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J. Biol. Chem., Vol. 275, Issue 47, 36803-36810, November 24, 2000

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Phosphatidylinositol 3-Kinase Function Is Required for Transforming Growth Factor -mediated Epithelial to Mesenchymal Transition and Cell Migration^*

Andrei V. Bakin^§, Anne K. Tomlinson^§, Neil A. Bhowmick^§¶, Harold L. Moses^§¶, and Carlos L. Arteaga^§¶**

From the Departments of  Medicine, ^¶ Cell Biology, and  Pathology, Vanderbilt University School of Medicine, ^** Department of Veteran Affairs Medical Center, and ^§ Vanderbilt-Ingram Cancer Center, Nashville Tennessee 37232

Received for publication, July 6, 2000, and in revised form, August 24, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have studied the role of phosphatidylinositol 3-OH kinase (PI3K)-Akt signaling in transforming growth factor  (TGF)-mediated epithelial to mesenchymal transition (EMT). In NMuMG mammary epithelial cells, exogenous TGF1 induced phosphorylation of Akt at Ser-473 and Akt in vitro kinase activity against GSK-3 within 30 min. These responses were temporally correlated with delocalization of E-cadherin, ZO-1, and integrin ₁ from cell junctions and the acquisition of spindle cell morphology. LY294002, an inhibitor of the p110 catalytic subunit of PI3K, and a dominant-negative mutant of Akt blocked the delocalization of ZO-1 induced by TGF1, whereas transfection of constitutively active p110 induced loss of ZO-1 from tight junctions. In addition, LY294002 blocked TGF-mediated C-terminal phosphorylation of Smad2. Consistent with these data, TGF-induced p3TP-Lux and p(CAGA)₁₂-Lux reporter activities were inhibited by LY294002 and transiently expressed dominant-negative p85 and Akt mutants in NMuMG and 4T1 cells. Dominant-negative RhoA inhibited TGF-induced phosphorylation of Akt at Ser-473, whereas constitutively active RhoA increased the basal phosphorylation of Akt, suggesting that RhoA in involved in TGF-induced EMT. Finally, LY294002 and neutralizing TGF1 antibodies inhibited ligand-independent constitutively active Akt as well as basal and TGF-stimulated migration in 4T1 and EMT6 breast tumor cells. Taken together, these data suggest that PI3K-Akt signaling is required for TGF-induced transcriptional responses, EMT, and cell migration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The transforming growth factor  (TGF)¹ family of secreted factors is involved in the control of different biological processes including cell proliferation, differentiation, and apoptosis (1). TGF signals through the activation of heteromeric complexes of TGF type I (TRI) and type II (TRII) receptors (1, 2). Activated TRI phosphorylates receptor-associated Smads (Smad2 and Smad3), which then bind Smad4 and translocate to the nucleus where they regulate transcription of target genes (3, 4). TGF exhibits a tumor suppressor activity, and components of its signaling pathway are frequently mutated or silenced in colon and pancreatic cancers (1, 5). However, accumulating data indicate that TGF can positively affect tumorigenesis and contribute to the progression and invasiveness of tumors (5-8). Moreover, it was recently reported that inhibition of autocrine TGF signaling in carcinoma cells reduces cell invasiveness and tumor metastases (9, 10). These effects of TGF are associated with its ability to induce an epithelial to mesenchymal transition (EMT) and stimulate cell migration.

The EMT induced by TGF results in the disruption of the polarized morphology of epithelial cells, formation of actin stress fibers, and enhancement of cell migration (8, 9). Two species of TRI, Alk2 and Alk5, have been implicated in the induction of EMT by TGF in mammary epithelial cells (11, 12). It has also been reported that high levels of ectopic Smad2 and Smad3 can induce some features of EMT in mammary epithelial cells in the context of expression of an activated type I receptor (12). However, considering the complexity of TGF signaling (3, 13-16), it is conceivable that other molecules can also contribute to EMT. For example, members of the AP-1 family of transcription factors have been shown to induce EMT and promote tumor invasiveness (17, 18). AP-1 complexes can be activated in response to TGF (19-21), physically interact with Smads (13, 14), and cooperate with Smads in the control of gene expression (19-21). In addition, several other downstream signaling pathways can also be activated by TGF, including p38Mapk (21), c-jun N-terminal kinase (22, 23), and phosphatidylinositol 3-OH kinase (PI3K) (24, 25). These signaling pathways can potentially contribute to TGF1-mediated EMT, but their significance for EMT and cell migration mediated by TGF remains unclear.

