Инноваци - Шинэ Санаа, Шинэ Нээлт, 2007, 1(4-1)
P38 mitogen-activated kinase and c-Jun terminal kinase, but not extracellular signal-regulated kinase, are required for the LPA- induced migration of glioma cells
( Судалгааны өгүүлэл )
1-Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan; 2-Department of Neurosurgery, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan; 3-Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
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Бид сэтгүүлийнхээ энэхүү булангаар та бүхэндээ хилийн чанадад амжилттай ажиллаж амьдран Монголынхоо нэрийг өндөрт өргөж яваа эрдэмтэд, тэдний бүтээлийг танилцуулах болно.
Энэ удаагийн дугаарт Япон улсын Гунма мужийн их сургуулийн Эс, молекулын зохицуулгын институтид ажиллаж буй АУ-ы доктор Малчинхүүгийн Энхзолыг танилцуулж байна.
A potential role for lysophosphatidic acid (LPA) in the regulation of malignant diseases has been widely considered. Migratory response to LPA in glioma cells was almost completely inhibited by either pertussis toxin, LPA1 receptor antagonists including Ki16425, or an inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin.
LPA action on migration was also suppressed, though incompletely by several specific inhibitors for intracellular signaling pathways such as Rac1, p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun terminal kinase (JNK), but not extracellular signal-regulated kinase.
Nearly complete inhibition of the migration response to LPA, however, required simultaneous inhibition of both the p38MAPK and JNK pathways. Inhibition of Rac1 suppressed JNK but not p38MAPK, and dominant-negative form of Cdc42 abrogated p38MAPK activity. These findings suggest that, in glioma cells, the PI3K/Cdc42/p38MAPK and PI3K/Rac1/JNK pathways are equally important for LPA1 receptor-mediated migration.
Introduction
Gliomas represent about half of all brain tumors and among them, glioblastoma multiforme is thought to be the most malignant and common intracranial tumor (Vandenberg, 1992). Although generally not metastatic, glioblastoma cells exhibit highly migratory and invasive behavior (Ishiuchi et al., 2002).
Due to their infiltrative nature, many of these high-grade neoplasms involve both cerebral hemispheres by invading through the corpus callosum and therefore rarely can be totally removed surgically, which makes the prognosis extremely poor. For this reason, determining the factors and mechanisms that regulate the migratory response in order to find new strategies to control the neoplastic process is of great interest.
Lysophosphatidic acid (LPA), one of the simplest natural phospholipids, originally known to act via classic second messenger pathways, is now recognized as an extracellular lipid mediator that evokes hormone- and growth-factor-like responses in almost every cell type, both normal and transformed.
Activating its cognate G-protein coupled receptors, four of which have been identified so far (LPA1-LPA4), LPA elicits diverse cellular responses ranging from cell proliferation and survival to induction of morphological changes and motility (Mills and Moolenaar, 2003).
A potential role of LPA and its receptors in the pathogenesis of human cancer, however, is a relatively new concept. LPA was implicated in human tumorigenesis from studies showing that LPA increases the motility and invasiveness of different types of cells (Imamura et al., 1993; Stam et al., 1998). LPA seems to be accumulated in ascitic fluids from patients with intraperitoneal malignancies, particularly ovarian cancer (Xu et al., 1995; Westermann et al., 1998; Yamada et al., 2004). A major part of extracellular LPA is known to be produced from lysophosphatidylcholine by autotaxin/lysophospholipase D, originally identified as an autocrine motility factor for melanoma cells and implicated in tumor progression (Umezu-Goto et al., 2002). LPA2 is over-expressed in differentiated thyroid cancer (Schulte et al., 2001).
Furthermore, in prostate cancer cells, LPA acts as an autocrine growth factor, and one or more LPA receptors are expressed in the prostate cancer cell line (Xie et al., 2002). In astroglial cells as well, extracellularly added LPA induces the migration of stimulated cells (Manning et al., 2000; Hama et al., 2004). Although there are enough studies related to LPA-stimulated cell migration, the details of LPA signaling in migration have not been elucidated so far.
