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[Cancer Research 65, 10881-10890, December 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Platelet-Derived Growth Factor–Induced p42/44 Mitogen-Activated Protein Kinase Activation and Cellular Growth Is Mediated by Reactive Oxygen Species in the Absence of TSC2/Tuberin

Geraldine A. Finlay1, Victor J. Thannickal3, Barry L. Fanburg1 and David J. Kwiatkowski2

1 Pulmonary and Critical Care Division, Department of Medicine, Tupper Research Institute, New England Medical Center; 2 Hematology Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts; and 3 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan

Requests for reprints: Geraldine Finlay, Pulmonary, Critical Care and Sleep Division, New England Medical Center, NEMC 257, 750 Washington Street, Boston, MA 02111. Phone: 617-636-7751; Fax: 617-636-5953; E-mail: gfinlay{at}tufts-nemc.org.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tuberous sclerosis complex (TSC) is a genetic disorder caused by inactivating mutations in the TSC1 or TSC2 genes, which encode hamartin and tuberin, respectively. TSC is characterized by multiple tumors of the brain, kidney, heart, and skin. Tuberin and hamartin inhibit signaling by the mammalian target of rapamycin (mTOR) but there are limited studies of their involvement in other pathways controlling cell growth. Using ELT-3 cells, which are Eker rat–derived smooth muscle cells, we show that ELT-3 cells expressing tuberin (TSC2+/+) respond to platelet-derived growth factor (PDGF) stimulation by activating the classic mitogen-activated protein (MAP)/extracellular signal-regulated kinase kinase (MEK)-1–dependent phosphorylation of p42/44 MAP kinase (MAPK) with nuclear translocation of phosphorylated p42/44 MAPK. In contrast, in tuberin-deficient ELT-3 cells (TSC2–/–), PDGF stimulation results in MEK-1–independent p42/44 MAPK phosphorylation with reduced nuclear localization of phosphorylated p42/44 MAPK. Moreover, in TSC2–/– cells but not in TSC2+/+ cells, cellular growth and activation of p42/44 MAPK by PDGF requires the reactive oxygen species intermediate, superoxide anion (O2·–). Both baseline and PDGF-induced O2·– levels were significantly higher in TSC2–/– cells and were reduced by treatment with rapamycin and inhibitors of mitochondrial electron transport. Furthermore, the exogenous production of O2·– by the redox cycling compound menadione induced MEK-1–independent cellular growth and p42/44 MAPK phosphorylation in TSC2–/– cells but not in TSC2+/+ cells. Together, our data suggest that loss of tuberin, which causes mTOR activation, leads to a novel cellular growth-promoting pathway involving mitochondrial oxidant–dependent p42/44 MAPK activation and mitogenic growth responses to PDGF.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome characterized by multiple tumors of the brain, kidney, heart, and skin (1, 2). Mutations in two genes, TSC1 and TSC2, are causally linked to the development of TSC. Accumulating data support the hypothesis that the major tumor suppressor effect of tuberin (TSC2 product) and hamartin (TSC1 product) occurs through their ability to inhibit one of the major cellular growth pathways, the phosphatidylinositol 3-kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR)–S6 kinase (S6K) pathway (3–6). In the current model, activated Akt phosphorylates tuberin (5), inhibiting its ability to act in a complex with hamartin as a GTPase-activating protein (GAP) for Rheb (7). Rheb is a major regulator of mTOR activity. Thus, in cells lacking functional hamartin or tuberin, elevated Rheb-GTP levels lead to constitutive activation of mTOR, resulting in hyperphosphorylation of p70 S6K and 4E-BP1 (8–10).

A second major growth pathway activated as a result of tyrosine kinase receptor phosphorylation is the ras–raf–mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)-1–MAP kinase (MAPK) pathway (11, 12). Activation of this pathway by growth factors leads to phosphorylation of two MAPKs, ERK-1(p44mapk) and ERK-2(p42mapk), which translocate to the nucleus to regulate gene transcription necessary for mitogenesis. Connections between the MAPK pathway and tuberin have been reported, including tuberin-dependent phosphorylation of B-raf and p42/44 MAPK in some cells (9) and p42/44 MAPK-dependent phosphorylation of tuberin both directly and through p90 ribosomal S6K (RSK1; refs. 13, 14). These data suggest that tuberin both regulates and is regulated by p42/44 MAPK.

A role for reactive oxygen species (ROS) as mediators of cell growth is increasingly recognized (15, 16). ROS can induce cellular growth in the absence of growth factors; ROS can also be generated following receptor stimulation by growth factors. Growth factor–generated ROS production has been coupled to downstream cytosolic signal transduction molecules, including members of the ras-raf-MEK-1-MAPK family pathway (17–20) and the PI3K-Akt-S6K pathway (21–24). It is unclear whether the effects of ROS on MAPK activation are indirect and due to upstream activators of MAPK or a direct effect of ROS on MAPK itself. Although ERK activation by ROS is reduced by inhibition of MEK-1 in some studies (17), other reports indicate that ROS activation of cell growth does not involve MAPK (18). Thus, growth factor–generated ROS can potentially play a role in mediating cell growth in a MAPK-dependent or MAPK-independent fashion.

