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Cancer Research 67, 3673, April 15, 2007. doi: 10.1158/0008-5472.CAN-06-3211
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Transforming Growth Factor ß Signaling via Ras in Mesenchymal Cells Requires p21-Activated Kinase 2 for Extracellular Signal-Regulated Kinase-Dependent Transcriptional Responses

Kaori Suzuki, Mark C. Wilkes, Nandor Garamszegi, Maryanne Edens and Edward B. Leof

Department of Biochemistry and Molecular Biology, Thoracic Diseases Research Unit, and Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, Minnesota

Requests for reprints: Edward B. Leof, Stabile 8-58, Mayo Clinic, 200 First Street, SW, Rochester, MN 55905. Phone: 507-284-5717; Fax: 507-284-4521; E-mail: leof.edward{at}mayo.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor ß (TGF-ß) signaling via Smad proteins occurs in various cell types. However, whereas the biological response to TGF-ß can be as distinct as growth promoting (i.e., mesenchymal cells) versus growth inhibiting (i.e., epithelial cells), few discernible differences in TGF-ß signaling have been reported. In the current study, we examined the role of Ras in the proliferative response to TGF-ß and how it might interface with Smad-dependent and Smad-independent TGF-ß signaling targets. TGF-ß stimulated Ras activity in a subset of mesenchymal, but not epithelial, cultures and was required for extracellular signal-regulated kinase (ERK)–dependent transcriptional responses. Although dominant negative Ras had no effect on TGF-ß internalization or Smad-dependent signaling (i.e., phosphorylation, nuclear translocation, or SBE-luciferase activity), it did prevent the hyperphosphorylation of the Smad transcriptional corepressor TG-interacting factor (TGIF). This was not sufficient, however, to overcome the mitogenic response stimulated by TGF-ß, which was dependent on signals downstream of p21-activated kinase 2 (PAK2). Moreover, although the initial activation of Ras and PAK2 are distinctly regulated, TGF-ß–stimulated PAK2 activity is required for Ras-dependent ERK phosphorylation and Elk-1 transcription. These findings show the requirement for crosstalk between two Smad-independent pathways in regulating TGF-ß proliferation and indicate that the mechanism(s) by which TGF-ß stimulates growth is not simply the opposite of its growth inhibitory actions. [Cancer Res 2007;67(8):3673–82]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß (TGF-ß) is the prototypic member of a superfamily of secreted proteins that regulate a wide range of cellular responses in a milieu- and cell type–dependent manner (1, 2). The vast majority of mammalian cells expresses the plasma membrane TGF-ß binding proteins referred to as the type I (TßRI), type II (TßRII), and type III (TßRIII or ß-glycan) receptors. Although the role of the type III receptor is not well understood, TßRI and TßRII are well characterized transmembrane proteins that possess serine/threonine kinase activity and mediate TGF-ß–generated signaling. Ligand binding to the constitutively active TßRII results in the formation of a heterotetrameric receptor complex on the cell surface and activation of TßRI through which the TGF-ß signal is propagated (3).

The best characterized means of TGF-ß signaling occurs through a family of cytoplasmic transducers referred to as Smad proteins (4, 5). There are three general subgroups of Smads, the receptor-associated Smads (R-Smads; Smad2 and Smad3 for TGF-ß signaling), a co-Smad (Smad4), and the inhibitory Smads (I-Smads; Smad6 and Smad7). The R-Smads bind transiently to the receptor complex and are direct targets of the activated TßRI kinase. This COOH-terminal phosphorylation leads to R-Smad activation, association with Smad4, and nuclear translocation where they assemble with DNA-binding elements to modulate gene transcription. The fact that a single ligand (TGF-ß) is capable of generating such divergent cellular responses is indicative of complex protein interactions at multiple levels in the signal transduction cascade. For instance, the differential combination of receptors that form the tetrameric receptor complex results in the activation of distinct R-Smad proteins, whereas the association of Smads with selective DNA-binding proteins modulates specific gene responses (4, 5).

In addition to the Smad cascade, TGF-ß activates a number of other signaling pathways (6, 7). Recently, we characterized the Smad-independent activation of p21-activated kinase 2 (PAK2), the c-Abl nonreceptor tyrosine kinase, and phosphoinositide-3-kinase (PI3K) by TGF-ß in mesenchymal, but not in epithelial cultures (8–11). Specifically, TGF-ß–activated PI3K/PAK2/c-Abl was required for the induction of morphologic transformation, proliferation, and anchorage-independent growth of fibroblasts in a Smad-independent manner. Identification of these cell type–specific pathways extends earlier studies documenting differential binding of TGF-ß to its receptors (12, 13) and the induction of distinct transcription factors of the Jun/activator protein 1 family in fibroblasts and keratinocytes, leading to distinct, even opposite, transcriptional outcomes (13, 14). Other Smad-independent signaling molecules include (but are not limited to) TGF-ß–activated kinase-1/p38, c-jun-NH2-kinase, Rho family proteins (Rac, cdc42, and RhoA), and Ras (15–19).

Ras is of particular interest due to its connection to PI3K and its well-established role in cell proliferation and transformation (20, 21). Ras is a small GTP binding protein in which activity is dictated by the ratio of GTP-bound (active) to GDP-bound (inactive) states. Ras-GDP is exchanged for GTP by the intervention of guanine nucleotide exchange factors (GEF), enabling it to interact with downstream effectors; conversely, GTPase-activating proteins return it to the inactive status. Once activated, the extracellular signal-regulated kinase (ERK) pathway is a common downstream route mediating Ras signaling. In general, activated Ras promotes association of Raf with the plasma membrane where other events facilitate Raf activation (22). This results in the phosphorylation/activation of mitogen-activated protein (MAP)/ERK kinases (MEK) 1 and 2 and the subsequent phosphorylation of ERK1 and ERK2 (23, 24). Once activated, ERKs phosphorylate a number of cytoplasmic proteins as well as translocate to the nucleus where they control the activity of various transcription factors and other nuclear proteins such as Elk-1, SAP1, and RNA polymerase II (24).

