Phosphatidylinositol 3-Kinase Is Required for Growth Factor–Induced Amino Acid Uptake by Vascular Smooth Muscle Cells
Abstract—Although accumulating evidence suggests that phosphatidylinositol 3-kinase (PI3K) is a common signaling molecule for growth factor–induced amino acid uptake by the cell, the role of PI3K in the uptake of different amino acids was not tested under the same conditions. In this study, we asked whether PI3K mediates platelet-derived growth factor (PDGF) –stimulated uptake of different amino acids that are taken up through 3 major amino acid transporters expressed in rat vascular smooth muscle cells and other cell types and whether PI3K mediates amino acid uptake stimulated with different growth factors and vasoactive substances. PDGF increased the uptake of [3H]leucine, [3H]proline, and [3H]arginine in a dose- and time-dependent fashion. Two different PI3K inhibitors, wortmannin (100 nmol/L) and LY294002 (10 μmol/L), completely inhibited the amino acid uptake stimulated by PDGF. Chinese hamster ovary cells expressing both PDGF receptor-β and a dominant-negative PI3K did not increase their leucine uptake when stimulated with PDGF, whereas the same cells expressing only PDGF receptor-β did. Transforming growth factor-β, as well as insulin-like growth factor-I and angiotensin II, increased leucine uptake by vascular smooth muscle cells. Wortmannin and LY294002 inhibited this increase. We also found that transforming growth factor-β stimulated PI3K activity and the phosphorylation of Akt, a downstream signaling molecule of PI3K. A similar effect of PI3K inhibitors on amino acid uptake was observed in Swiss 3T3 cells. We conclude that PI3K mediates the uptake of different amino acids by vascular smooth muscle cells and other cell types stimulated with a variety of growth factors, including transforming growth factor-β. Our findings suggest that PI3K may play an important role in vascular pathophysiology by regulating amino acid uptake.
- phosphatidylinositol 3-kinase
- amino acid uptake
- platelet-derived growth factor
- transforming growth factor-β
Current address of M.H., Medical Biology Research Lab, Fujisawa Pharma, Co, Ltd, Kashima, Osaka, Japan.
- Received October 9, 1998.
- Accepted January 26, 1999.
Various amino acids are taken up into the cell through different amino acid transport systems, with some overlapping of substrate specificity.1 2 In vascular smooth muscle cells (VSMCs), a few ubiquitous transporters have been identified, including system A,3 system L,4 and cationic amino acid transporters 1 and 2B (CAT-1 and CAT-2B).5 These transporters are regulated differently by various growth factors at the level of gene transcription and activation of protein, and have been implicated in different biological consequences. For example, platelet-derived growth factor (PDGF), insulin, insulin-like growth factor (IGF)-I, and transforming growth factor (TGF)-β stimulate amino acid uptake through systems A and L, and induce cell proliferation, cellular hypertrophy, and matrix synthesis.3 5 6 7 Angiotensin (Ang) II has similar effects on amino acid uptake by VSMCs.8 PDGF induces gene expression of CAT, and arginine uptake through CAT is indispensable for the mitogenic activity of PDGF.5 Despite the important role of amino acid uptake in growth factor action, the signaling pathway between the growth factor receptors and amino acid uptake has not been elucidated.
Involvement of phosphatidylinositol 3-kinse (PI3K) in amino acid uptake has been reported with different cell types and growth factors: insulin-stimulated uptake of α-aminoisobutyric acid in VSMCs and skeletal muscle3 9 ; uptake of α-aminoisobutyric acid and leucine in mouse 3T3 fibroblasts10 ; uptake of methyl α-aminoisobutyric acid in 3T3-L1 adipocytes11 ; and PDGF-induced uptake of leucine in Swiss 3T3 cells.12 These studies suggest that PI3K is a common signaling molecule in the uptake of various amino acids by various cell types. However, these studies were performed under different conditions with different cell types, and the notion was never tested in a single cell type. In this study, we asked whether PI3K is a common signaling molecule for the uptake of amino acids through different transporters expressed in VSMCs and Swiss 3T3 cells and whether PI3K is a common signaling molecule for different stimuli of amino acid uptake in these cells.
