Rapamycin Selectively Inhibits Angiotensin II–Induced Increase in Protein Synthesis in Cardiac Myocytes In Vitro
Potential Role of 70-kD S6 Kinase in Angiotensin II– Induced Cardiac Hypertrophy
Abstract It has been suggested that phosphorylation of a 40S ribosomal protein, S6, regulates protein synthesis. Two distinct families of S6 kinase have been identified, the rsk-encoded 85- to 92-kD S6 kinase (RSK) and the 70- or 85-kD S6 kinase (p70S6K). We have previously shown that hypertrophic stimuli, such as angiotensin II (Ang II), rapidly activate RSK in cardiac myocytes. However, RSK and p70S6K are regulated by distinct mechanisms, and p70S6K, but not RSK, is the physiological S6 kinase in vivo in other cell types. Using cultured neonatal rat ventricular myocytes, we examined whether Ang II activates p70S6K and investigated the effect of rapamycin, a potent yet indirect inhibitor of p70S6K, on the Ang II–induced hypertrophic response. Immunoblot analyses indicate that cardiac myocytes express the 70- and 85-kD forms of p70S6K. Ang II caused a rapid and sustained activation of p70S6K through the type I Ang II receptor. Rapamycin inhibited Ang II–induced activation of p70S6K in a dose-dependent manner, with an IC50 of 0.14 ng/mL (0.15 nmol/L). Rapamycin did not inhibit Ang II–induced activation of tyrosine kinase, mitogen-activated protein kinase, RSK, and protein kinase C. The effect of rapamycin is unlikely to be mediated by its effect on p34cdc2 and p33cdk2 because Ang II did not activate these cell cycle–dependent kinases in cardiac myocytes. In contrast, a dose-dependent inhibition of p70S6K by rapamycin is very closely correlated with its inhibition of the Ang II–induced increase in protein synthesis. Interestingly, rapamycin did not affect the Ang II–induced activation of specific gene expression, including the immediate-early gene c-fos and fetal type genes, such as atrial natriuretic factor and skeletal α-actin. Moreover, rapamycin did not suppress Ang II–induced phenotypic changes at the protein level, such as increased atrial natriuretic factor secretion, expression of β-myosin heavy chain, and organization of actin into sarcomeric units. These results indicate that p70S6K is activated by Ang II and that a rapamycin-sensitive signaling mechanism, most likely p70S6K, plays an essential role in the Ang II–induced increase in overall protein synthesis but not in Ang II–induced specific phenotypic changes in cardiac myocytes.
Cardiac hypertrophy is a fundamental adaptive process in response to hemodynamic overload. It is well known that external load plays a critical role in determining muscle mass and its phenotype, but accumulating evidence suggests that cardiac hypertrophy induced by growth factors mimics most, if not all, aspects of load-induced cardiac hypertrophy.1 2 3 For example, load-induced hypertrophy in ventricular myocytes in vitro and in vivo is associated with induction of immediate-early genes, such as c-fos, c-jun, and c-myc, which is followed by activation of fetal-type genes, such as ANF, skeletal α-actin, and β-MHC genes.1 2 3 4 5 Very similar phenotypic changes are observed in cardiac hypertrophy induced by Ang II, endothelin-1, basic fibroblast growth factor, and phenylephrine.3 6 7 8 9 We have previously shown that mechanical stretch of neonatal cardiac myocytes causes autocrine secretion of Ang II and that this initiates the hypertrophic response in vitro.10 Ang II, in turn, modulates the expression of various growth factors, such as transforming growth factor-β1.7 Thus, growth factors seem to play an essential role in the pathogenesis of load-induced cardiac hypertrophy.
In contrast to the wealth of knowledge of the hypertrophic effects of various growth factors, the specific roles of intracellular signal transduction molecules in mediating the phenotypic changes associated with cardiac hypertrophy remain poorly characterized. We11 and others12 have previously shown that mechanical stretch and Ang II13 14 activate multiple intracellular second messenger systems, such as phospholipases C, D, and A2, tyrosine kinases, p21ras, MAP kinases, RSK, and PKC in neonatal rat cardiac myocytes in vitro. However, the precise role of each second messenger system has not been established. Recently, by use of the microinjection technique, a “dominant negative” form of p21ras and a specific antibody against the Gq-type GTP binding protein have been shown to inhibit phenylephrine-induced cardiac hypertrophic responses (such as increase in cell size, transcriptional activation of ANF, and organization of actin filaments), thereby indicating that p21ras and Gq are essential in phenylephrine-induced hypertrophy.15 16 More recently, inhibition of the MAP kinase or Raf-1 by transfection or microinjection of a dominant negative form of MAP kinase or Raf-1 has been shown to inhibit phenylephrine-induced transcriptional activation of ANF and MLC-2 but not to suppress organization of actin filaments by phenylephrine.17 18 These results suggest that each second messenger system may have a different role in the cardiac hypertrophic response.
