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Vol. 19, Issue 12, 5435-5445, December 2008
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Departments of *Biochemistry and Molecular Biology and Human Genetics, and Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, VA 23298
Submitted March 26, 2008;
Revised August 20, 2008;
Accepted September 25, 2008
Monitoring Editor: Carl-Henrik Heldin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Sano et al., 2002). At least six G protein–coupled receptors (GPCRs) have been identified to mediate a wide range of biological functions of LPA (Hecht et al., 1996; An et al., 1998; Bandoh et al., 1999; Im et al., 2000; Noguchi et al., 2003; Lee et al., 2006; Pasternack et al., 2008). The LPA1/Edg2, LPA2/Edg4 and LPA3/Edg7 receptors are members of the endothelial cell differentiation gene (Edg) family, sharing 50–57% homology in their amino acid sequences (Hecht et al., 1996; An et al., 1998; Bandoh et al., 1999; Im et al., 2000). LPA4/GPR23/p2y9, a member of the purinergic receptor family, and the related LPA5/GPR92 and p2y5 were structurally distant from the Edg LPA1–3 receptors (Noguchi et al., 2003; Lee et al., 2006; Pasternack et al., 2008). LPA receptors couple to multiple G proteins, G12/13, Gi, Gq, and probably Gs that in turn, activate diverse pathways including stimulation of phospholipase C and D, small GTPases, mitogen-activated protein kinases, and phosphatidylinositol 3-kinase (PI3K; van der Bend et al., 1992; Howe and Marshall, 1993; An et al., 1998; Bandoh et al., 1999; Im et al., 2000; Mills and Moolenaar, 2003; van Meeteren and Moolenaar, 2007). Activation of these signaling events downstream of LPA receptors culminates in cytoskeleton remodeling, cell proliferation, survival and migration (Mills and Moolenaar, 2003; van Meeteren and Moolenaar, 2007). In addition to these cell surface GPCRs, LPA has been shown to bind and activate the peroxisome proliferator-activated receptor 
(PPAR) which plays critical roles in controlling fat and energy metabolism (McIntyre et al., 2003).
Only minor phenotypic changes were seen in LPA1 or LPA2 receptor-deficient mice (Contos et al., 2000, 2002). When the lpa1 knockout mice were studied under pathophysiological conditions, however, essential roles for LPA1 in the initiation of neuropathic pain and promotion of pulmonary and renal fibrosis were unraveled (Inoue et al., 2004; Pradere et al., 2007; Tager et al., 2008). Homozygous deletion of the LPA3 receptor leads to a delayed implantation and defective embryo spacing, associated with reduced uterine expression of Cox-2 mRNA (Ye et al., 2005). Although these roles for individual receptors have been identified, more profound effects such as early embryonic lethality have not been observed from single or even double receptor knockouts (Contos et al., 2000, 2002; Ye et al., 2005). These results are in contrast to deletion studies of the LPA-synthesizing enzyme autotoxin (ATX) where homozygous deletion results in embryonic lethality at embryonic day (E)9.5 due to impaired vessel formation in the yolk sac and embryo proper (Tanaka et al., 2006; van Meeteren, 2006). These results suggest involvement of LPA4, LPA5, or other unidentified receptors as effectors of ATX. In the current study, we disrupted the LPA4-encoding gene (lpa4/p2Y9/gpr23) in mice. Similar to deletion of lpa1 or lpa2 (Contos et al., 2000, 2002), LPA4-deficient mice did not show obvious abnormalities in embryonic development, fertility or normal physiology. However, analysis of LPA4-negative cells from lpa4 knockout (KO) mice revealed a novel role for LPA4 in the negative control of cell migration. Stimulation of cell motility is one of the major biological effects of LPA and its producing enzyme ATX (Umezu-Goto et al., 2002; Mills and Moolenaar, 2003; van Meeteren and Moolenaar, 2007). LPA-induced cell migration is mediated mainly by LPA1 although LPA2 or LPA3 may be also capable of evoking the response in various cellular contexts (Shida et al., 2003; Hama et al., 2004;Chan et al., 2007; Chen et al., 2007). However, little is known about how the migratory response to LPA is appropriately controlled in mammalian cells that usually coexpress multiple LPA receptor subtypes. The results of the present study using LPA4-deficient cells and other cellular models identify LPA4 as a suppressor of LPA-mediated cell migration and invasion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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lpa4 Targeting Vector
Similar to the human lpa4, the murine lpa4 gene is located on the X chromosome in the region 11953246-11969022 (GI no. 51772331). Unlike its intronless human locus (O'Dowd et al., 1997), the mouse lpa4 has three exons spanning 
12 kb, whereas the coding sequence is present in exon 3 only (NC_000086
[GenBank]
). The genomic sequences of the mouse lpa4 (C57BL/6) were isolated and PCR amplified from a BAC clone (RP23-343P30; BACPAC Resources, Oakland, CA). A 1.965-kb KpnI/EcoRI fragment containing the 3' part of exon 3 was PCR amplified from the BAC DNA and cloned as the short arm into the pKO Scrambler NTKV-1901 targeting vector that carries both PGK/neo/BGH cassette for positive selection of homologous recombinants with G418 and an MC1-tk cassette for negative selection of nonhomologous recombinants with gangcyclovir (Stratagene, La Jolla, CA). The 5.34-kb EcoRI/BstBI fragment containing exon 2 and the major part of intron 2 was cloned into pBluescript II SK(+) from the BAC DNA and recloned using the NotI and SalI sites into the targeting vector. Thus, a 2.591-kb fragment containing the 3' end of intron 2 and the 5' portion (with the complete coding sequence) of exon 3 was replaced with 1.603 kb of the PGK/neo/BGH cassette, creating the final targeting vector (Figure 1A).
