©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of p70 S6 Protein Kinase in Angiotensin II-induced Protein Synthesis in Vascular Smooth Muscle Cells (*)

(Received for publication, June 7, 1994; and in revised form, November 7, 1994)

Edith Giasson Sylvain Meloche (§)

From the Centre de Recherche, Hôtel-Dieu de Montréal and the Department of Pharmacology, University of Montreal, Montreal, Quebec H2W 1T8, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Angiotensin II (AII) is a growth factor which induces cellular hypertrophy in cultured vascular smooth muscle cells (SMC). To understand the molecular basis of this action, we have examined the role of the 70-kDa S6 kinases (p70) in the hypertrophic response to AII in aortic SMC. AII potently stimulated the phosphotransferase activity of p70, which reached a maximal value at 15 min and persisted for at least 4 h. This response was completely abolished when the cells were incubated in the presence of the AT(1)-selective receptor antagonist losartan. The enzymatic activation of p70 was associated with increased phosphorylation of the enzyme on serine and threonine residues. The immunosuppressant drug rapamycin was found to selectively inhibit the activation of p70 by AII, but not the activation of mitogen-activated protein kinase or the induction of c-fos mRNA expression. Treatment of aortic SMC with rapamycin also potently inhibited AII-stimulated protein synthesis with a half-maximal concentration similar to that required for inhibition of p70. These results provide strong evidence that p70 plays a critical role in the signaling pathways by which AII induces hypertrophy of vascular SMC.


INTRODUCTION

Angiotensin II (AII) (^1)is a peptide hormone that evokes a wide range of biological responses, including arteriolar vasoconstriction, stimulation of aldosterone secretion, and renal sodium reabsorption(1) . In addition, AII is a growth factor for diverse cell types, such as fibroblasts, adrenocortical cells, vascular SMC, and cardiac myocytes(2, 3) . In cultured aortic SMC derived from normal rats, AII induces cell hypertrophy as a result of increased protein synthesis, but not cell proliferation(4, 5, 6, 7) . This trophic response is associated with increased expression of mRNAs for early growth response genes such as c-fos and c-myc(8, 9, 10) .

AII exerts its physiological effects by interacting with two pharmacologically distinct subtypes of receptors, designated AT(1) and AT(2) (for review, see (11) and (12) ). Rat vascular tissues express predominantly the AT(1) subtype, although the rat aorta contains a small proportion of AT(2) receptors(13, 14) . Most of the major in vitro and in vivo responses to AII are mediated by AT(1) receptors (reviewed in Refs. 11, 12, and 15). The AT(1) subtype is also responsible for the growth promoting effects of AII in cultured cells (16, 17) . Both AT(1) and AT(2) receptors belong to the superfamily of seven transmembrane domain receptors, although G protein coupling has not been demonstrated for the AT(2) receptor(18, 19, 20, 21) . Activation of the AT(1) receptor triggers various G protein-mediated signaling pathways, including stimulation of phospholipases C, D, and A(2) and inhibition of adenylyl cyclase(12) . Furthermore, in common with other G protein agonists, AII stimulates tyrosine phosphorylation of multiple substrates in target cells(22, 23, 24) . (^2)However, the molecular basis of the hypertrophic response to AII is still largely unknown.

A common response of cells to both mitogenic and hypertrophic factors is the activation of protein synthesis. This event is thought to be controlled in part by multiple phosphorylation of the ribosomal protein S6(25, 26, 27) . Phosphorylation of S6 at five serine residues, all clustered at the carboxyl end of the protein, is correlated with an activation of protein synthesis at the level of initiation. Much effort has been directed toward identification of the enzymes controlling S6 phosphorylation and elucidation of their mode of regulation. Two distinct families of growth factor-regulated S6 kinases have been characterized at the molecular level: the 90-kDa S6 kinase family, referred to as p90(28, 29) ; and the 70-kDa S6 kinase family, referred to as p70(30, 31) . Both classes of S6 kinases are activated by serine/threonine phosphorylation, but their activities are regulated by distinct signaling pathways(27, 32, 33) . The p90 is phosphorylated and activated by the MAP kinase isoforms p42 and p44, which are themselves regulated by complex protein kinase cascades (for recent reviews, see (34, 35, 36) ). The upstream components responsible for p70 activation have not yet been identified. While both the p90 and the p70 S6 kinases are able to phosphorylate the 40 S ribosomal protein S6 in vitro, a number of experimental results indicate that p70 is the major S6 kinase in vivo. Indeed, the p70 is highly specific for S6, whereas p90 has wide substrate specificity(33, 37) . Additionally, inhibition of protein synthesis, which leads to S6 phosphorylation, is accompanied by activation of p70 but not p90(38) . Finally, the immunosuppressant drug rapamycin, which blocks serum-stimulated S6 phosphorylation, inhibits activation of p70 by mitogenic factors but has no effect on the activity of p90(39, 40, 41, 42) .

