From the Department of Neurobiology and Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294
Received for publication, December 23, 2002
, and in revised form, February 18, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One example of a growth factor receptor that has received particular attention in other malignancies is the erbB2 receptor. Specifically, in human breast cancer cell lines, ligand-dependent activation of erbB2 has been shown to promote cellular motility and invasiveness via activation of matrix proteases (9, 10), phosphorylation of paxillin (11), modulation of focal adhesion kinase (FAK)1 (12) and of a related kinase, Pyk2 (13), and regulation of the actin cytoskeleton (14). ErbB2 is a member of the type 1 family of receptor tyrosine kinases which also includes erbB1, erbB3, and erbB4. Ligand-dependent activation of erbB receptors results in homo- or heterodimerization. This stimulates receptor auto- and/or transphosphorylation on cytoplasmic tyrosine residues creating binding sites for adaptor proteins, kinases, and phosphatases. The downstream signaling cascades initiated depend on the identity of the erbB heterodimers, which is determined, in part, by the specific erbB receptor ligand (15).
Although erbB2 has no known ligand, it can be activated in a
ligand-dependent manner via heterodimerization with other erbB receptors
(16). Ligands for erbB1 are
numerous and include transforming growth factor- and epidermal growth
factor. ErbB3 and erbB4 bind with low and high affinity, respectively, to a
family of polypeptide growth factors called the neuregulins
(17). In the central nervous
system, neuregulin-1 (NRG-1) is expressed by both neurons and glia
(18,
19,
20). Moreover, the
extracellular domain of type I and type II NRG-1 isoforms contains an Ig-like
motif that enables them to adhere to the extracellular environment
(21). This allows them to
function in a paracrine manner. Interestingly, recent findings
(22) demonstrate that glioma
cells can produce NRG-1 isoforms. Furthermore, erbB2 has been repeatedly
reported to be overexpressed in human glioma biopsy samples and in a number of
human glioma cell lines (22,
23,
24,
25,
26). Despite this evidence,
relatively little is known regarding the role of the NRG-1/erbB receptors in
glioma cell biology.
In an attempt to fill this void, we examined the potential for NRG-1 to
modulate glioma cell motility. Our results demonstrate that the NRG-1
receptors erbB2 and erbB3 localize in regions of the plasma membrane involved
in cellular movement. In addition, their activation by a recombinant form of
NRG-1, NRG-1, stimulates a functional association between erbB2 and FAK,
thereby facilitating glioma cell migration.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ImmunocytochemistryFor our initial erbB receptor immunostaining, U251 cells on uncoated glass coverslips were rinsed with sterile PBS and fixed for 10 min with 4% paraformaldehyde in PBS. Fixed cells were rinsed three times with PBS, then placed in a blocking buffer, which consisted of PBS, 5% normal goat serum, and 0.1% Triton X-100. After 30 min, cells were incubated with the appropriate primary antibody diluted in blocking buffer overnight at 4 °C. Polyclonal antibodies for the erbB receptors (Santa Cruz Biotechnology, Santa Cruz, CA) were used at 1 µg/ml (1:200). The following day, the cells were rinsed three times for 10 min with blocking buffer and then incubated with Alexa 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) for 1 h at room temperature. Cells were washed three times for 10 min with PBS, mounted using mounting medium with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA), visualized (Leica DMRB fluorescence microscope, Heerbrugg, Switzerland), and digitally imaged (Spot RT, Diagnostic Instruments, Sterling Heights, MI).
To study erbB2 localization in actively migrating glioma cells, U251 cells were fixed after a migration assay (see below). The cells and cellular processes on the undersurface of the Transwell filter were immunostained for erbB2 as above. The filter was then removed from the insert using a scalpel blade, mounted on a glass microscope slide, visualized, and imaged as above.
For colocalization of erbB receptors with focal complex components, cells
were processed as for a migration assay (see below). After 4 h, cells were
fixed as above and double labeled with antibodies for erbB2 and one of the
following mouse monoclonal antibodies: erbB3 (Santa Cruz),
1-integrin (Santa Cruz), FAK (Upstate Biotechnology, Lake
Placid, NY), or phalloidin-586 (Molecular Probes). Antibodies and phalloidin
were used at the manufacturer's recommended dilution. Cells were visualized
and imaged as above.
