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Am J Physiol Heart Circ Physiol 289: H1814-H1820, 2005; doi:10.1152/ajpheart.00581.2005
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Angiotensin-converting enzyme regulates bradykinin receptor gene expression

Ivana Ignjacev-Lazich,1 Ekaterina Kintsurashvili,1 Conrado Johns,1 Olga Vitseva,2 Arvi Duka,1 Sherene Shenouda,2 Irene Gavras,1 and Haralambos Gavras1

1Hypertension and Atherosclerosis Section and 2Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts

Submitted 2 June 2005 ; accepted in final form 27 June 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 
The angiotensin-converting enzyme (ACE) is a membrane-bound peptidyl dipeptidase known to act on a variety of peptide substrates in the extracellular space. Its most notable functions are the formation of angiotensin II and the degradation of bradykinin. In the current experiments, we found that exogenous ACE added to vascular smooth muscle cell culture strongly induces and upregulates the genes of bradykinin receptors B1 and B2. This transcriptional regulatory property of ACE was shown to be unrelated to its known enzymatic properties. Indeed, ACE at 3.75 µg/ml added in the culture medium of vascular smooth muscle cells was found to cause marked upregulation of the mRNA expression of the genes for the B1 and B2 receptors of bradykinin by 22- and 11-fold, respectively. This phenomenon was not altered by the addition of specific angiotensin II antagonists for the AT1 or AT2 receptors. Moreover, the ACE inhibitor captopril, which inhibited ACE enzymatic activity, did not block its effect at the bradykinin receptor gene transcription level. Expression of both receptor genes was completely abolished by actinomycin D. Furthermore, transcriptional upregulation was inhibited by curcumin, suggesting involvement of different transcriptional factors in this phenomenon. Electrophoretic mobility shift assay revealed increase in NF-{kappa}B and activator protein-1 protein binding for consensus sequences, between ACE-treated cells versus untreated cells. The data indicate a novel biological function of the ACE unrelated to its well-known enzymatic function as a peptidyl dipeptidase.

vascular smooth muscle cells; peptidyl dipeptidase; nuclear factor-{kappa}B; activator protein-1

ANGIOTENSIN-CONVERTING ENZYME (ACE) inhibition is one of the most important advances in cardiovascular pharmacology in the last 35 years. After the first small clinical studies suggested the therapeutic potential of ACE inhibitors for the treatment of hypertension (9, 10) and heart failure (11), this class of drugs became the object of numerous large outcome trials that demonstrated their cardioprotective and nephroprotective properties and are now the most widely prescribed agents for these conditions. Yet, whereas the clinical benefits of ACE inhibition are well established, the multiple functions of ACE and the mechanisms by which its inhibitors exert these benefits are still being investigated.

The ACE is a membrane-bound enzyme that acts on a variety of peptide substrates. Skidgel and Erdos (32) have clarified that the peptidyl dipeptidase, which cleaves off His-Leu from angiotensin I to form angiotensin II, is identical to kininase II, which cleaves off Phe-Arg from bradykinin to form inactive residue. Therefore, its inhibition will block the generation of angiotensin II and potentiate the actions of bradykinin. A large body of literature is devoted to dissection of angiotensin-mediated and bradykinin-mediated effects of ACE inhibition, as well as to other aspects of ACE-related functions.

In a previous series of experiments studying the interactions of these two systems, we found that various experimental manipulations affect the gene expression of the bradykinin receptors B1 and B2, both in vivo and in vitro. The majority of the physiological effects of bradykinin, including its hemodynamic (vasodilation) and metabolic actions (insulin-dependent glucose transport and utilization) is mediated by the B2 receptor, whereas the B1 receptor is minimally expressed under normal conditions (26, 17, 15). In the absence of B2 receptors, such as in B2 receptor gene knockout animals, the B1 receptor becomes expressed and is capable of assuming the hemodynamic (8, 6) but not the metabolic functions of the B2 receptor (7). The B2 receptor gene is normally expressed constitutively in many tissues, whereas the B1 is inducible by various factors, notably lipopolysaccharides and tissue inflammation or damage (18). We recently found that angiotensin II in vivo and in vitro stimulates gene expression of both B1 and B2 receptors in cardiomyocytes and vascular smooth muscle cells (VSMC), whereas concurrent inhibition of the AT1 receptor with losartan can abolish this effect (14).

