RhoA Inhibits the Nerve Growth Factor-induced Rac1 Activation
through Rho-associated Kinase-dependent Pathway*
Yoshiaki
Yamaguchi,
Hironori
Katoh,
Hidekazu
Yasui,
Kazutoshi
Mori, and
Manabu
Negishi
From the Laboratory of Molecular Neurobiology, Graduate School of
Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received for publication, January 11, 2001, and in revised form, March 8, 2001
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ABSTRACT |
The Rho family of small GTPases has been shown to
be involved in the regulation of neuronal morphology, and Rac and Rho
exert antagonistic actions in neurite formation. In this study, we have examined the cross-talk between Rac and Rho in relation to the nerve
growth factor (NGF)-induced neurite outgrowth in PC12 cells. NGF
induced a rapid activation of Rac1 and suppression of RhoA activity.
Constitutively active RhoA, RhoAV14, or
constitutively active G
12-induced endogenous RhoA
activation inhibited the NGF-induced Rac1 activation without any effect
on the NGF-induced extracellular signal-regulated kinase activation. Moreover, Y-27632, an inhibitor of Rho-associated kinase, completely abolished the RhoA-induced down-regulation of the NGF-induced Rac1
activation. We also revealed that NGF induced a rapid recruitment of
Rac1 to the cell surface protrusion sites and formed filamentous actin-rich protrusions. Activation of RhoA and Rho-associated kinase
formed a thick ringlike structure of cortical actin filaments at the
cell periphery and then inhibited the NGF-induced recruitment of Rac1
to protrusions. These results indicate that RhoA down-regulates the
NGF- induced Rac1 activation through Rho-associated kinase, inhibiting the neurite formation.
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INTRODUCTION |
In the developing nervous system, formation of appropriate
connections between neurons is an essential process for the
establishment and maturation of neural circuits. Neurite extension and
retraction are very important processes in the formation of neuronal
networks, and these dynamic morphological changes of neuronal cells are largely decided by the actin cytoskeletal organization (1).
The Rho family of small GTPases, consisting of Cdc42, Rac, and Rho, has
been implicated in the reorganization of the actin cytoskeleton and
subsequent morphological changes in various cellular functions (2).
Among them, Rac is involved in membrane ruffling and formation of
lamellipodia, whereas Rho is responsible for regulating the assembly of
focal adhesion and stress fiber formation in fibroblasts (3, 4).
Similar to other GTPases of the Ras superfamily, they serve as a
molecular switch by cycling between an inactive GDP-bound state and an
active GTP-bound state. Activation of the Rho family proteins requires
GDP-GTP exchange catalyzed by various guanine nucleotide exchange
factors, and their activation is regulated by GTPase-activating
proteins, which stimulate the intrinsic GTPase activities of the G
proteins, leading to the cessation of their actions. In addition,
guanine nucleotide dissociation inhibitors inhibit the exchange of GDP
for GTP and might also serve to regulate their association with
membranes (5). In neuronal cells, Rho family proteins are involved in
axon and dendrite formation in various types of neurons (6-9), and
defects in the regulation of these GTPase activities have been reported
to affect the development of the nervous system (10-13). Rac has been
shown to be involved in the formation of lamellipodia of a growth cone and to be required for the outgrowth of neurites (14). On the other
hand, activation of Rho was reported to induce the collapse of the
growth cone and the retraction of neurites and to inhibit the neurite
outgrowth (14-16). As mentioned above, Rac and Rho are thought to
counteract each other's activity in neuronal cells (14, 17).
Therefore, the balance between Rac and Rho activities is likely to be a
crucial point for neuronal morphology. The cross-talk between Rac and
Rho has been studied in a variety of cell lines, and the physiological
significance of the cross-talk has been established in various cellular
functions (18, 19). However, little is yet known about the regulatory
mechanism of the cross-talk between Rac and Rho.
Rat pheochromocytoma PC12 cells have been used as a model system for
neuronal differentiation and neurite outgrowth. After stimulation with
nerve growth factor (NGF),1
the cells stop growing and begin to extend neurites. Rac plays an
important role in the regulation of the cytoskeletal changes required
for neurite outgrowth in response to NGF, whereas Rho has been shown to
inhibit neurite outgrowth by NGF (16). In this study, we have examined
the cross-talk between Rac and Rho in PC12 cells, and we showed that
NGF induced the Rac1 activation but also the inhibition of RhoA
activity and that the activation of RhoA down-regulated the NGF-induced
Rac1 activation via Rho-associated kinase.