In this study, we used the NMuMG mammary epithelial cell line as a model for TGF1-induced EMT (11). Two metastatic breast tumor cell lines, 4T1 and EMT6, that express high levels of TGF ligands and TGF receptors were used in transcription and migration studies. We report that TGF-induced EMT and cell migration depend on the PI3K-Akt pathway. We also show that the phosphorylation of Smad2 and transcriptional responses induced by TGF are inhibited by pharmacological and molecular antagonists of the PI3K-Akt pathway. TGF1 can induce phosphorylation and activation of Akt/PKB in a PI3K-dependent manner, and this activation requires the Rho GTPase function. Taken together, our data suggest that PI3K-Akt signaling is required for the morphogenic, transcriptional, and migratory activities of TGF.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Antibodies and Other Reagents-- TGF1 was from R & D Systems (Minneapolis, MN) and EGF from CLONTECH (Palo Alto, CA). Antibodies to E-cadherin and integrin ₁ were from Transduction Laboratories (Lexington, KY), to p85 from Upstate Biotechnology (Lake Placid, NY), and to ZO-1 from Chemicon (Temecula, CA). Phalloidin- fluorescein isothiocyanate (actin) was from Molecular Probes (Eugene, OR). The TGF1-neutralizing 2G7 monoclonal IgG₂ was a gift from B. Fendly (Genentech, Inc.) and has been described previously (26). Antibodies to phospho-Ser-473 Akt and total Akt were from New England BioLabs (Beverly, MA), to Smad2 (N19) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and to C-terminal phospho-Smad2 from Upstate Biotechnology. Antibodies to phospho-ERK1/2 and total ERK1/2 were from Promega (Madison, WI) and New England BioLabs, respectively. Mouse monoclonal antibodies 12CA5 and M2 to HA and Flag epitopes were from Roche Molecular Biochemicals and Sigma, respectively. Anti-Myc mouse monoclonal 9E10 antibody was a gift from J. F. Primus (Vanderbilt University). LY294002, ML7, okadaic acid, PD098059, rapamycin, U0126, and U73122 were purchased from Calbiochem (San Diego, CA). Curcumin was from Sigma. The Rac1 inhibitor SCH51344 was a kind gift from C. Kumar (Schering Research Institute, Kenilworth, NJ) (27). Adenovirus vectors encoding a dominant-negative mutant of Akt (AxAktK179D), a mutant regulatory subunit of p85 (Axp85), and a constitutively active myristoylated mutant of p110 (AxMyr-p110) were kindly provided by W. Ogawa (Kobe University School of Medicine, Kobe, Japan) (28). The pCMV6-AktK179M mutant was a gift from P. N. Tsichlis (Thomas Jefferson University, Philadelphia, PA). Plasmid vectors encoding Q61LRhoA and N19RhoA mutants were obtained from Dr. Lynn Cross (National Institutes of Health, Bethesda, MD). A plasmid vector encoding a GST-GSK3 peptide fusion protein was a gift from C. L. Van Den Berg (University of Colorado, Denver).

Cell Culture and Adenoviral Infection-- NMuMG cells were purchased from American Type Culture Collection (Manassas, VA) and maintained in DMEM supplemented with 10% FBS and 10 µg/ml insulin. 4T1 tumor cells were provided by F. Miller (Karmanos Cancer Center, Detroit, MI) and EMT6 tumor cells by B. Teicher (Lilly Research Laboratories, Indianapolis, IN); both were cultured in DMEM plus 10% FBS. For adenoviral infection of NMuMG and 4T1 cells, 10⁵ cells/well in 6-well plates were transduced with adenovirus vectors at 10-100 plaque-forming units/cell as described by Sakaue et al. (29). More than 90% of the NMuMG cells infected at a similar multiplicity of infection with an adenovirus expressing -galactosidase (Ad-Gal) exhibited blue staining. Infected cells were subjected to further treatment 24-48 h later.

Cell Lysis and Immunoblot Analysis-- Cells were lysed in EBC buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 20 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2 µg/ml leupeptin), and protein concentrations in cell lysates were determined by the Bradford method. Protein extracts (50 µg/lane) were separated by 12.5% SDS-PAGE and transferred to nitrocellulose membranes (100 mA, 2.5 h). Membranes were blocked with 5% milk in TBST buffer (containing 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20 (v/v)) for 1 h at room temperature and then incubated with primary antibodies in TBST plus 1% milk for 16 h at 4 °C followed by incubation with secondary antibodies for 1 h at room temperature. Membranes were washed three times in TBST and immunoreactive bands visualized by ECL (Pierce).