The aim of this study was to evaluate the involvement of p70 in the hypertrophic effect of AII on rat aortic SMC. We show that AII stimulates the phosphorylation and enzymatic activation of p70 in aortic SMC. In addition, we demonstrate that p70 activity may play a critical role in the signaling pathway leading to AII-stimulated protein synthesis.


EXPERIMENTAL PROCEDURES

Materials

AII and [Sar^1,Ile^8]AII were purchased from Hukabel Scientific. The receptor antagonists losartan and PD123319 were generous gifts of Du Pont Merck and Parke-Davis, respectively. [-P]ATP and [P]phosphoric acid were obtained from Amersham Corp. Protein A-Sepharose was obtained from Pharmacia Biotech Inc. Protease inhibitors and bovine MBP were obtained from Sigma. [^3H]Leucine and [^3H]thymidine were obtained from ICN. Rapamycin was a gift of Wyeth-Ayerst Research and was dissolved in ethanol to give 5 mg/ml stock solution. The p70 antiserum was raised against a synthetic peptide corresponding to amino acids 2-30 of rat p70(30) and was generously provided by Dr. Frederick Hall (Children's Hospital of Los Angeles). Antiserum S6K-III was from Upstate Biotechnology Inc. Antiserum SM1, produced by immunization of rabbits with purified recombinant glutathione S-transferase-p44 fusion protein, specifically immunoprecipitates p44(43) .

Cell Culture

Vascular SMC were isolated from the thoracic aortas of 12-week-old male Brown-Norway rats by an explant procedure as described(44) . Cells were grown in low glucose DMEM supplemented with 10% calf serum, 2 mML-glutamine, and antibiotics (50 units/ml penicillin and 50 µg/ml streptomycin). Cultures were maintained at 37 °C in a humidified atmosphere of 95% air, 5% CO(2). All experiments were conducted on cells at passage levels 9-14. Quiescent aortic SMC were obtained by incubation of 95% confluent cell cultures in serum-free DMEM-Ham's F-12 (1:1) supplemented with 15 mM Hepes (pH 7.4), 0.1% bovine serum albumin, and 5 µg/ml transferrin for 48 h. For experiments with rapamycin, the cells were treated with vehicle alone or with the indicated concentrations of rapamycin for 30 min before addition of AII.

Immune Complex Kinase Assay of p70

Quiescent rat aortic SMC in 60-mm dishes were stimulated with AII for the indicated times at 37 °C. The cells were then washed twice with ice-cold PBS and lysed in 0.4 ml of Triton X-100 lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 10M phenylmethylsulfonyl fluoride, 10M leupeptin, 10M pepstatin A, 1% Triton X-100) for 25 min at 4 °C. Cell lysates were clarified by centrifugation at 13,000 times g for 10 min at 4 °C, and normalized amounts of lysate proteins (100 µg) were incubated for 4 h at 4 °C with 2 µl of p70 antiserum preadsorbed to protein A-Sepharose beads. The immune complexes were washed three times with lysis buffer and once with S6 kinase assay buffer (20 mM Hepes, pH 7.4, 10 mM MgCl(2), 1 mM dithiothreitol, 10 mM beta-glycerophosphate). The beads were then resuspended in 25 µl of S6 kinase assay buffer containing 0.2 mM of the S6 peptide RRRLSSLRA (Upstate Biotechnology), 20 µM ATP, and 5 µCi of [-P]ATP (5,000 Ci/mmol). The reactions were initiated by the addition of ATP, incubated at 30 °C for 20 min, and terminated by applying the mixture to phosphocellulose P-81 paper (Whatman). The filters were washed four times for 15 min with 500 ml of 1% phosphoric acid, two times with distilled water, once in ethanol, and counted in a liquid scintillation counter. Blank reactions were processed identically in the absence of peptide substrate. Protein kinase activities are expressed as picomoles of phosphate incorporated into the S6 substrate/min/mg of lysate protein. The reaction was linear over the time of the assay and within the range of protein concentrations used.

