutes; B).

Using an antibody that specifically recognizes phosphorylated ACE Ser¹²⁷⁰, we assessed the effects of ramiprilat in ACE-overexpressing porcine endothelial cells. As in the primary endothelial cell cultures, ramiprilat elicited the rapid phosphorylation of ACE on Ser¹²⁷⁰; phosphorylation was increased by 2.1±0.1-fold over control values (n=7, P<0.05) 2 minutes after the addition of the ACE inhibitor (C). ACE phosphorylation decreased over the next 5 minutes (C) and reached control levels within 10 minutes, but a secondary increase in phosphorylation was detected after 12 to 24 hours and remained elevated for at least 48 hours (D). The acute (2 minutes) effect of ramiprilat on the phosphorylation of ACE Ser¹²⁷⁰ was also concentration-dependent with maximal effects being observed using 30 to 100 nmol/L (E).

Effect of Ramiprilat on the Activity of ACE-Associated CK2
In ACE-overexpressing endothelial cells, the CK2 inhibitor 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB; 100 µmol/L, 8 hours) attenuated the basal phosphorylation of ACE, as previously reported,¹⁴ and prevented the ramiprilat-induced phosphorylation of the enzyme (A). The amount of ACE recovered from the DRB-treated endothelial cells was slightly lower than that recovered from solvent-treated cells, an effect that can be attributed to the enhanced cleavage/secretion of dephosphorylated ACE.¹⁴ A second CK2 inhibitor, apigenin (20 µmol/L), also prevented the ramiprilat-induced increase in ACE phosphorylation (data not shown).

fig.ommitted Figure 2. Involvement of CK2 in the ramiprilat-induced phosphorylation of ACE. A, Effect of the CK2 inhibitor DRB (100 µmol/L) on the ramiprilat (100 nmol/L)-induced phosphorylation of ACE in porcine aortic endothelial cells overexpressing human somatic ACE. Similar results were obtained in 3 additional experiments. B, Effect of solvent (CTL) and ramiprilat (100 nmol/L) on the association of CK2 with ACE. C and D, Bar graphs showing the results of in vitro CK2 activity assays performed using the ACE immunoprecipitates from porcine endothelial cells overexpressing either ACE (C) or the S1270A mutant (D). Results are the mean±SEM of data obtained in 5 to 11 separate experiments. *P<0.05 vs CTL.

As CK2 is associated with and phosphorylates ACE in endothelial cells, we determined whether or not ramiprilat affects the association of the kinase with ACE. Exposure of ACE-overexpressing endothelial cells to ramiprilat (100 nmol/L) did not alter the association of the two proteins (B) but did increase the activity of ACE-associated CK2, as determined by an in vitro kinase assay (C).

Although the S1270A ACE mutant was not phosphorylated, CK2 was associated with the protein and the addition of ramiprilat resulted in the activation of S1270A-associated CK2 (D). Thus, the mutation of the CK2 consensus sequence affects the phosphorylation of ACE but not its association with, or the ACE inhibitor-induced activation of, CK2.

A second ACE inhibitor, perindoprilat (100 nmol/L), also elicited the time-dependent phosphorylation of ACE Ser¹²⁷⁰ as well as the activation of ACE-associated CK2 (). The time course of these effects was slightly different from those of ramiprilat with ACE phosphorylation and CK2 activity peaking between 5 and 7 minutes and a second peak in phosphorylation being detected after 12 to 24 hours. Neither perindoprilat (B) nor ramiprilat (data not shown) increased the activity of CK2 in ACE-deficient cells.

fig.ommitted Figure 3. Effect of perindoprilat on the phosphorylation of ACE Ser1270 and the activation of ACE-associated CK2 immunoprecipitated from porcine endothelial cells overexpressing human somatic ACE. A, Western blots showing the short time course (2 to 7 minutes) of ACE Ser1270 phosphorylation in response to perindoprilat (100 nmol/L). B, Effect of perindoprilat (hatched bars) on the activity of ACE-associated CK2 in ACE-expressing endothelial cells (left) and on global CK2 activity in ACE-deficient (-ACE) endothelial cells (right). Experiments were performed in the absence and presence of DRB (100 µmol/L, filled bars). Results are the mean±SEM of data obtained in 3 to 7 separate experiments. *P<0.05 vs control (CTL); #P<0.05 vs absence of DRB. C, Prolonged time course (6 to 24 hours) showing the effect of perindoprilat (100 nmol/L) on the phosphorylation of ACE Ser1270 in ACE-overexpressing endothelial cells. Similar results were obtained in 8 additional experiments.