Although Ras has been known to be modulated by TGF-ß ligands since the early 1990s (25), the role which the Ras/ERK pathway plays in TGF-ß signaling is unclear (6, 18). It has been shown that Ras opposes the effect of Smad-dependent TGF-ß signaling by (a) stimulating ERK phosphorylation of the Smad linker region, resulting in a block to nuclear translocation; (b) inducing degradation of Smad4; or (c) regulating the expression of the Smad transcriptional corepressor TG-interacting factor (TGIF) (26–28). Alternatively, Ras has been reported to promote TGF-ß signaling (29–31). It is currently unknown how such distinct responses can be integrated within the context of TGF-ß action. In that regard, because Ras signaling is primarily associated with providing a growth advantage, a possible explanation may be that the majority of studies used epithelial cell models where TGF-ß normally functions as a growth inhibitor. Given that TGF-ß is one of the most profibrotic cytokines known (8), the relevant target for TGF-ß–stimulated Ras might be the tumor stroma.

To that end, because tumor development and progression are dependent on the synergistic interplay of cancer cells and an activated stroma (32, 33), in the present manuscript, we have investigated the role of the Ras/ERK pathway with other Smad-dependent and Smad-independent signaling effectors in cells of fibroblastic lineage. The results indicate that (a) TGF-ß stimulates Ras and ERK activation in a subset of fibroblast, but not epithelial, cell lines; (b) Ras activity is not required for Smad2 or Smad3 phosphorylation, nuclear translocation, or SBE-luciferase activity; (c) inhibition of the Smad transcriptional corepressor TGIF by dominant negative Ras is not sufficient to overcome TGF-ß–stimulated proliferation; and (d) whereas PAK2 and Ras signaling are independently initiated, there is crosstalk between these two Smad-independent pathways such that PAK2 is required for Ras-dependent ERK phosphorylation and transcriptional responses via regulating Raf activity. Together, these findings not only document the complexity of the intracellular crosstalk required for regulated TGF-ß signaling, but emphasize the need to more critically integrate the action of cytokines on stromal elements when investigating tumor development and/or progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. AKR-2B, BALB/c 3T3, HeLa, MDCK, Mv1Lu, NIH/3T3, Smad3-knock-out mouse embryonic fibroblasts (S3KO) and 293-HEK cells were grown in high-glucose DMEM (Invitrogen, Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% (AKR-2B) or 10% (the rest of cell types) fetal bovine serum (FBS; Biosource International, Camarillo, CA).

Chemicals. Protein A-sepharose, myelin basic protein, DMSO, PI3K inhibitor LY294002, and MEK inhibitor PD098059 were purchased from Sigma-Aldrich (St. Louis, MO), whereas the Akt inhibitors were from Calbiochem (San Diego, CA).

Antibodies. Anti–phospho-Smad2 was purchased from Calbiochem; anti-Akt and anti-pS473Akt were purchased from Cell Signaling (Beverly, MA); goat anti-adenovirus hexon was from Chemicon International, Inc. (Temecula, CA); goat anti-6x His tag was from Novus Biologicals (Littleton, CO); anti-green fluorescent protein (GFP) was from Roche Applied Science (Indianapolis, IN); anti-Smad2 was from BD Transduction Labs-PharMingen (San Diego, CA); anti–phospho-ERK (E4), anti-ERK, anti-{gamma}PAK, horseradish peroxidase (HRP)–conjugated anti-mouse immunoglobulin G, HRP-conjugated anti-rabbit were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); anti-Flag M2 was from Sigma-Aldrich; anti-pT308Akt, anti-Raf, anti–phospho-Raf-1 (Ser338), and anti-Ras clone Ras10 were from Upstate Biotechnology (Charlottesville, MA); rabbit anti-Smad3 antibody was from Zymed Laboratories-Invitrogen, (Carlsbad, CA); and Alexa fluor 594-conjugated donkey anti-rabbit and Alexa fluor-488–conjugated donkey anti-goat antibody were from Invitrogen-Molecular Probes (Carlsbad, CA). The rabbit anti–phospho-Smad3 antibody to the peptide COOH-GSPSIRCSpSVpS was generated in our laboratory (10).

Western blot analysis. Cultures were treated overnight in serum-free DMEM before stimulation. Lysates were prepared [50 mmol/L Tris (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L Na3VO4, 1x complete protease inhibitor; Roche Applied Science] and equivalent protein (~100 µg determined by Pierce BCA Protein Assay kit; Pierce Biotechnology, Rockford, IL) probed with the indicated phospho or total antibody. Secondary goat anti-mouse (1:2,000 dilution) or donkey anti-rabbit (1:2,000 dilution) antibodies were from Santa Cruz Biotechnology and Amersham (Piscataway, NJ), respectively. As the adenoviral-expressed dominant negative Ras was not always observed as either an increase in total Ras protein or a slightly higher migrating protein (Fig. 2B), transduced His-RasN17 or eGFP protein was detected using anti-His or anti-GFP (Figs. 2B, 3A, 4B, 5A, and 6A).


