© 1996 American Heart Association, Inc.
Articles |
EJ-Ras Inhibits Phospholipase C
1 but Not Actin Polymerization Induced by Platelet-Derived Growth Factor-BB via Phosphatidylinositol 3-Kinase
From the Department of Medicine, Division of Cardiology (A.W.H., D.E.K., L.E.C., P.J.G.-C.), the Department of Oncology (R.W.T.), and the Department of Cell Biology and Anatomy (R.W.T., P.J.G.-C.) of the Johns Hopkins University School of Medicine, Baltimore, Md; the Department of Pathology (E.R.F.), Yale University School of Medicine, New Haven, Conn; and the Department of Cancer Research (K.S.K.), Merck Research Laboratories, West Point, Pa.
Correspondence to Dr Pascal J. Goldschmidt-Clermont, 1023 Ross, Johns Hopkins University, 720 Rutland Ave, Baltimore MD 21287.
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Abstract |
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 Top Abstract IntroductionMaterials and Methods Results Discussion References |
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Abstract Transformation of fibroblast-like cells (NIH 3T3) by a constitutively activated GTP-bound isoform of p21ras (EJ-Ras) produces morphogenic changes characterized by decreased attachment to the substratum, with retraction and rounding of the cell body. Transformed fibroblasts lose their "stressed" conformation and adopt a "relaxed" morphology. The specific molecular mechanisms responsible for these changes remain uncharacterized. We found that EJ-Ras transformation of NIH 3T3 cells decreased the cellular content of polymerized actin, particularly at the expense of actin stress fibers, but induced the accumulation of actin filaments in peripheral ruffling membranes. Polymerization of actin could be induced in EJ-Ras–transformed cells by exposure to platelet-derived growth factor (PDGF)-BB to an extent similar to that observed in wild-type NIH 3T3 cells. In EJ-Ras cells, actin polymerization was independent of phospholipase C
1 (PLC1) activity, because inositol
tris-phosphate (IP3) production observed in
control NIH 3T3 cells in response to PDGF-BB was absent. Although
PDGF-BB did stimulate tyrosine phosphorylation of
PLC1, the phospholipase was strongly inhibited by an
inhibitory factor present in the cytoplasm of
EJ-Ras–transformed cells. In addition, cytoplasmic extracts of
EJ-Ras, but not of control cells, inhibited phosphatidylinositol
4,5-diphosphate (PIP2) hydrolysis catalyzed by a
recombinant PLC1 in vitro. Although PIP2 hydrolysis
could not contribute to the reorganization of the actin cytoskeleton
induced by PDGF-BB in EJ-Ras–transformed cells,
phosphatidylinositol 3-kinase (PI3-K) was necessary for actin
polymerization. Wortmannin, a specific PI3-K inhibitor, not
only blocked actin polymerization in both control and
EJ-Ras–transformed cells but actually led to rapid actin
depolymerization when these cells were exposed to
PDGF-BB. Thus, in EJ-Ras–transformed cells, cell morphogenic
changes in response to PDGF-BB rely importantly on PI3-K and can occur
in the complete absence of IP3 production despite
tyrosine phosphorylation of PLC1.
Key Words: actin • platelet-derived growth factor receptor • tyrosine kinase • p21ras • EJ-Ras
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Migration of vascular cells from the media to the intima corresponds with an important step in the pathological processes of restenosis and atherogenesis.1 This migration is strongly influenced by PDGF, a potent chemotactic agent for smooth muscle cells and fibroblasts,2 3 and requires a rapid and reversible reorganization of the actin cytoskeleton.4 5 The characterization of intracellular signaling events regulating this coordinated membrane and cytoskeletal response remains incomplete, but in smooth muscle cells, PLC
1 seems to play a central
role.6 Site-directed mutagenesis analysis of
PDGF-R has demonstrated that the respective bindings of PI3-K and
PLC1 to phosphotyrosine residues 708 and 719 and residues 977 and
989 of PDGF-R are both important in the motile response stimulated by
PDGF.7 The effect of these initial activation events is
also modulated by the activity of small GTP-binding proteins of the
p21ras family (eg, Rho, Rac, and Cdc
42).8 9 10
Rac, in particular, is necessary for stabilization of specific actin
structures required for membrane ruffling and motility induced by
growth factors.9 How small GTP-binding proteins and inositol phospholipids interact in the regulation of the actin cytoskeleton is now better understood.11 12 13 Binding of the fibronectin receptor to its extracellular ligand triggers the activity of phosphatidylinositol 4-phosphate 5-kinase in a GTP-Rho–dependent manner.11 12 Rac association with GTP (the active conformation) is regulated by PI3-K.13 To help characterize further the relationships between these messenger systems, we studied the actin cytoskeleton of NIH 3T3 cells transformed with EJ-Ras, an oncogenic mutant of the cellular Ha-Ras, which produces a constitutive "p21ras-on" state of the transformed cells.14 Previous work has shown that EJ-Ras prevented PDGF signaling by inhibiting tyrosine phosphorylation (or by increasing dephosphorylation) of PDGF-R.15 16 However, new evidence suggested that reduced tyrosine phosphorylation of the PDGF-R could be secondary (because of the relaxed state of cell adhesion) instead of causative in the altered signaling system of EJ-Ras–transformed cells.17 Therefore, we decided to characterize this signaling system further and to study the consequences of signaling alterations in the ability of PDGF-BB to induce an actin response.