Akt/PKB in Vitro Kinase Assay-- Akt/PKB was precipitated from protein extracts (150 µg) with GST-GSK-3 fusion protein immobilized on agarose beads (Sigma) or GST-agarose beads for 2 h at 4 °C. An in vitro kinase reaction was performed by adding 10 µCi of [ -³²P]ATP (specific activity, 3000 Ci/mmol; PerkinElmer Life Sciences) for 20 min at 30 °C in the presence of 10 µM PKA peptide inhibitor (Calbiochem). Reaction was terminated by the addition of 5× Laemmli buffer and heating followed by 15% SDS-PAGE. Quantitative analysis of ³²P-labeled bands was performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Transcriptional Assays-- NMuMG, 4T1, and EMT6 cells (0.5 × 10⁶) were seeded in 60-mm dish and transfected the following day with 0.5 µg/ml p3TP-Lux (provided by J. Massague, Memorial Sloan-Kettering Cancer Center, New York, NY) or p(CAGA)₁₂-Lux (provided by J.-M. Gauthier, Laboratoire Glaxo Wellcome, Les Ulis Cedex, France), each with 0.002 µg/ml pCMV-Rl (Promega) using 4 µl of FuGENE6 reagent (Roche Molecular Biochemicals)/µg of DNA according to the manufacturer's protocol. The next day, cells were seeded in equal amounts in 24-well dishes and incubated for 16 h in low serum (0.5-2%) followed by treatment with 1 ng/ml TGF1 for 4 or 16 h. Firefly luciferase (Luc) and Renilla reniformis luciferase (RlLuc) activities in cell lysates were determined using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Luc activity was normalized to RlLuc activity and presented as relative luciferase units. All assays were done in triplicate wells, and each experiment was repeated at least twice.

Immunofluorescent Microscopy-- NMuMG cells (10⁵cells/well) were grown in DMEM, 5% FBS on glass coverslips (22 × 22 mm) for 24 h before treatment with 2 ng/ml TGF1. Cells were fixed with methanol for 10 min at 20 °C or with 2% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature. For permeabilization, cells were incubated with 0.1% Triton X-100 for 5 min at room temperature. Cells were washed three times in PBS after each treatment. Cells were blocked with 3% milk in PBS for 30 min at room temperature, incubated with primary antibodies diluted in 1% milk/PBS (1/300 for ZO-1, 1/500 for integrin ₁, and 1/2000 for E-cadherin), and then incubated with fluorescent secondary antibodies (1/500) for 1 h at room temperature. Coverslips were mounted onto 25 × 75-mm microslides (VWR Scientific, West Chester, PA) using AquaPolyMount (Polysciences, Warrington, PA). Fluorescent images were captured using a Princeton Instruments cooled CCD digital camera from a Zeiss Axiophot upright microscope.

Migration Assays-- 4T1 and EMT6 tumor cells (4 × 10⁴/well) were plated in DMEM, 10%FBS in the upper chamber of 8-µm pore (24-well) transwells (Corning Costar, Cambridge, MA) and incubated alone or with variable concentrations of TGF1 in the absence or presence of LY294002 or the TGF1-neutralizing 2G7 IgG₂. Three days later, the cells that had migrated through pores and reattached to the lower chamber were trypsinized and cell numbers measured in a Coulter counter.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The PI3K-Akt Pathway Is Involved in EMT Induced by TGF1-- TGF1 induced a mesenchymal transition in NMuMG cells within 24 h. Cells treated with 2 ng/ml TGF1 changed their shape from a cuboidal to a more elongated form (Fig. 1A, DIC). Concomitantly, TGF1 induced the delocalization of E-cadherin from adherens junctions, ZO-1 from tight junctions, and the delocalization of integrin ₁ from the cell surface (Fig. 1). There were no detectable differences in the intracellular staining of E-cadherin, ZO-1, and integrin ₁ between treated and untreated cells. In addition, no detectable changes in E-cadherin were found by immunoblot analysis of whole cell extract (Fig. 1B).

View larger version (98K): [in this window] [in a new window]   Fig. 1.   TGF1-mediated EMT in NMuMG cells. A, NMuMG mammary epithelial cells were grown on glass coverslips for 24 h and treated (bottom row) or not (top row) with 2 ng/ml TGF1 for an additional 24 h. Differential interference contrast (DIC) images show changes in cell morphology in response to TGF1. Antibodies to ZO-1 (1:300), E-cadherin (1:2000), and integrin 1 (1:500) were used to visualize cell junctions as indicated under "Experimental Procedures." Scale bars represent 15 µm. B, immunoblot analysis of E-cadherin (1:2000) and -catenin (1:2000) in protein extracts (50 µg/lane) from control NMuMG cells or from cells treated with 2 ng/ml TGF1 for 24 or 48 h.

To determine the signaling pathways that contribute to TGF-induced EMT, we examined the ability of different pharmacological agents to block the changes in cell morphology and in localization of epithelial markers at cell junctions. We found that LY294002, a synthetic inhibitor of the p110 catalytic subunit of PI3K (30), blocked the morphological transition, the delocalization of ZO-1 from cell junctions, and the reorganization of actin fibers (Fig. 2A). Inhibitors of MEK1/2 (PD098059 (Fig. 2A) and U0126), c-jun N-terminal kinase (curcumin), mTOR (mammalian target of rapamycin), phospholipase C (U73122), Rac1 (SCH51344), MLCK (myosin light chain kinase; ML7), and PP2A (okadaic acid) did not affect TGF-mediated transition (data not shown), suggesting that signaling pathways associated with these molecules may not contribute to EMT mediated by TGF1. Inhibition of EMT by LY294002 suggested that PI3K is involved in EMT induced by TGF1. To further test this hypothesis, NMuMG cells were infected with adenovirus encoding a constitutively active mutant of p110 (ca-p110), the catalytic subunit of PI3K. Cells expressing Myc-tagged ca-p110 showed a higher level phosphorylation of Akt at Ser-473, confirming its functional activity (Fig. 2B). Similar to exogenous TGF1, infection with the ca-p110 virus resulted in the delocalization of ZO-1 from tight junctions. However, the cells retained their epithelial morphology, whereas infection with a -galactosidase adenovirus (Ax-Gal) did not alter cell morphology nor ZO-1 staining at adherens junctions (Fig. 2B). Finally, we examined whether Akt/PKB, a downstream effector of PI3K, would affect EMT. Transduction of NMuMG cells using a dominant-negative mutant Akt (AktK179D) adenovirus inhibited TGF-induced delocalization of ZO-1 from tight junctions as well as changes in cell morphology (Fig. 2C). These data suggest that the PI3K-Akt pathway is required for some of the phenotypic hallmarks associated with TGF-mediated EMT.