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EXPERIMENTAL PROCEDURES |
Materials--
Y-27632 was a generous gift from Yoshitomi
Pharmaceutical Industries (Saitama, Japan). Agents obtained and
commercial sources were as follows: NGF, Promega; mouse
monoclonal anti-Rac1 antibody, Transduction Laboratories; mouse
monoclonal anti-RhoA antibody, Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA); mouse monoclonal anti-hemagglutinin (HA) antibody (clone
12CA5), Roche Molecular Biochemicals; rabbit antiphosphospecific
extracellular signal-regulated kinase (ERK) antibody, New England
Biolabs, Inc.; rabbit anti-ERK1 and mouse anti-ERK2 antibodies, Upstate
Biotechnology, Inc. (Lake Placid, NY); Alexa 488-conjugated phalloidin
and rhodamine-conjugated phalloidin, Molecular Probes, Inc.
(Eugene, OR); rhodamine-conjugated donkey anti-mouse immunoglobulin G
(IgG) antibody, Chemicon International Inc.; horseradish
peroxidase-conjugated goat anti-mouse IgG antibody and horseradish
peroxidase-conjugated swine anti-rabbit IgG antibody, DAKO; and
chemiluminescence ECL Western blotting system, Amersham Pharmacia
Biotech. Mammalian expression vector pcDNA3 carrying cDNA for a
variant of the Aequorea victoria green fluorescent protein
(GFP) was obtained as described previously (20). cDNA for the
catalytic domain of Rho-associated kinase, ROK
(CD-ROK
; Ref. 15),
was inserted into mammalian expression vector pEF-BOS. The sources of
other materials are as indicated.
Cell Culture and Transfection--
PC12 cells were cultured in
Dulbecco's modified Eagle's medium containing 10% horse serum, 5%
fetal bovine serum, 4 mM glutamine, 100 units/ml
penicillin, and 0.2 mg/ml streptomycin under humidified conditions in
95% air and 5% CO2 at 37 °C. PC12 cell line expressing RhoAV14 or G
12QL under the control of
isopropyl-
-D-thiogalactoside (IPTG) was obtained as
described previously (21). For transfection, cells were seeded onto
poly-D-lysine (Sigma)-coated glass coverslips (circular, 13 mm) in 24-well plates at a density of 1 × 104
cells/well and cultured for 15-18 h. Then cells were transfected with
0.8 µg of total DNA using LipofectAMINE 2000 (Life Technologies, Inc.) according to the manufacturer's instructions. Cells were fixed
24 h after transfection.
SDS Gel Electrophoresis and Immunoblotting--
After PC12 cells
were rinsed briefly with PBS, they were lysed for 5 min with the
respective ice-cold lysis buffers (Rac1 activity assay, 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin; RhoA activity assay, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 30 mM MgCl2, 0.1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin; ERK activity assay, 50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, 10 mM NaF, 1 mM Na3VO4, 10% glycerol, 1%
Nonidet P-40, 250 µM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin). The cell lysates were
immediately centrifuged for 5 min at 10,000 × g at 4 °C, and the supernatants were separated by SDS-polyacrylamide gel
electrophoresis. The proteins were transferred onto a polyvinylidene difluoride membrane (Millipore Corp.). The membrane was blocked with
3% low fat milk in Tris-buffered saline and then incubated at 4 °C
with primary antibodies. The immunoblots were detected using a
chemiluminescence ECL Western blotting system with horseradish peroxidase-conjugated secondary antibodies.
Rac1 and RhoA Activity Assays--
Measurement of Rac1 and RhoA
activities was performed as described previously (20, 21).