Previously, we have observed altered temporal patterns of MAPK phosphorylation in tuberin-null (TSC2–/–) compared with tuberin-expressing (TSC2+/+) ELT-3 cells (25). In this study, we extend these findings and show that in response to a standard growth stimulus, platelet-derived growth factor (PDGF), TSC2+/+ cellular growth, and activation of p42/44 MAPK are MEK-1-dependent. Moreover, following activation, MAPK rapidly and quantitatively localizes to the nucleus. In contrast, in TSC2–/– cells, we show that cellular growth and activation of MAPK is MEK-1-independent and nuclear localization is reduced. We also show in TSC2–/– cells that cell growth and p42/44 MAPK activation are redox sensitive. Furthermore, in the absence of tuberin expression, O2·– levels, both at baseline and in response to PDGF, are much higher in TSC2–/– than in TSC2+/+ cells. These data suggest that tuberin plays an integral role in regulating cell growth by regulating ROS production, activation, and location of p42/44 MAPK within the cell.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and growth factor stimulation. ELT-3 cells are uterine-derived leiomyoma tumor cells cultured from the Eker rat that were obtained from and characterized by Dr. Cheryl Walker (M.D. Anderson Cancer Center; ref. 26). ELT3 cells are TSC2 null and do not express tuberin. Reexpression of the TSC2 gene in ELT-3 cells and the creation of stable cell lines is described elsewhere (25). For descriptive purposes, tuberin-null ELT-3 cells infected with an empty vector are called TSC2–/– and tuberin-expressing ELT-3 cells infected with retrovirus containing TSC2 are called TSC2+/+.

For cell growth experiments, cells were seeded at a density of 0.5 x 104 to 1.0 x 104 cells/mL in 35 mm dishes. When cultures achieved 20% confluency, serum was withdrawn for 72 hours. Cells were then treated with PDGF (R&D Systems, Minneapolis, MN), menadione (Sigma, St. Louis, MO), and/or cotreated with inhibitors for specific time periods at the doses indicated in the text. After trypsinization, cells were diluted and counted in a Model Z1 Coulter Counter (Beckman Coulter, Miami, FL). Growth experiments were done in quadruplicate and repeated at least twice to ensure reproducibility. For all other experiments, cells were seeded at a density of 0.5 x 106 cells/mL on 100 mm Petri dishes. When cultures achieved 90% to 100% confluency, serum was withdrawn and cells were treated with PDGF, menadione, and/or inhibitors under serum-free conditions. After intervals specified in the text, cells were harvested and extracts were prepared as described below. All experiments were done at least in duplicate using appropriate vehicle controls.

Inhibitors. Inhibitors used in these studies included the MEK-1 inhibitor UO126 (Cell Signaling Technology, Cambridge, MA), PD98059 (Calbiochem, La Jolla CA), the PI3K inhibitor LY294002, the mTOR inhibitor rapamycin (Cell Signaling Technology), and the flavoprotein oxidase inhibitor diphenyliodonium (ICN Biomedicals, Aurora, OH). All other inhibitors were obtained from Sigma: the superoxide radical scavenger Tiron; glutathione precursor; N-acetylcysteine; polyethyleneglycol catalase (PEG-catalase); the PI3K inhibitor wortmannin; inhibitors of mitochondrial function potassium cyanide, rotenone, and antimycin; the cytochrome P450 inhibitor methoxypsoralen; the xanthine oxidase inhibitor allopurinol; and the nitric oxide inhibitor L-nitro-D-arginine methyl ester hydrochloride. Unless otherwise stated, pretreatments were for 1 hour at the doses indicated in the text.

Preparation of cell lysates was as previously described (25). Nuclear extracts were prepared by lysis in 10 mmol/L HEPES, 1 mmol/L KCl, 2 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L NaF, 1 mmol/L Na4P2O7, 0.1 mmol/L Na3VO4, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 5 µg/mL leupeptin, 10 µg/mL aprotinin. Octylphenoxypoly(ethyleneoxy)ethanol solution was added and samples were vortexed for 15 seconds and centrifuged at 1,400 rpm for 30 seconds at 4°C. The supernatant was removed and the pellet containing nuclear proteins was resuspended in buffer containing 50 mmol/L HEPES, 50 mmol/L KCl, 300 mmol/L NaCl, 0.1 mmol/L EDTA, 1 mmol/L NaF, 1 mmol/L Na4P2O7, 0.1 mmol/L Na3VO4, 0.2 mmol/L PMSF, 5 µg/mL leupeptin, 10 µg/mL aprotinin, and 10% glycerol. Samples were shaken vigorously for 20 minutes at 4°C and centrifuged at 1,400 rpm. Supernatants were removed and protein content was determined by Bradford assay.

Immunocytochemistry. MAPK translocation into the nucleus was identified by immunofluoresence. Cells were plated on glass coverslips precoated with gelatin. After serum starvation, cells were treated with PDGF for the times indicated in the text, fixed in 4% formaldehyde in PBS for 20 minutes, rinsed with PBS, and permeabilized with 0.4% Triton/PBS for 5 minutes before staining. Slides were blocked with 1.5% goat serum/1% bovine serum albumin (BSA)/PBS for 40 minutes. Slides were incubated with 1:100 diluted rabbit ERK-1/2 (p42/44 MAPK) polyclonal antibody overnight at 4°C. Coverslips were rinsed with PBS and stained with FITC-conjugated goat anti-rabbit IgG for 60 minutes at room temperature. Coverslips were then washed, mounted, and sealed. Slides were viewed on a Zeiss fluorescent microscope and photographs were obtained digitally with Metamorph software.