We found that EJ-Ras transformation did not completely prevent tyrosine
phosphorylation of PDGF-R and had little effect on
PLC1 tyrosine phosphorylation in response to
PDGF-BB. Instead, EJ-Ras transformation produced a soluble factor that
completely inhibited PLC1 activity and was responsible for
inhibiting PIP2 hydrolysis and downstream signaling events.
In spite of the absence of stimulated PIP2 hydrolysis in
EJ-Ras–transformed cells, PDGF-BB was able to produce actin
filament polymerization that was inhibitable by wortmannin, a specific
PI3-K inhibitor. In fact, inhibition of PI3-K in both
control and EJ-Ras–transformed cells led to
depolymerization of actin filaments upon exposure
to PDGF-BB. This paradoxical effect strongly suggests that PDGF
normally induces concurrent polymerization and
depolymerization of actin, with resulting net
polymerization. Wortmannin also prevented the increased baseline
motility of EJ-Ras–transformed cells and the motile response of
these cells to PDGF-BB (L.E.C., A.W.H., P.J.G.-C., R.W.T., unpublished
data). Thus, our data support the prime importance of the PI3-K pathway
in EJ-Ras–transformed cells in producing morphogenic changes
transiently induced by PDGF-BB and constitutively induced by EJ-Ras.
These results emphasize the interaction between small GTP-binding
proteins and specific enzymes of the inositol signaling pathway and
suggest new strategies for the prevention of restenosis and
atherogenesis.1
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Materials
DMEM (high glucose), CO2-independent medium, Dulbecco's PBS, penicillin-streptomycin, FCS, G418 sulfate, trypsin-EDTA, and Lipofectin reagent were from GIBCO. Agarose-conjugated anti-p21ras IgG, and monoclonal anti–Ha-Ras antibodies were from Santa Cruz Biotechnology. Recombinant human PDGF-BB, anti–PDGF-R antibody (type A/B), agarose-conjugated and soluble anti-phosphotyrosine IgG, anti-p85 of PI3-K, and anti-PLC
1 monoclonal IgG were from Upstate Biotechnology Inc. C4
monoclonal anti-actin antibody was from ICN. Protein A–Sepharose
was from Pharmacia. Rhodamine phalloidin was from Molecular Probes.
Wortmannin, BSA (radioimmunoassay grade), antipain, pepstatin,
chymostatin, and leupeptin were from Sigma Chemical Co. AEBSF was from
Calbiochem. Precoated thin-layer chromatography
silica H plates with 1% potassium oxalate were from Analtech. Glacial
acetic acid was from J.T. Baker, Inc.
[3H]PIP2 and [3H]inositol
were
from Amersham. En3Hance spray (surface
autoradiography enhancer) was from Dupont
(Biotechnology Systems, NEN Research Products). All chemicals were
first grade and, unless otherwise indicated, were from Sigma.
Recombinant PLC1 was prepared and characterized as previously
described.18 Polyacrylamide 4% to 20% gradient
gels were from Jule. Horseradish peroxidase–labeled IgGs were from
HyClone. The ECL technique (Amersham) was used to develop Western
blots. Densitometry was used for the analysis chemiluminograms
where quantitative data on the intensity of bands on Western blots are
provided.
Constructs and Cell Lines
To obtain EJ-Ras–transformed
cell lines, NIH 3T3 cells
(American Type Culture Collection) were cotransfected with 20 µg of
pUC19/EJ-Ras and 1 µg of pCMVNeo using Lipofectin reagent and the
manufacturer's instructions. G418-resistant control cell lines
were obtained by Lipofectin transfection of NIH 3T3 cells with 1 µg
of pCMVNeo. The plasmid pUC1-9/EJ-Ras contains a 6.6-kB
BamHI fragment with EJ-Ras cDNA and its promoter from the EJ
bladder carcinoma cell line.14 The pCMVNeo plasmid encodes
resistance to G418.19 Selection of clones was carried out
by dilution in 96-well plates in G418 sulfate at 600 mg/L and reduced
to 250 mg/L for maintenance of lines. The cell lines 5A2 and
5A6 are representative clonal cell lines expressing
EJ-Ras, whereas the EJ-Ras pool corresponds to a mixture of 
25
G418-resistant clones after transfection with pUC19/EJ-Ras and
pCMVNeo. The control cells (Neos) were expanded from 20 G418 clones
after transfection with pCMVNeo.
Cells were grown in DMEM, to which was added 1% penicillin-streptomycin, 5% FCS, and 250 mg/L G418 sulfate (complete medium). Cells were grown at 37°C in a water-jacketed incubator with a humid 5% CO2 atmosphere. Medium was exchanged twice weekly, and cells were passaged at subconfluence with 0.05% trypsin and 0.53 mmol/L EDTA. Before stimulation with PDGF-BB, cells were maintained for 48 hours in DMEM supplemented with 0.5% FCS (serum-starvation conditions).
