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Molecular and Cellular Biology, January 2006, p. 50-62, Vol. 26, No. 1
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.1.50-62.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Transactivation of Platelet-Derived Growth Factor Receptor by the GTPase-Deficient Activated Mutant of G₁₂

Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Received 12 September 2005/ Accepted 12 October 2005

	ABSTRACT

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The GTPase-deficient, activated mutant of G₁₂ (G₁₂Q229L, or G₁₂QL) induces neoplastic growth and oncogenic transformation of NIH 3T3 cells. Using microarray analysis, we have previously identified a role for platelet-derived growth factor receptor (PDGFR) in G₁₂-mediated cell growth (R. N. Kumar et al., Cell Biochem. Biophys. 41:63-73, 2004). In the present study, we report that G₁₂QL stimulates the functional expression of PDGFR and demonstrate that the expression of PDGFR by G₁₂QL is dependent on the small GTPase Rho. Our results indicate that it is cell type independent as the transient expression of G₁₂QL or the activation of G_12-coupled receptors stimulates the expression of PDGFR in NIH 3T3 as well as in human astrocytoma 1321N1 cells. Furthermore, we demonstrate the presence of an autocrine loop involving PDGF-A and PDGFR in G₁₂QL-transformed cells. Analysis of the functional consequences of the G₁₂-PDGFR signaling axis indicates that G₁₂ stimulates the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway through PDGFR. In addition, we show that G₁₂QL stimulates the phosphorylation of forkhead transcription factor FKHRL1 via AKT in a PDGFR- and PI3K-dependent manner. Since AKT promotes cell growth by blocking the transcription of antiproliferative genes through the inhibitory phosphorylation of forkhead transcription factors, our results describe for the first time a PDGFR-dependent signaling pathway involving PI3K-AKT-FKHRL1, regulated by G₁₂QL in promoting cell growth. Consistent with this view, we demonstrate that the expression of a dominant negative mutant of PDGFR attenuated G₁₂-mediated neoplastic transformation of NIH 3T3 cells.

	INTRODUCTION

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Heterotrimeric G proteins regulate diverse cellular responses by coupling heptahelical receptors to intracellular effectors (11, 25, 35). Of the different -subunits that have been analyzed thus far, the -subunit of the heterotrimeric G protein G12 (G₁₂) shows the most potent mitogenic and oncogenic activities (5, 35). The initial identification of G₁₂ as the transforming oncogene in Ewing's sarcoma cell lines indicated the critical role of G₁₂ in oncogenic signaling (5). Consistent with these observations, serum stimulation or mutational activation of G₁₂ has been shown to stimulate mitogenic pathways in different cell types in addition to inducing neoplastic transformation of Rat-1a and NIH 3T3 fibroblasts (11, 35). NIH 3T3 cells transformed by the GTPase-deficient activated mutant of G₁₂ (G₁₂Q229L, or G₁₂QL) show the characteristic oncogenic phenotype defined by the increased proliferation, anchorage-independent growth, reduced growth factor dependency, attenuation of apoptotic signals, and neoplastic cytoskeletal changes (35). Previously, we have shown that the neoplastic growth of NIH 3T3 cells mediated by the activated mutant G₁₂ involves the expression several unique genes, including platelet-derived growth factor receptor (PDGFR) (25). This finding is of critical interest since the signaling pathways involving PDGFR have been strongly correlated with cell proliferation and neoplastic transformation (18), thus pointing to a possible role of PDGFR in G₁₂QL-mediated oncogenic signaling pathways.

The receptor kinases PDGFR and PDGFRß are activated by dimers of PDGF isoforms, PDGF-A, PDGF-B, PDGF-C, and PDGF-D (18). Distinct homo- or heterodimers of PDGFs activate specific homo- or heterodimers of PDGFR and PDGFRß in a tissue-specific and context-specific manner (18). The activation of PDGFRs, leading to their dimerization and transphosphorylation at specific tyrosine residues, provides docking sites for different effector molecules such as Shp, phospholipase C-gamma, and phosphatidylinositol 3-kinase (PI3K) (18). Overexpression of PDGFR or PDGFRß and the associated autocrine signaling pathways appear to play an etiological role in tumorigenesis and tumorangiogenesis of different neoplasms including basal cell carcinoma (45), gastrointestinal stromal tumors (17), and ovarian cancers (19, 30). It has also been observed that PDGFR, specifically PDGFRß, is transactivated by G protein-coupled receptors (GPCRs) stimulated by lysophosphatidic acid (LPA) (20), sphingosine-1 phosphate (2), serotonin (33), and angiotensin II (39). However, the underlying signaling mechanisms and the role of the specific G proteins in mediating such transactivation are not fully understood.

In the present study, we demonstrate that G₁₂QL stimulates the expression and transactivation of PDGFR in NIH 3T3 cells. The ability of transiently expressed G₁₂QL to stimulate the expression of PDGFR in 1321N1 astrocytoma cells indicates that such a nexus between G₁₂ and PDGFR is not restricted to one specific cell type. Our results indicate that the expression of PDGFR stimulated by G₁₂QL involves the small GTPase Rho-dependent signaling pathway. We further determine that the transactivation of PDGFR by G₁₂QL involves an autocrine signaling loop involving PDGF-A. Transactivation of PDGFR by G₁₂ leads to the activation of PI3K and AKT-kinase (AKT), with the resultant inhibitory phosphorylation of forkhead transcription factors such as FKHRL1. This, in turn, leads to a decrease in the binding of FKHRL1 to its response elements. We also show that the coexpression of a dominant negative mutant of PDGFR inhibits neoplastic transformation of NIH 3T3 cells induced by the activated mutant of G₁₂. Taken together with the observation that the inhibition of FKHRL1 leads to an increase in cell proliferation and survival (29), our data presented here identify a novel PDGFR-dependent signaling pathway activated by G₁₂ in promoting neoplastic growth via the PI3K-AKT-FKHRL signaling conduit.