Assay of p44 Activity

The activity of p44 was analyzed by an immune complex kinase assay as described(43, 45) . Quiescent aortic SMC were stimulated with 100 nM AII for 5 min, washed twice with ice-cold PBS, and lysed in 0.4 ml of Triton X-100 lysis buffer for 25 min at 4 °C. Insoluble material was removed by centrifugation (13,000 times g for 10 min), and 100 µg of lysate proteins were incubated for 4 h at 4 °C with 5 µl of p44 antiserum SM1 preadsorbed to protein A-Sepharose beads. The beads were washed three times with lysis buffer and once with MAP kinase assay buffer (20 mM Hepes, pH 7.4, 10 mM MgCl(2), 1 mM dithiothreitol, 10 mMp-nitrophenylphosphate). MBP kinase activity was assayed by resuspending the beads in a total volume of 40 µl of MAP kinase assay buffer containing 0.25 mg/ml MBP, 50 µM ATP, and 5 µCi of [-P]ATP. Reactions were initiated with ATP, incubated at 30 °C for 10 min (linear assay conditions), and stopped by addition of 2 times Laemmli sample buffer. The samples were analyzed by SDS-gel electrophoresis on 12% acrylamide gels, and the band corresponding to MBP was excised and counted. Protein kinase activities are expressed as picomoles of phosphate incorporated into MBP/min/mg of lysate protein.

P Labeling and Immunoprecipitation

Quiescent rat aortic SMC in 100-mm dishes were metabolically labeled for 5 h at 37 °C in bicarbonate and phosphate-free Hepes-buffered DMEM containing 0.5 mCi/ml [P]phosphoric acid. The cells were stimulated by addition of AII to the medium for 15 min and quickly washed with ice-cold PBS. Cell lysates were prepared as described above. The lysates were then precleared for 1 h with 5 µl of normal rabbit serum and incubated for 4 h at 4 °C with 5 µl of p70 antiserum preadsorbed to protein A-Sepharose beads. Immune complexes were washed six times with Triton X-100 lysis buffer, once with 100 mM Tris-HCl (pH 7.4), 500 mM LiCl, 40 mM beta-glycerophosphate, 200 µM sodium orthovanadate, and once with 10 mM Tris-HCl (pH 7.4), 40 mM beta-glycerophosphate, 200 µM sodium orthovanadate. Protein complexes were eluted by heating at 95 °C for 5 min in denaturing sample buffer and analyzed by SDS-gel electrophoresis on 10% acrylamide gels. The proteins were then electrophoretically transferred to PVDF membranes (Millipore) in 25 mM Tris, 192 mM glycine, 20% methanol and visualized by autoradiography.

Immunoblot Analysis of p70

Cell lysates were prepared and equal amounts of lysate proteins were subjected to immunoprecipitation with p70 antiserum as described above. The proteins were resolved on 7.5% acrylamide gels and electrophoretically transferred to Hybond-C nitrocellulose membranes (Amersham Corp.) in 25 mM Tris, 192 mM glycine. After fixation for 15 min in 40% methanol, 7% acetic acid, 3% glycerol, the membrane was blocked for 1 h at 25 °C in TBS containing 3% nonfat dry milk. The membrane was then incubated overnight at 4 °C with antiserum S6K-III (1 µg/ml) in blocking solution. The membrane was washed with TBS, 0.05% Tween 20 prior to incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 1:10000 in blocking solution. After washing as above, the membrane was developed using enhanced chemiluminescence (Amersham Corp.).

Phosphoamino Acid Analysis

The p70 was immunoprecipitated from P-labeled cell lysates as described above. The proteins were resolved on 10% acrylamide gels and transferred to PVDF membranes. Following brief exposure of the membrane, the labeled bands corresponding to p70 and p85 were excised from the membrane and subjected to partial acid hydrolysis in 5.7 M HCl for 1 h at 110 °C (46) . The supernatants were lyophilized and resuspended in pH 1.9 electrophoresis buffer containing cold phosphoamino acid standards (0.2 mg/ml each of phosphoserine, phosphothreonine, and phosphotyrosine). The phosphoamino acids were separated by thin layer electrophoresis at pH 1.9 for 30 min in the first dimension and pH 3.5 for 20 min in the second dimension(47) . The plates were exposed by autoradiography.

