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J Biol Chem. 2008 October 3; 283(40): 27230–27238. doi: 10.1074/jbc.M804520200. | PMCID: PMC2555994 |
Copyright © 2008, The American Society for Biochemistry and
Molecular Biology, Inc. ABL2/ARG Tyrosine Kinase Mediates SEMA3F-induced RhoA Inactivation and
Cytoskeleton Collapse in Human Glioma
Cells *![[S with combining enclosing square]](/corehtml/pmc/pmcents/x0053x20DE.gif) Akio Shimizu, ‡§1 Akiko Mammoto, ‡§1 Joseph E. Italiano, Jr., ‡¶ Elke Pravda, ‡§ Andrew C. Dudley, ‡§ Donald E. Ingber, ‡§ and Michael Klagsbrun ‡§2‡Vascular Biology Program and Departments of §Surgery and Pathology, Children's Hospital Boston and Harvard Medical School, and the ¶Division of Translational Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115 Received June 13, 2008; Revised July 25, 2008. Class three semaphorins (SEMAs) were originally shown to be mediators of
axon guidance that repelled axons and collapsed growth cones, but it is now
evident that SEMA3F, for example, has similar effects on tumor cells and
endothelial cells (EC). In both human U87MG glioma cells and human umbilical
vein EC, SEMA3F induced rapid cytoskeletal collapse, suppressed cell
contractility, decreased phosphorylation of cofilin, and inhibited cell
migration in culture. Analysis of the signaling pathways showed that SEMA3F
formed a complex with NRP2 (neuropilin-2) and plexin A1. These interactions
eventually led to inactivation of the small GTPase, RhoA, which is necessary
for stress fiber formation and cytoskeleton integrity. A novel upstream RhoA
mediator was shown to be ABL2, also known as ARG, a membrane-anchored
nonreceptor tyrosine kinase. Within minutes after the addition of SEMA3F, ABL2
directly bound plexin A1 but not to a plexin A1 mutant lacking the cytoplasmic
domain. In addition, ABL2 phosphorylated and thereby activated p190RhoGAP,
which inactivated RhoA (GTP to GDP), resulting in cytoskeleton collapse and
inhibition of cell migration. On the other hand, cells overexpressing an ABL2
inactive kinase mutant or treated with ABL2 small interfering RNA did not
inactivate RhoA. Cells treated with p190RhoGAP small interfering RNA also did
not inactivate RhoA. Together, these results suggested that ABL2/ARG is a
novel mediator of SEMA3F-induced RhoA inactivation and collapsing
activity. Class 3 semaphorins (SEMA3A to -G) are secreted proteins that were first
shown to regulate axon guidance in the developing nervous system
( 1- 4)
and subsequently to regulate angiogenesis
( 5- 7).
SEMA3s bind to their receptors, NRP1 (neuropilin-1) and NRP2. However, to
convey a signal, SEMA3 and neuropilins
(NRPs) 3 need also to
interact with plexins, transmembrane proteins whose cytoplasmic domains are
substrates for nonreceptor kinases, such as Fyn or Fes
( 8- 10).
There are at least nine plexins: A1-A4, B1-B3, C1, and D1
( 8,
11). SEMA3F binds NRP2.
Plexins A1 and A2 form a complex with NRP2 when it binds SEMA3F
( 12). NRP2 signaling is also
mediated by plexin A3 in the mouse embryonic nervous system
( 13). An exception appears to
be SEMA3E, which is not dependent on NRPs but acts directly via plexin D1 to
repel blood vessels ( 14). Most of the SEMA3 mechanistic studies have been carried out in neurons.
Early studies showed that SEMA3A repelled axons and collapsed axonal growth
cones by depolymerizing F-actin and inducing lamellipodia retraction in dorsal
root ganglia ( 15,
16). We had demonstrated that
SEMA3A (originally known as collapsin-1) was an inhibitor of endothelial cell
(EC) motility, possibly the first study showing that a semaphorin could affect
nonneuronal cell types ( 5).