Figure 2
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  		Figure 2. Ras signaling is required for TGF-ß–induced ERK phosphorylation and Elk-1 luciferase activity. A, expression of transduced adenovirus. AKR-2B cells were left untreated (left column) or infected with MOI 400 of Ad-His-RasN17 (middle column) or Ad-eGFP (right column) for 24 h. Following fixation and permeabilization, cells were examined for anti-Hexon expression (middle row) or eGFP fluorescence (bottom row). Top row, phase image from the anti-Hexon panel (magnification, 200x). B, AKR-2B cells seeded in six-well plates were uninfected (–) or transduced (+; MOI 400) with Ad-eGFP or Ad.RasN17 for 24 h. Top four rows, following 10 min TGF-ß (10 ng/mL) treatment (+), cells were harvested and lysates prepared. The first three panels reflect Ras activity, total Ras protein, and the transduced dominant negative His-Ras, respectively, whereas the fourth panel shows expression of eGFP from the control adenovirus. Bottom four rows, similarly transduced AKR-2B cells were stimulated with 10 ng/mL TGF-ß for 45 min before lysate preparation and Western blot analysis for phospho-ERK (p-ERK1/pERK2), total ERK (ERK1/ERK2), transduced Ras (His-RasN17), or eGFP (GFP). Because the slightly higher migrating adenoviral-expressed dominant negative His-Ras is not always observed in a total Ras blot, expression and activity are documented by anti-His analysis and/or inhibition of phospho-ERK, respectively. C, AKR-2B cells were transduced as in (B) with increasing doses of Ad-eGFP (black line) or Ad.RasN17 (red line). Left, cultures were treated with TGF-ß (10 ng/mL) for 45 min, harvested, and analyzed by Western blot using anti–p-ERK and total ERK antibodies. The increase in p-ERK (normalized to total ERK) stimulated by TGF-ß in the presence of the indicated adenovirus MOI is depicted. Right, Elk-1 and ß-galactosidase reporter plasmids were transfected into AKR-2B cells in the presence of Ad-eGFP (black line) or Ad.RasN17 (red line) as described in Materials and Methods. Following treatment with TGF-ß (10 ng/mL) for 20 h, normalized luciferase expression was determined in duplicate samples. Points, mean for both ERK phosphorylation and Elk-1 luciferase of three separate experiments; bars, SD.

 

Figure 3
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  		Figure 3. Ras/ERK signaling and the Smad pathway are independently activated by TGF-ß. A, AKR-2B cells were untreated (–) or transduced (+) with a MOI 400 of Ad-His-RasN17 or Ad-eGFP and incubated in the absence (–) or presence (+) of TGF-ß (10 ng/mL) for 30 min. Western blot analysis was done for phosphorylated (p-Smad2 and p-Smad3) and total (Smad2 and Smad3) R-Smad or adenoviral expressed dominant negative Ras (His-RasN17) and eGFP (GFP) from the indicated lysate. B, nuclear translocation of Smad3 was studied by immunohistochemistry. AKR-2B cells were transduced with MOI 400 of Ad-eGFP (b, e, and g) or Ad-His-RasN17 (c, f, and h) as in (A) and left untreated (–; a–c) or stimulated (+, d–f) for 20 min with 10 ng/mL TGF-ß. Following fixation and permeabilization, cells were examined for eGFP fluorescence (g) or incubated with anti-Smad3 (a–f) or anti-Hexon (h) antibodies, followed by Alexa fluor 594 (a–f) or Alexa fluor 488 (h) conjugated secondary antibodies. Images were photographed using an inverted fluorescence microscope (magnification, 200x). C, TGF-ß (10 ng/mL) stimulated (+) SBE-luciferase activity was determined in AKR-2B cells in the absence (–) or presence (+) of transduced (MOI 200) Ad.eGFP or Ad.RasN17 as described in Fig. 2. Columns, mean of two separate experiments done in duplicate; bars, SD. D, AKR-2B or Smad3 KO MEFs expressing dominant negative Smad2 (Smad2S467A) were transfected with Gal4-Elk-1-307-428/Gal4-tk80-Luc (top) or SBE-luciferase (bottom) as in (C) and Fig. 2. Cultures were left untreated (–) or stimulated (+) with TGF-ß (10 ng/mL) or PDGF (25 ng/mL) for 20 h. Columns, mean of two experiments done in duplicate; bars, SD.

 

Figure 4
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  		Figure 4. Inhibition of the Smad transcriptional corepressor TGIF is not sufficient to prevent TGF-ß proliferation. A, Mv1Lu or AKR-2B cells were plated in 100-mm plates at 2.5 x 106 cells per well in 10% FBS/DMEM for 24 h. The medium was removed and replaced with 0.1% FBS/DMEM for an additional 24 h when the cultures were either left untreated (–) or stimulated (+) for 2 h with 10 ng/mL TGF-ß and/or 25 µmol/L PD098059 (30 min pretreatment). Western blot analysis for TGIF, phosphorylated (p-ERK1/p-ERK2), or total (ERK1/ERK2) ERK was then done. The slower migrating TGIF species has been shown to represent the phosphorylated/stabilized form of TGIF (27), and total ERK protein is used as a loading control. B, AKR-2B cells were treated and assessed for TGIF, phospho-ERK, and total ERK protein as in (A) following transduction (MOI 200) with Ad.RasN17 or Ad.eGFP. Bottom two panels, expression of the transduced dominant negative Ras (His-RasN17) or control eGFP (GFP), respectively. C, AKR-2B cells were plated at 2.5 x 105 per six-well dish and incubated at 37°C for 24 h. Confluent cultures were placed in serum-free DMEM alone (Control) or containing the indicated adenovirus (MOI 200), 20 µmol/L LY294002, or 0.5% DMSO for 48 h in the absence (–) or presence (+) of 10 ng/mL TGF-ß. Cell counts were done on triplicate wells; columns, mean of two experiments; bars, SD.

