From the Division of Molecular Cardiology, Cardiovascular Research Institute, The Texas A&M University System Health Science Center, College of Medicine, Temple, Texas 76504
Received for publication, June 26, 2002, and in revised form, October 2, 2002
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ABSTRACT |
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All-trans-retinoic acid (RA) plays a
crucial role in survival and differentiation of neurons. For
elucidating signaling mechanisms involved in RA-induced neuronal
differentiation, we have selected SH-SY5Y cells, which are an
established in vitro cell model for studying RA signaling.
Here we report that RA-induced neuronal differentiation of SH-SY5Y
cells is coupled with increased expression/activation of TGase and
in vivo transamidation and activation of RhoA. In addition,
RA promotes formation of stress fibers and focal adhesion complexes,
and activation of ERK1/2, JNK1, and p38 Retinoic acid (RA),1 a
metabolic derivative of vitamin A, plays a crucial role in the
development and differentiation of the nervous system (1). During
normal embryogenesis, there is a requirement for precise timing of the
exposure of embryonal structures to RA and a coordinated pattern of
expression of the retinoic acid receptor (RAR) and retinoic acid X
receptor (RXR) isoforms (2). In addition, RA plays an important role in
the function of the adult brain, which has been shown to synthesize RA
and express retinoid receptors, as well as cellular-binding proteins (3). Many studies on a variety of embryogenic neuronal cell types have
shown that RA can stimulate both neurite number and length (4) and
neurite outgrowth in amphibian spinal cord (5). SH-SY5Y cells, a
neuroblastic subclone of the neuroblastoma cell line SK-N-SH, withdraw
from the cell cycle and exhibit a distinct neuronal phenotype when
treated with different agents such as neurotrophic factors (6),
retinoic acid (6), phorbol ester (7), or staurosporine (8, 9). The
RA-induced change to a neuronal phenotype in SH-SY5Y cells is
associated with increased expression of tissue transglutaminase (TGase)
(10-12). TGase, a GTP-binding protein, participates in signal
transduction pathways as a nonconventional G-protein, and exhibits
distinct GTP-binding/hydrolyzing and transglutaminase activities
(13-15). Transglutaminase activity (or transamidation function), used
to cross-link polyamines such as putrescine, spermine, or spermidine to
target proteins, is regulated by the GTP binding activity of TGase (13,
16). The transamidation reaction of TGase has been implicated in a
number of biological processes such as axonal regeneration, cellular differentiation, and apoptosis (17-19).
The Rho family of small G-proteins, primarily RhoA, Rac1, and Cdc42,
are known to have a significant role in neuronal differentiation (20,
21). RhoA is an in vivo substrate of TGase and is known to
have an important role in cytoskeletal rearrangement and regulation of
cell morphology and differentiation (15, 22-25). Like other G-proteins, RhoA binds GTP in the active state, and following hydrolysis, returns to an inactive GDP-bound state (26). The glutamine
residue at position 63 of RhoA (switch II domain) is required for GTP
hydrolysis (27). After deamidation/transamidation, glutamine 63 is not
available for GTP hydrolysis, and RhoA is constitutively activated (28,
29). RA promotes activation of TGase and in vivo
transamidation of RhoA (at glutamine 63) in HeLa cells. After
transamidation, RhoA binds/activates RhoA-associated kinase-2 (ROCK-2),
a downstream target and an effector of GTP-bound RhoA, leading to
increased stress fiber and focal adhesion complex formation (15).
Mitogens, tumor promoters, and cell differentiation inducing agents
trigger an intracellular signaling cascade, which involves Ras and Rho
GTPases and leads to activation of mitogen-activated protein (MAP)
kinases (30). Previous studies have suggested that ERK/MAPK pathway is
crucial for NGF-induced neuronal differentiation of the cells, since
blockade of the ERK/MAPK activation inhibits neurite induction (31,
32), and constitutive activation of the ERK/MAPK pathway results in
neurite outgrowth (33, 34). However, some other findings demonstrate
that sustained activation of ERK/MAPK does not induce neurite outgrowth
in dorsal root ganglionic (DRG) sensory and sympathetic neurons and
SH-SY5Y cells (35, 36). These observations suggest the existence of an
additional signaling pathway(s) important for neuronal differentiation.
Activation of c-Jun amino-terminal kinase is required for RA-induced
neuronal differentiation of P19 embryonal carcinoma cells (37). The
subfamily of p38 MAP kinases includes p38 There is growing evidence that RA-induced neuronal differentiation in
SH-SY5Y cells is mediated by TGase. Using stable cell lines of SH-SY5Y,
overexpressing wild type, C277S mutant (transglutaminase-defective), and antisense TGase, it has been demonstrated that TGase is necessary and sufficient in promoting neurite outgrowth (12). However, the
signaling mechanisms involved during differentiation are not known.
Here, we present evidence that RA-induced neuronal differentiation in
SH-SY5Y cells is associated with increased expression/activation of
TGase, leading to transamidation of RhoA. After transamidation, RhoA is
activated and promotes formation of stress fibers and focal adhesion
complexes. The transamidated RhoA also promotes activation of ERK1/2
and p38 Materials--
Cell culture reagents were purchased from
Invitrogen. RA, common use reagents, and vinculin antibody were from
Sigma; RhoA, GAP-43, actin, ERK 1/2, JNK1, and p38 Cell Culture--
SH-SY5Y cells were grown in Dulbecco's
Modified Eagle's Medium supplemented with 10% FBS and antibiotics
(penicillin and streptomycin; 1% each) in a 5% CO2
humidified incubator at 37 °C. Stable cell lines of SH-SY5Y cells,
stably transfected with vector, wild-type TGase, an inactive TGase
mutant (C277S, without transamidating activity due to a point mutation
within the active site), or an antisense TGase construct (which blocks
endogenous and RA-induced expression of TGase) were obtained from Dr.