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). The embryos were separated from the uterine wall and amniotic sac and placed in Petri dishes containing small volumes of PBS. The head and heart tissues were removed. The remaining embryo were minced with surgical scissors and razor blade, digested with 0.25% trypsin/EDTA at 37°C for 25 min, and triturated through a Pasteur pipette. After digestion and removal of undigested tissues, the cells were spun briefly, plated into a 10-cm dish, and allowed to grow to subconfluence in DMEM + 10% FBS. The cells were then either frozen as passage 1 or subcultured at a 1:4 ratio for experiments. Immortalized cell lines were established from primary MEFs using standard protocols for generation of 3T3 cells (Jainchill et al., 1969).
Other Cells
The DLD1 colon cancer cell line was kindly provided by Dr. D. Shida (Virginia Commonwealth University) and maintained as described (Hama et al., 2004). The B103 rat neuroblastoma cell line lacking endogenous LPA receptors was obtained from Dr. J. Chun (Scripps Research Institute) and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (Ishii et al., 2000). The rat hepatoma cell line RH7777 was purchased from ATCC (Manassas, VA) and maintained in the same conditions as B103. These cell lines were frozen at early passages and used for <6 wk in continuous culture.
Fluorescence Microscopy
MEF lines in 60-mm dishes were starved overnight and stimulated with LPA (1 µM) for 30 min. The cells were washed with PBS, fixed with 3% formaldehyde/PBS, and permeabilized with 0.2% Triton X-100 before staining for F-actin with Alexa Fluor 488–labeled phalloidin (1:125 dilution; Invitrogen).
Recombinant Retroviruses and Infection of Cells
The Human LPA4 or LPA1 cDNA was inserted between BamHI and XhoI sites upstream of the internal ribosomal entry site of the Moloney murine leukemia retrovirus vector pLZRS-EGFP (a gift of J. Chun, Scripps Research Institute; Ishii et al., 2000). The structure and sequences of the cDNAs in these viral constructs were confirmed by restriction digestion and automatic sequencing. The Bosc23 packaging cell line (ATCC) was transfected with pLZRS-EGFP, pLZRS-EGFP-LPA4, or pLZRS-EGFP-LPA1 using Lipofectamine 2000 as described (Invitrogen; Fang et al., 2004). Approximately 20 h after the beginning of transfection, the cells were fed fresh DMEM + 10% FBS. Culture supernatants containing retrovirus were harvested 48 h later, cleared by centrifugation, and used to infect cells or stored at –80°C.
The LPA1 and LPA4 cDNAs were also cloned into the pLenti-TOPO lentivirus vector (Invitrogen) as an alternate expression system for LPA receptors. The pLenti-TOPO-LPA1, pLenti-TOPO-LPA4 or pLenti-TOPO-LacZ vector was transfected along with packaging plasmids into 293FT cells (Invitrogen) to replicate lentivirus using a protocol similar to that for retrovirus generation in Bosc23 cells. MEF and other cell lines in 35-mm dishes at around 50% confluence were incubated for 16–22 h with 1.5 ml of viral supernatants containing 8 µg/ml polybrene. The infected cells were harvested 72 h after infection. For the cells infected with the retrovirus, EGFP-positive cells were isolated by fluorescence-activated cell sorting (FACS). Lentivirus-infected cells were selected with blasticidin (10 µg/ml) for 10–14 d and pooled colonies were expanded for experiments. When coexpression of LPA1 and LPA4 was desired, the recipient cells were infected with the pLenti-TOPO-LPA1 lentivirus followed by infection with the pLZRS-EGFP-LPA4 retrovirus.
