Phosphatidylinositol 3-kinase but not tuberin is required for PDGF-induced cell migration

Carla Irani1, Elena A. Goncharova1, Deborah S. Hunter2, Cheryl L. Walker2, Reynold A. Panettieri1, and Vera P. Krymskaya1

1 Pulmonary, Allergy, and Critical Care Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and 2 University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957


    ABSTRACT
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ABSTRACT
INTRODUCTION
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The loss of function of the tumor suppressor gene TSC2 and its protein product tuberin promotes the development of benign lesions by stimulating cell growth, although the role of tuberin in regulating cell migration and metastasis has not been characterized. In addition, the role of phosphatidylinositol 3-kinase (PI 3-kinase), an important signaling event regulating cell migration, in modulating tuberin-deficient cell motility remains unknown. Using a tuberin-deficient rat smooth muscle cell line, ELT3, we demonstrate that platelet-derived growth factor (PDGF) stimulates cell migration by 3.2-fold, whereas vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-alpha , and basic fibroblast growth factor (bFGF) increase migration by 2.1-, 2.1-, and 2.6-fold, respectively. Basal and PDGF-induced migration in tuberin-deficient ELT3, ELT4, and ERC15 cells was not significantly different from that of tuberin-positive transformed rat kidney epithelial 2, airway smooth muscle, and pulmonary arterial vascular smooth muscle cells. Expression of tuberin in tuberin-deficient ELT3 cells also had little effect on cell migration. In parallel experiments, the role of PI 3-kinase activation in ELT3 cell migration was investigated. LY-294002, a PI 3-kinase inhibitor, decreased PDGF-induced migration in a concentration-dependent manner with an IC50 of ~5 µM. LY-294002 also abrogated ELT3 cell migration stimulated by bFGF and TGF-alpha but not by VEGF and phorbol 12-myristate 13-acetate. Furthermore, transient expression of constitutively active PI 3-kinase (p110*) was sufficient to induce ELT3 cell migration. However, the migration induced by p110* was less than that induced by growth factors, suggesting other signaling pathways are also critically important in modulating growth factor-induced cell migration. These data suggest that PI 3-kinase is required for growth factor-induced cell migration and loss of tuberin appears to have little effect on cell migration.

smooth muscle; airway; vascular; vascular endothelial growth factor; pulmonary lymphangioleiomyomatosis (LAM); remodeling


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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TUBEROUS SCLEROSIS COMPLEX 2 (TSC2), a tumor suppressor gene, and its product, tuberin, are implicated in the development of tuberous sclerosis complex (TSC), polycystic kidney disease (PKD), and pulmonary lymphangioleiomyomatosis (LAM; see Refs. 10, 11, 43). TSC is a systemic disorder characterized by benign lesions (hamartomas) of the brain, eyes, skin, heart, lung, and kidneys (16). Approximately one-third of the TSC cases are an inherited autosomal dominant disorder, and two-thirds are sporadic mutations. Autosomal dominant PKD is one of the most common human genetic disorders. A recent study has shown a functional link between the TSC2 gene and PKD in which tuberin determines the appropriate membrane localization of the PKD1 gene polycystin-1 and thus modulates its function (26). Studies also demonstrated that somatic mutations in the TCS2 gene are associated with pulmonary LAM (8), a rare lung disease that occurs almost exclusively in women of childbearing age. The disease is characterized by the abnormal proliferation, migration, and differentiation of smooth muscle-like cells that involve the entire lung, including the airways and the blood and lymph vessels (24). LAM can induce a chronic, progressive loss of respiratory function, for which there is no therapy. In animal models in which the TSC2 gene is inactivated, tumors occur in the uterus and kidney (benign leiomyomas), and renal cell carcinoma, which unlike in their human counterpart, fails to metastasize to extrarenal sites. Therefore, although loss of tuberin function affects diverse organ systems, both human and murine lesions manifest common phenotypes of abnormal cellular proliferation, but the neoplastic cell growth remains localized within target organs. Whether the loss of the TSC2 gene function affects motile properties of cells in the lesions has not been examined.