Eighteen-hour serum-starved PC12 cells (Rac1, 3 × 106
cells; RhoA, 2 × 107 cells) were lysed for 5 min with
the respective ice-cold cell lysis buffer containing 6 µg of the
glutathione S-transferase-fused Cdc42/Rac-interactive
binding domain of rat
PAK (GST-CRIB) for GTP-bound Rac1 or 16 µg
of GST-Rho-binding domain of mouse rhotekin (GST-RBD) for GTP-bound
RhoA. For preparing suspended cells, PC12 cells were detached and kept
in suspension in serum-free Dulbecco's modified Eagle's medium for 10 min before lysis. Cell lysates were then centrifuged for 5 min at
10,000 × g at 4 °C, and the supernatants were
incubated with glutathione-Sepharose beads for 30 min (Rac1) or 60 min
(RhoA) at 4 °C. After the beads had been washed with the cell lysis
buffer, the bound proteins were resolved on 12.5% SDS-polyacrylamide
gel electrophoresis and immunoblotted using a mouse monoclonal
anti-Rac1 antibody (1:1000) or a mouse monoclonal anti-RhoA antibody
(1:100). Densitometric analyses were performed using NIH Image
software, and the amounts of GTP-bound Rac1 and RhoA were normalized to
the total amounts of Rac1 and RhoA in cell lysates, respectively.
Immunofluorescence Microscopy--
PC12 cells were seeded onto
poly-D-lysine-coated glass coverslips in 24-well plates at
a density of 1 × 104 cells/well. All steps were
carried out at room temperature, and cells were rinsed with
phosphate-buffered saline (PBS) between each step. Cells on coverslips
were fixed with 3.7% formaldehyde/PBS for 20 min. After residual
formaldehyde had been quenched with 50 mM
NH4Cl/PBS for 10 min, cells were permeabilized in 0.2%
Triton X-100 for 10 min and incubated with 10% fetal bovine serum in PBS for 30 min to block nonspecific antibody binding. Endogenous Rac1
was stained with an anti-Rac1 monoclonal antibody in PBS at a 1:1000
dilution for 1 h followed by incubation with a
rhodamine-conjugated donkey anti-mouse IgG in PBS at a 1:500 dilution
for 1 h. Filamentous actin (F-actin) was stained with Alexa
488-conjugated phalloidin in PBS (0.5 units/ml) or rhodamine-conjugated
phalloidin in PBS (1 unit/ml) for 1 h. Cells were mounted in 90%
glycerol containing 0.1% p-phenylenediamine dihydrochloride
in PBS. Confocal microscopy was performed using an MRC-1024
laser-scanning confocal imaging system (Bio-Rad) equipped with a Nicon
Eclipse E800 microscope and a Nicon Plan Apo 60 × 1.4 oil
immersion objective.
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RESULTS |
Effects of NGF on Rac1 and RhoA Activities--
Rac has been shown
to be involved in the NGF-induced neurite outgrowth (17, 22), while
active Rho has been reported to prevent the NGF-induced neuritogenesis
(16). To assess more directly modulation of Rac1 and RhoA activities by
NGF, we measured the amounts of cellular GTP-bound Rac1 and RhoA using
the GST-fused CRIB domain of
PAK (GST-CRIB) and GST-fused
Rho-binding domain of rhotekin (GST-RBD), respectively. As shown in
Fig. 1, NGF induced a rapid increase in
the amount of cellular GTP-bound Rac1, the elevation reaching a maximum
at 3 min. The level decreased gradually but remained above the basal
level for over 60 min after the stimulation. On the other hand, NGF
induced a rapid decrease in the amount of cellular GTP-bound RhoA, the
level reaching a minimum at 3-10 min, and it took at least 30 min to
regain the basal level. Therefore, NGF oppositely regulated the
activities of Rac1 and RhoA, activation of Rac1 and suppression of
RhoA.

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Fig. 1.
Effects of NGF on Rac1 and RhoA
activities. Serum-starved PC12 cells were treated with 50 ng/ml
NGF for the indicated times. A, the cell lysates were
incubated with GST-CRIB, and the amounts of GTP-bound Rac1 were
determined by immunoblotting using a monoclonal antibody against Rac1
(inset). Total amounts of Rac1 in cell lysates are also
shown. B, the cell lysates were incubated with GST-RBD, and
the amounts of GTP-bound RhoA were determined by immunoblotting using a
monoclonal antibody against RhoA (inset). Total amounts of
RhoA in cell lysates are also shown. Rac1 and RhoA activities are
indicated by the amounts of GTP-bound Rac1 and GTP-bound RhoA
normalized to the amounts of Rac1 and RhoA in whole cell lysates,
respectively, and values are expressed as -fold of the value of
serum-starved cells at time 0 min. The results shown are the means ± S.E. of triplicate experiments.
