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Am J Physiol Heart Circ Physiol 291: H3144-H3153, 2006. First published August 18, 2006; doi:10.1152/ajpheart.00327.2006
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Small cytoplasmic domain peptides of natriuretic peptide receptor-C attenuate cell proliferation through Gi{alpha} protein/MAP kinase/PI3-kinase/AKT pathways

Shehla Hashim, Yuan Li, and Madhu B. Anand-Srivastava

Department of Physiology and Groupe de recherche sur le système nerveux autonome, Faculty of Medicine, University of Montreal, Montreal, Quebec, Canada

Submitted 29 March 2006 ; accepted in final form 10 August 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
The present studies were undertaken to investigate the effect of C-atrial natriuretic peptide (ANP)4–23 and several peptide fragments containing 12 amino acids from different regions of the cytoplasmic domain of natriuretic peptide receptor (NPR)-C on cell proliferation in the absence or presence of angiotensin (ANG) II, endothelin (ET)-1, and arginine vasopressin (AVP) in A-10 vascular smooth muscle cells (VSMC). The peptide fragments used have either complete Gi activator sequences K461-H472 (peptide 1) and H481-H492 (peptide 3) or partial Gi activator sequences R469-K480 (peptide 2) and I465-H472 (peptide Y) with truncated COOH or NH2 terminus, respectively. The other peptide used had no structural specificity (Q473-K480, peptide X) or was the scrambled peptide control for peptide 1 (peptide Z). ANG II, ET-1 and AVP significantly stimulated DNA synthesis in these cells as determined by [3H]thymidine incorporation that was inhibited by peptides 1, 2, and 3 and not by peptides X, Y, and Z in a concentration-dependent manner, with an apparent Ki between 1 and 10 nM. In addition, C-ANP4–23, which interacts with NPR-C, also inhibited DNA synthesis stimulated by vasoactive peptides; however, the inhibition elicited by C-ANP4–23 was not additive with the inhibition elicited by peptide 1. On the other hand, basal DNA synthesis in these cells was not inhibited by C-ANP4–23 or the peptide fragments. Furthermore, vasoactive peptide-induced stimulation of DNA synthesis was inhibited by PD-98059 and wortmannin, and this inhibition was potentiated by peptide 1. In addition, peptide 1 also inhibited vasoactive peptide-induced phosphorylation of ERK1/2 and AKT and enhanced expression of Gi{alpha} proteins. These data suggest that C-ANP4–23 and small peptide fragments containing 12 amino acids irrespective of the region of the cytoplasmic domain of NPR-C inhibit proliferative responses of vasoactive peptides through Gi{alpha} protein and MAP kinase/phosphatidylinositol 3-kinase/AKT pathways.

vasoactive peptides; DNA synthesis; extracellular signal-regulated kinase 1/2; peptide 1; vascular smooth muscle cell

THE NATRIURETIC PEPTIDES (NP) are a family of three peptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (13, 43, 44). All of these peptides are produced in mammalian hearts, including human hearts (29, 49). ANP regulates a variety of physiological parameters including blood pressure, progesterone secretion, renin release, vasopressin release, and endothelin release by interacting with receptors on the plasma membrane either to alter the levels of second messengers such as cAMP (1, 2, 4, 5, 9, 10) and cGMP (20, 46, 47) or to affect ion channels (3). The other members of the NP family act as antagonists to vasopressin, endothelin, and the renin-angiotensin-aldosterone system (13, 38). CNP has also been shown to be a vasodilator produced by endothelial cells (44, 45). When compared with ANP, BNP has an additional six-amino acid sequence at its NH2-terminal end (13, 43), whereas CNP lacks the COOH-terminal extension (44).

Molecular cloning techniques revealed three subtypes of natriuretic peptide receptors (NPR): NPR-A (17, 31), NPR-B (16, 42), and NPR-C (8, 19). NPR-A and NPR-B are membrane guanylyl cyclases, whereas NPR-C (clearance receptor) lacks guanylyl cyclase activity. NPR-A catalyzes the production of cGMP in response to ANP and BNP, whereas NPR-B is the target for CNP. NPR-C is coupled to adenylyl cyclase inhibition through inhibitory guanine nucleotide-regulatory protein Gi (6, 8) or to activation of phospholipase C (PLC) (22). However, we recently (32) showed that NPR-C-mediated decrease in cAMP levels contributes to the activation of PLC signaling and suggested a cross talk between NPR-C-mediated adenylyl cyclase and PLC signaling pathways.

