From the Departments of Neurology and Pediatrics and
§ Pharmacology, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104
Received for publication, December 30, 2002
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ABSTRACT |
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Lysophosphatidic acid (LPA;
1-acyl-sn-glycerol-3-phosphate), an abundant constituent of
serum, mediates multiple biological responses via G protein-coupled
serpentine receptors. Schwann cells express the LPA receptors
(Edg receptors), which, once activated, have the potential to
signal through G In the peripheral nervous system, Schwann cells
(SCs)1 form the myelin sheath
and play a key role in the maintenance of the normal
physiological function of the axon (1). The majority of SCs originate
from neural crest cells under the guidance of axonal cues that regulate
the proliferation, survival, and differentiation of precursor cells
into myelin-forming cells (1). During differentiation, SCs make contact
with a single axon, become spindle-shaped, and then form a myelin
sheath that contains specific proteins such as myelin P0
protein (2). The survival and differentiation of SCs are regulated by
extracellular cues from axons and the extracellular matrix (3-6). Some
of these factors are lysophosphatidic acid (LPA), All peripheral nerves have a blood-nerve barrier that normally excludes
large molecules contained in serum from entering the endoneurial space
where SCs reside (15). In some pathological states, this barrier is
broken, resulting in the entry of serum into the endoneurial space.
Whereas serum promotes SC survival, it also down-regulates myelin gene
expression (16). Interestingly, LPA, a normal constituent of serum
(concentration varies between 2 and 20 µM), replicates
many of the cellular effects of serum that include effects on SC
survival (7) and actin rearrangement (13). Some of the other effects of
LPA include stimulation of platelet aggregation, smooth muscle
contraction, stimulation of cell proliferation (fibroblasts, smooth
muscle cells, endothelial cells, and keratinocytes), differentiation
(keratinocytes, neuroblastoma, and myoblasts), induction of apoptosis
(neurons, T cells, and macrophages), collapse of axonal growth cones
with retraction of neurites, release of neurotransmitters, inhibition
of gap junction communication, induction of focal adhesions, induction
of stress fibers, invasion of tumor cells, and regulation of gene
expression (17).
The effects of LPA on target cells are mediated by activation of
specific G protein-coupled serpentine receptors. The first LPA receptor
identified, vzg-1 (ventricular zone
gene-1; also termed
Edg-2/rec1.3/lpA1) encodes a
41-kDa protein that is widely expressed in mouse tissue with the
highest levels seen in embryonic cortex (18). In postnatal life, the
LPA receptor lpA1/vzg-1 is predominantly
expressed in glial cells (mainly oligodendrocytes and SCs) (7, 19). The
appearance of the
lpA1/vzg-1/Edg-2 receptor in
SCs parallels the appearance of myelin formation in developing
peripheral nerves and is up-regulated after nerve injury (7, 13).
Although it is likely that
lpA1/vzg-1/Edg2 receptor is
responsible for mediating most of the effects of LPA, SCs from
lpA1 LPA receptors are coupled to at least three different G Rat SCs undergo apoptosis during normal development. They also undergo
apoptosis after nerve injury, in dysmyelinating disease, and during
demyelination in experimental allergic neuritis. In vitro,
SC apoptosis is observed after serum withdrawal (12, 22). Serum, LPA,
and Materials--
LPA, 4 Schwann Cell Isolation--
Rat Schwann cells were isolated from
the sciatic nerves of 2-day-old rat pups using the method described by
Brockes et al. (26). The cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated fetal bovine serum. Schwann cells were expanded using
2 µM forskolin and 25 ng/ml recombinant glial growth
factor (27). Prior to all of the studies, both forskolin and glial
growth factor were removed from the cell culture for 1 week.
Apoptosis Assay--
To assess morphological changes in
chromatin structure of SCs undergoing apoptosis, attached and floating
cells were collected and centrifuged. The pellet was resuspended in 100 µl of PBS (pH 7.4) containing 0.03% Nonidet P-40, 0.15 mM sodium citrate, and 0.014% propidium iodide (Sigma).
Cells were examined under a fluorescent microscope at ×400
magnification and scored for cells with normal and condensed chromatin.
The data are expressed as a percentage of the cells with condensed
chromatin (apoptotic cells).