Furthermore, SEMA3A depolymerized EC F-actin and retracted lamellipodia in a
manner similar to what occurs in neurons. Our subsequent studies in EC and
tumor cells showed that SEMA3F, in an NRP2-dependent manner, inhibited cell
adhesion, cell migration in vitro, and tumor angiogenesis and
metastasis in vivo
( 6). This activity is dependent
on plexins. We have found that SEMA3F had a striking effect on tumor cell and EC
morphology, causing cytoskeleton collapse, loss of stress fibers, loss of
adhesion, and depolymerization of F-actin, rapidly within minutes. Cell
migration was also inhibited. The loss of stress fibers suggested an
inactivation of RhoA. RhoA is a small GTPase that plays a central role in the
control of cell shape, cytoskeletal organization, and cell motility by
regulating actomyosin-based tension generation and actin polymerization that,
in turn, govern formation of stress fibers and focal adhesions
( 17). Activated RhoA
stimulated Rho kinase and thereby enhanced myosin light chain phosphorylation.
Contractility of the actomyosin network promoted actin filament alignment and
stress fiber formation. Rho kinase also activated LIM kinase, which
phosphorylated cofilin, inhibiting its ability to bind and depolymerize actin
( 18,
19). In the present study, we
found that SEMA3F inactivated RhoA and activated cofilin in tumor and
endothelial cells, consistent with similar effects of semaphorins on neurons.
However, little is known about the mechanisms by which SEMA3F inactivates
RhoA. Accordingly, we have analyzed the upstream signaling pathway that ends
up inactivating RhoA and causing cytoskeleton collapse of human U87MG glioma
cells and HUVEC. This pathway includes physical interactions between SEMA3F,
NRP2, plexin A1, p190RhoGAP, and, importantly, ABL2. The Abelson (ABL2) tyrosine kinase (v-Abl Abelson murine leukemia viral
oncogene homolog 2), which is a nonreceptor tyrosine kinase, is a novel
mediator of RhoA inactivation. Drosophila Abl and the mammalian
homologues Abl1 and Abl2, also known as ARG (Abelson-related gene), have a
role in axonogenesis and cancer
( 20,
21). The domain structure of
Abl has been analyzed and consists of an N terminus region that is a
myristylation site, Src homology 3, Src homology 2, and tyrosine kinase
domains, and a large C-terminal region
( 22). The Src homology 3
domain inhibits tyrosine kinase activity, and deletion of the Src homology 3
domain activates tyrosine kinase activity
( 22). In this report, we show that ABL2 and RhoA play key roles in mediating
SEMA3F-induced collapsing activity in tumor cells and EC. ABL2 bound to plexin
A1, on one hand, and to p190RhoGAP on the other. Upon binding, ABL2
phosphorylated p190RhoGAP and activated it, leading to the inactivation of
RhoA. Inactivation of RhoA affected several downstream kinase events
(e.g. dephosphorylation of cofilin), resulting in depolymerization
and severing of F-actin, thereby collapsing the cytoskeleton and inhibiting
cell migration. We conclude that ABL2/ARG is a novel mediator of
SEMA3F-induced RhoA inactivation and collapsing activity. Materials Antibodies—Anti-NRP1, -NRP2, -VEGFR1, -VEGFR2, -cofilin,
-ABL2, and -RhoA were purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA), and rabbit monoclonal anti-RhoA antibody was purchased from Cell
Signaling Technology (Danvers, MA). Anti-p190RhoGAP-A was purchased from BD
Biosciences. Anti-phosphotyrosine (4G10) was purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). Anti-phosphocofilin antibody was
provided by John H. Hartwig. Neutralizing antibody, anti-human NRP2, was
purchased from R&D Systems (Minneapolis, MN). Anti-HA antibody was from
Covance (Emeryville, CA). Anti-Rac1 and anti-Cdc42 were purchased from
Cytoskeleton (Denver, CO). Protein G-Sepharose was purchased from GE