NPR-C is a disulfide-linked homodimer of 64–66 kDa having a single transmembrane domain (19, 26, 40), an extracellular domain of ~440 amino acids, and a short 37-amino acid cytoplasmic domain or tail. We previously demonstrated (7) that the 37-amino acid peptide (R37A) corresponding to the cytoplasmic domain of the NPR-C inhibited adenylyl cyclase activity in rat heart particulate fractions, which was completely blocked by the polyclonal rabbit antisera raised against R37A. The cytoplasmic domain of NPR-C contains several Gi activator sequences (34) characterized by the presence of two basic amino acids at the NH2 terminal and B-B-X-B or B-B-X-X-B at the COOH terminal, where B and X denote basic amino acid and non-basic amino acid, respectively. We further reported (34) that the synthetic peptide fragments of the cytoplasmic domain of NPR-C with complete or partial Gi activator sequence inhibited adenylyl cyclase activity in heart particulate fractions and A-10 vascular smooth muscle cells (VSMC), whereas the peptide fragments with no Gi activator sequences were unable to exert any inhibitory effect on adenylyl cyclase activity.

The present studies were undertaken to investigate whether small active peptide fragments of 12 amino acids of the cytoplasmic domain of NPR-C that have been shown to inhibit adenylyl cyclase could also modulate the proliferative effect of vasoactive peptides such as angiotensin (ANG) II, endothelin (ET)-1, and arginine vasopressin (AVP) in A-10 VSMC. For these studies, the effect of small peptides of NPR-C on cell proliferation determined by [3H]thymidine incorporation was examined in the absence or presence of ANG II, ET-1, and AVP in A-10 VSMC. We have shown for the first time that small peptide fragments containing 12 amino acids from different regions of the cytoplasmic domain of NPR-C with complete or partial Gi activator sequence inhibit vasoactive peptide-induced cell proliferation through Gi{alpha}/MAP kinase/phosphatidylinositol 3-kinase (PI3-kinase)/AKT pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
Materials

A ring-deleted analog of ANP, C-ANP4–23, was from Peninsula Laboratories (Belmont, CA). ANG II, AVP, and ET-1 were purchased from Sigma (St. Louis, MO). Peptides 1, 2, 3, X, Y, and Z were synthesized by standard solid-phase techniques and were highly purified (95–99%) by high-performance liquid chromatography (Peninsula Laboratories and Chiron Technologies). Monoclonal phospho-specific-Tyr204 ERK1/2 antibody, polyclonal pAKT1/2/3 (Ser473)-R antibody, and Western blotting reagents were from Santa Cruz Biotechnology (Santa Cruz, CA).

Methods

Cell culture. The A-10 cell line from rat embryonic thoracic aorta was obtained from the American Type Culture Collection (Manassas, VA). The cells were plated in 75-cm2 flasks and incubated at 37°C in a humidified 95% air-5% CO2 atmosphere in Dulbecco's modified Eagle's medium (DMEM) (with glucose, L-glutamine, and sodium bicarbonate) containing antibiotics and 10% heat-inactivated fetal calf serum (FCS) as described previously (36). The cells were passaged on reaching confluence with 0.5% trypsin containing 0.2% ethylenediaminetetraacetic acid (EDTA) and were utilized between passages 5 and 15.

Cell permeabilization. Cells, after being washed once with streptolysin O (SLO) buffer containing (mM) 200 HEPES, 50 NaCl, 140 KCl, 5 MgCl2 and 50 ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, pH 7.4), were incubated with SLO (0.8 U/ml) at 37°C for 5 min. The cells were washed with DMEM without FCS. The permeabilized cells were treated with peptides in the presence or absence of hormones for 24 h. For each experiment nonpermeabilized cells were used as a control group.

Cell lysis and Western blotting. Cells incubated in the absence or presence of various agents were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in 100 µl of buffer [mM: 25 Tris·HCl (pH 7.5), 25 NaCl, 1 Na orthovanadate, 10 Na fluoride, 10 Na pyrophosphate, 2 EGTA, 2 EDTA, and 1 phenylmethylsulfonyl fluoride, with 10 µg/ml aprotinin, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and 0.5 µg/ml leupeptin] on ice. The cell lysates were centrifuged at 12,000 g for 10 min at 4°C. Protein concentrations were measured with the Bradford assay. Equal amounts of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride membranes (Millipore), and incubated with respective primary antibodies: monoclonal phospho-specific-Tyr204-ERK1/2 antibody (1:2,000) or polyclonal pAKT1/2/3 (Ser473)-R antibody (1:1,000). The antigen-antibody complex was detected by a horseradish peroxidase-conjugated second antibody (1:400), and protein bands were visualized by enhanced chemiluminescence as described previously (35). Quantitative analysis of specific bands was performed by densitometric scanning of the autoradiographs with an enhanced laserdensitometer (LKB Ultroscan XL) and quantified using gel scan evaluation software (version 2.1) from Pharmacia.