The current studies were designed to explore the role of ACE in the presence or absence of angiotensin II antagonists on the gene expression of the B1 and B2 receptors in VSMC from the rat aorta. We found that exogenous ACE added to cell culture medium can indeed produce significant upregulation of both receptor genes by up to 11- to 22-fold by a mechanism unrelated to its enzymatic properties. We also made the unexpected discovery that this enzyme, which is normally found as a type I integral membrane glycoprotein located on the surface of epithelial and endothelial cells or circulating as a free enzyme in body fluids (plasma and seminal fluid), has actually the capacity to trigger this change in gene expression level without being enzymatically active as a peptidyl dipeptidase. The signaling mechanism was shown to involve activation of two transcription factors in this process, NF-{kappa}B and activator protein (AP-1).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 
Materials. Dulbecco’s modified Eagle’s medium, penicillin-streptomycin mixture, and TRIzol were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum, Ponceau S solution, actinomycin D, curcumin, PD-123319, lisinopril, and captopril were purchased from Sigma-Aldrich (Milwaukee, WI). Losartan was generously provided by Merck Research Laboratories (Rahway, NJ). "DNA free" was purchased from Ambion (Austin, TX). Bradykinin B1 receptor antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and bradykinin B2 antibodies were purchased from Transduction Laboratories (Lexington, KY).

ACE purification and preparation. Preliminary experiments were done with the commercially available ACE. However, SDS-PAGE analysis of these preparations revealed impurity and contamination of the preparation with other proteins. To exclude involvement of any other contaminant in our results, we purified the ACE in our laboratory and used it in all subsequent experiments.

Briefly, porcine kidneys were obtained from a slaughterhouse. Kidney cortex was surgically removed and homogenized in 0.33 M sucrose, 50 mM HEPES, pH 7.5, buffer at +4°C. The homogenate was centrifuged several times. The final pellet was resuspended in 5 mM HEPES, pH 7.5, made 0.5% Triton X-100, stirred overnight, and centrifuged again. The supernatant was dialyzed and applied on a Q Sepharose (Amersham Biosciences) column, and collected fractions were applied on Epoxy-Activated Sepharose 6B (Amersham Biosciences) coupled with lisinopril (2). Elution was performed with 10 mM NaH2PO4 and 7 mM EDTA, pH 7. Fractions of 1 ml were collected and analyzed on 7.5% polyacrylamide SDS-PAGE gel using Bio-Safe Coomassie (Bio-Rad, Hercules, CA) staining to confirm purity of the eluate (Fig. 1). Furthermore, the samples were dialyzed to the final buffer PBS and 10 µM ZnCl2. The final sample was assayed for protein concentration with the DC Protein assay (Bio-Rad) and for enzyme activity with the modified Cushman and Cheung assay (5) where hippuryl-His-Leu was used as a substrate and one unit of enzyme produced 1 M of hippuric acid per minute in 50 mM HEPES and 300 mM NaCl at pH 8.3 at 37°C. All procedures involving protein handling, except protein concentration assay, activity assay, and SDS-PAGE gel analysis, were performed at +4°C.



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  		Fig. 1. Coomassie-stained SDS-PAGE gel showing 6 µg of purified angiotensin-converting enzyme (ACE, 180 kDa) after elution from lisinopril-coupled chromatography column.