Healthcare. Plasmids—The full-length, His-Myc-tagged, human SEMA3F
construct was provided by Marc Tessier-Lavigne (Genentech Inc., South San
Francisco, CA). Human ABL2b was provided by Gary Kruh (Fox Chase Cancer
Center, Philadelphia, PA). The plexin A1 HA-tagged construct was provided by
Shigeru Yanai (Kobe University, Japan). The cofilin construct was purchased
from Open Biosystems (Huntsville, AL). The human NRP2 construct was described
previously ( 23). Inhibitors—The Rho kinase inhibitor, Y27632, and Src kinase
inhibitor, PP2, were purchased from EMD Chemicals, Inc. (Darmstadt,
Germany). siRNA—ABL2 siRNA oligonucleotide pairs were
5′-CCUCGUCAUCUGUUGUUCCAU-3′ and
5′-AUGGAACAACAGAUGACGAGG-3′ (oligonucleotide pair 1) and
5′-CGGUCAGUAUGGAGAGGUUUA-3′ and
5′-UAAACCUCUCCAUACUGACCG-3′ (oligonucleotide pair 2). p190RhoGAP
siRNA has been described before
( 24). siRNAs of siGENOME SMART
pool NRP2 were purchased from Dharmacon (Lafayette, CO). As a control, an
siRNA duplex with an irrelevant sequence (Ambion, Inc., Austin, TX) was
used. Mutagenesis Primers—The cofilin mutagenesis primer pairs
were 5′-CGTTTCCGGAAACATGGCCGAAGGTGTGGCTGTCTCTGATG-3′ and
5′-CATCAGAGACAGCCACACCTTCGGCCATGTTTCCGGAAACG-3′. The ABL2
mutagenesis primer pairs were
5′-AGCCTTACAGTTGCTGTGAGAACATTGAAGGAAGATACC-3′ and
5′-GGTATCTTCCTTCAATGTTCTCACAGCAACTGTAAGGCT-3′. The PCR primers for
plexin A1 cytoplasmic domain deletion mutant were
5′-ATGCCACTGCCACCTCTGAGCTCT-3′ and
5′-ACTAGGATTTGCGTTTGTAGGCGAT-3′. Cell Culture Porcine aortic endothelial cells (PAE) were provided by Lena Claesson-Welsh
(Uppsala University, Uppsala, Sweden). PAE/NRP1 and PAE/NRP2 were described
previously ( 23). HUVEC
purchased from Lonza Inc. (Allendale, NJ) were cultured in EBM-2 (Lonza),
supplemented with EGM-2 Single Quote. Mutagenesis and Construct A site-directed mutagenesis kit (Qiagen, Valencia, CA) was used to mutate
cofilin and ABL2. The plexin A1 cytoplasmic domain-deleted mutant was
generated by using Pfu DNA polymerase (Stratagene). The sequences of
primers are described above. The amplified cDNA was inserted to pcDNA3.1 TOPO
vector (Invitrogen). Purification of Human Recombinant SEMA3F The SEMA3F construct was transfected into HEK293 cells to express SEMA3F
protein. After 16 h, the culture medium, including 10% fetal bovine serum, was
changed to CD293 serum-free medium (Invitrogen) for a further 48-h incubation.
The conditioned medium was collected and applied to a HiTrap HP Chelating
column (GE Healthcare) with nickel divalent cation. Proteins were eluted with
500 mm imidazole. The eluate was desalted by a PD-10 column (GE
Healthcare). The protein concentration was measured by a DC protein assay
(Bio-Rad). Approximately 3 mg of SEMA3F protein was purified from 15 tissue
culture dishes (15 cm). Videography Cells were pipetted into chambers formed by mounting a glass coverslip onto
a 35-mm glass bottom dish. Preparations were maintained at 37 °C with
constant 5% carbon dioxide gas infusion using an Incubator XL-3 incubation
chamber (Carl Zeiss) and examined on a Zeiss Axiovert 200 microscope equipped
with a ×63 objective (numerical aperture 1.4) and ×1.6 optivar.
Images were acquired with an Orca IIER cooled charged-coupled device camera
(Hamamatsu). Electronic shutters and image acquisition were under the control
of Metamorph software (Universal Imaging of Molecular Devices, Downington,
PA). Images were acquired every 1-5 min with an image capture time of 50-100
ms. Movies were generated using the Metamorph image analysis program. Confocal Microscopy SEMA3F (0-640 ng/ml) was added to HUVEC and U87MG cells, which were
cultured on coverglasses with a density of 1-2 × 104
cells/well in a 6-well plate. After 30 min, cells were fixed with 4%
paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 in PBS.