Northern Blot Analysis

Quiescent rat aortic SMC in 150-mm dishes were stimulated with 100 nM AII for 30 min at 37 °C. Total RNA was extracted with guanidinium thyocyanate as described(48) . Equal amounts of total RNA (10 µg) were denatured by heating for 15 min at 65 °C in 2.2 M formaldehyde and 50% formamide and resolved by electrophoresis in a 1% agarose gel containing 1.8% formaldehyde. The RNA was transferred to Hybond N (Amersham Corp.) nylon membranes by vacuum blotting, fixed, and hybridized with P-labeled c-fos cDNA. Hybridization was carried out in hybridization medium (5 times SSC (1 times SSC = 150 mM NaCl, 15 mM sodium citrate), 0.1% SDS, 5 times Denhardt's solution (1 times Denhardt's = 0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin), 50% formamide, and 50 µg/ml herring sperm DNA) containing the labeled probe (1 times 10^6 cpm/ml) for 16 h at 42 °C. The membranes were washed twice at 25 °C for 15 min in 2 times SSC, 0.1% SDS, once at 60 °C for 30 min in 2 times SSC, 0.1% SDS, and once at 60 °C for 30 min in 0.5 times SSC, 0.1% SDS. The extent of hybridization was visualized by autoradiography and quantitated by laser densitometry of the corresponding autoradiograms. The results were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA.

The probes used were a 0.9-kilobase pair PstI fragment of mouse c-fos cDNA (provided by Dr. Mona Nemer, Montreal) labeled by random priming and a DNA oligonucleotide derived from the rat glyceraldehyde-3-phosphate dehydrogenase RNA sequence (provided by Dr. Louis Ferland, Montreal) 5`-end-labeled using T4 polynucleotide kinase.

Protein Synthesis Measurements

Quiescent rat aortic SMC in triplicate wells of 24-well plates were stimulated with 100 nM AII in serum-free DMEM-Ham's F-12 medium containing 0.5 µCi/ml [^3H]leucine. After 24 h, the medium was aspirated and the cells were fixed for a minimum of 30 min with cold 5% trichloroacetic acid. The cells were then washed once with trichloroacetic acid and three times with tap water. The radioactivity incorporated into trichloroacetic acid-precipitable material was measured by liquid scintillation counting after solubilization in 0.1 M NaOH. For experiments with rapamycin, quiescent cells were pretreated for 30 min with the indicated concentrations of rapamycin and stimulated for 24 h with AII in the continuous presence of the drug.

Other Methods

Protein concentrations were measured using the BCA protein assay kit (Pierce) with bovine serum albumin as standard.


RESULTS AND DISCUSSION

Growth Response to AII in Aortic SMC

We first characterized the effects of AII on protein synthesis and DNA synthesis in quiescent aortic SMC derived from Brown-Norway rats. In agreement with previous observations(4, 5, 6, 7) , we found that AII potently stimulates protein synthesis in aortic SMC, with a half-maximal effect observed at 0.5 nM (n = 3) (Fig. 1A). No effect on DNA synthesis and cell proliferation was observed in these cells. To determine which subtype of AII receptor was linked to protein synthesis, aortic SMC were pretreated for 10 min with selective receptor antagonists prior to stimulation with AII. Fig. 1B shows that incubation with the non-selective peptide antagonist [Sar^1,Ile^8]AII and the AT(1)-selective antagonist losartan completely suppressed the AII-induced increase in protein synthesis, whereas PD123319 had no effect. Addition of the drug antagonists alone did not influence the basal rate of protein synthesis. These results indicate that the stimulatory effect of AII on protein synthesis in aortic SMC is mediated by the AT(1) receptor.