The tumor suppressor properties of the TSC2 gene have been established in both human and rodent tumors (9, 27). The DNA sequence of TSC2 and in vitro and in vivo studies suggest that tuberin may act as a GTPase-activating protein (10) for Rap1 (53) and Rab5 (54), which are components of the Ras superfamily of GTPases (34). Mutations in members of the Ras superfamily alter intrinsic GTPase function and modulate cell migration and growth (6). Ras, Rac, Rho, and, potentially, Rap1 or Rab5 are signaling regulators of phosphatidylinositol 3-kinase (PI 3-kinase), the activation of which is necessary for cell migration in many cell types (48). Although cell migration appears to be essential in promoting mesenchymal cell growth, the relative contribution of PI 3-kinase activation and tuberin expression in regulated cell migration has not been defined in cells that have lost tuberin function. Because cell migration is necessary for promoting tumorigenesis in some cell types, we hypothesize that loss of TSC2 function may modulate PI 3-kinase activation and growth factor-induced cell migration.

Cell motility and migration are among the hallmarks of neoplastic transformation and clearly are associated with metastasis to distant sites. Current evidence suggests that PI 3-kinase is necessary for cell migration in some cell types (20, 31, 32, 42, 47, 52). PI 3-kinase also modulates a variety of other cellular physiological functions such as mitogenesis, differentiation, transformation, and apoptosis (48). PI 3-kinase coordinates cellular events by generating 3-phosphoinositide lipids. These lipids, in turn, recruit signaling proteins that contain domains (usually pleckstrin homology domains) that bind directly to the phosphoinositides. Several downstream targets of PI 3-kinase have been identified, including two protein-Ser/Thr kinases, PDK1 and Akt (also called PKB), and a protein tyrosine kinase, BTK (38). PI 3-kinase appears to be critical for human airway smooth muscle (ASM; see Ref. 30) and human pulmonary arterial vascular smooth muscle (PAVSM) cell proliferation (28) and migration (55). PI 3-kinase also modulates unstimulated and growth factor-induced mitogenesis in ELT3 cells that are tuberin deficient (29).

To determine whether a linkage exists among loss of tuberin, cell migration, and PI 3-kinase signaling, we used tuberin-deficient tumor-derived cell lines (19, 22, 49) of epithelial and mesenchymal origin from the Eker rat, a naturally occurring animal model with a germ line TSC2 gene mutation (27). We found that the levels of basal and platelet-derived growth factor (PDGF)-induced cell migration of tuberin-deficient ELT3, ELT4, and ERC15 cell lines were comparable to levels found in tuberin-positive transformed rat kidney epithelial (TRKE) 2, ASM, and PAVSM cells. Importantly, inhibition of PI 3-kinase activity attenuated PDGF-induced migration in all cell lines, and transient expression of constitutively active PI 3-kinase (p110*) was sufficient to promote ELT3 cell motility. These studies indicate a requirement for PI 3-kinase activation in growth factor-induced smooth muscle cell migration and suggest that, although loss of the tumor suppressor tuberin promotes tumorigenesis, the loss of tuberin function does not promote cell migration, which underlies the benign nature of tuberin-deficient lesions.


    METHODS
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Materials. Ham's F-12 and DMEM were purchased from Life Technologies (Grand Island, NY). FBS was obtained from HyClone Laboratories (Logan, UT). Ferrous sulfate, vasopressin, triiodothyronine, insulin, cholesterol, hydrocortisone, transferrin, and BSA were purchased from Sigma Chemical (St. Louis, MO). Endothelial cell growth supplement (ECGS) was obtained from Becton-Dickinson (Bedford, MA). Transwells, 6.5 mm diameter, 8 µm pore size, were purchased from Corning Costar (Cambridge, MA). Effectene transfection reagent was obtained from QIAGEN (Valencia, CA). PDGF-BB, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-alpha were purchased from Calbiochem-Novabiochem (San Diego, CA). Anti-c-myc tag and anti-phosphotyrosine (PY) antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Protein A-Sepharose was obtained from Pharmacia Biotech (Uppsala, Sweden). [gamma -32P]ATP was purchased from NEN (Boston, MA). Thin-layer chromatography (TLC) plates (Silica Gel 60) were obtained from Merck KGaA (Darmstadt, Germany).