[Methyl-3H]thymidine incorporation: DNA synthesis was evaluated by incorporation of [3H]thymidine into cells. Subconfluent A-10 VSMC were plated in 24-well plates for 24 h and were serum deprived for 24 h to induce cell quiescence. The cells were then incubated with ANG II, AVP, or ET-1 (10–7 M) alone or in the presence of small fragments of cytoplasmic domain of NPR-C (10–9–10–6 M) for 24 h with or without SLO treatment. [3H]thymidine (1 µCi) was added and further incubated for 4 h before the cells were harvested. The cells were rinsed twice with ice-cold PBS and incubated with 5% trichloroacetic acid for 1 h at 4°C. After being washed twice with ice-cold water, the cells were incubated with 0.4 N sodium hydroxide solution for 30 min at room temperature, and radioactivity was determined by liquid scintillation counter. Cell viability was checked with the trypan blue exclusion technique.

Pertussis toxin treatment. The serum-deprived cells were pretreated with or without pertussis toxin (PT; 500 ng/ml) for 0.5 h at 37°C. The cells were further incubated in the absence or presence of ANG II, AVP, or ET-1 (10–7 M) for 24 h and used for thymidine incorporation.

Statistical Analysis

Results are expressed as means ± SE. Comparisons between groups were made with ANOVA in conjunction with the Newman-Keuls test. Results were considered significant at a value of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANT
 REFERENCES
 
Effect of C-ANP4–23 on Vasoactive Peptide-Induced Cell Proliferation

The A-10 vascular smooth muscle cell line has been shown to demonstrate characteristics similar to those of VSMC (25) and has been used as a model to study vascular cellular process. We previously showed (11, 12, 35) the presence of NPR-C and its modulation by vasoactive peptides such as ANG II, ET-1, and AVP in these cells. To investigate whether ANG II, AVP, ET-1, and C-ANP4–23 could modulate cell proliferation in these cells, we examined the effect of these peptides on DNA synthesis. As shown in Fig. 1, AVP, ET-1, and ANG II stimulated DNA synthesis in these cells by ~70%, 50%, and 25%, respectively, as determined by [3H]thymidine incorporation. However, C-ANP4–23, a ring-deleted peptide that interacts specifically with NPR-C, did not affect basal DNA synthesis but significantly inhibited AVP-, ET-1-, and ANG II-stimulated DNA synthesis. For example, AVP-induced increased DNA synthesis was inhibited by ~65% by C-ANP4–23, whereas ET-1- and ANG II-induced increased DNA synthesis were almost abolished by C-ANP4–23.


Figure 1
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  		Fig. 1. Effect of C-atrial natriuretic peptide (ANP)4–23 on vasoactive peptide-induced thymidine incorporation in A-10 vascular smooth muscle cells (VSMC). A-10 VSMC were incubated in the absence (control) or presence of C-ANP4–23 (10–7 M) alone or in combination with angiotensin (ANG) II (10–7 M), arginine vasopressin (AVP; 10–7 M), or endothelin (ET)-1 (10–7 M) for 24 h. Thymidine incorporation was determined as described in EXPERIMENTAL PROCEDURES. Results are expressed as % of control (taken as 100%). Values are means ± SE of 3 separate experiments. §P < 0.05, §§P < 0.01 vs. control; **P < 0.01 vs. vasoactive peptide-induced thymidine incorporation.