Western Blotting--
After the different treatment paradigms,
SCs were rinsed once with PBS and then with TBS (50 mM
Tris, pH 8.0, and 150 mM NaCl). Cells were lysed in
radioimmune precipitation buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM p-nitrophenyl phosphate, 20 nM
calyculin A, 1 mM sodium vanadate, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 10 µg/ml
aprotinin). The lysates were collected and clarified by centrifugation
for 15 min at 4 °C. Protein concentration of cell lysates was
measured by BCA protein assay (Pierce). 50 µg of cell lysate were
then electrophoresed through an SDS-polyacrylamide gel and then
transferred onto nitrocellulose membrane. The blots were then incubated
overnight at 4 °C with the primary antibody. The following day, the
blots were incubated with horseradish peroxidase-conjugated secondary
antibody (1:10,000 dilution; Amersham Biosciences) for 1 h at room
temperature. Bands were visualized by the ECL Western blot detection
system (Amersham Biosciences).
Transient Transfection and Chloramphenicol Acetyltransferase
(CAT) Assay--
SCs were transfected with 10 µg of
P0CAT reporter plasmid by the calcium phosphate
precipitation technique, and exposed to the precipitate for 8 h.
Cultures were washed with PBS and incubated with fresh medium. After
24 h, cells were harvested, and the protein concentration of cell
lysates was measured by the BCA method (Pierce). CAT activity was
determined by the conversion of [14C]chloramphenicol to
its acetylated product (28).
Infection of Schwann Cells with Dominant Negative PKC Purification of Recombinant Proteins and
Microinjection--
Plasmid containing glutathione
S-transferase-RhoA (V14) was expressed in
Escherichia coli, and the recombinant protein was purified using glutathione-Sepharose 4B beads (31). The botulinum C3
exoenzyme was purified as described (32). The protein concentrations of
the recombinant glutathione S-transferase fusion protein and C3 transferase were determined by using the BCA protein assay kit (Pierce).
For microinjection, SCs were plated onto poly-L-lysine (0.1 µg/ml; Sigma)-coated 12-mm diameter circular glass coverslips. The
coverslips were scored with a diamond pen to help identify the area
where the cells were microinjected. Before microinjection or specific
treatment paradigms, the cells were serum-starved for 16-20 h. Cells
were visualized using a Zeiss inverted microscope, and microinjections
were performed using an Eppendorf microinjector. For each experiment,
proteins were injected into the cytoplasm of 100 cells, together with
rabbit immunoglobulin (IgG at 4 mg/ml; Jackson Immunoresearch
Laboratories) to facilitate identification of the injected cells.
Immunofluorescence Staining--
SCs were fixed in 10% formalin
solution for 15 min at room temperature. After several washes with PBS,
the cells were permeabilized with 0.2% Triton X-100 in
phosphate-buffered saline for 5 min and then blocked with 2% bovine
serum albumin in PBS for 30 min. Cells were incubated with 0.2 µg/ml
TRITC-labeled phalloidin (Sigma) and Texas Red-labeled donkey
anti-rabbit IgG (Jackson Immunoresearch Laboratories) for 1 h at
room temperature and viewed using a Leitz microscope.
RNA Interference--
21-Nucleotide RNA duplexes with
2-nucleotide (2'-deoxy)thymidine 3' overhangs directed against
nucleotides 419-441 and 1186-1208 of the respective rat Edg2 and
mouse Edg4 coding sequences were obtained from Dharmacon Research Inc.
(Lafayette, CO). GL2 luciferase small interfering RNA (siRNA) duplex
was used as a nonspecific siRNA control (Dharmacon Research).
The day before transfection, cells were plated onto 60-mm dishes in
fresh medium without antibiotics. Transient transfection of siRNAs was
carried out using Oligofectamine (Invitrogen), following the
manufacturer's instructions. Cells were treated in 200 nM siRNA transfection solution (2 ml/dish) for 4 h and then incubated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for 24 h. Cells were then treated with LPA (10 µM in serum-free medium) for 24 h and harvested.
PKC LPA Is a Survival Factor for Schwann Cells--
SCs undergo
apoptosis upon serum withdrawal, characterized by DNA condensation in
the nucleus (Fig. 1A). About
50% of SCs underwent apoptosis following serum withdrawal for 24 h (Fig. 1B). The addition of serum to the medium completely
prevented apoptosis in SCs. When 10 µM LPA was added to
serum-starved SCs, apoptotic cells were dramatically reduced from 45 to
6% (Fig. 1B). Since LPA is a component of serum, this
finding indicates that LPA is an important constituent in serum-induced
SC survival.
Since the effects of LPA are mediated via its cognate receptor, we
examined the effects of different doses of LPA on its survival activity
in SCs. LPA prevented SC apoptosis in a dose-dependent manner, with the antiapoptotic effect being observed at a concentration as low as 0.1 µM of LPA with a maximal effect being
observed at 10 µM (Fig. 1C). Based on these
results, all subsequent experiments in this study were performed with
10 µM of LPA.