Increased protein synthesis is the cardinal feature of cardiac hypertrophy. Protein synthesis is regulated by various molecules that interact with the translational machinery of the ribosome.19 Among them, S6, a component of 40S ribosomal proteins, is located at the interface between 40S and 60S ribosomal proteins and may interact directly with mRNA.20 Accumulating evidence suggests that multiple serine phosphorylations of S6 at the carboxy terminus regulate the rate of protein synthesis by stimulating initiation and elongation of protein translation.20 The S6 phosphorylation at the carboxy terminus is mediated by a family of serine/threonine kinases, known as S6 kinases. The S6 kinases consist of two distinct families, RSK and p70S6K.20 21 22 Several lines of evidence suggest that RSK and p70S6K are regulated by distinct signaling pathways. For example, RSK is phosphorylated and activated by MAP kinases, but p70S6K is not.21 23 24 25 Although both RSK and p70S6K phosphorylate S6 at the same serine residues in vitro, recent studies suggest that p70S6K, not RSK, is the physiological kinase that phosphorylates S6 in vivo in Swiss 3T3 cells.25
Recently, it has been shown that a subnanomolar concentration of the macrolide rapamycin specifically binds to a cellular protein termed FKBP and blocks activation of p70S6K in response to cytokines.25 26 27 After microinjection of a polyclonal antibody against p70S6K or treatment with rapamycin, it has been suggested that p70S6K plays an essential role in G1 to S progression during the cell cycle in various cell types.25 26 28 29 However, rapamycin was also shown to inhibit two cell cycle–dependent kinases, p33cdk2 and p34cdc2.27 30 Therefore, at present, it is not clear whether rapamycin inhibits cell growth primarily by its effect on p70S6K or on these cell cycle–dependent kinases.
Neonatal cardiac myocytes in serum-free culture are in a terminally differentiated state and do not reenter the cell cycle. Upon growth factor stimulation, these cells undergo hypertrophy without cell division.1 2 3 Therefore, cardiac myocytes provide a unique opportunity to probe the function of p70S6K by rapamycin, without its confounding effect on G1-S and G2-M progressions, which are controlled by p33cdk2 and p34cdc2, respectively.31 Therefore, the present study was conducted to examine (1) whether Ang II activates p70S6K in cardiac myocytes, (2) the effects of rapamycin on Ang II–induced p70S6K activation as well as Ang II–induced activation of other signaling molecules, and (3) the effects of rapamycin on Ang II–induced increase in protein synthesis as well as phenotypic changes in cardiac myocytes.
Materials and Methods
Protein A–Sepharose was purchased from Pharmacia Biotech. S6 peptide (RRRLSSLRA) was from UBI. Phosphocellulose units were from Pierce. GS peptide (PLSRTLSVAAKK) and PKC pseudosubstrate peptide [PKC (19-36)] were from Bachem. Histone H1 was from Boehringer Mannheim. Immobilon-P was from Millipore. Ang II and the RIA kit for ANF were from Peninsula. Sep-Pak C18 columns were from Waters. Losartan and PD123319 were gifts from Du-Pont Merck and Parke-Davis, respectively. Rapamycin was a gift from Wyeth-Ayerst Research. The stock solution of rapamycin was freshly prepared with ethanol before experiments as 1000-fold concentrated solution. All other chemicals were purchased from Sigma Chemical Co. A recombinant anti-phosphotyrosine antibody (RC20H) was purchased from Transduction Laboratories. Anti-p70S6K polyclonal antibody (C-18), anti-RSK polyclonal antibody (C-21), and anti–MAP kinase polyclonal antibodies (anti-ERK-1 [K-23] and anti-ERK-2 [C-14]) were purchased from Santa Cruz Biotechnology. Another polyclonal anti-p70S6K antibody (C-2) was a gift from Dr J. Blenis, Harvard Medical School. Rabbit anti-p34cdc2 serum was purchased from GIBCO. Anti-p33cdk2 monoclonal antibody was a gift from Dr E. Harlow, Massachusetts General Hospital Cancer Center. Rabbit anti-mouse IgG antibody and normal rabbit serum (nonimmune serum) were purchased from Jackson Immuno Research.
Primary culture of the neonatal rat cardiac myocyte was prepared as previously described.5 7 After an enzymatic dissociation, the cells were preplated for 1 hour to selectively enrich for cardiac myocytes. The resultant suspension of myocytes was plated onto gelatin-coated 35- or 60-mm culture dishes at a density of 1.5×105 cells per square centimeter and cultured in DMEM/F-12 (GIBCO) (1:1 [vol/vol]) supplemented with 5% horse serum, 2 g/L bovine serum albumin (fraction V), 3 mmol/L pyruvic acid, 15 mmol/L HEPES (pH 7.6), 100 μmol/L ascorbic acid, 100 μg/mL ampicillin, 4 μg/mL transferrin, 0.7 ng/mL sodium selenium, and 100 μmol/L bromodeoxyuridine. The culture medium was changed 24 hours after seeding to a defined serum-free DMEM/F-12 medium that had the same composition as described above, except that 5% horse serum and bromodeoxyuridine were not added. The use of the preplating procedure, mitogen-poor serum for plating (5% horse serum), bromodeoxyuridine, high cell-plating density, and a prompt switch to the serum-free medium enabled us to routinely obtain myocyte cultures in which >90% were myocytes, as assessed by immunofluorescence staining with a monoclonal antibody against sarcomeric MHC (MF-20).32 Cardiac fibroblast culture was prepared as previously described.5 7 All experiments were performed in the serum-free conditions 48 hours after changing to the serum-free medium.
