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First published online 5 August 2003
doi: 10.1242/jcs.00679

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Mechanisms in LPA-induced tumor cell migration: critical role of phosphorylated ERK

¹ Department of Pharmacology and Toxicology, University of Ulm, 89069 Ulm, Germany
² MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK

View larger version (95K): [in a new window]   Fig. 1. LPA-stimulated cell migration. For wound healing experiments, cells were incubated until a confluent monolayer was formed. Cells were then serum-starved overnight in DMEM containing 0.1% BSA, treated for 2 hours with 10 µg/ml mitomycin C to inhibit cell proliferation, and a cell-free space was created by scraping through the monolayer. Migration was induced by addition of 10 µM LPA. Results from one representative experiment of three independent studies are shown.

View larger version (21K): [in a new window]   Fig. 2. RT-PCR analysis of LPA receptor expression and LPA-stimulated activation. (A) Total RNA was prepared from PANC-1, BxPC-3 and MiaPaCa-2 cells and used as template for RT-PCR using specific oligonucleotides for LPA1/Edg2, LPA2/Edg4 and LPA3/Edg7. The amounts of cDNA used as template were adjusted to similar levels according to the amount of a -actin fragment present in the samples determined by semi-quantitative PCR. A sample containing no single-stranded cDNA was analyzed for comparison (Control). The amplified DNA fragments were fractionated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. The sizes for LPA receptor PCR-products are: LPA1/Edg2: 349 bp; LPA2/Edg4: 798 bp; LPA3/edg7: 382 bp. (B) LPA-stimulated [35S]GTP[S] binding to cell membranes. PANC-1 and BxPC-3 cells were incubated for 16 hours prior to membrane preparation in DMEM containing 10% FCS with or without PTX (50 ng/ml). Binding of [35S]GTP[S] to 6 µg of membrane protein was assayed after addition of LPA at the indicated concentrations for 2 hours at 30°C in assay buffer containing 100 mM NaCl and 10 µM GDP. Fold stimulation of [35S]GTP[S] binding as compared to membrane preparations treated without ligand is expressed as mean values ± s.e.m. (n=4).

View larger version (31K): [in a new window]   Fig. 3. Time-course of LPA-induced Ras, RhoA and Rac activation. PANC-1 cells were incubated overnight in DMEM without supplements and then treated for the indicated time periods with 10 µM LPA. GTP-bound GTPases were recovered from RIPA-cell lysates (0.5 mg protein for Ras detection, 1 mg protein for Rac detection, or 2 mg protein for K-Ras, N-Ras and RhoA detection) using GTPase-specific GST-fusion proteins in in vitro precipitation experiments. Proteins were separated by 12.5% SDS-PAGE and the amounts of bound GTP-Ras, GTP-Rac and GTP-RhoA proteins were analyzed in immunoblots (left panel). Pan-Ras antibody was used to detect all Ras-isoforms, and antibodies specifically reactive against N-Ras, K-Ras, Rac and RhoA were used to detect the individual GTPases. The right panel shows aliquots (30 µg of protein for Ras, 50 µg of protein for Rac, and 80 µg of protein for RhoA) of total cell lysates to control for equal GTPase loading. For direct comparison, activation of N- and K-Ras was determined in the same lysate. One blot representative for at least three independent experiments for each GTPase is shown.

View larger version (40K): [in a new window]   Fig. 4. Activation of ERK2 by LPA. Confluent, serum-starved PANC-1 cells were treated with 10 µM LPA. ERK2 activity was determined by using a synthetic MBP-peptide as ERK-substrate in in vitro phosphorylation assays as described in Materials and Methods. (A) For time-course analysis, PANC-1 cells were incubated with 10 µM LPA for the indicated time periods and ERK2 was immunoprecipitated from 1 mg of cell lysate protein. Results of one representative experiment out of four are given in counts per minute (cpm). (B) To determine the influence of activated MEK1 and activated Gi/o-proteins on ERK activity, cells were either pretreated with 25 µM PD98059 for 15 minutes or with 100 ng/ml PTX overnight, and then incubated for 10 minutes with 10 µM LPA, 30 ng/ml EGF, or carrier. Means ± s.e.m. (n=3) of fold stimulation relative to solvent-treated cells are shown. (C) The influence of dominant negative GTPases on LPA-induced ERK2 activity was determined in transient cotransfection studies. PANC-1 cells were cotransfected with 8 µg of pcDNA3/HA-ERK2 and either 8 µg of pEGFP-C1/H-Ras(S17N), pEGFP-C2/K-Ras(S17N), pEGFPC3/Rac1(T17N), or pEGFP-C3/RhoA(T19N). After 24 hours in growth medium, cells were serum-starved for 4 hours in DMEM with 0.1% FCS and then treated for 10 min with 10 µM LPA. HA-ERK2 was immunoprecipitated using anti-HA antibody (12CA5) and the kinase activity was determined by in vitro radioactive phosphorylation of the MBP peptide. The right diagram shows the results of one representative assay of three experiments with the phosphorylation given in cpm. The corresponding immunoblot (upper-left panel, WB: anti-GFP) shows the expression of ectopically expressed EGFP- and EGFP-GTPase-fusion proteins, as detected using an anti-GFP antibody. The lower panel (IP: anti-HA) shows the corresponding anti-ERK2 immunoblot of one-third of the immunoprecipitated HA-ERK2, as detected using an anti-ERK2 antibody. IP, immunoprecipitation; WB, western blot.