	MATERIALS AND METHODS

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Cell lines, plasmids, and transfection. Parental NIH 3T3 cells and the previously described pcDNA3-NIH 3T3 and G₁₂Q229L-NIH 3T3 cell lines were maintained as previously described (44). 1321N1 astrocytoma cells were kindly provided by Stephen Cosenza (Fels Institute, Temple University, PA) and were maintained in Dulbecco's modified Eagle's medium (Cellgro, NJ) containing 5% fetal bovine serum. A C terminus-truncated dominant negative mutant of PDGFR in pLXSN2 vector (22) was a kind gift from Andrius Kazlauskas (Schepens Eye Research Institute, Harvard Medical School, Boston, MA). The cDNA insert encoding truncated PDGFR was excised from pLXSM2 vector and shuttled into pcDNA3(+) vector at NotI-BamHI sites. Transient expression of G₁₂QL and RhoA-N19 in NIH 3T3 cells was carried out by transfecting the cells (0.7 x 10⁶ cells/60-mm dish) with appropriate expression vectors using Lipofectamine Plus reagent (Invitrogen Technologies, CA). Transient expression using the adenoviral vectors encoding G₁₂QL, RhoA-V14, or RhoA-N19 was carried out by infecting cells (0.5 x 10⁶ cells/60-mm dish) with a multiplicity of infection of viral particles of 600. Transient expression of different constructs in 1321N1 astrocytoma cells was carried out using the Fugene 6 reagent (Roche Diagnostics, IN) according to the manufacturer's protocol. Cells were collected and lysed with modified RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM sodium fluoride, 1 mM sodium vanadate, 2 µg/ml leupeptin, 4 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) at 24 h posttransfection.

Construction of the adenoviral vectors expressing G₁₂QL. A cDNA insert encoding G₁₂QL (1,800 bp) was excised from pcDNA3-G₁₂QL vector (44) using the restriction nucleases KpnI and XbaI. The cohesive ends were blunted and cloned in the EcoRV site of pShuttle-IRES-GFP2 vector (Stratagene, CA) (where IRES is internal ribosome entry site and GFP is green fluorescent protein). The resultant plasmid was linearized using PmeI before it was transformed into Escherichia coli BJ5183, in which the homologous recombination event with the plasmid containing adenoviral backbone takes place. The recombinant clones were selected by analysis of the PacI-digested DNA from these clones. The positive recombinant DNA was amplified by transforming it on a suitable E. coli strain. The recombinant DNA was cut with PacI and then purified before transfection onto AD293 cells for the virus production. Isolation of virus was carried out by following the standard freeze-thaw protocol. In some instances, further amplification of the virus was carried out by infecting AD293 cells. These recombinant adenoviruses were titrated by plaque assay before target cells were infected. Recombinant adenoviral vectors expressing the constitutively activated RhoA-G14V (RhoA-V14) and the dominant negative RhoA-T19N (RhoA-N19) mutants were kindly provided by Aviv Hassid, University of Tennessee, Memphis, TN (6).

Cell proliferation assay. The CyQUANT Cell proliferation assay kit (Molecular Probes, Inc., Eugene, OR) was used to monitor cell proliferation. Equal numbers of cells (5 x 10³ cells/well) were grown in 96-well plates for 24 h and then serum starved for 24 h. The cells were then incubated with the CyQUANT reagent as described by the manufacturer, and fluorescence was monitored using a microplate reader with 485-nm excitation and 535-nm emission filters. A reference standard curve was created as described by the manufacturer for converting the sample fluorescence values into cell numbers.

Semiquantitative RT-PCR. An aliquot of the total RNA (2 µg) was converted into cDNA using a ThermoScript RT-PCR System (Invitrogen Life Technologies, CA). The reverse-transcribed cDNA was subjected to PCR analysis. The following primers were used for the semiquantitative reverse transcription-PCR (RT-PCR): PDGF-A, 5'-GAGATACCCCGGGAGTTGAT and 3'-CTGTCTCCTCCTCCCGATG; PDGF-B specific, 5'-ATCGCCGAGTGCAAGACG and 3'-TCCGAATGGTCACCCGAG; PDGF-C specific, 5'-ACAAGGAACAGAACGGAGT and 3'-TCAGATACAAATCTTATCCT. The PCR conditions were 2 min of denaturation at 94° followed by cycles of denaturation at 94° for 30 s, annealing at 58° for 1 min, and elongation at 72° for 1 min. To define the optimal number of PCR cycles for linear amplification, PCR products were removed at the end of 20, 24, and 28 cycles. The mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific forward (5'-GTGAAGGTCGGTTGTGAACGG-3') and reverse (5'-GATGCAGGGATGATGTTCTG-3') primers were used as controls. The amplification products were analyzed by 1% agarose gel electrophoresis.

Immunoprecipitation and immunoblot analysis. Immunoprecipitation of PDGFR was carried out by incubating cell lysate protein (1 mg each) with 1 µg of PDGFR antibodies for 16 h at 4°C, followed by incubation with 20 µl of 50% protein A-Sepharose beads (Amersham Biosciences Corp., Piscataway, NJ) for 2 h at 4°C. After repeated washes with cell lysis buffer, the immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto polyvinylidene difluoride membranes for immunoblot analysis. Immunoblot analyses of lysate or immunoprecipitated proteins were carried out according to previously published procedures (5). Antibodies to G₁₂ (sc-409), PDGFR (sc-338), PDGFRß (sc-432), PDGF-A (sc-7958), ROCK-1 (sc-6056), and AKT (sc-1618) were purchased from Santa Cruz Biotechnology, Inc., CA. Antibodies to phospho-FKHRL1-Thr32 (no. 06-952), FKHRL1 (no. 6-951) and phosphotyrosine (clone 4G10, no. 05-321) were purchased from Upstate Signaling Solutions (Charlottesville, VA). Antibodies to GAPDH (no. 4300) and p-AKT Ser473 (no. 9271) were purchased from Ambion, Inc. (Austin, TX) and Cell Signaling Technology, Inc. (Beverly, MA), respectively. Peroxidase-conjugated anti-mouse immunoglobulin G and rabbit immunoglobulin G were purchased from Amersham Biosciences UK, Ltd. (Buckinghamshire, England) and Promega (Madison, WI), respectively.