F-actin and nuclei were stained with Alexa Fluor 488 phalloidin (Invitrogen)
and bis-benzimide (Hoescht 33258; Sigma), respectively. The mounted samples on
slide glasses were imaged on a Leica TCS SP2 confocal laser-scanning
microscope equipped with a ×63 objective (numerical aperture 1.4),
488-nm argon ion laser (F-actins), and 405-nm diode (nuclei). Leica Confocal
Software and NIH Image software (ImageJ) were used to scale recorded
images. siRNA Knockdown Transient transfection of siRNAs was performed using SILENTFECT reagent
(Bio-Rad) according to the manufacturer's directions. After 48-70 h, cells
were analyzed for effects of the knockdown. Silencing efficiency was confirmed
by Western blotting and reverse transcription-PCR. Inhibition of NRP2 siRNA for NRP2 and an irrelevant sequence were transfected into U87MG
cells. After a 48-h incubation, SEMA3F (80 ng/ml) was added. Anti-NRP2
antibody (20 μg/ml) and normal goat immunoglobulin as a control were added
to U87MG cells. After a 30-min incubation, SEMA3F was added to the cells,
which were then analyzed by confocal microscopy. Migration Migration assays were performed in Transwells® (Corning Glass) with an
8.0-μm pore size. The polycarbonate membrane of the upper wells was coated
with a 0.5% gelatin solution. Cells (2.5 × 104) in serum-free
minimum essential medium were added to upper wells. Increasing concentrations
of SEMA3F, including 1% fetal bovine serum, were added to the lower wells.
Cells that had migrated through the filter after 14 h at 37 °C were
stained with Difquick (Dade Behring Inc., Newark, DE) and counted by phase
microscopy. Cell Contractility Cell contractility was measured by the traction force microscopy method
using fluorescent beads. The bead displacements that accounted for
contractility were visualized and quantitated as previously described
( 25,
26). Briefly, U87MG cells (1
× 10 5 cells) were plated on a flexible polyacrylamide gel
(0.25% bis and 2% acrylamide; <70 mm thick) coated with fibronectin (0.1
mg/ml) that contained red fluorescent nanobeads (200-nm diameter; Invitrogen)
as markers. After the addition of SEMA3F (320 ng/ml), fluorescent microscope
images were recorded at 0-30 min. The traction field was calculated from the
displacement field using Mat-Lab software. RhoA Activity RhoA activity assays were performed and quantified using the RhoA
activation assay kit based on rhotekin pull-down according to the
manufacturer's instructions (Cytoskeleton). U87MG cells (1.5 ×
106 cells) were incubated with SEMA3F (320 ng/ml) for 0-30 min. The
cells were washed with PBS and extracted in 600 μl of cell lysis buffer (25
mm Tris, pH 7.5, 150 mm NaCl, 5 mm
MgCl2, 1% Triton X-100). Samples were centrifuged for 5 min at 8000
rpm, and the supernatant was incubated with rhotekin beads for 1.5 h at 4
°C. After washing the beads with buffer (25 mm Tris, pH 7.5, 40
mm NaCl, 15 mm MgCl2), proteins were removed
from the beads in Laemmli buffer and analyzed by Western blotting. Similarly,
Rac1 and Cdc42 levels were examined using a Rac activation kit
(Cytoskeleton). RhoA Lentivirus The HA-tagged wild type and constitutively active (G14V) forms of RhoA were
constructed and subcloned into the pHAGE lentiviral backbone vector as
described ( 27,
28). U87MG cells were
incubated with wild type or constitutively active (G14V) RhoA lentiviruses in
the presence of 5 μg/ml Polybrene (Sigma). About 90-100% infection
efficiency was achieved by 3 days. Immunoprecipitation One mg of U87MG cell lysates lysed in 20 mm HEPES, 1% Triton
X-100, and 150 mm sodium chloride was incubated with either anti-HA
(plexin A1), anti-p190A, or anti-ABL2 antibody for 16 h. Alternatively,
ice-cold radioimmune precipitation buffer (Boston Bioproducts, Worcester, MA)
supplemented with complete protease inhibitor mixture (Roche Applied Science)
was incubated with anti-phosphotyrosine antibody (4G10). Protein G-Sepharose
(GE Healthcare) was used to pull down antibodies. After washing the beads with
the lysis buffer, proteins were removed from the beads in Laemmli buffer and
analyzed by Western blotting. Immunoblot U87MG cells and HUVEC were lysed with ice-cold radioimmune precipitation
buffer (Boston Bioproducts) supplemented with complete protease inhibitor
mixture (Roche Applied Science) and phosphatase inhibitor mixture I and II
(Sigma). Proteins were transferred onto polyvinylidene fluoride membranes and
immunoblotted with antibodies. Horseradish peroxidase-conjugated secondary
antibodies (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) and
chemiluminescent substrates (PerkinElmer Life Sciences) were used to detect
primary antibodies. Apoptosis SEMA3F (640 ng/ml) was added to U87MG cells. After 24 h, caspase activity
was measured by fluorescence-activated cell sorting
( 29) using the Vybrant FAM
polycaspases kit (Invitrogen), according to the manufacturer's instructions.