 

Figure 5
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  		Figure 5. TGF-ß–stimulated Ras activation and downstream signaling are differentially regulated by PI3K and PAK2. A, uninfected control and Ad-eGFP or Ad-RasN17 (MOI 400) transduced AKR-2B cells were left untreated (–) or stimulated (+) with 10 ng/mL TGF-ß. At the indicated times for detection of optimal phospho-ERK (left, 45 min), PAK2 kinase activity (middle, 60 min), or phosphorylated AKT (right, 90 min), cell lysates were prepared. PAK2 kinase activity (PAK2 activity) or Western blot analysis for phosphorylated ERK (p-ERK1/pERK2), total ERK (ERK1/ERK2), total PAK2 (PAK2), phosphorylated T308 AKT (p-AKT), and total AKT (AKT) was done as described (10). Bottom, expression of adenoviral-transduced eGFP (GFP) and His-Ras (His-RasN17) was determined in the lysate from the corresponding kinase assay or Western blot. B, control (Untreated) or AKR-2B cells transduced (MOI 400) with Ad.RasN17, Ad.PAK2K278 or Ad.eGFP were incubated in the absence (–) or presence (+) of 10 ng/mL TGF-ß for 45 min. Western blotting for phosphorylated (p-ERK1/p-ERK2) or total (ERK1/ERK2) ERK protein was determined as in (A). Parallel plates were pretreated for 30 min with 20 µmol/L LY294002 or 0.3% DMSO (vehicle) before TGF-ß stimulation. C, AKR-2B cells were treated as in (B) and luciferase activity from an Elk-1 reporter done following 20 h incubation in the absence (–) or presence (+) of 10 ng/mL TGF-ß. Columns, mean of two experiments done in duplicate; bars, SD. D, GTP-loaded Ras was determined following 10 min stimulation (+) with 10 ng/mL TGF-ß in AKR-2B cells treated as described in (B). Columns, mean of three separate experiments expressed as fold increase in Ras activity following TGF-ß treatment compared with the corresponding unstimulated cells; bars, SD.

 

Figure 6
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  		Figure 6. Raf is the point of crosstalk between TGF-ß–stimulated PI3K and Ras pathways. A, AKR-2B cells were left untreated (–) or stimulated (+) with 10 ng/mL TGF-ß for 45 min. Endogenous Raf kinase activity (top), Ser338 phosphorylation (p-Raf), and total protein (Raf) were determined following infection with the indicated adenovirus or treatment with LY294002 (20 µmol/L) or PD098059 (25 µmol/L) as in Fig. 5B (legend) and Materials and Methods. Bottom two rows, adenoviral transduced dominant negative His-RasN17 (His) or PAK2 (eGFP-PAK2K279R) expression. B, a model summarizing our current understanding of cell type–specific TGF-ß signaling is depicted. Following ligand binding to the TGF-ß receptor complex, fibroblasts and epithelial cells differentially integrate Smad-dependent and Smad-independent signals. Although Smad2/3 activation occurs in both cell types and is dependent on receptor endocytic activity, PI3K and Ras pathways represent cell type–specific TGF-ß signaling responses initiated upstream of dynamin action (9, 37). PI3K functions as a branch point in TGF-ß signal propagation leading to independent activation of Akt or PAK2 (9). Although PAK2 is necessary for continued PI3K signaling via c-Abl (11) as well as interfacing with Ras-dependent signaling at the level of c-Raf activation, the signals downstream of Akt are currently unknown. Arrows, not necessarily a direct interaction or the only point of intersection.

 
Kinase assays. For PAK2 and Raf kinase assays, cells were grown to confluence in 10% DMEM and serum starved overnight (10). Cultures were treated as indicated and lysed for 30 min at 4°C in 750 µL of kinase lysis buffer [50 mmol/L Tris (pH 7.4), 5 mmol/L EDTA, 250 mmol/L NaCl, 0.1% Triton X-100, 50 mmol/L NaF, 0.1 TIU/mL aprotinin, 50 µg/mL phenylmethylsulfonyl fluoride, 100 µmol/L sodium vanadate, and 1 µg/mL leupeptin]. Extracts were clarified, and equivalent protein (500–700 µg) was incubated overnight at 4°C with antibody. Immune complexes were collected with protein A-sepharose (Sigma) and washed twice in kinase lysis buffer and twice in kinase buffer [25 mmol/L Tris (pH 7.4), 10 mmol/L MgCl2, 1 mmol/L DTT] before incubation in 50 µL kinase buffer containing 5 µmol/L ATP, 5 µg myelin basic protein (Sigma), and 5 µCi/µL [{gamma}32P]ATP or 5 µmol/L ATP, 5 µg MEK1-GST (Chemicon International), and 1 µCi/µL [{gamma}32P]ATP for PAK2 and Raf kinase assays, respectively. The kinase reaction was allowed to proceed for 10 min at 37°C, stopped with 50 µL 2x Laemmli buffer, and following SDS-PAGE visualized by autoradiography.

Adenoviruses. His tagged-dominant negative Ras adenovirus (Ad-His-RasN17) was constructed as follows: the insert SpeI-6x His-N17-NheI was synthesized by PCR using pCMV-RasN17 (BD Biosciences Clontech, Palo Alto, CA) as the template and the primer pair SpeI-His-Ras5' and NheI-Ras-wt3'. The PCR product was cloned into the shuttle vector pSV8, digested with BstBI, and transfected into 293Cre cells. The generated Ad-His-RasN17 was plaque purified, and cesium chloride gradient–purified virus was used for the experiments. Construction of enhanced GFP (Ad-eGFP) and dominant negative PAK2 (Ad-PAK2K278R) expressing adenoviruses was previously described (10).

Ras activation. Cultures were grown to ~90% confluency in 100-mm dishes when the media was changed to serum-free DMEM. Following 24 h incubation, the cells were treated with TGF-ß2 (10 ng/mL) for the indicated times. Control and treated groups were washed twice in cold PBS and 0.5 mL of lysis buffer (EZ-Detect Ras Activation kit; Pierce) supplemented with Complete protease inhibitor (Roche Applied Science). The lysate was incubated on ice for 20 min and spun at 16,060 x g (4°C for 15 min). An aliquot was used for Western blot analysis to determine total Ras (8–10), and ~500 µg of total protein per sample was processed for determining GTP-loaded Ras as per kit instructions.