Rho activation assay. Bacterial expression vector pGEX-2T encoding a glutathione S transferase (GST)-fused Rho-binding domain (GST-RBD) of rhotekin (amino acids 8 to 89) was kindly provided by Gary Bokoch, Scripps Research Institute, La Jolla, CA. GTP-bound Rho was precipitated using GST-RBD according to previously published methods (3, 36, 38). Briefly, cells were lysed in Rho-binding buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl₂, 1 mM EDTA, 10% glycerol, and protease inhibitor cocktail), and the clarified cell lysates were incubated with GST-RBD-bound glutathione-Sepharose 4B beads (30 µl of 50% slurry) at 4°C for 20 min. The beads were washed four times with ice-cold Rho-binding buffer and resuspended in Laemmli's sample buffer. The pulled-down Rho-GTP was identified by immunoblot analysis using Rho-A antibodies.

PDGFR immunocomplex kinase assay. An immunocomplex kinase assay to monitor the kinase activity of PDGFR was carried out using previously published methods with appropriate modifications (46). To monitor the autophosphorylating kinase activity of PDGFR, PDGFR was immunoprecipitated from cell lysates using antibodies to PDGFR (as described above). After repeated washes, the immunoprecipitates were resuspended in 50 µl of kinase buffer (25 mM HEPES, pH 7.4, 5 mM MgCl₂ and 0.2 mM EDTA) and subjected to an auto-kinase assay by incubating them with 20 µM [-³²P]ATP (5,000 cpm/pmol) for 30 min at 30°C. The phosphorylated proteins were separated by SDS-PAGE, followed by autoradiography.

p160ROCK immunocomplex assay. p160ROCK was immunoprecipitated from 50 µg of cell lysates using antibodies to p160ROCK (sc-6056). The immunoprecipitates were washed twice with the lysis buffer (25 mM HEPES pH 7.6, 0.1% Triton X-100, 300 mM NaCl, 20 mM ß-glycerophosphate, 1.5 mM MgCl₂, 0.2 mM EDTA, 2 µM dithiothreitol [DTT], 1 mM sodium vanadate, 2 µg/ml leupeptin, 4 µg/ml aprotinin) followed by two washes with reaction buffer (20 mM HEPES, pH 7.6, 20 mM ß-glycerophosphate, 1 mM MgCl₂, and 1 mM sodium vanadate). The kinase reaction was carried out by resuspending the immunoprecipitates in 40 µl of reaction buffer containing 20 µM [-³²P]ATP (5,000 cpm/pmol) and 5 µg of myelin basic protein (Sigma-Aldrich, St. Louis, MO) as a substrate according to previously published procedures (26). The reaction mixture was incubated for 30 min at 30°C. The phosphorylated myelin basic protein bands were visualized by SDS-PAGE, followed by autoradiography. The radioactive myelin basic protein bands were excised and quantified in a liquid scintillation counter.

Immunocomplex AKT assay. AKT was immunoprecipitated from 500 µg of cell lysates using antibodies to AKT (sc-1618). The immunoprecipitates were washed twice with the lysis buffer (25 mM HEPES, pH 7.6, 0.1% Triton X-100, 300 mM NaCl, 20 mM ß-glycerophosphate, 1.5 mM MgCl₂, 0.2 mM EDTA, 2 µM DTT, 1 mM sodium vanadate, 2 µg/ml leupeptin, 4 µg/ml aprotinin), followed by two washes with reaction buffer (20 mM HEPES, pH 7.6, 20 mM ß-glycerophosphate, 1 mM MgCl₂, and 1 mM sodium vanadate). The kinase reaction was carried out by resuspending the immunoprecipitates in 40 µl of reaction buffer containing 20 µM [-³²P]ATP (5,000 cpm/pmol) and 2 µg of purified recombinant FKHR protein (14-343; Upstate, NY) as a substrate. The reaction was incubated for 30 min at 30°C. The phosphorylated FKHR bands were visualized by SDS-PAGE, followed by autoradiography. The radioactive FKHR bands were excised and quantified in a liquid scintillation counter.

PI3K assay. PI3K assays were carried out according to published procedures (34). Cell lysates were prepared by lysing the cells in PI3K lysis buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, MgCl₂, 5 mM, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 4 µg/ml aprotinin) for 20 min at 4°C and removing the cell debris by centrifuging at 18,000 x g for 10 min. PDGFR was immunoprecipitated from 200 µg of lysate protein (as described above). The immunoprecipitates were sequentially washed with PI3K lysis buffer, followed by three washes with PI3K lysis buffer lacking Triton X-100. The PI3K reaction was carried out by resuspending the PDGFR immunocomplex in 35 µl of PI3K reaction buffer (25 mM HEPES, pH 7.4, 5 mM MgCl₂, and 0.2 mM EDTA) and incubating with 50 µM ATP, 5 µCi of [-³²P]ATP, and 5 µg of phosphotidylinositol plus 5 µg of phosphoserine for 5 min at 25°C. The reaction was stopped by the addition of 300 µl of CH₃OH · 1 N HCl (1:1). The phosphorylated inositols were separated on a 1% potassium oxalate-coated thin-layer chromatography (TLC) plate using CHCl₃ · CH₃OH · 4 M NH₄OH (9:7:2) as the developing solvent (34).

Electrophoretic mobility shift assay. Nuclear extracts from pcDNA3-and G₁₂QL-NIH 3T3 cells were prepared according to previously published procedures (29). The oligonucleotides used were the following: forward, 5'-TTAAATAAATAAGTAAATAAATAAAC-3'; reverse, 5'-GTTTATTTATTTACTTATTTATTTAA-3'. The annealed double-stranded oligonucleotides (25 pmol) were end labeled with 20 µM [-³²P]ATP (5,000 cpm/pmol) and purified by G-25 spin columns (Amersham Pharmacia, MA). A total of 5 µg of nuclear extracts was mixed with 1 µg of salmon sperm DNA (Gibco BRL, CA) and radiolabeled probes (50,000 cpm) with or without unlabeled competitor probes. For supershift assays, nuclear extracts were preincubated with 10 µg of FKHRL1-antibodies (06-951) from Upstate Signaling Solutions, (Charlottesville, VA) for 30 min at 25°C prior to the labeled probes. DNA-protein complexes were resolved on a 5% nondenaturing PAGE gel in 0.25x TBE buffer (50 mM Tris, 50 mM boric acid, and 1 mM EDTA). The gels were dried and autoradiographed.

