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
Author for correspondence (e-mail:
c.deroanne{at}ulg.ac.be)
Accepted 5 December 2002
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Summary |
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Key words: EphrinA1, RhoGTPases, siRNA, Lamellipodia, Integrin, Laminin
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Introduction |
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While integrin activation is required in various biological processes, such
as cell-cycle progression, growth and cell survival, a dynamic in the adhesion
process is associated with either physiological or pathological situations.
Decreased adhesion is important in the destabilization of blood vessel
structures during angiogenesis
(Yancopoulos et al., 2000).
EphrinA1 and its cognate receptor, EphA2, are strongly expressed at sites of
neovascularization, and vascular endothelial growth factor (VEGF), one of the
most-potent angiogenic factors, induces the expression of ephrinA1 in
endothelial cells (Cheng et al.,
2002
; Ogawa et al.,
2000
). Therefore, it is possible that the pro-angiogenic effects
of Eph/ephrin are mediated not only through local modulation of cell adhesion
and motility of endothelial cells but also of perivascular cells. To test this
hypothesis, we analyzed the impact of EphA activation by ephrinA1 on VSMC
adhesion and morphology.
Investigation of the signaling pathways involved in ephrinA1-induced
morphological alterations, in particular spreading inhibition, underlined the
key role played by the Rac/PAK pathway. While specific ablation of RhoA using
the double-stranded siRNA approach had a limited effect, decreased levels of
Rac1 using siRNA or decreased activation of Rac1 using S1P led to a decrease
in cell spreading and amplification of the action of ephrinA1. We therefore
conclude that the Rac activity in VSMCs is essential in mediating the action
of ephrinA1, a control point that could be reinforced in vivo by the lipid
mediator S1P (Ryu et al.,
2002).
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Materials and Methods |
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Cell adhesion assay
To analyze cell adhesion and spreading on immobilized proteins, tissue
culture dishes were coated overnight with laminin (0.25 µg/cm2)
and Fc (1 µg/cm2), or with laminin (0.25 µg/cm2)
and ephrin A1/Fc (1 µg/cm2) in PBS. In some experiments, laminin
was replaced by fibronectin (0.125 µg/cm2). Nonspecific sites
were blocked with 1% BSA for 30 minutes. Starved VSMCs were detached
non-enzymatically with CDS, washed twice in DMEM+0.1% BSA and seeded on coated
dishes in the same medium. As already published by others, we observed that
coating with only ephrin A1/Fc only permits cell adhesion but does not allow
cells spreading.
Immunoprecipitation and immunoblotting
Subconfluent VSMCs were chilled on ice, rinsed twice with ice-cold PBS and
lysed in buffer containing 1% Triton X-100, 50mM Tris pH 7.4, 100 mM NaCl, 50
mM NaF, 40 mM ß-glycerophosphate, 1 mM Na3VO4, 0.1
mM AEBSF, 4 µg/ml aprotinin, 1 µg/ml leupeptin and 1 µg/ml pepstatin.
Immunoprecipitations were carried out using 1 µg of rabbit anti-EphA2.
After 1 hour of incubation at 4°C, 40 µl of protein-A-Sepharose beads
were added for 1 hour to capture immune complexes. Beads were washed four
times with lysis buffer and boiled in 60 µl SDS-PAGE lysis buffer. For
immunoblotting, samples were separated on a 6% gel and transferred to
Immobilon-P PVDF membrane. EphA2 and phosphotyrosines were immunodetected with
the corresponding antibodies. The bands were visualized with the
electrochemiluminescence (ECL) system.
siRNA transfection
21-nucleotide RNAs were chemically synthesized and purified by
reverse-phase HPLC (Eurogentec). To inhibit Rac1 synthesis, we used the
5'-GUUCUUAAUUUGCUUUUCCTT-3' and
5'-GGAAAAGCAAAUUAAGAAC-3' oligoribonucleotides and, to inhibit
RhoA, we used the 5'-GAAGUCAAGCAUUU-CUGUCTT-3' and
5'-GACAGAAAUGCUUGACUUCTT-3' oligoribonucleotides. Control siRNA
primers corresponded to 21-nucleotide RNAs from Drosophilia hypoxia
inducible factor 1. Each pair of oligoribonucleotides was annealed at a
concentration of 20 µM in 100 mM potassium acetate, 2 mM magnesium acetate,
30 mM HEPES-KOH pH 7.4. Calcium phosphate-mediated transfection was performed
in 6-well plates with a final concentration of 20 nM of siRNA. VSMCs were used
in the adhesion assay 48 hours post-transfection.