View larger version (83K): [in this window] [in a new window]   Fig. 2.   The PI3K-Akt pathway is involved in TGF-induced EMT. A, NMuMG cells were treated or not with 2 ng/ml TGF1 for 24 h in the presence 20 µM PD098059 or 20 µM LY294002 where indicated. Cells were stained with antibodies to ZO-1 (1:300) or phalloidin-fluorescein isothiocyanate (1:100) to visualize actin filaments. B, localization of ZO-1 in NMuMG cells infected with adenovirus vectors encoding -galactosidase or the constitutively active mutant of p110 (ca-p110) for 48 h at a multiplicity of infection of 100 plaque-forming units/cell. By phase contrast (DIC, differential interference contrast), cells infected with the ca-p110 virus retained their epithelial morphology. The immunoblot analysis shows expression of Myc-tagged ca-p110 (lane 2) in cells infected with ca-p110 compared with control virus (lane 1). The lower panel shows the level of phospho-Ser-473 Akt in control cells (lane 1), cells infected with dn-p85 (lane 2), or cells infected with ca-p110 (lane 3). C, NMuMG cells were infected with AxAkt-K179D (dn-Akt) or Ax-Gal (control) adenoviruses at a multiplicity of infection of 40; 48 h later, cells were treated with 2 ng/ml TGF1 for an additional 24 h followed by immunostaining for ZO-1 (1:300) as indicated under "Experimental Procedures." The immunoblot shows expression of Flag-tagged dn-Akt in cells infected with AxAkt-K179D (lane 2) compared with the control virus (lane 1). Scale bars represent 15 µm.

Activation of the PI3K-Akt Pathway in Response to TGF1-- To further test that the PI3K pathway is activated by TGF1, we examined the phosphorylation status and kinase activity of Akt. Immunoblot analyses with antibodies specific to the phosphorylated form of Akt showed that TGF induced phosphorylation of Akt at Ser-473 within 30 min, achieving a detectable maximum at 2 h (Fig. 3A). Phosphorylation of Ser-473 Akt was inhibited by 20 µM LY294002 (Fig. 3A, last lane), indicating that Akt activation requires PI3K function. The activity of Akt/PKB was measured using an in vitro kinase assay with GST-GSK3 fusion protein containing GSK-3 peptide in frame with GST and immobilized on agarose beads as a substrate. Treatment of NMuMG cells with TGF1 for 2 h stimulated a 4-fold induction in the incorporation of ³²P into GST-GSK3 (Fig. 3B). Next, we tested the TGF1 dose dependence of phosphorylation of Akt and Smad2. Treatment with 0.5 ng/ml (20 pM) TGF1 was sufficient to induce a maximal phosphorylation for both Ser-473 Akt and Smad2 (Fig. 3C). TGF1 and EGF, a known agonist of PI3K, induced similar levels of Ser-473 Akt phosphorylation. EGF induced activating phosphorylation of ERK1/2, whereas TGF1 did not stimulate ERK activation at any concentration tested (Fig. 3C).

View larger version (57K): [in this window] [in a new window]   Fig. 3.   TGF1 activates the Akt/PKB kinase in NMuMG cells. A, NMuMG cells were stimulated with 2 ng/ml TGF1 for the indicated times. After lysis in EBC buffer, protein extracts (50 µg/lane) were subjected to SDS-PAGE followed by immunoblot analysis with antibodies for phospho-Ser-473 Akt (1:1000) or total Akt (1:1000). B, Akt/PKB kinase activity in protein extracts (150 µg) from NMuMG cells treated with 2 ng/ml TGF1 was determined by an in vitro kinase assay as described under "Experimental Procedures." Phosphorylated GST-GSK-3 was resolved by SDS-PAGE, and 32P incorporation was analyzed by PhosphorImager. C, immunoblot analysis of protein extracts from NMuMG cells treated with different concentration of TGF1 or 2 ng/ml EGF for 60 min using phospho-specific antibodies to phospho-Smad2, Akt, and ERK1/2. GST, glutathione S-transferase. D, immunoblot analysis of phospho-Ser-473 Akt and total Akt in cells transiently transfected with a dominant-negative RhoA mutant (dn-RhoA). E, immunoblot analysis of phospho-Ser-473 Akt and total Akt in cells transiently transfected with a constitutively active RhoA mutant (ca-RhoA).