Wortmannin was purchased from Kyowa Medics. LY294002 was from Biomol Research Laboratories Inc. Monoclonal antibodies against phosphotyrosine (PY-20) and phospholipase C-γ were from Signal Transduction Laboratories. Antisera against human PDGF-β receptor and PI3K were from Upstate Biotechnology Inc. Antiserum against the p110 subunit of PI3K was from Santa Cruz Biotechnology Inc. Monoclonal antibody against bovine p85α (G12) was kindly provided by Dr Masato Kasuga from Kobe University. PI was from Sigma. Recombinant human PDGF-BB was from Pepro Tech Inc. IGF-I was from Genzyme Diagnostic; Ang II was from the Peptide Institute, Inc; and recombinant human TGF-β was from King Brewing Co, Ltd.
VSMCs were prepared from the aortas of Sprague-Dawley rats and cultured as reported previously.13 Hill-and-valley–type VSMCs were used for the experiments between the fourth and sixth passage. Swiss 3T3 cells were obtained from the Japan Cancer Research Resources Bank (Osaka, Japan) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS. To obtain Chinese hamster ovary (CHO) cells stably expressing PDGF receptor-β (PDGFR-β), CHO-K1 cells were transfected with pDX–PDGFR-β14 with lipofectamine (Gibco) according to the manufacturer’s instructions; G418- (Geneticin, Wako) resistant cells were screened by Western blotting with anti–PDGFR-β antiserum and cloned by limiting dilution. To obtain CHO/PDGFR cells stably expressing a dominant-negative PI3K (Δp85), CHO/PDGFR cells were transfected with SRα-Δp8515 with lipofectamine; hygromycin-resistant cells were screened by Western blotting with anti-bovine p85 antibody and cloned by limiting dilution. CHO cells were cultured in F12 medium supplemented with 10% FCS.
Amino Acid Uptake
Amino acid uptake was measured by the method previously described, with a slight modification.12 In brief, confluent cells in a 24-well plate were cultured in DMEM containing 0.1% BSA for 48 hours. Quiescent cells were cultured in DMEM supplemented with tritiated amino acids (leucine, arginine, or proline; 92.5 kBq per well) and 0.1% BSA for 30 minutes, and then a growth factor was added. After a 6-hour incubation, cells were washed 3 times with cold PBS and lysed with 1 mL of 0.25N NaOH. The radioactivity in the whole-cell lysate was counted with a Packard Tri-Carb 2700TR liquid scintillation analyzer.
Western Blot Analysis
Western blot analysis was conducted by using an enhanced chemiluminescence Western blotting kit (Amersham) as reported previously.12 In brief, cells incubated with or without PDGF-BB for 5 minutes were lysed with ice-cold RIPA buffer (10 mmol/L Tris-HCl, pH 7.4; 150 mmol/L NaCl; 5 mmol/L EDTA; 1% Triton X-100; 1% deoxycholic acid; 1% Trasylol; 0.1% SDS; 1 mmol/L ABSF; and 1 mmol/L Na3VO4) and centrifuged at 15 000 rpm for 20 minutes at 4°C. The supernatant was incubated with antibody coupled with protein G–Sepharose (Pharmacia) for 60 minutes at 4°C. The immunoprecipitate was applied to an SDS-polyacrylamide gel. After electrophoresis, the immunoprecipitate was electrotransferred to a nitrocellulose membrane (Atto Co). The membrane was incubated with the primary antibodies at room temperature for 60 minutes. After 3 washes, specific bands were detected by an enhanced chemiluminescence Western blotting kit according to the manufacturer’s instructions.