Focus formation assay. An NIH 3T3 cell focus formation assay was carried out as previously described (43). Parental NIH 3T3 cells were transfected with pcDNA3 vectors encoding G₁₂QL (5 µg) with or without dominant negative PDGFR (DN-PDGFR; 5 µg) using the calcium phosphate transfection method. The control group included transfections with empty pcDNA3 vector (10 µg). Transfected NIH 3T3 cells were cultured in the presence of 5% serum, and transformed foci were stained and scored after 14 days.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activated mutant of G₁₂ stimulates cell proliferation and PDGFR expression. Expression of constitutively activated mutants of G₁₂ stimulates proliferation in many cell types including NIH 3T3 cells (35). NIH 3T3 cells overexpressing G₁₂QL (G₁₂QL-NIH 3T3) show increased proliferation even under reduced serum growth conditions (Fig. 1A). Since our previous transcriptional profiling data of G₁₂QL-NIH 3T3 cells has indicated a role for PDGFR in G₁₂QL-mediated oncogenic growth of NIH 3T3 cells (25), we analyzed the expression of PDGFR in G₁₂QL-NIH 3T3 cells. Results from such analyses indicated that the protein levels of PDGFR are increased by fourfold in G₁₂QL-NIH 3T3 cells compared to the pcDNA3-NIH 3T3 vector control cells (Fig. 1B). Next, we examined the possibility that the observed increased expression of PDGFR was due to the presence of G₁₂QL and not to the nonspecific phenotype of the transformed or highly proliferating cells. NIH 3T3 cells transformed by activated G₁₃ (G₁₃QL) can be used to assess this possibility, since the activated mutant of G₁₃, which shares 67% amino acid identity with G₁₂, has been shown to transform NIH 3T3 cells readily through signaling pathways distinctly different from those of G₁₂ (36). With this view, we analyzed the expression of PDGFR in NIH 3T3 cells transformed by the activated mutant of G_13. Results from such analysis indicated that the increased expression of PDGFR is observed only in cells transformed by G₁₂QL (Fig. 1C). Thus, our results demonstrate that the increased expression of PDGFR is not a generic function of a transformed or highly proliferating cellular phenotype but, rather, is due to specific pathways activated by G₁₂QL.

View larger version (41K): [in this window] [in a new window]   FIG. 1. Activation of G12 stimulates the expression of PDGFR in NIH 3T3 cells. (A) G12QL mediates serum-independent growth of NIH 3T3 cells. Vector control and G12QL-transformed NIH 3T3 cells (4 x 105) were plated on 100-mm plates. Lysates (100 µg) from these cells were subjected to immunoblot analysis with antibodies to G12 for the overexpression of G12QL and also stripped and reprobed with GAPDH to monitor equal loading (top). Proliferation of these cells in serum-free medium was monitored as described under in Materials and Methods (bottom). (B) G12QL mediates increase in PDGFR expression. Lysates from 24-h serum-starved pcDNA3- and G12QL-NIH 3T3 cells were subjected to immunoblot analysis using antibodies to PDGFR (top). The expression levels were quantified using Kodak 1D image analysis software. Results are presented as the increase in expression (n-fold) over the vector control cells (mean ± SEM; n = 10). (C) Activated G12, not G13, stimulates the expression of PDGFR. pcDNA3-NIH 3T3 cells, G12QL-NIH 3T3 cells, and G13QL-NIH 3T3 were plated (1 x 106 cells per 100-mm culture dish) and allowed to grow for 24 h, after which the cells were serum starved for 24 h. Lysates were prepared and subjected to SDS-PAGE. Immunoblot analyses were carried out using anti-PDGFR. Immunoblot analysis was carried out with antibodies to G12 or G13 to monitor the expression of the respective -subunits. The blot was stripped and reprobed with GAPDH to monitor equal protein loading. Results are from a representative experiment (n = 3). (D) PDGFR levels increase upon stimulation of WT G12 (G12WT) with LPA. pcDNA3-NIH 3T3 cells, G12QL-NIH 3T3 cells, and G12WT-NIH 3T3 cells and were plated (1 x 106 cells per 100-mm culture dish) and allowed to grow for 24 h. The cells were then serum starved for 24 h and stimulated with 20 µM LPA. After 6 h, the lysates were prepared and subjected to SDS-PAGE. Immunoblot analyses were carried out using anti-PDGFR. The blot was stripped and reprobed with antibodies to G12 and GAPDH to monitor G12 expression and equal protein loading, respectively. Results from a typical (n = 3) experiment are presented. IB, immunoblot.

G₁₂QL stimulates the expression of PDGFR through Rho. We next investigated the signaling mechanism that couples G₁₂ to PDGFR expression and associated transactivation. Studies from our laboratory as well as others have shown that G₁₂ regulates diverse sets of signaling pathways primarily through the activation of the Rho family of GTPases (11). It has been observed that G₁₂QL stimulates the expression of many different growth-promoting genes through the signaling pathways regulated by the small GTPase Rho (14). In this context, it is interesting that PDGFR promoter contains several Rho-responsive elements such as NF-B, GATA, CCAAT, and AP-1 sites (24). Therefore, we examined whether the upregulation and subsequent transactivation of PDGFR by G₁₂QL involved a Rho-dependent signaling pathway. G₁₂QL-NIH 3T3 cells were treated with 10 µM Y27632, an inhibitor of the Rho effector kinase p160ROCK for 16 h (conditions which effectively block the p160ROCK enzymatic activity [23]), and the expression as well as the phosphorylation levels PDGFR was monitored. The ability of Y27632 to inhibit p160ROCK in these lysates was also monitored using an in vitro p160ROCK assay. Results indicated that Y26732 treatment completely inhibited a G₁₂QL-mediated increase in PDGFR levels (Fig. 2A), thus pointing to a role for the Rho-p160ROCK signaling pathway in the transactivation of PDGFR by G₁₂QL.