Rho-like GTPases Mediate Activation of the PI3K-Akt Pathway in Response to TGF1-- Recent studies have suggested that RhoA is involved in TGF1-mediated transcription (22, 23) and that TGF1 can activate RhoA in NMuMG cells.² Therefore, we tested whether RhoA GTPase affected the activation of PI3K-Akt mediated by TGF1. NMuMG cells transiently transfected with a dominant-negative RhoA mutant (N19RhoA) showed a significantly reduced level of Akt phosphorylation compared with a control (Fig. 3D). Transfection of the constitutively active form of RhoA (Q61LRhoA) resulted in an increase of basal phosphorylation of Akt (Fig. 3E). These results suggest that RhoA may be involved in TGF1-mediated activation of the PI3K-Akt pathway.

Transcriptional Responses to TGF1 Involve the PI3K-Akt Pathway-- TGF transcriptional responses can be controlled through the subcellular localization of Smads. It has been shown that SARA, a recently identified mediator of TGF signaling, controls recruitment of Smad2 to TGF receptors (31). The function of SARA depends on its FYVE homology domain, which binds phosphatidylinositols phosphorylated by PI3K (31). In addition, recent data have suggested that microtubules (MTs) may control Smad-dependent TGF1 transcriptional responses (32). It has been shown that PI3K associates tightly with - and -tubulins (33), and it is involved in the function of MTs (34). Therefore, we next examined whether PI3K is involved in the regulation of TGF-mediated transcription. Two TGF-responsive reporter constructs were used in transcriptional assays: p3TP-Lux, containing the firefly luciferase reporter gene under the control of three 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements and a fragment of the PAI-1 promoter (1), and p(CAGA)₁₂-Lux, a reporter gene containing 12 repeats of Smad binding sequences from the PAI-1 promoter (35). In NMuMG cells transiently transfected with p3TP-Lux, TGF-mediated induction of luciferase was inhibited by LY294002 in a dose-dependent manner at 4 and 16 h (Fig. 4A). Similar results were obtained with 4T1 and EMT6 mammary tumor cell lines (data not shown). LY294002 also inhibited TGF-stimulated reporter activity in both NMuMG and 4T1 cells transfected with p(CAGA)₁₂-Lux (Fig. 4B). We next examined whether an adenovirus vector encoding a dominant-negative mutant of p85 (dn-p85), the regulatory subunit of PI3K, would emulate the effects of LY294002. Expression of dn-p85 significantly reduced a basal phosphorylation of Ser-473 Akt (Fig. 2B), confirming its functional activity. TGF-induced p3TP-Lux reporter activity was reduced by 75% in both NMuMG and 4T1 cells infected with the dn-p85 adenovirus vector but not with a control adenovirus encoding -galactosidase (Fig. 4C). Finally, transient transfection of a dominant-negative mutant of Akt (AktK179M) markedly inhibited TGF-induced p3TP-Lux transcription (Fig. 4D). These data suggest that the PI3K-Akt pathway is involved in TGF transcriptional responses.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Blockade of PI3K-Akt abrogates transcriptional responses to TGF1. A, NMuMG were transfected with p3TP-Lux and pCMV-Rl vectors, starved for 16 h in 1% FBS, and stimulated with 1 ng/ml TGF1. Cells were lysed 4 or 16 h thereafter and assayed for dual-luciferase activity as described under "Experimental Procedures." Relative luciferase units represent the ratio of firefly to Renilla luciferase activities. Each data point represents the mean ± S.D. of 3 wells. B, relative luciferase units in NMuMG and 4T1 cells transfected with p(CAGA)12-Lux and pCMV-Rl vectors and treated with 1 ng/ml TGF1 for 16 h in the absence or presence of LY294002. Each bar represents the mean ± S.D. of 3 wells. C, analysis of luciferase activity in NMuMG or 4T1 cells transduced with an adenoviral vector encoding a dominant-negative mutant of p85 (dn-p85) or -galactosidase (-gal) and subsequently transfected with the p3TP-Lux vector. Luciferase activity was measured as indicated under "Experimental Procedures" and normalized to the protein concentration. Each data point represents the mean ± S.D. of 6 wells. Immunoblot analysis shows expression of dn-p85 in cells infected with a control virus (first lane) or with dn-p85 virus (second lane). D, NMuMG cells were transfected with reporter vectors and the indicated amounts of plasmid encoding AktK179M, a dominant-negative Akt mutant (dn-Akt), and/or pcDNA3 empty vector (control) for a combined total of 667 ng of ectopic plasmid DNA. After treatment with 1 ng/ml TGF1 or no treatment for 16 h, the relative luciferase units from triplicate wells were measured as described under "Experimental Procedures." The immunoblot detects HA-tagged dn-Akt in cells transfected with a control plasmid or with 250 ng (lane 2) or 667 ng (lane 3) of dn-Ak.