Figure 1: AII increases the rate of protein synthesis in rat aortic SMC. Rat aortic SMC were made quiescent by incubation in serum-free medium for 48 h. The cells were then stimulated for 24 h with indicated concentrations of AII. Protein synthesis was measured by [^3H]leucine incorporation. Each value represents the mean of triplicate determinations. A, dose-response curve for the stimulatory effect of AII on protein synthesis. B, effect of AII receptor antagonists. Quiescent aortic SMC were pretreated for 10 min with medium alone or with the non-selective antagonist [Sar^1,Ile^8]AII (sarile, 10M), the AT(1)-selective antagonist losartan (10M), or the AT(2)-selective antagonist PD123319 (3 times 10M). The cells were then stimulated with medium or 100 nM AII for 24 h.



AII Stimulates p70 S6 Kinase Activity in Aortic SMC

To gain insight in the mechanism by which AII activates protein synthesis, we examined the ability of the hormone to regulate the activity of p70, the major in vivo S6 kinase. The enzymatic activity of p70 was assayed in an immune complex kinase assay using a specific antiserum raised against a N-terminal peptide from the predicted rat p70 sequence (30) . This antiserum recognizes p70 and the minor isoform p85, which is encoded by the same gene(49, 50) . Aortic SMC were made quiescent by serum deprivation, stimulated with AII, and lysed prior to incubation with p70 antiserum. Phosphotransferase activity was determined using a synthetic peptide derived from ribosomal protein S6 as substrate. As shown in Fig. 2, addition of AII caused a potent stimulation of the enzymatic activity of p70, resulting in up to 6-fold activation at 15 min. Time course experiments revealed that AII-stimulated p70 activity was detectable at 1 min, reached a maximum at 15 min, and slowly declined thereafter. The activity was still elevated above basal level (1.5-fold) 4 h after stimulation. Previous studies have shown that activation of p70 is biphasic following mitogenic stimulation of Swiss 3T3 cells (51, 52) and CCL39 fibroblasts(53) . The two phases of enzyme activity appear to be controlled by distinct signaling pathways, although the components involved remain uncertain(51, 54) . It will be of interest in future studies to determine whether the activation of p70 by AII in SMC also involves multiple signaling pathways.


Figure 2: AII stimulates the enzymatic activity of p70 in rat aortic SMC. Quiescent rat aortic SMC were treated for the indicated times with 100 nM AII. Cell lysates were prepared and subjected to immunoprecipitation with p70 antiserum preadsorbed to protein A-Sepharose beads. Immune complexes were washed, and p70 phosphotransferase activity was assayed using an S6 peptide as substrate (see ``Experimental Procedures''). The enzymatic activities are expressed as picomoles of PO(4) incorporated into the substrate/min/mg of lysate protein. The data presented are representative of three independent experiments with similar results.



AII has been shown previously to stimulate S6 phosphorylation in vascular SMC (55) and renal proximal tubular cells(56) . However, the identity of the S6 kinases implicated has not been investigated. Our present findings clearly established that AII potently stimulates the enzymatic activity of the 70-kDa S6 kinase family in aortic SMC. In this respect, we have also observed that AII activates the two MAP kinase isoforms p44 and p42 in these cells. However, the kinetic of activation of these enzymes is different from the one of p70. The activity of p44 and p42 increases rapidly to reach a maximum at 5 min and then declines rapidly to low value at 30 min. The activity of the enzymes remains negligible thereafter. These observations are consistent with the notion that MAP kinases and p70 lie on distinct signaling pathways(57) .

To determine which subtype of AII receptors is involved in the activation of p70, rat aortic SMC were pretreated for 10 min with selective receptor antagonists prior to stimulation with AII. Fig. 3shows that incubation with the AT(1)-selective antagonist losartan completely abolished p70 activation, whereas the AT(2) antagonist PD123319 had no effect. Thus, these results demonstrate that stimulation of p70 activity, like most of the known biochemical responses to AII, is mediated by AT(1) receptors.


Figure 3: Effect of AII receptor antagonists on AII stimulation of p70 activity. Quiescent rat aortic SMC were pretreated for 10 min with medium alone or with the non-selective AII receptor antagonist [Sar^1,Ile^8]AII (sarile, 10M), the AT(1)-selective antagonist losartan (10M), or the AT(2)-selective antagonist PD123319 (3 times 10M). The cells were then stimulated with medium(-) or 100 nM AII for 15 min. The p70 was immunoprecipitated from the cell lysates and S6 kinase activity was measured as described in Fig. 2. Similar results were obtained in three independent experiments.