Cell culture. The Eker rat uterine leiomyoma-derived cell lines ELT3 and ELT4 were described previously (22). These cell lines express smooth muscle-specific actin and desmin, and ELT3 cells are tumorigenic in nude mice. The ERC15 cell line was derived from Eker rat renal carcinoma (19). The TRKE2 cell line was derived from primary kidney epithelial cells (49). ELT3, ELT4, ERC15, and TRKE2 were maintained in DF8 medium consisting of equal amounts of Ham's F-12-DMEM with 1.6 × 10-6 M ferrous sulfate, 1.2 × 10-5 U/ml vasopressin, 1.0 × 10-9 M triiodothyronine, 0.025 mg/ml insulin, 1.0 × 10-8 M cholesterol, 2.0 × 10-7 M hydrocortisone, and 10 pg/ml transferrin supplemented with 10% FBS. Human ASM cells and PAVSM cells were obtained as previously described (36). ASM cells were maintained in Ham's F-12 with 10% FBS. PAVSM were maintained in Ham's F-12 with 10% FBS supplemented with 15 µg/ml ECGS. ELT3, ELT4, ERC15, and TRKE2 were growth arrested for 48 h in DF8 basal media containing 1% BSA. ASM and PAVSM cells were growth arrested in serum-free Ham's supplemented with 0.1% BSA.

Migration assay. For the cell migration assays, cells were added to the upper well of a Transwell at 2.0 × 105 cells/well. Growth factors were then added to the lower chamber at the concentrations indicated, and cells were allowed to migrate for 4 h. In the experiments with PI 3-kinase inhibitor LY-294002, cells were preincubated with LY-294002 in the upper well of a Transwell for 30 min followed by the addition of growth factors to the lower chamber. Migration was quantified by performing microscopic cell counts at ×200 on 6-12 random fields in each well.

Transient transfection. ELT3 cells were transiently transfected using the Effectene transfection reagent according to the manufacturer's protocol. The plasmid directing the expression of a myc-tagged constitutively active form of PI 3-kinase p110* was a generous gift from Dr. A. Klippel. Constitutively active p110* PI 3-kinase is an activated mutant generated by the covalent attachment of the inter-Src homology region 2 of the p85 regulatory subunit to the NH2 terminus of the p110 catalytic subunit (23). The construct containing a full-length TSC2 cDNA was obtained from Dr. R. Yeung and was described previously (26, 54). All assays were performed 72 h after transfection. Cells were growth arrested for 48 h in serum-free media before migration or PI 3-kinase activity assays. Transient transfection of myc-tagged constitutively active p110* PI 3-kinase plasmid was verified by immunoprecipitation using anti-c-myc tag (3 µg/ml) antibodies followed by the PI 3-kinase activity assay.

PI 3-kinase activity assay. Nearly confluent ELT3 cells were growth arrested in serum-free media supplemented with 1% BSA for 48 h. Cells were stimulated with PDGF, bFGF, TGF-alpha , VEGF, and phorbol 12-myristate 13-acetate (PMA) or treated with 30 µM of LY-294002 at 37°C for the times indicated. PI 3-kinase activity assays were performed as previously described (30). Briefly, PI 3-kinase was immunoprecipitated using anti-PY (2 µg/ml) or anti-c-myc tag (2 µg/ml) antibodies. Immunocomplexes were collected using protein A-Sepharose for 2 h at 4°C, followed by the in vitro kinase activity assay. Sonicated phosphatidylinositol in Tris · HCl/EGTA (0.2 mg/ml, final concentration) was added to immunoprecipitates, and the phosphorylation reactions were started by the addition of MgCl2, ATP, and [gamma -32P]ATP (30 µCi/sample) for 10 min at 30°C. Reactions were stopped by the addition of 100 µl of 1 N HCl and were extracted with 160 µl of chloroform-methanol (1:1). Lipids were separated on oxalate-coated TLC plates using a solvent system of chloroform-methanol-water-ammonium hydroxide (60:40:11.3:2), and then they were detected by autoradiography. The position of [32P]phosphatidylinositol monophosphate was determined by the position of a phosphatidylinositol standard that is separated on a TLC in parallel and developed in iodine vapor.