View larger version (38K): [in this window] [in a new window]   FIG. 2. G12QL stimulation of PDGFR-expression involves Rho-dependent signaling pathway. (A) G12QL stimulates the expression of PDGFR through Rho kinase p160ROCK. G12QL-NIH 3T3 and pcDNA3-NIH 3T3 cells were serum starved for 24 h. Prior to lysis, the cells were treated with the p160ROCK inhibitor, Y27632 (20 µM), for 16 h along with nontreated controls. The lysates (50 µg) were subjected to immunoblot analysis with anti-PDGFR antibodies. The blot was also reprobed with anti-GAPDH to monitor equal loading of proteins. The ability of Y26732 to inhibit p160ROCK was monitored using a p160ROCK immunocomplex kinase assay as described in Materials and Methods using myelin basic protein (MBP) as a substrate. The phosphorylated myelin basic protein was separated by 12% SDS-PAGE and visualized by autoradiography. (B) G12QL requires functional Rho for the expression of PDGFR. NIH 3T3 cells were infected with the adenoviral expression vectors encoding G12QL, G12QL plus three-HA-tagged N19-RhoA (where HA is hemagglutinin) or HA-tagged V12-RhoA (multiplicity of infection of 600). After 24 h, the lysates from these cells (100 µg) were subjected to immunoblot analysis with antibodies to PDGFR. The blot was further probed with antibodies to G12 and HA epitope to monitor the expression of the exogenous cDNAs. Please note that the difference in the mobility of RhoA-N19 is due to the three-HA epitope. Rho-inhibition by RhoA-N19 and the constitutively activated status of RhoA-V14 mutants were monitored by a Rho activation assay using a GST-RBD binding assay as described in Materials and Methods. The blot was reprobed with GAPDH antibodies to monitor equal loading of proteins. IB, immunoblot.

Activated mutant of G₁₂ stimulates the activity of PDGFR. Activation of PDGFR involves ligand-dependent dimerization of the receptor tyrosine kinase followed by the transphosphorylation of specific tyrosine residues (18). Therefore, we sought to investigate whether the G₁₂QL-upregulated PDGFR undergoes such activation. Analyses of the expression levels and the tyrosine phosphorylation profiles of PDGFR in G₁₂QL-NIH 3T3 cells indicated that G₁₂QL stimulated the expression as well as phosphorylation of PDGFR in these cells (Fig. 3A). The phosphorylation levels of PDGFR in relation to its expression levels were increased by 60% ± 9% (mean ± standard error of the mean [SEM]; n =6) over control values (P = 0.01). Since previous studies have shown that such an increase in the phosphorylation levels of PDGFR can elicit mitogenic responses in different cell types (32, 42), our results identify PDGFR as a novel player in G₁₂-mediated mitogenic signaling. In this context, it is of interest to analyze whether G₁₂ also stimulates the expression and activation of closely related PDGFRß. Lysates from cells expressing G₁₂QL were subjected to parallel immunoblot analysis with antibodies to PDGFR and PDGFRß. Results from these studies showed that G₁₂QL had no effect on PDGFR expression or activity (Fig. 3B), thus clearly establishing the ability of G₁₂ to specifically stimulate and transactivate PDGFR (Fig. 3A) and not PDGFRß (Fig. 3B).

View larger version (34K): [in this window] [in a new window]   FIG. 3. G12 and GPCRs that activate G12 stimulate the expression and transactivation of PDGFR. (A) G12QL stimulates the expression and activation of PDGFR. Lysates (500 µg) from G12QL-NIH 3T3 cells along with vector control were immunoprecipitated with antibodies to PDGFR and subjected to immunoblot analysis with antibodies to P-Tyr. After stripping, the blot was sequentially reprobed with PDGFR and GAPDH. (B) G12QL does not stimulate the expression of PDGFRß. Lysates (500 µg) from G12QL-NIH 3T3 cells along with vector control were subjected to immunoprecipitation with antibodies to PDGFRß, followed by immunoblot analysis with antibodies to P-Tyr. The blot was stripped and reprobed with PDGFRß and GAPDH. (C) Transient expression of G12QL stimulates the expression and transactivation of PDGFR in NIH 3T3 cells. NIH 3T3 cells were transfected with the expression vector pcDNA3 or pcDNA3 encoding G12QL (8 µg) using Lipofectamine Plus reagent. After 24 h, the lysates (100 µg) from the transfectants were subjected to immunoprecipitation using antibodies to PDGFR, following which an immunoblot analysis was carried out with antibodies to P-Tyr. The blot was stripped and reprobed with antibodies to PDGFR. The blot was further probed with antibodies to G12 and GAPDH to monitor G12QL-expression and equal loading, respectively. (D) Transient expression of G12QL stimulates the expression and transactivation of PDGFR in 1321N1 astrocytoma cells. 1321N1 astrocytoma cells were transfected with vectors encoding G12QL or pcDNA3 vector (5 µg) using Fugene 6 reagent. After 24 h, the lysates (100 µg) from the transfectants were subjected to immunoprecipitation using antibodies to PDGFR, following which an immunoblot analysis was carried out with antibodies to P-Tyr. The blot was stripped and reprobed with antibodies to PDGFR. The blot was stripped and reprobed with antibodies to G12 and GAPDH to monitor G12QL-expression and equal loading, respectively. (E) GPCR ligands, LPA and TRAP, stimulate the expression and transactivation of PDGFR in 1321N1 astrocytoma cells. 1321N1 astrocytoma cells were serum starved for 24 h. After starvation, the cells were stimulated with 20 µM LPA or 5 µM TRAP for 6 h. The cell lysates (100 µg) were subjected to immunoblot analysis with antibodies to P-Tyr. The blot was further reprobed with antibodies to GAPDH to monitor equal loading of proteins. IP, immunoprecipitate; IB, immunoblot.