As a positive control, the U87MG cells were irradiated with UV (100 mJ). SEMA3F Induces Cytoskeletal Collapse in HUVEC and U87MG
Cells—HUVEC and U87MG glioma cells were stimulated with SEMA3F and
analyzed by time lapse videomicroscopy. The complete videos are shown in the
supplemental data (Movies S1 and S2). Analysis of selected frames showed that
within 30 min after the addition of SEMA3F, the cells rounded up, were less
spread and less adherent ( for HUVEC; e and f for
U87MG cells). Both cell types decreased dramatically in size, up to 30-40% in
total area by 25 min. There were retraction fibers remaining on the
extracellular matrix. Confocal microscopy showed that HUVEC
() and U87MG
glioma cells () were adherent and well spread and displayed abundant actin
stress fibers, indicative of an intact F-actin cytoskeleton. However, in the
presence of SEMA3F, HUVEC () and U87MG cells () were less adherent and had lost their stress
fibers, indicative of F-actin cytoskeleton collapse
(). Thus, the time lapse videography and confocal
microscopy were consistent. After overnight incubation with no further SEMA3F
treatment, the cells recovered their stress fibers and were able to
proliferate, which suggests that SEMA3F treatment did not appear to be toxic
for the cells. In addition, lack of caspase activity indicated that these
cells were not undergoing apoptosis (Fig. S4). The Cytoskeleton-collapsing Effects of SEMA3F Are Mediated by
NRP2—Immunoblot analysis showed that U87MG cells and HUVEC
expressed NRP2, the functional receptor for SEMA3F
(). HUVEC and
U87MG cells also expressed NRP1, the functional receptor for SEMA3A. HUVEC,
but not U87MG cells, expressed both VEGF receptor tyrosine kinases, VEGFR-1
and VEGFR-2. Thus, in HUVEC, VEGF 165 could bind to NRPs and VEGF
RTKs, whereas in U87MG cells, VEGF 165 could bind NRPs only (not
shown). U87MG cells expressed plexins A1, A2, A3, and A4 (not shown). Plexin
A1 is required for SEMA3F signaling
( 12). The addition of SEMA3F
induced the interaction of NRP2 and plexin A1 within 5 min
(). The SEMA3F
induction of cytoskeleton collapse was NRP2-dependent, as shown by loss of
collapsing activity in the presence of siRNA (not shown) or in the presence of
anti-NRP2 antibody (). SEMA3F Inhibits U87MG Cell Contractility and Migration—To
explore the consequence of SEMA3F-induced F-actin cytoskeleton collapse on
U87MG cell behavior, the effects on cell motility and contractility were
measured (). SEMA3F suppressed U87MG migration in a Transwell®
assay by up to 75% in a dose-dependent manner
(). Cells need
to exert traction forces on their extracellular matrix adhesions in order to
migrate ( 30,
31). Accordingly, U87MG cell
contractility in control and SEMA3F-treated cells using traction force
microscopy was measured (). SEMA3F significantly decreased cell traction force
(total strain energy) in U87MG cells within 5 min after treatment relative to
controls. The rapid kinetics of contractility loss was consistent with that of
rapid cytoskeleton collapse shown in . SEMA3F Inactivates RhoA and Activates Cofilin—The small
GTPase RhoA stimulates cell contractility, actin polymerization, and stress
fiber formation that are required for cell movement
( 32,
33). SEMA3F effects on RhoA
activity were analyzed using a rhotekin pull-down assay, which measures levels
of active RhoA. SEMA3F decreased GTP-RhoA levels by 90% within 5 min after the
addition (),
consistent with the rapid cytoskeleton collapse and loss of contractility.