Apoptosis analysis. Analysis of apoptosis was performed using a CaspaTag fluorescein caspase activity kit according to the manufacturer's protocol (Intergen, Purchase, NY). Briefly, cells were growth arrested for 48 h in serum-free media; they were then incubated with 30 µM LY-294002 or diluent (0.03% DMSO) or left untreated (control) for 4 h in the presence of FAM-VAD-FMK, a carboxyfluorescein (FAM) derivative of benzyloxycarbonyl valylalanylaspartic acid fluoromethyl ketone (VAD-FMK), which binds to activated caspases. Next, cells were fixed with 2% formaldehyde; nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI), and cells were observed under a microscope using a filter with excitation 460 nm and emission 560 nm to view green fluorescence of caspase-positive cells and an ultraviolet filter for viewing DAPI nuclei staining. As a positive control, we used an overnight incubation of cells with 50 µM C2-ceramide. apoptotic cells were counted as the percentage of caspase-positive cells relative to the total number of cells.

Data analysis. Data points from individual assays represent the mean values ± SE. Statistically significant differences among groups were assessed with ANOVA (Bonferroni-Dunn test), with values of P < 0.05 sufficient to reject the null hypothesis for all analyses. All experiments were designed with matched control conditions within each experiment to enable statistical comparison as paired samples.


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Growth factors induce smooth muscle cell migration. Using ELT3 cells, a tuberin-deficient rat smooth muscle cell line, we examined whether growth factors that have been reported to induce smooth muscle cell motility, such as PDGF, VEGF, TGF-alpha , and bFGF (40, 44), promote ELT3 cell migration. Growth-arrested ELT3 cells were plated in Transwell plates and stimulated with 10 ng/ml of each growth factor added to the lower chamber. This particular concentration of growth factors was chosen since it has been reported to induce migration in other cell types (1, 4, 5, 45). Because preliminary experiments established that ELT3 cells maximally migrate after 4 h of growth factor stimulation, migration assays were performed using 4-h incubation intervals. All growth factors significantly stimulated cell migration (Fig. 1). These data indicate that growth factors promote migration of tuberin-deficient ELT3 cells.


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Fig. 1.   Growth factors induce ELT3 cell migration. Growth-arrested cells were plated on Transwell filters and allowed to migrate toward 10 ng/ml of either platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-alpha , or basic fibroblast growth factor (bFGF) for 4 h. Filters were then fixed and counted as described in METHODS. Values are means ± SE of 6 replicates in 2 separate experiments. * P < 0.005 by ANOVA (Bonferroni-Dunn test).

Mutations of TSC2 gene or loss of function of its product tuberin may promote the development of benign tumors. To examine whether loss of tuberin affects cell migration, tuberin-deficient cell migration was compared with the migration of tuberin-positive cells. Basal levels of migration of tuberin-deficient ELT3, ELT4, and ERC2 cell lines were not significantly different from those of tuberin-positive TRKE15, ASM, and PAVSM cells (Fig. 2A), and PDGF significantly stimulated the migration of each of the cell lines. Additionally, transient expression of tuberin in tuberin-deficient ELT3 cells had little effect on cell migration when compared with the migration of cells transfected with control plasmid or untransfected cells (Fig. 2B). These data suggest that loss of tuberin function has little effect on cell migration.