Next, we investigated whether the observed expression of PDGFR stimulated by the activated mutant of G₁₂ is cell type dependent. Since it has been shown that G₁₂ plays a dominant role in the proliferation of astrocytoma cells (1, 7) and PDGFRs play a critical role in the growth of astrocytomas (12, 27), we analyzed the role of G₁₂ in the expression of PDGFR in astrocytoma cell line 1321N1. An activated mutant of G₁₂ was transiently expressed in astrocytoma cells using pcDNA3 vectors encoding G₁₂QL for 24 h, and the expression of PDGFR was monitored using immunoblot analysis. Results indicated that the transient expression of G₁₂QL in these cells stimulated the expression of PDGFR (Fig. 3D), further validating the ability of G₁₂QL to stimulate the expression of PDGFR in two distinctly different cell types.

Previous studies have shown that thrombin stimulates a mitogenic response in 1321N1 astrocytoma cells by coupling to G₁₂ (1). Microinjection of antibodies to G₁₂ blocked the thrombin-stimulated mitogenic response in 1321N1 astrocytoma cells, thus indicating that G₁₂ is critically required for thrombin-mediated proliferation of these cells (1). It has also been shown that LPA can stimulate the proliferation of these cells by coupling to G₁₂ (40). Therefore, we investigated whether LPA or thrombin stimulates the expression of PDGFR in these cells. 1321N1 astrocytoma cells were serum starved for 24 h, following which they were stimulated with 20 µM LPA or 5 µM thrombin receptor-activating peptide (TRAP) for 6 h. Lysates from these cells were subjected to immunoblot analysis using antibodies to PDGFR and P-Tyr. Results from such analysis indicated that activation of LPA as well as thrombin receptors by their cognate ligands stimulated the expression and tyrosine phosphorylation of PDGFR in 1321N astrocytoma cells (Fig. 3E). Together with the observation that LPA stimulates the expression of PDGFR in cells expressing WT G₁₂, (Fig. 1D), these results demonstrate that GPCRs that link to G₁₂ stimulate the expression and activation of PDGFR in two distinctly different cell types.

G₁₂QL-mediated neoplastic growth involves the PDGF-PDGFR autocrine loop. It has been observed that neoplastic transformation often involves the formation of autocrine signaling pathways through which the transformed cells become less dependent on exogenous growth factors (16). Such oncogene-specific autocrine loops involving different receptor tyrosine kinases including PDGFR have been well characterized (9, 41). To determine whether any of the known PDGFR ligands are involved in such an autocrine signaling loop stimulated by the activated mutant of G₁₂, we investigated the expression of the known PDGFR ligands, namely, PDGF-A, PDGF-B, and PDGF-C in G₁₂QL-expressing NIH 3T3 cells. Using RNAs isolated from G₁₂QL-NIH 3T3 cells along with the pcDNA3-vector controls, semiquantitative RT-PCR analyses were carried out to monitor the expression of the PDGF-A, PDGF-B, or PDGF-C. These results indicate that the expression of PDGF-A is increased in G₁₂QL cells by twofold in comparison with the vector control cells (Fig. 4A). In addition, a relatively modest increase in the expression of PDGF-C can also be seen with little or no change in the expression of PDGF-B.

View larger version (29K): [in this window] [in a new window]   FIG. 4. G12QL stimulates a PDGF-PDGFR-mediated autocrine loop. (A) Expression of PDGF-A and PDGF-C in G12QL-NIH 3T3 cells. Semiquantitative RT-PCR was carried out using total RNA in G12QL-NIH 3T3 and pcDNA3-NIH 3T3 cells with primers specific to PDGF-A, PDGF-B, and PDGF-C using conditions described in Material and Methods. RT-PCR using GAPDH-specific primers was used as loading control. (B) Expression of PDGF-A in G12QL-NIH 3T3 cells. Lysates from 24-h serum-starved pcDNA3- and G12QL-NIH 3T3 cells were subjected to immunoblot analysis using antibodies to PDGF-A and G12. The blot was reprobed with antibodies to GAPDH to monitor equal loading of proteins. (C) Effect of G12QL-CM on PDGFR phosphorylation. G12QL-NIH 3T3 cells and pcDNA3-NIH 3T3 cells (4 x 105 cells/100-mm plate) were grown for 16 h, followed by serum starvation for 24 h. The medium from pcDNA3-NIH 3T3 cells was replaced with CM from G12QL-NIH 3T3 cells and incubated for 10 min. Lysates from serum-starved pcDNA3-NIH 3T3 cells (lane 1), pcDNA3-NIH 3T3 cells treated with CM (lane 2), and pcG12QL-NIH 3T3 cells treated with CM in which PDGF-A was immunodepleted (lane 3) were subjected to immunoprecipitation with antibodies to PDGFR antibodies, followed by immunoblot analysis with antibodies to P-Tyr (top). The same blot was reprobed with antibodies to PDGFR after stripping (bottom). IB, immunoblot; Ab, antibody.

G₁₂-transactivated PDGFR stimulates the PI3K-AKT signaling pathway. In vitro autophosphorylating activity of PDGFR has been widely used as an index of its functional activation (46). To further demonstrate that the expressed receptors are functional, an in vitro immunocomplex auto-kinase assay was carried out using PDGFR immunoprecipitated with antibodies to PDGFR. Results from this analysis of the autophosphorylation of PDGFR indicated that there was an increase in the auto-kinase activity due to the transphosphorylation by PDGFR in G₁₂QL-NIH 3T3 cells (Fig. 5A). PDGFR stimulates a multitude of downstream effector molecules including PI3K, phospholipase C-gamma, Grb2/Sos1, Stats, Src, and SHP-2 (18). These molecules often integrate with each other in costimulating pathways involved in cell proliferation and cell survival while attenuating apoptotic pathways. While it is possible that the transactivation of PDGFR by G₁₂QL generates such multiple signaling inputs, the PDGFR-mediated activation of a PI3K-AKT signaling pathway is of great interest. It has been known that the pathways regulated by PI3K and AKT stimulate the expression of molecules involved in cell proliferation while downregulating the ones involved in apoptosis (29). Therefore, it can be speculated that G₁₂QL promotes serum-independent neoplastic cell growth by stimulating PI3K and AKT activities through PDGFR. To examine such a role for PI3K in the G₁₂QL-PDGFR signaling alliance, we first determined whether PI3K is associated with PDGFR and is activated in G₁₂QL-expressing cells. PDGFR was immunoprecipitated from G₁₂QL-NIH 3T3 or the vector control cells using antibodies to PDGFR and subjected to a PI3K assay using phosphotidylinositol as the substrate. The phosphorylated phospho-inositols were then separated using TLC. An increase in PDGFR-associated PI3K activity, which can be inhibited by AG1296, a PDGFR-specific inhibitor, was seen in G₁₂QL cells (Fig. 5B and C). Quantification of the results showed that G₁₂QL stimulates PI3K activity by threefold (Fig. 5C). More interestingly, this increase could be inhibited strongly (70%) by treating the cells with the PDGFR inhibitor AG1296 (Fig. 5C).