Immunoprecipitation and Immunoblotting
For immunoblotting of p70S6K, cell lysates were prepared by an addition of 1 mL of ice-cold lysis buffer A containing (mmol/L) potassium phosphate 10 (pH 7.4), EDTA 1, EGTA 5, MgCl2 10, β-glycerophosphate 50, sodium orthovanadate 1, dithiothreitol 2, and AEBSF 1, along with 10 nmol/L okadaic acid, 100 μmol/L leupeptin, 10 μg/mL aprotinin, and 0.5% Triton X-100. Lysates containing equal amounts of protein (750 μg) were incubated with 1 μg of polyclonal antibody against p70S6K (C-18) raised against C terminus (485-502) of the rat 70-kD S6K for 2 hours at 4°C. In some experiments, a preabsorbed antibody was used for immunoprecipitation. Preabsorbtion of the antibody was performed by incubating the antibody with a 10-fold higher concentration of the antigen peptide for 12 hours at 4°C. The immune complex was precipitated by protein A–Sepharose, and the immunoprecipitates were electrophoresed on an 8% polyacrylamide gel and were then transferred to Immobilon-P membranes. Membranes were blocked with 5% bovine serum albumin in TBST solution (containing 20 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, and 0.05% Tween 20) for 2 hours. Membranes were then incubated with the same anti-p70S6K antibody at 1 μg/mL in blocking solution or with polyclonal antisera raised against the predicted C terminus (502-525) of the p70S6K (C-2)25 at a dilution of 1:500 in TBST. Immunoreactive bands were probed with 125I–protein A, and blots were subjected to autoradiography. Immunoblotting of phosphotyrosine was performed as described previously by using a horse radish peroxidase–conjugated recombinant anti-phosphotyrosine antibody (RC20H).14 An enhanced chemiluminescence system (Amersham) was used as a detection system.
Immune Complex S6 Kinase Assay
RSK and p70S6K activities were measured with an immune complex kinase assay using S6 peptide (RRRLSSLRA) corresponding to amino acids 231 to 239 of human S6 as a substrate. This peptide has been shown to be a good substrate for RSK.33 However, it contains the consensus recognition sequence of p70S6K [R-(R)-R-X-X-S-X],34 and it has been shown to be a good substrate for p70S6K as well.35 The cell-free lysates were prepared with buffer A. The lysates containing equal amounts of protein (750 μg) were incubated with 1 μg of anti-RSK antibody (C-21) or 1 μg of anti-p70S6K antibody (C-18 or C-2) for 2 hours at 4°C. Protein A–Sepharose was then added, and the immunoprecipitates were washed with buffer A three times. Kinase reaction (25 μL) was performed in conditions inhibitory to cyclic nucleotide–dependent protein kinases and Ca2+-dependent protein kinases by incubating the immunoprecipitates with 12.5 μL of 2× kinase buffer containing (mmol/L) MOPS 50 (pH 7.2), β-glycerophosphate 120, p-nitrophenylphosphate 60, EGTA 10, MgCl2 30, dithiothreitol 2, and sodium orthovanadate 2, along with 2 μmol/L protein kinase inhibitor (rabbit sequence), 6.25 μL of ATP mixture containing 40 μmol/L cold ATP and 5 μCi [γ32P]ATP (6000 Ci/mmol), and 1.25 μL of 5 mmol/L S6 peptide for 20 minutes at 30°C. To terminate the reaction, samples were spotted onto phosphocellulose units (Pierce) and washed twice by centrifugation with 75 mmol/L phosphoric acid. The filters were then placed into scintillation vials, and the radioactivity was counted.
MAP Kinase, PKC, and Histone H1 Kinase Assays
The immune complex in-gel MAP kinase assay, the PKC assay in permeabilized cells, and the immune complex histone H1 kinase assay were performed as described previously.13 14 36 The specificity of the PKC assay has been described previously.37
Measurement of Protein Content, Rate of Protein Synthesis, and Protein Degradation
Cardiac myocytes were plated on 35-mm dishes and cultured in the serum-free condition for 48 hours before the experiments. After stimulation of myocytes, each dish was rinsed three times with PBS. The cell layer was scraped with 1 mL of 1× standard sodium citrate containing 0.25% (wt/vol) SDS and vortexed extensively. Total cell protein and the DNA content were determined by the Lowry method and the Hoechst dye method, respectively, and the protein content was normalized by the DNA content to correct for small differences in the cell number between dishes as described previously.5
As an index of protein synthesis, [3H]phenylalanine incorporation was measured as described previously.5 Cardiac myocytes were pretreated with rapamycin or vehicle (0.1% ethanol) for 30 minutes. The myocytes were then incubated with 5 μCi/mL of [3H]phenylalanine (120 Ci/mmol) and unlabeled phenylalanine (0.36 mmol/L) in the medium and treated with or without Ang II in the presence of rapamycin or vehicle. The cells were washed with PBS, and 10% TCA was added at 4°C for 60 minutes to precipitate protein. The precipitate was washed three times with 95% ethanol and then resuspended in 0.15N NaOH. Aliquots were counted by a scintillation counter.
Protein degradation was determined as described previously.38 To radiolabel cellular proteins, cardiac myocytes were incubated with 10 μCi/mL of [3H]phenylalanine for 4 hours. The myocytes were then washed with nonradioactive medium containing excess concentrations (500 μg/mL) of phenylalanine three times at 30-minute intervals. Some dishes were then harvested with 10% TCA to determine “zero time” values. After pretreatment with rapamycin (10 ng/mL) or vehicle (0.1% ethanol) for 30 minutes, some dishes were maintained for an additional 24 hours in control medium containing excess nonradioactive phenylalanine (500 μg/mL) to minimize reutilization of label, and others were maintained for an identical period in the same medium plus Ang II (100 nmol/L) and/or rapamycin (10 ng/mL). After 24 hours of incubation, all dishes were harvested with 10% TCA. TCA precipitates were treated as described above and counted by scintillation counter.