Western Blot
Cells were lysed in SDS sample buffer or in ice-cold Triton X-100 lysis buffer (1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 100 mM NaF, 10 mM Na PPi, and protease inhibitor cocktail). Total cellular proteins were resolved by SDS-PAGE, transferred to Immun-Blot membrane [poly(vinylidene difluoride)]; Bio-Rad, Hercules, CA), and immunoblotted with antibodies following the protocols provided by manufacturers. Immunocomplexes were visualized with an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ), using horseradish peroxidase–conjugated secondary antibodies (Cell Signaling).
Migration and Invasion Assays
Cell migration was measured in Transwell chambers (pore size 8 µm; BD Biosciences, San Jose, CA; cat. no. 354578). Transwells were coated with 10 µg/ml collagen 1 and placed in the lower chamber containing serum-free DMEM supplemented with LPA or EGF. Cells suspended in serum-free DMEM containing 0.1% fatty acid-free BSA were added to the upper chamber at 2.5 x 104 cells/well or 1.0 x 104 cells/well as indicated. Cells were allowed to migrate for 4 or 6 h at 37°C. Nonmigrated cells were removed from the top filter surface with a cotton swab. Migrated cells attached to the underside of the transwells were washed with PBS and stained with crystal violet and counted under a microscope. The invasion of tumor cells were measured using Transwells coated with growth factor–reduced Matrigel Basement Membrane Matrix (pore size 8 µM; BD Biosciences; cat. no. 354483). The assays were performed as migration assays except that the cells were incubated for 20–24 h before termination of the experiments.
Rho and Rac Activation Assays
Activation of Rho and Rac was analyzed by glutathione S-transferase (GST) pulldown assays (Bernard et al., 1999; Ren and Schwartz, 2000). The cells were grown in 10-cm dishes to subconfluence, starved overnight, and stimulated with LPA or vehicle for the indicated periods of time. The cells were lysed in Magnesium-containing lysis buffer (MLB; 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP40, 10% glycerol, 10 mM MgCl2, 1 mM EDTA, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 mM NaF). Clarified lysates were incubated for 1 h at 4°C with GST-Rhotekin-RBD (Rho binding domain of Rhotekin, residues 7-89; Ren and Schwartz, 2000) or GST-PAK-PBD (p21 binding domain of PAK, residues 67-150; Bernard et al., 1999) produced in Escherichia coli and immobilized on glutathione-coupled Sepharose beads. Beads were washed in MLB three times, eluted with SDS sample buffer, and analyzed by Western blotting using monoclonal anti-Rac antibody (BD Biosciences; cat. no. 610650) or rabbit anti-RhoA antibody (Santa Cruz Biotechnology, SC-418).
Statistics
The 2 test of goodness-of-fit was used to determine statistical difference between observed and expected numbers of mice. The null hypothesis is that the number of mice in each category is equal to that predicted by the Mendelian inheritance rule. Numerical results from chemotaxis and invasion experiments were presented as average cell numbers ± SD. The statistical significances of differences were analyzed using Student's t test, where p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 test of goodness-of-fit. In addition, heterozygous females (X+X–), homozygous females (X–X–), and hemizygous males (X–Y) were born at statistically expected Mendelian rule, reflecting that loss of LPA4 does not cause embryonic lethality or impose a detrimental effect on embryonic development (Table 1). The lpa4 KO mice were grossly indistinguishable from their WT or heterozygous littermates in appearance, size, and behavior. They did not show any defects in mating, pregnancy, or litter sizes. There were no gross abnormalities in the internal organs of LPA4-deficient adults (data not shown).
Expression of LPA4 in Murine Tissues and MEFs
To delineate the tissue distribution of LPA4, we examined its mRNA expression in a number of adult tissues including the liver, heart, skeletal muscle, and ovary or testis. As analyzed by RT-PCR, LPA4 mRNA was present in the heart, skeletal muscle, and ovary but was seen weakly in the liver or testis (Figure 2A). The expression of LPA4 mRNA in these tissues was absent from lpa4 KO mice.