In this study, employing rat and human glioma cell lines, we explored the role of mitogen-activated protein (MAP) kinases in LPA-stimulated cell migration. LPA stimulated the migratory response in a manner dependent on p38MAP kinase (p38MAPK) and c-Jun terminal kinase (JNK).
Both enzymes were located downstream of phosphatidylinositol 3-kinase (PI3K) and were activated in parallel. However, activation of p38 MAPK occurred depending on Cdc42, while JNK was activated through Rac1. Thus, the PI3K/Cdc42/p38MAPK and PI3K/Rac1/JNK pathways may be involved in LPA-induced migration in glioma cells.
Methods
Materials
1-Oleoyl-sn-glycero-3-phosphate (LPA) was purchased from Cayman Chemical Co. (Ann Arbor, MI); fatty acid-free BSA and U0126 were from Calbiochem-Novabiochem Co. (San Diego, CA); dioctylglycerol pyrophosphate (DGPP 8:0) was from Avanti Polar Lipids, Inc. (Alabaster, AL); PTX was from List Biological Laboratories, Inc. (Campbell, CA); SP600125 was from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA); PDGF-BB was from Pepro Tech House (London, England). Ki16425 (3- (4-[4-([1-(2-chlorophenyl)ethoxy]carbonylamino)-3-methyl-5-isoxazolyl] benzylsulfonyl)propanoic acid) was synthesized by Kirin Brewery Co. (Takasaki, Japan), and VPC12249 was a generous gift from Prof. Kevin R. Lynch (University of Virginia School of Medicine). The sources of all other reagents were the same as described previously in (Malchinkhuu et al., 2003).
Cell culture
A rat C6 glioma cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). GNS-3314 and CGNH-89 cells, established from human glioblastoma surgical specimen, were grown in Minimal essential medium Eagle (MEM) containing 10% FBS. Glioma cells were plated on 6-cm dishes for migration assay and for evaluation of MAPKs activity. When cells had become 80-90% confluent, the culture medium was changed to fresh DMEM or MEM containing 0.1% (w/v) BSA to make them quiescent and the cells were cultured for one day. PTX (100ng/ml) was added to starvation medium 24 h before experiments.
Construction of adenoviral vector
The cDNAs for p38MAPK (GeneBank NM 031020) were cloned from rat astrocytes by PCR. The dominant-negative form of p38MAPK (p38-DN) was employed to replace phosphorylation sites with Ala and Phe according to (Raingeaud et al., 1995). The cDNAs for Cdc42 (GeneBank NM 001791) were cloned from human kidney cDNA library (NIPPON GENE CO., LTD., Toyama, Japan) by PCR. The dominant-The cell migration was quantified using a blind Boyden chamber apparatus (Neuro Probe Inc., Gaithersburg, MD) as described previously (Kon et al., 1999). Briefly, the lower chambers were filled with starvation medium containing the indicated concentrations of test agents, and subsequently, covered with 8-mm pores (Neuro Probe) membrane filters coated with type I collagen. The cells were trypsinized, washed once with DMEM containing 0.1% BSA. Re-suspended cells, after being incubated for 20 min without or with LPA receptor antagonists, and for 30 min with or without different inhibitors at 37°C, were loaded into the upper chamber. Migration was allowed to proceed for 4 h at 37°C under a humidified air/CO2 (19:1) atmosphere. Cells that had migrated to the lower surface were counted in four microscopic fields at 400x magnification.