Northern Blot Analysis
Isolation of total cellular RNA and Northern blot analysis were performed as described previously.5 7 The probes for skeletal α-actin, ANF, and GAPDH were used as described previously.5 The hybridization signals of specific mRNAs were normalized to those of GAPDH mRNA to correct for differences in loading and/or transfer.7
Cardiac myocytes were plated on 35-mm dishes (7×105 cells per dish). After 48 hours of culture in the serum-free medium, myocytes were treated with rapamycin (10 ng/mL) or vehicle (0.1% ethanol) for 30 minutes and then stimulated with Ang II (100 nmol/L) for 24 hours. ANF production was defined as the amount of ANF released into the medium during a 60-minute period after 24 hours of incubation with Ang II as described previously.39 At 24 hours, the medium was removed from the culture and replaced with 2.5 mL of fresh medium. After 60 minutes, 2 mL of the medium was collected. EDTA (1 mg/mL) was added, and after a 5-minute centrifugation at 1000g, the supernatant was stored at −20°C. The sample was partially purified by using a Sep-Pak C18 column as described previously10 and lyophilized. The samples were redissolved in the RIA buffer, and RIA was performed according to the manufacturer’s instructions (Peninsula). Repeated RIAs of a control sample indicate that the interassay variations were <5%.
Analysis of MHC Isoforms
MHCs were analyzed by using a method described by Fauteck and Kandarian,40 which enabled separation of α- and β-MHCs on a 5% denaturing SDS-PAGE gel. Because considerable expression of β-MHC protein is still observed in the neonatal rat heart,41 T3 (2 ng/mL) was applied throughout the experiment to suppress the basal expression of β-MHC protein. From day 4, Ang II (100 nmol/L) was applied every 12 hours in the presence of rapamycin (10 ng/mL) or vehicle (0.1% ethanol) for 5 days. Rapamycin was applied every other day when the culture media were changed. On day 9, myocytes grown on six-well plates were washed with PBS three times and scraped with 200 μL per well of PBS containing 1 mmol/L EDTA. Approximately 30 000 cells (10 μL of the PBS suspension) were treated with Laemmli loading buffer and boiled for 10 minutes. SDS-PAGE was performed at 4°C by using a 5%-separating gel that was 20 cm in length. A running buffer was recirculated to keep its pH constant. The gel was then transferred to the Immobilon-P filter, and immunoblot analysis was performed by using antibody against sarcomeric myosin (MF-20, 1:25 dilution).32 The heart homogenates from adult and fetal rats were used to identify α- and β-MHCs, respectively. Densitometric analysis was performed with Intelligent Quantifier (Bio Image).
Cells were cultured on gelatin-coated glass coverslips. Cells were treated with rapamycin (10 ng/mL) or vehicle alone (0.1% ethanol) for 30 minutes and then Ang II (100 nmol/L) as required. The cells were maintained for 24 hours before fixation. Cells were washed with PBS, fixed with 3.6% formaldehyde in PBS for 10 minutes, permeabilized with 0.3% Triton X-100 in PBS for 10 minutes, and blocked in 1% ovalbumin plus 0.1% Tween 20 for 30 minutes. The cells were stained with fluorescein isothiocyanate–conjugated phalloidin (Sigma, 40 μg/mL in PBS, 0.5% NP-40, and 2 mg/mL bovine serum albumin), washed in PBS and 0.1% Tween 20, and mounted for fluorescence microscopy as described previously.17
Data are given as mean±SEM. Statistical analysis was performed by ANOVA as appropriate. Post-test multiple comparison was performed by the method of Bonferroni. Significance was accepted at the P<.05 level.
Expression of p70S6K in Cardiac Myocytes
We first examined expression of p70S6K protein in cardiac myocytes. Immunoprecipitation was performed by using a specific polyclonal anti-p70S6K antibody (C-18), and after SDS-PAGE of the immunoprecipitates, immunoblot analysis was performed by using the same antibody. In the control state (Fig 1⇓, lane 1), multiple bands were observed at ≈70 kD. After longer exposure (bottom panel), bands were also detected at ≈85 kD. Both of these bands were significantly attenuated when the immunoprecipitation was performed with the anti-p70S6K antibody preabsorbed with 10-fold excess of the antigen peptide (lane 5). The remaining band at 70 kD in lane 5 is unlikely to be a nonspecific band, because no band was seen when the immunoprecipitation was performed with a rabbit polyclonal antibody against RSK (C-21, lane 6). These bands were not observed at all when the preabsorbed antibody was used in immunoblotting (data not shown). This suggests that these bands correspond to 70-kD (αII) and 85-kD (αI) forms of p70S6K.25 Interestingly, after a 2-minute stimulation of cardiac myocytes with Ang II (100 nmol/L), multiple bands having reduced mobility were observed above the 70- and 85-kD bands (lanes 2 to 4). It has been shown that p70S6K is observed as multiple bands (≈70 and 85 kD) on immunoblotting, depending on its phosphorylation state of several residues.25 Therefore, it is likely that both 70- and 85-kD forms of p70S6K are phosphorylated upon stimulation with Ang II. We did not detect bands at ≈90 kD, confirming that the antibody we used does not cross-react with RSK.