View larger version (18K): [in this window] [in a new window]   FIG. 5. G12QL-stimulated PDGFR activates PI3K. (A) G12QL stimulates the autophosphorylation of PDGFR. NIH 3T3 cells stably expressing G12QL or pcDNA3 vector were serum starved for 24 h and PDGFR was immunoprecipitated from the lysates using antibodies to PDGFR. An immunocomplex kinase assay was carried out as described in Materials and Methods. The phosphorylated proteins were separated by SDS-PAGE and visualized by autoradiography. The band corresponding to the molecular size of PDGFR is labeled. (B) G12QL stimulates PDGFR-associated PI3K. G12QL-NIH 3T3 and pcDNA3-NIH 3T3 cells were serum starved for 24 h. They were pretreated with AG1296 (10 µM) or the vehicle dimethyl sulfoxide for 18 h, and the lysates were prepared using PI3K lysis buffer (see Materials and Methods). PDGFR in the lysates (200 µg) was precipitated using antibodies to PDGFR and assayed for PI3K activity using 5 µg of phosphotidylinositol as a substrate. The phosphorylated inositols were resolved by ascending TLC on a silica gel plate developed by CHCl3 · CH3OH · NH4OH (9: 7: 2) and visualized by autoradiography. A representative autoradiographic analysis of a chromatogram is presented. (C) G12QL-stimulated PI3K activity is inhibited by PDGFR inhibitor. PI3K activity in the presence and absence of AG1296 (10 µM) in G12QL-NIH 3T3 and pcDNA3-NIH 3T3 cells was analyzed. The phosphorylated inositols were resolved by ascending TLC on a silica gel plate developed by CHCl3 · CH3OH · NH4OH (9:7:2) and visualized by autoradiography. The PI3-P spots were quantified using Kodak 1D Image analysis software. The amount of phosphatidylinositol 3-phosphate formed, an index of PI3K activity, is presented as the percent increase over the vector control cells (mean ± SEM; n = 3). IP, immunoprecipitate.

View larger version (17K): [in this window] [in a new window]   FIG. 6. G12QL stimulates AKT via PDGFR and PI3K. (A) G12QL stimulates the phosphorylation of AKT. G12QL-NIH 3T3 and pcDNA3-NIH 3T3 cells were serum starved for 24 h. Prior to lysis the cells were treated with 10 µM AG1296 (for 18 h) or 500 µM Wortmannin (2 h) or the vehicle dimethyl sulfoxide (0.1%). Immunoblot analysis was carried out with antibodies to phospho-AKT, followed by stripping and reprobing with antibodies to AKT. The blot was further analyzed for the expression of PDGFR and GAPDH using antibodies to PDGFR and GAPDH, respectively. Results from a typical experiment are presented. (B) G12QL-mediated stimulation of AKT is inhibited by PDGFR and PI3K inhibitors. G12QL-NIH 3T3 and pcDNA3-NIH 3T3 cells were serum starved for 24 h. Prior to lysis the cells were treated with 10 µM AG1296 (for 18 h) or 500 nM Wortmannin (2 h) or the vehicle dimethyl sulfoxide (0.1%). Immunoblot analysis was carried out with antibodies to phospho-AKT. The expression levels of phospho-AKT bands were quantified using Kodak 1D image analysis software. AKT activity is presented as the percent increase over the vector control group (mean ± SEM; n = 3). (C) G12QL stimulates the kinase activity of AKT. AKT was immunoprecipitated with antibodies to AKT from the lysates of serum-starved G12QL- and pcDNA3-NIH 3T3 cells. An immunocomplex kinase assay was carried out using purified recombinant FKHR as a substrate. The phosphorylated FKHR were separated by SDS-PAGE and visualized by autoradiography (top). Phosphorylated FKHR bands were excised and counted in a scintillation counter. AKT activity is expressed as the percent increase over the pcDNA3-NIH 3T3 control group (bottom). IB, immunoblot.

To further demonstrate that inhibitory phosphorylation of forkhead transcription factors occurs in G₁₂QL-NIH 3T3 transformants in vivo, an immunoblot analysis was carried out using antibodies directed against forkhead transcription factor in rhabdomyosarcoma-like factor 1 (FKHRL1) or FOXO3a, a member of the forkhead family of transcription factors. Lysates from G₁₂QL-NIH 3T3 cells along with vector control cells were examined for the phosphorylation of FKHRL1 by immunoblot analysis using antibodies to phospho-FKHRL1. The immunoblot analysis clearly indicated that the phosphorylation of FKHRL1 is increased in G₁₂QL-NIH 3T3 cells (Fig. 7A). To define the role of PI3K and PDGFR in G₁₂QL-stimulated phosphorylation of FKHRL1, we examined the phosphorylation levels of FKHRL1 in G₁₂QL cells with the PI3K inhibitor Wortmannin or the PDGFR inhibitor AG1296. As expected, treatment of cells with Wortmannin completely inhibited the G₁₂QL-stimulated phosphorylation of FKHRL1 (Fig. 7B). More significantly, treatment of cells with AG1296 drastically decreased the levels of phosphorylation in FKHRL1 (Fig. 7C), similar to its effect on AKT phosphorylation (Fig. 6A and B). Taken together, these results suggest a linear pathway involving PDGFR, PI3K, and AKT in G₁₂QL-mediated phosphorylation of FKHRL1.