p21ras Immunoassay
The preparation of cell extract
was performed according to our
standard procedure20 ; briefly, cells were lysed in melting
ice-cold buffer containing 145 mmol/L NaCl, 0.1 mmol/L
MgCl2, 15 mmol/L HEPES (pH 7.0), 5 mmol/L EGTA, 1
mmol/L AEBSF, 20 mg/L each of chymostatin, leupeptin, antipain, and
pepstatin, 1.0 mmol/L sodium vanadate, and 0.5% Triton X-100 (lysis
buffer). Cell lysates of 5A2 and 5A6 clones, of the EJ-Ras pool, and of
neomycin-resistant controls were normalized for total
protein at 2 g/L. Cell lysates (2 mL) were incubated with 15 µL of
agarose bead–conjugated anti–pan-p21ras IgG
for 12 hours at 4°C. Beads were washed with ice-cold PBS and
boiled in SDS buffer,20 and the supernatants were
electrophoresed on SDS-polyacrylamide gels and then transferred
onto nitrocellulose.21 Western blots were incubated with
1:40 mouse monoclonal anti–pan-p21ras IgG or 1:50
rat monoclonal anti–Ha-Ras IgG and then with 1:400
peroxidase-labeled goat anti-mouse or goat anti-rat,
respectively; we used the ECL detection system for all Western
blots.20
Filamentous Actin Assay
Confluent cells were treated with 30
µg/L PDGF-BB for 1, 3, 9,
or 27 minutes or with vehicle buffer (120 mmol/L NaCl, 4 mmol/L
KCl, 1 mmol/L KH2PO4, 1 mmol/L
MgSO4, 1.3 mmol/L CaCl2,
and 1.2 mmol/L HEPES [Krebs/HEPES]+0.1% BSA) for 27 minutes,
followed immediately by fixation with 3.7% formaldehyde in PBS for 30
minutes. Fixed cells were permeabilized using 0.2%
Triton X-100, and F-actin was stained with 0.2 µmol/L rhodamine
phalloidin, a concentration shown to be saturating.20
Unbound fluorescence was washed away, and bound
fluorescent indicator was extracted in 0.1 mol/L NaOH
overnight. Fluorescence corresponding to the rhodamine
phalloidin that was bound to actin filaments was measured at 580-nm
(slit, 5 nm) emission and 550-nm (slit, 2 nm) excitation, then was
normalized to the total protein content measured by Bradford
assay21 in identically treated cells, and was expressed as
a fraction of the fluorescent signal measured in unstimulated
control cells.
F-actin was also measured using a biochemical assay on microsamples.20 Briefly, 5x105 cells were extracted in ice-cold lysis buffer (150 µL) supplemented with rhodamine phalloidin (0.6 µmol/L, shown to be saturating).20 The extracts were homogenized and normalized and then centrifuged at 150 000g for 60 minutes. Pelleted actin filaments were rinsed once with plain lysis buffer (200 µL). Rhodamine phalloidin was solubilized in methanol (200 µL) overnight at 4°C and then transferred into 96-well microtiter plates (Corning, flat bottom, low protein binding). Fluorescence was measured in a CytoFluor 2300 (Millipore), using excitation at 530 nm and emission at 590 nm.
Total actin was determined for cell lysates by reacting Western blots of normalized extracts with 1:1000 C4 monoclonal anti-actin antibody and 1:500 peroxidase-labeled goat anti-mouse. The blots were developed with the ECL method. The densities of actin bands were compared with those of actin standards to calculate cellular actin concentration.
Fluorescence Microscopy and Confocal Analysis of
the Actin Cytoskeleton
Cells cultured on coverslips were treated with
30 µg/L PDGF-BB
for 1 or 5 minutes, and the F-actin cytoskeleton was stained with 0.2
µmol/L rhodamine phalloidin. Photomicrography was performed using a
Zeiss Axiophot microscope and Ektachrome Elite (ASA 400). Laser
scanning confocal microscopy was performed with a BioRad MRC 600, and
rhodamine fluorescence was detected with a BioRad GHS filter
block. Sequential images (z series) represented slices
1.5 µm thick and 1 µm apart in the z axis (into/out
of the plane of the slide). Images were stored on optical disk, and
artificial color hard copies were generated on a color video
printer.
PDGF-R Tyrosine Phosphorylation Assay
PDGF-BB was dissolved
(10 mg/L) in aqueous 0.1 mol/L acetic acid
and 0.1% BSA and was used at a final concentration of 30 µg/L in PBS
to stimulate confluent cells. Serum-starved cells were treated at
37°C with PDGF-BB for 1 to 20 minutes or were treated with vehicle
alone for 20 minutes. Stimulation was stopped by scraping cells into
melting ice-cold lysis buffer.20 Lysates were
sonicated for 10 seconds (at energy level 1 to 2; Sonifier 450,
Branson, Inc) and centrifuged at 14 000 rpm for 3 minutes
(Eppendorf centrifuge 5415C). Supernatants were normalized for
total protein with lysis buffer and incubated with rabbit polyclonal
anti-human PDGF-R type A/B antibody for 10 minutes at 4°C and
then with protein A–Sepharose for 12 hours. Electrophoresis and
Western blotting were performed as described above,21 and
blots were reacted with 1:200 anti–PDGF-R antibody and 1:1000
peroxidase-labeled goat anti-rabbit IgG for quantification of
total PDGF-R or with 1:1000 mouse monoclonal anti-phosphotyrosine
and 1:1000 peroxidase-labeled goat anti-mouse to detect the
fraction of tyrosine-phosphorylated receptor.