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Fig. 2.   A: PDGF-induced migration of tuberin-deficient ELT3, ELT4, and ERC15 and tuberin-positive transformed rat kidney epithelial (TRKE) 2, human airway smooth muscle (HASM), and human pulmonary arterial vascular smooth muscle (HPAVSM) cells. ELT3, ELT4, ERC15, TRKE2, HASM, and HPAVSM cells were growth arrested for 48 h, plated on Transwells, and migrated toward 10 ng/ml PDGF in the lower chamber. Data are means ± SE of 6 replicates in 2 separate experiments. * P < 0.001 by ANOVA (Bonferroni-Dunn test). B: expression of tuberin in tuberin-deficient ELT3 cells has little effect on cell migration. ELT3 cells were transiently transfected with pcDNA3-TSC2 mammalian expression vector expressing tuberin, with empty pcDNA3 plasmid, or mock transfected (Control). Cells were then growth-arrested, plated on Transwells, and migrated for 4 h. Data are means ± SE of 6 replicates in 2 separate experiments.

PI 3-kinase modulates growth factor-induced ELT3 cell migration. Because PI 3-kinase activation is required for migration in some cell types, we investigated whether PI 3-kinase mediates growth factor-induced smooth muscle cell migration. Pretreatment of growth-arrested ELT3 cells with a PI 3-kinase inhibitor, LY-294002, over a range of concentrations for 30 min before stimulation with PDGF attenuated ELT3 cell migration compared with diluent-treated cells (IC50 value of 5 µM; Fig. 3A). LY-294002 (30 µM) also attenuated TGF-alpha - and bFGF-induced ELT3 migration but had no significant effect on VEGF-stimulated migration (Fig. 3B). Cell migration stimulated by PMA (200 nM), which directly activates protein kinase C (PKC; see Ref. 35), was not altered by LY-294002 (Fig. 3B). This was expected, since studies suggest that PMA-induced activation of PKC is independent of PI 3-kinase activation (35). Interestingly, LY-294002 did not abrogate basal levels of ELT3 migration; this is consistent with PI 3-kinase activation being necessary only for cell migration induced by some growth factors. ELT3 cell viability over the duration of this assay was not changed by LY-294002, as determined by trypan blue staining (data not shown) and CaspaTag fluorescein caspase activity assay (Fig. 3C). In addition to attenuation of ELT3 cell migration, LY-294002 also attenuated PDGF-induced ELT4, ERC2, TRKE15, ASM, and PAVSM cell migration (Fig. 3D). Taken together, these data suggest that PI 3-kinase is required for PDGF-induced migration of tuberin-deficient ELT3, ELT4, and ERC2 and tuberin-positive TRKE15, ASM, and PAVSM cells but is not required for PMA- or VEGF-induced cell migration.


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Fig. 3.   Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) attenuates cell migration. A: ELT3 cells were plated on Transwells in the media containing either diluent or 1, 10, or 30 µM LY-294002. After 30 min, 10 ng/ml PDGF or diluent were added to lower chamber, and cells were allowed to migrate for 4 h. Cell migration was subsequently assessed as described in METHODS. Data represent means ± SE from 6 replicates for each condition in 3 separate experiments. * P < 0.003 for 10 and 30 µM LY-294002 + PDGF vs. PDGF. B: ELT3 cells were treated with 30 µM LY-294002 followed by stimulation with 10 ng/ml of TGF-alpha , bFGF, or VEGF and 1 µM phorbol 12-myristate 13-acetate (PMA). Data represent means ± SE from 6 replicates in 3 separate experiments. * P < 0.001 for 30 µM LY-294002 + PDGF vs. PDGF. C: growth-arrested cells were treated with 30 µM LY-294002 or diluent (DMSO) or left untreated (Control) for 4 h or with 50 µM C2-ceramide overnight. Apoptosis was subsequently assessed by fluorescein caspase activity assay (Intergen). Data represent the percentage of caspase-positive cells compared with the total number of cells. D: growth-arrested ELT3, ELT4, ERC15, TRKE2, ASM, and PAVSM cells were pretreated with diluent or 30 µM LY-294002 and then cells were allowed to migrate for 4 h in the presence of 10 ng/ml PDGF. Data are means ± SE of 6-12 replicates in 2 separate experiments.