Luciferase assays. Cells were plated in six-well plates at 2 x 105 per well. The next day, media were changed to OPTI-MEM, and cells were transfected with 0.5 µg of CMV–ß-galactosidase and SBE-Luc (2.5 µg) or a mix of Gal4-Elk-1-307-428 (0.5 µg) + Gal4-tk80-Luc (2.0 µg) using Gene Juice (Novagen/EMD Biosciences, San Diego, CA). After 4 h, media were changed to DMEM–5% FBS, and the cells were allowed to recover overnight. When adenovirus was employed, cultures were transduced with the corresponding viral vectors in OPTI-MEM for 4 h and then placed in serum-free DMEM for 20 h. On day 3, TGF-ß2 (10 ng/mL) was added in the absence or presence of the indicated drug or vehicle. Luciferase activity was measured 24 h after TGF-ß2 or platelet-derived growth factor (PDGF) treatment (8).

Statistical analysis. Student's t test was done, with a P value <0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TGF-ß stimulates Ras pathway signaling in fibroblasts but not epithelial cells. The role(s) of activated Ras in TGF-ß signaling is unclear. Because actions of TGF-ß are known to be dependent on cell context, we wished to determine, first, if Ras activity is modulated by TGF-ß and, second, if this might occur in a cell type–specific manner. To address both questions, we stimulated three representative fibroblast (AKR-2B, BALB/c, NIH-3T3) and epithelial (HeLa, Mv1Lu, MDCK) cells with TGF-ß and determined whether there was any effect on Ras activation with an assay whereby the Ras-binding domain (RBD) of Raf1 specifically pulls down the GTP-bound form of Ras. Because preliminary experiments indicated Ras activation beginning after 2 to 5 min of TGF-ß treatment (data not shown), to ensure greater reproducibility, Ras activity was routinely determined following 10 min TGF-ß addition (Fig. 1A ). Although activated Ras was stimulated ~2-fold in the three mesenchymal lines, there was no significant effect of TGF-ß on Ras activation in any of the epithelial cultures from 1 to 240 min TGF-ß stimulation (Fig. 1A and data not shown).


Figure 1
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  		Figure 1. Ras signaling is activated by TGF-ß in fibroblasts but not in epithelial cells. A, mesenchymal (AKR-2B, BALB/c, NIH-3T3) and epithelial (HeLa, Mv1Lu, MDCK) cell lines were left untreated (–) or stimulated (+) for 10 min with 10 ng/mL TGF-ß and GTP-loaded Ras determined. Columns, mean of two separate experiments expressed as fold induction compared with unstimulated cells for each condition (stippled columns, control; solid columns, TGF-ß treated); bars, SD. Bottom, a representative Western blot of the pulled down GTP-bound form of Ras (Ras Activity) and total Ras in the lysate (Ras) for each cell line. B, the time course of ERK phosphorylation by TGF-ß was compared in AKR-2B and Mv1Lu cells. Cultures were incubated with TGF-ß (10 ng/mL) for the indicated times, and total (ERK1/ERK2) and phosphorylated (p-ERK1/p-ERK2) ERK were assessed by Western blot analysis. Top, points, mean of three separate experiments of TGF-ß–stimulated ERK induction (p-ERK1 plus p-ERK2 relative to total ERK1 plus ERK2) in AKR-2B (solid line) and Mv1Lu (dashed line) cells; middle (AKR-2B cells) and bottom (Mv1Lu cells) show representative ERK Westerns; bars, SD. C, AKR-2B and Mv1Lu were transiently cotransfected with plasmids Gal4-Elk-1-307-428/Gal4-tk80 and normalized luciferase activity was determined following incubation with TGF-ß (10 ng/mL) for 20 h. The data are depicted as the fold increase of luciferase activity in TGF-ß–treated cultures (solid columns) relative to untreated cells (stippled columns) and represent the mean ± SD of three separate experiments done in duplicate.

 
Once activated, Ras couples to a number of downstream targets of which the MAPK pathway is one of the most extensively studied, leading to gene activation (24). As such, we next determined whether the cell type–specific activation of Ras was similarly observed for both ERK phosphorylation and ERK-dependent Elk-1 luciferase expression. ERK phosphorylation became detectable between 20 and 40 min TGF-ß treatment and remained elevated for at least 120 min in mesenchymal AKR-2B cells. However, when a similar kinetic study was done in Mv1Lu epithelial cultures, there was no significant effect of TGF-ß on the basal levels of ERK1 or ERK2 phosphorylation (Fig. 1B). Quantitation of this differential cell type response in TGF-ß–stimulated ERK activity is shown in Fig. 1B (top). Furthermore, TGF-ß increased expression of the ERK-dependent Elk-1 luciferase reporter ~3-fold in AKR-2B cells, whereas there was no difference between the untreated and treated Mv1Lu groups (Fig. 1C). Similar responses were observed in the other mesenchymal and epithelial lines (data not shown).