Constitutively active RhoA administered by lentivirus rescued the cytoskeletal
collapse by SEMA3F (), whereas the Rho kinase inhibitor, Y27632, mimicked
SEMA3F-induced collapse (). SEMA3F also inactivated Rac1 activity (Fig. S1),
consistent with retraction of lamellipodia observed in the video (Movies S1
and S2). On the other hand, Cdc42 activity was not changed (Fig. S1). Active RhoA promotes formation of actin bundles by activating multiple
downstream effectors. In addition to stimulating tension generation through
Rho kinase, RhoA also activated LIM kinase
( 34), which in turn
phosphorylated cofilin, also known as actin depolymerizing factor.
Phosphorylated cofilin was inactive, whereas dephosphorylated cofilin was
active. Active cofilin monomerizes F-actin and severed F-actin chains, leading
to cytoskeleton collapse ( 35).
Cofilin dephosphorylation in U87MG cells and HUVEC in response to SEMA3F
occurred rapidly, within 15 min (), consistent with the rapid kinetics of collapse and
loss of contractility shown above. Mutating wild type Ser 3 to
anionic Glu 3 mimicked phosphorylation and resulted in cofilin
inactivation ( 36). We found
that SEMA3F inhibited U87MG migration 2-fold
(). However,
mutant cofilin acting as a dominant negative diminished the SEMA3F
antimigratory activity by about 1.7-fold, suggesting that cofilin plays an
important role in the SEMA3F-induced collapse, migration, and signal
transduction pathways. ABL2 Mediates SEMA3F-induced Interactions with Plexin A1 and
p190RhoGAP—The upstream steps leading to RhoA inactivation by
SEMA3F are not fully understood; however, ABL2 appears to play a central role.
Immunoprecipitation and immunoblotting analysis showed that in response to
SEMA3F, ABL2 bound plexin A1 within 5 min
(). Deletion of
the plexin A1 cytoplasmic domain abrogated this interaction
(). ABL2 and
p190RhoGAP interacted but were preformed
(). ABL2 siRNA
abolished the interaction of p190RhoGAP and plexin A1, showing that these
interactions are ABL2-dependent (). Two different siRNAs for ABL2 were tested (Fig. S3),
and both siRNAs showed the same results. SEMA3F Induces p190RhoGAP Phosphorylation via ABL2—SEMA3F
induced phosphorylation of p190RhoGAP (), which was abrogated by knockdown of ABL2
(). A mutant
tyrosine kinase-inactive form of ABL2 was prepared by mutation of
Lys 317 to Arg 317, as described previously
( 37). The kinase-inactive
ABL2, acting as a dominant negative, when overexpressed in U87MG cells,
inhibited the SEMA3F-induced phosphorylation of p190RhoGAP
(). ABL2 Activity and p190RhoGAP Are Necessary for SEMA3F-induced RhoA
Inactivation, Cytoskeleton Collapse, and Inhibition of
Migration—p190RhoGAP siRNA treatment of U87MG cells resulted in the
failure of RhoA to be inactivated in response to SEMA3F
(). Similarly,
ABL2 siRNA abrogated the ability of SEMA3F to inactivate RhoA
(). Silencing
of p190RhoGAP () or silencing of ABL2
() in both
cases inhibited the ability of SEMA3F to induce cytoskeleton collapse. In
addition, when these two genes were silenced, the ability of SEMA3F to inhibit
U87MG migration was diminished (). Two different siRNAs for ABL2 were
tested (supplemental Fig. S3), and both siRNAs showed the same results. SEMA3F Signaling—The various steps in the SEMA3F signaling
pathway are shown in a schematic (). SEMAs were first described as negative regulators of axonal guidance that
repel axons and collapse growth cones
( 3,
4,
38,
39). SEMA3F was subsequently
found to be an inhibitor of tumor angiogenesis and of tumor progression and
metastasis ( 6,
40- 42).