PLC1 Phosphorylation and Enzymatic
Assay
Extracts of PDGF-stimulated and of control cells (conditions as
above) were immunoprecipitated with agarose-conjugated mouse
monoclonal anti-phosphotyrosine (for
tyrosine-phosphorylated PLC1, 5 µL of beads
for 500 µL of extract) or with multimonoclonal antibodies for PLC1
and protein A–Sepharose beads (for total PLC1, 2 µg of
anti-PLC1 and 10 µL of beads); the precipitated immune complexes
were electrophoresed and blotted, and blots were reacted with 1:1000
multimonoclonal anti-PLC1 and 1:1000 peroxidase-labeled goat
anti-mouse antibody.
To test for the presence of an inhibitor of
PLC1 in the
cytosol of control and EJ-Ras–transformed cells, cell extracts
were added to purified recombinant PLC1 (0.14 nmol/L), and total
resulting PLC activity (intrinsic to the extract plus exogenous
recombinant PLC1) was measured on SSLVs.22 23
PLC
activity was measured in the presence of cellular extracts obtained
from control and EJ-Ras–transformed cells using a lysis buffer
containing lower Triton X-100 concentration (0.05%) and, as the
substrate, SSLVs containing PIP2 (50 µmol/L, with traces
of [3H]PIP2) and
phosphatidylethanolamine (1:10 ratio). Each sample contained 5
µL of SSLVs, various volumes of cell extract (as indicated, 1.0 g/L),
and 40 µL of vesicle buffer containing 5 mmol/L Tris (pH 7.0), 0.4
mmol/L EGTA, and 0.2 mmol/L CaCl2.22 23
Released [3H]IP3 was isolated by lipid
extraction at various time points and quantified in a scintillation
counter.22 23
IP3 Competitive Binding Assay
IP3 mass
measurements were made using
IP3-binding protein prepared from rat cerebellum. Each rat
cerebellum was added to 10 mL of Tris-EDTA buffer (50 mmol/L Tris-HCl
[pH 7.7], 1 mmol/L EDTA, and 1 mmol/L ß-mercaptoethanol). After
centrifugation at 13 000 rpm (SS-34 rotor) for 15
minutes, membrane preparations were obtained after washing the pellets
twice with Tris-EDTA buffer, followed by resuspension in the same
buffer. A standard curve was obtained with known concentrations of
[3H]IP3 in Tris-EDTA buffer (adjusted to pH
8.4), such that a linear relationship from 1 to 100 nmol/L
IP3 was obtained. Each binding reaction consisted of adding
50 µL of IP3-binding protein preparation to 50 µL of
IP3 standards and 400 µL of Tris-EDTA buffer (pH 8.4) in
Eppendorf tubes. The reactions were vortexed vigorously for 1 second,
incubated for 20 minutes at 0°C to 4°C, and centrifuged for
5 minutes at 14 000 rpm in Eppendorf tubes; the supernatants were then
discarded. The pellets were resuspended in 0.5 mL of Tris-EDTA buffer
(pH 8.4), washed twice in the same buffer, and then added to
scintillation fluid to be counted.
Measurements of IP3 at various time points of cultured cell assays (performed in 35-mm cuvettes) were performed by stopping the reactions with 1 mL of ice-cold 10% trichloroacetic acid, scraping, and sonicating for 10 seconds. The suspensions were then placed on ice and centrifuged for 15 minutes (in Eppendorf tubes, 14 000 rpm). Supernatants were assayed for IP3 content. For the competitive assays, unlabeled IP3 produced by NIH 3T3 cells (both control and EJ-Ras–transformed cells, either quiescent or activated) was mixed with [3H]IP3 (100 nmol/L) in 50-µL samples before mixing with the aliquot of IP3-binding protein. A decrease in 3H count in the pellets indicated that more cold IP3 competed with labeled IP3 for the binding protein.
Inositol Phospholipid Mass Measurements
The mass of major
inositol phospholipids in NIH 3T3 cells (PI,
PIP, and PIP2) was determined by thin-layer
chromatography of extracted lipids. The inositol
phospholipids of confluent NIH 3T3 cells in six-well plates were
labeled by starving the cells from inositol for 48 hours
(inositol-free DMEM, 5% dialyzed FBS) and then adding
[3H]inositol to the cultures (10 to 50 µCi). At
various
time points, the cells were extracted by scraping the cells in
methanol/chloroform (2:1 [vol/vol]). The organic extracts were dried
under nitrogen, resuspended in 750 µL of methanol/chloroform (2:1),
vortexed, added to 500 µL chloroform/1 mol/L HCl (1:1
[vol/vol]),
and centrifuged for 5 minutes at 3000 rpm in a Beckman tabletop
centrifuge. The lower phase was collected (containing inositol
phospholipids), dried under nitrogen, and then resuspended in 50 µL
of methanol/chloroform (5:95 [vol/vol]). The samples were spotted at
the base (2.5 cm from bottom) of thin-layer
chromatography silica H plates, 2 cm apart, with
constant drying by warm air to reduce the size of the spots.
Chromatography was initiated by immersing the bottom (1
cm) of the plates in organic solvent
(CHCl3/MetOH/NH4OH [4N], 9:7:2
[vol/vol/vol] for a total of 180 mL) in a vapor glass chamber.
Chromatography was stopped when the yellow dye reached
the top of the plate. The position of individual inositol lipids was
determined by labeled standards. A 2x2-cm area of the thin gel
corresponding to each phospholipid spot was scraped and counted in
scintillation fluid.