Inhibitors of G LPA Stimulates Phosphorylation of MAPK through a PTX-sensitive and
PI3K-dependent Pathway--
To further study the signaling
pathways that mediate the survival effect of LPA, MAPK and Akt
phosphorylation were measured. LPA stimulated MAPK phosphorylation
(activation) in a time-dependent manner (Fig.
2A). Activation of MAPK was
maximal at 5 min followed by a decline to basal level by 30 min. LPA
also stimulated phosphorylation of Akt with kinetics similar to MAPK;
however, the degree of activation as estimated by the amount of
phosphorylated Akt was small as compared with MAPK. With continuous
exposure of SCs to LPA, a consistent finding was a second peak of
activation of MAPK and Akt at 8 and 16 h. The significance of the
biphasic activation of the two kinases is unclear.
To analyze the relevant signaling pathways leading to activation of
mitogen-activated protein and Akt kinases by LPA, several inhibitors
were used. LPA-stimulated phosphorylation of MAPK and Akt was partially
blocked by pertussis toxin, LY249002, and wortmannin but not by
rapamycin (Fig. 2B). Pretreatment of the cells with PD98059
prevented activation of MAPK while having no effect on activation of
Akt (Fig. 2B). These results, combined with the use of the
same inhibitors in the SC survival assays, confirm the importance of
the G LPA Increases Myelin Protein P0 Expression--
The
LPA receptor is expressed in SC during postnatal development of
peripheral nerves and parallels the formation of the myelin sheath (7).
To demonstrate a correlation between LPA signaling and myelination, we
examined the effect of LPA on expression of the major myelin protein,
P0. Western blot analysis demonstrated that LPA increased
myelin P0 protein levels within 24 h after the
addition of LPA (Fig. 3A).
During normal differentiation of SCs, an increase in myelin
P0 expression is paralleled by a decrease in p75 nerve
growth factor receptor expression. However, LPA did not alter the basal
expression of p75 nerve growth factor receptors on SCs (data not
shown). Since LPA can alter protein turnover, the increase in myelin
P0 protein may simply reflect a decrease in protein
turnover. To address this question, transient transfection assays using
a plasmid containing the P0 promoter regulating expression of the bacterial CAT gene (pP0cat) were performed. SCs
transiently transfected with pP0cat plasmid were treated
for 24 h with LPA, at which time the cells were harvested, and CAT
activity was measured. LPA increased CAT activity by 2-fold as compared
with the basal expression of CAT activity (Fig. 3B),
indicating that LPA increased P0 expression from the myelin
P0 promoter.
Edg4 Mediates LPA-induced P0 Expression--
Recent
studies have identified three G protein-coupled receptors for LPA,
Edg2, Edg4, and Edg7. Among them, Edg2 and Edg4 are expressed in SCs
(13). To identify the Edg receptors mediating LPA-induced
P0 expression, we carried out RNA interference study to
knock down the expression of a given Edg receptor (36). Cells were
transfected with either target-specific siRNA duplexes (for Edg2 or
Edg4, respectively) or a nonspecific siRNA control (GL2 luciferase
siRNA duplex) (36) or buffer only. Gene silencing was measured by
immunoblot analysis. In cells treated with Edg4-specific siRNA for
48 h, there was significant decrease in Edg4 protein expression
(Fig. 4B). A decrease in
LPA-induced P0 expression was also observed in cells
treated with Edg4-specific siRNA (Fig. 4C). However, no
inhibition in LPA-induced P0 expression was also observed
in cells treated with Edg2-specific siRNA (Fig. 4C), although Edg2 protein expression was down-regulated by such treatment (Fig. 4A). These results demonstrate a role of LPA receptor
Edg4 in mediating LPA induced P0 expression.
PKC LPA and Serum Stimulate Actin Stress Fiber Formation through the
Rho/Rho Kinase Pathway--
During differentiation, SCs undergo
morphological changes and become spindle-shaped. To investigate these
morphological changes, the reorganization of actin cytoskeleton in SCs
after serum and LPA treatment was studied. Both serum and LPA induced
actin stress fiber formation within 15 min (Fig.
7, A-C). To determine whether these changes in the actin cytoskeleton were dependent on the Rho
pathway, cells were injected with C3 transferase (1 mg/ml), an enzyme
that specifically inhibits RhoA by ADP-ribosylation of the protein (37)
(Fig. 7, G and H). Control cells were injected only with rabbit IgG (Fig. 7, E and F). 30 min
after injection, SCs were treated with 10% fetal bovine serum for 15 min. C3 transferase-injected cells failed to form stress fibers, unlike
control cells injected with IgG. To further confirm a role for Rho in
regulating stress fiber formation, serum-starved SCs were injected with
1 mg/ml recombinant constitutively active RhoA (V14). As expected,
constitutively active RhoA rapidly increased the formation of actin
stress fibers (within 15 min after injection) (Fig. 7J).