TGF1-mediated Phosphorylation of Smad2 Requires PI3K-- The transcriptional data using the p(CAGA)₁₂Lux reporter (Fig. 4B) suggested that PI3K is involved in the control of Smad-dependent transcription. Therefore, we examined the effect of PI3K blockade on TGF-induced phosphorylation of Smad2. Immunoblot analysis with antibodies specific to Smad2 phosphorylated at the C terminus showed that C terminus phosphorylation of Smad2 was induced by TGF1 within 15 min, reaching a maximum by 1 h. However, co-incubation with 20 µM LY294002 markedly reduced ligand-mediated Smad2 phosphorylation without detectable changes in total Smad2 protein levels (Fig. 5A). At the same time, phosphorylation of Ser-473 Akt was completely blocked by LY294002 (Fig. 5B). The induction of the C-terminal phosphorylation of Smad2 and phosphorylation of Ser-473 Akt in response to TGF1 appears to occur with similar kinetics and TGF1 dose dependence (Figs. 3 and 5). To test whether the PI3K-Akt pathway is directly involved in the C-terminal phosphorylation of Smad2, NMuMG cells were transfected with dn-Akt followed by TGF1 treatment and immunoblot analysis of C terminus phosphorylation of Smad2. The level of Smad2 phosphorylation was similar in control cells and cells transfected with dn-Akt, suggesting that Akt is not involved in C-terminal phosphorylation of Smad2 (Fig. 5C). Infection of cells with ca-p110 also did not induce ligand-independent phosphorylation of Smad2 (Fig. 5D).

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Blockade of PI3K inhibits TGF-mediated C-terminal phosphorylation of Smad2. NMuMG cells were treated with 2 ng/ml TGF1 for the indicated times in the presence or absence of 20 µM LY294002. Protein extracts (50 µg/lane) were separated by 12.5% SDS-PAGE followed by immunoblot analysis for phospho-Smad2 (1:500) and total Smad2 (1:500) (A) or phospho-Ser-473 Akt (1:1000) and total Akt (1:1000) (B), as indicated under "Experimental Procedures." C, immunoblot analysis of phospho-Smad2 and total Smad2 in cells transiently transfected with AktK179M, a dn-Akt mutant. D, immunoblot analysis of phospho-Smad2 in cells infected with a ca-p110. -Gal, -galactosidase.

TGF1-induced Cell Migration Requires PI3K Activity-- TGF1 can stimulate the migration of tumor and nontumor cells (7, 36, 37). PI3K has been implicated in the regulation of cell migration and chemotaxis of human neutrophils (38-40). Therefore, we examined whether PI3K is involved in TGF-induced cell migration. We used 4T1 and EMT6 mouse tumor cells, which exhibit high levels of TGF receptors that mediate transcriptional responses (Fig. 4) but are not growth inhibited by exogenous TGF1.³ TGF1 enhanced migration of both cell lines in a dose-dependent manner with an EC₅₀ of approximately 0.1 ng/ml (4 pM). LY294002 blocked both basal and TGF-stimulated cell migration (Fig. 6A) without an effect on tumor cell proliferation (data not shown). The TGF1-neutralizing 2G7 monoclonal antibody also reduced basal cell migration, suggesting that this phenotypic response was partially dependent on autocrine TGF signaling (Fig. 6B). Furthermore, both LY294002 and 2G7 reduced the basal level of phosphorylation at Ser-473 Akt in 4T1 and EMT6 cells (Fig. 6C), suggesting a causal association between autocrine TGF signaling with basal PI3K-Akt signaling and the subsequent migration of tumor cells.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Basal and TGF-stimulated tumor cell migration and Akt kinase activity are reduced by LY294002. A, 4T1 and EMT6 tumor cells (4 × 104 cells/well) were seeded in the upper chamber of 8-µm pore transwells and incubated with TGF1 in the absence or presence of LY294002. Cells that migrated through the polycarbonate filters and attached to the bottom chamber were counted 3 days later. Each bar represents the mean ± S.D. of 3 wells. B, 4T1 and EMT6 cells were seeded under identical conditions as described in A in the absence or presence of the TGF-neutralizing monoclonal antibody 2G7. Cells migrating through the 8-µm pores were counted 3 days later. Data represent the mean ± S.D. of 3 wells. C, exponentially growing 4T1 and EMT6 cells in DMEM, 5% FBS were incubated with 1 ng/ml TGF1 with or without 20 µM LY294002 for 4 h. Where indicated, the 2G7 monoclonal antibody (10 µg/ml) was added for 24 h. Whole cell lysates were prepared, and 50 µg of total protein/lane were subjected to SDS-PAGE followed by immunoblot analyses for phospho-Ser-473 Akt and total Akt as indicated under "Experimental Procedures."