Gail V. Johnson, University of Alabama at Birmingham,
Alabama. Cells were grown in the presence of 150 µg/ml G418 and 10%
FBS. Cells grown to subconfluence (20%) were untreated or treated with
RA at 5 µM or vehicle (Me2SO), in
medium containing 3% FBS for 1-4 days, as indicated in the figures.
Medium with or without different agents was replaced every day.
Kinase Reaction--
Untreated and RA treated SH-SY5Y cells were
lysed in buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
Immunoblotting--
The expression level of TGase, neuronal
marker growth-associated protein 43 (GAP-43), and neurofilament B
(NF-B) were determined by immunoblotting. Cells were treated with or
without RA in medium containing 10% FBS for the indicated times. After
washing with cold PBS, the cells were harvested in lysis buffer
containing 25 mM Tris-HCl (pH 7.4), 100 mM
NaCl, 1 mM EDTA, 1mM dithiothreitol, 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin and leupeptin. Insoluble material was removed by
sedimentation at 10,000 × g for 15 min at 4 °C. The
protein concentration was determined according to the Bio-Rad method.
Lysates (50 µg of protein) were electrophoresed on SDS-PAGE,
transferred to nitrocellulose membrane, blotted with anti-TGase,
GAP-43, and NF-B antibody. The blots were reprobed with anti-actin
antibody to determine the loading difference between samples. Scanning
densitometry was used for semiquantitative analysis of the data.
Rhotekin Binding Assay--
RhoA activation was determined using
the Rhotekin-binding assay, as previously described (43). In brief,
cells were lysed with lysis buffer containing 50 mM Tris,
pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, 10 µg/ml
each of leupeptin and aprotinin, and 1 mM
phenylmethylsulfonyl fluoride. The lysate was sedimented at 14,000 × g for 5 min. Equal volume of cell lysates was incubated
at 4 °C, for 45 min with 30 µg of GST-RBD (GST fusion protein
containing the RhoA binding domain of Rhotekin) immobilized on
glutathione-Sepharose beads (Sigma). The beads were suspended in
Laemmli sample buffer, and eluted proteins were separated on 15%
SDS-PAGE. Activated RhoA was detected by Western blotting with
monoclonal antibody against RhoA. The amount of RBD-bound Rho was
normalized to the total amount of RhoA in cell lysates for the
comparison of Rho activity (activated Rho) in different samples.
Indirect Immunofluorescence Microscopy--
Immunoflourescence
studies on stress fiber and focal adhesion complex formation were
performed using SH-SY5Y cells grown on chamber slides (Lab-Tek II,
Nalgen Nunc Int.) in medium with 3% FBS. After washing with cold PBS,
cells were fixed with 3% paraformaldehyde at 21 °C for 20 min.
Cells were exposed to 0.2% Triton X-100 for 3 min after washing with
PBS. To block nonspecific binding, cells were first incubated with 100 mM glycine for 20 min at room temperature, and then
incubated with primary antibody at 21 °C for 30 min (dilutions: vinculin monoclonal antibody, 1:400; Texas Red-X phalloidin, 1:40). After washing three times with PBS, fluorescein-labeled secondary antibody (1:400) was added and incubated for an additional 30 min and
washed two times with PBS. Coverslips were mounted with anti-fade
medium (ProLong®, Molecular Probes) and slides viewed with an Olympus
Provis fluorescence microscope. Pictures of the slides were taken with
a digital color camera (Olympus UltraPix 2000 RGB).
Analysis of Cell Morphology--
To study the effect of RhoA and
MAPK pathways on RA-induced neurite outgrowth, SH-SY5Y cells were
pretreated with C-3 exoenzyme, monodansylcadaverine (MDC), PD98059
(specific MEK inhibitor), SB202190 (specific inhibitor of p38 MAPK),
and JNK inhibitor or Me2SO before addition of 5 µM RA in medium with 10% FBS. Medium with or without
different inhibitors was replaced every day. After 4 days, the cell
photographs were taken using a Nikon digital camera. The cell lysates
were used for determining neuronal marker expression and
transglutaminase activity.
Transglutaminase Activity Assay--
Untreated and RA-treated
SH-SY5Y cells were lysed, and 100 µg of protein from the lysate was
used for the assay of transglutaminase activity, as previously
described (13, 44).
Transamidation Reaction--
SH-SY5Y cells, grown to 40%
subconfluence in Dulbecco's modified Eagle's medium containing 3%
FBS, were treated (or untreated) with 5 µM RA for 2 days.
Labeling (in vivo) of [14C]putrescine (0.5 µCi/ml) was performed overnight in the presence of aminoguanidine
(100 mM), as described previously (15). Cells were lysed
and precipitated with RhoA antibody (or GST-RBD beads). Samples were
subjected to SDS-PAGE (15%), and the gel was dried after incubating
with a signal enhancer kit. Transamidated proteins were detected by autoradiography.
Statistical Analysis--
Data presented are mean ± S.E.