Measurement of superoxide anion production. Measurement of intracellular superoxide anion (O2·–) production was done using lucigenin-enhanced chemiluminescence. This method is based on the reaction between reduced lucigenin and O2·–, resulting in the emission of photons that can be quantified using a luminometer. ELT-3 cells were cultured in 35 mm Petri dishes and serum starved for 72 hours. Cells were treated for 15 minutes with PDGF and other reagents at the doses indicated in the text in phenol red–free medium. Cells were then trypsinized, pelleted by gentle centrifugation, and resuspended into a cuvette in 1 mL PBS containing 10 mmol/L glucose and 1 mg/mL BSA. Cellular suspensions were quickly loaded into a Turner two-dimensional luminometer (Turner Designs, Sunnyvale, CA) and lucigenin (Sigma; final concentration 500 µmol/L) was injected to start the reaction. A 15-second dark-adaptive period was done before each sample reading. Consecutive readings of photoemission were recorded with a 15-second integration for a total of 10 minutes or until maximum-level readings were reached. Buffer blank with lucigenin produced negligible chemiluminescence and values were subtracted from peak readings. Cell samples were counted using a hemocytometer and results were expressed as lucigenein chemiluminesence per 106 cells. Menadione (Sigma) at the dose indicated in the text was used as a positive control for the production of O2·–. The specificity of the reaction was determined by the inhibition of the luminescent signal by Tiron, a O2·– radical scavenger.

Measurement of hydrogen peroxide release. Hydrogen peroxide (H2O2) release by ELT-3 cells was measured using a modification of the method by Ruch et al. (27). This fluorimetric method is based on horseradish peroxidase (HRP)–mediated oxidation of phenol red by H2O2 to its fluorescent dimer. Cells were grown on 96-well plates until confluent. Cells were then washed with PBS (pH 7.4) and incubated with a reaction mixture containing 100 µL of phenol red solution (0.56 mol/L), HRP (19 units/mL), and PDGF or vehicle control. Cells were incubated at 37°C for 1 hour and the reaction was stopped by the addition of 20 µL of 1 mol/L NaOH per well. Fluorescence was measured using a Spectrafluor Plus microplate reader (Tecan Co., Salzburg, Austria) at 610 nm. All readings were standardized for the reaction mixture alone. The exact H2O2 concentrations were calculated using a standard curve for H2O2. Results were expressed as nanomoles per liter of H2O2 produced per milliliter.

Immunoblot analysis was done as described (25). Antibodies used were against phospho-p42/44 MAPK, p42/44 MAPK, phospho-Akt, Akt, phospho-S6, S6 (Cell Signaling Technology), tuberin, and nucleolin (Santa Cruz Biotechnology, Santa Cruz, CA). Secondary HRP-conjugated antibody (Cell Signaling Technology) was used with chemiluminescent signal detection by enhanced chemiluminescence (Cell Signaling Technology). Semiquantitative densitometric analysis was done by ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Data were expressed as fold activation relative to control.

Results are expressed as mean ± SD. Statistical analysis using a nonparametric Kruskal-Wallis ANOVA with Dunn's multiple comparison test for follow-up analysis was done using GraphPad Prism 3.0 software.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Platelet-derived growth factor–induced mitogen-activated protein kinase activation and cellular growth is similar in tuberin-expressing and tuberin-deficient cells. We examined the growth rates of TSC2+/+ and TSC2–/– cells in the presence of serum and following serum withdrawal. We observed that cell growth rates were similar in both cell lines in the presence of 10% serum (Fig. 1A). However, TSC2+/+ cells seem to undergo growth arrest following 72 hours of serum withdrawal, whereas TSC2–/– cells continue to grow in the absence of serum (Fig. 1A). PDGF-stimulated cellular growth responses were not significantly different between TSC2+/+ and TSC2–/– cells (Fig. 1B); we observed a 2- to 3-fold induction of growth after 3 days in both cell lines, indicating that rates of cell growth in response to PDGF are not significantly altered by the presence or absence of tuberin.



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  		Figure 1. PDGF-induced growth and p42/44 MAPK activation is similar in tuberin-null and tuberin-expressing cells. A, cell counts for TSC2+/+ cells (black columns) and TSC2–/– cells (clear columns). Cells were seeded on day 0. Cells were counted and serum was withdrawn on day 3. Cells were counted for 3 consecutive days thereafter (days 4-6). B, after serum withdrawal, cells were treated with PDGF (10 ng/mL) or vehicle (Veh) and counted 3 days later. C, representative immunoblots (IB) for phospho-p42/44 MAPK, phospho-Akt, phospo-S6, the respective total proteins, and tuberin are shown. TSC2+/+ and TSC2–/– cells are compared following stimulation with PDGF (10 ng/mL) for the times indicated. Densitometric analysis of phospho-p42/44 MAPK levels as fold increase compared with time 0 is shown.