 
Cell culture. VSMC were isolated from the rat thoracic aorta of 8-wk-old male Wistar rats by an explant method (28) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in humidified atmosphere of 95% O2-5% CO2. They were characterized as smooth muscle cells by immunohistochemical detection of characteristic {alpha}-actin protein. After 5–10 passages, VSMC were grown to 70–80% confluence and incubated in serum-free media for 24 h before experimental manipulations and isolation of total RNA, membrane, and cytosolic protein.

In the first set of experiments, cells were incubated with porcine kidney ACE for 3 or 4 h. In the second set of experiments, cells were incubated with porcine kidney ACE plus 10–4 mol/l captopril mix (which was previously incubated at 37°C) for 3 h. The third set of experiments was performed by incubating VSMC with porcine kidney ACE after the preincubation with 10–5 mol/l of the AT1 receptor blocker losartan or 10–5 mol/l of the AT2 receptor blocker PD-123319 for 30 min. In the fourth set of experiments, VSMC were preincubated for 30 min with 8 x 10–3 mol/l actinomycin D [a transcriptional inhibitor (3)] and were then incubated for 3 h with porcine kidney ACE. In the last set of experiments, cells were preincubated for 1 h with the transcriptional inhibitor curcumin (1.5 x 10–5 mol/l) and were then incubated for 3 h with porcine kidney ACE.

Actinomycin D and curcumin were dissolved in DMSO, and to exclude DMSO interference with the ACE effect, we performed a set of experiments with DMSO + ACE without inhibitors and used them for purposes of comparison.

RNA isolation and gene expression analysis of B1 and B2 receptor genes. Total RNA was isolated from rat VSMC with the use of TRIzol reagent according to the manufacturer’s instructions. Elimination of genomic DNA contamination from the RNA isolate was done with "DNA free."

Expression of bradykinin B1 and B2 receptor genes was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) as described before (14) with few changes. Briefly, from each cell plate, 1.7 µg of total RNA were reverse transcribed into cDNA that was PCR amplified as described. Internal standard used was glyceraldehyde-3-phosphate dehydrogenase, which was amplified with 5'-TGCACCACCAACTGCTTAG-3' and 5'-GGATGCAGGGATGATGTTC-3' primers producing a 117-bp fragment. The PCR products were separated on 2.2% agarose gel and visualized by ethidium bromide staining. The resulting gels were scanned with Pdi scanner (model 420oe, Pdi; Huntington Station, NY) and analyzed with the use of NIH Image software.

Results obtained were analyzed more precisely by quantitative real-time PCR. cDNA prepared as described above was used for Q-RT-PCR reactions. Q-RT-PCR was performed with the ABI Prism 7900HT Sequence Detection System using an iTaq SYBR Green-based protocol (Bio-Rad). Oligonucleotide primers for RT-PCR were designed with the Primer Express 2.0 software program (Applied Biosystems) and manufactured by Integrated DNA Technologies (Coralville, IA). The sequences of the primers used in expression analysis were for the bradykinin B1 receptor gene 5'-TCCTGGTCCAGGTGAGAGTGA-3' and 5'-GGTTCAGGCAGCTGTTGCCA-3'; for the bradykinin B2 receptor gene 5'-TGCACTGTGGCCGAGATCT-3' and 5'-TATTGGCGATGGTGATGGC-3'; and for the GAPDH gene 5'-TGCACCACCAACTGCTTAG-3' and 5'-GGATGCAGGGATGATGTTC-3'. All reactions were run in triplicate and included negative controls. The concentrations of the forward and reverse primers were 3 x 10–7 mol/l. After initial denaturation at 95°C for 3 min, the cDNA products were amplified for 40 cycles consisting of denaturation at 95°C for 15 s, and annealing and extension were performed in a single step at 60°C for 45 s. The SDS 2.1 software generated standard curves from 10-fold serial cDNA dilutions, and the threshold cycle (Ct) was normalized for each standard curve. The slopes were between –3.19 and –3.57, where –3.33 corresponds to 100% efficiency of the PCR reaction. The copy numbers for all samples were normalized with the data obtained from GAPDH endogenous controls.