Statistical differences were determined by a Student's t
test for statistical significance (p < 0.05).
RA Promotes Expression of TGase and Neuronal
Markers--
RA-induced cellular differentiation is coupled with
increased expression/activation of TGase in many cell lines (12, 16). To further study the role of TGase in RA-induced neuronal
differentiation in SH-SY5Y cells, we determined the expression level of
TGase and neuronal markers in response to RA treatment. As shown in Fig. 1, A and B,
up-regulation of TGase expression was observed after 1 day of RA
treatment, which was associated with increased transglutaminase
activity (~10-fold, specific activity: 110 nmol of putrescine
incorporated/mg of casein/mg of protein, compared with 10.5 nmol of
putrescine incorporated/mg of casein/mg of protein, Fig.
1C). Under similar conditions, RA
time-dependently induced the expression of NF-B and GAP-43
from 1 to 4 days of treatment (Fig. 1, A and B),
unlike actin (negative control of neuronal marker expression). These
data demonstrated that RA-induced expression of neuronal markers was
also associated with increased expression/activation of TGase, which
might be involved in promoting neuronal differentiation.
RA Promotes Transamidation/Activation of
RhoA--
Previously we demonstrated that RhoA is an in
vivo substrate of TGase and RA promotes transamidation of RhoA in
HeLa cells (15). To address the question of whether RA-induced
expression of TGase in SH-SY5Y cells promotes transamidation of RhoA,
we performed in vivo labeling of SH-SY5Y cells using
[14C]putrescine. Incorporation of radiolabeled putrescine
into RhoA was detected by immunoprecipitation and subsequent
autoradiography of RhoA. As shown in Fig.
2A, there was increased
labeling of [14C]putrescine into RhoA (in vivo
transamidation) after 24 h of RA treatment, which was consistent
with the increased transglutaminase activity of TGase (Fig.
1C). To examine whether transamidated RhoA is activated, the
Rhotekin binding assay was performed. There was a putrescine-labeled
protein of 24 kDa, precipitated by GST-RBD beads from RA (24 h)-treated
cells (Fig. 2B, upper panel) identified as RhoA
(lower panel), indicating that transamidated RhoA functions as an activated form. As a negative control, cell lysates prepared from
in vivo transamidated cells were incubated with GST beads, and putrescine incorporation and activation of RhoA were examined. As
shown in Fig. 2C, we could not detect pull-down of
[14C]RhoA or activated RhoA (using GST beads),
demonstrating the specificity of GST-RBD assays.
RA-induced Activation of RhoA Is Mediated by TGase--
To
determine the role of TGase in activation of RhoA, we have used
monodansylcadaverine (MDC), a known TGase inhibitor (45), and as shown
in Fig. 3A, MDC
dose-dependently inhibited RA-induced transglutaminase
activity. SH-SY5Y cells were pretreated with RA in the absence or
presence of MDC (50 µM for 2 h) and were subjected
to the Rhotekin binding assay. RA promoted the pull-down of RhoA (or
activation of RhoA) after 1 day of treatment and peaked in 2 days. The
RA-induced activation of RhoA was blocked by MDC treatment (Fig.
3B, upper panel). We further used stable SH-SY5Y cell lines overexpressing vector, wild-type, C277S mutant, and antisense TGase (12) for determining the role of TGase in RhoA activation (Fig. 3, C and D). Overexpression of
wild-type TGase resulted in a significant activation of RhoA (25-fold
increase compared with vector), unlike the C277S mutant- or
antisense-overexpressing cell lines, where no change in RhoA activation
was observed. RA treatment markedly induced activation of RhoA in a
vector cell line (20-fold), which is similar to that of normal SH-SY5Y
cells. Only a 1.5-fold increase of RhoA activation was observed in wild type, and no significant changes in RhoA activation were observed in
C277S mutant and antisense cell lines after RA treatment. Further, we
determined the expression level of TGase in different cell lines
(lowermost panel). RA induced up-regulation of TGase
expression in vector-transfected cells, and did not result in any
detectable changes in TGase expression in wild-type and C277S mutant
cell lines. We could not detect any TGase expression in the absence or
presence of RA in antisense TGase-overexpressing cells. These results
indicated that TGase (transglutaminase activity) was required for
RA-induced activation of RhoA in SH-SY5Y cells.
Activation of RhoA by Transamidation Induces Formation of Stress
Fibers and Focal Adhesion Complexes--
One of the hallmarks of RhoA
activation is increased cyotskeletal rearrangement (25). To determine
the effects of RhoA transamidation/activation on cytoskeletal
rearrangement, SH-SY5Y cells were treated with RA in the presence or
absence of C-3 exoenzyme (RhoA inhibitor) or MDC for 24 h, and
immunofluoresence staining was performed with Texas Red-X phalloidin
(for stress fiber formation) and vinculin (for focal adhesion complex
formation). There was increased stress fiber formation (Fig.
4A) and focal adhesion complex
formation (Fig. 4B) in response to RA treatment. A minimum
of 24 h of RA treatment was required to induce the changes in
stress fiber and focal adhesion complex formation. Treatment of cells
with C-3 or MDC blocked RA-induced formation of stress fibers (Fig.
4A) and focal adhesion complexes (Fig. 4B). These
results indicated that transamidation of RhoA was required for
activation and inducing cytoskeletal rearrangement in response to RA in
SH-SY5Y cells.