Motility and Migration AssaysThe scratch motility assay was
used to measure two-dimensional movement. U251 cells were grown to confluence
in 24-well plates. A scratch was then made on the monolayer using a sterile
200-µl pipette tip. The monolayer was rinsed three times with migration
assay buffer (MAB) consisting of serum-free medium plus 0.1% fatty-acid free
bovine serum albumin and placed in MAB without or with 1 ng/ml NRG-1. At
the initiation of the experiment (t = 0) a digital image of the scar
was taken at a magnification of x10 (Axon Imaging, Foster City, CA). 24
h later (t = 24) the same region of the scar was imaged again. The
images were imported into the Scion imaging program (available online at
scioncorp.com).
This software allowed us to quantify the two-dimensional movement of the cells
by measuring the surface area of the scar at t = 0 and comparing it
with the surface area at t = 24. Experiments were done at least three
times. Measurements were made in triplicate.
To measure three-dimensional movement, the Transwell migration assay was
used as described previously
(27). Transwell filters (B-D
Falcon, Bedford, MA) with 8-µm pores were coated on the undersurface with
10 µg/ml laminin. The following day, they were washed three times with PBS
and blocked for 1 h with 1% fatty acid-free bovine serum albumin. In addition
to providing a necessary substrate for migration, the laminin undercoating
filled the 8-µm pores of the Transwell, creating a liquid diffusion barrier
effectively dividing the well into two chambers, upper and lower, to which
factor and/or inhibitors were added. Subconfluent U251 cells in 10-cm dishes
were serum starved overnight. On the day of the experiment, they were lifted
from the plate using 0.5 mM EDTA in PBS, pelleted, and resuspended
in MAB to attain a concentration of 100,000 cells/ml. Equal numbers of cells
(40,000/filter) were aliquoted onto the top surface of the Transwell filter
and allowed to adhere for 30 min prior to the addition of any drugs.
Inhibitors (AG 825, PP2, and PP3) were added for 30 min prior to the addition
of NRG-1
. The cells were returned to the incubator for 46 h,
rinsed with PBS, fixed, and stained with an ethanol-based crystal violet
solution. Cells on the upper surface of the filter were removed using a
cotton-tipped applicator. Cells on the lower surface of the filter were
visualized and imaged as above for immunostaining. Experiments were repeated
at least three times.
Adhesion Assay96-well plates were coated overnight with 10
µg/ml laminin. Cells were maintained as for a migration assay, plated at
25,000 cells/well using MAB, and treated with MAB without or with NRG-1
or NRG-1
plus AG 825. After 30 min, the cells were washed three times
with PBS, fixed, and stained with crystal violet. The plates were quantified
using an enzyme-linked immunosorbent assay plate reader at 590 nm and
analyzed. Experiments were repeated at least four times.
Immunoprecipitation and Western BlottingImmunoprecipitation
experiments were performed on confluent U251 cells grown on 10-cm dishes.
Cells were serum starved overnight. The following day, the cells were placed
in MAB for at least 1 h prior to the addition of drugs. AG 825 was added for
30 min prior to the addition of NRG-1. After 30 additional min, cells
were rinsed with ice-cold PBS and lysed for 30 min with ice-cold radioimmune
precipitation assay buffer (150 mM NaCl, 1% Nonidet P-40, 0.5%
deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, plus protease and
phosphatase inhibitors (Sigma)) on a shaker at 4 °C. Lysates were placed
in microcentrifuge tubes and spun for 4 min at 12,000 rpm to pellet the DNA.