 
The p42/44 (ERK-1/2) MAPKs constitute a highly conserved protein kinase pathway that mediates extracellular growth factor signaling responses. We examined the ability of PDGF to activate the p42/44 MAPK pathway in TSC2+/+ and TSC2–/– cells (Fig. 1C). We observed rapid and time-dependent p42/44 MAPK phosphorylation with peak activation within 15 minutes of PDGF treatment [fold phosphorylation (mean ± SD) for TSC2+/+ = 4.4 ± 0.77 and TSC2–/– = 2.9 ± 1.33, P > 0.05]. Similarly, the temporal pattern of p42/44 MAPK activation was not significantly different between TSC2+/+ and TSC2–/– cells as phosphorylation levels returned to baseline by 24 hours in both cell lines. Although there seemed to be a trend toward increased activation of MAPK by PDGF in TSC2+/+ cells compared with TSC2–/– cells, this observation was not consistent nor was it statistically significant. The activation of Akt was markedly reduced in TSC2–/– cells following PDGF treatment compared with TSC2+/+ cells (Fig. 1C), consistent with previous observations in TSC2 null murine fibroblasts (28). Constitutive phosphorylation of S6 was evident in TSC2–/– cells but not TSC2+/+ cells. PDGF treatment rapidly induced S6 phosphorylation in TSC2+/+ cells but had little effect on p-S6 levels in TSC2–/– cells (Fig. 1C). These results indicate that tuberin expression does not inhibit PDGF-induced p42/44 MAPK activation and cellular growth in ELT-3 cells. Moreover, in the absence of tuberin, PDGF stimulation has reduced effect in stimulating Akt activation or S6 phosphorylation, suggesting that the Akt pathway is not likely to be directly involved in PDGF-induced mitogenic signaling.

Nuclear translocation of p42/44 mitogen-activated protein kinase in response to platelet-derived growth factor is altered in tuberin-deficient cells. Classically, receptor tyrosine kinase activation by mitogenic growth factors, such as PDGF, leads to MEK-dependent phosphorylation of p42/44 MAPK (11, 12). Upon phosphorylation, p42/44 MAPK dissociates from MEK to translocate to the nucleus and activate transcription of multiple genes required for cell cycle progression. We assessed the potential role of tuberin in the nuclear translocation of p42/44 MAPK in response to PDGF. Western blot analysis of nuclear extracts showed that both total and phosphorylated p42/44 MAPK was less abundant in TSC2–/– nuclei than in TSC2+/+ nuclei both at baseline and following PDGF stimulation (Fig. 2A). These differences match the trend to reduced MAPK activation in response to PDGF in TSC2–/– cells noted above (Fig. 1), but are greater in magnitude and statistically significant (P < 0.05; Fig. 2A). Concordant qualitative results were obtained by immunolocalization studies that showed that following treatment with PDGF, more p42/44 MAPK translocates to the nucleus in TSC2+/+ cells than in TSC2–/– cells (Fig. 2B). Thus, we conclude that in the absence of tuberin expression, there is reduced translocation of activated p42/44 MAPK to the nucleus.



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  		Figure 2. Tuberin regulates PDGF-induced nuclear translocation of p42/44 MAPK. A, representative immunoblots of phospho-p42/44 MAPK, total p42/44 MAPK, and nucleolin, which was used as a loading control, are shown in nuclear extracts in TSC2+/+ cells ({05-1394fx01g}) and compared with TSC2–/– cells ({05-1394fx02g}). Cells were treated with PDGF (10 ng/mL) for the times indicated. Densitometric analysis of nuclear phospho-p42/44 MAPK in response to PDGF expressed as fold activation relative to untreated cells is shown. *, statistically significant difference in the amount of nuclear phospho-p42/44 MAPK in TSC2+/+ cells compared with TSC2–/– cells measured at 2 minutes. B, localization of total p42/44 MAPK was determined using immunofluoresence. For each cell line, phase contrast (top) and immunofluoresence (bottom) images are shown. Cells were treated with PDGF (10 ng/mL) for the times indicated.

 
Platelet-derived growth factor–induced p42/44 mitogen-activated protein kinase activation and cell growth is mediated by a mitogen-activated protein/extracellular signal-regulated kinase kinase-1–independent mechanism in cells lacking tuberin. We next examined the effect of tuberin expression on the mechanism of activation of MAPK by treating TSC2+/+ and TSC2–/– cells with PDGF in the presence and absence of the MEK-1/2 inhibitor, UO126. As expected, UO126 inhibited PDGF-induced p42/44 MAPK activation in a dose-dependent manner in TSC2+/+ cells, suggesting that this effect is mediated by the classic MEK-dependent mechanism (Fig. 3A). In contrast, UO126 had no effect on PDGF-induced p42/44 MAPK activation in TSC2–/– cells, suggesting that a MEK-independent pathway may be responsible for p42/44 MAPK activation in cells deficient in tuberin. Identical results were obtained using the MEK-1 inhibitor, PD98059 (dose range 1-50 µmol/L; results not shown).