RA Promotes Activation of MAP Kinases--
Recent studies suggest
that MAPK pathways play a role in neuronal differentiation (34). To
determine whether the MAPK pathway is involved in RA-induced neuronal
differentiation of SH-SY5Y cells, MAP kinase (ERK1/2, JNK, and p38)
activity was determined by in vitro kinase assay. As shown
in Fig. 5, RA induced significant activation of ERK1/2, JNK1, and p38 TGase Mediates RA-induced Activation of MAP Kinases--
To
address the question, whether RA-induced activation of MAP kinases are
mediated by TGase and resulting transamidation/activation of RhoA, we
determined the effect of MDC and C-3 exoenzyme on activation of MAP
kinases. As shown in Fig. 6, A
and B, RA-induced activation of ERK1/2, JNK, and p38
Using stable SH-SY5Y cell lines (overexpressing wild-type, C277S
mutant, and antisense TGase), we further examined the role of TGase in
activation of MAP kinases. Overexpression of wild-type TGase promoted
the activation of ERK1/2, JNK1, and p38 RA Induced Neuronal Differentiation Is Mediated by TGase--
To
determine the role of TGase and transamidated RhoA in RA-induced
neurite outgrowth, SH-SY5Y cells were treated with RA in the presence
or absence of MDC or C-3 exoenzyme and any changes in phenotype
observed. Four days after RA treatment, extensive neuronal
differentiation was observed, as shown in Fig.
8, by the shrinkage of the cell body and
the extension of neurites, which made cell-to-cell connections
(B). These morphological signs of differentiation were first
evident after 2 days of RA treatment. RA-induced neurite outgrowth was
blocked by MDC treatment (C), and no phenotype change was
observed in C-3-treated cells (D). To further determine
whether transamidated RhoA mediates neuronal marker expression, we
treated cells with MDC or C-3 exoenzyme for detecting the expression of
GAP-43 and NF-B by Western blotting. RA-induced expression of GAP-43
and NF-B was inhibited by MDC, and no change was observed in
C-3-treated cells (Fig. 9A),
indicating that TGase is required in RA-induced neuronal
differentiation, but RhoA may not be involved in RA-induced neurite
outgrowth and neuronal marker expression.
MAP Kinase Pathways Are Involved in RA-induced Neuronal
Differentiation--
To elucidate the role of MAP kinases in
RA-induced neuronal differentiation, SH-SY5Y cells were treated with RA
or RA plus PD98059 (MEK inhibitor), JNK inhibitor, or SB202190
(p38 We have previously demonstrated that RA induced TGase GTP binding
and transglutaminase activities without affecting the protein expression level of TGase in Hela cells, indicating post-translational modification and/or involvement of other TGase-interacting proteins (14). Unlike HeLa cells, where there was no change in the expression level of TGase, and the inactive form of TGase was converted to the
active form, RA promoted expression and activation of TGase in SH-SY5Y
cells (Fig. 1). Another important feature, which distinguishes RA
signaling in SH-SY5Y cells, is the phenotype change and expression of
the neuronal markers. SH-SY5Y cells rapidly underwent neuronal differentiation and up-regulation of NF-B and GAP-43 expression in
response to RA treatment (Figs. 1, 8, and 9). GAP-43 is the most
abundant neuron-specific protein in the growth cones, and NF proteins
form major fibrillar elements of the neurite cytoskeleton required for
growth of axons and dendrites. NF-B and GAP-43 have been shown to be
critical for neuronal differentiation (46, 47). Our data suggested that
RA-induced neuronal differentiation was associated with increased
expression/activation of TGase. The labile state of differentiation
(Go-arrest) of neurons in human brain is suggested to make
them more vulnerable to neuronal degeneration (48). Recent findings
demonstrating that TGase is involved in providing protection against
apoptosis (16) and promoting neurite outgrowth (12) are consistent with
our results, indicating that RA-induced expression/activation of TGase
may play a crucial role in promotion of neuronal differentiation.
For studying the signaling events that are initiated by TGase in
RA-induced neuronal differentiation, we have used MDC, which acts as a
pseudo-substrate inhibitor of TGase and is extensively used for
inhibition of in vivo transamidation reactions (15, 49). It
competes with polyamines for transglutamination and prevents their
incorporation into proteins. MDC is toxic at higher doses and induces
apoptosis in HL60 cells (400 µM for more than a 4-day
treatment) and prevents the anti-apoptotic function of TGase at lower
doses and shorter periods of treatment (16). In SH-SY5Y cells, MDC
dose-dependently inhibited RA-induced transglutaminase activity (Fig. 3A), and induced apoptosis when used at 200 µM (12 h) in low serum (data not shown). RhoA is an
in vivo substrate of TGase, and it has been shown that RA
treatment of Hela cells leads to transamidation of RhoA and promotes
formation of stress fibers and focal adhesion complexes, which are
markers of RhoA activation (15). In SH-SY5Y cells, RA promoted
transamidation of RhoA leading to activation, as shown by Rhotekin
binding assay (Figs. 2 and 3B). RA-induced activation of
RhoA was blocked by MDC (50 µM for 2 h) treatment
(Fig. 3B), indicating a role of transglutaminase activity in
activation of RhoA. The apparent difference between MDC inhibition of
transglutaminase activity (>50%, Fig. 3A), and complete
inhibition of RhoA activation at a 50 µM concentration
(Fig. 3B) may be due to the in vitro and in
vivo nature of corresponding transamidation reactions. To further determine the role of TGase in RhoA activation, we used SH-SY5Y stable
cell lines, overexpressing vector, wild-type, C277S mutant, and
antisense TGase. The overexpression of wild-type TGase induced dramatic
activation of RhoA (Fig. 3, C and D); however, no
significant change was observed after RA treatment of wild-type
TGase-overexpressing cells. RA promoted activation of RhoA in the
vector-transfected cells but not C277S mutant- or antisense
TGase-overxpressing cells. Our results suggest that transglutaminase
activity is required for RA-induced transamidation/activation of RhoA
in SH-SY5Y cells.