After determining the protein concentration using a detergent-compatible
protein assay (Dc protein assay kit, Bio-Rad), lysates were diluted
to equal concentrations (1 mg/ml) using radioimmune precipitation assay
buffer. Samples were precleared with 5 µl of normal rabbit serum plus
protein A beads or with beads alone. Primary antibodies were added to the
precleared lysates at the following concentrations: 2 µg/ml erbB2, 6
µg/ml
1-integrin, and 5 µg/ml FAK. Lysates with primary
antibodies were incubated with constant rotation overnight at 4 °C. The
next day, 50 µl of agarose-conjugated protein A beads (Roche Applied
Science) was added to each sample for an additional 2 h at 4 °C. For
anti-phosphotyrosine immunoprecipitations, 30 µl/ml agarose-conjugated
anti-PY 99 was used (Santa Cruz). Immunoprecipitates were pelleted at low
speed at 4 °C and rinsed three times with ice-cold radioimmune
precipitation assay buffer. To release the immunoprecipitates from the beads,
2x sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2%
SDS, 0.1% brom-phenol blue, and 600 mM 2-mercaptoethanol) was
added, and the samples were placed in boiling water for 5 min. Proteins were
separated using 7.5% polyacrylamide ready gels (Bio-Rad), transferred to
polyvinylidene difluoride membranes (Millipore, Bedford, MA), and blocked
overnight with 5% nonfat milk. The membranes were subsequently incubated in
primary antibodies diluted in 5% nonfat milk for 2 h, rinsed with
Tris-buffered saline with 0.19% Tween 20 three times for 10 min each, and
probed with an horseradish peroxidase-conjugated goat anti-rabbit (1:3,000)
(Sigma) or mouse (1:2,000) (Santa Cruz) secondary antibody for 1 h. Membranes
were rinsed three times for 10 min each, incubated with ECL Plus according to
the manufacturer's instructions (Amersham Biosciences), exposed, and
developed.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
NRG-1 Increases Motility of U251 Human Glioma
Cells Because erbB2 and erbB3 receptors localized in regions of
the cell important for movement, we set out to determine whether their
activation could modulate motility, defined herein as movement in a
two-dimensional plane. This was accomplished by using a scratch motility assay
in which the repopulation of cells into a cell-free region (scar) could be
examined quantitatively. U251 cells were maintained until they reached
confluence. Subsequently, a scar was created in the monolayer, and the cells
were washed and placed in MAB without or with 1 ng/ml NRG-1
. We have
demonstrated previously that exogenous NRG-1
can activate erbB2 and
erbB3 receptors in U251 glioma cells as measured by induction of tyrosine
phosphorylation (29). After 24
h, the surface area of the scarred region was quantified using imaging
software (see "Experimental Procedures") and compared with the
surface area of the scarred region at the initiation of the experiment.
Movement of cells into the scarred region resulted in a decrease in the
surface area of the scar. Fig.
2A is a digital image of the scarred region before
(t = 0) and after the 24-h incubation period (t = 24) from a
typical experiment. The same experiment is quantified in
Fig. 2B. The overall
change in the surface area of the scar for control, untreated samples was
31.83% ± 0.23%, whereas the change in the surface area of the scar for
samples treated with 1 ng/ml NRG-1
was 52.48% ± 0.88%, an
increase of about 20% (p < 0.001).
|
NRG-1 Increases Transwell Migration of U251 Human Glioma
CellsOur results from the scratch motility assay indicated that
NRG-1
could modulate glioma cell movement in a two-dimensional plane. In
the brain, such movement is not likely. Instead, glioma cells must be able to
migrate and invade through the three-dimensional spatial constraints of the
surrounding brain tissue. To replicate this type of environment more closely,
we employed a Transwell migration assay
(Fig. 3). To induce glioma cell
migration, it was necessary to coat the undersurface of the filter with an
extracellular matrix (ECM) protein (data not shown). A similar dependence for
ECM proteins is thought to occur with glioma cells in vivo
(30). We performed our
migration assays using laminin, a potent inducer of U251 cell migration
(27). Serum-starved U251
glioma cells were plated onto the top of the Transwell filter and allowed to
migrate for 46 h before they were fixed, stained, and counted.
Fig. 3, A and
B, shows representative digital images of cells on the
undersurface of a filter which migrated in response to laminin alone (control)
or in response to laminin plus 1 ng/ml NRG-1
added to the lower chamber.
These images show that U251 cells migrated in response to laminin but that the
number of migrating cells increased markedly in the presence of NRG-1
.