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Enhancement of Migratory Response to LPA by lpa4 Deletion
Previous studies using MEFs from lpa1 and lpa2 double knockouts revealed relative contributions of each of these receptors to activation of intracellular signaling cascades by LPA (Contos et al., 2002). Furthermore, analysis of lpa1–/– fibroblasts provided compelling evidence that the LPA1 receptor was the most critical mediator of the migratory response to LPA in these cells (Hama et al., 2004).
Both LPA4-negative and WT MEFs showed morphological characteristics of fibroblasts (Figure 3A). However, LPA4-deficient MEFs exhibited an apparently more flattened shape distinguishable from WT MEFs, which were more heterogeneous in size and shape (Figure 3, A and B). Because morphology and migratory potential of mammalian cells are closely associated and coordinately regulated by Rho GTPases (Nobes and Hall, 1999; Van Leeuwen et al., 2003; Yanagida et al., 2007), we examined whether lack of LPA4 affects migratory response to LPA. We compared WT and LPA4-negative MEFs for LPA-induced cell migration using the transwell chambers. Surprisingly, the LPA4-deficient MEFs exhibited remarkably enhanced migratory response to LPA (Figure 3C) compared with WT cells that only weakly responded to LPA. Consistent with this difference in cell migration, LPA stimulated lamellipodia formation in more LPA4-deficient cells than WT control cells (Figure 3B). Of interest, the LPA4-deficient MEFs were also more motile than WT MEFs in unstimulated conditions (Figure 3C), suggesting that some endogenous LPA may exist or accumulate in the cellular microenvironment during the course of the experiments. Consistent with the greater basal migratory potential associated with loss of LPA4, more lpa4 KO MEFs than WT cells migrated toward EGF (Figure 3C). However, the net increase by EGF over unstimulated conditions was similar between lpa4 KO and WT MEFs. Thus lack of LPA4 specifically sensitized MEFs to LPA-induced cell migration. Furthermore, the enhancement of LPA-mediated cell migration in the absence of LPA4 was consistent among multiple pairs of LPA4 WT and KO MEFs (data not shown) and was observed in both primary MEFs and immortalized MEF lines (Figure 3C).
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), the Gi inhibitor PTX, and the PI3K inhibitor LY294002 (Figure 3D), indicating that the prominent Akt activation was mediated through the LPA1-Gi-PI3K signaling route in LPA4-deficient cells. Moreover, LPA-induced migration of LPA4-deficient cells was suppressed by Ki16425, PTX, or LY294002 but only slightly reduced by an Akt inhibitor (Akti II; Kozikowski et al., 2003), suggesting that an LPA1-Gi-PI3K–dependent pathway mediates the robust migratory response to LPA in LPA4-deficeint cells.
We next examined LPA-induced activation of Rac, a well-documented effector of PI3K, and the G12/13 downstream target Rho (van Leeuwen et al., 2003; van Meeteren and Moolenaar, 2007; Yanagida et al., 2007). Rac and Rho mediate cell migration in a coordinate manner. Rac promotes lamellipodia protrusion and forward movement, whereas RhoA regulates actomyosin-driven cytoskeleton contraction and detachment of the rear of migrating cells (Nobes and Hall, 1999). LPA-induced Rac activation in fibroblasts is generally weak and has been best demonstrated in LPA receptor-overexpressing cells (van Leeuwen et al., 2003; Pilquil et al., 2006). As shown in Figure 3E, Rac activation in response to LPA was barely detectable in WT MEFs, which correlated with the weak migratory response to LPA in WT cells. In contrast, LPA evoked prominent increases in Rac-GTP levels in the lpa4 KO MEFs (Figure 3E).
In contrast to Rac activation, LPA-induced Rho activation was oppositely affected by LPA4 deficiency. As shown in Figure 3E, LPA induced immediate and sustained increases in Rho-GTP levels in both WT and lpa4 KO MEFs as measured by pulldown with GST-Rhotekin Sepharose beads. However, the magnitude of Rho activation induced by LPA was significantly reduced in lpa4 KO cells, suggesting that signaling through LPA4 contributes to the overall Rho activation in LPA-stimulated cells. The result was consistent with the previous observation that in skin fibroblasts of LPA1 and LPA2 double knockouts, LPA remained capable of stimulating partial activation of Rho (Hama et al., 2004), which could be attributed to the input of the LPA4 receptor.