 
Effect of Small Peptide Fragments of Cytoplasmic Domain of NPR-C on Vasoactive Peptide-Stimulated DNA Synthesis

Small peptide fragments containing 12 amino acids from different regions of the cytoplasmic domain of NPR-C with partial or complete Gi activator sequences that have been shown to inhibit adenylyl cyclase activity (34) were used to investigate whether these peptides could also mimic the effect of C-ANP4–23 on vasoactive peptide-induced DNA synthesis. The peptide fragments used represent different parts of the cytoplasmic domain as shown in Fig. 2. These consist of 12 amino acids (peptides 1, 2, 3, and Z) or 8 amino acids (peptides X and Y). Peptides 1 and 3 possess the required Gi activator sequences: two basic amino acids at the NH2 terminus and BBXB, BXB at the COOH terminus (where B represents a basic amino acid and X represents a non-basic amino acid), whereas peptide 2 has two basic amino acids at the NH2 terminus but does not have the consensus sequence at the COOH terminus. On the other hand, peptide Y has only the consensus sequence of the COOH terminus and peptide X lacks the Gi activator sequence, whereas peptide Z is the scrambled peptide and serves as the control for peptide 1.


Figure 2
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  		Fig. 2. Sequence of the entire cytoplasmic domain of natriuretic peptide receptor (NPR)-C and the various synthetic peptides corresponding to different regions of the cytoplasmic domain. B, basic amino acid; X, non-basic amino acid; aa, amino acids.

 
As shown in Fig. 3, peptides 1, 2, and 3 (10–7 M) inhibited ANG II-stimulated DNA synthesis (Fig. 3A) in a concentration-dependent manner in SLO-treated permeabilized VSMC. The maximal inhibition observed was ~60%; however, peptide 1 was more potent than peptides 2 and 3 and inhibited DNA synthesis with an apparent Ki of 5 nM, whereas a Ki of ~10 nM was observed for peptides 2 and 3. On the other hand, the scrambled peptide Z and peptides X and Y with no Gi activator sequences were unable to inhibit ANG II-stimulated DNA synthesis in A-10 VSMC. In addition, none of the peptides used had any effect on vasoactive peptide-stimulated cell proliferation in nonpermeabilized cells (data not shown).


Figure 3
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  		Fig. 3. Effect of different concentrations of small peptide fragments of cytoplasmic domain of NPR-C on vasoactive peptide-induced thymidine incorporation in permeabilized A-10 VSMC. Permeabilized A-10 VSMC were incubated in the presence of 10–7 M ANG II (A), 10–7 M AVP (B), and 10–7 M ET-1 (C) alone or in combination with different peptide fragments, and thymidine incorporation was determined as described in EXPERIMENTAL PROCEDURES. Results are expressed as % of control (taken as 100%). Values are means ± SE of 3 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

 
Similarly, AVP-stimulated DNA synthesis was also inhibited in a concentration-dependent manner by peptides 1, 2, and 3. The three peptides were almost equipotent and inhibited DNA synthesis with an apparent Ki of ~1 nM; however, peptide 1 inhibited DNA synthesis by ~45%, whereas ~30% inhibition was observed for peptides 2 and 3 (Fig. 3B). On the other hand, the scrambled peptide Z and inactive peptides (X and Y) without Gi activator sequences did not inhibit AVP-induced DNA synthesis in A-10 VSMC. In addition, peptides 1, 2, and 3 but not X, Y, and Z were also able to inhibit ET-1-stimulated DNA synthesis in a concentration-dependent manner (Fig. 3C). The maximal inhibition of DNA synthesis by peptide 1 was ~60% and by peptides 2 and 3 was ~30% with an optimal Ki between 5 and 10 nM. However, the peptide fragments did not have any effect on basal DNA synthesis (data not shown).

To investigate whether NPR-C ligand C-ANP4–23 and NPR-C peptide 1 act via the same pathway, the effect of both C-ANP4–23 (10–7 M) and peptide 1 (10–7 M) on AVP-induced enhanced DNA synthesis was examined together. The results shown in Fig. 4 indicate that C-ANP4–23 as well as peptide 1 inhibited AVP-stimulated DNA synthesis by ~30%; however, no additive effect was observed when both ligands were added together.


Figure 4
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  		Fig. 4. Effect of C-ANP4–23 and peptide 1 on AVP-induced thymidine incorporation in permeabilized A-10 VSMC. Permeabilized A-10 VSMC were incubated in the presence of 10–7 M AVP alone or in combination with 10–7 M C-ANP4–23, 10–7 M peptide 1 (Pep-1) or both, and thymidine incorporation was determined as described in EXPERIMENTAL PROCEDURES. Results are expressed as % of control (CTL, taken as 100%). Values are means ± SE of 3 separate experiments. **P < 0.01, ***P < 0.001.