PI3-K Inhibition
PI3-K activity was blocked with the
Penicillium-derived metabolite wortmannin. Wortmannin has
been shown to inhibit the activity of PI3-K in a noncompetitive and
irreversible fashion with an IC50 of <4
nmol/L.24 Serum-starved cells were incubated for 45
minutes with wortmannin (11 or 22 nmol/L) before exposure to PDGF-BB.
The effect of PI3-K inhibition on actin polymerization after the
addition of PDGF-BB was measured by rhodamine phalloidin assays (see
above) and expressed as percent change in F-actin concentration
relative to that of untreated cells.
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Results |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Morphogenic Effects of EJ-Ras Transformation
NIH 3T3 clonal cell lines transfected with EJ-Ras cDNA, encoding an oncogenic mutant (valine substituted for glycine at amino acid 12) of the cellular Ha-Ras, appeared morphologically in a relaxed conformation when compared with control cells transfected with pCMVNeo vector only. Retracted, fusiform, and highly polarized cell bodies (with occasional long cytoplasmic extensions) having the tendency to cluster within foci (similar to stratified epithelium) were characteristic of the transformed phenotype. These morphological changes were associated with altered growth characteristics of EJ-Ras–transformed NIH 3T3 cells, as the growth and DNA synthesis activities of EJ-Ras–transformed cells were not inhibited at confluence or in medium containing low (0.5%) serum supplementation (data not shown). In contrast, Neo control cells formed monolayers of flattened and stressed cells and were growth-inhibited at confluence and/or in low serum.
Demonstration of EJ-Ras on Western Blots
To demonstrate
further that these phenotypic changes observed in
the transfected cells were associated with EJ-Ras expression, we
assessed the overexpression of EJ-Ras protein by Western blot assay
(Fig 1
). Lysates of two Ras-transformed cell lines
(clones 5A2 and 5A6) and of EJ-Ras pool clones contained a band of
p21ras immunoreactivity, not present in control cells
(Neos), with an electrophoretic motility on 4% to 20% gradient
SDS-PAGE that was slightly faster than the cellular p21ras
present in all cell lines studied. This band was detectable both
with anti–pan-p21ras and with
anti–Ha-Ras–specific antibodies.
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Changes in Actin Cytoskeleton in EJ-Ras Cells, With and
Without PDGF-BB
Organization and distribution of the actin network in
EJ-Ras–transformed cells was strikingly different from that in
control cells (compare panels A and B of Fig 2). When
incubated in 0.5% FCS, Neo control cells contained multiple stress
fibers, stable cell-spanning bundles of F-actin and associated
regulatory proteins (stressed cells).17 These stress
fibers were nearly absent in EJ-Ras–transformed clones; instead,
F-actin was concentrated under the cell membrane, particularly at sites
of membrane extension or ruffling.
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PDGF-BB stimulation induced rapid redistribution of actin filaments in control cells, with loss of stress fibers from the cell center and concentration of F-actin under the plasma membrane, particularly in areas where membrane ruffling appeared prominent. In EJ-Ras–transformed cells, F-actin was already concentrated at the cell periphery, and further redistribution after PDGF-BB treatment could not be detected by confocal microscopy (data not shown).
F-actin
quantification revealed that at baseline, control cells
contained more polymerized actin than did EJ-Ras–transformed cells
(Fig 3). Thus, despite having a more motile
phenotype, the EJ-Ras–transformed cells contained lower
basal concentrations of F-actin (43% that of control cells). However,
a marked actin polymerization response to PDGF-BB in
EJ-Ras–transformed cells was observed, similar in extent and
kinetics to that observed in control cells (Fig 3). After 3
minutes in
PDGF-BB, the concentration of polymerized actin peaked in all cell
types studied, independent of the p21ras status, and was
increased 60% over baseline in Neo control cells and 87% in
EJ-Ras–transformed cells. Total actin, assessed by densitometric
analysis of Western blots, was not affected by
p21ras transformation (Fig 3, inset), and was
70.3±7.5
µmol/L, or 1.4±0.2% of total cellular protein (mean±SEM).
Calculations are based on estimated cell volume of 1923
µm3 as previously described.20
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We concluded from this series of experiments that EJ-Ras transformation of NIH 3T3 cells induced marked morphological changes through reorganization of the actin cytoskeleton. Despite the effect of EJ-Ras on the actin cytoskeleton, we observed that the actin cytoskeleton of EJ-Ras–transformed cells was still responsive to PDGF-BB. To characterize the signaling pathways that regulate these morphological changes, we examined signaling reactions after exposure to PDGF-BB, starting with the tyrosine phosphorylation of the receptor itself.
PDGF-R Tyrosine Phosphorylation
In the absence of agonist, no
phosphotyrosine immunoreactivity was
detected on PDGF-R (Fig 4, top) in either control or
EJ-Ras–transformed cells. After agonist binding, tyrosine
phosphorylation of the PDGF-R was detected within 1
minute; in control cells, PDGF-R phosphotyrosine immunoreactivity
(density on Western blot developed by ECL) increased 16-fold over
background, whereas 5A2 and 5A6 cells' immunoreactivity at 1 minute
increased only 6- and 4-fold, respectively. In control cells, continued
PDGF-BB exposure resulted in a further marked increase in tyrosine
phosphorylation; both the early response and the late
increase of PDGF-R tyrosine phosphorylation were
reduced in the EJ-Ras–transformed cell lines. After 20 minutes of
exposure to PDGF-BB, PDGF-R phosphotyrosine immunoreactivity was
increased 63-fold over baseline in control cells, whereas the activity
of 5A2 and 5A6 cells increased only 10±5-fold over baseline. Total
PDGF-R cellular content, assessed by Western blot, was similar in all
cell types (data not shown).