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  		Figure 3. Tuberin regulates MEK-1 dependence of p42/44 MAPK activation and cellular growth. A, phospho-p42/44 MAPK, total p42/44 MAPK, and tuberin were determined by Western immunoblot in TSC2+/+ and TSC2–/– cells. Following serum withdrawal, cells were treated with PDGF (10 ng/mL) for 15 minutes in the presence and absence of the MEK-1 inhibitor UO126 (UO) at the doses indicated. Pretreatments with UO 126 were for 1 hour before the administration of PDGF. B, cell growth (as assessed by Coulter counting) of TSC2+/+ cells (black columns) and TSC2–/– cells (clear columns) is shown following treatment with PDGF (10 ng/mL) in the presence and absence of UO126 under serum-free conditions at the doses indicated. Results are expressed as fold growth compared with vehicle control. *, P < 0.05. C, phospho-p42/44 MAPK, total p42/44 MAPK, phospho-Akt, total Akt, and tuberin were determined by Western immunoblot in TSC2+/+ and TSC2–/– cells. Following serum withdrawal, cells were treated with PDGF (10 ng/mL) for 15 minutes in the presence and absence of the PI3K inhibitor LY294002 (LY; 10 µmol/L). Pretreatments with LY294002 were for 1 hour before the administration of PDGF. D, cell growth is shown for TSC2+/+ cells (black columns) and TSC2–/– cells (clear columns) after a one time treatment with PDGF (10 ng/mL) in the presence and absence of LY294002 (10 µmol/L) under serum-free conditions. Cells counts were done on Coulter counter on day 3. Results are expressed as fold growth compared with vehicle control. Vehicle control, LY294002, and PDGF treatments are indicated. E, phospho-p42/44 MAPK and total p42/44 MAPK were done in TSC2–/– and TSC2+/+ cells. Cells were treated with PDGF (10 ng/mL) for 15 minutes in the presence and absence of the mTOR inhibitor rapamycin (Rap; 10 nmol/L). Pretreatments with rapamycin were for 1 hour (Rap 1 hr) and 24 hours (Rap 24 hr) before the administration of PDGF. Vehicle, rapamycin, and PDGF treatments are indicated. F, cell growth is shown for TSC2+/+ cells (black columns) and TSC2–/– cells (clear columns) after a one time treatment with PDGF (10 ng/mL) in the presence and absence of rapamycin (10 nmol/L) under serum-free conditions. Cells counts were done on a Coulter counter on day 3. Results are expressed as fold growth compared with vehicle control. Vehicle control, rapamycin, and PDGF treatments are indicated. *, P < 0.01.

 
To assess whether the observed differences in the mechanism of p42/44 MAPK activation in these cells correlated with growth responses, the effect of MEK inhibition on PDGF-induced growth in TSC2+/+ and TSC2–/– cells was examined. Inhibition of MEK-dependent p42/44 MAPK activation with UO126 resulted in significant inhibition of PDGF-induced growth of TSC2+/+ cells; in contrast, UO126 mediated relatively little inhibition of PDGF-induced cell growth in TSC2–/– cells (Fig. 3B). These results suggest that, in the absence of tuberin, PDGF-induced activation of p42/44 MAPK and cellular growth is independent of MEK.

To investigate other potential upstream regulators of p42/44 MAPK activation in TSC2–/– cells, we examined the effect of PI3K and mTOR inhibition on p42/44 MAPK activation and cellular growth. The inhibition of PI3K by LY294002 had no effect on PDGF-induced MAPK activation (Fig. 3C) or cellular growth (Fig. 3D). The ability of this compound to inactivate PI3K activity was confirmed by demonstrating blockade of Akt phosphorylation induced by PDGF (Fig. 3C). Similar results were observed using wortmannin to inhibit PI3K (results not shown). Inhibition of mTOR with rapamycin had little effect on PDGF-induced activation of p42/44 MAPK in either TSC2+/+ or TSC2–/– cells (Fig. 3E) when cells were pretreated with rapamycin for 1 hour. However, pretreatment with rapamycin for longer periods of time (24 hours) before the addition of PDGF resulted in significant inhibition of PDGF-induced p42/44 MAPK in TSC2–/– cells and had no effect in TSC2+/+ cells. Similarly, rapamycin had little effect on the PDGF growth response of TSC2+/+ cells, but completely abolished the PDGF-induced growth of TSC2–/– cells (Fig. 3F). Together, these results indicate that in cells expressing tuberin, PDGF-stimulated p42/44 MAPK activation and cellular growth is mediated by the classic MEK-1-dependent pathway. In contrast, in TSC2–/– cells, PDGF-stimulated p42/44 MAPK activation and cellular growth are independent of MEK-1 and PI3K but are mTOR dependent.

Activation of p42/44 mitogen-activated protein kinase and cellular growth responses by platelet-derived growth factor are mediated by the generation of superoxide anion in tuberin-deficient cells. ROS, such as O2·– and H2O2, have been reported to activate p42/44 MAPK. The mechanisms that underlie this process are poorly understood. As the mechanism of p42/44 MAPK activation by PDGF in TSC2–/– cells was unknown, we examined the possibility that ROS might regulate p42/44 MAPK activation in these cells. We compared the effects of the antioxidants, Tiron (an O2·– scavenger), N-acetylcysteine (a glutathione precursor that reduces intracellular H2O2 levels), and PEG-catalase (an enzyme that localizes intracellularly and reduces H2O2 to H2O), and diphenyliodoniun (an inhibitor of flavoenzymes, which are required for O2·– production), on PDGF-induced p42/44 MAPK activation and cell growth in TSC2+/+ and TSC2–/– cells. None of the antioxidants or diphenyliodonium inhibited p42/44 MAPK activation in TSC2+/+ cells (Fig. 4A). Interestingly, Tiron and diphenyliodonium completely abolished PDGF-induced p42/44 MAPK activation in TSC2–/– cells, whereas N-acetylcysteine and PEG-catalase had minimal effects (Fig. 4A). These observations suggested that O2·– participates as an intermediate signaling molecule in the activation of p42/44 MAPK by PDGF in TSC2–/– cells. Previous studies have implicated ROS in the regulation of the PI3K-Akt-S6K pathway (21–24). However, none of the antioxidants tested had any effect on PDGF-induced phosphorylation of Akt or S6 in either TSC2+/+ or TSC2–/– cells (Fig. 4A).