Similar results were seen with two other human glioma cell lines, U87-MG and
U118-MG (data not shown), suggesting that this was not a cell type-specific
phenomenon. These experiments were repeated with the U251 glioma cell line
using a concentration range of NRG-1
from 0.1 to 100 ng/ml. The
resulting dose-response relationship is shown in
Fig. 3C and
Table I.
|
|
To determine whether NRG-1 effects on migration were permissive
(chemokinetic) or instructive (chemotactic), we designed a series of Transwell
experiments with 1) NRG-1
in the lower chamber, 2) NRG-1
in the
upper chamber, or 3) NRG-1
in both the lower and upper chambers. The
results for these studies are summarized in
Fig. 3D and
Table I. In each experimental
configuration, NRG-1
significantly increased the number of cells
successfully migrating through to the undersurface of the filter, implying
that NRG-1
acted in a permissive manner. However, although migration was
enhanced in each configuration, more cells were induced to migrate when
NRG-1
was present in the bottom of the chamber (2.36 ± 0.02-fold
increase) versus in the top (1.42 ± 0.07-fold increase),
suggesting an instructive role (see "Discussion").
Although our data clearly indicated that NRG-1 could enhance the
Transwell migration of glioma cells, a role for erbB2 in the effect was not
conclusive. Therefore, we examined NRG-1
effects in the presence of the
erbB2-specific pharmacological inhibitor, AG 825
(31,
32,
33). For these experiments, 1
ng/ml NRG-1
and increasing concentrations of AG 825 were included in
both the upper and lower chambers. As demonstrated
(Fig. 3E) AG 825
dose-dependently inhibited the NRG-1
-induced stimulation of Transwell
migration with an apparent IC50 of 12.28 µM. Results
for the migration experiments are summarized in
Table I.
Because the ability of cells to migrate depends critically on their
interactions with the ECM, we next examined the potential effect of
NRG-1 on the adhesion of U251 cells to laminin. Serum-starved cells were
plated onto a laminin-coated 96-well plate and allowed to adhere for 30 min to
1 h. Subsequently, the cells were washed, fixed, and stained. The number of
adherent cells was quantified using an enzyme-linked immunosorbent assay plate
reader. As indicated (Fig.
3F), the ability of the U251 cells to adhere to laminin
was not altered by exposing the cells to a range of NRG-1
concentrations, or to 50 µM AG 825.
ErbB2 Receptor Immunoreactivity Localizes to the Invadipodia of
Migrating Cells and Colocalizes with the Focal Complex Proteins
1-Integrin and FAKThus far, our
results indicated that stimulation of glioma cells by NRG-1
could
enhance motility and migration. Our initial immunostaining
(Fig. 1) demonstrated that the
erbB2 and erbB3 receptors localized in regions of the plasma membrane called
lamellipodia. We hypothesized that this localization might help facilitate the
ability of the receptors to modulate migration. To investigate further, we
performed erbB2 immunostaining in actively migrating cells. For these studies,
we used the same Transwell migration assays; however, instead of fixing and
staining the cells for cell counting, we processed them for immunostaining of
the erbB2 receptor. As with the migration assays (see "Experimental
Procedures"), cells on the upper surface of the filter were removed,
leaving only the cells and the invading processes on the undersurface. The
invading processes of the migrating cells, called invadipodia
(34), contain the components
necessary for migration into the pore. The digital image displayed in
Fig. 4 is a composite image
made by combining sample images taken from the undersurface of one Transwell
filter. For this experiment, the cells were exposed to 1 ng/ml NRG-1
in
both the upper and lower chambers. It is clear from this composite that the
erbB2 receptor localized in the invadipodia. The morphology of the invadipodia
varied depending on the temporal status of the cells in the migration process.
We saw no significant difference in the erbB2 staining pattern from cells on
filters that were unstimulated, exposed to NRG-1
in the lower chamber
only, or exposed to NRG-1
plus AG 825 (data not shown), suggesting the
constitutive and invariant expression of these receptors on glioma cells.
|
In light of our immunostaining results localizing erbB2 in invadipodia and
based on evidence from the literature indicating the presence of focal complex
proteins in these processes
(34), we decided to examine
whether erbB2 could colocalize with other molecules implicated in the
migration process. Specifically, we examined the localization of erbB2 with
F-actin, the polymerized form of actin which is present in these cellular
extensions (35);
1-integrin, an ECM receptor that mediates engagement with the
laminins (36); and FAK, a
nonreceptor protein-tyrosine kinase that has been implicated in mediating both
integrin- and growth factor-induced cellular migration
(37). In addition, we examined
whether the erbB3 receptor also colocalized with erbB2 in these invading
processes. These experiments were performed as described above, by
immunostaining the fixed processes of U251 cells actively migrating through a
Transwell filter in the absence or presence of NRG-1
or NRG-1
plus
AG 825. Images displayed in Fig.