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Tyrosine Phosphorylation of PLC1
Because tyrosine
phosphorylation of the PDGF-R in
EJ-Ras cells was partially inhibited, we suspected that the
phosphorylation of PLC1 might be impaired as well.
Tyrosine phosphorylation of PLC1 is required for
PIP2 hydrolysis in response to PDGF-BB and has been linked
to the actin cytoskeletal response to growth
factors.25 26
Western blot analysis of anti-PLC1 immunoprecipitates
stained for phosphotyrosine residue revealed only mildly (30%)
decreased tyrosine phosphorylation of PLC1 in EJ-Ras
cells stimulated by PDGF-BB (30 µg/L) (Fig 4
, bottom).
Actually, the
level of tyrosine phosphorylation of PLC1 in EJ-Ras
cells induced by 30 µg/L of PDGF-BB was between that induced by 10
and 30 µg/L PDGF-BB in control cells (Table), and 10
µg/L of PDGF-BB was sufficient in control cells to induce
IP3 production. Since the tyrosine
phosphorylation of PLC1, which is required for
PLC1 activity, was nearly intact in EJ-Ras cells, we studied the
hydrolysis of PIP2 in these cells exposed to PDGF-BB.
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PDGF Stimulation of IP3 Production Was Lacking
in EJ-Ras–Transformed Cells
The time course of IP3
production measured by
competitive binding assay revealed that PDGF-BB stimulated a 3.7-fold
IP3 increase, peaking at 40 to 60 seconds in control NIH
3T3 cells (Table). IP3 production was dependent on
the dose of PDGF-BB, such that 3 µg/L of PDGF-BB stimulated no
detectable IP3 increase, whereas 10 and 30 µg/L
stimulated significant IP3 production (Table). In
contrast, in EJ-Ras–transformed NIH 3T3 cells, addition of PDGF-BB
(30 µg/L) did not stimulate any IP3 production
(Table). Even after exposure of EJ-Ras–transformed cells
to
lithium (to block IP3 recycling), PDGF-BB–induced
production of IP3 was still undetectable (data not
shown). The absence of IP3 production in
transformed cells is paralleled by the calcium transient
response, which is present in control cells but absent in
transformed cells (L.E.C., A.W.H., P.J.G.-C., R.W.T., unpublished
data). Lack of IP3 increase did not result from
constitutively elevated IP3 content in
EJ-Ras–transformed cells (Table). We next determined
whether a
lack of substrate (PIP2) could account for the absence of
PDGF-stimulated IP3 production in
EJ-Ras–transformed NIH 3T3 cells.
Phosphoinositide Content Is Similar in Control and
EJ-Ras–Transformed Cells
The cellular content of
PIP2, PIP, and PI was
measured by thin-layer chromatography in control
and EJ-Ras–transformed NIH 3T3 cells. Relative amounts of the
phosphoinositides were similar in control and in
EJ-Ras–transformed NIH 3T3 cells (Fig 5).
Phosphoinositide content was measured by scintillation
counting of the scraped thin-layer chromatography
gels corresponding to individual inositol phospholipid spots. Whether
counts per minute values corresponding to the PIP and PIP2
spots were normalized to total protein or to the PI mass, no difference
could be detected between control and EJ-Ras–transformed cells.
Thus, the lack of PIP2 hydrolysis in PDGF-stimulated
EJ-Ras–transformed cells was not a result of limited substrate
availability.
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Recombinant PLC1 Inhibition by EJ-Ras Cell
Extracts
In spite of nearly normal tyrosine phosphorylation
of PLC1 in PDGF-activated EJ-Ras–transformed cells, we
found no detectable PLC activity in extracts from the
Ras-transformed cells obtained before or after PDGF stimulation.
This finding is consistent with our observation that the
production of IP3 by EJ-Ras–transformed cells
in response to PDGF-BB is negligible (Table). Moreover, the
activity of
a purified recombinant PLC1 was markedly reduced upon the addition
of cytosolic proteins from EJ-Ras–transformed cell extracts but
not by the cytosolic proteins extracted from control cells (Fig
6). As shown in Fig 6B, 50 µg/mL of
extracted protein
was nearly able to block the activity of 14 nmol/L of recombinant
PLC1. Together, these data support the concept that the cytosol of
Ras-transformed cells contained an inhibitory factor
(or factors) for PLC1 and that this inhibitor was
responsible for the complete suppression of PLC1 activity in
EJ-Ras–transformed cells.
|
) and EJ-Ras–transformed (
) cells. Time course of
[3H]IP3 production by recombinant
PLC
) and EJ-Ras–transformed (
) cells. No native
PLC activity was detected in EJ-Ras–transformed cell extracts.
Recombinant PLC
PI3-K and Actin Response to PDGF
Since EJ-Ras transformation
prevented PIP2 hydrolysis
induced by PDGF, we investigated whether another aspect of inositol
phospholipid turnover was involved with actin changes induced by PDGF.