Activation of p70 S6 Kinase by AII Is Associated with the Phosphorylation of the Enzyme on Serine/Threonine Residues

Activation of p70 by growth factors is accompanied by multiple phosphorylation of the protein on serine/threonine residues(58, 59) . Removal of these phosphate groups by type 2A protein phosphatase leads to inactivation of the enzyme (60) . p70 activation is associated with phosphorylation of four sites displaying the motif Ser/Thr-Pro and clustered in a putative autoinhibitory domain(61) . The upstream kinases responsible for regulating the phosphorylation of these sites remains to be identified, although the sequence surrounding the phosphoacceptor sites suggest proline-directed kinases as potential candidates. To confirm that AII activation of p70 is correlated with increased phosphorylation of the protein, quiescent aortic SMC were labeled with P(i), stimulated with AII, and p70 was immunoprecipitated from cell lysates. Little phosphorylation of p70 was detected in quiescent cells (Fig. 4A). Treatment of the cells with 100 nM AII for 15 min caused a large increase in the phosphorylation of p70, which was accompanied by a characteristic decrease in electrophoretic mobility. We also observed an augmented phosphorylation of a minor band migrating at M(r) 85,000, which may correspond to the p85 isoform(49, 50) . However, it must be emphasized that the identity of this protein has not been firmly established. The phosphorylation of p70 was also examined by immunoblot analysis following immunoprecipitation with the same p70 antiserum. AII stimulation of quiescent cells resulted in reduced electrophoretic mobility of p70 on SDS-polyacrylamide gels (Fig. 4B). This mobility shift is indicative of increased phosphorylation of the enzyme(39, 58) .


Figure 4: AII stimulates the phosphorylation of p70 in rat aortic SMC. A, quiescent rat aortic SMC were labeled with [P]phosphoric acid for 5 h and then stimulated or not (Cont) with 100 nM AII for 15 min. The cells were lysed, and p70 was immunoprecipitated using a specific p70 antiserum preadsorbed to protein A-Sepharose beads. The immunoprecipitated proteins were resolved by SDS-gel electrophoresis on 10% acrylamide gels and transferred to PVDF membranes prior to autoradiography. Molecular weight standards are shown on the left. The positions of p70 and the minor isoform p85 are indicated. B, quiescent rat aortic SMC were stimulated or not (Cont) with 100 nM AII for 15 min. Equal amounts of cell lysate protein (500 µg) were then subjected to immunoprecipitation with either p70 antiserum (lanes1 and 2) or normal rabbit serum (lane3). The proteins were resolved on 7.5% acrylamide gels and transferred to nitrocellulose membrane. The membrane was probed with S6K-III antiserum and the proteins visualized by chemiluminescence detection. The positions of p70 and the minor p85 isoform are indicated. The p85 was hard to visualize on these immunoblots. Similar results were obtained in two independent experiments.



To further analyze the changes in the phosphorylation state of p70, the P-labeled bands corresponding to p70 and to the 85-kDa protein were subjected to phosphoamino acid analysis. In quiescent aortic SMC, p70 was phosphorylated exclusively on serine residues (Fig. 5). Stimulation of the cells with AII resulted in a significant increase in the phosphoserine and to a much lesser extent phosphothreonine content of p70 (Fig. 5). The increased phosphorylation of the 85-kDa protein was also characterized by an augmentation of serine phosphorylation, but the signal was too low to detect any change in threonine phosphorylation (Fig. 5). Therefore, our results demonstrate that activation of p70 by AII is associated with increased phosphorylation of the enzyme mainly on serine but also on threonine residues. It is noteworthy that stimulation of p70 by either epidermal growth factor in Swiss 3T3 cells (61) or insulin in H4 hepatoma cells (59) is associated with a similar content of phosphorylated amino acids, thereby suggesting that G protein-coupled receptors and receptor tyrosine kinases might use the same upstream kinases to regulate the activity of p70.


Figure 5: Phosphoamino acid analysis of AII-stimulated p70. Quiescent rat aortic SMC were labeled with [P]phosphoric acid for 5 h and then stimulated or not (Cont) with 100 nM AII for 15 min. The p70 was immunoprecipitated from the cell lysates and analyzed as described in Fig. 4. The P-labeled protein bands corresponding to p70 and the 85-kDa protein were excised from the PVDF membrane and subjected to partial acid hydrolysis. The phosphorylated amino acids were separated by two-dimensional thin layer electrophoresis. The positions of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) are indicated.