5 are of cells treated with NRG-1
. There was no significant
difference in the pattern of staining between cells that were untreated,
treated with NRG-1
or with NRG-1
plus AG 825 (data not shown). As
demonstrated, erbB2 and erbB3 receptors colocalized in the invadopodia
(Fig. 5, AC).
Additionally, the erbB2 immunostaining appeared to overlap with that of
phalloidin (Fig. 5,
DF),
1-integrin
(Fig. 5, GI),
and FAK (Fig. 5,
JL).
|
ErbB2 Coimmunoprecipitates with
1-Integrin and FAKTo establish
further a functional interaction between erbB2 and components of the focal
complex, we performed coimmunoprecipitation experiments. First, using
antibodies against
1-integrin, we immunoprecipitated proteins
from whole cell lysates of serum-starved U251 cells that were treated
previously without or with NRG-1
or NRG-1
plus AG 825. The
immunoprecipitates were separated by SDS-PAGE, transferred to polyvinylidene
difluoride membrane, probed for erbB2, then stripped and reprobed for
1-integrin. Bands at the appropriate molecular masses
(
1-integrin,
130 kDa; erbB2,
185 kDa) were detected
(Fig. 6A). The reverse
immunoprecipitation was performed yielding similar results (data not shown).
Although not quantified, the level of association between erbB2 and
1-integrin did not appear to change in the presence of
NRG-1
or NRG-1
plus AG 825
(Fig. 6A and data not
shown).
|
A second focal complex protein, FAK, was immunoprecipitated from U251
lysates and analyzed by immunoblotting for erbB2. Subsequently, the blot was
stripped and reprobed for FAK. Bands at the appropriate molecular masses (FAK,
125 kDa; erbB2,
185 kDa) were detected
(Fig. 6B). The reverse
immunoprecipitation of treated and untreated U251 lysates with erbB2-specific
antibodies was performed and immunoblotted for FAK
(Fig. 6C).
Interestingly, as indicated, the amount of FAK associating with erbB2 appeared
to increase when the receptors were stimulated by NRG-1
. Likewise, the
level of association appeared to decrease when the receptors were inhibited by
AG 825 (in the presence of NRG-1
).
NRG-1 Activates FAKOur results described in
Fig. 6 allowed us to formulate
the hypothesis that erbB receptor activation by NRG-1
led to the
stimulation of FAK. Such modulation of FAK has been described downstream from
NRG-1 treatment in human breast cancer cells
(12) and in cultured Schwann
cells (38). Therefore, we
examined, in more detail, whether erbB2 could modulate the activation status
of FAK in human glioma cells. This was accomplished by measuring the level of
tyrosine-phosphorylated FAK in U251 cells that had been treated with
increasing concentrations of NRG-1
or NRG-1
plus increasing
concentrations of AG 825 (Fig.
7A). Lysates were immunoprecipitated with
agarose-conjugated phosphotyrosine antibodies then immunoblotted with
antibodies specific for FAK. As demonstrated, NRG-1
dose dependently
increased the level of tyrosine phosphorylation of FAK, and AG 825 (in the
presence of NRG-1
) dose dependently decreased the level of tyrosine
phosphorylation of FAK.
|
FAK contains at least six tyrosine residues that can be phosphorylated
resulting in kinase activation
(39). Tyr-397 is the major
phosphorylation (activation) site on FAK
(40). Phosphorylation of this
residue recruits another nonreceptor protein-tyrosine kinase, Src, to FAK.
Subsequently, Src is believed to activate FAK completely through additional
tyrosine phosphorylation events
(41). In this way, the
FAK·Src complex is thought to activate other key proteins involved in
migration such as p130Cas
(42,
43). Therefore, we took our
analysis one step further by investigating whether a Src-specific inhibitor
(PP2) could decrease the effects of NRG-1 on glioma cell migration.
Indeed, our results (Fig.
