Lysophosphatidic Acid Promotes Survival and Differentiation of Rat Schwann Cells*

Yiwen LiDagger , Marco I. Gonzalez§, Judy L. Meinkoth§, Jeffrey Field§, Marcelo G. Kazanietz§, and Gihan I. TennekoonDagger

From the Departments of Dagger  Neurology and Pediatrics and § Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received for publication, December 30, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Galpha i to activate p21ras and phosphatidylinositol 3-kinase, through Galpha 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 Galpha i/phosphatidylinositol 3-kinase/MEK/MAPK-dependent manner. In addition, LPA increased the expression of myelin protein P0 in Schwann cells in a Galpha i-independent manner but dependent on protein kinase C. By means of pharmacological and overexpression approaches, we found that the novel isozyme protein kinase Cdelta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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), beta -neuregulin, and insulin-like growth factor-I for SC survival; beta -neuregulin, platelet-derived growth factor, epidermal growth factor, and tumor growth factor-beta for SC proliferation; and tumor growth factor-beta 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.

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-/- 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).

LPA receptors are coupled to at least three different Galpha proteins: Galpha i, Galpha q, and Galpha 12/13 (20, 21). Activation of Galpha 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 Galpha i decreases intracellular cAMP by inhibiting adenylyl cyclase and also induces MAPK/extracellular signal-regulated kinase activity. LPA activation of Galpha 12/13 stimulates the Rho pathway, which in turn induces serum response element-mediated transcription, actin cytoskeletal reorganization, and activation of PI3K.

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 beta -neuregulin all promote SC survival. In previous studies investigating the signaling pathways responsible for LPA- and beta -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 Galpha i protein, PI3K, and MAPK. We also demonstrate that LPA, acting through receptor lpA2/Edg4 and its downstream effector protein kinase Cdelta (PKCdelta ), 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- LPA, 4beta -phorbol 12-myristate 13-acetate (PMA), pertussis toxin (inhibitor of Galpha 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-PKCdelta 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).

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 PKCdelta , PKCalpha , and LacZ Adenoviruses-- Adenovirus (AdV)-expressing dominant negative PKCalpha and PKCdelta were generated as described (29, 30). SCs grown in 60-mm dishes were infected with dominant negative PKCdelta , dominant negative PKCalpha , 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.

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.

PKCdelta -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 PKCdelta -GFP fusion protein (30) using FuGENE 6 (Roche Molecular Biochemicals). Translocation of PKCdelta -GFP was monitored under a confocal laser-scanning fluorescence microscope (30).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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 Galpha 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 Galpha 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 PKCdelta 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 Galpha i/PI3K/MEK/MAPK-dependent manner.

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.


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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.

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 Galpha 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. beta -Neuregulin (GGF) rescued SCs from apoptosis in an Akt-dependent manner (12). However, when we compared beta -neuregulin and LPA activation of Akt, there was significantly less activation of Akt by LPA as compared with beta -neuregulin (Fig. 2B).

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.


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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 beta -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.

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.


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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 beta -actin was used as a control of protein loading in each panel.

PKCdelta 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 PKCalpha , -beta , and -gamma , had no effect on P0 induction, whereas rottlerin, an inhibitor of PKCdelta , blocked induction of P0 by LPA. Pretreatment of SCs with 100 nM PMA (for 8 h) to down-regulate PKCs (including PKCdelta ) also inhibited the induction of P0 expression by LPA (Fig. 5C). As an index of activation, PKCdelta translocation was monitored by transfecting SCs with a plasmid carrying a DNA insert encoding for a PKCdelta -GFP fusion protein (30). When transfected cells were treated with 10 µM of LPA, translocation of PKCdelta to the plasma membrane was observed (Fig. 6). To confirm the role of PKCdelta in regulating expression of P0 in SCs, cells were infected with adenoviruses for kinase-inactive PKCdelta or PKCalpha . These PKCs have been mutated in the ATP-binding site and function as dominant negative mutants (29, 30). Infection of SCs with dominant negative PKCdelta 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 PKCalpha AdV or control LacZAdV. Thus, it appears that the induction of P0 expression by LPA is mainly mediated via PKCdelta and that both Rho kinase and MAPK are also likely to be involved in myelin gene expression.


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Fig. 5.   PKCdelta 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-PKCdelta AdV, DN-PKCalpha , or LacZAdV and 24 h later either treated with or without LPA (10 µM) for an additional 24 h. The amount of beta -actin was used as a control of protein loading in each panel.


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Fig. 6.   Effect of LPA on translocation of PKCdelta -GFP. SCs were transfected with a plasmid pPKCdelta -GFP. 48 h later, cells were treated with 10 µM LPA for the indicated times under a confocal fluorescence laser-scanning microscope. Translocation of PKCdelta -GFP fusion protein to focal spots at the plasma membrane was observed after LPA treatment.

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 Galpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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 beta -neuregulin mediated SC survival via a PI3K/Akt pathway (7). We find that the LPA effects on SC survival require the involvement of Galpha i, PI3K, MEK, and MAPK. Since phosphorylation of MAPK was inhibited by pretreatment of SCs with PTX or wortmannin, MAPK was downstream of Galpha 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 beta -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 Galpha 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 alpha -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 (ROKalpha and p160ROCK/ROKbeta ) 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 Galpha i 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 Galpha q protein (21), which can activate PKC, we investigated the role of PKC in P0 expression. Several isoforms of PKC are found in SCs, including PKCalpha , -beta , -epsilon , and -delta (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 PKCalpha , -beta , -epsilon , and -delta protein; data not shown), suggesting an essential role for PKCs in this effect. Gö6976, an inhibitor of "classical" PKCs (PKCalpha , -beta , and -gamma ), had no effect on LPA induction of P0 expression, which rules out the involvement of "classical" PKCs. However, rottlerin, a specific inhibitor for PKCdelta , significantly attenuated the effect of LPA on P0 expression. Also, overexpression of dominant negative PKCdelta in SCs inhibited LPA-induced P0 expression, whereas dominant negative PKCalpha had no inhibitory effect. Together, these data support the concept that PKCdelta 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 PKCdelta , 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.

    ACKNOWLEDGEMENTS

We thank Dr. Paula Stein for designing siRNA duplexes and Andrew Coyle for technical assistance.

    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

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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