Although the preceding data document Ras/ERK/Elk-1 activation following TGF-ß treatment of mesenchymal cells, the dependence of downstream targets such as ERK and Elk-1 on Ras had not, as yet, been determined. As such, to investigate the role of Ras in this TGF-ß–regulated pathway, we constructed an adenovirus encoding a His-tagged dominant negative Ras (Ad-His-RasN17; ref. 34). Following the documentation of AKR-2B infectivity (Fig. 2A ) and inhibition of TGF-ß–stimulated Ras activity (Fig. 2B, top), cells were transduced with Ad-His-RasN17 or control Ad-eGFP, and ERK phosphorylation and Elk-1 transcriptional activity were determined. Ad.RasN17 decreased both the level of p-ERK (Fig. 2B, bottom and Fig. 2C, left) and Elk-1–luciferase (Fig. 2C, right) in direct relation with increasing doses of the adenovirus, reducing the TGF-ß–induced p-ERK and luciferase levels to 25% to 30% of TGF-ß–stimulated control cells. Although high doses of control adenovirus (Ad-eGFP) had some nonspecific effects in the Elk-1 transcriptional assay (Fig. 2C, right), these effects were minimal for ERK phosphorylation. Because the Elk-1 transcription factor is phosphorylated by MAPKs (such as ERK) to stimulate transcription (24, 35), the data indicate that Ras-dependent gene activation is similarly induced as ERK phosphorylation by TGF-ß in a subset of mesenchymal (but not epithelial) cell lines (Figs. 1 and 2).

The Smad pathway is crucial to many aspects of the biological actions of TGF-ß. As such, we next investigated the relation (if any) between Ras/ERK and Smad signaling as studies have shown both antagonistic as well as cooperative interactions (6). Initial experiments addressed the general question whether Ras activity is required for Smad2 and Smad3 signaling. Accordingly, AKR-2B cells were infected with adenovirus expressing eGFP or dominant negative Ras and the level of R-Smad phosphorylation and ligand internalization assessed following addition of TGF-ß (Fig. 3A and data not shown). Because Smad activation was unchanged by dominant negative Ras (Fig. 3A), the observed similar lack of an effect on TGF-ß internalization (data not shown) by Ad.His.RasN17 was anticipated because Smad phosphorylation is dependent on TGF-ß endocytic activity in the cell models used (36, 37). However, as recent publications have indicated that Smad nuclear translocation and/or effects on gene expression can be dissociated from Smad phosphorylation (38), we also examined whether Ras signaling would be necessary for either response. As shown in Fig. 3B and C, Ad.RasN17 had no discernible effect on Smad3 nuclear translocation or SBE luciferase activity induced by TGF-ß.

Although the preceding data show that Ras-dependent signaling is not required for R-Smad phosphorylation, nuclear translocation, or gene activation, it does not address the converse (i.e., whether Ras-dependent signaling requires Smad activity). Consequently, expression of the Ras/ERK-regulated Elk-1 reporter was examined in control AKR-2B cells and a MEF line from a Smad3 knock-out mouse transfected with dominant negative Smad2. Although TGF-ß was unable to activate the Smad3 responsive SBE promoter in the Smad knock-out cells, Elk-1 luciferase activity was similarly induced by TGF-ß as was observed with the known Elk-1 activator PDGF (ref. 39; Fig. 3D). The previous data (Figs. 1–3) show that TGF-ß stimulation of Ras activity (a) is differentially regulated in fibroblast and epithelial cell types; (b) results in ERK-dependent gene activation; and (c) is mediated independent of Smad2 and Smad3.

Inhibition of TGIF is not sufficient to prevent TGF-ß proliferation. The mechanisms controlling TGF-ß growth inhibition have been extensively investigated (1, 4, 5). Although a number of cytoplasmic and/or nuclear pathways have been implicated, a common theme associated with the growth inhibitory response is the ability of the Smads to recruit DNA binding transcription factors as well as coactivators or corepressors to the transcriptional complex. Because we have been focusing on processes regulating the proliferative side of TGF-ß action most relevant to the tumor stroma, an unanswered question is whether TGF-ß growth promotion is mediated via a reciprocal action on those intermediaries required for growth arrest. One such target proposed to antagonize TGF-ß growth inhibition through ERK-dependent phosphorylation/stabilization, preventing cyclin-dependent kinase inhibitor gene expression, is the Smad corepressor TGIF (27). As would be expected for a negative regulator of TGF-ß growth inhibition, the addition of TGF-ß to Mv1Lu cells (growth inhibited by TGF-ß) had no discernible effect on TGIF phosphorylation (Fig. 4A, left ). However, consistent with a model whereby TGF-ß–mediated growth arrest and cell proliferation are regulated through inverse effects on similar targets, TGF-ß treatment of AKR-2B cells (growth stimulated by TGF-ß) resulted in a significant increase in phosphorylated/stabilized TGIF (Fig. 4A, right).

TGIF phosphorylation by epidermal growth factor (EGF) has been shown to be controlled by Ras/ERK signaling (27). In agreement with that finding, addition of the MEK inhibitor PD098059 or dominant negative Ras abrogated the effect of TGF-ß on TGIF phosphorylation in AKR-2B cells (Fig. 4A and B). However, whereas inhibiting PI3K or its downstream target PAK2 abolished the stimulatory effect of TGF-ß on AKR-2B growth (Fig. 4C; refs. 9, 10), contrary to our expectations, inhibition of Ras activity had no effect on TGF-ß–stimulated proliferation (Fig. 4C). Thus, whereas stabilization of the Smad transcriptional corepressor TGIF may be critical for overcoming TGF-ß growth inhibition by diminishing p15Ink4B expression, it is not sufficient to account for the growth-promoting effects of TGF-ß in fibroblasts.