Estimation of JNK activation by Western blot analysis
Polyclonal antibodies specific for the dually phosphorylated and hence active forms of JNK family members (p-p54 and p-p46) were used. These antibodies, together with antibodies to JNKs (p54 and p46) that recognize kinases independent of their phosphorylation state, were all obtained from Cell Signaling Technology (Beverly, MA). The cells were serum-starved for more than 18 h before incubation with a Hepes buffered medium composed of 20 mM HEPES, pH 7.4, 134 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 2.5 mM NaHCO3, 5 mM glucose, and 0.1% (wt/vol) BSA (fraction V) containing various test substances for the indicated times at 37°C. Reactions were terminated by washing twice with ice-cold PBS and adding 0.2 ml of the lysis buffer (Malchinkhuu et al., 2003). Cell lysates were centrifuged at 12.000 x g to remove the insoluble material. Equal amounts of protein were electrophoretically resolved on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBS-T. For detection of the phosphorylated forms of the kinases, the membranes were incubated with 1:500 dilution of anti-phosphospecific antibodies overnight at 4°C. Membranes were incubated for 1h with appropriate second antibodies conjugated with horseradish peroxidase. Then, the membranes were visualized using enhanced chemiluminescence detection system, according to the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ). The scanned films were quantified by NIH image software. In order to substantiate the consistency of protein content between the treatment groups the membranes were re-probed with phosphorylation state independent antibodies to JNK (1:1000 dilution) for overnight and processed as above.
Evaluation of p38 MAP kinase activity
Cells were treated in a way similar to that shown in western blot experiments, except that the glioma cells were stimulated for 10 min instead of 2.5 min in case of JNK. For measurement of p38 MAPK activity, the phosphorylated p38 MAPK in the lysate was immunoprecipitated and its activity was evaluated by its ability to phosphorylate the activating transcription factor-2 (ATF-2) fusion protein according to the manufacturer’s instructions for p38 MAP kinase assay kit from Cell Signaling Technology (Beverly, MA). In brief, the phosphorylated ATF-2 was separated by electrophoresis and detected by an antiphospho-ATF-2 antibody. Phosphorylation of ATF-2 was quantified by densitometry. The expression of total p38 MAPK proteins was also examined with polyclonal antibodies against p38 MAPK obtained from Cell Signaling Technology (Beverly, MA).
Data presentation
All experiments were performed in duplicate or triplicate. The results of multiple observations are presented as the mean ± SEM or as representative results from more than three different batches of cells unless otherwise stated.
Abbreviations
LPA, 1-oleoyl-sn-glycero-3-phosphate or lysophosphatidic acid; PI3K, phosphatidylinositol 3-kinase; MAP kinase, mitogen-activated protein kinase; p38 MAP kinase, p38 MAPK; p38-DN, dominant negative mutant for p38 MAPK; JNK, c-Jun terminal kinase; JIP1-JBD, JNK binding domain of JNK-interacting protein-1; ERK, extracellular signal-regulated kinase; ATF-2, activating transcription factor-2; T17NRac1, dominant negative mutant for Rac1; PDGF, platelet-derived growth factor; DGPP 8:0, dioctylglycerol pyrophosphate; G-potein, GTP-binding regulatory protein; PTX, pertussis toxin; BSA, bovine serum albumin; PBS, phosphatebuffered saline; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GAPDH, glyceroaldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein.
