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• W2061649562 abstract "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 Gαi to activate p21ras and phosphatidylinositol 3-kinase, through Gαq 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 Gαi/phosphatidylinositol 3-kinase/MEK/MAPK-dependent manner. In addition, LPA increased the expression of myelin protein P0 in Schwann cells in a Gαi-independent manner but dependent on protein kinase C. By means of pharmacological and overexpression approaches, we found that the novel isozyme protein kinase Cδ 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. 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 Gαi to activate p21ras and phosphatidylinositol 3-kinase, through Gαq 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 Gαi/phosphatidylinositol 3-kinase/MEK/MAPK-dependent manner. In addition, LPA increased the expression of myelin protein P0 in Schwann cells in a Gαi-independent manner but dependent on protein kinase C. By means of pharmacological and overexpression approaches, we found that the novel isozyme protein kinase Cδ 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. In the peripheral nervous system, Schwann cells (SCs) 1The 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 form the myelin sheath and play a key role in the maintenance of the normal physiological function of the axon (1Gould R.M. Jessen k.R. Mirsky R. Tennekoon G. The Cell of Schwann: An Update. CRC Press, Inc., Boca Raton, FL1992: 123-171Google Scholar). 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 (1Gould R.M. Jessen k.R. Mirsky R. Tennekoon G. The Cell of Schwann: An Update. CRC Press, Inc., Boca Raton, FL1992: 123-171Google Scholar). 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 P0protein (2Fernandez-Valle C. Gorman D. Gomez A.M. Bunge M.B. J. Neurosci. 1997; 17: 241-250Google Scholar). The survival and differentiation of SCs are regulated by extracellular cues from axons and the extracellular matrix (3Aguayo A.J. Peyronnard J.M. Terry L.C. Romine J.S. Bray G.M. J. Neurocytol. 1976; 5: 137-155Google Scholar, 4Jessen K.R. Mirsky R. Ann. N. Y. Acad. Sci. 1999; 883: 109-115Google Scholar, 5Bunge M.B. Bunge R.P. Ann. N. Y. Acad. Sci. 1986; 486: 241-247Google Scholar, 6Bunge R.P. Bunge M.B. Eldridge C.F. Annu. Rev. Neurosci. 1986; 9: 305-328Google Scholar). Some of these factors are lysophosphatidic acid (LPA), β-neuregulin, and insulin-like growth factor-I for SC survival; β-neuregulin, platelet-derived growth factor, epidermal growth factor, and tumor growth factor-β for SC proliferation; and tumor growth factor-β and jagged/delta for regulating myelin gene expression (7Weiner J.A. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5233-5238Google Scholar, 8Grinspan J.B. Marchionni M.A. Reeves M. Coulaloglou M. Scherer S.S. J. Neurosci. 1996; 16: 6107-6118Google Scholar, 9Trachtenberg J.T. Thompson W.J. Nature. 1996; 379: 174-177Google Scholar, 10Mews M. Meyer M. Glia. 1993; 8: 208-217Google Scholar, 11Davis J.B. Stroobant P. J. Cell Biol. 1990; 110: 1353-1360Google Scholar). 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 (12Li Y. Tennekoon G.I. Birnbaum M. Marchionni M.A. Rutkowski J.L. Mol. Cell. Neurosci. 2001; 17: 761-767Google Scholar, 13Weiner J.A. Fukushima N. Contos J.J. Scherer S.S. Chun J. J. Neurosci. 2001; 21: 7069-7078Google Scholar). Additionally, agents that elevate intracellular cAMP levels stimulate myelin gene expression (10Mews M. Meyer M. Glia. 1993; 8: 208-217Google Scholar,14Sobue G. Pleasure D. Science. 1984; 224: 72-74Google Scholar). 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 (15Wadhwani K.C. Rapoport S.I. Prog. Neurobiol. 1994; 43: 235-279Google Scholar). 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 (16Cheng L. Mudge A.W. Neuron. 1996; 16: 309-319Google Scholar). 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 (7Weiner J.A. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5233-5238Google Scholar) and actin rearrangement (13Weiner J.A. Fukushima N. Contos J.J. Scherer S.S. Chun J. J. Neurosci. 2001; 21: 7069-7078Google Scholar). 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 (17Moolenaar W.H. Ann. N. Y. Acad. Sci. 2000; 905: 1-10Google Scholar). 