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Inhibition of NGF-induced Rac1 Activation by RhoA--
Active Rho
has been known to prevent NGF-induced neuritogenesis (16). We next
examined the effect of RhoA activation on NGF-induced Rac1 activation,
using the PC12 cell line expressing constitutively active HA-tagged
RhoA, HA-RhoAV14, under the control of IPTG, which we had
previously established (referred to as "RhoAV14-inducible
PC12 cells"; Ref. 21). The expression of RhoAV14 by IPTG
completely inhibited the NGF-induced Rac1 activation without any effect
on the basal Rac1 activity in RhoAV14-inducible PC12 cells
(Fig. 2). We recently demonstrated that the G12 family of heterotrimeric G proteins
induced activation of endogenous RhoA, causing neurite
retraction in PC12 cells (21). We then examined the effect of
endogenous RhoA activation on the NGF-induced Rac1 activation using the
PC12 cell line expressing constitutively active G
12,
G
12QL, under the control of IPTG (21). The expression of
G
12QL by IPTG completely inhibited the NGF-induced Rac1
activation (Fig. 3), indicating that the RhoA activation pathway is a negative regulator of the NGF-induced Rac1
activation.

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Fig. 2.
Inhibition of the NGF-induced Rac1 activation
by RhoAV14. A, serum-starved
RhoAV14-inducible PC12 cells were treated with or without 5 mM IPTG for 12 h and then they were stimulated with or
without 50 ng/ml NGF for 3 min. The cell lysates were incubated with
GST-CRIB, and the amounts of GTP-bound Rac1 were determined by
immunoblotting using a monoclonal antibody against Rac1
(upper panel). Total amounts of Rac1 in
cell lysates (middle panel) and expression of
HA-tagged RhoAV14 (bottom panel) are
also shown. The results shown are representative of three independent
experiments that yielded similar results. B, quantification
of the effect of RhoAV14 on Rac1 activity. The Rac1
activity is indicated by the amount of GST-CRIB-bound Rac1 normalized
to the amount of Rac1 in whole cell lysates, and values of Rac1
activity are expressed as -fold increase over the value of cells that
were not treated with either IPTG or NGF. Data are the means ± S.E. of triplicate experiments.
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Fig. 3.
Inhibition of the NGF-induced Rac1 activation
by G 12QL. A,
serum-starved G 12QL-inducible PC12 cells were treated
with or without 5 mM IPTG for 12 h, and then they were
stimulated with or without 50 ng/ml NGF for 3 min. The cell lysates
were incubated with GST-CRIB, and the amounts of GTP-bound Rac1 were
determined by immunoblotting using a monoclonal antibody against Rac1
(upper panel). Total amounts of Rac1 in cell
lysates (middle panel) and expression of
HA-tagged G 12QL (bottom panel,
arrow) are also shown. The results shown are representative
of three independent experiments that yielded similar results.
B, quantification of the effect of G 12QL on
Rac1 activity. The Rac1 activity is indicated by the amount of
GST-CRIB-bound Rac1 normalized to the amount of Rac1 in whole cell
lysates, and values of Rac1 activity are expressed as -fold increase
over the value of cells that were not treated with either IPTG or NGF.
Data are the means ± S.E. of triplicate experiments.
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Recent studies have shown that cell adhesion to the extracellular
matrix induces activation of Rac (23, 24). On the other hand,
activation of Rho has been shown to induce cell rounding in PC12 cells,
rendering the cells less adherent (21, 25). Therefore, down-regulation
of the NGF-induced Rac1 activation by RhoAV14 might be a
secondary effect of RhoAV14-induced reduction of cell
adhesion activity. To address this issue, we examined the effect of
RhoAV14 on the NGF-induced Rac1 activation in suspended
RhoAV14-inducible PC12 cells. NGF induced a rapid increase
in the amount of GTP-Rac1 in suspended PC12 cells as well, but the
level reached a maximum at 1 min (data not shown). As shown in Fig.
4, the expression of RhoAV14
by IPTG inhibited the NGF-induced Rac1 activation in the suspended PC12
cells. Thus, the RhoAV14-induced down-regulation of the
Rac1 activation is not due to the reduction of cell adhesion
activity.

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Fig. 4.