Nuclear extract preparation and electrophoretic mobility shift assay. VSMC were washed with cold PBS, and nuclear protein was extracted following the manufacturer’s protocol of BD Trans Factor Extraction Kit (San Jose, CA). The resulting nuclear extract was assayed for protein concentration and stored at –80°C until use. Gel mobility-shift assay was done with the Promega gel shift assay kit. Double-stranded oligonucleotides with consensus sequences of AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3'), NF-{kappa}B (5'-AGT TGA GGG GAC TTT CCC AGG C-3'), and cAMP response element binding protein (CREB) (5'-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3') were purchased from Promega. Oligonucleotides were end labeled in 10 µl of reaction buffer [in mM: 70 Tris·HCl (pH 7.6) 10 MgCl2, and 5 dithiothreitol] along with T4 polynucleotide kinase and [{gamma}-32P]ATP (3,000 Ci/mmol at 10 mCi/ml) for 10 min at 37°C. The reaction was stopped by the addition of 1 µl of 0.5 M EDTA and 89 µl Tris-EDTA buffer. Unincorporated label was removed with G-25 spin column. Nuclear extracts (10 µg) were preincubated for 15 min at room temperature with binding buffer [poly(dI-dC)·poly(dI-dC), 10 mM Tris·HCl (pH 7.5), 4% (vol/vol) glycerol, 0.1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, and 50 mM NaCI]. Labeled probe (107,500 counts/min) was then added, and the binding reaction was incubated for a further 20 min at room temperature. Unlabeled AP-1, NF-{kappa}B, CREB, and SP-1 competitor oligonucleotides (18x, 36x, 88x-fold molar excess) were added to binding reactions at the start of the preincubation period. Protein-DNA complexes were resolved on 4% (wt/vol) native polyacrylamide gels in 0.5x Tris-borate-EDTA run at 175 V room temperature. Gels were then dried and autoradiographed at –70°C.

Western blot analysis. Rat VSMC were grown as described earlier, incubated with porcine kidney ACE for 4 h, washed with ice-cold PBS, and scraped in ice-cold lysis buffer (in mM: 50 Tris, 5 EDTA, and 150 NaCl) without detergent and with added cocktail of protease inhibitors (Sigma-Aldrich). Cells were prepared by homogenization followed by centrifugation for 30 min at 51,500 g and +4°C. The supernatant was discarded and the pellet resuspended and homogenized again in the lysis buffer supplemented with 0.5% Triton X-100. Protein concentrations were determined by DC Protein assay (Bio-Rad). Isolated protein (30 µg) was loaded per lane of 10% polyacrylamide gel and separated by electrophoresis followed by transfer to nitrocellulose membrane. After the membrane was blocked with 5% nonfat dried milk, nitrocellulose blots were washed in PBS 0.05%-Tween 20 and incubated with primary antibodies for bradykinin B1 receptor overnight at +4°C (1:200) or bradykinin B2 receptor for 1 h and 30 min at room temperaure (1:1,000). Secondary horseradish peroxidase-conjugated antibody was applied for 1 h. Antibody binding was visualized with enhanced chemiluminescence Western blotting analysis (Amersham Biosciences). All blots were stripped with Western blot stripping buffer for 2 h (Pierce, Rockford, IL) and then stained with Ponceau S solution for 1 h.

Measurement of intracellular cAMP. Rat VSMC were exposed to 3.75 µg/ml of ACE for 20 min and 3 h and scraped in 0.1 M HCl and then centrifuged at 1,000 g for 10 min. The supernatant was collected and acetylated together with standards before the analysis. cAMP contained in each sample was determined by specific kit cAMP EIA Kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions.

