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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2127-2132.)
© 1999 American Heart Association, Inc.

Vascular Biology

Phosphatidylinositol 3-Kinase Is Required for Growth Factor–Induced Amino Acid Uptake by Vascular Smooth Muscle Cells

Masahide Higaki; Kentaro Shimokado

From the National Cardiovascular Center Research Institute, Osaka, Japan.

Correspondence to Kentaro Shimokado, MD, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail kshimoka{at}res.ncvc.go.jp

	Abstract

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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-ß and a dominant-negative PI3K did not increase their leucine uptake when stimulated with PDGF, whereas the same cells expressing only PDGF receptor-ß did. Transforming growth factor-ß, 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-ß 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-ß. Our findings suggest that PI3K may play an important role in vascular pathophysiology by regulating amino acid uptake.

Key Words: phosphatidylinositol 3-kinase • amino acid uptake • platelet-derived growth factor • transforming growth factor-ß

	Introduction

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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)-ß 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 -aminoisobutyric acid in VSMCs and skeletal muscle^{3 9} ; uptake of -aminoisobutyric acid and leucine in mouse 3T3 fibroblasts¹⁰ ; uptake of methyl -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.

	Methods

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Materials
Wortmannin was purchased from Kyowa Medics. LY294002 was from Biomol Research Laboratories Inc. Monoclonal antibodies against phosphotyrosine (PY-20) and phospholipase C- were from Signal Transduction Laboratories. Antisera against human PDGF-ß 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 p85 (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-ß was from King Brewing Co, Ltd.

Cell Culture
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-ß (PDGFR-ß), CHO-K1 cells were transfected with pDX–PDGFR-ß¹⁴ with lipofectamine (Gibco) according to the manufacturer's instructions; G418- (Geneticin, Wako) resistant cells were screened by Western blotting with anti–PDGFR-ß antiserum and cloned by limiting dilution. To obtain CHO/PDGFR cells stably expressing a dominant-negative PI3K (p85), CHO/PDGFR cells were transfected with SR-p85¹⁵ 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.

PI3K Assay
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 [-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
Statistical analysis was conducted by using Student's t test.

	Results

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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).

View larger version (15K): [in this window] [in a new window]   Figure 1. PDGF stimulated amino acid uptake in a time- and dose-dependent fashion. A, Time-dependent uptake. VSMCs were incubated in DMEM containing 0.1% BSA and [3H]leucine (•), [3H]proline (), or [3H]arginine (x) for 30 minutes, and then the cells were stimulated with PDGF-BB (10 ng/mL). Amino acid uptake was measured at the indicated times as described in Methods. Each point represents the mean±SE of triplicate values. *#P<0.01 compared with each respective control. The experiment was repeated twice with similar results. B, Dose-dependent amino acid uptake. VSMCs were incubated in DMEM containing 0.1% BSA and [3H]leucine (•), [3H]proline (), or [3H]arginine (x), and then the cells were stimulated with PDGF-BB at the indicated concentrations. After 6 hours, amino acid uptake was measured as described in Methods. Each point represents the mean±SE of triplicate values. *#P<0.01 compared with each respective control; ##P<0.05 compared with each respective control. The experiment was repeated twice with similar results.

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.

View larger version (28K): [in this window] [in a new window]   Figure 2. Wortmannin (WT) and LY294002 (LY) inhibited PDGF-induced amino acid uptake. VSMCs (A) and Swiss 3T3 cells (B) were incubated in DMEM containing 0.1% BSA and [3H]leucine (open bars), [3H]arginine (closed bars), or [3H]proline (hatched bars) in the presence or absence of inhibitors (100 nmol/L wortmannin or 10 µmol/L LY294002) for 30 minutes, and then PDGF-BB (2 ng/mL) was added to the medium. After 6 hours, amino acid uptake was measured as described in Methods. Values are the mean±SE of 3 independent experiments, each conducted in triplicate. *,,P<0.01 compared with each PDGF-treated group.

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-ß 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-ß alone (CHO/PDGFR) and CHO cells expressing both PDGFR-ß and a dominant-negative p85 subunit of PI3K (CHO/PDGFR/p85) had functional PDGFR-ß; PDGF-BB tyrosine-phosphorylated PDGFR-ß (Figure 3A) and activated phospholipase C-, a downstream signal, in these cells (Figure 3D). The p85 subunit cotransfected with PDGFR-ß 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/p85 cells (Figure 3C). PI3K activity measured with PI as the substrate was suppressed in CHO/PDGFR/p85 cells to <5% of that in CHO/PDGFR cells (Figure 3E). PDGF-BB did not stimulate leucine uptake in CHO/PDGFR/p85 cells but stimulated it in CHO/PDGFR cells in a dose-dependent fashion (Figure 4), confirming the findings obtained with the inhibitors.

