First published online 12 February 2003
doi: 10.1242/jcs.00308
EphrinA1 inactivates integrin-mediated vascular smooth muscle cell spreading via the Rac/PAK pathway
Christophe Deroanne1,*,
,
Valérie Vouret-Craviari1,
Bingcheng Wang2 and
Jacques Pouysségur1
1 Institute of Signaling, Developmental Biology and Cancer Research, CNRS-UMR
6543, Centre Antoine Lacassagne, 33 Avenue de Valombrose, 06189 Nice,
France
2 Rammelkamp Center for Research, R421 and Department of Pharmacology and
Ireland Cancer Center, Case Western Reserve University School of Medicine,
2500 MetroHealth Drive, Cleveland, OH 44109, USA
* Present address: Laboratory of Connective Tissues Biology, University of
Liège, Tour de Pathologie, B23/3, 4000 Liège, Sart Tilman,
Belgium
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Fig. 1. EphrinA1 represses spreading of VSMCs on laminin. (A) Activation of EphA2
by immobilized ephrinA1. Quiescent VSMCs plated on tissue culture dishes
coated with laminin (0.25 µg/cm2) and 1 µg/cm2 of
either Fc or ephrinA1 for the indicated times were lysed as described. EphA2
was precipitated (IP) with an anti-EphA2 antibody. Immunoblotting was
performed using an anti-phosphotyrosine to determine the activation of EphA2
(Phospho-EphA2) or an anti-EphA2 to control loading. The fold induction is
calculated from densitometric measurements of phosphotyrosine signal
normalized to EphA2 loading in lysates of ephrinA1-stimulated cells compared
with Fc control at each time point. (B) Representative phase-contrast
microscopy of quiescent VSMCs allowed to spread for 16 hours on tissue culture
dishes coated with laminin (0.25 µg/cm2) and the indicated
amounts of control Fc fragment (L+Fc) or ephrinA1/Fc (L+A1). Reversion of the
effect was initiated by trypsinizing VSMCs attached on dishes coated with
laminin and 1 µg/cm2 of either Fc or A1, seeding them on another
dish coated with laminin alone and allowing spreading for 24 hours. Bar, 40
µm. (C) Quiescent VSMCs were allowed to spread for the indicated times on
tissue culture dishes coated with laminin (0.25 µg/cm2) and 1
µg/cm2 of either Fc (L+Fc) or ephrinA1/Fc (L+A1). Bar, 40
µm.
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Fig. 2. ROCK inhibitor Y27632 antagonizes the ephrinA1-mediated repression of VSMC
spreading whereas ephrinA1 does not modulate RhoA activity. (A) Representative
phase-contrast microscopy of quiescent VSMCs allowed to spread on tissue
culture dishes coated with laminin (0.25 µg/cm2) and 1
µg/cm2 of either Fc (L+Fc) or ephrinA1/Fc (L+A1), in the
presence of 7 µM Y27632 or vehicle alone. Bar, 20 µm. (B) Quiescent
VSMCs were seeded on tissue culture dishes coated with laminin and either Fc
or ephrinA1/Fc (A1). Cells were harvested at the indicated times and processed
for pull-down experiments. RhoA activity is determined by the amount of
GST-RBD-bound RhoA (RhoA-GTP) normalized to total RhoA in whole cell lysates.
(C) Quiescent VSMCs were stimulated (FCS) or not (C) with 10% FCS. After 5
minutes, cells were harvested and processed for pull-down experiments. RhoA
activity is determined as described above (B).
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Fig. 3. Effect of RhoA silencing on cell spreading. (A) An siRNA targeting RhoA
strongly reduced the RhoA protein level without a significant effect on Rac1
and Cdc42 protein levels. Western blot analysis of whole cell lysates of VSMCs
transfected with 20 nM of a control siRNA (Control) or an siRNA targeting RhoA
or left untransfected (C). 48 hours post-transfection, cells were lysed in
SDS-PAGE loading buffer and analyzed by immunoblotting with specific
antibodies to RhoA, Rac1, Cdc42 and p42 MAP kinase. (B) Inhibition of RhoA
with an siRNA stabilizes process formation. Representative phase-contrast
microscopy of quiescent VSMCs allowed to spread for 4 hours (a-d) on tissue
culture dishes coated with laminin and either Fc (a,b) or A1 (c,d). VSMCs
transfected with control siRNA (a,c) or an siRNA targeting RhoA (b,d) were
used 48 hours post-transfection. Bar, 20 µm.