Bradykinin but not Angiotensin I Activates CK2 and Increases ACE Phosphorylation in Endothelial Cells
To determine whether the activation of CK2 and the phosphorylation of ACE could be modulated by ACE substrates/products, we assessed the effects of bradykinin, angiotensin I, and angiotensin II on ACE phosphorylation and CK2 activity. To avoid potential complications arising from the cross-talk between ACE and the kinin and angiotensin receptors, these experiments were performed using ACE-overexpressing cells that lack B₂ and AT₁ receptors. Bradykinin (100 nmol/L) increased the phosphorylation of ACE (A) as well as the activity of ACE-associated CK2 (B), both of which peaked within 2 to 5 minutes of cell stimulation. Neither angiotensin I (C), nor angiotensin II (data not shown), effected the phosphorylation of ACE Ser¹²⁷⁰.

fig.ommitted   Figure 4. Bradykinin, but not angiotensin I, enhances ACE phosphorylation and ACE-associated CK2 activity. A, Representative Western blots showing the effect of bradykinin (100 nmol/L) on the phosphorylation of ACE Ser1270 in ACE-overexpressing porcine endothelial cells. Bar graph shows data from 7 separate experiments. B, Effect of bradykinin on the activity of ACE-associated CK2. Bar graph shows data from 4 separate experiments. C, Western blots showing the effect of angiotensin I on the phosphorylation of ACE Ser1270 in ACE-overexpressing porcine endothelial cells. Identical results were obtained in 2 additional experiments. *P<0.05 and **P<0.01 vs control (CTL).

Activation of JNK and Phosphorylation of c-Jun by ACE Inhibitors
Analysis of ACE immunoprecipitates revealed that additional proteins of approximately 46 and 55 kDa associated with the cytoplasmic tail of ACE (data not shown). Using immunoprecipitation and Western blot analysis, we identified MAP kinase kinase 7 (MKK7; A) and the c-Jun N-terminal kinase (JNK) as ACE-associated kinases (B). Identical results were obtained using primary cultures of human umbilical vein endothelial cells and the porcine endothelial cells that overexpress human somatic ACE.

fig.ommitted Figure 5. Association of ACE with MKK7 and JNK and activation of JNK by ramiprilat. A, Representative Western blots (in duplicate) showing the association of MEK7 with ACE immunoprecipitated from ACE-overexpressing endothelial cells. Left lane shows the results of preclearing (Pre) a sample with protein A/G before immunoprecipitation. B, Representative Western blots (in duplicate) showing the association of ACE with JNK (left) and the association of JNK with ACE (right) immunoprecipitated from ACE-overexpressing endothelial cells. C, Autoradiography (32P c-Jun) and statistical analysis showing the effect of solvent (CTL, open bars) and ramiprilat (Rami, 100 nmol/L, 7 minutes, hatched bars) on the activity of ACE-associated JNK immunoprecipitated from endothelial cells overexpressing ACE or the S1270A mutant. Bar graph summarizes data from 3 separate experiments. D, Autoradiography (32P c-Jun) and statistical analysis showing the effect of solvent (S) and ramiprilat (R, 100 nmol/L, 7 minutes) on the activity of JNK immunoprecipitated from ACE-deficient endothelial cells (-ACE) or endothelial cells overexpressing either ACE or the S1270A mutant. Anisomycin (An, 1 µmol/L, 10 minutes) was used as a positive control. Bar graph shows data from 7 separate experiments. *P<0.05 vs solvent or control (CTL).