View larger version (31K): [in this window] [in a new window]   Figure 3. Characterization of CHO/PDGFR/p85 cells. Quiescent cells were treated with PDGF-BB (10 ng/mL) or vehicle (PBS) for 5 minutes, and the cleared cell lysate was subjected to immunoprecipitation with anti–PDGFR-ß antiserum (A) or anti-phosphotyrosine monoclonal antibody (B, C, and D). Western blotting was conducted as described in Methods with the following antibodies: A, anti-phosphotyrosine antibody; B, anti-bovine PI3K p85 antibody (G12); C, anti-PI3K p110 antibody; and D, anti–phospholipase C- (PLC-) antibody. The experiment was repeated twice with similar results. PI3K was immunoprecipitated with anti-phosphotyrosine monoclonal antibody from CHO/PDGFR and CHO/PDGFR/p85 cells that were stimulated with 10 ng/mL PDGF-BB for 10 minutes, and PI3K activity was then assayed as described in Methods (E). PI(3)P indicates PI 3-monophosphate.

View larger version (25K): [in this window] [in a new window]   Figure 4. p85 subunit inhibited PDGF-induced leucine uptake in CHO/PDGFR cells. Quiescent CHO/PDGFR/p85 and CHO/PDGFR cells were obtained by culturing in F12 medium containing 0.1% BSA for 48 hours. The quiescent cells were cultured in F12 medium containing 0.1% BSA and [3H]leucine for 30 minutes, and then PDGF was added at the indicated concentrations. After 6 hours, amino acid uptake was measured as described in Methods. Values are the mean±SE of 3 independent experiments, each conducted in triplicate. *P<0.01 compared with CHO/PDGFR cells at the same concentration of PDGF.

PI3K Mediates Leucine Uptake Stimulated With TGF-ß as Well as IGF-I and Ang II
As reported previously with other cells,^{7 17 18} TGF-ß (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-ß and IGF-I, but not Ang II, increased leucine uptake, and this increase was inhibited by the inhibitors (data not shown).

View larger version (45K): [in this window] [in a new window]   Figure 5. Wortmannin (WT) and LY294002 (LY) inhibited leucine uptake induced by various growth factors. VSMCs were incubated in DMEM containing 0.1% BSA and [3H]leucine either in the presence or absence of inhibitors (100 nmol/L wortmannin or 10 µmol/L LY294002) for 30 minutes, and the cells were then stimulated with either 1 ng/mL TGF-ß (open bars), 10 ng/mL IGF-I (closed bars), or 100 nmol/L Ang II (AG-II, hatched bars). After 6 hours, amino acid uptake was measured as described in Methods. Values are the mean±SE of 3 independent experiments, each conducted in triplicate. *,,P<0.01 compared with each PDGF-treated group.

Although TGF-ß had not been reported to stimulate PI3K activity, our finding that PI3K inhibitors blocked the effect of TGF-ß suggested that TGF-ß might stimulate PI3K activity. Therefore, we studied whether TGF-ß stimulates PI3K activity by using Swiss 3T3 cells expressing more TGF-ß receptors than do VSMCs. TGF-ß 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-ß also increased phosphorylation of Akt, a downstream signaling molecule of PI3K¹⁹ (Figure 7).

View larger version (45K): [in this window] [in a new window]   Figure 6. TGF-ß activated PI3K in Swiss 3T3 cells. PI3K was immunoprecipitated with anti-PI3K antibody from Swiss 3T3 cells that were stimulated with 5 ng/mL TGF-ß for 10 minutes. PI3K activity in the immunoprecipitate was determined with PI as the substrate as described in Methods. PI(3)P indicates PI 3-monophosphate.

View larger version (45K): [in this window] [in a new window]   Figure 7. TGF-ß phosphorylated Akt. Swiss 3T3 cells were treated with TGF-ß for 30 minutes before being lysed with SDS sample buffer, and then they were subjected to Western blot analysis. The membrane was probed sequentially with anti-Akt rabbit IgG that recognizes total Akt, regardless of its phosphorylation state (A), and anti-phospho-Akt rabbit IgG that recognizes Akt phosphorylated at serine 473 (B).

	Discussion

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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-ß transmits its signal through non–tyrosine kinase–type receptors and through unique signaling molecules, such as SMAD and TAK1 (TGF-ß–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-ß and a dominant-negative PI3K. These cells expressed more PDGFR and responded more to PDGF than did the CHO/p85 cells that we had reported previously.¹² The amount of PDGFR protein and the tyrosine phosphorylation of PDGFR and phospholipase C- are comparable between CHO/PDGFR/p85 and CHO/PDGFR. PI3K activation and leucine uptake were completely suppressed in CHO/PDGFR/p85 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-ß 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-ß increases PI3K activity. TGF-ß 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-ß did not increase PI3K activity in the anti-phosphotyrosine immunoprecipitate, whereas PDGF increased it 20-fold, suggesting that PI3K activation with TGF-ß is not mediated by tyrosine phosphorylation. Because we could not immunoprecipitate PI3K with an anti–TGF-ß type II receptor, TGF-ß may stimulate PI3K indirectly. Activation of PI3K by TGF-ß 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.

	Acknowledgments

 
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 SR-p85, Dr Daniel F. Bowen-Pope (University of Washington) for pDX-PDGFR-ß, and H. Sugita for her technical assistance.

	Footnotes

 
Current address of M.H., Medical Biology Research Lab, Fujisawa Pharma, Co, Ltd, Kashima, Osaka, Japan.

Received October 9, 1998; accepted January 26, 1999.

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