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Fig. 4. EphrinA1 inhibits the activation of Rac1 but does not affect the activation
of Cdc42. (A) Quiescent VSMCs were seeded on tissue culture dishes coated with
laminin and either Fc or ephrinA1/Fc (A1). Cells were harvested at the
indicated times and processed for pull-down experiments. Rac1 activity is
determined by the amount of GST-PBD-bound Rac1 (Rac1-GTP) normalized to total
Rac1 in whole cell lysates. (B) Densitometric analysis of (A). Results are the
mean±s.d. of three independent experiments. (C) Quiescent VSMCs were
seeded on tissue culture dishes coated with laminin and either Fc or
ephrinA1/Fc (A1). Cells were harvested at the indicated times and processed
for pull-down experiments. Cdc42 activity is determined by the amount of
GST-PBD-bound Cdc42 (Cdc42-GTP) normalized to total Cdc42 in whole cell
lysates.
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Fig. 5. EphrinA1 inhibits the activation of PAK1, an effector of Rac1 signaling.
(A) Quiescent VSMCs were seeded on tissue culture dishes coated with laminin
and either Fc or ephrinA1/Fc (A1). Cells were lysed in SDS-PAGE loading buffer
at the indicated times. PAK1 activity is indicated by the amount of
phosphorylated PAK1 (phosphoPAK1) normalized to the total PAK1 in each cell
lysate. (B) Densitometric analysis of (A). Results are the mean±s.d. of
three independent experiments.
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Fig. 6. Constitutively active Rac antagonizes the ephrinA1-mediated repression of
VSMC spreading. (A-B) VSMCs expressing either GFPRacN17 or GFPRacV12 cultured
on tissue culture dishes coated with laminin and either Fc (A) or ephrinA1/Fc
(B) were fixed 4 hours after seeding in the adhesion assay and analyzed by
immunofluorescence labeling with DAPI and phalloidin-TRITC. Bar, 20 µm. (C)
The morphology of GFPRacN17 (RacN17)- and GFPRacV12 (RacV12)-expressing cells
cultured on tissue culture dishes coated with laminin and ephrinA1/Fc from ten
randomly chosen fields were scored (spread or retracted). Data represent the
mean±s.d. of three independent experiments. Non-infected cells (N.I.)
in the same fields were also scored and used as a control for the ephrinA1
activity.
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Fig. 7. Repression of Rac1 synthesis amplifies the ephrinA1-mediated inhibition of
VSMC spreading. (A) Western blot analysis of whole cell lysates of VSMCs
transfected with calcium phosphate alone (Control), with an siRNA targeting
Rac1 (Rac1) or with an siRNA targeting RhoA (RhoA). 48 hours
post-transfection, cells were lysed in SDS-PAGE loading buffer and analyzed by
immunoblotting with specific antibodies to RhoA, Rac1, Cdc42 and p42 MAP
kinase. (B) Representative phase-contrast microscopy of quiescent VSMCs
cultured for 4 hours on tissue culture dishes coated with laminin (0.25
µg/cm2) and 0.5 µg of either Fc (L+0.5Fc) or ephrinA1/Fc
(L+0.5A1). VSMCs were transfected with calcium phosphate alone (Control), with
20 nM siRNA Rac1 (Rac1) or 20 nM siRNA RhoA (RhoA). Bar, 20 µm. (C) The
morphology of control (Control), siRNA Rac1 (Rac1) or siRNA RhoA
(RhoA)-treated cells from eight randomly chosen fields were scored (spread or
retracted). Data represent the mean±s.d. of three independent
experiments.
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Fig. 8. S1P amplifies the ephrinA1-mediated repression of VSMC spreading.
Representative phase-contrast microscopy of quiescent VSMCs cultured for 16
hours in the adhesion assay with vehicle (a-d) or with 0.5 µM S1P (e-h).
VSMCs where seeded on tissue culture dishes coated with laminin (0.25
µg/cm2) and either 1 µg/cm2 of Fc (a,e) or 0.2
(b,f), 0.5 (c,g), 1.0 (d,h) µg/cm2 of ephrinA1/Fc. Bar, 20
µm.
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© The Company of Biologists Ltd 2003