Inhibition of the NGF-induced Rac1 activation
by RhoAV14 in suspended cells. A, after
serum-starved RhoAV14-inducible PC12 cells had been treated
with or without 5 mM IPTG for 12 h, they were detached
and kept in suspension in serum-free Dulbecco's modified Eagle's
medium. After they were stimulated with 50 ng/ml NGF for 1 min, the
cells were lysed and incubated with GST-CRIB, and the amounts of
GTP-bound Rac1 were determined by immunoblotting using a monoclonal
antibody against Rac1 (upper panel). Total
amounts of Rac1 in cell lysates (middle panel)
and expression of HA-tagged RhoAV14 (bottom
panel) are also shown. The results shown are representative
of three independent experiments that yielded similar results.
B, quantification of the effect of RhoAV14 on
Rac1 activity in suspended cells. The Rac1 activity is indicated by the
amount of GST-CRIB-bound Rac1 normalized to the amount of Rac1 in whole
cell lysates, and values of Rac1 activity are expressed as -fold
increase over the value of cells that were not treated with either IPTG
or NGF. Data are the means ± S.E. of triplicate
experiments.
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NGF has been known to differentiate PC12 cells through the ERK signal
transduction pathway (26, 27). RhoAV14-induced
down-regulation of the Rac1 activation might result from perturbation
of the NGF-induced ERK activation. To exclude this possibility, we
examined the effect of RhoAV14 on the NGF-induced ERK
activation by visualizing phosphorylation of endogenous ERK with an
antiphospho-ERK antibody. As shown in Fig.
5, the expression of RhoAV14
by IPTG did not affect the NGF-induced ERK activation in
RhoAV14-inducible PC12 cells, indicating that the
RhoAV14-induced down-regulation occurred irrespective of
the ERK signal transduction pathway.

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Fig. 5.
RhoA has no effect on the NGF-induced ERK
activation. Serum-starved RhoAV14-inducible PC12 cells
were treated with or without 5 mM IPTG for 12 h, and
then they were stimulated with or without 50 ng/ml NGF for 5 min.
Phosphorylated ERK in cell lysates was determined by immunoblotting
using a rabbit polyclonal antiphosphospecific ERK antibody
(upper panel). Total amounts of ERK1 and ERK2 in
cell lysates (middle panels) and expression of
HA-tagged RhoAV14 (bottom panel) are
also shown. The results shown are representative of three independent
experiments that yielded similar results.
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Involvement of Rho-associated Kinase in RhoAV14-induced
Down-regulation of the Rac1 Activation--
Rho-associated kinase, one
of the downstream targets of RhoA, has been shown to be involved in a
variety of actions of Rho (28). To determine whether Rho-associated
kinase was involved in the RhoAV14-induced down-regulation
of the Rac1 activation, the cells were treated with a Rho-associated
kinase-selective inhibitor, Y-27632 (29). As shown in Fig.
6, Y-27632 had little effect on the basal and NGF-induced Rac1 activation. However, Y-27632 completely abolished the RhoAV14-induced down-regulation of the Rac1 activation
in both adherent and suspended RhoAV14-inducible PC12
cells. In addition, Y-27632 also abolished the G
12QL-induced down-regulation of the Rac1 activation in
G
12QL-inducible PC12 cells (data not shown). On the
other hand, Y-27632 did not affect the time course profile of the
NGF-induced Rac1 activation (Fig. 6E). These results
indicate that the RhoAV14-induced down-regulation of the
Rac1 activation is mediated by Rho-associated kinase.

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Fig. 6.
Effect of a Rho-associated kinase inhibitor
on the RhoAV14-induced inhibition of Rac1 activation.
A and B, serum-starved
RhoAV14-inducible PC12 cells were treated with or without 5 mM IPTG for 12 h in the absence or presence of 10 µM Y-27632. After attached (A) or suspended
(B) cells had been stimulated with or without 50 ng/ml NGF
for 3 (A) or 1 (B) min, the cell lysates were
incubated with GST-CRIB, and the amounts of GTP-bound Rac1 were
determined by immunoblotting using a monoclonal antibody against Rac1
(upper panels). Total amounts of Rac1 in cell
lysates (middle panels) and expression of
HA-tagged RhoAV14 (bottom panels) are
also shown. The results shown are representative of three independent
experiments that yielded similar results. C and
D, quantification of the effect of Y-27632 on Rac1 activity
in attached (C) or suspended (D)
RhoAV14-inducible PC12 cells. The Rac1 activity is
indicated by the amount of GST-CRIB-bound Rac1 normalized to the amount
of Rac1 in whole cell lysates, and values of Rac1 activity are
expressed as -fold increase over the value of cells that were not
treated with either IPTG or NGF or Y-27632. Data are the means ± S.E. of triplicate experiments. E, after serum-starved PC12
cells had been exposed to vehicle or 10 µM Y-27632, they
were treated with 50 ng/ml NGF for the indicated times. The cell
lysates were incubated with GST-CRIB, and the amounts of GTP-bound Rac1
were determined by immunoblotting using a monoclonal antibody against
Rac1 (upper panel). Total amounts of Rac1 in cell
lysates (lower panel) are also shown.