Integration of Ras and PAK2 in TGF-ß signaling. Because TGF-ß growth inhibition and proliferation are uniquely regulated, defining the proliferative signals and determining the nodes of intersection between the various pathways are critical to understanding this aspect of TGF-ß biology. To that end, we have recently shown that PI3K and PAK2 are spatially dependent targets activated within 15 min TGF-ß treatment of mesenchymal cells that have a crucial role in the induction of morphologic transformation, proliferation, and anchorage-independent growth (Fig. 4C and refs. 9, 10). As PI3K is a known Ras effector, we examined whether TGF-ß activation of Ras/ERK signaling was upstream, dependent on, and/or coupled to PI3K/PAK2 activity. To determine if Ras activity was required for PI3K-dependent signaling, AKR-2B cells were transduced with Ad-eGFP or Ad-His-RasN17 and assessed for Akt phosphorylation and PAK2 kinase activity following TGF-ß addition (Fig. 5A, right and middle ). Despite reducing TGF-ß–stimulated ERK1 and ERK2 phosphorylation to basal levels (Fig. 5A, left), dominant negative Ras had no detectable effect on either Akt or PAK2 activation by TGF-ß, indicating that Ras is not directly upstream.

PI3K has also been reported to be a Ras activator (20, 40); as such, the requirement for PI3K/PAK2 activity on Ras signaling was also examined. Interestingly, inhibition of PI3K with LY294002 or infection with a dominant negative PAK2 adenovirus abolished TGF-ß–stimulated ERK phosphorylation and Ras-dependent Elk-1 transcriptional activity (Fig. 5B and C). This requirement for PI3K and PAK2 activity in the TGF-ß stimulation of Ras signaling could indicate a role for these proteins in regulating Ras activation, per se, and/or within the downstream kinase cascade leading to ERK phosphorylation. That this control is not occurring at the level of Ras activation is shown in Fig. 5D. Cells were treated with the indicated reagents, and the increase in GTP-loaded Ras was determined. Although TGF-ß stimulated an approximately 3- to 3.5-fold increase in Ras activity, and Ad.RasN17 reduced this to basal, inhibiting PI3K or PAK2 with LY294002 or dominant negative Ad.PAK2K278R, respectively, had no significant effect.

The most proximal target downstream of Ras required for ERK phosphorylation is Raf. Raf activation is complex and depends on diverse interactions with various accessory and regulatory proteins (22). Moreover, group I PAKs have been shown to have a role in Raf activation by phosphorylating Ser338 (41–43). To determine whether PAK2 was fulfilling a similar requirement in TGF-ß signaling via Ras, the studies reported in Fig. 6A were done. As expected, TGF-ß stimulated both Raf Ser338 phosphorylation and kinase activity, and this was unaffected by the downstream MEK inhibitor PD098059 (Fig. 6A, compare lanes 1 and 2 with lanes 5 and 6). In addition, consistent with the data presented in Fig. 5B and C, inhibition of either PI3K or PAK2 activity abolished both responses to a similar extent as dominant negative Ad.RasN17 (Fig. 6A, compare lanes 1 and 2 with lanes 4, 10, and 12). Based on these observations, we propose a model whereby Ras signaling is activated by TGF-ß in mesenchymal cells independent of the Smad and PI3K pathways (Fig. 6B and refs. 9, 10). Although no condition for integrating with Smad signaling has currently been identified, PI3K-dependent PAK2 activity is coupled to the Ras cascade because it is necessary for TGF-ß activation of Raf and subsequent ERK-dependent transcriptional responses. Although it is currently unknown whether there are additional nodes of intersection, what is clear, however, is the need for crosstalk between a number of signaling pathways to mediate the diverse biological response generated by TGF-ß.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Identification and characterization of the Smad proteins have been critical to understanding a large extent of TGF-ß action. However, when one considers the diversity of phenotypes controlled by TGF-ß, it would seem reasonable that additional Smad-independent pathways are also operative. Although it is difficult to completely separate Smad-dependent from Smad-independent signaling (7), a number of responses have been reported to occur independent of primary Smad regulation (7, 9). One such pathway that has received a significant amount of attention is MAPK signaling following Ras activation (6, 18).

TGF-ß has been reported to stimulate, inhibit, and/or have no effect on the Ras/ERK pathway (6, 31, 44, 45). A possible explanation to account for such divergent findings is that a variety of cell types or differentiation models have been employed. One means by which that issue might be more clearly addressed is to identify/characterize TGF-ß pathways that function in a cell type–specific manner. For instance, we have determined that PI3K represents a branch point in the mesenchymal cell response to TGF-ß (9). Following the addition of TGF-ß to a subset of fibroblast (but not epithelial) cultures, PI3K activity is required for independent activation of PAK2 and Akt (9, 10). Although the targets downstream of Akt are currently under investigation, PAK2 is downstream of PI3K, upstream of the c-Abl nonreceptor tyrosine kinase, and essential for TGF-ß–stimulated proliferation and morphologic transformation (8, 9, 11). Because Ras and PI3K signaling have been coupled in a number of models, we wished to address the general question whether Ras would show a similar cell tropism and association with PI3K in TGF-ß signaling. To that end, initial studies directly examined TGF-ß–stimulated Ras GTP loading in both mesenchymal and epithelial cell lines (Fig. 1A). Similar to what we had observed for PI3K, each of the mesenchymal cultures (but not epithelial) showed Ras activation within minutes of TGF-ß addition. These kinetics are consistent with that observed for other Ras activators (46) and support a direct interaction of the TGF-ß receptor complex on Ras. Although it is currently unknown how TGF-ß receptor/Ras coupling occurs, it was further shown that this interaction leads to the activation of the MAPK cascade and subsequent stimulation of an ERK-dependent luciferase reporter independent of Smad2 or Smad3 (Figs. 1–3). Although we did not observe Ras or ERK activation by TGF-ß in a number of epithelial cultures (Fig. 1 and data not shown), this finding should not be extended beyond the cell types studied. Similarly, the biological phenotype being assessed needs to also be considered as ERK responses in the context of a TGF-ß–driven EMT may be reflecting the "mesenchymal transition" in contrast to an inherent property of epithelia (29, 31, 45).