PDGF, TGF-alpha , and bFGF activate PI 3-kinase in ELT3 cells. Because LY-294002 attenuated PDGF-, TGF-alpha -, and bFGF-induced ELT3 cell migration, studies were then performed to confirm that PDGF, TGF-alpha , and bFGF directly activate PI 3-kinase in ELT3 cells. Growth-arrested cells were stimulated with 10 ng/ml of PDGF, TGF-alpha , or bFGF for 5 min, in the presence or absence of 30 µM LY-294002, and PI 3-kinase activity was measured by an in vitro kinase activity assay. As shown in Fig. 4A, all growth factors significantly stimulated PI 3-kinase activity. Interestingly, the magnitude of PI 3-kinase activation by PDGF, TGF-alpha , or bFGF was markedly different. PDGF stimulation induced robust activation of PI 3-kinase by 108-fold compared with PI 3-kinase activity in unstimulated cells, whereas PI 3-kinase activity stimulated by TGF-alpha or bFGF was increased by 8- and 12-fold, respectively, compared with control. Pretreatment of cells with 30 µM LY-294002 for 30 min before stimulation with PDGF, TGF-alpha , or bFGF markedly attenuated growth-factor-induced PI 3-kinase activation (Fig. 4C). The inhibition of PI 3-kinase activity by LY-294002 at 30 µM was not complete (Fig. 4C). Although LY-294002 (30 µM) is relatively specific for inhibiting PI 3-kinase (30), higher concentrations will likely have nonspecific effects. Therefore, we could not conclusively state that some component of PDGF-induced ELT3 cell migration may be PI 3-kinase independent. VEGF and PMA, however, had little effect on PI 3-kinase activity (Fig. 4B). These data demonstrate that PDGF-, TGF-alpha -, or bFGF-induced migration involves direct activation of PI 3-kinase in ELT3 cells and VEGF- and PMA-induced migration is PI 3-kinase-independent.


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Fig. 4.   PI 3-kinase activity in ELT3 cells. Growth-arrested ELT3 cells were stimulated with either 10 ng/ml PDGF, TGF-alpha , bFGF (A) or 10 ng/ml VEGF or 200 nM PMA (B) or were pretreated with 30 µM LY-294002 for 30 min and then stimulated for 5 min with either PDGF, TGF-alpha , or bFGF (A) or VEGF, 200 nM PMA, or diluent (B). PI 3-kinase activity was measured in anti-phosphotyrosine immunoprecipitates as described in METHODS. Autoradiographs are representative of 2 separate experiments that yielded similar results. C: quantitative analysis of inhibition of PI 3-kinase activity by LY-294002. PDGF-, TGF-alpha -, and bFGF-induced phosphatidylinositol monophosphate (PI-3-P) accumulation was measured by densitometry analysis of autoradiographs using Scion Image software.

PI 3-kinase is sufficient to induce ELT3 cell migration. To test whether PI 3-kinase could directly stimulate ELT3 cell migration, we transiently transfected ELT3 cells with pCG-p110* plasmid expressing a constitutively active form of class IA PI 3-kinase, which activates PI 3-kinase signaling pathways independent from receptor stimulation (23). To confirm that p110* expression was present in transiently transfected cells, cell lysates were immunoprecipitated with an anti-myc-tag specific antibody, and PI 3-kinase activity was measured. ELT3 cells transfected with pCG-p110* plasmid demonstrated robust intrinsic PI 3-kinase activity of expressed p110* protein compared with cells transfected with control plasmid (Fig. 5A). Next, we examined whether transient expression of p110* modulated basal and PDGF-induced ELT3 cell migration. Expression of p110* alone was sufficient to promote ELT3 cell migration (Fig. 5B). Migration of pCG-p110*-transfected cells was 24.9 ± 1.8 cells/field compared with 11.7 ± 1.4 cells/field in control cells (P < 0.0001 by Bonferroni/Dunn). Expression of p110* also significantly augmented PDGF-induced cell migration. Interestingly, constitutively active PI 3-kinase enhanced basal and PDGF-induced migration by 13.2 ± 1.2 and 11.1 ± 2.7 cells/field, respectively, compared with cells transfected with control plasmid. Collectively, these data show that PI 3-kinase directly promotes ELT3 cell migration.