Exposure of ACE-overexpressing endothelial cells to ramiprilat (100 nmol/L, 7 minutes) enhanced the activity of ACE-associated JNK, assessed in an in vitro kinase assay using GST-c-Jun as a substrate (C). Although JNK associated with the nonphosphorylatable S1270A ACE mutant, ramiprilat was unable to activate the kinase in S1270A overexpressing cells (C). A similar effect was observed when the activity of JNK was assessed in total cell lysates; ramiprilat pretreatment increased the activity of JNK immunoprecipitated from ACE-overexpressing cells whereas no such increase was observed in cells expressing the S1270A mutant or in ACE-deficient cells (D). Bradykinin, but not angiotensin I, also increased ACE-associated JNK activity (data not shown).

The activation of JNK is normally associated with the nuclear accumulation of phosphorylated c-Jun.¹⁹ Therefore, ACE-deficient cells and cells overexpressing ACE or the S1270A mutant were stimulated with ramiprilat, and the nuclear accumulation of phosphorylated c-Jun was assessed by immunohistochemistry. Ramiprilat (100 nmol/L) stimulated the nuclear accumulation of phosphorylated c-Jun in cells overexpressing ACE but not in cells overexpressing the S1270A mutant or in ACE-deficient cells (). In the latter cell types, anisomycin (1 µmol/L) was used as a positive control for the phosphorylation of c-Jun. Nuclear accumulation of phosphorylated c-Jun was also stimulated by perindoprilat (100 nmol/L).

fig.ommitted Figure 6. Immunohistochemical staining of phosphorylated c-Jun in porcine aortic endothelial cells overexpressing human somatic ACE, the S1270A ACE mutant (S1270A) or in ACE-deficient (-ACE) endothelial cells. Cells were incubated with solvent (CTL), ramiprilat (Rami, 100 nmol/L), or perindoprilat (Perindo, 100 nmol/L) for up to 30 minutes. In some experiments, anisomycin (1 µmol/L, 10 minutes) was used as a positive control for the activation of JNK and phosphorylation of c-Jun. Bars represent 10 µm, and the results are representative of data obtained in 2 further experiments.

Effect of ACE Inhibitors on the Expression of ACE in Cultured Endothelial Cells
ACE inhibitors are reported to increase the expression of ACE in endothelial cells in vitro and in vivo by a mechanism that is independent of either angiotensin or bradykinin.^20,21 Because the formation of c-Jun homodimers can affect ACE expression,²² we assessed whether or not the ramiprilat-induced expression of ACE could be attributed to "ACE signaling" via JNK.

In primary cultures of human umbilical vein endothelial cells, ramiprilat induced a time-dependent increase in ACE expression (A), which was prevented by pretreating the cells with the JNK inhibitor, SP600125 (5 µmol/L; B).

fig.ommitted   Figure 7. Effect of ramiprilat on the expression of ACE in primary cultures of human endothelial cells. A, Time course of the ramiprilat (Rami, 100 nmol/L)-induced increase in ACE expression and the effect of the JNK inhibitor, SP600125 (5 µmol/L) on ramiprilat-induced ACE expression (B). Expression of ACE was normalized to the endothelial cell marker protein PECAM-1. Bar graphs show data obtained in 4 to 9 independent experiments. *P<0.05, **P<0.01, and *** P<0.001 vs control (CTL).

Effect of a JNK Inhibitor on the ACE Inhibitor–Induced Increase in ACE Expression In Vivo
To assess the relevance of this signaling pathway in vivo, we assessed the effects of ramiprilat on JNK activity and ACE expression in the mouse lung. The acute administration of ramiprilat (250 µg/mouse, i.p. 15 minutes) resulted in the activation of JNK immunoprecipitated from lung homogenates (A) as well as in the activation of ACE-associated JNK (ramiprilat increased ACE-associated JNK activity by 34±9%, n=4; P<0.05). Prolonged treatment of mice with the prodrug ramipril (5 mg/kg per day for 5 days) markedly increased ACE expression in the lung (B). Pretreatment of mice with the JNK inhibitor (30 mg/kg per day, i.p. daily for 7 days, ie, beginning 2 days before ramipril) attenuated JNK activity in lung homogenates (activity was 70±7% of that detected in lung homogenates from vehicle-treated mice, n=5; P<0.05) as well as the ramipril-induced increase in ACE expression (B).