Ang II Activates p70S6K
To examine whether p70S6K is activated upon stimulation with Ang II, in vitro kinase assays were performed with S6 peptide used as a substrate. To eliminate other kinases that phosphorylate S6 peptide, such as RSK and PKC,33 the kinase assay was performed after immunoprecipitation with the antibody against p70S6K (C-18 or C-2). As shown in Fig 2⇓, an increase in phosphorylation of S6 peptide was observed after 3 minutes of stimulation with Ang II (100 nmol/L), and this persisted for >60 minutes. S6 peptide phosphorylation observed in immunoprecipitates with an antibody preabsorbed with the antigen peptide was as low as the background level and did not increase after stimulation with Ang II, suggesting that the S6 peptide phosphorylation observed in the immunoprecipitate with the anti-p70S6K antibody was specific to p70S6K.
The Ang II (100 nmol/L)–induced increase in p70S6K activity was suppressed by losartan (5 μmol/L), an Ang II type-1 receptor antagonist, but not by PD123319 (5 μmol/L), an Ang II type-2 receptor antagonist (Fig 2B⇑). We used 50-fold higher doses of the antagonists because they have lower affinity to the receptors than does Ang II. The result suggests that Ang II–induced p70S6K activation is mediated by the Ang II type-1 receptor.
Rapamycin Suppresses p70S6K Activation by Ang II
We next examined effects of rapamycin on Ang II–induced p70S6K activation. Cardiac myocytes were treated with various concentrations of rapamycin (0.03 to 30 ng/mL [33 pmol/L to 33 nmol/L]) for 30 minutes and then treated with or without Ang II (100 nmol/L) for 10 minutes, and the p70S6K activity was measured by the immune complex S6 peptide kinase assay. As shown in Fig 3A⇓, rapamycin inhibited both basal and Ang II–induced p70S6K activity in a dose-dependent manner, with half-maximum inhibition doses at 0.68 and 0.14 ng/mL, respectively. The Ang II–induced increase in p70S6K activity was completely suppressed at >1 ng/mL of rapamycin.
To examine the effect of rapamycin on the phosphorylation state of p70S6K, p70S6K immunoprecipitates were immunoblotted with the anti-p70S6K antibody (C-18) after SDS-PAGE. As shown in Fig 3B⇑, in rapamycin-treated myocytes, faster migrating bands were observed at ≈70 kD (and also at ≈85 kD after long exposure of the blot [not shown]), and the upward shift of the bands by the Ang II treatment was abolished. This suggests that rapamycin induces dephosphorylation of p70S6K in the basal condition and also suppresses Ang II–induced phosphorylation of p70S6K.
Rapamycin Does Not Affect Ang II–Induced Activation of Tyrosine Kinase, MAP Kinase, RSK, or PKC
To examine the specificity of rapamycin, effects of the drug on Ang II–induced activation of other second messenger systems were examined. We have shown that Ang II causes tyrosine phosphorylation of several proteins at least in part by activating tyrosine kinase activity in cardiac myocytes.14 Rapamycin, however, did not affect the Ang II–induced increase in tyrosine phosphorylation of proteins such as p42, p44, and p75-80 (peak phosphorylation at ≈5 minutes [Fig 4A⇓]) and p120-130 (peak phosphorylation at ≈1 minute [Fig 4A⇓ and data not shown]). We have also shown that Ang II activates MAP kinases and subsequently another distinct S6 kinase, RSK.14 However, it has been suggested that p70S6K and RSK are regulated by different signaling mechanisms in other cell systems.21 23 24 25 Therefore, we examined whether rapamycin affects activation of MAP kinases and RSK by Ang II. MAP kinase activity was assessed by the immune complex in-gel MAP kinase assay. As shown in Fig 4B⇓, rapamycin did not affect the Ang II–induced increase in MAP kinase activity at 44- and 42-kD proteins, which are ERK-1– and ERK-2–related MAP kinases, respectively.14 RSK activity was assessed by the immune complex kinase assay using S6 peptide as a substrate. RSK was immunoprecipitated with an anti-RSK antibody (C-21). Rapamycin did not significantly affect the basal activity of RSK or the Ang II–induced activation of RSK (Fig 4C⇓), indicating that rapamycin specifically inhibits p70S6K among the family of S6 kinases.
We have previously shown that Ang II activates PKC and that PKC plays an essential role in Ang II–induced c-fos gene expression.13 Therefore, we examined effects of rapamycin on Ang II–induced activation of PKC. PKC activity was assessed by the permeabilized cell assay using a synthetic peptide, glycogen synthase (GS) peptide, as a substrate. As shown in Fig 5A⇓, a 2-minute stimulation of cardiac myocytes with Ang II significantly increased phosphorylation of the GS peptide, whereas this increase in phosphorylation was largely inhibited in the presence of the PKC pseudosubstrate peptide. Ang II–induced increase in phosphorylation of GS peptide was not affected by the treatment with rapamycin (10 ng/mL), suggesting that rapamycin does not inhibit PKC.
In other cell systems, signaling molecules (other than p70S6K) thus far reported to be sensitive to rapamycin are two cyclin-dependent kinases, p34cdc2 and p33cdk2.27 30 We examined whether Ang II activates these kinases in cardiac myocytes. Myocytes were treated with or without Ang II (100 nmol/L) for 18 hours. Immunoprecipitation was performed with anti-p34cdc2 or -p33cdk2 antibody, and the immunoprecipitates were subjected to the histone H1 kinase assays. As shown in Fig 5B⇑, control myocytes had very low activity of these kinases, and its level did not change after stimulation with Ang II (lanes 1, 2, 6, and 7). In contrast, an 18-hour treatment of cardiac fibroblasts with fetal calf serum (FCS, 20%) significantly increased kinase activity of p34cdc2 and p33cdk2 toward histone H1 (lanes 3, 5, 8, and 10). This increased kinase activity was not observed in the immunoprecipitates prepared with nonimmune serum (lanes 4 and 9). This suggests that kinase activity in the immunoprecipitates was in fact due to p34cdc2 or p33cdk2 and that the apparent absence of induction of histone H1 kinase activity of p34cdc2 and p33cdk2 in cardiac myocytes after Ang II stimulation was not due to a technical problem. These results suggest that the effects of rapamycin are not mediated by inhibition of these cyclin-dependent kinases in cardiac myocytes.