Statistical analysis. All data are expressed as means ± SE. Student’s t-test for paired and unpaired data was used as appropriate. Differences at P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 
Effects of ACE on B1 and B2 bradykinin receptor gene expression. Addition of ACE in the culture medium of rat aortic VSMC upregulated the B1 receptor gene expression after 3 h of incubation. Cells were depleted of serum for 24 h and were subsequently exposed to 3.75 µg/ml of ACE with specific activity between 90 and 100 U/mg. mRNA levels of B1 receptor were significantly upregulated compared with baseline (Fig. 2A).



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  		Fig. 2. Bradykinin B1 receptor (B1R) gene expression (A) and bradykinin B2 receptor (B2R) gene expression (B) upregulation under 3.75 µg/ml ACE stimulation alone and with added captopril (Capt), losartan (Los), and PD-123319. Top: representative RT-PCR. Bottom: real-time PCR analysis of B1R and B2R gene expression. *P < 0.05 ACE-treated cells compared with unstimulated cells.

 
With the same conditions, we analyzed levels of mRNA for the B2 receptor, which is already constitutively expressed in VSMC in basal conditions. Addition of 3.75 µg/ml ACE significantly stimulated expression of both bradykinin receptor genes: expression of the B1 receptor gene increased by 22-fold and of the B2 receptor gene by 11-fold (Fig. 2B).

A control set of experiments was performed by analyzing gene expression levels of both bradykinin B1 and B2 receptors after stimulation for 3 h with the ACE buffer. Analysis for mRNA levels showed no difference from the unstimulated control (data not shown).

To separate the enzymatic activity from other possible properties of the ACE, we repeated the above experiments after coincubation of ACE with 10–4 mol/l ACE inhibitor captopril. ACE specific activity was determined to be almost completely blocked to 0.03–0.07 U/mg. In both cases, the enzymatic blockade did not interfere with the ACE effect on B1 and B2 receptor gene expression (Fig. 2, A and B).

To investigate the possibility that the ACE effect might be mediated by generation of angiotensin II (acting via its receptors AT1 and AT2) and convincingly exclude any influence of enzymatic activity of the ACE on this process, we repeated the above experiments with addition of the specific AT1 receptor antagonist losartan (10–5 mol/l) or the AT2 receptor antagonist PD-123319 (10–5 mol/l).

In neither case was the ACE effect on B1 and B2 receptor gene expression altered by these angiotensin II antagonists (Fig. 2, A and B). In previous studies we have determined that PD-123319 and losartan given alone have no effect on B1 and B2 receptor mRNA expression (14).

The final set of experiments was designed to confirm that any changes in B1 and B2 receptor gene expression represent a transcriptionally regulated event. In cells preincubated for 10 min with the transcriptional inhibitor actinomycin D, the 3-h incubation with ACE failed to produce induction and upregulation in B1 and B2 receptor mRNA levels (Fig. 3, A and B).



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  		Fig. 3. B1R gene expression (A) and B2R gene expression (B) under 3.75 µg/ml ACE stimulation alone and in presence of DMSO. ACE effect was inhibited with 8 x 10–3 mol/l actinomycin D (ActD) and 1.5 x 10–5 mol/l curcumin. Top: representative RT-PCR. Bottom: real-time PCR analysis of B1R and B2R gene expression. *P < 0.05 compared with control cells; **P < 0.05 compared with DMSO + ACE-treated cells.

 
Pretreatment of cells with curcumin, an inhibitor that affects several different factors that are involved in gene expression regulation, including the NF-{kappa}B and AP-1 factor, also blocked the ACE effect on both genes as shown by the decrease of B1 and B2 receptor gene expression to basal levels (Fig. 3, A and B).

Induction of NF-{kappa}B and AP-1 transcriptional factors by ACE. The electrophoretic mobility shift assay (EMSA) was used to study the involvement of different transcriptional factors that were shown to be responsible in the regulation of bradykinin receptor gene expression (18, 30, 25).