Rapamycin Blocks AII-induced p70 Activation and Inhibits the Hypertrophic Response to the Hormone

In order to explore the role of p70 in the hypertrophic effect of AII on aortic SMC, we have used the immunosuppressant drug rapamycin to inhibit the cellular activity of the enzyme. Rapamycin is a macrolide antibiotic with potent immunosuppressive activity that has been shown to inhibit yeast and mammalian cell proliferation(39, 62, 63) . Recently, rapamycin was shown to selectively block the activation of p70 by a number of mitogens, without interfering with the activity of MAP kinases or p90(39, 40, 41, 42) . Although the exact target of rapamycin has not yet been identified, its selective action on the p70 pathway makes it a useful tool to investigate the involvement of p70 in cellular responses.

We first tested the effects of rapamycin on p70 and MAP kinase activity in AII-stimulated aortic SMC. Consistent with previous studies using different growth factors or cell types, pretreatment of quiescent aortic SMC with 10 ng/ml rapamycin completely abolished the activation of p70 by AII (Fig. 6A). Half-maximal inhibition was observed at a concentration of 0.5 ng/ml rapamycin (n = 2) (Fig. 8). In contrast, rapamycin did not affect AII-induced p44 activity as measured by the phosphorylation of MBP (Fig. 6B). To further demonstrate the selectivity of rapamycin action, we evaluated the effect of the drug on induction of the early-immediate gene c-fos in aortic SMC. AII stimulation leads to increased c-fos mRNA expression in SMC and other cell types. As shown in Fig. 7, AII-induced c-fos gene induction was not inhibited by rapamycin. The normalized values of c-fos induction, expressed as -fold stimulation above control, were 10.1 in untreated cells stimulated with AII and 13.1 or 9.7 in cells treated with 10 or 30 ng/ml rapamycin, respectively. These findings also indicate that p70 activity is not required for induction of c-fos gene expression. Similar observations were made in an interleukin 2-dependent T-cell line where rapamycin was found to block p70 activation by interleukin 2, but not p90 activation, c-fos and c-myc mRNA expression, or early tyrosine phosphorylation events(64) . Taken together, these data indicate that rapamycin selectively inhibits the activation of p70 among the early signaling events triggered by AII in aortic SMC.


Figure 6: Rapamycin selectively inhibits AII-stimulated p70 activation in rat aortic SMC. Quiescent rat aortic SMC were pretreated for 30 min with vehicle alone or with the indicated concentrations (ng/ml) of rapamycin (rap). The cells were then stimulated or not (Cont) with 100 nM AII for 15 min (p70 assays) or 5 min (p44 assays). A, enzymatic activity of p70. The p70 was immunoprecipitated from the cell lysates and S6 kinase activity was measured as described in Fig. 2. B, enzymatic activity of p44. The p44 was immunoprecipitated from the cell lysates with antiserum SM1, and phosphotransferase activity was assayed using MBP as substrate (see ``Experimental Procedures''). The enzymatic activities are expressed as picomoles of PO(4) incorporated into the substrate/min/mg of lysate protein. The data presented are representative of three independent experiments with similar results.




Figure 8: Rapamycin potently inhibits AII-stimulated protein synthesis in rat aortic SMC. Quiescent rat aortic SMC were pretreated for 30 min with the indicated concentrations of rapamycin and then stimulated for 24 h with 100 nM AII in the continuous presence of the inhibitor. Protein synthesis was measured by [^3H]leucine incorporation (bullet). Each value represents the mean of triplicate determinations. In the experiment shown, the extent of stimulation of protein synthesis in response to AII was 2.0-fold above basal level. The activity of p70 S6 kinase (circle) was determined as described in Fig. 6. Each point represents the average from two independent experiments. AII stimulated the S6 kinase activity of the enzyme 2.9-fold in the absence of rapamycin. Similar results were obtained in four separate experiments.