7B) indicated that PP2 but not PP3 (an inactive analog of
PP2) could inhibit the stimulatory effects of NRG-1
on U251 glioma cell
migration. Specifically, NRG-1
increased migration 1.95 ±
0.11-fold, relative to control. PP3 treatment in the presence of NRG-1
did not significantly alter cell migration (1.76 ± 0.08-fold increase),
whereas PP2 treatment markedly reduced NRG-1
effects (0.51 ±
0.05-fold decrease, p < 0.001).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Evidence That ErbB2 Is Localized in Regions of the Cell Involved in
Motility and MigrationLamellipodia in motile cells and invadipodia
in migrating or invading cells are dynamic processes that contain members of
the focal complex (44). Growth
factor receptors have been reported to associate with components of this
complex. In particular, the erbB2 receptor coimmunoprecipitated with integrins
1 and
4
(45,
46,
47) and with FAK
(12,
39). In our study, we
demonstrated the localization of erbB2 receptors in lamellipodia and
invadipodia. Importantly, we evidenced the coimmunostaining of erbB2 with
members of the focal complex. Our immunostaining results demonstrated similar
localization of components in cells treated without or with NRG-1
or
NRG-1
plus AG 825. This suggested that the activation status of erbB2
did not alter its subcellular localization or the localization of actin,
1-integrin, vinculin, or FAK. Instead, it appeared that the
colocalization between these components was constitutive and represented a
coclustering (48) in the same
region of the plasma membrane. Indeed, coimmunoprecipitation experiments
between erbB2 and
1-integrin confirmed a level of association
which did not change after stimulation or inhibition of erbB2.
Interestingly, our coimmunoprecipitation experiments between erbB2 and FAK demonstrated an association status that was dependent upon the activation of erbB2. In this case, we believe that FAK, already localized in the same region of the cell as erbB2, was stimulated to complex either directly or indirectly with erbB2 in a manner that was dependent upon the activation status of erbB2. Such an association between FAK and activated platelet-derived growth factor receptors has recently been demonstrated in transiently transfected human 293T cells (49). These studies place the erbB receptors, specifically erbB2, in regions of the membrane which are important for cellular motility and may facilitate their ability to modulate movement.
Activation of ErbB2 and ErbB3 Receptors by a Specific Ligand,
NRG-1, Can Modulate Motility and MigrationErbB2 and
erbB3 receptors were selectively activated using the specific ligand
NRG-1
(29). This
recombinant form of NRG-1 contains only the active epidermal growth
factor-like domain. The endogenous NRG-1 protein is synthesized as a
transmembrane-bound proprotein by both neurons and glia
(18,
19,
20) and by glioma cells
themselves (22). It is
believed that this membrane-bound form of NRG-1 can stimulate erbB receptors
on the same (autocrine) or surrounding cells (juxtacrine). For example,
membrane-bound NRG-1 expressed by radial glial cells has been shown to induce
the migration of cortical neurons
(50).
In addition to producing NRG-1 as a membrane-bound form, cells of the central nervous system and glioma cells (data not shown) can proteolytically cleave and release the functional portion of the NRG-1 protein. Included in the domains of this released form of NRG-1 is an Ig-like domain that allows it to adhere to the surrounding ECM (21). In this way, released NRG-1 appears to function in a paracrine manner to stimulate erbB receptors (51). We speculate that the ability of NRG-1 to adhere to the ECM in vivo places it in a position to activate erbB receptors present on the invading processes of glioma cells.
In our cellular movement experiments we demonstrated that the motility and
migration of glioma cells could be enhanced by exogenous application of
NRG-1. Notably, we saw that glioma cells could migrate in the absence of
NRG-1
but that the magnitude of movement under a variety of experimental
conditions could be increased in response to NRG-1
. This meant that
activation of erbB2 and erbB3 by NRG-1
could increase a process that was
already activated in the glioma cells. More specifically, in our migration
experiments, we saw that laminin could induce migration; however, the effect
was enhanced in the presence of NRG-1
. Indeed, NRG-1
appeared to
function in both a chemokinetic and chemoattractant manner.
Laminin induces migration of glioma cells in a manner that is dependent
upon the activation of integrin receptors and subsequently, the activation of
FAK (52). We believe that the
enhancement seen with NRG-1 was caused by the concomitant activation of
FAK by signals generated downstream from both integrins and erbB2 receptors.
Such a convergence onto FAK has been demonstrated in the coactivation of
integrins and the epidermal growth factor receptor (erbB1)
(49).