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Fig. 5.   Expression of constitutively active p110* PI 3-kinase is sufficient for ELT3 cell migration. A: ELT3 cells were transiently transfected with pCG and pCG-p110* plasmids. Cell lysates were immunoprecipitated with anti-myc-tag specific antibody, and PI 3-kinase activity was assessed as described in METHODS. C, control pCG plasmid; p110*, pCG-p110* plasmid expressing constitutively active PI 3-kinase. B: ELT3 cells were transiently transfected with control pCG or pCG-p110* plasmids and growth arrested for 48 h, and the migration assay was performed as described in METHODS. Data represent means ± SE from 6 replicates in 3 separate experiments. * P < 0.0001 for pCG vs. p110; ** P < 0.001 for p110* + PDGF vs. PDGF.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Accumulation of smooth muscle-like cells with somatic mutations of the TCS2 gene may be the result of migration of these cells in pulmonary LAM. In this study, we showed that PDGF, VEGF, TGF-alpha , and bFGF promote cell migration in tuberin-deficient and tuberin-positive cell lines. Interestingly, tuberin-deficient and tuberin-positive cells have comparable levels of basal and PDGF-induced cell migration. Furthermore, transient expression of tuberin in tuberin-deficient ELT3 cells had little effect on cell migration. We also demonstrated that PI 3-kinase appeared to modulate cell migration. Inhibition of PI 3-kinase attenuated PDGF-, TGF-alpha -, and bFGF- but not VEGF-induced ELT3 cell motility. Inhibition of PI 3-kinase also attenuated PDGF-induced migration of ELT4, ERC15, TRKE2, ASM, and PAVSM cells. Furthermore, transient expression of constitutively active p110* PI 3-kinase promoted the migration of quiescent ELT3 cells and increased PDGF-stimulated ELT3 cell motility.

In some cell types, PI 3-kinase is essential for PDGF-induced chemotaxis (31, 52). Our finding that PI 3-kinase is sufficient to induce ELT3 cell migration supports published observations that confirm the role of PI 3-kinase in cell motility. Mutation of PI 3-kinase binding sites in the cytoplasmic domain of the PDGF receptor abrogated chemotactic responses to PDGF in porcine aortic endothelial cells and NIH/3T3 cells (31, 52). Evidence, however, also suggests that PDGF-induced chemotactic signal transduction is cell and tissue specific (18, 37). Although inhibition of PI 3-kinase attenuated PDGF-induced migration in rat thoracic aorta vascular smooth muscle (37) and in canine pulmonary artery smooth muscle (55), expression of dominant-negative PI 3-kinase mutants had little effect on PDGF-induced rat aorta vascular smooth muscle and Chinese hamster ovary cell migration (18). These seemingly conflicting data may be because of PI 3-kinase-dependent and PI 3-kinase-independent signal transduction pathways regulating cell migration in a species- and cell-specific manner. Our data support this notion. LY-294002 attenuated but did not completely inhibit PDGF-induced ELT3 cell migration, and VEGF-induced migration was unaffected by PI 3-kinase inhibition. Furthermore, migration induced by the transient expression of constitutively active p110* PI 3-kinase was less than that induced by growth factor stimulation, suggesting that other parallel or upstream signaling pathways, in addition to PI 3-kinase activation, are important in regulating cell motility. A possible limitation of our approach may be that transient transfection of highly differentiated cell lines using lipofection technologies usually has relatively low and variable transfection efficiencies. Our findings together with the reports of others suggest the existence of PI 3-kinase-dependent and PI 3-kinase-independent pathways for cell migratory signaling that are cell type and tissue specific.