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 zonegene-1; also termedEdg-2/rec1.3/lp A1) encodes a 41-kDa protein that is widely expressed in mouse tissue with the highest levels seen in embryonic cortex (18Hecht J.H. Weiner J.A. Post S.R. Chun J. J. Cell Biol. 1996; 135: 1071-1083Google Scholar). In postnatal life, the LPA receptor lp A1/vzg-1 is predominantly expressed in glial cells (mainly oligodendrocytes and SCs) (7Weiner J.A. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5233-5238Google Scholar, 19Weiner J.A. Hecht J.H. Chun J. J. Comp. Neurol. 1998; 398: 587-598Google Scholar). The appearance of thelp A1/vzg-1/Edg-2 receptor in SCs parallels the appearance of myelin formation in developing peripheral nerves and is up-regulated after nerve injury (7Weiner J.A. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5233-5238Google Scholar, 13Weiner J.A. Fukushima N. Contos J.J. Scherer S.S. Chun J. J. Neurosci. 2001; 21: 7069-7078Google Scholar). Although it is likely thatlp A1/vzg-1/Edg2 receptor is responsible for mediating most of the effects of LPA, SCs fromlp A1−/− mice were able to respond to LPA, indicating that other LPA receptors such aslp A2/Edg4 orlp A3/E7 may be involved in mediating the LPA effects, since SC also expresseslp A2/Edg4 (13Weiner J.A. Fukushima N. Contos J.J. Scherer S.S. Chun J. J. Neurosci. 2001; 21: 7069-7078Google Scholar). LPA receptors are coupled to at least three different Gα proteins: Gαi, Gαq, and Gα12/13 (20Fukushima N. Chun J. Prostaglandins Other Lipid Mediators. 2001; 64: 21-32Google Scholar,21Swarthout J.T. Walling H.W. Cell. Mol. Life Sci. 2000; 57: 1978-1985Google Scholar). Activation of Gαq 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 Gαidecreases intracellular cAMP by inhibiting adenylyl cyclase and also induces MAPK/extracellular signal-regulated kinase activity. LPA activation of Gα12/13 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 (12Li Y. Tennekoon G.I. Birnbaum M. Marchionni M.A. Rutkowski J.L. Mol. Cell. Neurosci. 2001; 17: 761-767Google Scholar, 22Syroid D.E. Zorick T.S. Arbet-Engels C. Kilpatrick T.J. Eckhart W. Lemke G. J. Neurosci. 1999; 19: 2059-2068Google Scholar). Serum, LPA, and β-neuregulin all promote SC survival. In previous studies investigating the signaling pathways responsible for LPA- and β-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 (7Weiner J.A. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5233-5238Google Scholar, 12Li Y. Tennekoon G.I. Birnbaum M. Marchionni M.A. Rutkowski J.L. Mol. Cell. Neurosci. 2001; 17: 761-767Google Scholar). Moreover, LPA has been shown to induce actin rearrangement in SCs (13Weiner J.A. Fukushima N. Contos J.J. Scherer S.S. Chun J. J. Neurosci. 2001; 21: 7069-7078Google Scholar). In this study, we provide evidence that LPA promotes SC survival through a signal transduction pathway involving PTX-sensitive Gαi protein, PI3K, and MAPK. We also demonstrate that LPA, acting through receptorlp A2 /Edg4 and its downstream effector protein kinase Cδ (PKCδ), 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. LPA, 4β-phorbol 12-myristate 13-acetate (PMA), pertussis toxin (inhibitor of Gαi), and wortmannin (PI3K inhibitor) were obtained from Sigma. PKC inhibitors, bisindolylmaleimide II and rottlerin, were obtained from Calbiochem(23Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Google Scholar). 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) (24Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Google Scholar). The Rho kinase (ROCK) inhibitor, Y27632, was obtained from Yoshitomi Pharmaceutical Industries (Saitama, Japan) (25Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Science. 1999; 285: 895-898Google Scholar). Rabbit antibodies against phosphorylated MAPK, total MAPK, phosphorylated Akt, and total Akt were obtained from New England Biolabs. Rabbit anti-PKCδ antibody and goat anti-Edg4 antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-Edg2 antibody was fromBIOSOURCE (Camarillo, CA). Rabbit anti-P0 antibody was provided by Dr. Bruce Trapp (Cleveland Clinic, Cleveland, OH). Rat Schwann cells were isolated from the sciatic nerves of 2-day-old rat pups using the method described by Brockes et al. (26Brockes J.P. Fields K.L. Raff M.C. Brain Res. 1979; 165: 105-118Google Scholar). 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 (27Porter S. Clark M.B. Glaser L. Bunge R.P. J. Neurosci. 1986; 6: 3070-3078Google Scholar). Prior to all of the studies, both forskolin and glial growth factor were removed from the cell culture for 1 week. 