SEMA3F has profound effects on the morphology of endothelial and tumor cells.
As shown by time lapse photography, within 5 min, the cells began to retract,
were less adherent, and spread less. Confocal microscopy showed rapid collapse
of the F-actin cytoskeleton with greatly diminished stress fiber formation.
Cell culture studies showed loss of contractility within 5 min and subsequent
inhibition of cell motility. These events are similar to those in SEMA-induced
axonal collapse ( 3,
15). However, the mechanisms
by which SEMAs produce these cytoskeletal changes and alter cell behavior,
especially in nonneuronal cells, are not fully understood. The loss of stress
fibers implicates inactivation of RhoA, a small GTPase, that stabilizes these
structures. Indeed, we have shown that in response to SEMA3F, RhoA-GTP is
inactivated to be RhoA-GDP. This effect can be rescued by constitutively
active expression of RhoA. On the other hand, a Rho kinase inhibitor, Y27632,
mimics SEMA3F-induced collapse. Of significance, several Rho kinase inhibitors
have been shown to prevent tumor progression in mouse models
( 43- 46). Signaling downstream from RhoA has been studied in detail
( 32,
33). Our major goal was to
identify those upstream events that led to SEMA3F-induced RhoA inactivation.
Signaling in response to SEMA3F began by formation of complex containing
SEMA3F, its NRP2 receptor, and plexin A1, which transduced the SEMA3F signal.
A significant finding was that ABL2 (v-Abl Abelson murine leukemia viral
oncogene homolog 2), a tyrosine kinase, plays an important role in SEMA3F
signaling. ABL2 (ARG (Ableson-related gene)) was first identified as a
mammalian homologue of Drosophila Abl, which has been known to have a
role in axonogenesis ( 20,
21). Subsequently, ABL2 has
also been shown to contribute to chronic myelogenous leukemia and other blood
neoplasias ( 47). Recently, it
was reported that ABL2 tyrosine kinase inhibited fibroblast migration by
attenuating actomyosin contractility
( 48). We have found that
SEMA3F is an inhibitor of cell migration and contractility, making it
plausible that SEMA3F and ABL2 share signaling pathways. The critical role of
ABL2 in SEMA3F-induced F-actin cytoskeleton collapse and in inhibition of
migration was shown in ABL2 knockdown experiments in which the cytoskeleton no
longer collapsed and migration was no longer inhibited. ABL2 expression is needed for SEMA3F-induced activities, such as collapse
and inhibition of migration, but how does ABL2 function to produce this
phenotype? One possibility is that ABL2 acts by activation of p190RhoGAP in a
SEMA3F-dependent manner. P190RhoGAP is a GTPase-activating protein (GAP) which
inactivates RhoA to a GDP-bound state. Knockdown of p190RhoGAP inhibited
SEMA3F-induced cytoskeleton collapse and abrogated the SEMA3F-induced
inhibition of migration. P190RhoGAP has been shown to be a substrate for ABL2
and is tyrosine-phosphorylated on Tyr 1105
( 49). Fibroblasts from
arg-/- mice do not phosphorylate p190RhoGAP. In our
studies, SEMA3F-induced tyrosine phosphorylation of p190RhoGAP was abolished
by ABL2 siRNA treatment and also by overexpression of an ABL2 tyrosine
kinase-inactive mutant (K317R) acting as a dominant negative.