Rapamycin Inhibits the Ang II–Induced Increase in Protein Content
We next examined the effects of rapamycin on the Ang II–induced increase in protein synthesis. Cardiac myocytes were pretreated with various concentrations of rapamycin or vehicle alone (0.1% ethanol) for 30 minutes and were then stimulated with or without Ang II (100 nmol/L) for 24 hours. Ang II caused a modest but highly consistent increase in the protein content over 24 hours. Ang II–induced increase in protein content was observed at 1 nmol/L (13.8±2.1%, n=4, P<.05), the lowest concentration we examined, and reached a peak at 100 nmol/L (19.3±0.1%, n=7, P<.01; Fig 6A⇓, left). The results confirm our previous observations that Ang II causes hypertrophy of cardiac myocytes. Interestingly, rapamycin at >0.3 ng/mL completely suppressed the Ang II (100 nmol/L)–induced increase in the protein content. Rapamycin at 10 ng/mL reduced the protein content in the basal level as well. This is not a general toxic effect of rapamycin, because an increase in the protein content caused by FCS was not affected by rapamycin (10 ng/mL) (Fig 6A⇓, right).
We next examined the effects of rapamycin on the rate of protein synthesis and protein degradation. The rate of protein synthesis was determined by [3H]phenylalanine incorporation over 24 hours. As shown in Fig 6B⇑, rapamycin at 1 ng/mL significantly suppressed the Ang II–induced increase in [3H]phenylalanine incorporation, and rapamycin at 10 ng/mL significantly suppressed both basal and Ang II–induced [3H]phenylalanine incorporation. Rapamycin, however, did not significantly change protein degradation over 24 hours in the presence or absence of Ang II (Fig 6C⇑). These results indicate that rapamycin inhibits the Ang II–induced increase in protein content by inhibiting the rate of protein synthesis but not by increasing the rate of protein degradation.
Rapamycin Does Not Affect Other Ang II–Induced Hypertrophic Responses
We next examined whether rapamycin affects other hypertrophic phenotypes mediated by Ang II, such as induction of immediate-early genes and activation of the “fetal”-type gene program.7 As shown in Fig 7A⇓, the rapid accumulation of c-fos mRNA in response to Ang II was not affected by rapamycin. Ang II–induced upregulation of the fetal type genes, such as ANF and skeletal α-actin, was also observed even in the presence of rapamycin (10 ng/mL) (Fig 7B⇓), which completely suppressed the Ang II–induced increase in p70S6K activity (Fig 3A⇑) as well as cardiac protein content (Fig 6A⇑).
To examine the effect of rapamycin on the Ang II–induced phenotypic change in protein level, the level of ANF production in cardiac myocytes was determined by measuring the rate of accumulation of ANF in the culture media over 60 minutes.39 Conditioned media were collected from myocytes treated with or without Ang II in the presence of rapamycin or vehicle alone, and the ANF content was determined by RIA after a partial purification with Sep-Pak C18 columns. As shown in Fig 7C⇑, the rate of ANF secretion by myocytes treated with Ang II for 24 hours was significantly higher than that in control myocytes treated with vehicle. This increase in ANF accumulation was observed in the presence of rapamycin (10 ng/mL), suggesting that rapamycin does not inhibit the increased expression of ANF protein.
We next examined the effect of rapamycin on the Ang II–induced isoform switch of MHC. Because a significant amount of β-MHC is expressed in the control state in neonatal myocytes, we added a physiological concentration of T3 (2 ng/mL) to the medium throughout the experiments in order to accelerate “maturation” of neonatal myocytes in vitro.41 Myocyte extracts were subjected to SDS-PAGE on 5% gel, which enabled separation of α- and β-MHC on denaturing conditions,40 and then immunoblotted with anti–sarcomeric myosin antibody (Fig 7D⇑). In control myocytes, almost all MHC protein was α-isoform (lane 1). Ang II stimulation increased the expression of β-MHC (lane 4), indicating that Ang II promotes an isoform switch of MHC in cardiac myocytes in vitro, although a more extensive isoform switch was not possible in this experimental condition. Treatment of myocytes with rapamycin (10 ng/mL) did not affect basal (lane 2) or Ang II–induced increase in β-MHC expression (lane 3).
We finally examined the effect of rapamycin on Ang II–induced reorganization of contractile proteins into sarcomeric units, another important characteristic of load-induced and growth factor–induced cardiac hypertrophy.2 17 This effect was examined by staining filamentous actin with fluorescently labeled phalloidin. Fig 8a⇓ shows control myocytes, and Fig 8c⇓ shows rapamycin (10 ng/mL)–treated myocytes maintained in a serum-free medium without Ang II. The actin of these myocytes is generally not well organized. Fig 8b⇓ shows myocytes treated with Ang II for 24 hours. After Ang II treatment, the number of myocytes that show organized sarcomeric actin (visible as discrete fluorescent bands) significantly increased. This Ang II–induced organization of actin was also observed in myocytes treated with Ang II in the presence of rapamycin (10 ng/mL) (Fig 8d⇓). Highly organized sarcomeric actin was observed in 17, 63, 14, and 54 of 100 randomly selected myocytes in each staining shown in Fig 8a⇓, 8b⇓, 8c⇓, and 8d⇓, respectively.