Inhibition of Cell Motility by Reconstitution of LPA4
To verify the critical role for LPA4 in restraining Rac activation and cell migration induced by LPA, we infected an LPA4-negative MEF line with LPA4-lentivirus to reconstitute LPA4 expression (Figure 4A). The LPA-induced migration in control virus-infected and LPA4-reconstituted cells was compared. As shown in Figure 4B, LPA4-reexpressing cells migrated in response to LPA less efficiently than the control virus-infected, LPA4-negative cells, confirming that LPA4 functions as a suppressor of LPA-dependent cell migration. Furthermore, the inhibition of LPA-dependent cell migration by reexpression of LPA4 was accompanied by increased Rho but decreased Rac activation induced by LPA (Figure 4C).
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). Submicromolar concentrations of LPA were sufficient to trigger significant migratory response to LPA in DLD1 cells. DLD1 cells expressed LPA1 and LPA2, but lacked LPA3 or LPA4 mRNA as analyzed by RT-qPCR (Figure 5A). We introduced LPA4 into DLD1 cells via lentivirus-mediated transduction (Figure 5B). LPA-, but not EGF-induced migration in DLD1 cells was dramatically inhibited by ectopic expression of LPA4 (Figure 5C). LPA was also effective in stimulating invasion of DLD1 cells through the Matrigel Basement Membrane Matrix. The invasion induced by LPA was similarly suppressed by expression of LPA4 (Figure 5C). The inhibitory effect of LPA4 on migration and invasion of DLD1cells was not due to interference with endogenous LPA1 or LPA2 receptor expression. In fact, after introduction of LPA4, LPA2 mRNA expression was not altered and LPA1 mRNA level was modestly increased as determined by RT-qPCR (Figure 5B). Collectively, these results establish a general role for LPA4 in negative regulation of motility-stimulating activity of LPA in both normal and neoplastic cells.
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). Although it was recently shown that there was slight expression of LPA4 mRNA in B103 cells, the cell line did not respond to LPA stimulation (Yanagida et al., 2007). Therefore, we infected B103 with retrovirus carrying LPA4 or LPA1, or with control retrovirus. The transduced, GFP-positive cells were isolated by FACS and expanded as stable lines expressing LPA4 or LPA1. In agreement with LPA4 being a functional receptor for LPA, LPA4-expressing B103 cells underwent neurite retraction and cell rounding upon exposure to LPA (Figure 6A), similar to the response seen in LPA1-expressing B103 cells. However, only LPA1-expressing cells showed chemotactic response to LPA, whereas the LPA4-expressing cells and vector control cells did not (Figure 6A). The observation indicates that LPA4 is not capable of driving cell migration in spite of its ability to promote Rho-dependent neurite retraction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; An et al.,1998; Bandoh et al., 1999; Im et al., 2000; Noguchi et al., 2003; Lee et al., 2006; Pasternack et al., 2008). It remains elusive why there are multiple receptor subtypes of two distant families for the small lipid mediator. The LPA4 receptor is linked to Gq, G12/13 and probably Gs, but not Gi, whereas LPA1–3 receptors all couple to Gi (Hecht et al., 1996; An et al., 1998; Bandoh et al., 1999; Im et al., 2000; Noguchi et al., 2003; Lee et al., 2006). The differences in receptor structure and G protein partnership between the Edg LPA receptors and the LPA4 receptor prompted us to study biological functions of LPA4 by developing knockout mice. Although the interim characterization has not revealed apparent abnormalities in LPA4-deficient mice, the truly LPA4-negative MEFs isolated from LPA4-deficient embryos have allowed us to evaluate the role of LPA4 in LPA signal transduction and migratory response to LPA. The results presented in the current work clearly demonstrate that knockout of LPA4 sensitizes the cells to LPA-induced migration. In WT cells, the presence of LPA4 may thus serve as a barrier preventing overreaction to LPA. The specific role of LPA4 in limiting LPA-induced cell motility was confirmed by LPA4 reconstitution experiment showing reexpression of LPA4 desensitizes KO MEFs to LPA-induced migration. Moreover, heterologous expression of LPA4 in rat neuroblastoma and human colon cancer cell lines suppressed LPA-triggered cell migration and invasion, antagonizing the motility-promoting effect of the LPA1 receptor. It will be of interest to examine in the future whether LPA4 expression was deregulated in human tumors contributing to the invasive and metastatic potential of cancer cells.