We found that bFGF, TGF-alpha , and VEGF also promote ELT3 cell motility. Interestingly, bFGF- and TGF-alpha -induced migration appeared to be PI 3-kinase dependent, whereas VEGF-induced migration was unaffected by LY-294002 in ELT3 cells. VEGF has been implicated in the normal development of smooth muscle cells surrounding coronary arteries, renal vessels, and retinal vasculature (7) and also in the migration of smooth muscle cells from human and (51) bovine aorta (17) and rat cavernous (33). Data on the role of PI 3-kinase activation in VEGF-induced signaling and migration are often contradictory. For example, VEGF-dependent migration of M21 melanoma cells is mediated by activation of the VEGF receptor 2 (VEGFR2) and is regulated by PI 3-kinase (5). PI 3-kinase activation is also an early event in VEGF-induced endothelial cell survival (15). Overexpression of dominant-negative Akt, the downstream effector of PI 3-kinase, abrogates VEGF-induced endothelial cell migration (14). Other reports, however, showed that stimulation of endothelial cells with VEGF does not induce PI 3-kinase activation (2, 46, 50). A more recent report also demonstrates that activation of VEGFR2 stimulates PI 3-kinase in porcine aortic endothelial cells and that activation of PI 3-kinase modulated VEGFR2-mediated cell growth (12). Our findings suggest that inhibition of PI 3-kinase has little effect on VEGF-induced migration and that VEGF-induced cell migration in ELT3 cells is PI 3-kinase independent.

The mechanism of PI 3-kinase-mediated cell motility is not well understood. Studies suggest that cell migration may involve p21-activated kinase (PAK)1 and PAK-interacting exchange factor (3, 56), FAK (39), PKC-zeta (13), and also the activation of small GTP-binding proteins Rho, Cdc42, and Rac (21, 25, 41, 57). PI 3-kinase and its downstream effectors Rho and Rac have been implicated in PDGF-induced stress-fiber reorganization (21, 57). The activation of Cdc42 and Rac1 disrupts the normal polarization of mammary epithelial cells and promotes cell motility and invasion in a PI 3-kinase-dependent manner (25). PI 3-kinase-dependent activation of PAK1 induces reorganization of the cortical actin cytoskeleton in heregulin-induced MCF7 breast cancer cell migration (3). PI 3-kinase also activates PAK-interacting exchange factor, leading to the activation of the kinase activity of PAK, and the migration of mesodermal cells on the extracellular matrix (56). We found that PDGF-induced activation of PI 3-kinase is necessary in modulating PDGF-induced effects on migration of rat smooth muscle ELT3 and ELT4, kidney endothelial ERC15 and TRKE2, and human ASM and PAVSM cells. The mechanism of PI 3-kinase-mediated motility in these cell types remains unknown and requires further study.

Regulation of cell motility is one of the critical steps in carcinogenesis. Our results suggest that some, but not all, growth factors activate PI 3-kinase and stimulate smooth muscle and epithelial cell motility in a PI 3-kinase-dependent manner. Such studies are important for fostering an understanding of the role of PI 3-kinase in regulating cell functions such as migration. Our comparative approach using a number of cell lines may provide insight into differences among signaling pathways and functions of the TSC2 gene product, tuberin, in tuberin-deficient and tuberin-positive primary airway and vascular cells. Our data suggest that loss of tuberin seemingly does little to modulate tumor-derived smooth muscle and epithelial cell migration; this is consistent with the benign nature and target-organ location of tuberin-deficient lesions. Further studies on the linkage between cell motility and cellular proliferation in these cell types may yield new therapeutic approaches at blocking signal transduction processes that promote cell migration in diseases such as LAM, pulmonary hypertension, asthma, and cancer.


    ACKNOWLEDGEMENTS

We thank Dr. Anke Klippel for pCG and pCG-p110* expression vectors, Dr. R. Hoffman for expert assistance with the CaspaTag assay, Dr. Margaret M. Chou for helpful discussion, and Dr. Ellen Puré for a critical reading of the manuscript.


    FOOTNOTES

This work was supported by grants from the American Heart Association and the Lymphangioleiomyomatosis Foundation to V. P. Krymskaya and National Heart, Lung, and Blood Institute Grants HL-55301 and HL-64063 to R. A. Panettieri.

Address for reprint requests and other correspondence: V. P. Krymskaya, Pulmonary, Allergy, and Critical Care Division, Univ. of Pennsylvania, Rm. 847 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160 (E-mail: krymskay{at}mail.med.upenn.edu).

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

10.1152/ajplung.00291.2001

Received 28 July 2001; accepted in final form 14 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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