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). After the different treatment paradigms, SCs were rinsed once with PBS and then with TBS (50 mmTris, 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 nmcalyculin A, 1 mm sodium vanadate, 1 mmphenylmethylsulfonyl 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). 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 (28Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Google Scholar). Adenovirus (AdV)-expressing dominant negative PKCα and PKCδ were generated as described (29Fujii T. Garcia-Bermejo M.L. Bernabo J.L. Caamano J. Ohba M. Kuroki T. Li L. Yuspa S.H. Kazanietz M.G. J. Biol. Chem. 2000; 275: 7574-7582Google Scholar, 30Garcia-Bermejo M.L. Leskow F.C. Fujii T. Wang Q. Blumberg P.M. Ohba M. Kuroki T. Han K.C. Lee J. Marquez V.E. Kazanietz M.G. J. Biol. Chem. 2002; 277: 645-655Google Scholar). SCs grown in 60-mm dishes were infected with dominant negative PKCδ, dominant negative PKCα, 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. Plasmid containing glutathioneS-transferase-RhoA (V14) was expressed inEscherichia coli, and the recombinant protein was purified using glutathione-Sepharose 4B beads (31Miller M.J. Prigent S. Kupperman E. Rioux L. Park S.H. Feramisco J.R. White M.A. Rutkowski J.L. Meinkoth J.L. J. Biol. Chem. 1997; 272: 5600-5605Google Scholar). The botulinum C3 exoenzyme was purified as described (32Jalink K. van Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Google Scholar). 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. 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. 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 nmsiRNA 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. 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 PKCδ-GFP fusion protein (30Garcia-Bermejo M.L. Leskow F.C. Fujii T. Wang Q. Blumberg P.M. Ohba M. Kuroki T. Han K.C. Lee J. Marquez V.E. Kazanietz M.G. J. Biol. Chem. 2002; 277: 645-655Google Scholar) using FuGENE 6 (Roche Molecular Biochemicals). Translocation of PKCδ-GFP was monitored under a confocal laser-scanning fluorescence microscope (30Garcia-Bermejo M.L. Leskow F.C. Fujii T. Wang Q. Blumberg P.M. Ohba M. Kuroki T. Han K.C. Lee J. Marquez V.E. Kazanietz M.G. J. Biol. Chem. 2002; 277: 645-655Google Scholar). SCs undergo apoptosis upon serum withdrawal, characterized by DNA condensation in the nucleus (Fig. 1 A). About 50% of SCs underwent apoptosis following serum withdrawal for 24 h (Fig. 1 B). 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. 1 B). 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. 1 C). Based on these results, all subsequent experiments in this study were performed with 10 μm of LPA. The role of the PI3K/Akt and MAPK pathways in SC survival has been demonstrated (12Li Y. Tennekoon G.I. Birnbaum M. Marchionni M.A. Rutkowski J.L. Mol. Cell. Neurosci. 2001; 17: 761-767Google Scholar, 34Maurel P. Salzer J.L. J. Neurosci. 2000; 20: 4635-4645Google Scholar, 35Xiao G.H. Jeffers M. Bellacosa A. Mitsuuchi Y. Vande Woude G.F. Testa J.R. Proc. Natl. Acad Sci. U. S. A. 2001; 98: 247-252Google Scholar). 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 Gαi, blocked the antiapoptotic effect of LPA (Fig. 1 B), as did the PI3K inhibitors wortmannin and LY249002 (Fig. 1 B). Furthermore, inhibition of MEK by PD98059 also blocked the antiapoptotic effect of LPA (Fig.1 B). Rapamycin, an inhibitor of mTOR that prevents activation of p70 S6 kinase, had no effect on LPA-mediated survival (Fig. 1 B). Of interest, LY249002 in the absence of LPA increased apoptosis (Fig. 1 B), probably due to the inhibition of basal PI3K activity in SCs (12Li Y. Tennekoon G.I. Birnbaum M. Marchionni M.A. Rutkowski J.L. Mol. Cell. Neurosci. 2001; 17: 761-767Google Scholar). Furthermore, neither inhibition of PKCδ 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 Gαi/PI3K/MEK/MAPK-dependent manner. 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.2 A). 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. 2 B). Pretreatment of the cells with PD98059 prevented activation of MAPK while having no effect on activation of Akt (Fig. 2 B). These results, combined with the use of the same inhibitors in the SC survival assays, confirm the importance of the Gαi/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. β-Neuregulin (GGF) rescued SCs from apoptosis in an Akt-dependent manner (12Li Y. Tennekoon G.I. Birnbaum M. Marchionni M.A. Rutkowski J.L. Mol. Cell. Neurosci. 