Both bradykinin B1 and B2 receptor genes have been shown to have a responsive element in the promoter region for AP-1 transcriptional factor (24). Collected nuclear extracts from untreated VSMC have shown fairly abundant binding for DNA consensus sequence of AP-1 factor; however, after a 3-h period of stimulation with ACE, we observed increase of signal for AP-1 presence in nuclear protein extracts as shown in Fig. 4A. The specificity of the observed DNA-protein interaction was confirmed with the ability of excess unlabeled AP-1 oligonucleotide (specific competitor) to inhibit binding in different concentrations, whereas the addition of nonspecific unlabeled oligonucleotide (SP-1 in our case) did not block the binding (Fig. 4A).



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  		Fig. 4. A: increase of transcriptional factor activator protein 1 (AP-1) levels by ACE in vascular smooth muscle cells (VSMC) compared with untreated control evaluated by electrophoretic mobility shift assay (EMSA) of nuclear extracts. Cells were treated with 3.75 µg/ml ACE for 3 h. Labeled AP-1 consensus sequence was competed with the fold molar excess of unlabeled AP-1 probe and with excess of unlabeled SP-1 probe. B: transcriptional factor cAMP response element binding protein (CREB) levels in nuclear extracts of VSMC under stimulation with ACE in VSMC compared with untreated control evaluated by EMSA of nuclear extracts. Cells were treated with 3.75 µg/ml ACE for 3 h. Labeled CREB consensus sequence was competed with the fold molar excess of unlabeled CREB probe and with excess of unlabeled SP-1 probe. C: induction of transcriptional factor NF-{kappa}B by ACE in VSMC compared with untreated control evaluated by EMSA of nuclear extracts. Cells were treated with 3.75 µg/ml ACE for 3 h. Labeled NF-{kappa}B consensus sequence was competed with the fold molar excess of unlabeled NF-{kappa}B probe and with excess of unlabeled SP-1 probe.

 
Studies of the promoter region for bradykinin B1 and B2 receptor gene suggest the presence of cAMP response element-binding protein site (25, 23). Therefore, we performed EMSA with nuclear extracts from control and ACE-treated VSMC and CREB DNA consensus sequence. As shown in Fig. 4B, there was no difference in CREB binding intensity between control and stimulated cells. As for AP-1 studies, CREB studies were confirmed by using specific unlabeled competitors and nonspecific (SP-1) unlabeled competitor.

The bradykinin B1 receptor gene is an easily inducible gene, and one of the transcriptional factors most likely to be involved is NF-{kappa}B. To explore the role of NF-{kappa}B in this phenomenon, we compared binding of nuclear protein from ACE-treated and ACE-nontreated cells for DNA consensus sequence corresponding to NF-{kappa}B binding site. ACE treatment resulted in markedly upregulated NF-{kappa}B DNA bindings compared with control (Fig. 4C). The result was confirmed by inhibiting the level of binding with different concentrations of unlabeled specific competitor and unlabeled nonspecific competitor (SP-1).

Intracellular cAMP levels in ACE-treated VSMC. As one of the most important second messengers in cellular signaling on activation of G protein-coupled receptors, cAMP was assayed as described in EXPERIMENTAL PROCEDURES. There was no difference in cellular levels of cAMP between cells that were not treated with ACE and cells that were treated with ACE. Additionally, we explored the possible time frame of cAMP activation in cells following the stimulation; again, there was no difference between a 20-min stimulation period versus a 3-h stimulation period (data not shown).

Bradykinin B1 and B2 receptor protein expression under ACE stimulation in VSMC. Western blot analysis of membrane protein extracts from rat VSMC with anti-B1 receptor antibodies revealed very low basal expression of bradykinin B1 receptor protein in the 45-kDa range. This signal was not significantly changed after ACE stimulation for 4 h in serum-free media. Densitometric analysis and normalization with equal amount protein loading of the immunoreactivity signal from protein extracts from treated and untreated cells showed no statistically significant differences in the level of protein expression between the two groups (data not shown).