fig.ommitted Figure 8. Effect of ramiprilat on the activity of JNK and ACE expression in mouse lung. A, Autoradio-graphy (32P c-Jun) and bar graph showing the effect of acute administration of ramipril (250 µg/mouse IP, 15 minutes) in vivo on the activity of JNK immunoprecipitated from mouse lung. B, Western blots and bar graph showing the effect of vehicle (CTL) or prolonged ramipril treatment (Rami, 5 mg/kg per day for 5 days) on the expression of ACE in the lungs of mice pretreated with and without SP600125 (30 mg/kg per day, i.p. daily for 7 days). Blots were reprobed with a tubulin antibody to demonstrate the equal loading of protein in each lane. Bar graphs show data obtained using 5 to 9 mice. **P<0.01 vs CTL.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have shown that the binding of an ACE inhibitor to ACE elicits outside-in signaling in endothelial cells. The pathway activated includes CK2, the phosphorylation of ACE Ser¹²⁷⁰, the activation of ACE-associated JNK, and the subsequent nuclear accumulation of phosphorylated c-Jun. This signaling pathway ultimately resulted in an increase in ACE expression in vitro and in vivo.

Because we recently demonstrated that Ser¹²⁷⁰ is phosphorylated by CK2 in primary cultures of human endothelial cells as well as porcine aortic endothelial cells overexpressing ACE,¹⁴ we hypothesized that this residue may be the start point for an ACE inhibitor and/or ACE substrate-activated signal transduction cascade in endothelial cells. Although the cross-talk with the B₂ kinin receptor and in particular the events controlling receptor sequestration¹⁰ may represent the best known effect of ACE signaling, we performed most of the experiments in endothelial cells that no longer express the B₂ receptor to avoid confusion with the effects of classical bradykinin signaling. However, each step in the ACE inhibitor-activated signal transduction cascade was confirmed in primary cultures of human endothelial cells, ie, in cells expressing normal levels of ACE as well as receptors for angiotensin II and bradykinin and, where possible, also in lungs isolated from ramipril-treated mice.

Our finding that two ACE inhibitors (ramiprilat and perindoprilat) as well as the preferred ACE substrate (bradykinin) enhance the activity of ACE-associated CK2, increase the phosphorylation of ACE, and lead to the activation of JNK as well as the accumulation of phosphorylated c-Jun in the endothelial cell nucleus provide conclusive evidence for a role of ACE as a signal transduction molecule. Moreover, because the effects described were not observed in cells either lacking ACE or overexpressing S1270A ACE, it appears that the CK2-mediated phosphorylation of Ser¹²⁷⁰ is a key event in the activation of JNK, and the transmission of ACE signaling from the plasma membrane to other intracellular compartments. Our observation that bradykinin but not angiotensin I was able to activate ACE-signaling may be the consequence of the difference in K_m values for angiotensin I and bradykinin,⁷ or differential binding to the two catalytic sites.²³

Although we have demonstrated that the binding of an ACE inhibitor to ACE enhanced the activity of ACE-associated CK2, the phosphorylation of Ser¹²⁷⁰, and the activation of JNK, the exact steps that link the binding of the inhibitor to the extracellular domain of ACE to the activation of the aforementioned kinases remains to be elucidated. CK2 can phosphorylate JNK on Ser407 and Thr404,²⁴ and there is circumstantial evidence that indicates that a CK2-JNK pathway exists in some cell types.²⁵ However, it is unlikely that the ACE inhibitor–induced activation of CK2 directly results in the phosphorylation of JNK because comparison of the responses obtained in expressing wild-type ACE and cells expressing the S1270A ACE mutant indicates that the activation of JNK is a process strictly dependent on the phosphorylation of ACE Ser¹²⁷⁰. The activation of CK2 by the ACE inhibitors was, on the other hand, unaffected by the mutation of Ser¹²⁷⁰. Because the JNK protein kinases are activated via phosphorylation on threonine and tyrosine residues by MKK4 and MKK7,²⁴ and MKK7 coprecipitated with ACE, it is tempting to suggest that the activation of MKK7 is involved in ACE signaling. Exactly how the binding of an ACE inhibitor is able to enhance the activity of ACE-associated CK2 also remains to be resolved. However, it is conceivable that the ACE inhibitor–induced signaling cascade we have described involves ACE clustering. Indeed, ACE has been recovered as a dimer and even as an oligomer under certain conditions.^26,27