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The tumor-promoting activity of TGF1 associated with the induction of EMT has been documented for different tumor types (5-9). Several reports have shown that TGF can induce a reversible mesenchymal transition in mammary epithelial NMuMG cells (11, 12). In this study, we present data to support the role of the PI3K-Akt pathway in TGF-mediated EMT. We found that either the blockade of PI3K activity by a synthetic inhibitor, LY294002, or by expression of dn-Akt significantly inhibited EMT (Fig. 2). These observations led us to hypothesize that the PI3K-Akt pathway is directly involved in this transition. Similar to TGF1, forced expression of constitutively active PI3K (ca-p110) was sufficient to promote the disruption of cellular junctions but did not induce per se the changes in cell morphology associated with EMT (Fig. 2B). The dissolution of tight junctions and the disruption of adherent junctions induced by TGF1 are relatively early processes, occurring within 4-8 h after the addition of TGF1, whereas changes in the cell shape occur later. This result suggests that PI3K function is required for the early changes during TGF-mediated EMT but that other events associated with the reorganization of cytoskeleton leading to changes in cell morphology may not depend on the PI3K-Akt pathway. The observed delocalization of E-cadherin, integrin ₁, and ZO-1 from cellular junctions occurred without detectable changes in their cellular content, suggesting that these TGF-mediated effects may involve PI3K-dependent endocytosis. These observations are consistent with the studies implicating PI3K in endocytosis and vesicular trafficking (41-43). Similar to TGF, hepatocyte growth factor can also disrupt epithelial cell-cell junctions and induce the delocalization of E-cadherin from cell junctions (44). In this process, hepatocyte growth factor induces the delocalization of both E-cadherin and the hepatocyte growth factor receptor, c-Met, via PI3K-mediated co-endocytosis (44). This co-endocytosis can be blocked by dominant-negative mutants of RhoA and Rab5, a component of early endosomes (44). In addition, Rab5-mediated endocytosis is also regulated by Akt/PKB (45). Thus, TGF-mediated delocalization of epithelial markers from cell junctions may involve the function of PI3K-Akt and Rho-like GTPases.

The activation of PI3K in response to TGF has been reported in two other cell systems (24, 25). In NMuMG cells, TGF1 induced phosphorylation and activation of Akt/PKB with kinetics similar to the C-terminal phosphorylation of Smad2 (Figs. 3 and 5). Activation of Akt depends on PI3K, since it can be blocked by a synthetic inhibitor of PI3K (Figs. 3 and 5) and by expression of dn-p85 (Fig. 2B, inset). These results suggest that the PI3K-Akt pathway is activated directly by TGF1. This conclusion is further supported by recent reports showing co-precipitation of p85, the regulatory subunit of PI3K, with TGF receptors and stimulation of PI3K activity by TGF1 in other cell types (24, 25). We also confirmed a direct association p85 with both type I and type II TGF receptors in NMuMG cells.⁴

Because of the reported role of Rho family GTPases in TGF1 signaling and their interaction with the PI3K pathway (46), we tested the role of the RhoA GTPase in TGF-mediated activation of Akt. Expression of dominant-negative N19RhoA mutant disrupted ligand-induced phosphorylation of Akt at Ser-473. On the other hand, expression of a constitutively active mutant, Q63LRhoA, resulted in an increase of the basal phosphorylation of Akt. These findings suggest that RhoA GTPase is involved in TGF1-mediated activation of Akt, which is consistent with recent reports that Rho-like GTPases can synergize with TGF signaling (22, 23). Therefore, RhoA may function as an upstream effector of Akt activation in response to TGF1.

Using two reporter constructs, p3TP-Lux and p(CAGA)₁₂-Lux, we found that TGF1 transcriptional responses in NMuMG and two tumor cell lines are inhibited by both pharmacological and molecular antagonists of the PI3K-Akt pathway, including dominant-negative p85 and Akt mutants (Fig. 4, A-D). The fact that a blockade of the PI3K-Akt pathway affected Smad-dependent transcriptional responses suggested the involvement of PI3K and Akt in TGF intracellular signal transduction. Consistent with this idea, we found that LY294002 significantly reduced TGF-mediated C-terminal phosphorylation of Smad2 in NMuMG cells (Fig. 5). However, neither PI3K nor Akt is involved in C-terminal phosphorylation of Smad2, since introduction of ca-p110 or dn-Akt did not affect it. These results, coupled with the inhibitory effect of LY294002 on Smad2 phosphorylation (Fig. 5), suggest that PI3K is involved indirectly in TGF-mediated C-terminal phosphorylation of Smad2.