 
Implication of MAP Kinase in Vasoactive Peptide-Stimulated Cell Proliferation

Since C-ANP4–23 has been shown to inhibit cell proliferation through the MAP kinase pathway in astrocytes (37), it was of interest to determine whether the inhibition of the vasoactive peptide-stimulated VSMC proliferation by the peptide fragments of cytoplasmic domain of NPR-C is also mediated through the MAP kinase pathway. For these studies, the effect of peptide 1 on vasoactive peptide-stimulated ERK1/2 phosphorylation was examined in A-10 VSMC. The results shown in Fig. 5 indicate that ANG II, AVP, and ET-1 significantly enhanced the ERK1/2 phosphorylation by ~40%, 40%, and 50% (n = 6), respectively, which was completely abolished by peptide 1. However, peptide 1 alone did not have any significant effect on ERK1/2 phosphorylation in these cells. In addition, PD-98059, a MEK inhibitor, also abolished completely the ERK1/2 phosphorylation stimulated by all three vasoactive peptides (data not shown). These results suggest that peptide 1 may inhibit MAP kinase activity through a mechanism similar to that of PD-98059. To investigate this possibility, the effect of PD-98059 on vasoactive peptide-induced DNA synthesis was examined in the absence or presence of peptide 1. The results shown in Fig. 6 indicate that peptide 1 inhibited ANG II (Fig. 6A)-, AVP (Fig. 6B)-, and ET-1 (Fig. 6C)-induced DNA synthesis by ~65%, 50%, and 80%, respectively. PD-98059 at 10 µM, on the other hand, completely inhibited the stimulated DNA synthesis, and this inhibition was further potentiated by peptide 1, reaching below control levels. These results suggest that peptide 1-induced inhibition of cell proliferation may also involve some other mechanisms in addition to the MAP kinase pathway.


Figure 5
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  		Fig. 5. Effect of peptide 1 on vasoactive peptide-induced ERK1/2 phosphorylation of in permeabilized A-10 VSMC. Permeabilized A-10 VSMC were pretreated in the presence of 10–7 M ANG II, 10–7 M AVP, or 10–7 M ET-1 alone or in combination with peptide 1. Phosphorylated (p-)ERK1/2 protein expression was determined as described in EXPERIMENTAL PROCEDURES. Results are expressed as % of control (taken as 100%). Values are means ± SE of 6 separate experiments. NS, no significant difference. **P < 0.01, *** P < 0.001 vs. control; {dagger}{dagger}{dagger}P < 0.001.

 

Figure 6
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  		Fig. 6. Effect of PD-98059 (PD) on vasoactive peptide-induced thymidine incorporation in the absence or presence of peptide 1 in permeabilized A-10 VSMC. Permeabilized A-10 VSMC were pretreated in the absence or presence of PD-98059 for 30 min and were further incubated in the presence of 10–7 M ANG II (A), 10–7 M AVP (B), or 10–7 M ET-1 (C) alone or in combination with peptide 1, and thymidine incorporation was determined as described in EXPERIMENTAL PROCEDURES. Results are expressed as % of control (taken as 100%). Values are means ± SE of 3 separate experiments. §P < 0.05, §§P < 0.01 vs. control; *P < 0.05, **P < 0.01, ***P < 0.001.

 
Implication of Gi{alpha} Protein in Vasoactive Peptide-Stimulated Cell Proliferation

Gi{alpha} proteins have been shown to be implicated in cell proliferation (33). We recently showed (30) that the enhanced cell proliferation of VSMC from spontaneously hypertensive rats (SHR) was attributed to the enhanced levels of Gi{alpha} proteins, because the treatment of VSMC from SHR with PT restored the enhanced cell proliferation to control levels. To investigate whether the Gi{alpha} protein is also implicated in vasoactive peptide-induced enhanced cell proliferation, the effect of PT treatment on ANG II-, AVP-, and ET-1-induced DNA synthesis was examined, and the results are shown in Fig. 7. As reported above, ANG II, AVP, and ET-1 increased DNA synthesis to various degrees, and this increase was significantly attenuated by PT. Thus it may be possible that the additive antiproliferative effect of peptide 1 and MAP kinase inhibitor PD-98059 on vasoactive peptide-stimulated proliferation of VSMC (Fig. 6) may be attributed to an ability to attenuate vasoactive peptide-induced increased expression of Gi{alpha} proteins. To investigate this possibility, we examined the effect of peptide 1 on the expression of Gi{alpha} proteins, and the results are shown in Fig. 8. As reported previously (11), ET-1 increased the expression of Gi{alpha}-2 and Gi{alpha}-3 protein by ~45% and 30%, which was completely abolished by peptide 1. However, peptide 1 did not alter the expression of Gi{alpha} proteins in control cells. A similar effect of peptide 1 on AVP- and ANG II-induced increased expression of Gi{alpha} protein was observed (data not shown). It may be possible that peptide 1-induced decreased expression of Gi{alpha} protein results in decreased ERK1/2 phosphorylation and thereby decreased cell proliferation. To investigate the relationship between the Gi{alpha} protein and MAP kinase activation, we tested the effect of PT on ANG II-induced increased ERK1/2 phosphorylation. The results shown in Fig. 9 indicate that PT treatment was ineffective in altering ANG II-induced increased phosphorylation of ERK1/2, suggesting that Gi{alpha} protein is not implicated in MAP kinase activation. However, ERK1/2 phosphorylation and not AKT phosphorylation was inhibited by PD-98059, whereas wortmannin, an inhibitor of PI3-kinase, was able to inhibit phospho-AKT and not phospho-ERK1/2 in these cells.