Pretreatment of Schwann cells for 30 min with 10 µM
Y27632, an inhibitor of Rock, a downstream target for of RhoA, blocked
serum-induced (data not shown) and LPA-induced actin stress fiber
formation (Fig. 7D). Moreover, actin stress fiber formation
induced by LPA was insensitive to pertussis toxin, wortmannin, PD98059,
and bisindolylmaleimide II, ruling out roles for G During normal development and after nerve injury, SCs undergo
dramatic changes in cell shape, motility, survival, and gene expression
(1). Many of these processes are tightly controlled by axons and by
signals that arise from the basal lamina (3, 4, 6). The axonal membrane
provides mitogenic and differentiation signals to the SCs. Moreover,
the nerve cell body and the axon secrete soluble factors that govern SC
proliferation, migration, and survival. The basal lamina also provides
differentiation cues to the SC. Although some of these molecules have
been identified, the signal transduction pathways activated by cell
surface receptors that mediate these processes are largely unknown. In
SC cell culture, serum promotes cell survival and alters myelin gene
expression. When we addressed the effects of serum, we found that only
some were mediated by LPA. In the present work, we found that LPA
exerts its effects on SC survival and differentiation using different G
proteins coupled to the LPA receptor. A scheme of the pathways involved
in LPA signaling is shown in Fig. 8.
i to activate p21ras and
phosphatidylinositol 3-kinase, through G
q to activate phospholipase C, or through Gq12/13 to activate the Rho
pathway. We found that the addition of serum or LPA to serum-starved
Schwann cells rapidly (10 min) induced the appearance of actin stress fibers via a Rho-mediated pathway. Furthermore, LPA was able to rescue Schwann cells from apoptosis in a
G
i/phosphatidylinositol 3-kinase/MEK/MAPK-dependent manner. In addition, LPA
increased the expression of myelin protein P0 in Schwann
cells in a G
i-independent manner but dependent on
protein kinase C. By means of pharmacological and overexpression
approaches, we found that the novel isozyme protein kinase C
was
required for myelin P0 expression. Thus, the multiple
effects of LPA in Schwann cells (actin reorganization, survival, and
myelin gene expression) appear to be mediated through the different G
protein-dependent pathways activated by the LPA receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-neuregulin, and
insulin-like growth factor-I for SC survival;
-neuregulin,
platelet-derived growth factor, epidermal growth factor, and tumor
growth factor-
for SC proliferation; and tumor growth factor-
and jagged/delta for regulating myelin gene expression (7-11).
The factors that regulate SC survival do so through activation of Akt
and MAPK signaling pathways, whereas actin reorganization is regulated
through the Rho family of GTPase (12, 13). Additionally, agents that
elevate intracellular cAMP levels stimulate myelin gene expression (10,
14). However, aside from the effects of cAMP on myelin gene expression,
there is little information on the signaling pathways involved in
regulating myelin gene expression.
/
mice were able to respond to LPA,
indicating that other LPA receptors such as
lpA2/Edg4 or
lpA3/E7 may be involved in mediating the
LPA effects, since SC also expresses
lpA2/Edg4 (13).
proteins:
G
i, G
q, and G
12/13 (20,
21). Activation of G
q stimulates phospholipase C
activity, thereby increasing the amount of diacylglycerol and inositol
3-phosphate. Diacylglycerol, in turn, activates protein kinase C (PKC),
whereas inositol 3-phosphate mobilizes intracellular calcium. LPA
activation of pertussis toxin (PTX)-sensitive G
i
decreases intracellular cAMP by inhibiting adenylyl cyclase and also
induces MAPK/extracellular signal-regulated kinase activity. LPA
activation of G
12/13 stimulates the Rho pathway, which
in turn induces serum response element-mediated transcription, actin
cytoskeletal reorganization, and activation of PI3K.
-neuregulin all promote SC survival. In previous studies
investigating the signaling pathways responsible for LPA- and
-neuregulin-induced SC survival, PI3K was shown to play a pivotal
role, although the relative importance of MAPK and protein kinase B
(Akt) in this process is unclear (7, 12). Moreover, LPA has been shown
to induce actin rearrangement in SCs (13). In this study, we provide
evidence that LPA promotes SC survival through a signal transduction
pathway involving PTX-sensitive G
i protein, PI3K, and
MAPK. We also demonstrate that LPA, acting through receptor
lpA2/Edg4 and its downstream effector
protein kinase C
(PKC
), induces expression of myelin P0 protein and that the Rho pathway regulates the actin
cytoskeleton reorganization. These results suggest multiple roles for
LPA in SC survival and differentiation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phorbol 12-myristate 13-acetate (PMA),
pertussis toxin (inhibitor of G
i), and wortmannin (PI3K
inhibitor) were obtained from Sigma. PKC inhibitors,
bisindolylmaleimide II and rottlerin, were obtained from Calbiochem
(23). The MEK inhibitor, PD98059, was from New England Biolabs
(Beverly, MA). The PI3K inhibitor, LY294002, and PKC inhibitor,
Gö6976, were obtained from Biomol (Plymouth Meeting, PA) (24).