The activity of PI3K in the anti-PI3K antiserum immunoprecipitate was assayed by the method reported previously.12 In brief, confluent cells in a 9-cm-diameter dish were lysed and then centrifuged at 15 000 rpm for 20 minutes at 4°C. The supernatant was incubated with anti-PI3K antiserum coupled with protein G–Sepharose (Pharmacia) for 60 minutes at 4°C. The immunoprecipitate was washed with kinase buffer and then suspended in the same buffer. Sonicated PI was then added. The reaction was started by the addition of 37 kBq of [γ-32P]ATP, and the samples were incubated at 30°C for 10 minutes. The reaction was stopped by the addition of 1N HCl and chloroform/methanol (2:1, vol/vol). Phospholipids were recovered from the lower organic phase, which was dried under N2 gas, dissolved in chloroform, spotted on a silica-gel 60 plate (Merck), impregnated with 1% potassium oxalate, and developed in chloroform/methanol/28% NH3/water (70:100:15:25, vol/vol/vol/vol). The radioactivity of PI 3-monophosphate on the dried plate was visualized and quantified by a Fuji BAS2000 Bioimaging Analyzer.
Phosphorylation of Akt
Phosphorylation of Akt at serine 473 was detected with the PhosphoPlus Akt antibody kit (New England Biolabs, Inc) according to the manufacturer’s instructions.
Statistical analysis was conducted by using Student’s t test.
PDGF Stimulates VSMC Uptake of 3 Different Amino Acids Through PI3K
PDGF-BB significantly stimulated the uptake by VSMCs of leucine, arginine, and proline, which are mainly taken up by system L, CAT, and system A, respectively1 (Figure 1⇓). The time course and dose dependency were similar to those reported previously by others.16 The amino acid uptake by PDGF-stimulated VSMCs reached a plateau at 4 hours, with maximum values of a 1.4-fold increase over controls for leucine, 1.7-fold for arginine, and 1.8-fold for proline (Figure 1A⇓). The uptake of all amino acids was stimulated with 1 to 5 ng/mL PDGF-BB, and higher concentrations did not further increase the uptake (Figure 1B⇓). A similar increase was observed in Swiss 3T3 cells (data not shown).
Wortmannin (100 nmol/L) and LY294002 (10 μmol/L), 2 inhibitors of PI3K with different modes of action, completely inhibited uptake of the 3 amino acids in both VSMCs (Figure 2A⇓) and Swiss 3T3 cells (Figure 2B⇓) at concentrations sufficient to inhibit PI3K activity in these cells,12 indicating that PI3K mediates amino acid uptake through different amino acid transporting systems.
CHO Cells Expressing PDGFR and a Dominant-Negative Subunit of PI3K
To rule out the possibility that the above findings were due to nonspecific effects of the inhibitors, we prepared CHO cells expressing PDGFR-β and a dominant-negative p85 subunit of PI3K, and studied the effect of PI3K suppression on PDGF-induced leucine uptake. A stable cell line of CHO cells expressing PDGFR-β alone (CHO/PDGFR) and CHO cells expressing both PDGFR-β and a dominant-negative p85 subunit of PI3K (CHO/PDGFR/Δp85) had functional PDGFR-β; PDGF-BB tyrosine-phosphorylated PDGFR-β (Figure 3A⇓) and activated phospholipase C-γ, a downstream signal, in these cells (Figure 3D⇓). The Δp85 subunit cotransfected with PDGFR-β effectively inhibited the PDGF-induced activation of PI3K (Figure 3B⇓); the p110 subunit became associated with PDGFR in CHO/PDGFR cells, whereas it did not in CHO/PDGFR/Δp85 cells (Figure 3C⇓). PI3K activity measured with PI as the substrate was suppressed in CHO/PDGFR/Δp85 cells to <5% of that in CHO/PDGFR cells (Figure 3E⇓). PDGF-BB did not stimulate leucine uptake in CHO/PDGFR/Δp85 cells but stimulated it in CHO/PDGFR cells in a dose-dependent fashion (Figure 4⇓), confirming the findings obtained with the inhibitors.