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Inhibition by RhoAV14 and CD-ROK
of NGF-induced
Recruitment of Rac1 to Protrusions--
We examined the subcellular
distribution of Rac1 and F-actin after the stimulation with NGF.
Immunofluorescence staining of Rac1 using an anti-Rac1 antibody
revealed that in unstimulated RhoAV14-inducible PC12 cells,
Rac1 was present mainly in the cytoplasm (Fig.
7). At 3 min after the addition of NGF,
RhoAV14-inducible PC12 cells produced a few protrusions
from the cell membrane, and Rac1 and F-actin were accumulated at these
protrusions. We recently showed that the constitutively active Rac1,
Rac1V12, induced membrane ruffles where Rac1V12
and F-actin were accumulated, but Rac1V12 failed to induce
protrusions in PC12 cells (20). Furthermore, Rac1V12 also
induced membrane ruffles in NGF-treated cells and abrogated the
NGF-induced straightforward neurite formation (data not shown). However, the expression of dominant negative Rac1, Rac1N17,
blocked the NGF-induced F-actin redistribution and the protrusion formation, indicating that Rac1 activation is required for the formation of F-actin-rich protrusion (data not shown). On the other
hand, the expression of RhoAV14 gave rise to cell rounding
accompanied by formation of a thick ringlike structure of cortical
actin filaments at the cell periphery. When the cells expressing
RhoAV14 were stimulated with NGF, the thick ring of
cortical actin filaments remained unchanged, and formation of
protrusions enriched with Rac1 and F-actin was not observed. We next
examined the effect of Rho-associated kinase on the NGF-induced
accumulation of Rac1 and F-actin by transient transfection of the
constitutively active form of Rho-associated kinase, CD-ROK
.
Transfected cells were identified by cotransfection of GFP. As shown in
Fig. 8, CD-ROK
also induced the cell
rounding with cortical actin filaments and inhibited the NGF-induced
accumulation of Rac1 and F-actin at the protrusions. Moreover, Y-27632
completely abolished the RhoAV14-induced inhibition of the
accumulation of Rac1 and F-actin (Fig. 9). These results indicate that
RhoAV14 inhibited the NGF-induced accumulation of Rac1 and
F-actin at the protrusions and eventually suppressed the NGF-induced
neurite outgrowth via Rho-associated kinase.

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Fig. 7.
Effect of RhoAV14 on the
NGF-induced subcellular distribution of endogenous Rac1 and
F-actin. Serum-starved RhoAV14-inducible PC12 cells
were treated with or without 5 mM IPTG for 12 h. The
cells stimulated or not stimulated with 50 ng/ml NGF for 3 min were
fixed and double-stained with an anti-Rac1 monoclonal antibody
(left panels) and Alexa 488-conjugated phalloidin
(right panels). The results shown are
representative of three independent experiments. Bar, 10 µm.
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Fig. 8.
Effect of CD-ROK on
the NGF-induced subcellular distribution of endogenous Rac1 and
F-actin. PC12 cells were transiently transfected with an empty
vector (a--d) or an expression vector encoding
CD-ROK (e--h) along with an expression vector
encoding GFP. At 24 h after transfection, cells were stimulated
with 50 ng/ml NGF for 3 min and were fixed. Transfected cells were
identified by the fluorescence of GFP (a, c,
e, and g). Cells were stained with an anti-Rac1
monoclonal antibody (b and f) or
rhodamine-conjugated phalloidin (d and h). The
results shown are representative of three independent experiments.
Bar, 10 µm.
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Fig. 9.