Results
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In C6 cells, LPA significantly stimulated chemotaxis as well as chemokinesis (Figure 1a) in a manner dependent on a concentration gradient of LPA. To examine the LPA receptor subtypes responsible for the migration response, we employed rat and human glioma cell lines and compared the effects of several LPA receptor antagonists (Figure 1b). Ki16425 has a preference for LPA1 and LPA3 over LPA2 (Ohta et al., 2003); similarly, VPC12249 also prefers LPA1 and LPA3 but not LPA2 (Heise et al., 2001), whereas DGPP 8:0 shows a preference for only LPA3 (Fischer et al., 2001). In both cell lines, VPC12249 as well as Ki16425, antagonists for LPA1 and LPA3, significantly inhibited the migratory response to LPA in a dose-dependent manner, but DGPP 8:0, an LPA3-specific antagonist, had no effect on cell migration. To get further convincing, we employed siRNAs for LPA1 receptor in CGNH-89 cells (Figure 1c). The siRNA transfection resulted in nearly 50% reduction of LPA1 expression without significant change in S1P2 expression (data not shown). .jpg)
The reduction was accompanied by a remarkable inhibition of LPA-, but not PDGF-induced migration. PTX treatment inhibited the response to LPA but not to PDGF (Figure 1d), suggesting an involvement of the PTX-sensitive G-protein-coupled LPA receptor in (Figure 2b). To further evaluate the role of Rac1, Cdc42, p38MAPK, and JNK in cell migration, we employed adenovirus containing dominant-negative mutants for Rac1 (T17NRac1), Cdc42 (T17NCdc42), and p38MAPK (p38-DN) or JNK-binding domain of JNK-interacting protein-1 (JIP1-JBD). Infection of GNS-3314 cells with the p38MAPK mutant led to the inhibition of migration to an extent similar to that caused by (Figure 2c). Treatment of GNS-3314 cells with T17NCdc42 mutant attenuated the migratory response to LPA (Figure 2d). The response to LPA, but not to PDGF, was attenuated in cells over-expressing JIP1-JBD (Figure 2e). When we added SB203580 to cells infected with T17NRac1 (Figure 2f), the number of migrating cells diminished to a level close to the basal value. Consistent with previous results (Matsumoto et al., 1999), we could not detect the activation of JNK by PDGF (Figure 4a), which would explain the negative effect of JIP1-JBD over-expression upon PDGF-stimulated cell migration.
The reduction was accompanied by a remarkable inhibition of LPA-, but not PDGF-induced migration. PTX treatment inhibited the response to LPA but not to PDGF (Figure 1d), suggesting an involvement of the PTX-sensitive G-protein-coupled LPA receptor in (Figure 2b). To further evaluate the role of Rac1, Cdc42, p38MAPK, and JNK in cell migration, we employed adenovirus containing dominant-negative mutants for Rac1 (T17NRac1), Cdc42 (T17NCdc42), and p38MAPK (p38-DN) or JNK-binding domain of JNK-interacting protein-1 (JIP1-JBD). Infection of GNS-3314 cells with the p38MAPK mutant led to the inhibition of migration to an extent similar to that caused by (Figure 2c). Treatment of GNS-3314 cells with T17NCdc42 mutant attenuated the migratory response to LPA (Figure 2d). The response to LPA, but not to PDGF, was attenuated in cells over-expressing JIP1-JBD (Figure 2e). When we added SB203580 to cells infected with T17NRac1 (Figure 2f), the number of migrating cells diminished to a level close to the basal value. Consistent with previous results (Matsumoto et al., 1999), we could not detect the activation of JNK by PDGF (Figure 4a), which would explain the negative effect of JIP1-JBD over-expression upon PDGF-stimulated cell migration.
LPA as well as PDGF activated p38MAPK within 10 minutes (Figure 3a), and the activation was sustained for 30 min (data not shown).
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The LPA-induced p38MAPK activation was 4-5-fold that of basal level and was attenuated by treatment with wortmannin (Figure 3b). Effect of adenovirus carrying T17NRac1 (Figure 3c) or JIP1-JBD (data not shown) on migration was marginal. On the other hand, LPA-induced p38MAPK activation was almost completely suppressed by T17NCdc42 (Figure 3d), suggesting that Cdc42 could mediate p38MAPK activation. These results suggest that PI3K/Cdc42, but not Rac1, is upstream of p38MAPK activation stimulated by LPA.