Figure 7
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  		Fig. 7. Effect of pertussis toxin (PT) treatment on vasoactive peptide-induced thymidine incorporation in A-10 VSMC. A-10 VSMC were pretreated in the absence or presence of PT as described in EXPERIMENTAL PROCEDURES and were further incubated with 10–7 M ANG II (A), 10–7 M AVP (B), or 10–7 M ET-1 (C), and thymidine incorporation was determined as described in EXPERIMENTAL PROCEDURES. Results are expressed as % of control (taken as 100%). Values are means ± SE of 3 separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

 

Figure 8
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  		Fig. 8. Effect of peptide 1 on ET-1-induced increased expression of Gi{alpha} proteins in A-10 VSMC. Permeabilized A-10 VSMC were pretreated in the absence or presence of 0.1 µM ET-1 alone or in combination with 0.1 µM peptide 1 for 24 h. The cell lysates were used for Western blotting as described in EXPERIMENTAL PROCEDURES. Results are expressed as % of control (taken as 100%). Values are means ± SE of 3 separate experiments. *P < 0.05, **P < 0.01.

 

Figure 9
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  		Fig. 9. Effect of PT, MEK inhibitor (PD-98059), and phosphatidylinositol 3-kinase (PI3-kinase) inhibitor (wortmannin) on ANG II-induced enhanced ERK1/2 phosphorylation (left) and AKT phosphorylation (right) in A-10 VSMC. A-10 VSMC were pretreated in the absence or presence of 500 ng/ml PT, 10 µM PD-98059, and 5 µM wortmannin (WM) for 30 min and then challenged with 10–7 M ANG II for 15 min. Phosphorylated ERK1/2 and AKT protein expression was determined as described in EXPERIMENTAL PROCEDURES. Results are expressed as % of control (taken as 100%). Values are means ± SE of 3 experiments. ***P < 0.001 vs. control; §§§P < 0.001.

 
Implication of PI3-Kinase/AKT in Peptide 1-Induced Attenuation of Vasoactive Peptide-Stimulated Cell Proliferation

Because PI3-kinase is an upstream regulator of the ERK1/2 activation cascade and has been shown to be activated by ANG II (39), it was of interest to assess whether the PI3-kinase pathway is also implicated in mediating the peptide 1-induced inhibition of vasoactive peptide-stimulated cell proliferation of A-10 cells. The results shown in Fig. 10 indicate that wortmannin and peptide 1 inhibited ANG II-, AVP-, and ET-1-stimulated cell proliferation to various degrees. However, when the effect of both wortmannin and peptide 1 was examined together, the inhibition was potentiated further. Since AKT/PKB is a downstream signaling molecule of PI3-kinase (18), it was of interest to investigate whether peptide 1 could inhibit the activity of AKT/PKB. The results shown in Fig. 11 demonstrate that ANG II, AVP, and ET-1 (but not peptide 1) enhanced the phosphorylation of AKT1/2 by ~80%, 40%, and 30%, respectively. Peptide 1, on the other hand, completely abolished the AVP- and ET-1-induced augmented phosphorylation of AKT1/2, whereas ~85% attenuation of ANG II-induced enhanced AKT1/2 phosphorylation was observed.