PI3K Mediates Leucine Uptake Stimulated With TGF-β as Well as IGF-I and Ang II
As reported previously with other cells,7 17 18 TGF-β (1 ng/mL), IGF-I (10 ng/mL), and Ang II (100 nmol/L) significantly stimulated leucine uptake by VSMCs to an extent similar to that obtained with PDGF. Wortmannin (100 nmol/L) and LY294002 (10 μmol/L) completely inhibited this growth factor–stimulated amino acid uptake in VSMCs (Figure 5⇓), indicating that PI3K is involved in amino acid uptake stimulated by these growth factors. In Swiss 3T3 cells, TGF-β and IGF-I, but not Ang II, increased leucine uptake, and this increase was inhibited by the inhibitors (data not shown).
Although TGF-β had not been reported to stimulate PI3K activity, our finding that PI3K inhibitors blocked the effect of TGF-β suggested that TGF-β might stimulate PI3K activity. Therefore, we studied whether TGF-β stimulates PI3K activity by using Swiss 3T3 cells expressing more TGF-β receptors than do VSMCs. TGF-β significantly increased PI3K activity in the immunoprecipitate of anti-PI3K antibody to an extent similar to that stimulated with PDGF (127±9; n=3, P<0.05; Figure 6⇓). This increase was completely inhibited by 100 nmol/L wortmannin or 10 μmol/L LY294002 (data not shown). TGF-β also increased phosphorylation of Akt, a downstream signaling molecule of PI3K19 (Figure 7⇓).
An important finding of the current study is that PI3K is a common signaling molecule that transmits the stimulus from the growth factor receptor to different amino acid transport systems. Different amino acids are taken up through different amino acid transporters that are regulated differently and have been implicated in different biological consequences. Leucine is mainly taken up by VSMCs through the system L amino acid transporter,20 which is 1 of the ubiquitous transporters regulated partly by nonhormonal mechanisms and that plays a role in general protein synthesis in VSMCs.4 Arginine is taken up by VSMCs through 2 subtypes of a CAT, CAT-1 and CAT-2B.5 This transporter activity is important in polyamine synthesis required for PDGF-induced mitogenesis5 and in providing arginine to NO synthase located at the caveolae.21 In mammals, proline is taken up through system A, another ubiquitous transporter serving for most bipolar amino acids.2 Previous reports from our laboratory and others have indicated that PI3K plays an important role in amino acid uptake through an individual transport system.3 9 10 11 12 However, the uptake of different amino acids was tested with different cell types but was never compared in any single cell type. Neither had a role for PI3K in amino acid transport through CAT been documented. The current study demonstrates that PI3K is indispensable for the stimulation of different amino acid transport systems, including CAT. Because previous studies had indicated that amino acid uptake is important for cellular proliferation,5 vascular hypertrophy,6 8 vascular remodeling,3 and regulation of vascular tone,22 PI3K may be a key enzyme in a wide range of VSMC functions.
Another important finding is that different growth factors and vasoactive substances share PI3K as a common signaling molecule in their stimulation of amino acid uptake. PDGF and IGF-I have receptors coupled with tyrosine kinases and transmit their signal through tyrosine phosphorylation of signaling molecules that have src-homology domains in them.23 TGF-β transmits its signal through non–tyrosine kinase–type receptors and through unique signaling molecules, such as SMAD and TAK1 (TGF-β–activated kinase-1).24 Ang II transmits its signal through receptors coupled with G proteins and protein kinase C. Despite all of these differences in signal transduction, amino acid uptake with different stimuli was blocked by the PI3K inhibitors. The role of PI3K in amino acid uptake is not limited to VSMCs but also occurs in other cell types, such as Swiss 3T3 and CHO cells.