Bradykinin B2 receptor was detected by using anti-B2 receptor antibodies, displaying a strong signal in the 42- to 50-kDa range. After densitometric analysis between two different groups of protein isolate, from ACE-stimulated and unstimulated cells, we concluded that the B2 receptor protein level was not altered (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURE
 REFERENCES
 
There are two isoforms of the ACE, both transcribed from the same gene (20). The somatic form is a large glycoprotein composed of a single polypeptide chain of about 1,300 amino acids and has been isolated from endothelial, pulmonary, and renal cells of various mammals, having an 80–90% identity between species. The germinal form, consisting of about 700 amino acids, which is almost identical to the COOH-terminal half of the somatic ACE, is only present in sperm cells of the testis (4). Although mainly bound on the surface of epithelial and endothelial cells, the ACE is also found in soluble form in many body fluids, where it is released via the proteolytic action of a membrane protein secretase (22). Whether membrane bound or in the extracellular fluid, the known role of the ACE is to hydrolyze a variety of short peptide substrates by removing a dipeptide from their COOH-terminal. Various carboxypeptidases participate in the release and metabolism of polypeptides and hormones in the circulation. Some are known to act intracellulary, either being endogenous or possibly entering the cell by receptor-mediated endocytosis, where their role seems to be processing of peptide hormones and influencing of signal transduction by acting on peptide ligands (32).

In the current experiments, we found that in vascular smooth muscle cell culture free from all plasma ingredients and their competing influences, the addition of exogenous ACE induced a highly significant upregulation of the gene expression of both the B1 and B2 receptors of bradykinin. This upregulation was more pronounced for the B1 receptor gene. Furthermore, we explored bradykinin B1 and B2 receptor expression on the cell membrane after 4 h of ACE stimulation. However, we found no changes in the protein level for these receptors. There are several possible explanations for this discrepancy, all of them speculative: they include the possibility that for B1 receptor, antibodies may not be sensitive and reliable enough as the B2 receptor antibodies (which are specific monoclonal antibodies against this receptor), or the possibility that upregulation of protein generation requires a longer interval of stimulation with ACE, or the possibility that the ACE acts to both initiate induction of gene expression and postranscriptionally downregulate gene activity, hence maintaining balance in protein generation.

The fact that ACE inhibitors did not interfere with this upregulation of B1 and B2 receptor genes suggests that this is an additional new function of the ACE, unrelated to its enzymatic properties as a peptidyl dipeptidase. Recently, it was reported that indeed ACE does exert another function that is independent of its activity as peptidyl dipeptidase: its ability to cleave glycosylphosphatidylinositol (GPI)-anchored protein (16). This new property was shown to play a major role in the fertilization process.

Further in our experiments, we showed that the presence or absence of other humoral factors, including selective AT1 or AT2 receptor antagonists, did not affect ACE-induced upregulation of bradykinin receptor gene expression. Blockade of angiotensin II receptors was necessary to prove that this effect was a direct action of the ACE and not an intracrine effect of locally generated angiotensin II.