The relation between TGF-ß activation of the Smad pathway and Ras/ERK signaling is complex. Although it is clear that the linker region of R-Smads can be phosphorylated by ERK, the functional consequence of that phosphorylation is not straightforward. For instance, linker phosphorylation has been shown to prevent (26) as well as enhance Smad nuclear translocation (30, 47). Although we did not directly investigate the relation between Smad linker region phosphorylation and nuclear translocation, in the mesenchymal cell models used where Ras and Smad signaling are both activated, these responses seem to be independently regulated (Fig. 3). However, whereas the results suggest that Ras has no direct effect on the Smad pathway, this should not be extended beyond the cells and biological end points studied.

The majority of studies investigating TGF-ß action have focused on the growth inhibitory actions of TGF-ß in epithelial cultures (4, 5). Because Ras activity is primarily associated with cell proliferation, it was of interest to investigate whether there was a reciprocal relation between the mechanisms associated with TGF-ß growth inhibition and growth stimulation. One such target reported to modulate the sensitivity of cells to TGF-ß growth inhibition is the Smad transcriptional corepressor TGIF (27). Stabilization of TGIF by Ras/ERK phosphorylation in response to EGF has been shown to favor the formation of a Smad2-TGIF corepressor complex (resulting in diminished TGF-ß growth inhibition), and the E3 ubiquitin ligase Tiul1 can associate with TGIF to target Smad2 for degradation (27, 48). As we were investigating cytokine activity in a cell context where TGF-ß both stimulated cell proliferation and induced Ras/ERK signaling, we addressed two general questions: first, would TGF-ß (similar to EGF) promote TGIF phosphorylation/stabilization under conditions where it is growth promoting; and second, if TGF-ß signaling induced TGIF phosphorylation, would preventing that response be associated with inhibiting TGF-ß proliferation? Although TGF-ß treatment resulted in Ras-dependent TGIF phosphorylation and p15Ink4b luciferase expression (Fig. 4A and B and data not shown), preventing TGIF phosphorylation by dominant negative Ras (i.e., avoiding the loss of growth-repressive targets such as p15Ink4B) was not sufficient to overcome TGF-ß–stimulated proliferation (Fig. 4B and C). Although the lack of association between TGIF phosphorylation and TGF-ß proliferation was unexpected given previous data relating TGIF to TGF-ß growth inhibition and p15Ink4b mRNA up-regulation (27), it clearly indicates that cell context–dependent responses of TGF-ß are regulated in a variety of distinct as well as overlapping levels.

Over the past few years, we have been developing a model of TGF-ß signaling that distinguishes the growth-promoting from the growth-inhibitory aspects of TGF-ß action (8–10). A central component of this model is PI3K (Fig. 6B and ref. 9). Addition of TGF-ß to mesenchymal cultures results in PI3K-dependent phosphoinositide production and the generation (to the best of our understanding) of two independent branches leading to either Akt phosphorylation or PAK2 and c-Abl activation (9, 11). Because TGF-ß–stimulated Ras/ERK activation displayed a similar cell tropism and Smad independence as PI3K, we next investigated whether it represented a unique signaling target and/or was integrated within the PI3K pathway(s). Although the inhibition of Ras had no effect on PAK2 activity or Akt phosphorylation (Fig. 5A), preventing PI3K or PAK2 signaling with LY294002 or dominant negative PAK2, respectively, abrogated TGF-ß induced ERK phosphorylation and Elk-1 luciferase activity (Fig. 5B and C). However, inhibiting Akt responses with Akt inhibitor II (SH-5), IV, or V had no discernible effect on ERK phosphorylation (data not shown). Because Ras activation is independent of PI3K or PAK2 (Fig. 5D), these findings are consistent with the hypothesis (a) that Ras and PI3K represent distinct arms downstream of the TGF-ß receptor; and (b) of crosstalk between Ras and PI3K signaling at the level of PAK2 for propagating the TGF-ß signal(s). Further evidence in support of that proposal was the demonstration that infection with dominant negative PAK2 prevents Raf activation following TGF-ß treatment (Fig. 6A). Because a similar requirement for group I PAKs in PI3K-dependent Raf phosphorylation has been reported previously (41, 43, 49), the results suggest that PAK proteins represent a common intermediary that integrates Ras and PI3K signaling downstream of numerous effectors.

It was unexpected that inhibition of Ras/ERK signaling with dominant negative Ras did not prevent TGF-ß–stimulated cell proliferation (Fig. 4C). Because this response is dependent on signals downstream of PI3K (8–10), it is clear that a direct linear relation between Ras and PI3K is not operative. However (as discussed above), although independently initiated, these pathways intersect in the PAK2 requirement for activation of Raf and subsequent ERK signaling. A model depicting these findings is presented in Fig. 6B. Although it is unknown whether there are additional junctures between PI3K and Ras or Smad signaling, the absence of detectable effects of Ras on TGF-ß–stimulated proliferation provides further evidence that distinct signaling programs control normal and deregulated growth. Moreover, because an activated stroma plays a critical role in regulating tumor development and/or progression (32, 33), the current study further documents that understanding TGF-ß action in both the "seed and the soil" (50) is essential for effective intervention strategies.


   Acknowledgments
 
Grant support: Public Health Service Grants GM54200 and GM55816 from the National Institutes of General Medical Sciences and the Mayo Foundation.

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. Anita Roberts for the Smad3–/– MEF cell line, Dr. Philip Howe for TGF-ß2, and Elizabeth Bruinsma for assistance in the early phases of the study.


   Footnotes
 
Note: K. Suzuki and M.C. Wilkes contributed equally to this work.

Current address for N. Garamszegi: Department of Orthopaedics and Rehabilitation, University of Miami School of Medicine, 1400 NW 12th Ave, East Building 4th Floor, Miami, FL 33136.

Received 8/30/06. Revised 2/ 2/07. Accepted 2/14/07.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online