The Rho kinase (ROCK) inhibitor, Y27632, was obtained from Yoshitomi
Pharmaceutical Industries (Saitama, Japan) (25). Rabbit antibodies
against phosphorylated MAPK, total MAPK, phosphorylated Akt, and total
Akt were obtained from New England Biolabs. Rabbit anti-PKC
antibody
and goat anti-Edg4 antibody were from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Rabbit anti-Edg2 antibody was from
BIOSOURCE (Camarillo, CA). Rabbit
anti-P0 antibody was provided by Dr. Bruce Trapp (Cleveland Clinic, Cleveland, OH).
, PKC
,
and LacZ Adenoviruses--
Adenovirus (AdV)-expressing dominant
negative PKC
and PKC
were generated as described (29, 30). SCs
grown in 60-mm dishes were infected with dominant negative PKC
,
dominant negative PKC
, or LacZ adenovirus for 16 h at
multiplicities of infection of 300 plaque-forming units/cell in
Dulbecco's modified Eagle's medium with 2% fetal bovine serum.
Infection of SCs with a multiplicity of infection of 300 plaque-forming
units/cell was determined in preliminary studies by maximizing for
GFP-expressing cells using GFPAdV. After removal of the virus, cells
were incubated in Dulbecco's modified Eagle's medium with 2% fetal
bovine serum for 24 h and then treated with LPA. 24 h later,
the cells were lysed in radioimmune precipitation buffer and analyzed.
-GFP Translocation--
Cells were plated onto 35-mm glass
bottom culture dishes (MarTek Corp., Ashland, MA) and transfected with
a plasmid carrying a DNA insert encoding for a PKC
-GFP fusion
protein (30) using FuGENE 6 (Roche Molecular Biochemicals).
Translocation of PKC
-GFP was monitored under a confocal
laser-scanning fluorescence microscope (30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
LPA inhibits SC apoptosis. SCs were
cultured in serum-free medium for 20 h either in the absence or
presence of LPA (10 µM). A, cells were stained
with propidium iodide and scored under ×400 microscopy for
normal or condensed nuclei. The arrows point to
apoptotic cells with condensed, fragmented nuclei. B,
cells were incubated for 20 h in serum-free medium in the absence
or presence of LPA with PTX (100 ng/ml), LY294002 (50 µM), wortmannin (200 nM), rapamycin (100 nM), or PD98059 (50 µM). Data from three
separate experiments (mean ± S.D.) are shown. C, LPA
decreases apoptosis in a dose-dependent manner. Data from
three separate experiments (mean ± S.D.) are shown.
i PI-3 Kinase and MEK Prevent LPA
Protection from Apoptosis--
The role of the PI3K/Akt and MAPK
pathways in SC survival has been demonstrated (12, 34, 35). To evaluate
the contribution of different signaling pathways in mediating
LPA-induced SC survival, several specific inhibitors were used. The
selection of the inhibitors used in this study was based on knowledge
of the G proteins implicated in LPA signaling. Pertussis toxin, an
inhibitor of G
i, blocked the antiapoptotic effect of LPA
(Fig. 1B), as did the PI3K inhibitors wortmannin and
LY249002 (Fig. 1B). Furthermore, inhibition of MEK by
PD98059 also blocked the antiapoptotic effect of LPA (Fig. 1B). Rapamycin, an inhibitor of mTOR that prevents
activation of p70 S6 kinase, had no effect on LPA-mediated survival
(Fig. 1B). Of interest, LY249002 in the absence of LPA
increased apoptosis (Fig. 1B), probably due to the
inhibition of basal PI3K activity in SCs (12). Furthermore, neither
inhibition of PKC
by rottlerin nor Rho kinase by Y27632 had any
effect on the ability of LPA to promote SC survival (data not shown).
These results indicate that the survival effect of LPA on serum-starved
SCs is mediated in a
G
i/PI3K/MEK/MAPK-dependent manner.
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Fig. 2.
LPA activates MAPK and Akt kinase.