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  		Figure 4. Tuberin regulates baseline and PDGF-inducible levels of ROS. A, phospho-p42/44 MAPK, total p42/44 MAPK, phospho-Akt, total Akt, phospho-S6, and total S6 were determined by immunoblot in TSC2+/+ and TSC2–/– cells in response to PDGF (10 ng/mL; 15 minutes), in the presence and absence of Tiron (Tir; 1 mmol/L), diphenyliodonium (10 mmol/L), N-acetylcysteine (100 µmol/L), and PEG-catalase (pCAT; 300 units/mL). All pretreatments were for 1 hour. B, O2·– production was measured in TSC2+/+ (black columns) and TSC2–/– (clear columns) cells using lucigenin-enhanced chemiluminescence at baseline (Ctl) and following PDGF stimulation (10 ng/mL) for 15 minutes. *, P < 0.05 O2·– generated from cells treated with menadione (Men; 1 µmol/L) was used as a positive control. Tiron (1 mmol/L) was used to confirm that the luminescence detected was specifically due to O2·–. *, P < 0.05. C, similar measurements were made in serum-starved, PDGF-stimulated (10 ng/mL), and menadione-treated (1 µmol/L) TSC2+/+ (black columns) and TSC2–/– (clear columns) cells in the presence and absence of the mTOR inhibitor rapamycin (10 nmol/L). Pretreatment with rapamycin was for 1 hour. D, extracellular H2O2 was measured in TSC2+/+ (black columns) and TSC2–/– (clear columns) cells in response to PDGF (0-50 ng/mL) using fluorimetry. E, cell growth was assessed in TSC2+/+ (black columns) and TSC2–/– (clear columns) cells following PDGF (10 ng/mL) treatment in the presence and absence of antioxidants at concentrations identical to those used in (A).

 
Next, we determined if PDGF induces generation of O2·– in ELT-3 cells. PDGF significantly increased O2·– production in TSC2–/– cells compared with TSC2+/+ cells. In addition, basal levels of O2·– production were significantly higher in TSC2–/– cells (Fig. 4B). Menadione, a vitamin K analogue that is known to generate intracellular O2·– by redox cycling, also induced elevated O2·– levels that were inhibited by Tiron (Fig. 4B). In the presence of rapamycin, both baseline and PDGF-induced O2·– production in TSC2–/– cells were significantly reduced; however, rapamycin did not inhibit menadione-induced O2·– production, suggesting that rapamycin does not act as a general antioxidant in these cells and that the observed reduction in PDGF-stimulated O2·– production in TSC2–/– cells is likely a specific mTOR-dependent effect (Fig. 4C). Next, to determine if intracellular O2·– production results in the release of extracellular H2O2, the release of H2O2 by cells was measured with a peroxidase-based assay that detects only extracellular H2O2. PDGF stimulation of TSC2–/– cells resulted in a significant dose-dependent increase in extracellular H2O2 release, whereas TSC2+/+ cells failed to show an increase in H2O2 production (Fig. 4D). The quantities of detectable extracellular H2O2 (picomolar and nanomolar concentrations) were significantly lower than levels of O2·– generated by TSC2–/– cells, suggesting that the primary site for O2·– generation may be in an intracellular compartment with subsequent dismutation to H2O2 and extracellular diffusion.

We next examined whether the generation of ROS in TSC2–/– cells is required for PDGF-induced cell growth in TSC2–/– cells by using antioxidants and diphenyliodonium to block O2·– and/or H2O2 production. We observed that Tiron, a O2·– radical scavenger, and diphenyliodonium, a flavoprotein oxidase inhibitor, significantly inhibited PDGF-induced growth of TSC2–/– cells but had no effect on TSC2+/+ cells (Fig. 4E). N-acetylcysteine and PEG-catalase, antioxidants that primarily target H2O2, had no effect on PDGF-induced growth in either TSC2+/+ or TSC2–/– cells (Fig. 4E). These results indicate that loss of tuberin expression or function is associated with elevated levels of O2·–, both at baseline and in response to PDGF stimulation. PDGF-induced generation of intracellular O2·– is required for both the activation of p42/44 MAPK and growth in TSC2–/– cells.