Figure 7: Effect of rapamycin on AII-induced c-fos gene expression. Quiescent rat aortic SMC were pretreated for 30 min with vehicle or with the indicated concentrations (ng/ml) of rapamycin (rap). The cells were then stimulated or not (Cont) with 100 nM AII for 30 min. Total RNA was extracted from the cells and analyzed by Northern hybridization using a P-labeled c-fos cDNA fragment as described under ``Experimental Procedures.'' The results were normalized by rehybridization of the blots with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) oligonucleotide probe. The extent of hybridization was visualized by autoradiography and quantitated by laser densitometry. Similar results were obtained in two independent experiments.



We next examined the effect of rapamycin on AII-stimulated protein synthesis in aortic SMC. For these experiments, quiescent SMC were preincubated for 30 min with rapamycin prior to stimulation with 100 nM AII for 24 h in the continuous presence of the drug. Fig. 8shows that rapamycin very significantly inhibited AII-induced protein synthesis with over 75% inhibition observed at 3 ng/ml of the drug. Half-maximal inhibition of protein synthesis was observed in the presence of 0.3 ng/ml rapamycin (n = 4), identical to the concentration required for inhibition of p70 S6 kinase. The ED value found for inhibition of AII-induced protein synthesis is comparable to those observed for inhibition of interleukin 2-stimulated DNA synthesis in T lymphoid cell lines(40, 64) . These findings demonstrate that there is a close correlation between the stimulation of p70 activity by AII and its hypertrophic effect in SMC. It should be noted, however, that although rapamycin totally abrogated AII-stimulated p70 activity, the drug never completely inhibited (60-80% inhibition) the increase in protein synthesis. This suggests that additional signaling pathways are recruited by AII to regulate the rate of protein synthesis. Future work will be required to identify these other signaling pathways and determine their relationship with the p70 pathway.


CONCLUSIONS

The present data provide strong evidence that activation of the p70 signaling pathway by AII represents a critical event in the hypertrophic response of SMC to the hormone. We showed that AII potently stimulates the enzymatic activity of p70 in aortic SMC. This activation is mediated by AT(1) receptors and is associated with increased phosphorylation of the enzyme on serine and threonine residues. The AII-induced p70 activation is completely blocked by rapamycin, whereas MAP kinase activation or c-fos gene induction is not affected. Rapamycin also potently inhibited AII-stimulated protein synthesis in these cells. Considering the role of p70 as the major in vivo S6 kinase, our findings strongly suggest that this enzyme plays a critical role in the hypertrophic response of vascular SMC to AII.

Another conclusion provided by these results is that stimulation of MAP kinase activity and c-fos mRNA expression are not sufficient to significantly increase the rate of protein synthesis in SMC. Activation of MAP kinase isoforms is required for cell cycle progression and S phase entry of fibroblast cells in response to mitogenic factors(43, 65) . These enzymes are rapidly translocated into the nucleus after mitogenic stimulation of quiescent cells where they can phosphorylate and regulate the activity of several transcription factors(66) . It has been suggested that MAP kinases may also play a crucial role in the growth promoting activity of AII(67, 68) . Our results do not support the conclusion that MAP kinases alone are sufficient intermediates in the signaling pathway between the AT(1) receptor and the ribosomes. The exact role of the MAP kinase signaling system in the response of vascular SMC to hypertrophic hormones such as AII remains to be determined.


FOOTNOTES

*
This work was supported in part by grants from the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Scholar of the Medical Research Council of Canada. To whom correspondence should be addressed. Tel.: 514-843-2733; Fax: 514-843-2715.

(^1)
The abbreviations used are: AII, angiotensin II; SMC, smooth muscle cell(s); MAP kinase, mitogen-activated protein kinase; MBP, myelin basic protein; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PVDF, polyvinylidene fluoride; TBS, Tris-buffered saline.

(^2)
I. Leduc and S. Meloche, manuscript submitted.

(^3)
E. Giasson and S. Meloche, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Dr. Frederick Hall for generous supply of p70 antiserum, Dr. Ronald Smith (Du Pont Merck) and Dr. Joan Keiser (Parke-Davis) for supply of losartan and PD 123319, respectively, and Dr. Patrice Larose (Wyeth-Ayerst) for providing rapamycin. We are grateful to members of Dr. Pavel Hamet's laboratory for their initial help and advice on the culture of rat aortic SMC. We also thank Elisabeth Pérès for preparation of the figures and Irène Rémillard for secretarial assistance.


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