A, SCs were treated with LPA for the times as indicated, and
cell lysates were analyzed by Western blotting using antibodies against
phospho-MAPK (p-Erk1 and p-Erk2), MAPK
(Erk1 and Erk2), and phospho-Akt. B,
cells were treated with LPA (10 µM) or GGF (100 ng/ml)
for 5 min, pretreated with PTX (200 ng/ml) for 3 h, or pretreated
with either LY249002 (50 µM), wortmannin (200 nM), rapamycin (100 nM), or PD98059 (50 µM) for 30 min and then exposed to LPA for 5 min.
Phosphorylation of MAPK and Akt was assessed by Western blotting.
Similar results were observed on three separate experiments.
i/PI3K/MAPK pathway in LPA-mediated SC survival.
Although MAPK activation is important for LPA-mediated SC survival, we
cannot completely exclude a role for Akt in this process.
-Neuregulin (GGF) rescued SCs from apoptosis in an
Akt-dependent manner (12). However, when we compared
-neuregulin and LPA activation of Akt, there was significantly less
activation of Akt by LPA as compared with
-neuregulin
(Fig. 2B).
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Fig. 3.
LPA increases myelin P0 protein
expression in SCs. A, SCs were treated with LPA (10 µM) for 24 or 48 h, and P0 protein
levels were measured by Western blotting. The amount of -actin was
used as a control of protein loading. B, SCs were
transiently transfected with a P0-CAT reporter plasmid (10 µg of pP0cat) and then treated with LPA for 24 h.
CAT activity was measured using [14C]chloramphenicol.
Bars, mean ± S.D. from three independent
experiments.
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Fig. 4.
Effects of Edg2 and Edg4 siRNAs in SCs.
A, SCs were transfected with Edg2 siRNA duplex, GL2
luciferase siRNA duplex (nonspecific siRNA control), or buffer only.
48 h after transfection, Edg2 protein were assessed by Western
blotting. B, SCs were transfected with Edg4 siRNS duplex,
GL2 luciferase siRNA duplex (nonspecific siRNA control), or buffer
only. 48 h after transfection, Edg4 protein was assessed by
Western blotting. C, SCs were transfected with siRNA duplex
for Edg2, Edg4, GL2 luciferase, or buffer only. 24 h after
transfection, cells were treated with 10 µM LPA for
24 h. P0 protein was assessed by Western blotting. The
amount of -actin was used as a control of protein loading in each
panel.
Mediates P0 Induction by LPA--
To elucidate
the signaling pathways through which LPA increased P0
expression, specific inhibitors were used. Fig.
5A shows that the induction of
P0 protein expression by LPA was not inhibited by PTX (200 ng/ml). Instead, PTX enhanced LPA-induced P0 protein levels. The Rock inhibitor Y27632 and the MEK inhibitor PD98059 both
partially blocked the induction of P0 protein expression. The broadly acting PKC inhibitor, bisindolylmaleimide II, blocked LPA-induced myelin P0 expression. Since SCs express various
isoforms of protein kinase C, more selective PKC inhibitors were used
to identify the specific isoform of PKC responsible for P0
induction (Fig. 5B). Gö6976, an inhibitor of classical
PKC
, -
, and -
, had no effect on P0 induction,
whereas rottlerin, an inhibitor of PKC
, blocked induction of
P0 by LPA. Pretreatment of SCs with 100 nM PMA
(for 8 h) to down-regulate PKCs (including PKC
) also inhibited
the induction of P0 expression by LPA (Fig. 5C).
As an index of activation, PKC
translocation was monitored by
transfecting SCs with a plasmid carrying a DNA insert encoding for a
PKC
-GFP fusion protein (30). When transfected cells were treated
with 10 µM of LPA, translocation of PKC
to the plasma
membrane was observed (Fig. 6). To
confirm the role of PKC
in regulating expression of P0
in SCs, cells were infected with adenoviruses for kinase-inactive PKC
or PKC
. These PKCs have been mutated in the ATP-binding site
and function as dominant negative mutants (29, 30). Infection of SCs
with dominant negative PKC
AdV dramatically decreased LPA induction
of P0 expression (Fig. 5D). In contrast, there
were no changes in LPA-induced P0 expression in cells
infected with dominant negative PKC
AdV or control LacZAdV. Thus, it
appears that the induction of P0 expression by LPA is
mainly mediated via PKC
and that both Rho kinase and MAPK are also
likely to be involved in myelin gene expression.
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Fig. 5.
PKC mediates the
effect of LPA on P0 protein expression. A, SCs
were treated with LPA (10 µM) in the presence or absence
of either PTX (200 ng/ml), PD98059 (50 µM),
bisindolylmaleimide II (10 µM), or Y27632 (10 µM) for 24 h. B, SCs were treated with
LPA and Gö6976 (1 µM) or rottlerin (10 µM) for 24 h. C, SCs were pretreated with
or without PMA (100 nM) for 12 h and then treated with
LPA for 24 h. D, SCs were infected with DN-PKC
AdV,
DN-PKC
, or LacZAdV and 24 h later either treated with or
without LPA (10 µM) for an additional 24 h. The
amount of
-actin was used as a control of protein loading in each
panel.