Figure 10
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  		Fig. 10. Effect of wortmannin on vasoactive peptide-induced thymidine incorporation in the absence or presence of peptide-1 in permeabilized A-10 VSMC. Permeabilized A-10 VSMC were pretreated in the absence or presence of wortmannin for 30 min and were further incubated in the presence of 10–7 M ANG II (A), 10–7 M AVP (B), or 10–7 M ET-1 (C) alone or in combination with peptide 1, and thymidine incorporation was determined as described in EXPERIMENTAL PROCEDURES. Results are expressed as % of control (taken as 100%). Values are means ± SE of 3 separate experiments. §P < 0.05, §§P < 0.01 vs. control; *P < 0.05, **P < 0.01, ***P < 0.001.

 

Figure 11
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  		Fig. 11. Effect of peptide 1 on vasoactive peptide-induced AKT1/2 phosphorylation in permeabilized A-10 VSMC. Permeabilized A-10 VSMC were pretreated in the presence of 10–7 M ANG II, 10–7 M AVP, or 10–7 M ET-1 alone or in combination with peptide 1. Phosphorylated AKT1/2 protein expression was determined as described in EXPERIMENTAL PROCEDURES. Results are expressed as % of control (taken as 100%). Values are means ± SE of 3 separate experiments. **P < 0.01, ***P < 0.001 vs. control; {delta}{delta}P < 0.01, {delta}{delta}{delta}P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANT
 REFERENCES
 
NPs have been shown to inhibit the proliferation of several cell types including VSMC (14, 23, 28). Prins et al. (37) showed that C-ANP4–23 inhibits mitogenic factor-stimulated cell proliferation of astrocytes via the MAP kinase pathway. Recently, Lelièvre et al. (27) reported proliferative and antiproliferative action of ANP in neuroblastoma cells; lower concentrations of ANP and CNP stimulate cell proliferation in a cGMP-dependent mechanism, and higher concentrations of ANP and CNP and low concentration of C-ANP4–23 exert antiproliferative action mediated through the MAP kinase pathway. However, the present studies demonstrate for the first time that the small peptide fragments of cytoplasmic domain containing 12 amino acids of NPR-C having complete or partial Gi activator sequences inhibit vasoactive peptide-induced cell proliferation in A-10 VSMC, but these peptides did not have any effect on basal cell proliferation. The antiproliferative effect of peptide 1 may not be attributed to apoptosis, because cell viability checked by trypan blue exclusion indicated that >90–95% cells were viable and, second, peptide 1 did not inhibit DNA synthesis in control cells.

The small peptide fragments of the cytoplasmic domain of NPR-C containing 12 amino acids from different regions, NH2-terminal K461-H472 (peptide 1), middle region R469-R480 (peptide 2), and COOH-terminal H481-H492 (peptide 3) with consensus sequence for Gi activation at both NH2 and COOH termini that have been reported to inhibit adenylyl cyclase activity (34), inhibited cell proliferation stimulated by ANG II, AVP, and ET-1 in A-10 VSMC. As shown previously (34), the inhibitory effect of these peptides on cell proliferation was also not due to the net positive charge present (i.e., amino acid composition), since the scrambled peptide Z with the same composition as that of peptide 1, but lacking the Gi activator sequence at the NH2 and COOH termini, did not inhibit vasoactive peptide-induced cell proliferation in these cells. On the other hand, the absence or presence of partial COOH-terminal motif (BXB) but intact NH2-terminal motif (BB) in peptides 2 and 3, respectively, did not significantly change the potency of the peptide to inhibit cell proliferation, suggesting that a COOH-terminal motif in the peptide may not be required to exert an inhibitory effect on cell proliferation. These results are in agreement with the studies of Kanwal et al. (24), who showed that a 15-amino acid peptide fragment of NPR-C (Arg1-Gln15) lacking the COOH-terminal motif attenuated dopamine efflux in pheochromocytoma cells (PC12). However, the truncation of the NH2-terminal motif of a peptide (peptide Y) that has been shown to inactivate the peptide to inhibit adenylyl cyclase (34) was also unable to inhibit cell proliferation stimulated by ANG II, AVP, and ET-1 in A-10 cells, suggesting that the NH2-terminal motif is important for the activation of the peptide and to exert physiological functions. Our results showing that C-ANP4–23, like small peptide fragments, also inhibited vasoactive peptide-induced cell proliferation in A-10 VSMC without affecting basal levels are in accordance with the studies of other investigators (14) who were also unable to show any effect of C-ANP4–23 on basal cell proliferation. In addition, our results showing that the inhibitory effects of C-ANP4–23 and peptide 1 on AVP-induced DNA synthesis were not additive further suggest that both NPR-C ligand and NPR-C peptide 1 act via the same pathway.