The outgrowth of neurites toward the proper targets in response to
several environmental cues is guided by growth cones (50). Growth cones
advance through cyclical extension of filopodia and lamellopodia, and
the shapes are largely determined by the organizaion of the actin
cytoskeleton (51). The Rho family of G-proteins regulating various
aspects of the actin cytoskeleton, are important components of the
signaling pathways that link the reception of extracellular cues to the
regulation of the cytoskeleton in neuronal growth cones (52).
Microinjection of Cdc42 and Rac1 promoted the formation of filopodia
and lamellipodia in N1E-115 neuroblastoma cells. These actin-containing
structures were also induced by injection of C-3 exoenzyme, which
abolishes RhoA-mediated functions such as neurite retraction (53). The
increased formation of stress fibers and focal adhesion complexes in RA
treated SH-SY5Y cells were inhibited by MDC and C-3 exoenzyme (Fig. 4),
indicating that RhoA transamidation is required for cytoskeletal
rearrangement. Similarly, RA treatment of SiHa carcinoma cells was
shown to promote stress fiber formation and increased cell adhesion
(54). RhoA is a regulatory participant in the process of cytoskeletal
remodeling, and in the transamidated state (analogous to the GTP-bound
form), may induce formation of stress fibers and focal adhesion
complexes in SH-SY5Y cells (25). Unlike focal adhesion complex
formation, RA-induced stress fiber formation was not detected in growth
cones (data not shown). It is possible that following activation by transamidation, RhoA exhibits differentiation effects toward assembly of focal adhesion complexes and formation of stress fibers in growth cones.
RA promoted activation of ERK1/2, JNK1, and p38 To test whether RA-induced neurite outgrowth and neuronal marker
expression in SH-SY5Y cells is mediated by TGase and resulting RhoA
transamidation, we treated SH-SY5Y cells with RA, RA plus C-3
exoenzyme, and RA plus MDC. As shown in Figs. 8, and 9, RA promoted
neurite outgrowth and expression of neuronal markers (NF-B and GAP-43),
which were inhibited by MDC, indicating that TGase is involved in
RA-induced neuronal differentiation, which is consistent with previous
studies (12). On the other hand, C-3 exoenzyme did not prevent neurite
outgrowth, or inhibit expression of NF-B and GAP-43, indicating that
activation of RhoA does not participate in RA-induced neurite
outgrowth, or expression of neuronal markers. Increased expression and
phosphorylation of neurofilament proteins is thought to contribute to
the extension and stabilization of neuronal processes (47, 57). The
neurite extension, as observed in our studies, may be due to the
contribution of other signaling components such as Cdc42 and Rac1,
which were also activated by RA treatment of SH-SY5Y cells (data not
shown). There is also a possibility that after transamidation, RhoA
promotes axonal guidance, as suggested by studies performed in
Drosophila (58).
Treatment of cells with JNK and MEK inhibitors did not affect
RA-induced neurite outgrowth and expression of NF-B, but inhibited the
expression of GAP-43. On the other hand, inhibition of p38 RA-induced activation of MAPKs raises the possibility that the MAPK
pathway may be involved in up-regulation of expression/activation of
TGase. To address this question, we used specific inhibitors of MEK,
JNK, and p38 In summary, our studies suggest that RA elicits dual effects on TGase
activation. It induces the expression/activation of TGase in SH-SY5Y
cells, unlike in HeLa cells, where RA promotes activation of TGase
without affecting the expression level (14). By using a stable cell
line of SH-SY5Y cells (overexpressing wild-type, C277S mutant, and
antisense TGase) and TGase inhibitor (MDC), we have demonstrated that
TGase is required for promoting neurite outgrowth and expression of
neuronal markers. We propose a novel mechanism of RA signaling, which
involves activation of TGase, leading to transamidation of RhoA. After
transamidation, RhoA is activated and promotes cytoskeletal
rearrangement and activation of MAP kinases. RA-induced activation of
TGase and resulting transamidation of RhoA may serve as a convergence
point for various signaling pathways, which are proposed to play an
important role in neuronal differentiation of SH-SY5Y cells (Fig.
10).
/
/
MAP kinases. Using
C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38
MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S
mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38
MAP kinases. Activated MAP kinases differentially regulate RA-induced
neurite outgrowth and neuronal marker expression. The results of our
studies suggest a novel mechanism of RA signaling, which involves
activation of TGase and transamidation of RhoA. RA-induced
activation of TGase is proposed to induce multiple signaling pathways
that regulate neuronal differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(38), p38
(39), p38
(40), and p38
(41). Sustained activation of p38
promotes neuronal differentiation, and inhibition of p38
/
by a specific inhibitor SB203580 or by expression of dominant-negative constructs of the p38
pathway blocks neurite outgrowth in PC12 cells (42). Studies have
demonstrated that p38
is highly expressed in human skeletal muscle
and appears to function as a signal transducer during differentiation of myoblasts to myotubes (40). However, its role in neuronal differentiation is not demonstrated.