Effect of a Rho-associated kinase inhibitor
on the RhoAV14-induced inhibition of endogenous Rac1 and
F-actin distribution. Serum-starved RhoAV14-inducible
PC12 cells were treated with or without 5 mM IPTG for
12 h in the presence of 10 µM Y-27632. The cells
stimulated or not stimulated with 50 ng/ml NGF for 3 min were fixed and
double-stained with an anti-Rac1 monoclonal antibody (left
panels) and Alexa 488-conjugated phalloidin
(right panels). The results shown are
representative of three independent experiments. Bar, 10 µm.
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DISCUSSION |
The cross-talk between Rac and Rho GTPases has been extensively
studied (18, 30), and the actions of Rac and Rho have been shown to
counteract each other in a variety of cell lines (14, 31-33).
Contractility by myosin activation has been shown to be
antagonistically regulated by Rac1 and RhoA through the action of PAK
on myosin light chain kinase and that of Rho-associated kinase on
myosin phosphatase, respectively (18). Thus, the cross-talk is
coordinated regulation of downstream functions of Rac1 and RhoA,
usually referred to as signal convergence (34). Such signal-convergent regulation by Rac1 and RhoA was also reported in neuronal cells. Activation of Rho promotes contractility by activation of
Rho-associated kinase, leading to neurite retraction, cell rounding,
and the inhibition of neurite outgrowth, while activation of Rac
enhances cell spreading and neurite extension and interferes with
Rho-mediated cell rounding through phosphorylation of the myosin-II
heavy chain, suggesting antagonistic roles for Rho and Rac in the
control of neuronal morphology (35). In contrast to the
signal-convergent system, we here demonstrated that constitutively
active RhoA or G
12QL-induced endogenous RhoA activation
down-regulated the NGF-induced Rac1 activation via a Rho-associated
kinase-dependent pathway. This down-regulation of Rac1
activity by RhoA can be categorized into the different regulatory
mechanisms, upstream signal regulation, referred to as signal
divergence (34). Concerning this system, down-regulation of Rho
activity by Rac has been reported in NIH3T3 cells (36). However, this
is the first report of down-regulation of Rac1 activity by RhoA. Rho
activation induces cell rounding, decreasing cell adhesion activity in
PC12 cells (21, 25). However, the RhoA-induced down-regulation of Rac1
activity is not a secondary effect of this RhoA-mediated reduction of
cell adhesion activity, because the down-regulation was observed with suspended PC12 cells as well. Therefore, the site of action of RhoA is
on a step(s), involved in the NGF-induced Rac1 activation signaling
pathway. Ras is a critical component in the signaling pathway of the
NGF-induced neurite outgrowth in PC12 cells (37-39), and activation of
Ras was shown to induce neurite outgrowth (20, 40, 41). Recently, Rac1
was revealed to act downstream of Ras in neurite outgrowth independent
of Ras-mediated ERK activation in N1E-115 neuroblastoma cells (42).
Furthermore, NGF was shown to activate ERK in PC12 cells in the
presence of RhoAV14 expression (16). We also observed here
that active RhoA did not perturb the NGF-induced ERK activation.
Therefore, active RhoA may modulate the signaling pathway from Ras to
Rac1 but not from Ras to ERK. The activity of Rac1 is known to be
regulated by a variety of proteins, such as guanine nucleotide exchange factors, GTPase-activating proteins, and guanine nucleotide
dissociation inhibitors (28). A Rac guanine nucleotide exchange factor,
Sos, was shown to be implicated in coupling Ras to Rac (43, 44). RhoA
may modulate the activities of these molecules, such as Sos. Molecular
mechanisms of the RhoA-induced down-regulation of Rac1 activity are
currently under investigation in our laboratory.
We further investigated the effect of active RhoA on actin
reorganization. We revealed here that NGF recruited Rac1 to the protrusion sites and accumulated F-actin, initiating the neurite formation. Active RhoA inhibited the NGF-induced Rac1 recruitment to
the protrusion sites and resultant process formation through Rho-associated kinase. Our result showed that the cytoskeletal action
of RhoA and its downstream effector, Rho-associated kinase, was the
stabilization of cortical actin filaments in the cell periphery of PC12
cells. Since Rho-associated kinase is known to increase phosphorylation
of myosin light chain, consequently elevating contractile activity of
myosin, this myosin activation may enhance the stabilization of
cortical actin filaments. In neuronal cells, disruption of the cortical
actin filament network was shown to induce neurite formation,
suggesting that contractile activity by cortical actin filaments
inhibits the neuritogenesis and neurite extension (45, 46). Therefore,
Rho-associated kinase-induced formation of the ringlike structure of
cortical actin filaments in PC12 cells may interfere with the
NGF-induced Rac1 recruitment and F-actin reorganization, which are
initial events for the neurite formation. In this study, we
showed that Y-27632, a Rho-associated kinase inhibitor, completely
abolished the RhoA-induced down-regulation of the NGF-induced Rac1
activation, suggesting that the RhoA-induced down-regulation is
mediated by Rho-associated kinase. The inhibition of Rac1 recruitment
by Rho-associated kinase may contribute to the RhoA-induced
down-regulation of the Rac1 activation.