GTPase assay
After seeding on coated dishes, VSMCs suspended in DMEM+0.1% BSA were
harvested every 30 minutes, until 2 hours. Cells were chilled on ice and lysed
in ice-cold buffer containing 1% Triton X-100, 25 mM HEPES pH 7.3, 150 mM
NaCl, 4% glycerol, 0.1 mM AEBSF, 4 µg/ml aprotinin, 1 µg/ml leupeptin
and 1 µg/ml pepstatin. Lysates were centrifuged for 8 minutes at 13,000
g. Supernatants were immediately frozen in liquid nitrogen and
stored at -80° until further use. An aliquot of each supernatant was
denatured in SDS-PAGE lysis buffer before freezing to measure the total
rhoGTPase content. For pull-down assays, supernatants were incubated with 30
µg of GST-PBD protein (containing the Cdc42- and Rac-binding region of
PAK-1B) or GST-RBD protein (containing the Rho-binding region of rhotekin)
affinity linked to glutathione Sepharose beads for 30 minutes
(Ren et al., 1999;
Sander et al., 1999
). The
beads were washed four times in lysis buffer and boiled in 60 µl SDS-PAGE
lysis buffer.
Retroviral infection
Retroviral supernatants were generated by transient transfection of Phoenix
cells with pBabeGFPRacN17 or pBabeGFPRacV12 and were used to infect VSMCs. Two
days post-infection, VSMCs were starved and tested on tissue-culture-coated
dishes.
Immunofluorescence labeling
VSMCs cultured for 4 hours on tissue-culture-coated dishes were fixed with
3% paraformaldehyde in PBS for 20 minutes and permeabilized with 0.2% Triton
X-100 in PBS for 2 minutes. The dishes were blocked with 1% BSA in PBS for 30
minutes and incubated with phalloidin-TRITC (60 ng/ml) for 30 minutes.
Fluorescence was analyzed with a Leica DM-R microscope equipped with a DC-200
digital camera.
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Results |
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Implication of the small GTPase RhoA
It was reported that some biological effects of ephrinA5, a member of the
ephrin family, are mediated through the Rho-Rho kinase pathway
(Wahl et al., 2000). We
therefore wanted to determine if such pathways are implicated in
ephrinA1-induced integrin inactivation. First, VSMCs were cultured in the
absence or presence of the Rho kinase (ROCK) inhibitor Y-27632. As seen in
Fig. 2A, the cells attached and
spread as efficiently with or without Y-27632 under control conditions. By
contrast, the ROCK inhibitor partially reversed the ephrinA1/Fc effect on VSMC
spreading, suggesting the involvement of the Rho-ROCK pathway. Similar results
were obtained when laminin was replaced by fibronectin (not shown). However,
we did not observe any activation of RhoA by ephrinA1/Fc
(Fig. 2B), whereas our
pull-down assay revealed a strong increase in RhoA activation with 10% FCS
(Fig. 2C). In an attempt to
define further the implication of Rho in our model, we tried to transfect by
various means an expression vector for RhoN19, a dominant-inactive form of
Rho, carrying a GFP-tag, but its expression was nearly undetectable in our
VSMCs (not shown). Hence, we used siRNA
(Elbashir et al., 2001
) to
ablate specifically RhoA in VSMCs. As seen in
Fig. 3, a 90% reduction in the
RhoA protein level was observed 48 hours post-transfection, without
significant modulation of the Rac1, Cdc42 and p42MAPK levels. These cells were
seeded on dishes coated with laminin and either control Fc or ephrinA1/Fc.
Interestingly, repression of RhoA synthesis markedly increases the length of
processes (Fig. 3B). However, a
lack of RhoA expression does not reverse the effect of ephrinA1 on cell
spreading. These results suggest that RhoA has a limited involvement in this
cellular model.