View larger version (27K): [in this window] [in a new window]   FIG. 7. G12QL inhibits FKHRL1 activity. (A) G12QL stimulates the inhibitory phosphorylation of forkhead transcription factor FKHRL1. Lysates from serum-starved G12QL- and pcDNA3-NIH 3T3 cells were subjected to immunoblot analysis with antibodies to phospho-FKHRL1. The blot was sequentially stripped and reprobed with antibodies to FKHRL1 and GAPDH. The levels of FKHRL1 phosphorylation were quantified by Kodak 1D image analysis software. FKHRL1 phosphorylation is presented as the increase over the pcDNA3-NIH 3T3 cell control group. (B) Phosphorylation of FKHRL1 by G12QL is dependent on PI3K. G12QL-NIH 3T3 and pcDNA3-NIH 3T3 cells were serum starved (24 h). Prior to lysis the cells were treated with Wortmannin (2 h) along with the vehicle control (0.1% dimethyl sulfoxide). Immunoblot analysis was carried out with the cell lysates using antibodies to phospho-FKHRL1, followed by sequential stripping and reprobing with antibodies to FKHRL1 and GAPDH, respectively (top). The levels of phosphorylated FKHRL1 bands were quantified by Kodak 1D image analysis software. The results are presented as the increase in the levels of phosphorylated FKHRL1 over the untreated pcDNA3-NIH 3T3 vector control cells (bottom). (C) Phosphorylation of FKHRL1 by G12QL is PDGFR dependent. G12QL-NIH 3T3 and pcDNA3-NIH 3T3 cells were serum starved (24 h). Prior to lysis the cells were treated with 10 µM AG1296 (18 h) along with the vehicle control (0.1% dimethyl sulfoxide). Immunoblot analysis was carried out with the cell lysates using antibodies to phospho-FKHRL1 followed by sequential stripping and reprobing with antibodies to FKHRL1 and GAPDH, respectively (top) The levels of phosphorylated FKHRL1 bands were quantified by Kodak 1D image analysis software. The results are presented as the increase in the levels of phosphorylated FKHRL1 over the untreated pcDNA3-NIH 3T3 vector control cells (bottom). (D) FKHRL1 binding to its response elements is attenuated in cells expressing G12QL. Electromobility shift assays were performed with nuclear extracts from 24-h serum-starved G12QL- and pcDNA3-NIH 3T3 cells and radiolabeled oligonucleotide probes containing FKHRL1 binding sites as described in Materials and Methods. The nuclear extracts prepared from pcDNA3- and G12QL-NIH 3T3 cells were incubated with radiolabeled oligonucleotide containing FKHRL1 response elements alone (lanes 3 and 4), with competing cold oligonucleotide (lanes 1 and 2) or with antibodies to FKRHL1 (lanes 5 and 6).

Dominant negative mutant of PDGFR inhibits G₁₂QL-mediated focus formation. Our results presented here thus far have shown the following: (i) that the activated mutant of G₁₂ increases the expression as well as the activation of PDGFR (Fig. 1 to 4), (ii) that activated PDGFR in turn activates the PI3K/AKT pathway (Fig. 5 and 6), and (iii) that the activated PI3K-AKT pathway stimulates the inhibitory phosphorylation of forkhead transcription factor FKHRL1, a gene that has been previously shown to be involved in the transcription of antiproliferation genes (Fig. 7). Based on these findings, it can be speculated that the increased expression and activation of PDGFR may play a role in G₁₂-mediated oncogenic pathways. G₁₂-transformed fibroblasts, in addition to exhibiting increased proliferation, show other transformed phenotypes such as anchorage-independent cell growth and loss of contact inhibition (5, 35). The focus formation assay has been widely used to define the signaling nodes involved in oncogenic pathways (8, 10). Therefore, we further explored whether PDGFR plays a role in G₁₂-mediated focus formation. It has been demonstrated that the deletion of the C terminus of PDGFR results in a dominant negative phenotype (22). Using this dominant negative mutant of PDGFR (DN-PDGFR), we investigated whether functional signaling by PDGFR is required for G₁₂QL-mediated focus formation of NIH 3T3 cells. NIH 3T3 cells were transfected with vector containing G₁₂QL or the empty vector pcDNA3, along with pcDNA3 carrying the truncated mutant of PDGFR. The numbers of foci were scored 10 to 14 days posttransfection. The results show that while G₁₂QL induces focus formation in NIH 3T3 cells (Fig. 8A and B), the number of foci induced by G₁₂QL is reduced in the presence of DN-PDGFR (Fig. 8C). Quantification of results from repeat experiments indicated that the number of foci induced by G₁₂QL was reduced by 40% in the presence of DN-PDGFR (Fig. 8D). These results suggest that PDGFR is involved in G₁₂QL-mediated loss of contact-inhibited growth of NIH 3T3 cells.

View larger version (39K): [in this window] [in a new window]   FIG. 8. Dominant negative mutant of PDGFR inhibits G12QL-mediated focus formation. (A) Parental NIH 3T3 cells were transfected with the activated mutant of G12 (G12QL), empty vector (pcDNA3), DN-PDGFR, or G12QL plus DN-PDGFR (G12QL+DN-PDGFR) using the calcium phosphate transfection method as described in Materials and Methods. The representative foci formed at 14 days were photographed at x4 magnification. (B) The number of foci formed from two independent experiments were scored and plotted (mean ± standard deviation).

	DISCUSSION

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View larger version (34K): [in this window] [in a new window]   FIG. 9. Schematic representation of G12-mediated activation of PDGFR signaling pathway. Activated G12 stimulates the expression of PDGFR via a Rho-dependent signaling pathway. The increased expression of PDGF-A in these cells stimulates PDGFR, thus forming an autocrine signaling loop. The G12-stimulated PDGFR engages the downstream PI3K-AKT signaling pathway to inhibit FKHRL1-mediated transcription of apoptotic and antiproliferative genes to promote neoplastic cell growth.