Phosphorylation of S6 by p70S6K has been shown to stimulate incorporation of mRNA into polysomes, thereby stimulating initiation of protein translation.19 20 In the present study, we show that Ang II causes a rapid and sustained activation of p70S6K in cardiac myocytes. Interestingly, the macrolide rapamycin inhibited Ang II–induced activation of p70S6K without affecting Ang II–induced activation of known signaling pathways. Rapamycin suppressed the Ang II–induced increase in the protein content with a dose dependence that was similar to that for its inhibition of p70S6K. On the other hand, rapamycin did not suppress Ang II–induced phenotypic changes at both the mRNA and protein levels. These results suggest that the increase in overall protein synthesis and specific phenotypic changes in cardiac hypertrophy are regulated by different mechanisms and that activation of a rapamycin-sensitive signaling molecule, most likely p70S6K, may be involved in the Ang II–induced increase in the protein synthesis in cardiac myocytes.
Rapamycin exerts its effect by high-affinity binding to a cellular receptor protein designated FKBP, one of the immunophilins.27 FKBP is an evolutionarily conserved protein and the only known cellular receptor for rapamycin (Reference 2727 and S.L. Schreiber, Harvard University, personal communication, 1995). Recently, cellular targets of the rapamycin-FKBP complex have been identified in yeast and mammals. These proteins, referred to as TORs or RAFTs, have considerable amino acid sequence similarity to PI-3 kinase, and it has been postulated that the effect of rapamycin might be mediated by inhibition of RAFT1 PI-3 kinase activity.27 42 43 Thus, the point of action by a subnanomolar concentration of rapamycin is specific, and p70S6K and cyclin-dependent protein kinases (p33cdk2 and p34cdc2) are the only signaling molecules thus far reported to be inhibited by the FKBP-rapamycin complex (References 27 and 3027 30 and S.L. Schreiber, personal communication, 1995). We found that rapamycin suppresses p70S6K activity at very low concentrations (IC50, 0.14 ng/mL [0.15 nmol/L]) but does not suppress Ang II–induced activation of tyrosine kinase, MAP kinase, RSK, or PKC, even at concentrations that completely suppress Ang II–induced activation of p70S6K. Furthermore, Ang II does not activate p33cdk2 or p34cdc2 in cardiac myocytes, as determined by the immune complex histone H1 kinase assay. Thus, it is unlikely that the effect of rapamycin is mediated by inhibition of these cyclin-dependent kinases. Therefore, as far as we examined, the inhibitory effect of rapamycin is best explained by its effects on p70S6K among the Ang II–regulated early signaling molecules in cardiac myocytes. However, we cannot formally exclude the possibility that rapamycin may affect an as-yet-unidentified signaling molecule(s) that is regulated by RAFTs.
As an alternative approach to obtain a specific inhibition of p70S6K, transfection of a dominant negative form of the kinase or microinjection of a blocking antibody could be considered. To our knowledge, however, a dominant negative form of p70S6K has not been reported. Furthermore, microinjection of a blocking antibody against p70S6K may not be informative in this case, because p70S6K inhibition is expected not to suppress the known markers of Ang II–induced hypertrophy that can be used at a single cell level (ie, increases in ANF expression and sarcomeric assembly). Therefore, despite its potential limitation, rapamycin is a highly useful tool for studying the signal transduction mechanism of cardiac hypertrophy, because it is able to segregate tyrosine kinase–dependent, MAP kinase–dependent, RSK-dependent, and PKC-dependent signaling pathways from the rapamycin-sensitive signaling mechanism, most likely p70S6K, in cardiac myocytes.
Microinjection of activated p21ras into cardiac myocytes has been shown to cause hypertrophic responses, such as an increase in cell size and induction of c-fos, ANF, and MLC-2.15 In other cell systems, a p21ras-dependent protein kinase cascade has been shown to play a critical role in growth factor–induced MAP kinase activation.21 In cardiac myocytes, it has been recently shown that transfection or microinjection of a dominant negative form of MAP kinase (ERK-1) inhibits phenylephrine-induced gene expression, such as c-fos, ANF, and MLC-2.15 Therefore, it is possible that the p21ras-induced phenotypic changes in cardiac myocytes may require MAP kinase and its downstream S6 kinase, RSK. However, our results suggest that activation of MAP kinase is not sufficient to initiate an increase in protein content, because Ang II–induced MAP kinase activation was still observed in the presence of rapamycin. Because p70S6K is activated in Ras-transformed cells,44 it is possible that the increase in cell size induced by p21ras may be mediated by p70S6K. It has been shown that RSK has wide substrate specificity and phosphorylates transcription factors, such as Fos and serum response factor in addition to S6.20 21 On the other hand, p70S6K phosphorylates almost exclusively S6.20 Therefore, it is likely that each signaling molecule has different roles and that activation of various signaling molecules in concert mediate the hypertrophic response.