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Fig. 6.
Effect of LPA on translocation of
PKC -GFP. SCs were transfected with a
plasmid pPKC
-GFP. 48 h later, cells were treated with 10 µM LPA for the indicated times under a confocal
fluorescence laser-scanning microscope. Translocation of PKC
-GFP
fusion protein to focal spots at the plasma membrane was observed after
LPA treatment.
i,
PI3K, MAPK, or PKC in this process (data not shown). These results
indicate that both serum- and LPA-induced actin cytoskeleton
reorganization in SCs are mediated by a pertussis toxin-insensitive G
protein/RhoA/ROCK-dependent pathway.
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Fig. 7.
LPA induces Rho-dependent actin
stress fiber formation. Serum-starved SCs (A) were
treated with fetal calf serum (10%) (B) and LPA (10 µM) (C) for 15 min or pretreated with Y27632
(10 µM) for 30 min prior to LPA treatment (D).
Cells were fixed and stained with fluorescein isothiocyanate-conjugated
phalloidin. Magnification was ×400. Serum-starved SCs were
microinjected with rabbit IgG (5 mg/ml) alone (E and
F) or co-injected with C3 transferase protein (1 mg/ml) and
rabbit IgG (G and H). 15 min after injection, the
cells were treated with fetal calf serum (10%) for 30 min. Cells were
fixed and double-stained with fluorescein isothiocyanate-conjugated
phalloidin (E and G) and Texas Red-conjugated
donkey anti-rabbit IgG antibody (F and H) to help
identify the injected cells. The arrows indicate cells
injected with C3 transferase that failed to form stress fibers in
response to serum (magnification, ×400). Serum-starved SCs were
microinjected with rabbit IgG alone (I) or co-injected with
RhoAV12 (1 mg/ml) (J) for 30 min. Cells
were fixed and stained with fluorescein isothiocyanate-conjugated
phalloidin (magnification, ×1000).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
The signaling cascade involved in LPA-induced
changes in SCs.
Both serum and LPA elicit antiapoptotic effects on SCs. Previously, it
had been demonstrated that LPA and -neuregulin mediated SC survival
via a PI3K/Akt pathway (7). We find that the LPA effects on SC survival
require the involvement of G
i, PI3K, MEK, and MAPK.
Since phosphorylation of MAPK was inhibited by pretreatment of SCs with
PTX or wortmannin, MAPK was downstream of G
i and PI3K.
Additionally, the phosphorylation of Akt was also seen, albeit the
extent of phosphorylation was small when compared with the robust
phosphorylation of MAPK by LPA. When comparing LPA- with
-neuregulin-mediated SC survival, both Akt and MAPK have key roles,
with PI3K having a central role. In the case of LPA-induced SC
survival, we believe that G
i/PI3K/MAPK plays a more
significant role than PI3K/Akt because of the effects of the MEK
inhibitor PD98059 in abrogating the cell survival effects of LPA
(Fig. 1).
SCs in peripheral nerves are spindle-shaped, whereas in cell culture
they can assume different shapes. In cell culture, differentiation of
SCs is accompanied by a change in cell shape from a flat to a
spindle-shaped cell. Serum or LPA treatment of SCs induces a change to
spindle-shaped cell due to the reorganization of the actin cytoskeleton
and the appearance of stress fibers. The stress fibers form and
traverse the length of the cell in bundles. They contain F-actin,
myosin, and actin-binding proteins that are anchored to the plasma
membrane by focal adhesions composed of -actin, vinculin, talin, and
focal adhesion kinase (38). In our studies, induction of stress fibers
by RhoA in SCs was not accompanied by the appearance of focal adhesion
complexes at the plasma membrane (detected by staining with
anti-vinculin antiserum), probably because cells were plated on
poly-L-lysine-coated coverslips. Fibroblasts under similar
plating conditions are unable to form focal adhesion complexes,
although they continued to form stress fibers (39).
In fibroblasts, the reorganization of the actin cytoskeleton through
Rho proteins has been extensively studied (40-42). LPA or serum
induced stress fiber formation through a similar pathway, since
activated Rho (RhoV14) induced formation of actin stress
fibers in SCs, an effect that was mimicked by the addition of serum or
LPA to these cells. Moreover, inhibiting Rho A with C3 transferase
blocked serum- and LPA-induced actin changes in SCs (37).