Generation of intracellular superoxide anion by a redox-cycling compound induces mitogen-activated protein/extracellular signal-regulated kinase kinase-1–independent activation of p42/44 mitogen-activated protein kinase and cellular growth in tuberin-deficient cells. Our studies show that PDGF-induced cell growth and p42/44 MAPK activation in TSC2–/– cells are independent of MEK-1 but are O2·– sensitive; in contrast, growth of TSC2+/+ cells in response to PDGF is dependent on MEK-1 and independent of O2·– modulation. To further characterize the growth-promoting effects of O2·– in TSC2–/– cells, we examined whether treating cells with menadione augments cellular growth. In TSC2–/– cells, menadione had a dose-dependent effect on cell growth and resulted in the activation of p42/44 MAPK, effects that were inhibited by the O2·– scavenger, Tiron (Fig. 5A). In TSC2+/+ cells, however, menadione had no effect on p42/44 MAPK activation nor did it induce cellular growth (Fig. 5A). In fact, menadione at higher doses reduced the survival and viability of TSC2+/+ cells. In addition, similar to the effects of PDGF, the activation of p42/44 MAPK and cellular growth by menadione in TSC2–/– cells was independent of MEK-1 as assessed by a lack of effect of UO126 (Fig. 5B).



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  		Figure 5. Tuberin regulates menadione-induced p42/44 MAPK activation and cellular growth. A, representative immunoblots of phospho-p42/44 MAPK, total p42/44 MAPK, and tuberin are shown in TSC2+/+ and TSC2–/– cells. Cells were treated with menadione (1-10 µmol/L) for 15 minutes at the doses indicated in the presence and absence of Tiron (1 mmol/L). Cell growth was assessed at 72 hours in TSC2+/+ and TSC2–/– cells following a one time treatment with menadione (10 µmol/L) at the doses indicated in the presence and absence of Tiron (1 mmol/L). B, similar experiments were done in TSC2–/– cells following treatment with menadione (10 µmol/L) in the presence and absence of UO126 (10 µmol/L).

 
Mitochondrial electron transport contributes to platelet-derived growth factor–induced generation of intracellular superoxide anion in tuberin-deficient cells. We have shown that O2·– levels were increased in cells lacking tuberin and that both basal and PDGF-stimulated O2·– generation were inhibited by the flavoenzyme inhibitor diphenyliodonium. A number of cellular enzymatic systems use flavins in oxidation-reduction reactions and may be inhibited by diphenyliodonium; these include plasma membrane NAD(P)H oxidoreductases, nitric oxide synthase, xanthine oxidase, the endoplasmic reticulum–associated cytochrome P450 system, and the mitochondrial electron transport system. Inhibition of nitric oxide synthase (with L-nitro-D-arginine methyl ester hydrochloride), xanthine oxidase (with allopurinol), or the endoplasmic reticulum–associated cytochrome P450 system (with methoxypsoralen) failed to block O2·– generation in response to PDGF in TSC2–/– cells (Fig. 6). Blockade of mitochondrial electron transport with rotenone (which blocks NADH dehydrogenase, site I), antimycin A (which blocks electron transport from cytochrome b to c, site II) and potassium cyanide (which blocks the terminal cytochrome a3, site III) resulted in significant inhibition of O2·– production and p42/44 MAPK activation (Fig. 6). These results suggest that the regulation of O2·– production by PDGF in tuberin-deficient cells occurs at the mitochondrial level and contributes to the activation of p42/44 MAPK.



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  		Figure 6. O2·– production and p42/44 MAPK activation by PDGF is inhibited by altering mitochondrial electron transport. O2·– production was assessed by lucigenin-enhanced chemiluminescence (LCL). Phospho-p42/44 MAPK, total p42/44 MAPK, and tuberin were determined by immunoblot in TSC2+/+ and TSC2–/– cells. Pretreatment with the indicated inhibitors was for 1 hour followed by PDGF (10 ng/mL) treatment for 15 minutes. *, significant inhibition of chemiluminescence compared with vehicle alone or PDGF treatment alone. KCN, potassium cyanide; L-NAME, L-nitro-D-arginine methyl ester hydrochloride.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Major progress has occurred in recent years in understanding a critical role of the tuberous sclerosis proteins TSC1 and TSC2 in the regulation of mTOR activity. In the current model, TSC2 is phosphorylated and regulated by several kinases, some of which (Akt, MAPK, and p90Rsk1) inhibit its GAP activity, whereas others (AMP-activated protein kinase) activate its GAP activity toward Rheb. Inactivation of the GAP activity of TSC2 leads to high levels of GTP-bound Rheb, which subsequently activates mTOR by an uncertain mechanism (29). In normal cells, the phosphorylation and inactivation of TSC1/TSC2 increased Rheb-GTP levels and activation of mTOR are transient responses to growth factor stimulation. However, in cells lacking TSC1 or TSC2, there is constitutive high-level activation of mTOR due to the lack of GTPase activity and high Rheb-GTP levels. This leads to downstream phosphorylation and activation of the S6Ks and phosphorylation of 4E-BP1 that release eIF4E (29). The net effect of these alterations is increased protein synthesis, cell size, and cell growth. In this study, we describe significant, major effects of TSC2 loss on the MAPK signaling pathway, ROS generation, and cell growth regulation.