The discovery of negative regulation of LPA-dependent migration by LPA4 provides novel insights into the cross-talk among the multiple LPA receptors that are frequently coexpressed in mammalian cells. We have recently described that LPA induced cyclooxygenase-2 expression in ovarian cancer cells through a mechanism requiring LPA1, LPA2, and LPA5 receptors (Oyesanya et al., 2008). Integration of signals from multiple LPA receptors may be necessary for the optimal activation of LPA-mediated responses. There are many other examples of functional complementation or redundancy among LPA receptors (Ishii et al., 2000; Fang et al., 2004; Lin et al., 2007; Oyesanya et al., 2008). However, the functional antagonism between LPA receptors has rarely been demonstrated (Ishii et al., 2000). The opposing effects of LPA1 and LPA4 receptors on LPA-induced cell migration and invasion described in the current work represent such an example of functional inhibition among LPA receptors. This inhibitory cross-talk is likely critical to ensure physiologically appropriate responses to LPA. It is yet to be determined whether LPA4 also negatively regulate some other biological functions of LPA. In B103 cells, LPA4 mimics LPA1 in promoting neurite retraction and cell rounding, suggesting that LPA4 is indeed a functional LPA receptor that mediates certain cellular effects of LPA while inhibiting others, depending on signaling pathways involved.
Although the molecular mechanism for LPA4 down-regulation of cell motility remains to be fully elucidated, our results provide some clues to the potential players in the process. As discussed above, the cell motility is tightly controlled by activities of Rho and Rac in a coordinate manner (Nobes and Hall, 1999). The balance of Rho and Rac activities are critical determinants of cell movement (Nobes and Hall, 1999). LPA4 contributes to the total Rho activation in MEFs likely through G12/13 (Noguchi et al., 2003). Our results demonstrate that loss of LPA4 decreased LPA-stimulated Rho activation as anticipated but simultaneously enhanced LPA-induced Rac activation. The observation suggests that LPA4 may exert its inhibitory effect on cell migration through increasing the relative ratios of active Rho versus active Rac in LPA-treated cells. LPA4 seems to interfere with activation of Rac by inhibiting PI3K. This is supported by the fact that LPA4 expression attenuates other PI3K effectors such as Akt. In addition to this possibility, Rac activation could be inhibited by excessive Rho activity in WT cells as has been proposed in other cell systems (Arikawa et al., 2003; Sugimoto et al., 2003, 2006). It is also possible that LPA4 directly desensitizes the LPA1 receptor that is known to stimulate Rac activation through Gi (van Leeuwen et al., 2003). The role of LPA4 in negative control of cell motility is reminiscent of S1P2, one of S1P receptors, that has been shown to inhibit cell migration (Arikawa et al., 2003; Sugimoto et al., 2003). Similar to LPA4, S1P2 is coupled to G12/13 and Gq, but not Gi (Arikawa et al., 2003; Sugimoto et al., 2003). Several studies suggest the negative effect of S1P2 on cell motility is attributed to G12/13-mediated Rho activation (Arikawa et al., 2003; Sugimoto et al., 2003). Activation of Rho in the absence of appropriate Rac input seems to be sufficient to confer inhibition of cell motility (Arikawa et al., 2003; Sugimoto et al., 2003, 2006).
It is not surprising that the LPA4-deficient mice do not show obvious phenotypic abnormalities at least at early ages. LPA1 and LPA2 knockouts are also dispensable from normal development and physiology (Contos et al., 2000, 2002). However, these knockout animals have proved to be valued models to link LPA signaling to pathophysiological processes (Inoue et al., 2004; Pradere et al., 2007; Tager et al., 2008). The backup and/or redundant receptor subtypes of LPA may suffice to compensate for the loss of individual LPA receptors in vivo. Alternatively, LPA, as one of phospholipids present in the circulation and tissues, may not be the only or rate-limiting mediator physiologically required in vivo. Instead, LPA signaling may be more critical in pathophysiological settings when levels of the lipid mediator are locally and temporally altered. Recent studies of LPA1-deficeint mice demonstrated involvement of LPA1 in abnormal wound healing and fibrosis formation (Pradere et al., 2007; Tager et al., 2008), supporting a major role for this LPA receptor subtype in chemotactic recruitment of fibroblasts to the site of wound. In light of the opposing effects of LPA4 and LPA1 on migratory response of various cell types to LPA, it is interesting to study roles of LPA4 in wound healing and other pathophysiological conditions using LPA4-deficient mice developed in the current study.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Xianjun Fang (xfang{at}vcu.edu).
Abbreviations used: LPA, lysophosphatidic acid; GPCR, G protein–coupled receptor; KO, knockout; MEF, mouse embryonic fibroblast.
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