At first sight, ACE seems an unlikely candidate for a signal transduction molecule as it is a zinc metallopeptidase and the cytoplasmic domain of the enzyme is short (only 29 amino acids). However, at least two other ectoenzymes, matrix metalloproteinase-1 (MMP-1)²⁸ and ADAM12,²⁹ have recently been associated with a signaling function. MMPs have well documented effects on intracellular signaling as a consequence of the extracellular cleavage of proteins such as big endothelin-1,³⁰ calcitonin gene–related peptide,³¹ and thrombin.³² However, a direct role as a signal transduction molecule has recently been attributed to MMP-1 in platelets on the basis of the observation that its activation markedly increases tyrosine phosphorylation as well as the targeting of ß₃ integrin to areas of cell contact.²⁸ Moreover, in addition to liberating active signaling molecules such as EGF from inactive precursor forms in response to a number of stimuli,^33,34 members of the ADAM (A Disintegrin and A Metalloprotease) family may also play an active role in signal transduction. Indeed, ADAM12 is capable of initiating intracellular signaling via the binding of the tyrosine kinase Src to specific domains within its short cytoplasmic tail.²⁹

The data presented in this article clearly demonstrate that the binding of an ACE inhibitor to ACE results in the activation of JNK as well as the phosphorylation of c-Jun and its translocation to the nucleus. This signaling pathway is therefore likely to affect the expression of several proteins and in particular the expression of ACE itself. Indeed, the expression of ACE in phorbol ester–stimulated human endothelial cells can be attributed to the binding of an AP-1 complex containing a c-Jun homodimer to the cAMP-responsive element/12-O-tetradecanoylphorbol 13-acetate responsive element sequence of the ACE promoter.²² Although it is tempting to claim that the signaling via ACE that is initiated by the binding of an ACE inhibitor is protective/beneficial to vascular cell function and or the development of cardiovascular disease, this is currently speculation because the end point identified in the present study, ie, an increase in the expression of ACE itself, could also be expected to have deleterious effects by leading to the enhanced generation of angiotensin II. However, it should be noted that the latter phenomenon has been demonstrated in lung tissue and plasma from ACE inhibitor–treated rats^21,35 and in serum from patient populations which distinctly benefit from ACE inhibitor therapy.^36,37 Additional investigation is therefore required to identify additional components of the ACE signaling cascade that influence endothelial cell function.

Taken together, the results of the present investigation indicate that ACE can be added to the list of nonreceptor cell surface proteins that play a role in endothelial cell signaling. The identification of ACE as a signaling molecule that can be activated by the binding of ACE inhibitors may account for some of the beneficial effects of this class of compounds on the cardiovascular system.

　

　

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (FL 364/1), the Heinrich and Fritz Riese-Stiftung, and a research grant from Aventis. The authors are indebted to Marie von Reutern, Tanja-Maria Mareczek, and Isabel Winter for expert technical assistance.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Jaspard E, Costerousse O, Wei L, Corvol P, Alhenc-Gelas F. The angiotensin I-converting enzyme (kininase II): molecular and regulatory aspects. Agents Actions Suppl. 1992; 38: 349–358.

2. Mancini GBJ, Henry GC, Macaya C, O’Neill BJ, Pucillo AL, Carere RG, Wargovich TJ, Mudra H, Luscher TF, Klibaner MI, Haber HE, Uprichard ACG, Pepine CJ, Pitt B. Angiotensin-converting enzyme inhibition with quinapril improves endothelial vasomotor dysfunction in patients with coronary artery disease: The TREND (Trial on Reversing ENdothelial Dysfunction) study. Circulation. 1996; 94: 258–265.

3. Schartl M, Bocksch WG, Dreysse S, Beckmann S, Hunten U. Remodeling of myocardium and arteries by chronic angiotensin converting enzyme inhibition in hypertensive patients. J Hypertens. 1994; 12: S37–S42.

4. Lonn EM, Yusuf S, Dzavik V, Doris CI, Yi Q, Smith S, Moore-Cox A, Bosch J, Riley WA, Teo KK. Effects of ramipril and vitamin E on atherosclerosis: the study to evaluate carotid ultrasound changes in patients treated with ramipril and vitamin E (SECURE). Circulation. 2001; 103: 919–925.