Connections between mTOR activation and the MAPK pathway have been examined by others in TSC2 null cells with inconsistent results. Some have observed increased activation of MAPK (30), whereas others have seen reduced or no effect under basal conditions in TSC2 null cells (9, 28). However, immunohistochemical analysis of human TSC-associated tumors shows high levels of activated p42/44 MAPK (31, 32), suggesting that, in vivo, there is activation of this pathway in cells lacking tuberin. In the conventional model, p42/44 MAPK is phosphorylated by its upstream activator MEK-1, following a sequence of events in which ras-GTP is generated, raf is recruited to the membrane and phosphorylated, and there is downstream phosphorylation and activation of MEK-1 (33, 34). Consistent with this model, we observed MEK-1-dependent phosphorylation of p42/44 MAPK in response to stimulation with PDGF in TSC2+/+ cells and following activation MAPK rapidly and quantitatively moves to the nucleus. In contrast, in TSC2–/– cells, PDGF-induced p42/44 MAPK phosphorylation was mediated by the regulation of the intracellular redox state and not by MEK-1 and nuclear localization of activated MAPK was reduced. Interestingly, long-term treatment with rapamycin was required to reduce both O2·– levels and activation of p42/44 MAPK in TSC2–/– cells. This suggests that the effect of TSC2 loss on p42/44 MAPK activation is due to translational effects of mTOR activation that do not reverse as rapidly as phosphorylation events. Furthermore, the reduced nuclear localization of p42/44 MAPK suggests that in the context of activated mTOR, TSC2–/– cells may be primed to grow and the amount of nuclear p42/44 MAPK that is required to mediate PDGF-induced growth is less than in tuberin-expressing cells.

Previous reports have implicated PI3K/Akt in the MEK-1-independent activation of p42/44 MAPK (22, 35). We observed in TSC2–/– cells that p42/44 MAPK activation was not regulated by PI3K. Additionally, activation of p-Akt by PDGF in TSC2–/– cells was not regulated by ROS, suggesting that the MEK-1-independent but ROS-sensitive activation of p42/44 MAPK in our system is not through ROS-mediated activation of PI3 kinase. Interestingly, both menadione and H2O2 have been implicated in the activation of p42/44 MAPK in a MEK-1-independent fashion, postulating phosphatases as targets for ROS (36, 37). Growing evidence now suggests that signal amplitude and duration of p42/44 MAPK is significantly altered by the activation of MAPK-directed phosphatases (38, 39). Interestingly, the negative regulation of p42/44 MAPK by phosphatases can occur at the MEK-1 level or directly at the p42/44 MAPK level (36, 39). Several phosphatases have been shown to be redox sensitive due to the presence of multiple cysteine residues in their active sites that render them highly susceptible to oxidation (40, 41). Inhibition of MAP kinase phosphatase-3, a tyrosine phosphatase that is specific for p42/44 MAPK, has been shown to play a key role in mediating the activation and nuclear localization of p42/44 MAPK in states of oxidative stress (42). In addition, the inhibition of the protein tyrosine phosphatases SHP-1 and HePTP and the serine/threonine phosphatase PP2A have also been shown to mediate p42/44 MAPK activation by H2O2 (43, 44). As there is some evidence that PP2A is a substrate of mTOR and the state of phosphorylation of the S6Ks and S6 are exquisitely sensitive to rapamycin inhibition of mTOR activity (45), it is possible that phosphatase inhibition directly due to mTOR activation partially accounts for the MAPK activation that occurs in TSC2–/– cells in response to PDGF. However, the exquisite sensitivity of PDGF-mediated MAPK activation to Tiron and diphenyliodonium treatment argues that ROS may also directly contribute to this phenomenon, possibly by blocking phosphatase activation.

A striking finding in this study was the increase in both basal and PDGF-stimulated ROS levels in cells lacking tuberin. Increased O2·– levels in TSC2–/– cells were dramatically reduced by rapamycin and inhibitors of mitochondrial electron transport to levels similar to those observed in control cells. These observations suggest that in the absence of tuberin, O2·– is being generated due to mTOR activation and its effects on mitochondrial function. mTOR is increasingly recognized as a central element in the pathway that regulates cellular energy stores and is sensitive in normal cells to conditions of starvation and oxidative stress (46, 47). It is possible that, as part of this role in cellular metabolism, mTOR regulates the production of ROS at the mitochondrial level. Indeed, there is evidence that mTOR colocalizes with and binds to the outer mitochondrial membrane (48). Although further study is required, the current observations provide further support for the importance of tuberin and mTOR in the regulation of cellular energy metabolism and in particular the production of ROS.

In conclusion, we have shown that tuberin-null cells are primed to grow due to high baseline and PDGF-stimulated levels of ROS and that elevated ROS levels are responsible for p42/44 MAPK activation. The observations are significant in that they provide a link between the PI3K-Akt-mTOR signaling pathway and the receptor tyrosine kinase–raf-MEK-MAPK signaling pathway. The results also suggest that ROS may contribute to the growth potential of TSC hamartomas in vivo, providing another potential target, in addition to mTOR itself, for therapeutic attack.


   Acknowledgments
 
Grant support: NIH grants K08 HL74113 (G. Finlay), R01 HL67967 (V. Thannickal), HL032723 (B. Fanburg), and NS3153 (D. Kwiatkowski).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Cheryl Walker for the provision of ELT-3 cells.

Received 4/22/05. Revised 8/19/05. Accepted 9/14/05.


   References
Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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