MAP kinase, which may regulate nuclear events for promoting
gene expression during neuronal differentiation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
MAPK
antibodies, ATF-2, c-Jun, and protein A/G-agarose were from Santa Cruz
Biotechnology; p38
,
MAP kinase antibody, myelin basic protein
(MBP) were from Upstate Biotechnology; TGase and neurofilament B (NF-B)
antibodies were purchased from Neomarkers. Texas Red-X phalloidin and
fluorescein-labeled anti-mouse secondary antibody were from Molecular
Probes; [32P]ATP and [14C]putrescine were
from PerkinElmer Life Sciences and the signal enhancer kit used for
autoradiography of 14C-labeled proteins was from Amersham
Biosciences. C-3 exoenzyme, PD98059, SB202190, and JNK inhibitor were
from CalBiochem.
-glycerophosphate, 1 mM sodium vanadate, 10 µg/ml
leupeptin and aprotinin, and 1 mM phenylmethylsulfonyl
fluoride. The lysate was sedimented at 15,000 × g for 10 min at 4 °C, and used for immunoprecipitation of
p38
/
, -
, -
MAP kinase, JNK1, or ERK 1/2. Immunoprecipitates were washed three times with the lysis buffer and two times with kinase
buffer (25 mM HEPES, pH 7.4, 5 mM
-glycerophosphate, 2 mM dithiothreitol, and 10 mM MgCl2) and resuspended in 40 µl of kinase
buffer containing 4 µg of GST-ATF-2, GST-c-jun, or 20 µg of MBP, 50 µM ATP, and 10 µCi of [
-32P]ATP. The
reaction mixture was incubated at 30 °C for 20 min, terminated by
the addition of 5× Laemmli's sample buffer. Samples were subjected to
electrophoresis on SDS-PAGE and proteins were transferred to
nitrocellulose membrane and exposed to Kodak x-ray film. Loading
differences (between samples) were determined by blotting the membrane
with anti-ERK, JNK1, or p38
/
, -
, -
antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
RA promotes expression of GAP-43, NF-B, and
TGase. SH-SY5Y cells grown to 20% confluence in medium with 10%
FBS were treated with RA (5 µM) for 1-4 days as
indicated. Cell lysates were used for Western blotting of GAP-43, NF-B,
and TGase. The membrane was blotted with actin antibody to normalize
the expression level of GAP-43, NF-B, and TGase (A). The
expression of GAP-43, NF-B, and TGase was quantified by scanning
densitometry and were expressed relative to actin. Results are
means ± S.E. for three separate experiments (B).
SH-SY5Y cells were treated with RA for the times indicated. The lysate
(100 µg of protein) was used for the determination of
transglutaminase activity as described under "Experimental
Procedures." Results are means ± S.E. for four separate
experiments (C).
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Fig. 2.
RA promotes in vivo
transamidation and activation of RhoA. SH-SY5Y cells grown
to 40% subconfluence in Dulbecco's modified Eagle's medium
containing 3% FBS were treated with 5 µM RA for the
times indicated. In vivo labeling of
[14C]putrescine (0.5 µCi/ml) was performed as described
under "Experimental Procedures." Cell lysates were
immunoprecipitated with RhoA antibody (A), or precipitated
with GST-RBD beads (B), or GST-only beads (C).
Samples were separated on SDS-PAGE, and the gel soaked in signal
enhancer solution. After drying the gel, the transamidation of RhoA was
visualized by autoradiography (A-C, upper panels). Parallel
sets of samples were processed for Western blotting for RhoA
(A-C, lower panels).
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Fig. 3.
TGase mediates transamidation and activation
of RhoA. For studying the role of TGase, we first determined the
effect of MDC on RA-induced transglutaminase activity. RA-pretreated
cells (2 days) were treated with MDC for 2 h at different
concentrations. Cell lysates were used for assaying transglutaminase
activity (A). For determining the role of TGase in RhoA
activation, SH-SY5Y cells treated with RA in the presence or absence of
MDC (50 µM for 2 h) were used for the Rhotekin
binding assay (B, upper panel). Activated RhoA is
indicative of the amount of RBD-bound RhoA normalized to the amount of
RhoA in whole cell lysates (B, lower panel). A
control of GST-RBD beads (without incubating with lysate) was included
(B, last lane both panels). Stable
SH-SY5Y cell lines overexpressing vector, wild type, C277S mutant, and
antisense TGase were treated (or untreated) with RA for different
times, and activation of RhoA was determined as described above
(C, upper panel). Equal amounts of proteins from
different samples were blotted for determining RhoA and TGase
expression (C, lower panels). Activated-RhoA
(C, upper panel) was quantified by scanning
densitometry and was expressed relative to total RhoA in lysates
(C, middle panel). Results are means ± S.E.
for three separate experiments (D).
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Fig. 4.
MDC and C-3 exoenzyme inhibit RA-induced
stress fiber and focal adhesion complex formation. RA-pretreated
cells (24 h) were treated with MDC (50 µM, 2 h) or
C-3 exoenzyme (5 µg/ml, 24 h) and used for visualization of
stress fibers and focal adhesion complex formation. As shown in
A, C-3 exoenzyme and MDC blocked RA-induced formation of
stress fibers. Similarly, treatment with C-3 exoenzyme or MDC also
inhibited RA-induced formation of focal adhesion complexes
(B).