We found particularly intriguing the fact that this normally extracellular or membrane-bound enzyme could markedly affect the gene expression of the receptors of one of its main substrates and therefore tried to elucidate further the underlying mechanism. The transcriptional inhibitor actinomycin D completely prevented ACE-induced upregulation of the B1 and B2 receptor gene, indicating that this was a transcriptionally regulated event rather than a posttranscriptional one. Another pharmacological inhibitor, curcumin, which interacts on the level of production and activation of the transcriptional factors (notably AP-1 and NF-{kappa}B), abolished increase in the mRNA level for the bradykinin B1 and B2 receptor genes. Curcumin is a widely used compound in vivo and in vitro that interferes with a broad range of intracellular signaling pathways (1). It has been shown to suppress the activation of NF-{kappa}B by possibly inhibiting a kinase involved in I{kappa}B{alpha} phosphorylation. Curcumin has also been shown to block AP-1 formation and action (13). The bradykinin B1 receptor gene was shown to have possibly two promoter regions, which have, among many others, potential binding sites for NF-{kappa}B and AP-1. Under stimulation with inflammatory stimuli and growth factors, the transcriptional factor NF-{kappa}B is usually activated and responsible for B1 gene upregulation (29, 21). Furthermore, an AP-1 binding site was shown to play an important role in B1 receptor gene upregulation (34). The B2 receptor gene promoter regions have been identified to have, among others, AP-1 binding sites, NF-{kappa}B binding sites, and CREB binding sites (25). cAMP activation by different stimuli was shown to modulate B2 receptor gene expression, possibly through CRE binding protein (23). EMSA performed with nuclear protein samples from ACE-treated VSMC showed increase of NF-{kappa}B and AP-1 nuclear accumulation compared with untreated cells, confirming indeed involvement of these two transcriptional factors in the ACE-mediated stimulation of B1 and B2 receptor genes expression. We also explored the possibility that CREB is increased under ACE stimulation but found no increase. VSMC were assayed for cAMP levels under ACE stimulation and presented no change, further excluding that particular pathway.

The discovery that ACE has another biological property in addition to its known enzymatic activity is intriguing. From the current experiments, we suggest that the ACE effect is exerted in the intracellular level through activation of NF-{kappa}B and AP-1 transcriptional factors, which seem to be involved in the increase of the expression of the bradykinin receptor genes. The level of transcriptional activation of these genes was very high and similar to that obtained by other inflammatory stimuli (i.e., LPS, TNF-{alpha}, and IL-1) as reported in the literature (21, 31, 24). It is tempting to speculate about the mechanisms involved. One possibility (which we are currently exploring) is the existence of a binding protein/receptor on the cell membrane surface that triggers the intracellular response of ACE stimulation. Another is that ACE acts intracellularly following endocytosis through the cell membrane perhaps by interacting with a currently unknown binding site responsible for endocytosis. In our studies, purified porcine kidney ACE was solubilized from kidney cells with Triton X-100, and hence it probably contains the COOH-terminal fragment (19) that represents a membrane anchor for this enzyme; therefore, we could assume that the enzyme itself gets reconstituted in the cell membrane under unknown conditions and as a result triggers an intracellular signaling cascade. A third possibility is that ACE might act via influence on another humoral factor unrelated to its peptidyl dipeptidase activity.

The ACE is already known to be one of the most widespread enzymes acting on a multitude of substrates in the extracellular space with new ones being discovered all the time. For example, in addition to its best known enzymatic effect (conversion of angiotensin I to angiotensin II and degradation of kinins), and its ability to cleave GPI-anchored protein (16), it has also been shown to act on a novel peptide with antifibrotic and antiproliferative properties (27). Moreover, a novel non-ACE-mediated property of the ACE inhibitors was recently described, the direct activation of the B1 receptor on the cell surface without an intermediate peptide ligand (12). The current data suggest that ACE has an additional property unrelated to its peptidyl dipeptidase activity. This novel biological activity involves activation of intracellular signaling cascades that produce increase in transcriptional factors NF-{kappa}B and AP-1 and result in upregulation of the bradykinin B1 and B2 receptor genes. The biological significance of this effect remains to be elucidated.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURE
 REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants R01 HL-58807 and P50 HL-55001.


   DISCLOSURE
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
REFERENCES
 
None of the authors reports conflict of interest.


   ACKNOWLEDGMENTS
 
Part of this work was presented at the American Society of Hypertension, 19th Annual Scientific Meeting, May 18–22, 2004, New York, NY.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Gavras, Hypertension & Atherosclerosis Section, Boston Univ. School of Medicine, 715 Albany St., Boston, MA (e-mail: hgavras{at}bu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 

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