A role of cAMP in cell proliferation has been reported (21, 41); however, cAMP, which has been shown to be decreased by C-ANP4–23 as well as by small peptide fragment in A-10 VSMC (34), does not appear to contribute to the antiproliferative effect of the peptides, because decreased cAMP levels have been reported to cause hyperproliferation of the cells (21, 41). This notion is supported by the studies of other investigators who have also shown that C-ANP4–23 inhibits cell proliferation by an cAMP-independent mechanism (37). The implication of MAP kinase and PI3-kinase in cell proliferation has been reported (48). Our results showing that peptide 1 attenuated the vasoactive peptide-induced enhanced ERK1/2 and AKT phosphorylation suggest that the antiproliferative effect of peptide 1 may also be mediated through the MAP kinase/PI3-kinase/AKT signaling pathway. However, the fact that peptide 1 further potentiated the inhibitory effect of the MEK inhibitor PD-98059 and the PI3-kinase inhibitor wortmannin on vasoactive peptide-induced cell proliferation suggests that mechanisms other than MAP kinase and PI3-kinase signaling may also contribute to the antiproliferative effect of peptide 1. In this regard, we have shown that treatment of A-10 cells with peptide 1 resulted in the decreased expression of Gi{alpha} protein stimulated by vasoactive peptides, which may also be responsible for the antiproliferative effect of peptide 1. This notion is substantiated by our studies showing that PT treatment that inactivates Gi{alpha} proteins attenuated vasoactive peptide-evoked enhanced cell proliferation in A-10 cells. The implication of Gi{alpha} protein in the cell proliferation has also been demonstrated by other investigators (30, 33). On the other hand, the peptide 1-induced decreased expression of Gi{alpha} protein may not be responsible for the decreased ERK1/2 phosphorylation, because PT treatment was ineffective in decreasing ANG II-induced enhanced ERK1/2 phosphorylation in A-10 VSMC. The implication of MAP kinase signaling in ANG II-induced enhanced expression of Gi{alpha} protein in A-10 VSMC has been reported (15). However, the results showing that PT treatment inhibits ANG II-induced Gi protein expression, DNA synthesis but not ERK1/2 phosphorylation suggest that MAP kinase-induced increased DNA synthesis may not be mediated through Gi proteins. In light of these findings, it may be suggested that peptide 1-induced inhibition of both MAP kinase/PI3-kinase/AKT signaling and decreased expression of Gi{alpha} protein may be responsible for the antiproliferative effect of peptide 1 (Fig. 12).


Figure 12
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  		Fig. 12. Schematic model showing potential mechanisms responsible for the antiproliferative effect of small peptide fragment of cytoplasmic domain of NPR-C receptor (peptide 1). Vasoactive peptide (ANG II, ET-1, and AVP)-induced enhanced expression of Gi{alpha} protein as well as ERK1/2 and AKT activation contribute to the enhanced cell proliferation (DNA synthesis) of VSMC. Peptide 1 exerts an antiproliferative effect through its ability to attenuate the enhanced Gi{alpha} protein expression as well as ERK1/2 and AKT activity. Solid lines denote activation; dashed lines denote inhibition. PI3K, PI3-kinase.

 
In conclusion, we have provided the first evidence to demonstrate that the short cytoplasmic domain peptide consisting of 12 amino acids with Gi activator sequence irrespective of the region of NPR-C inhibits the proliferative responses of vasoactive peptides through Gi{alpha} protein, MAP kinase, and PI3-kinase/AKT signaling mechanisms in A-10 VSMC. From these studies, it may be suggested that the antimitogenic properties of the small peptide fragments of NPR-C can be used in designing the therapies in the treatment of cardiovascular complications.


   GRANT
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANT
REFERENCES
 
This study was supported by a grant from the Canadian Institutes of Health Research (MOP 13661).


   ACKNOWLEDGMENTS
 
We thank Christiane Laurier for valuable secretarial help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. B. Anand-Srivastava, Dept. of Physiology, Faculty of Medicine, Univ. of Montreal, C.P. 6128, Succ. Centre-ville, Montreal, QC, Canada H3C 3J7 (e-mail: madhu.anand-srivastava{at}umontreal.ca)

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
 GRANT
 REFERENCES
 

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