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|
Implication of the Rac and Cdc42 pathways
The morphological effects of ephrinA1/Fc, mainly characterized by a lack of
lamellipodia formation and a transient extension of processes
(Fig. 1C), suggested an
impairment of Rac1 and/or an increase in Cdc42 signaling
(Hall, 1998). Hence, we
measured the activation level of these small RhoGTPases with a pull-down
assay. VSMCs were harvested every 30 minutes until 120 minutes after seeding.
In control dishes, the level of activated Rac1 increased progressively to
reach a maximum 90 minutes after seeding before returning to basal level. By
contrast, ephrinA1 prevented this Rac1 activation mediated by either spreading
on laminin (Fig. 4A) or
fibronectin (not shown). As shown in Fig.
4C, we were unable to detect any significant modulation of Cdc42
activity by ephrin A1/Fc even though we were able to detect strong activation
of Cdc42 in VSMCs stimulated with bradykinin (not shown). However, it is
interesting to note that a basal activation of Cdc42 is detected in all tested
conditions.
|
To evaluate further the changes in the Rac1 signaling pathway, we measured the activation level of PAK1, the immediate effector of Rac1, with an anti-phospho-PAK1 antibody. As shown in Fig. 5, ephrinA1/Fc strongly impaired the phosphorylation of PAK1, suggesting that EphA activation represses VSMC spreading through inhibition of the Rac/PAK pathway. To evaluate the importance of the inactivation of this pathway, VSMCs were infected with retroviral vectors encoding the GFP-tagged constitutive active and inactive forms of Rac, respectively RacV12 and RacN17. The constitutive activity of RacV12 was assessed in the pulldown assay used previously (not shown). 48 hours post-infection, the cells were used in the adhesion assay. More than 95% of infected or non-infected cells spread on a control coat. As seen in Fig. 6A, VSMCs expressing RacV12 or RacN17 spread as efficiently as non-infected cells on tissue culture dishes coated with laminin and control Fc. On tissue culture dishes coated with laminin and ephrinA1/Fc, 65% of the RacV12-expressing cells spread, whereas most of the RacN17-expressing cells and non-infected cells were unable to spread (Fig. 6B,C).
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|
S1P or a decreased level of Rac (via siRNA) amplify the action of
ephrinA1/Fc
To analyze further the involvement of Rac1 in the ephrinA1 signaling
pathway, we took advantage of interfering RNA technology. An siRNA targeting
Rac1 specifically was designed and transfected into VSMCs. 48 hours
post-transfection, a 60-70% decrease in the Rac1 protein level was observed,
without significant modulation of the RhoA and Cdc42 levels, in VSMCs
(Fig. 7A). The siRNA of RhoA
was used as a positive control (Fig.
7A, lane 3). These cells were tested in the adhesion assay in the
presence of a suboptimal concentration of ephrinA1/Fc. As illustrated in
Fig. 7B and 7C, the repression
of Rac1 synthesis significantly enhanced ephrinA1-mediated inhibition of
spreading, further supporting the involvement of Rac1 in this phenomenon.
|
S1P is a lipid mediator exerting various effects on cytoskeletal
organization depending on the cell type. In VSMCs, S1P is known to inhibit Rac
and to increase Rho activity (Ryu et al.,
2002). 0.5 µM S1P did not modulate the ability of VSMCs to
spread on a control tissue culture dish coated with laminin and Fc
(Fig. 8a,e). However, S1P
significantly reduced the spreading of VSMCs with a suboptimal concentration
of ephrinA1/Fc whereas, in the presence of the highest concentration of
ephrinA1/Fc, all the cells were round (Fig.