In Figure 2b and 2e, we showed that SP600125 and JIP1-JBD over-expression in glioma cells significantly though incompletely inhibited cell migration. We further examined the ability of LPA and other growth factors to phosphorylate the JNK proteins. Only LPA could phosphorylate p54 and p46 JNK in contrast to S1P and PDGF, both of which were unable to activate the JNK isoforms (Figure 4a). LPA-stimulated phosphorylation of JNK was completely inhibited by wortmannin but not by the p38 MAPK inhibitor SB203580 (Figure 4b). .jpg)
Dominant negative Rac1-infected cells showed no activation of JNK response to LPA (Figure 4c). Taken together, these results suggest that LPA may stimulate JNK through LPA1-mediated and PI3K/Rac1-dependent mechanisms.
Dominant negative Rac1-infected cells showed no activation of JNK response to LPA (Figure 4c). Taken together, these results suggest that LPA may stimulate JNK through LPA1-mediated and PI3K/Rac1-dependent mechanisms.
Experimental results related to LPA-induced glioma cell migration are summarized in Figure 5. LPA, utilizing the Gi-protein-coupled LPA1 receptor, stimulates the migration of glioma cells in a PI3K/Cdc42/p38MAPK- and PI3K/Rac1/JNK-dependent manner. Based on our findings, we can conclude that p38MAPK and JNK are equally important for sufficient LPA action on glioma cell motility.
Data from a variety of laboratories have indicated that several
.jpg)
signal transduction
molecules participate in
the regulation of migration.
The small molecular weight G-proteins, i.e., Rho, Rac and Cdc42, regulate the formation of the focal adhesions, lamellipodia and filipodia, respectively (Nobes and Hall, 1999). These Rho-like G-proteins are known principally for their pivotal role in regulating the actin cytoskeleton in cell migration (Burridge and Wennerberg, 2004). PI3K has been thought to be a key molecule in the induction of cell migration through Rac signaling (Chandrasekar et al., 2003; van Leeuwen et al., 2003). MAP kinases, including JNK (Xia et al., 2000; Huang et al., 2003; Kawauchi et al., 2003) and p38MAPK (Rousseau et al., 1997; Cara et al., 2001; Kimura et al., 2003; Pichon et al., 2004), have also been reported to be involved in cell migration. Consistent with these reports, our results indicate that activation of PI3K is required for the induction of glioma cell migration as well as for the activation of p38MAPK and JNK in response to LPA. In Figure 2a and 2b, we defined that wortmannin, SB203580 or SP600125 inhibited LPA-induced cell migration. However, Rac1 signaling is not enough to account for LPA-induced migration via p38MAPK. Two lines of evidence support the role of p38MAPK without Rac1 in the motility of glioma cells caused by LPA. First, LPA-induced p38MAPK activation was not affected by dominant-negative mutant of Rac1 expression (Figure 3c). Second, the motility of cells expressing dominant-negative mutant of Rac1 decreased to the basal level by treatment of cells with SB203580 (Figure 2f).
Discussion
Our results showed that Cdc42 might be essential for migration and p38MAPK activation induced by LPA (Figure 2d and 3d). This hypothesis was supported by the studies showing that Cdc42 could also be important for p38MAPK-dependent T-cell chemotaxis (Shi et al., 2003) and the VEGF-induced formation of stress fibers in endothelial cells (Lamalice et al., 2004). In prostate carcinoma cells (Edlund et al., 2003), Cdc42 as well as RhoA could participate in not only transforming growth factor-β-induced formation of stress fibers, but also in membrane ruffling. However, it is still unclear how MAPKs participate in the regulation of cytoskeleton in cell migration, and it is important further to elucidate molecular mechanisms by which MAPKs regulate cytoskeleton downstream of Rho-like G-proteins.
In glioma cells, though LPA stimulated ERK activation (data not shown), the migration was not affected by treatment of cells with the ERK kinase inhibitors. These data are consistent with the previous observation of Bian et al. (2004) and suggest that the MEK/ERK pathway does not play a significant role in LPA-stimulated glioma cell migration. This information is of interest given the study showing that ERK activation is essential for LPA-induced pancreatic cancer cell migration (Stahle et al., 2003).