5. Yusuf S, Sleight P, Pogue J, Bosch J, Davies R, Dagenais G. Effects of an angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. N Engl J Med. 2000; 342: 145–153.

6. Heart Outcomes Prevention Evaluation Study Investigators. Effects of ramipril on cardiovascular and microvascular outcomes in people with diabetes mellitus: results of the HOPE study and MICRO-HOPE substudy. Lancet. 2000; 355: 253–259.

7. Soubrier F, Hubert C, Testut P, Nadaud S, Alhenc-Gelas F, Corvol P. Molecular biology of the angiotensin I converting enzyme, I: biochemistry and structure of the gene. J Hypertens. 1993; 11: 471–476.

8. Yang XP, Liu YH, Mehta D, Cavasin MA, Shesely E, Xu J, Liu F, Carretero OA. Diminished cardioprotective response to inhibition of angiotensin-converting enzyme and angiotensin II type 1 receptor in B₂ kinin receptor gene knockout mice. Circ Res. 2001; 88: 1072–1079.

9. Hecker M, Porsti I, Bara AT, Busse R. Potentiation by ACE inhibitors of the dilator response to bradykinin in the coronary microcirculation: interaction at the receptor level. Br J Pharmacol. 1994; 111: 238–244.

10. Benzing T, Fleming I, Blaukat A, Müller-Esterl W, Busse R. The ACE inhibitor ramiprilat prevents the sequestration of the B₂ kinin receptor within the plasma membrane of native endothelial cells. Circulation. 1999; 99: 2034–2040.

11. Hecker M, Blaukat A, Bara AT, Muller-Esterl W, Busse R. ACE inhibitor potentiation of bradykinin-induced venoconstriction. Br J Pharmacol. 1997; 121: 1475–1481.

12. Marcic B, Deddish PA, Jackman HL, Erdös EG. Enhancement of bradykinin and resensitization of its B₂ receptor. Hypertension. 1999; 33: 835–843.

13. Minshall RD, Tan F, Nakamura F, Rabito SF, Becker RP, Marcic B, Erdös EG. Potentiation of the actions of bradykinin by angiotensin I-converting enzyme inhibitors: the role of expressed human bradykinin B₂ receptors and angiotensin I-converting enzyme in CHO cells. Circ Res. 1997; 81: 848–856.

14. Kohlstedt K, Shoghi F, Müller-Esterl W, Busse R, Fleming I. CK2 phosphorylates the angiotensin-converting enzyme and regulates its retention in the endothelial cell plasma membrane. Circ Res. 2002; 91: 749–756.

15. Popp R, Bauersachs J, Hecker M, Fleming I, Busse R. A transferable, ß-naphthoflavone-inducible, hyperpolarizing factor is synthesized by native and cultured porcine coronary endothelial cells. J Physiol (Lond). 1996; 497: 699–709.

16. Wei L, Alhenc-Gelas F, Soubrier F, Michaud A, Corvol P, Clauser E. Expression and characterization of recombinant human angiotensin I-converting enzyme: evidence for a C-terminal transmembrane anchor and for a proteolytic processing of the secreted recombinant and plasma enzymes. J Biol Chem. 1991; 266: 5540–5546.

17. Potente M, Michaelis UR, Fisslthaler B, Busse R, Fleming I. CYP 2C9-induced endothelial cell proliferation involves induction of MAP kinase phosphatase 1, inhibition of the c-Jun N-terminal kinase and upregulation of cyclin D1. J Biol Chem. 2002; 277: 15671–15676.

18. Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L, Manning AM, Firestein GS. c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest. 2001; 108: 73–81.

19. Dunn C, Wiltshire C, MacLaren A, Gillespie DA. Molecular mechanism and biological functions of c-Jun N-terminal kinase signaling via the c-Jun transcription factor. Cell Signal. 2002; 14: 585–593.

20. King SJ, Oparil S. Converting-enzyme inhibitors increase converting-enzyme mRNA and activity in endothelial cells. Am J Physiol. 1992; 263: C743–C749.