View larger version (50K): [in a new window]   Fig. 5. Inhibition of LPA-stimulated cell migration. (A) Influence of dominant negative GTPases. PANC-1 cells were incubated overnight in DMEM without supplements and subjected to wound healing experiments. Microinjection of plasmids into the cell nuclei was performed 1 hour after wounding with either 0.1 µg/µl pEGFP-C3, pEGFP-C1/H-Ras(S17N), pEGFP-C3/Rac1(T17N) or pEGFP-C3/RhoA(T19N). Protein expression was allowed to proceed for 3 hours in medium without supplements. Migration was induced by addition of 10 µM LPA. Cells were fixed after 24 hours, and well-spread, green-fluorescent cells were counted. Cells, which had migrated into the cell-free space, were calculated as per cent of injected cells. Means ± s.e.m. of three independent experiments are shown. The inset shows pEGFP-injected cells 3 hours after microinjection. (B,C) Inhibition of LPA-stimulated cell migration by pertussis toxin and PD98059. PANC-1 and BxPC-3 cells were seeded on top of trans-well cell culture inserts (8 µm pore size) in DMEM with 10% FCS, and treated for 2 hours with 10 µg/ml mitomycin C to inhibit cell proliferation. Chemoattractants [10% FCS (open bars in C), 10 µM LPA (solid bars in C) or 20 ng/ml HGF (hatched bars in C) in DMEM with 0.1% BSA] were added to the lower chamber. The upper chamber was filled with DMEM with 0.1% BSA. PTX (25 ng/ml), PD98059 (25 µM), U0126 (10 µM) or SP600125 (25 µM) was added to both chambers; controls were treated with solvent. PANC-1 and BxPC-3 cells were incubated for 40 hours. Afterwards, cells were fixed, stained with hematoxylin and cells migrated to the bottom side of the filter were photographed. For quantification, cells in three independent visual fields were counted. (B) Representative microphotographs of the bottom sides of the filters. (C) Quantitative analysis of one representative assay out of three experiments. Means ± s.d. of three independent visual fields are shown.

View larger version (76K): [in a new window]   Fig. 6. LPA-induced changes in actin cytoskeleton and focal contact organization. PANC-1 cells were starved overnight in DMEM + 0.1% BSA, wounded and treated for 3 hours with DMEM supplemented with 0.1% BSA (a,b), 10 µM LPA (c,d), 10 µM LPA, 25 ng/ml PTX (e,f), or 10 µM LPA, 25 µM PD98059 (g,h). PTX and PD98059 were added 16 hours and 15 minutes, respectively, prior to wounding. Paraformaldehyde-fixed cells were costained with TRITC-conjugated phalloidin to visualize filamentous actin and with anti-vinculin and Alexa488-conjugated antibodies to visualize vinculin. Microscopy was performed with an inverse fluorescence microscope. Arrows in panel c point to actin-rich lamellipodia, arrows in panel d point to focal contacts. Images in g' and h' represent enlargements of the indicated sections in g and h, respectively. The illustration in i shows the merged images of g' and h'. The yellow color indicates overlapping of red and green structures. Bars, 25 µm.

View larger version (82K): [in a new window]   Fig. 7. (A) Targeting of active ERK1/2 to focal contacts. Confluent PANC-1 cells were incubated overnight in DMEM without supplements. After wounding, cells were treated with DMEM without supplements (a-a"), with 10 µM LPA (b-b"), or with 10 µM LPA and 25 µM PD98059 (c-c"). Cells were fixed after 3 hours and were costained for phosphorylated ERK1/2 (pTpY-ERK1/2 antiserum and Alexa488-conjugated antibodies) and vinculin (anti-vinculin and Cy3-conjugated antibodies). Merged images are shown in a", b" and c". Arrows point to focal contact structures. Microscopy was performed with an inverse fluorescence microscope. Bars, 10 µm. (B) Localization of active ERK1/2 during LPA treatment. Serum-starved PANC-1 cells were wounded, treated for 3 hours, 5 hours and 8 hours with 10 µM LPA, fixed and then stained for phosphorylated ERK1/2 using pTpY-ERK1/2 antiserum and Alexa488-conjugated antibodies. Bars, 20 µm.

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