PI3K activity may also be required for the function of intracellular mediators of TGF signaling. Recently, two factors regulating C-terminal phosphorylation of Smad2 were described (31, 32). First, the intracellular localization of Smad2 is controlled by SARA, a recently cloned Smad2-binding protein (31). SARA co-localizes with EEA1, an early endosome marker,⁵ and this co-localization depends on the FYVE domain of SARA, which binds phosphatidylinositol 3-phosphates (47, 48). It has been shown that deletion of the FYVE domain results in the mislocalization of Smad2 and inhibition of TGF transcriptional responses (31). We found that Smad2 co-localizes with EEA1 in the absence of TGF in NMuMG cells.⁶ Thus, it is conceivable that the blockade of PI3K activity in NMuMG cells with LY294002, similar to wortmannin (49), will reduce the levels of phosphatidylinositol 3-phosphate, resulting in the mislocalization of Smad2. This is a potential explanation of the inhibitory effect of LY294002 on TGF-induced phosphorylation of Smad2 (Fig. 5A), whereas neither ca-p110 nor dn-Akt can directly modulate Smad2 phosphorylation (Fig. 5C, D). In addition, a recent report provides evidence that endogenous Smad2, Smad3, and Smad4 are stored in the MT network (32). It has been suggested that upon TGF treatment, Smad2 and Smad3 dissociate from MT, become phosphorylated by TRI, and translocate to the nucleus where they regulate the transcription of TGF target genes. Moreover, destabilization of MTs with nocodazole can facilitate Smad-mediated TGF transcriptional responses per se in the absence of exogenous TGF1 (32). On the other hand, TGF has been reported to stabilize MTs (50), potentially limiting Smad signaling. PI3K has also been shown to control the dynamics of the MT network, which is important for intracellular trafficking, cell motility, and other cell functions (51). Therefore, PI3K antagonists may affect the MT network and interfere with TGF signaling. To formally demonstrate that PI3K blockade inhibits TGF signaling via its effects on MTs will require further investigation.

Both TGF and PI3K have been implicated in chemotaxis and cell migration (7, 36-40). Here, we show that pM concentrations of TGF1 enhanced the basal migration of tumor cells, whereas blockade of PI3K with LY294002 reduced both basal and TGF-stimulated cell migration (Fig. 6, A and B). These data are in agreement with a critical role of PI3K in cell motility and migration via the modulation of cytoskeletal organization (47, 51). These results were generated with tumor cells that exhibit high levels of TGF expression and TGF receptors as well as constitutive activation of Akt in the absence of added TGF ligand. Similar to LY294002, TGF1-neutralizing monoclonal antibodies reduced basal cell migration and Ser-473 phosphorylation of Akt, suggesting an association between autocrine TGF signaling with both constitutively activated Akt/PKB and cell invasiveness. Neither exogenous TGF, anti-TGF antibodies, nor LY294002 had any effect on 4T1 or EMT6 cell proliferation. These data coupled with the transcription data using TGF reporters in 4T1 and EMT6 cells imply that EMT can be dissociated from the anti-mitogenic effects of TGF. In summary, the results presented provide evidence that the PI3K-Akt pathway is causally involved in the morphogenic, transcriptional, and migratory activities of TGF.

	ACKNOWLEDGEMENTS

We thank Teresa Dugger and Sorena Nadaf for excellent technical assistance, Michael Engel for critical reading of the manuscript, W. Ogawa for the adenovirus vectors, C. Kumar for the Rac1 inhibitor SCH51344, C. L. Van Den Berg for the GST-GSK expression construct, P. N. Tsichlis for the mutant AktK179M plasmid, and J. Massague and J.-M. Gauthier for the TGF reporter constructs.

	FOOTNOTES

^* This work was supported by Public Health Service (PHS) Grant R01 CA62212, U. S. Department of Defense, U. S. Army Medical Research Material Command Grant DAMD17-98-1-8262, a Clinical Investigator Award from the Department of Veterans Affairs (to C. L. A.), PHS Grant R35 CA42572 (to H. L. M.), National Institutes of Health Training Grant CA09592 (to N. A. B.), and Vanderbilt-Ingram Cancer Center NCI National Institutes of Health Support Grant CA68485.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Div. of Hematology-Oncology, Vanderbilt University School of Medicine, 22nd Ave. South, 1956 TVC, Nashville, TN 37232-5536. E-mail: carlos.arteaga@ mcmail.vanderbilt.edu.

Published, JBC Papers in Press, August 31, 2000, DOI 10.1074/jbc.M005912200

² N. A. Bhowmick, M. Ghiassi, A. V. Bakin, M. Aakre, C. A. Lundquist, M. E. Engel, C. L. Arteaga, and H. L. Moses, submitted for publication.

³ C. L. Arteaga, unpublished data.

⁴ N. Dumont and A. Bakin, unpublished data.

⁵ J. Wrana (University of Toronto), personal communication.

⁶ A. Bakin, unpublished data.

	ABBREVIATIONS

The abbreviations used are: TGF, transforming growth factor ; TRI, TGF type I; EMT, epithelial to mesenchymal transition; PI3K, phosphatidylinositol 3-OH kinase; EGF, epidermal growth factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; MT, microtubule; ca, constitutively active; dn, dominant-negative; PKB, protein kinase B; SARA, Smad activator for receptor activation; FYVE domain, domain found in Fab1p, YOTB, Vac1p, and EEA1 proteins.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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