Signals from ErbB2 and Integrins Converge on FAKFAK is a
nonreceptor protein-tyrosine kinase that can localize in the focal complex
through a direct (53) or
indirect (54) association with
the 1-integrin cytoplasmic tail. Upon
1-integrin engagement with an ECM such as laminin, FAK
becomes autophosphorylated and initiates signaling cascades that ultimately
lead to a variety of effects including cytoskeletal reorganization
(39). ErbB2 has been shown to
associate with and modulate FAK after stimulation with NRG-1. As discussed
previously, we observed colocalization between erbB2 and FAK in human glioma
cells. Additionally, we were able to coimmunoprecipitate erbB2 and FAK in a
manner that appeared to be dependent upon the activation status of the erbB2
receptor. In subsequent experiments we were able to demonstrate that
NRG-1
dramatically increased the amount of tyrosine-phosphorylated FAK
in U251 glioma cells. This stimulation was dependent upon the erbB2 receptor
as demonstrated by a pharmacological inhibitor, AG 825. It is possible that
FAK is a substrate for erbB2 kinase, implying a direct association between the
two proteins. Alternatively, erbB2 has been shown to associate and/or activate
a number of proteins such as Src and Grb2
(16), which also associate
with FAK. Therefore, erbB2 may indirectly modulate FAK by stimulating a
kinase, which subsequently modulates its activity.
As a first step to elucidate the downstream effects of NRG-1
stimulation of FAK, we demonstrated that the Src-specific inhibitor PP2, but
not its inactive analog (PP3), could abrogate NRG-1
effects on
migration. At least two possible scenarios could explain the PP2 effects: 1)
Src is upstream from FAK activation by erbB2, and its inhibition prevents FAK
stimulation; or 2) Src is downstream from FAK activation, and its inhibition
prevents the formation of the FAK·Src complex. The exact mechanisms
mediating erbB2 activation of FAK and Src inhibition of migration remain to be
clarified and will be the subject of future studies.
In conclusion, our results demonstrate that the activation of erbB
receptors by NRG-1 enhances glioma cell migration. Such a function for
the NRG-1/erbB receptor cascade has not been demonstrated previously in human
brain malignancies. These findings are intriguing, however, in light of NRG-1
effects on the development of the brain. In fact, NRG-1 is believed to serve a
number of important functions related to cellular migration in the nervous
system. For example, during embryonic development, GGF2 (an isoform of NRG-1)
promotes the migration of mammalian neural crest cells
(55). In the developing
central nervous system, GGF/NRG-1 expression on radial glial cells was
documented to be necessary for cortical neuronal migration
(50), and NRG-1 expression on
migrating granule cells is necessary for radial glia formation in the
developing cerebellum (56). In
the peripheral nervous system, NRG-1 was shown to enhance the motility of
Schwann cells (57). One study
reported that this function was dependent upon the activation of the
mitogen-activated protein kinase cascade
(58). In addition, Vartanian
et al. (38)
demonstrated that NRG-1 could induce the rapid association of FAK with erbB2
and erbB3 in cultured Schwann cells. Our report, demonstrating a similar
effect of NRG-1 on glioma cells, may provide additional support to the theory
that gliomas recapitulate many features of cells of the developing brain
(59,
60). Indeed, it is still
unclear whether gliomas arise from differentiated glial cells or from their
progenitors. Regardless of their origin, it is apparent from our report that
glioma cells respond to NRG-1 in a manner that is similar to immature neural
cells migrating through the brain. A better understanding of the mechanisms
involved in this effect would not only contribute to our knowledge of the
events that lead to the spread human malignant gliomas but would also apply to
the way brain cells, in general, migrate within the developing brain.
![]() |
FOOTNOTES |
---|
To whom correspondence should be addressed: Dept. of Neurobiology, University
of Alabama, 1719 6th Ave. South, CIRC 589, Birmingham, AL 35294-0021. Tel.:
205-975-5805; Fax: 205-975-5518; E-mail:
sontheimer{at}uab.edu.
1 The abbreviations used are: FAK, focal adhesion kinase; ECM, extracellular
matrix; MAB, migration assay buffer; NRG-1, neuregulin-1; PBS,
phosphate-buffered saline; PP, protein phosphatase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|