MAP kinase. ERK1/2 and p38
MAP kinase activation peaked at 24 h, and JNK activation peaked at
48 h. Under similar conditions, RA induced mild activation of
p38
/
, but had no effect on p38
activation. Corresponding Western blots of ERK1/2, JNK1, and p38
/
, -
, and -
showed an equal amount of protein in different samples.
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Fig. 5.
RA promotes activation of MAP kinases.
SH-SY5Y cells were treated with RA for 1-3 days as indicated. Cell
lysates were used for immunoprecipitation of ERK1/2, JNK1, and
p38 /
,
, and
MAP kinases. A kinase reaction was performed
using the immunoprecipitates in the presence of MBP, c-Jun, and ATF-2,
respectively. The samples were run on SDS-PAGE, transferred to
nitrocellulose membrane, and exposed to x-ray film for autoradiography.
The membrane was Western blotted for total ERK1/2, JNK1, p38
/
,
and
MAP kinases.
was
blocked by MDC treatment, indicating a role of transglutaminase
activity in their activation. On the other hand, treatment of cells
with C-3 exoenzyme blocked RA-induced activation of p38
, and
partially inhibited the activation of ERK1/2, but had no effect on JNK
activation, indicating that transamidated RhoA may not be involved in
RA-induced JNK activation. Under similar conditions, we demonstrated
that MDC or C-3 exoenzyme had no effect on activation of p38
/
(data not shown) or RA-induced up-regulation of TGase expression (Fig.
6A, bottom panel).
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Fig. 6.
Effect of MDC and C-3 exoenzyme on RA-induced
activation of MAP kinases. SH-SY5Y cells, untreated or treated
with RA (5 µM, 24 h), RA plus C-3 exoenzyme (5 µg/ml, 24 h), or RA plus MDC (50 µM, 2 h)
were used for determination of MAP kinase activities. For JNK assay,
cells were treated with RA for 48 h. Cell lysates were used for
in vitro kinase assay as described in the legend to Fig. 5.
The activities of ERK1/2, JNK1, and p38 MAPKs were determined by
autoradiography and the amount by Western blotting. Another set of
equal amounts of protein were blotted for TGase expression
(A). The bands corresponding to 32P-MBP,
32P-c-Jun, and 32P-ATF-2 were scanned by
densitometry, analyzed by Image'Quant software, and expressed relative
to total ERK1/2, JNK, and p38 . Results are means ± S.E. for
three separate experiments. *, p < 0.05 versus RA treatment without inhibitor (B).
MAP kinase (2-fold, 2-fold,
and 3-fold, respectively, compared with vector) as shown in Fig.
7, A and B. Only
basal level activation was observed in the C277S mutant- and
antisense-overexpressing cells, indicating transglutaminase activity of
TGase plays an important role in their activation. Treatment with RA
induced significant activation of ERK1/2, JNK1, and p38
MAP kinase
in vector (4-fold, 3-fold, and 2.5-fold increase, respectively,
compared with untreated vector cell line) and wild-type TGase
(2.5-fold, 4-fold, and 1.2-fold increase, respectively, compared with
untreated wild-type TGase cell line) cell lines. Overexpression of
C277S mutant or antisense TGase inhibited RA-induced activation of
ERK1/2, JNK1, and p38
. Under similar conditions, overexpression of
TGase had no effect on the activation of p38
/
and
MAP
kinases. Treatment with RA induced a mild activation of p38
/
in
vector and wild-type TGase-overexpressing cell lines, and had no effect
on activation of p38
MAP kinase in all three cell lines. These data
indicated that RA-induced activation of ERK1/2, JNK1, and p38
MAP
kinases might be mediated by the TGase-dependent
pathway.
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Fig. 7.
TGase is involved in RA-induced activation of
MAP kinases. SH-SY5Y cells, stably transfected with vector,
wild-type TGase (WT), C277S mutant (C277) or
antisense TGase, were treated with Me2SO (0.1%) or RA (5 µM) for 24 h in medium with 3% FBS. An in
vitro kinase assay for ERK1/2, JNK1, p38 , -
/
, and -
kinase was performed, as described in the legend to Fig. 5. Another set
of equal amount of proteins was blotted for TGase to determine
expression level (A). The bands corresponding to
32P-MBP, 32P-c-Jun, and 32P-ATF-2
were scanned by densitometry and analyzed by Image'Quant software.
Results are means ± S.E. for three separate experiments
(B).
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Fig. 8.
Role of RhoA and MAP kinases in RA-induced
neurite outgrowth. SH-SY5Y cells grown to 40% confluence,
untreated (A) and treated with RA (B); RA plus
MDC, 50 µM (C); RA plus C-3 exoenzyme, 5 µg/ml (D); RA plus PD98059, 50 µM
(E); RA plus JNK inhibitor, 20 µM
(F); and RA plus SB202190, 10 µM
(G) in medium with 10% FBS for 4 days and photographed for
visualization of neurite outgrowth. Under similar conditions SH-SY5Y
cells treated only with inhibitors did not affect neurite outgrowth
(data not shown).
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Fig. 9.