These conclusions were based on experiments with both PI3K inhibitors and with CHO cells expressing a dominant-negative PI3K.Wortmannin, a noncompetitive and irreversible inhibitor of PI3K,25 and LY294002, a competitive inhibitor,26 have been used to inhibit PI3K activity in various cells and to study the physiological role of PI3K. Both compounds inhibit PI3K activity of the purified enzyme and in cultured cells at 100 nmol/L,12 a concentration insufficient to inhibit phospholipase A2 or myosin light-chain kinase.25 27 To further rule out the possibility that the inhibition of amino acid uptake was due to nonspecific effects of the inhibitors, we prepared CHO cells expressing both PDGFR-β and a dominant-negative PI3K. These cells expressed more PDGFR and responded more to PDGF than did the CHO/Δp85 cells that we had reported previously.12 The amount of PDGFR protein and the tyrosine phosphorylation of PDGFR and phospholipase C-γ are comparable between CHO/PDGFR/Δp85 and CHO/PDGFR. PI3K activation and leucine uptake were completely suppressed in CHO/PDGFR/Δp85 cells, confirming the findings obtained with the inhibitors.
Although PDGFR and insulin receptor substrate-1 (IRS-I), a downstream signaling molecule of the IGF-I receptor, directly bind to PI3K and increase its activity, it is not clear how the receptors for Ang II and TGF-β stimulate PI3K activity. Ang II increases tyrosine phosphorylation of IRS-I and the subsequent association of IRS-I and PI3K in the rat heart.28 However, Ang II does not increase PI3K activity in that model.29 Recently, it was revealed that Ang II phosphorylates and activates growth factor receptors, such as the receptor for epidermal growth factor.30 Ang II may activate PI3K indirectly by activating other growth factor receptors as well.
So far as we are aware, this is the first report showing that TGF-β increases PI3K activity. TGF-β increased PI3K activity in anti-PI3K immunoprecipitates by 20%. This rather small increase is similar to that obtained with PDGF stimulation and reflects a large pool of PI3K that is unaffected by a single growth factor.31 TGF-β did not increase PI3K activity in the anti-phosphotyrosine immunoprecipitate, whereas PDGF increased it 20-fold, suggesting that PI3K activation with TGF-β is not mediated by tyrosine phosphorylation. Because we could not immunoprecipitate PI3K with an anti–TGF-β type II receptor, TGF-β may stimulate PI3K indirectly. Activation of PI3K by TGF-β was further substantiated by phosphorylation of a downstream signaling molecule of PI3K, Akt, which is phosphorylated at serine 473 by PI 3,4-bisphosphate, a product of activated PI3K.19
In summary, we report that PI3K is necessary for the uptake of different amino acids stimulated with various growth factors. PI3K activity may therefore affect various aspects of VSMC functions through amino acid uptake.
This study was supported by grants from the Science and Technology Agency of Japan, the Ministry of Health and Welfare, and the Organization for Pharmaceutical Safety and Research (to K.S.). We thank Dr Masato Kasuga (Kobe University) for SRα-Δp85, Dr Daniel F. Bowen-Pope (University of Washington) for pDX-PDGFR-β, and H. Sugita for her technical assistance.
Christensen HN. Role of amino acid transport and countertransport in nutrition and metabolism. Physiol Rev. 1990;70:43–77.
Obata T, Kashiwagi A, Maegawa H, Nishio Y, Ugi S, Hidaka H, Kikkawa R. Insulin signaling and its regulation of system A amino acid uptake in cultured rat vascular smooth muscle cells. Circ Res. 1996;79:1167–1176.
Low BC, Ross IK, Grigor MR. Glucose deprivation and acute cycloheximide treatment stimulate system L amino acid transport in cultured vascular smooth muscle cells. J Biol Chem. 1994;269:32098–32103.
Durante W, Liao L, Iftikhar I, Cheng K, Schafer AI. Platelet-derived growth factor regulates vascular smooth muscle cell proliferation by inducing cationic amino acid transporter gene expression. J Biol Chem. 1996;271:11838–11843.
Boerner P, Resnick RJ, Racker E. Stimulation of glycolysis and amino acid uptake in NRK-49F cells by transforming growth factor β and epidermal growth factor. Proc Natl Acad Sci U S A. 1985;82:1350–1353.