Rho-dependent kinases (ROK and p160ROCK/ROK
) are
involved in the formation of stress fibers and focal adhesions in
fibroblasts (43, 44). In our studies, LPA-induced actin stress fiber
formation was prevented by the Rho kinase inhibitor Y27632. How Rho
kinase participates in the reorganization of actin is unclear. It has
been proposed that phosphorylation of myosin light chain phosphatase by
Rho kinase may decrease phosphatase activity, thereby increasing the
concentration of phosphorylated myosin light chains (42, 45) that bind
to actin and promote formation of stress fibers (46, 47).
Alternatively, Rho kinase may induce actin stress fiber formation by
promoting the bundling of preexisting filaments (41, 48) or perhaps by
altering the intracellular levels of phosphatidylinositol
4,5-bisphosphate (49, 50) that causes uncapping of barbed ends of actin
and thus the rapid polymerization of actin (51, 52). LIM kinase, a
substrate for p160ROCK, also mediates reorganization of the actin
cytoskeleton (25). However, expression of LIM kinase in SCs did not
stimulate the formation of stress fibers (data not shown), suggesting
that activation of LIM kinase by p160ROCK is not important in the
formation of stress fibers in SCs.
In addition to morphological alterations, LPA induced the expression of
myelin protein P0 in SCs. Our siRNA experiments showed that
knockdown of Edg4 expression but not Edg2 inhibited LPA-induced P0 expression, suggesting that Edg4 mediates this effect.
Interestingly, pretreatment of SCs with PTX enhanced P0
protein expression. Since P0 expression in SCs is
stimulated by cAMP analogs and adenylyl cyclase activators, the
increase in LPA-induced P0 expression by PTX may involve an
increase in intracellular cAMP levels, since Gi
negatively regulates adenylyl cyclase.
Since changes in cell shape can influence gene expression, it was
possible that LPA-induced morphological changes were sufficient to
increase P0 expression. Y27632, an inhibitor of Rho kinase, partially blocks LPA-induced expression of P0. Furthermore,
since survival of SCs may be linked to differentiation, the effects of
the MEK inhibitor PD980959 on LPA-induced expression of P0 protein was studied. PD98059 also partially blocked this LPA effect. Finally, since LPA-mediated effects can occur via the Gq protein (21), which can activate PKC, we investigated the role of PKC
in P0 expression. Several isoforms of PKC are found in SCs,
including PKC
, -
, -
, and -
(33), and it is likely that each
individual PKC isoform may have a different function in SCs. Pretreatment of SCs with bisindolylmaleimide II, a broad spectrum inhibitor of PKC, completely blocked LPA-induced expression of P0, indicating an important role for the PKC pathway. This
was substantiated by PKC down-regulation with PMA (decreased expression of PKC
, -
, -
, and -
protein; data not shown), suggesting an essential role for PKCs in this effect. Gö6976, an inhibitor of
"classical" PKCs (PKC
, -
, and -
), had no effect on LPA
induction of P0 expression, which rules out the involvement
of "classical" PKCs. However, rottlerin, a specific inhibitor for
PKC
, significantly attenuated the effect of LPA on P0
expression. Also, overexpression of dominant negative PKC
in SCs
inhibited LPA-induced P0 expression, whereas dominant
negative PKC
had no inhibitory effect. Together, these data support
the concept that PKC
is critical in the regulation of P0
expression by LPA.
In conclusion, our findings suggest that LPA has multiple effects on SC
survival and differentiation that are mediated via coupling of the
receptor to different G proteins (Fig. 8). The survival effects are
largely mediated through PI3K/MAPK, the cytoskeletal changes via RhoA,
and myelin P0 expression via PKC, although other
pathways such as RhoA/Rock, MEK/MAPK, and cAMP/protein kinase A are
likely to play a role, indicating the complex network of pathways
activated by LPA in SCs.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Paula Stein for designing siRNA duplexes and Andrew Coyle for technical assistance.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R01 NS21700 (to G. T.) and R01 CA89202 (to M. G. K.) and National Multiple Sclerosis Society Grant RG2780 (to G. T.).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: 514 Abramson Bldg., 3400 Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-5177; Fax: 215-590-5195; E-mail: tennekoon@email.chop.edu.
Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M213244200
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ABBREVIATIONS |
---|
The abbreviations used are: SC, Schwann cell; LPA, lysophosphatidic acid; PMA, phorbol 12-myristate 13-acetate; PI3K, phosphatidylinositaol 3-kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PTX, pertussis toxin; PKC, protein kinase C; AdV, adenovirus; siRNA, small interfering RNA; ROCK, Rho kinase; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; CAT, chloramphenicol acetyltransferase.
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