TGase and MAP kinases mediate RA-induced
expression of GAP-43 and NF-B. SH-SY5Y cells untreated or treated
with different inhibitors, as noted in the legend to Fig. 8, were lysed
and blotted for GAP-43, NF-B, and TGase as described in the legend to
Fig. 1. The membrane was blotted for actin as a negative control
(A). The cell lysates were further used for the
determination of transglutaminase activities. Results are means ± S.E. for three separate experiments (B).
/
inhibitor), and the morphological changes and neuronal
marker expression were observed. RA-induced neurite outgrowth and
up-regulation of NF-B expression was inhibited by SB202190 treatment
(Figs. 8G and 9), while PD98059 (Fig. 8E) or JNK
inhibitor (Fig. 8F) had no effect on RA-induced neurite
outgrowth, but inhibited RA-induced expression of GAP-43. To address
the question of whether the MAPK pathway is involved in RA-induced
expression/activation of TGase, we examined the effect of MAPK
inhibitors on TGase expression and activation. RA-induced up-regulation
of TGase expression was not inhibited by any of the inhibitors (Fig.
9A), and RA-induced TGase activity was only inhibited by
MDC, but not the other inhibitors (Fig. 9B). From these
studies, it is clear that RA induced extension of neurites, and
expression of neuronal markers is regulated by induced expression of
TGase and the resulting activation of MAP kinases.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in SH-SY5Y cells,
which was inhibited by MDC, indicating that transglutaminase activity
is involved in the activation (Figs. 5 and 6). By using the C-3
exoenzyme, which inactivates the effector domain of RhoA by
ribosylating Asp-41 (55), we determined the role of RhoA in RA-induced
activation of MAP kinases. The inhibitory effect of the C-3 exoenzyme
on ERK1/2 and p38
indicates a direct involvement of RhoA in their
activation (Fig. 6B). ERK1/2 is shown to regulate neuronal
differentiation by modulating gene expression (34), and p38
stimulates the transcription factors ATF-2 and MEF-2, which act on the
c-jun promoter through jAP1 and MEF-2 responsive elements
(40, 56). By activating ERK1/2, JNK, and p38
, RA may promote
expression of new genes required for neuronal differentiation. Overexpression of wild type (not the C277S mutant or antisense TGase)
promotes activation of ERK1/2, JNK1, and p38
MAP kinase, indicating
that transglutaminase activity is required (Fig. 7). The activation of
ERK1/2, JNK1, and p38
/
/
in wild-type TGase overexpressing
cells by RA treatment may be due to the participation of another
co-factor, which may be functioning downstream to TGase and activated
in response to RA treatment.
/
prevented neurite outgrowth as well as the expression of NF-B, but not
GAP-43. SB compound treatment was stressing to cells (as shown by their
morphological appearances), raising the possibility that SB compound
may promote apoptosis over a longer time treatment (more than 4 days).
The differential regulation of neurite outgrowth and expression of
neuronal markers by MAP kinase inhibitors, indicates the involvement of
multiple signaling components in RA-induced neuronal differentiation. A
previous report (59) showing that SB202190 does not inhibit p38
,
still leaves open the possibility that p38
mediates neurite
outgrowth and expression of GAP-43 and NF-B in RA-induced neuronal differentiation.
/
, and examined the expression/activation of TGase in
response to RA. As shown in Fig. 9, pretreatment of SH-SY5Y cells with
PD98059, SB202190, and JNK inhibitor had no effect on RA-induced
expression or activation of TGase, indicating that MAPKs are not involved.
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Fig. 10.
Model depicting the role of TGase in
RA-induced neuronal differentiation. RA treatment of SH-SY5Y cells
leads to increased expression/activation of TGase, resulting in
transamidation/activation of RhoA and formation of the RhoA-ROCK-2
complex. Transamidated RhoA functions as a constitutively active
G-protein, and with ROCK-2, translocates to the plasma membrane
initiating downstream effects and promoting formation of stress fibers
and focal adhesion complexes. Activation of TGase and resulting
transamidation of RhoA is also responsible for activation of MAP
kinases, which by stimulating transcription factors, may induce
expression of new genes required for neuronal differentiation.
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ACKNOWLEDGEMENTS |
---|
We thank Shubha Bagrodia (Sugen Inc.) for providing cDNA of the GST-RBD construct; Gail V Johnson (University of Alabama at Birmingham, Alabama) for providing stable cell lines of SH-SY5Y that overexpress wild type, C277S mutant, and antisense TGase, and Michael Brinkman (Department of Veteran Affairs, Central Texas) for providing technical support.
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FOOTNOTES |
---|
* This work was supported in part by Veteran Affairs Grant VISN-17 and National Institutes of Health Grants HL-44883 and HL-58439.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 correspondence should be addressed: Division of Molecular
Cardiology, Cardiovascular Research Inst., Bldg. 162, 1901 South 1st
St., Temple, Texas 76504. Tel.: 254-778-4811 ext. 6620; Fax:
254-899-6165; E-mail: Usingh@medicine.tamu.edu.
§ Both authors contributed equally to the article.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M206361200
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ABBREVIATIONS |
---|
The abbreviations used are: RA, retinoic acid; MAP, mitogen-activated protein; MAPK, MAP kinase; ERK, extracellular-regulated kinase; FBS, fetal bovine serum; JNK, c-Jun amino-terminal kinase; GST, glutathione S-transferase; MBP, myelin basic protein; PBS, phosphate-buffered saline; TGase, transglutaminase; MDC, monodansylcadaverine.
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REFERENCES |
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