Studies using a dominant negative Rac1 show that Rac1 is involved in
the NGF-induced neurite outgrowth in PC12 cells (17, 22). Furthermore,
activation of Rac was shown to produce a loss of contractility
associated with cell spreading and the formation of neurite-like
extensions in N1E-115 cells (42). On the other hand, active Rho
suppresses the NGF-induced neurite outgrowth in PC12 cells (16).
Therefore, Rac and Rho exert antagonistic actions in neurite formation.
We here demonstrated that NGF activated Rac1 but inversely suppressed
the RhoA activity in PC12 cells. In the light of antagonistic actions
of Rac1 and RhoA in neuronal morphology, the NGF-induced opposite
regulation of Rac1 and RhoA activities may play an important role in
the NGF-induced neurite outgrowth. The expression of the active RhoA
did not affect the basal level of Rac1 activity, and Y-27632 did not
increase the basal Rac1 activity in PC12 cells. Effects of RhoA and
Y-27632 appear to be restricted to the NGF-induced Rac1 activation.
Although the regulation of this basal Rac1 activity is not understood, the activation of Rac1 by NGF and the basal activity may be regulated by different signaling mechanisms. Suppression of RhoA activity by NGF
may enhance the duration of the NGF-induced Rac1 activation. Recently,
NGF was shown to abolish Rho activation by binding to the neurotrophin
receptor p75 (47). Considering the expression of p75 in PC12 cells
(48), the NGF-induced down-regulation of RhoA may be mediated by p75.
Furthermore, inactivation of Rho signaling pathway has been reported to
promote axon regeneration in the central nervous system (8), and
inhibition of the Rho/Rho-associated kinase pathway was suggested to
act as a gate critical for the initiation of axonal outgrowth (49).
Thus, negative regulation of Rho activity is critical for neurite
outgrowth. In sharp contrast to NGF, ephrin-A5 was shown to induce
collapse of growth cone by opposite regulation of Rho and Rac, Rho
activation and Rac down-regulation (50). Neuronal morphology appears to
be critically balanced between Rho and Rac activities.
In conclusion, we demonstrate here that NGF oppositely regulates Rac1
and RhoA activities, activation of Rac1 and suppression of RhoA, and
that activation of RhoA blocks the NGF-induced neurite formation by the
inhibition of Rac1 activity via Rho-associated kinase in PC12 cells.
This work takes a close-up of an important role of Rho family GTPases
in neurite formation and will help to elucidate the molecular mechanism
of neurite outgrowth.
 |
ACKNOWLEDGEMENTS |
We thank Yoshitomi Pharmaceutical Industries
and Dr. S. Nagata of Osaka University for supplying Y-27632 and
pEF-BOS, respectively.
 |
FOOTNOTES |
*
This work was supported in part by Ministry of Education,
Science, Sports and Culture of Japan Grants-in-aid for Scientific Research 10470482, 11780579, 12053244, and 12210078 and grants from the
Uehara Memorial Foundation and the Naito Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom all correspondence should be addressed. Tel.:
81-75-753-4547; Fax: 81-75-753-7688; E-mail:
mnegishi@pharm.kyoto-u.ac.jp.
Published, JBC Papers in Press, March 15, 2001, DOI 10.1074/jbc.M100254200
 |
ABBREVIATIONS |
The abbreviations used are:
NGF, nerve growth
factor;
HA, hemagglutinin;
ERK, extracellular signal-regulated kinase;
GFP, green fluorescent protein;
IPTG, isopropyl-
-D-thiogalactoside;
GST, glutathione
S-transferase;
CRIB, Cdc42/Rac interactive binding;
RBD, Rho-binding domain;
PBS, phosphate-buffered saline;
CD-ROK
, catalytic domain of Rho-associated kinase.
 |
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