Immunoprecipitation experiments indicated that ABL2 bound both plexin A1 and
p190RhoGAP; thus, it is an important intermediary in the SEMA3F signaling
cascade. Although ABL2 is a candidate as the activator of p190RhoGAP, it
cannot be ruled out that other tyrosine kinases could be active as well. For
example, p190RhoGAP has been shown to be phosphorylated by Src tyrosine
kinases, leading to inactivation of RhoA
( 50). However, in our studies,
SEMA3F-induced U87MG cell collapse was not inhibited by Src kinase inhibitors,
such as PP2 (Fig. S1). Another important regulator of cytoskeleton collapse is cofilin. Cofilin,
an actin depolymerization factor, is inactive when phosphorylated. SEMA3F
induced dephosphorylation of cofilin within 15 min to become active. Active
cofilin depolymerized and severed F-actin. A mutagenic change of wild type
Ser3 to anionic Glu3 mimicked phosphorylation and
resulted in constitutively inactive cofilin. This mutant acted as a dominant
negative cofilin. Overexpression of dominant negative cofilin abrogated the
SEMA3F-induced inhibition of migration, suggesting that cofilin directly
contributes to cellular collapse. SEMA3F also inactivated Rac1 (Fig. S1), a regulator of lamellipodia
formation ( 32). Lamellipodia
retraction in response to SEMA3F was observed in videography and confocal
microscopy. Cdc42, which regulates filopodia
( 33), did not appear to be
inactivated by SEMA3F (Fig. S1), consistent with no change in filopodia in
response to SEMA3F. Signaling has been studied in response to other semaphorins, including
SEMA3A, SEMA3E, and SEMA4D. Our initial studies on the effects of SEMA3A on
nonneuronal cells demonstrated that SEMA3A depolymerized EC F-actin, retracted
EC lamellipodia, and inhibited EC migration and capillary sprouting
( 5). Those results suggested
that SEMA3A might inactivate both RhoA and Rac1, as does SEMA3F. However,
previous studies with chick DRG neurons concluded that SEMA3A did not
inactivate RhoA but did activate Rac1 in the induction of growth cone collapse
( 51). Thus, SEMA3A signaling
pathways might show some differences in neuronal cells versus EC. SEMA3E acts differently. It has been shown that SEMA3E does not interact
with NRPs but interacts directly with plexin D1 on endothelial cells
( 14), suggesting a pathway
different from those of SEMA3A and SEMA3F. Unlike SEMA3A and SEMA3F, SEMA3E
induced both migration and growth-promoting activity on EC which resulted in
prometastatic activity to lung
( 52). Subiculomammilary
neurons expressing both plexin D1 and NRP1 were attracted to SEMA3E; however,
if NRP1 was knocked down, these neurons were repelled by SEMA3E
( 53). These phenotypic results
suggest that SEMA3E and SEMA3A/F have different signaling pathways, to some
degree. SEMA4D is a transmembrane class 4 semaphorin that does not interact with
NRPs but instead binds directly to plexin B1
( 11). As a transmembrane
protein, it is not clear how SEMA4D signals naturally. However, recombinant
soluble SEMA4D induces tumor, endothelial, and fibroblast cellular collapse
( 54). Although SEMA4D induces
collapse, it is not known whether the complex of plexin B1-p190RhoGAP is
mediated by ABL2. Furthermore, since SEMA4D has a cytoplasmic domain, it might
show bidirectional signaling like SEMA6D
( 55) and ephrin B2
( 56). Brain tumors are highly vascular. Glioblastoma multiforme, the most common
primary brain tumor, is highly aggressive, with median survival of 40-50 weeks
from the time of diagnosis
( 57). The disease is
characterized by dissemination and infiltration of tumor cells into the brain
stroma. SEMA3F, an inhibitor of glioma cells and EC migration in
vitro, might be a candidate for glioma therapy. Preliminary studies in
mice indicated that overexpression of SEMA3F in glioma tumors reduced the
growth rate and appeared to collapse the tumor blood
vessels. 4In summary, a multistep signaling pathway for SEMA3F leading to tumor cell
and EC collapse has been elucidated. The results indicate that ABL2, in
response to SEMA3F, is recruited to bind plexin A1 and to phosphorylate and
activate p190RhoGAP directly, resulting in RhoA inactivation and
cofilin-mediated cytoskeleton collapse. We thank E. Geretti, D. R. Bielenberg, and J. H. Hartwig for valuable
discussions. We thank J. H. Hartwig for providing cofilin antibody; M.
Tessier-Lavigne, G. Kruh, and S. Yanai for providing plasmids; L.
Claesson-Welsh for providing PAE cells; and T. Polte for technical assistance
in analyzing traction force by microscopy. We thank K. Johnson and M. Herman
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