2001; 17: 761-767Google Scholar). However, when we compared β-neuregulin and LPA activation of Akt, there was significantly less activation of Akt by LPA as compared with β-neuregulin (Fig. 2 B). The LPA receptor is expressed in SC during postnatal development of peripheral nerves and parallels the formation of the myelin sheath (7Weiner J.A. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5233-5238Google Scholar). 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. 3 A). 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. 3 B), indicating that LPA increased P0 expression from the myelin P0 promoter. Recent studies have identified three G protein-coupled receptors for LPA, Edg2, Edg4, and Edg7. Among them, Edg2 and Edg4 are expressed in SCs (13Weiner J.A. Fukushima N. Contos J.J. Scherer S.S. Chun J. J. Neurosci. 2001; 21: 7069-7078Google Scholar). 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 (36Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Google Scholar). Cells were transfected with either target-specific siRNA duplexes (for Edg2 or Edg4, respectively) or a nonspecific siRNA control (GL2 luciferase siRNA duplex) (36Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Google Scholar) 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. 4 B). A decrease in LPA-induced P0 expression was also observed in cells treated with Edg4-specific siRNA (Fig. 4 C). However, no inhibition in LPA-induced P0 expression was also observed in cells treated with Edg2-specific siRNA (Fig. 4 C), although Edg2 protein expression was down-regulated by such treatment (Fig. 4 A). These results demonstrate a role of LPA receptor Edg4 in mediating LPA induced P0 expression. To elucidate the signaling pathways through which LPA increased P0expression, specific inhibitors were used. Fig.5 A 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 P0induction (Fig. 5 B). Gö6976, an inhibitor of classical PKCα, -β, and -γ, had no effect on P0 induction, whereas rottlerin, an inhibitor of PKCδ, blocked induction of P0 by LPA. Pretreatment of SCs with 100 nm PMA (for 8 h) to down-regulate PKCs (including PKCδ) also inhibited the induction of P0 expression by LPA (Fig. 5 C). As an index of activation, PKCδ translocation was monitored by transfecting SCs with a plasmid carrying a DNA insert encoding for a PKCδ-GFP fusion protein (30Garcia-Bermejo M.L. Leskow F.C. Fujii T. Wang Q. Blumberg P.M. Ohba M. Kuroki T. Han K.C. Lee J. Marquez V.E. Kazanietz M.G. J. Biol. Chem. 2002; 277: 645-655Google Scholar). When transfected cells were treated with 10 μm of LPA, translocation of PKCδ to the plasma membrane was observed (Fig. 6). To confirm the role of PKCδ in regulating expression of P0in SCs, cells were infected with adenoviruses for kinase-inactive PKCδ or PKCα. These PKCs have been mutated in the ATP-binding site and function as dominant negative mutants (29Fujii T. Garcia-Bermejo M.L. Bernabo J.L. Caamano J. Ohba M. Kuroki T. Li L. Yuspa S.H. Kazanietz M.G. J. Biol. Chem. 2000; 275: 7574-7582Google Scholar, 30Garcia-Bermejo M.L. Leskow F.C. Fujii T. Wang Q. Blumberg P.M. Ohba M. Kuroki T. Han K.C. Lee J. Marquez V.E. Kazanietz M.G. J. Biol. Chem. 2002; 277: 645-655Google Scholar). Infection of SCs with dominant negative PKCδAdV dramatically decreased LPA induction of P0 expression (Fig. 5 D). In contrast, there were no changes in LPA-induced P0 expression in cells infected with dominant negative PKCαAdV or control LacZAdV. Thus, it appears that the induction of P0 expression by LPA is mainly mediated via PKCδ and that both Rho kinase and MAPK are also likely to be involved in myelin gene expression.Figure 6Effect of LPA on translocation of PKC δ-GFP. SCs were transfected with a plasmid pPKCδ-GFP. 48 h later, cells were treated with 10 μm LPA for the indicated times under a confocal fluorescence laser-scanning microscope. Translocation of PKCδ-GFP fusion protein to focal spots at the plasma membrane was observed after LPA treatment.View Large Image Figure ViewerDownload" @default.
• W2061649562 created "2016-06-24" @default.
• W2061649562 creator A5007470485 @default.
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• W2061649562 title "Lysophosphatidic Acid Promotes Survival and Differentiation of Rat Schwann Cells" @default.
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