Low BC, Grigor MR. Angiotensin II stimulates system y+ and cationic amino acid transporter gene expression in cultured vascular smooth muscle cells. J Biol Chem. 1995;270:27577–27583.
Su TZ, Wang M, Syu LJ, Saltiel AR, Oxender DL. Regulation of system A amino acid transport in 3T3–L1 adipocytes by insulin. J Biol Chem. 1998;273:3173–3179.
Higaki M, Sakaue H, Ogawa W, Kasuga M, Shimokado K. Phosphatidylinositol 3-kinase-independent signal transduction pathway for platelet-derived growth factor-induced chemotaxis. J Biol Chem. 1996;271:29342–29346.
Shimokado K, Yokota T, Umezawa K, Sasaguri T, Ogata J. Protein tyrosine kinase inhibitors inhibit chemotaxis of vascular smooth muscle cells. Arterioscler Thromb. 1994;14:973–981.
Gronwald RG, Grant FJ, Haldeman BA, Hart CE, O‘Hara PJ, Hagen FS, Ross R, Bowen-Pope DF, Murray MJ. Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: evidence for more than one receptor class. Proc Natl Acad Sci U S A. 1988;85:3435–3439.
Hara K, Yonezawa K, Sakaue H, Ando A, Kotani K, Kitamura T, Kitamura Y, Ueda H, Stephens L, Jackson TR, Hawkins PT, Dhand R, Clark AE, Holman GD, Waterfield MD, Kasuga M. 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci U S A. 1994;91:7415–7419.
Owen AJ 3d, Geyer RP, Antoniades HN. Human platelet-derived growth factor stimulates amino acid transport and protein synthesis by human diploid fibroblasts in plasma-free media. Proc Natl Acad Sci U S A. 1982;79:3203–3207.
Moran A, Brown DM, Kim Y, Klein DJ. Effect of IGF-I and glucose on protein and proteoglycan synthesis by human fetal mesangial cells in culture. Diabetes. 1991;40:1346–1354.
McDonald KK, Zharikov S, Block ER, Kilberg MS. A caveolar complex between the cationic amino acid transporter 1 and endothelial nitric-oxide synthase may explain the ‘arginine paradox.’ J Biol Chem. 1997;272:31213–31216.
Kikuta K, Sawamura T, Miwa S, Hashimoto N, Masaki T. High-affinity arginine transport of bovine aortic endothelial cells is impaired by lysophosphatidylcholine. Circ Res. 1998;83:1088–1096.
Powis G, Bonjouklian R, Berggren MM, Gallegos A, Abraham R, Ashendel C, Zalkow L, Matter WF, Dodge J, Grindey G, Vlahos CJ. Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase. Cancer Res. 1994;54:2419–2423.
Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994;269:5241–5248.
Yano H, Agatsuma T, Nakanishi S, Saitoh Y, Fukui Y, Nonomura Y, Matsuda Y. Biochemical and pharmacological studies with KT7692 and LY294002 on the role of phosphatidylinositol 3-kinase in Fc εRI-mediated signal transduction. Biochem J. 1995;312:145–150.
Saad MJ, Velloso LA, Carvalho CR. Angiotensin II induces tyrosine phosphorylation of insulin receptor substrate 1 and its association with phosphatidylinositol 3-kinase in rat heart. Biochem J. 1995;310:741–744.
Velloso LA, Folli F, Sun XJ, White MF, Saad MJ, Kahn CR. Cross-talk between the insulin and angiotensin signaling systems. Proc Natl Acad Sci U S A. 1996;93:12490–12495.
Eguchi S, Numaguchi K, Iwasaki H, Matsumoto T, Yamakawa T, Utsunomiya H, Motley ED, Kawakatsu H, Owada KM, Hirata Y, Marumo F, Inagami T. Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells. J Biol Chem. 1998;273:8890–8896.
Domin J, Dhand R, Waterfield MD. Binding to the platelet-derived growth factor receptor transiently activates the p85α-p110α phosphatidylinositide 3-kinase complex in vivo. J Biol Chem. 1996;271:21614–21621.