In this study, we demonstrated that LPA1 receptor antagonist Ki16425 could be one of the potential agents to be used as a new tool for treatment of brain tumors. The hallmark of malignant astrocytic tumors is their ability to infiltrate surrounding normal brain tissue. This phenotype makes complete surgical resection difficult and focal therapy ineffective. Thus, understanding the mechanisms of astrocytic tumor cell migration in the brain is essential for the development of new strategies to control this malignancy.
Ном зүй
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Chandrasekar N, Mohanam S, Gujrati M, Olivero WC, Dinh DH and Rao JS. (2003). Oncogene, 22, 392-400.
Edlund S, Landstrom M, Heldin CH and Aspenstrom P. (2002). Mol. Biol. Cell., 13, 902-914.
Fischer DJ, Nusser N, Virag T, Yokoyama K, Wang Da, Baker DL, Bautista D, Parrill AL and Tigyi G. (2001). Mol. Pharmacol., 60, 776-784.
Hama K, Aoki J, Fukaya M, Kishi Y, Sakai T, Suzuki R, Ohta H, Yamori T, Watanabe M, Chun J and Arai H. (2004). J. Biol. Chem., 279, 17634-17639.
Heise CE, Santos WL, Schreihofer AM, Heasley BH, Mukhin YV, Macdonald TL and Lynch KR. (2001). Mol. Pharmacol., 60, 1173-1180.
Huang C, Rajfur Z, Borchers C, Schaller MD and Jacobson K. (2003). Nature, 424, 219-223.
Imamura F, Horai T, Mukai M, Shinkai K, Sawada M and Akedo H. (1993). Biochem. Biophys. Res. Commun.,193, 497-503.
Ishiuchi S, Tsuzuki K, Yoshida Y, Yamada N, Hagimura N, Okado H, Miwa A, Kurihara H, Nakazato Y, Tamura M, Sasaki T and Ozawa S. (2002). Nat. Med., 8, 971-978.
Kawauchi T, Chihama K, Nabeshima Y and Hoshino M. (2003). EMBO J., 22, 4190-4201.
Kimura T, Sato K, Malchinkhuu E, Tomura H, Tamama K, Kuwabara A, Murakami M and Okajima F. (2003). Arterioscler. Thromb. Vasc. Biol., 23, 1283-1288.
Kon J, Sato K, Watanabe T, Tomura H, Kuwabara A, Kimura T, Tamama K, Ishizuka T, Murata N, Kanda T, Kobayashi I, Ohta H, Ui M and Okajima F. (1999). J. Biol. Chem., 274, 23940-23947.
Lamalice L, Houle F, Jourdan G, Huot J. (2004). Oncogene., 23, 434-445.
Malchinkhuu E, Sato K, Muraki T, Ishikawa K, Kuwabara A and Okajima F. (2003). Biochem. J., 370, 817-827.
Manning TJ Jr, Parker JC and Sontheimer H. (2000). Cell Motil. Cytoskeleton, 45, 185-199.
Maruyama Y, Nishida M, Sugimoto Y, Tanabe S, Turner JH, Kozasa T, Wada T, Nagao T and Kurose H. (2002). Circ. Res., 91, 961-969.
Matsumoto T, Yokote K, Tamura K, Takemoto M, Ueno H, Saito Y and Mori S. (1999). J. Biol. Chem., 274, 13954-13960.
Mills GB and Moolenaar WH. (2003). Nat. Rev. Cancer, 3, 582-591.
Nobes CD and Hall A. (1999). J. Cell. Biol., 144, 1235-1244.
Ohta H, Sato K, Murata N, Damirin A, Malchinkhuu E, Kon J, Kimura T, Tobo M, Yamazaki Y, Watanabe T, Yagi M, Sato M, Suzuki R, Murooka H, Sakai T, Nishitoba T, Im DS, Nochi H, Tamoto K, Tomura H and Okajima F. (2003). Mol. Pharmacol., 64, 994-1005.
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