21. Costerousse O, Allegrini J, Clozel J-P, Menard J, Alhenc-Gelas F. Angiotensin I-converting enzyme inhibition but not angiotensin II suppression alters angiotensin I-converting enzyme gene expression in vessels and epithelia. J Pharmacol Exp Ther. 1998; 284: 1180–1187.

22. Eyries M, Agrapart M, Alonso A, Soubrier F. Phorbol ester induction of angiotensin-converting enzyme transcription is mediated by Egr-1 and AP-1 in human endothelial cells via ERK1/2 pathway. Circ Res. 2002; 91: 899–906.

23. Georgiadis D, Beau F, Czarny B, Cotton J, Yiotakis A, Dive V. Roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin I and bradykinin: insights from selective inhibitors. Circ Res. 2003; 93: 148–154.

24. Fleming Y, Armstrong CG, Morrice N, Paterson A, Goedert M, Cohen P. Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7. Biochem J. 2000; 352: 145–154.

25. Min YK, Park JH, Chong SA, Kim YS, Ahn YS, Seo JT, Bae YS, Chung KC. Pyrrolidine dithiocarbamate-induced neuronal cell death is mediated by Akt, casein kinase 2, c-Jun N-terminal kinase, and IB kinase in embryonic hippocampal progenitor cells. J Neurosci Res. 2003; 71: 689–700.

26. Bull HG, Thornberry NA, Cordes EH. Purification of angiotensin-converting enzyme from rabbit lung and human plasma by affinity chromatography. J Biol Chem. 1985; 260: 2963–2972.

27. Kost OA, Balyasnikova IV, Chemodanova EE, Nikolskaya II, Albrecht RF, Danilov SM. Epitope-dependent blocking of the angiotensin-converting enzyme dimerization by monoclonal antibodies to the N-terminal domain of ACE: possible link of ACE dimerization and shedding from the cell surface. Biochemistry. 2003; 42: 6965–6976.

28. Galt SW, Lindemann S, Allen L, Medd DJ, Falk JM, McIntyre TM, Prescott SM, Kraiss LW, Zimmerman GA, Weyrich AS. Outside-in signals delivered by matrix metalloproteinase-1 regulate platelet function. Circ Res. 2002; 90: 1093–1099.

29. Kang Q, Cao Y, Zolkiewska A. Metalloprotease-disintegrin ADAM 12 binds to the SH3 domain of Src and activates Src tyrosine kinase in C2C12 cells. Biochem J. 2000; 352: 883–892.

30. Fernandez-Patron C, Radomski MW, Davidge ST. Vascular matrix metalloproteinase-2 cleaves big endothelin-1 yielding a novel vasoconstrictor. Circ Res. 1999; 85: 906–911.

31. Fernandez-Patron C, Stewart KG, Zhang Y, Koivunen E, Radomski MW, Davidge ST. Vascular matrix metalloproteinase-2-dependent cleavage of calcitonin gene-related peptide promotes vasoconstriction. Circ Res. 2000; 87: 670–676.

32. Fernandez-Patron C, Radomski MW, Davidge ST. Role of matrix metalloproteinase-2 in thrombin-induced vasorelaxation of rat mesenteric arteries. Am J Physiol Heart Circ Physiol. 2000; 278: H1473–H1479.

33. Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C, Ullrich A. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature. 1999; 402: 884–888.

34. Yan Y, Shirakabe K, Werb Z. The metalloprotease Kuzbanian (ADAM10) mediates the transactivation of EGF receptor by G protein-coupled receptors. J Cell Biol. 2002; 158: 221–226.

35. Fyhrquist F, Forslund T, Tikkanen I, Gronhagen-Riska C. Induction of angiotensin I-converting enzyme rat lung with captopril (SQ 14225). Eur J Pharmacol. 1980; 67: 473–475.

36. Boomsma F, de Bruyn JH, Derkx FH, Schalekamp MA. Opposite effects of captopril on angiotensin I-converting enzyme "activity" and "concentration": relation between enzyme inhibition and long-term blood pressure response. Clin Sci (Lond). 1981; 60: 491–498.

37. Sassano P, Chatellier G, Billaud E, Alhenc-Gelas F, Corvol P, Menard J. Treatment of mild to moderate hypertension with or without the converting enzyme inhibitor enalapril: results of a six-month double-blind trial. Am J Med. 1987; 83: 227–235.