PI3-K, a key mediator of the signaling response orchestrated by
activated PDGF-R, phosphorylates inositol lipids
and contributes to the actin response to PDGF in nontransformed
cells.7 27 We used a selective PI3-K
inhibitor, wortmannin,24 to test whether PI3-K
activation is required for PDGF stimulation of actin changes in
EJ-Ras–transformed cells. Pretreatment of EJ-Ras–transformed
cells with wortmannin completely abolished the polymerization of actin
induced by PDGF-BB (Fig 7). Indeed, exposure of both
control cells and EJ-Ras–transformed cells to wortmannin led to
rapid depolymerization of actin filaments, a
response quite the opposite of that seen in cells not treated with
wortmannin. Even higher concentrations of PDGF-BB (up to 100 µg/L)
did not overcome wortmannin inhibition (Fig 7). Using
immunofluorescence microscopy, we confirmed the
presence of PI3-K within the cytoplasm of control and
Ras-transformed cells and its concentration within the ruffling
area of the plasma membrane (data not shown). After treatment with
wortmannin and PDGF, rhodamine phalloidin microscopy showed that what
little F-actin remained was organized in spotty clusters; these cells
were not rounded, however, and resembled untreated cells except that
ruffles were not present after exposure to PDGF (data not shown).
These experiments suggest that unlike PLC1, PI3-K plays an important
role in the regulation of the actin cytoskeleton and morphological
changes observed in NIH 3T3 cells transformed with EJ-Ras.
|
|
Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Actin Organization in EJ-Ras–Transformed Cells
F-actin concentration (normalized for total protein) is reduced in the EJ-Ras–transformed cell lines, consistent with the observation of Rao et al,28 although total actin content does not differ between transformed and control cells. The increased basal and PDGF-stimulated motility of the transformed cells therefore does not correlate with the cellular concentration of F-actin. Rather, it is the distribution of F-actin, and in particular its local concentration within membrane ruffles, which correlates best with the dynamic morphological changes associated with the transformed phenotype and with PDGF-BB stimulation.
The EJ-Ras–transformed phenotype suggests a crucial role for Rac in the organization of the actin cytoskeleton of these cells8 9 10 29 30 ; the morphology of EJ-Ras–transformed cells is similar to that obtained with a dominant positive Rac mutant microinjected in fibroblasts.9 10 The involvement of Rac in the transformed phenotype is further suggested by the response to PI3-K inhibition, since PI3-K controls the association of Rac with GTP.13 Wortmannin not only inhibits PI3-K and blocks the expected actin response to PDGF of control and EJ-Ras–transformed cells but actually reverses the response to PDGF. In wortmannin-treated EJ-Ras and control cells, PDGF exposure leads to actin depolymerization, suggesting that the regulation of balance between monomeric versus filamentous actin by PDGF is complex and involves concurrent production and disruption of actin filamentous structures, with production under the influence of PI3-K.
Little is known about reactions downstream from small GTP-binding proteins that connect these proteins to the actin cytoskeleton. Several actin-binding proteins are regulated by inositol phospholipids in their interaction with actin.27 Profilin is an essential actin-binding protein that regulates actin monomers and filament stability and binds to polyphosphoinositides (PIP and PIP2).31 The distribution of F-actin beneath the plasma membrane and within the cortical cytoskeleton of EJ-Ras–transformed cells is reminiscent of the distribution of actin filaments in cells overexpressing profilin20 or microinjected with profilin32 and of actin filaments that have incorporated microinjected G-actin subunits having a high affinity for profilin.33
Thus, profilin may represent a downstream
effector protein for
the p21ras pathway,27 directly regulating the
organization of the actin cytoskeleton. Such a hypothesis is also
supported by experiments performed in yeast, showing that
overexpression of profilin can rescue S. cerevisiae mutants
null for the C-terminal of the p21ras-regulated
adenylate cyclase associated protein.34
Adenylate cyclase–associated protein C-terminal null
cells display altered morphology that can be corrected by profilin
overexpression. Thus, the ability of profilin to stabilize cortical
actin filaments may contribute to the actin cytoskeleton organization
in EJ-Ras cells. Importantly, the effect of profilin is unlikely to be
related to its ability to inhibit PLC1,26 since this
enzyme is inactive in EJ-Ras–transformed cells (Fig 7).
Other actin regulatory proteins have been implicated in the genesis of
the EJ-Ras–transformed actin phenotype. Overexpression of
tropomyosin, whose binding to actin stabilizes filaments in vivo, has
been shown to suppress the neoplastic growth of Ras-transformed
cells, possibly by causing reversion to a phenotype with more
F-actin.35 Flat revertant clones of
EJ-Ras–transformed NIH 3T3 cells have been found to contain a
gelsolin mutant with increased affinity for
polyphosphoinositides, resulting in stronger
inhibition of PLC1 in vitro.36 37 Competitive
inhibition by polyphosphoinositides of gelsolin's
capping function for actin might decrease the actin critical
concentration in these revertants, resulting in more actin
polymerization and in a flat revertant cytoskeletal
phenotype.36 37 38 However, the mechanism
whereby
EJ-Ras might affect filament capping remains to be characterized.