We have shown that rapamycin inhibits the Ang II–induced increase in overall protein content but not other Ang II–induced hypertrophic responses. For example, rapamycin did not inhibit Ang II–induced expression of an immediate-early gene, c-fos, or fetal-type genes, such as ANF and skeletal α-actin. Interestingly, rapamycin does not affect Ang II–induced phenotypic changes at the protein level either; increases in ANF production, β-MHC induction, and reorganization of actin filaments were observed in the presence of rapamycin. Interestingly, it has been suggested that phosphorylation of S6 at the carboxy terminal sites might induce translation of only a specific class of mRNAs, which are characterized by having a polypyrimidine tract at their 5′ cap sites. These mRNAs include elongation factors and ribosomal protein mRNAs.45 It is conceivable that inhibition of p70S6K by rapamycin might exert its effect by selectively inhibiting translation of mRNAs that encode regulators of protein translation whose increased expression is required for the Ang II–induced increase in the protein content. It is also interesting to note that mRNAs of ANF, skeletal α-actin, and β-MHC lack a 5′ polypyrimidine tract,46 47 48 raising the possibility that their translation may be regulated by a different mechanism.
At present, we do not know how p70S6K is activated in response to Ang II stimulation in cardiac myocytes. Although a wide variety of stimuli have been shown to activate p70S6K in other cell systems, the mechanism of p70S6K activation is not well understood.23 25 49 50 Although a 10-minute treatment of cardiac myocytes with phorbol 12-myristate 13-acetate (1 μmol/L), an activator of PKC, activated p70S6K (2.75-fold increase versus control, n=3; authors’ unpublished data, 1995), Ang II–induced activation of p70S6K was not affected by high concentrations of the general protein kinase inhibitor H-7 (100 μmol/L; authors’ unpublished data, 1995), which we have shown to inhibit the Ang II–induced activation of PKC activity.13 Therefore, at least H-7–sensitive isoforms of PKC may not be essential for Ang II–induced p70S6K activation. Similarly, both p21ras-dependent and -independent mechanisms have been reported for p70S6K activation in other cell systems.44 50 We have recently shown that Ang II activates p21ras (authors’ unpublished data, 1995), but at present, we do not know whether Ang II–induced p70S6K activation is p21ras -dependent or not. It has been recently shown that PI-3 kinase plays an essential role in p70S6K activation induced by platelet-derived growth factor.49 Considering the recent report that the βγ subunit of the G protein directly mediates PI-3 kinase activation in myeloid-derived cells,51 it is possible that agonists for G protein–coupled receptors directly activate PI-3 kinase, thereby causing activation of p70S6K. Therefore, it would be interesting to examine the role of PI-3 kinase in Ang II–induced p70S6K activation and Ang II–induced increase in protein content in cardiac myocytes.
Our results indicate that induction of c-fos is not sufficient to cause the Ang II–induced increase in protein synthesis in cardiac myocytes. Schunkert et al52 have recently shown that in the isolated perfused adult rat heart, Ang II causes an increase in the rate of protein synthesis without inducing c-fos expression. These results indicate that c-fos expression may not be essential for the increase in protein synthesis. However, induction of c-fos gene expression may play a role in other aspects of cardiac hypertrophy, such as induction of the genes regulated by the AP-1 transcription factor complex.5
Finally, it should be noted that different growth factors seem to use different mechanisms for regulating protein synthesis. We found that the increased protein content stimulated by Ang II is rapamycin-sensitive, but that stimulated by FCS is not. We confirmed that FCS also activates p70S6K, which is inhibited by rapamycin in cardiac myocytes (authors’ unpublished data, 1995). This suggests that molecules other than p70S6K also mediate the serum-induced increase in the protein content. It is interesting to note that MAP kinase has been implicated in the regulation of protein translation by phosphorylating the rat PHAS-I (phosphorylated heat- and acid-stable proteins regulated by insulin) after exposure of adipocytes to insulin. These proteins are thought to prevent initiation of protein translation by interacting with the translation initiation factor eIF-4E, but their phosphorylation relieves the translation inhibition.53 It has been also shown that Ang II induces phosphorylation of eIF-4E itself through a PKC-dependent mechanism in vascular smooth muscle.54 Because rapamycin did not affect activation of MAP kinase or PKC in cardiac myocytes, it is unlikely that rapamycin affects these signaling mechanisms in our system. However, it is likely that protein translation is regulated by multiple mechanisms, and further studies are necessary to elucidate the precise mechanism of the increase in protein synthesis by hypertrophic stimuli in cardiac myocytes.
Selected Abbreviations and Acronyms
|ANF||=||atrial natriuretic factor|
|Ang II||=||angiotensin II|
|ERK||=||extracellular signal–related kinase|
|FKBP||=||FK506 binding protein|
|MHC||=||myosin heavy chain|
|MLC||=||myosin light chain|
|p34cdc2 and p33cdk2||=||cyclin-dependent kinases|
|p70S6K||=||70- or 85-kD form of S6 kinase|
|PKC||=||protein kinase C|
|RAFTs||=||rapamycin and FKBP targets|
|RSK||=||rsk-encoded 85- to 92-kD ribosomal S6 kinase|
|TORs||=||targets of rapamycin|
A part of this study was supported by a Charles King Trust Fellowship of the Medical Foundation (Dr Sadoshima) and an Established Investigator Award of the American Heart Association (Dr Izumo). We thank Drs J. Blenis and E. Harlow for antibodies, Dr T. Kulik for the critical reading of the manuscript, Dr S. Schreiber for advice on the specificity of rapamycin, Dr S. Sehgal for providing rapamycin, and Dr J. Metzger for advice on MHC isoform analysis.
- Received July 24, 1995.
- Accepted September 6, 1995.
- © 1995 American Heart Association, Inc.
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