8, compare b-d with f-h). These observations suggest that S1P
could amplify the effect of ephrinA1/Fc on VSMCs through modulation of Rac,
and perhaps Rho, activity.
|
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Discussion |
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In the present study, we have shown that the morphological effects of EphA
activation in VSMCs are associated with inhibition of the Rac/PAK pathway and
can be rescued by constitutive activation of Rac. Moreover, repression of Rac1
synthesis by means of an siRNA strategy amplifies the ephrinA1-mediated
spreading inhibition, further supporting the key role played by Rac1 in this
process. These observations are in agreement with the lack of lamellipodia
formation in the presence of ephrinA1/Fc as the establishment of this cell
structure is dependent on Rac activity
(Hall, 1998). In this way,
ephrins can compete with integrin-mediated signaling, but could also interfere
with the optimal localization of activated integrins, which is mediated in
some cases by activated Rac (Kiosses et
al., 2001
). EphrinA1 can also mediate its effect partly through
decreased phosphorylation of the myosin light chain (C.D. and J.P.,
unpublished), a protein activated by PAK1
(Sells et al., 1999
). This
molecule is localized to lamellipodia and proposed to contribute to membrane
extensions at the leading edge (Sells et
al., 1999
; Sells et al.,
2000
). Cdc42 is also able to activate PAK1. However, this GTPase
is probably not involved in the morphological effect of ephrinA1 as we did not
observe any modulation of its activity by ephrinA1/Fc, whereas our pull-down
assay allowed the detection of a strong increase of Cdc42 activity by
bradykinin treatment. As recently reported by Carter et al.
(Carter et al., 2002
),
spreading of NIH3T3 on ephrinA1/Fc was also observed in our culture model
while VSMCs only bind to immobilized ephrinA1/Fc and do not spread.
Differences between NIH3T3 and other cell types have been previously reported.
In a recent review, Coleman and Marshall discussed the discrepancies between
NIH3T3 and endothelial cells in Rac signaling for cyclin D1 expression
(Coleman and Marshall, 2001
).
Among several proposals, they suggested: "NIH3T3 can have undergo
genetics changes that liberate them from Rac signaling". Differences in
sensitivity to Rac signaling between the cells used in Carter's study and our
VSMCs could explain our antagonistic observations. In agreement with recent
reports proposing that some biological effects of ephrins are due to a
Rho-mediated increase of acto-myosin contraction, we observed an enhancement
of VSMC spreading on ephrinA1/Fc by the ROCK inhibitor Y-27632
(Lawrenson et al., 2002
;
Wahl et al., 2000
). However,
repression of RhoA synthesis using an siRNA strategy antagonizes this
phenomenon only partly, suggesting that RhoA has a limited role in this
cellular model. Nonetheless, the stabilization of process formation in the
absence of RhoA expression observed in VSMCs and in HeLa cells (not shown)
cultured on a laminin+ephrinA1/Fc coat suggested that other biological effects
of ephrinA1, like outgrowth of neurites or regression of melanoma cell
processes, could be antagonized by an inhibition of RhoA synthesis.
Alternatively, Y-27632 could also mediate its effect in our model through Rac,
as it was proposed that ROCK inhibition can enhance Rac activity
(Hall et al., 2001
) and a
recent study reported the activation of Rac by Y27632 in VSMCs
(Katsumi et al., 2002
).
Moreover, in VSMCs cultured on laminin+ephrinA1/Fc, we observed that Y-27632
induces a modest but reproducible increase of PAK1 phosphorylation level (C.D.
and J.P., unpublished). By contrast, it was also reported that Y-27632 did not
exclusively inhibit ROCK. At micromolar concentration, as it is commonly used,
Y-27632 can inhibit other kinases (Davies
et al., 2000
). These side effects can also account for the partial
reversion of the ephrinA1-induced phenotype independently of its action on the
Rho pathway. Finally, the cooperativity between S1P and ephrinA1/Fc reported
here could represent a supplementary control level of the ephrinA1 action in
vivo, not only by amplification of the effect but also by differential
regulation on endothelial and smooth muscle cells through opposite regulation
of RhoGTPases (Ryu et al.,
2002
).
In conclusion, our study underlines the implication of the Rac/PAK pathway in ephrinA1-mediated integrin inactivation in VSMCs whereas RhoA has a limited effect. These mechanisms are potentially regulated by S1P, which could participate in the early steps of angiogenesis. EphrinA1 can operate through various proteins regulating the activity of Rac1. Among them, GAPs like chimaerin and the p190RhoGAP, or the recently identified GEF ephexin, are potentially interesting candidates. Investigations are now in progress to analyze the implications of some of these proteins in our model.
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Acknowledgments |
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