Intact Actin Response to PDGF in EJ-Ras–Transformed
Cells
The p21ras mutations, such as the one found in the
EJ-Ras isoform, generate a constitutive GTP-bound conformation of the
small GTP nucleotide triphosphatase14 and
represent dominant mutations that substantially alter the
biology of the affected cells in a concentration-dependent
fashion.39 For example, transforming p21ras
isoforms alter normal growth regulation, such that cells do not become
quiescent (G0) at confluence or in the presence of sparse
nutrient supplies. EJ-Ras–transformed cells are also remarkable
for their decreased adhesion leading to relaxed
morphology40 and reduced tyrosine kinase response to
growth factors like PDGF. The morphology of these cells strongly
resembles that of fibroblasts attached to an extracellular matrix that
has been mechanically relaxed.17
In this context, we had anticipated that the actin response to growth factors such as PDGF would be markedly altered, if not absent, as a result of EJ-Ras transformation. Although we confirmed that EJ-Ras–transformed cells have increased motile activity in the absence of agonist, we found that, against expectations, the actin response to PDGF is essentially intact in EJ-Ras–transformed cells.
Actin Response to PDGF Occurs Despite PLC1 Inhibition in
EJ-Ras Cells
Two phospholipid regulatory enzymes, PI3-K and PLC1,
are
targets for the tyrosine kinase of the PDGF-R and contribute to the
actin response to PDGF.7 41 Although we confirmed
that
EJ-Ras transformation reduced the tyrosine
phosphorylation of the PDGF-R
itself,15 16 42 43 the
tyrosine
phosphorylation of PLC1 was nearly
intact.44 In spite of normal
phosphorylation of PLC1, PIP2 hydrolysis
in EJ-Ras cells stimulated by PDGF was undetectable (Table).
Although
we did not rule out the possibility that qualitative alterations of
PLC1 phosphorylation may have occurred (four
tyrosine residues can be phosphorylated on
PLC1)45 as a result of EJ-Ras transformation,
quantitatively, the net tyrosine phosphorylation of
this substrate could not account for the lack of PLC activity in the
transformed cells.
Instead, the cytosol of EJ-Ras–transformed
cells contained an
inhibitor (or inhibitors) that suppressed the
activity of cytoplasmic PLC and prevented the hydrolysis of
PIP2 by recombinant PLC1 added to the extracts.
Inhibition of PLC1 in EJ-Ras–transformed cells was not due to
profilin, because profilin inhibits only the
unphosphorylated conformation of PLC1 and not
tyrosine-phosphorylated PLC1.26
Although we are presently working on identifying the PLC1
inhibitor induced by EJ-Ras expression, our data are
consistent with a catalytic mechanism of inactivation (Fig 7).
PI3-K Is Required for the Cytoskeletal Response to
PDGF
There is growing evidence that PI3-K plays a pivotal role in the
motile (and mitogenic) responses of cells exposed to growth
factors.46 Indeed, we found that an intact PI3-K function
was necessary to generate a normal actin response to PDGF in
EJ-Ras–transformed (and control) cells. Wortmannin, an
inhibitor for PI3-K that is specific when used at low
nanomolar concentrations,24 47 efficiently prevents
PDGF-stimulated polymerization of the actin cytoskeleton. PI3-K is
present within ruffles of PDGF-activated NIH 3T3
cells48 49 50 and in
EJ-Ras–transformed cells, with and
without growth factor stimulation (data not shown).
Four mechanisms may contribute to the association of PI3-K with the plasma membrane. First, the SH2 domain of the p85 docking subunit of PI3-K binds to tyrosine-phosphorylated residues 708 and 719 of the activated PDGF-R.7 Second, GTP-bound p21ras that is membrane associated (through its farnesyl group) binds to PI3-K.51 The latter mechanism is likely to play an important role in the association of PI3-K with the ruffles of EJ-Ras–transformed cells, even in the absence of growth factor stimulation. In support of this idea, farnesyl-transferase inhibitors reverse the phenotype of cells transformed with EJ-Ras to flattened cells displaying normal actin stress fibers.52 Third, PI3-K may bind directly to its phospholipid substrates in the membrane. Fourth, PI3-K stabilizes the association of Rac with GTP.13 In turn, GTP-Rac may stabilize the interaction between p21ras and PI3-K, or alternatively, Rac may promote the generation of oxygen reactive species, through its interaction with the NADPH-oxidase complex,53 which could affect the stabilization of actin filaments within ruffles.
These findings may be of importance for
atherosclerosis
and restenosis. The intracellular pathways regulating cell
motility are affected by context: the migration of smooth muscle cells
to the intima is highly dependent on the activity of
PLC1.6 41 However, p21ras mutations
leading
to a p21ras-on state of these cells can block the PLC
pathway. In such cells, the PI3-K pathway becomes essential for the
actin reorganization in response to growth factors. Although our
understanding of the role of p21ras in proliferation and
migration of vascular cells is still scanty, it is likely that
modulation of this pathway will become an important therapeutic option
for the management of plaque formation and restenosis.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by grants from the American Heart Association, Maryland Affiliate, Inc (Grant-in-Aid); from Syntex; from the Bernard A. and Rebecca S. Bernard Foundation; and from Friends of Bobbie Burnett. Dr Heldman was the recipient of a 1993 and 1994 Research Fellowship from the American Heart Association, Maryland Affiliate, Inc. Dr Goldschmidt-Clermont was selected as a Syntex Scholar in 1992.
Received August 3, 1995; accepted November 6, 1995.
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