Restoration of beta 1A Integrins is Required for Lysophosphatidic Acid-induced Migration of beta 1-null Mouse Fibroblastic Cells*

Takao SakaiDagger , Olivier PeyruchaudDagger , Reinhard Fässler§, and Deane F. MosherDagger

From the Dagger  Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706 and the § Department of Experimental Pathology, Lund University, 221 85 Lund, Sweden

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Cells lacking the beta 1 integrin subunit or expressing beta 1A with certain cytoplasmic mutations have poor directed cell migration to platelet-derived growth factor or epidermal growth factor, ligands of receptor tyrosine kinases (Sakai, T., Zhang, Q., Fässler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538). We investigated the effect of expression of beta 1A integrins on lysophosphatidic acid (LPA)-induced migration of fibroblastic cells derived from beta 1-null mouse embryonic stem cells. These cells expressed edg-2, a G-protein-linked receptor for LPA, as well as the related edg-1 receptor. Cells expressing wild type beta 1A demonstrated enhanced cell migration across filters coated with gelatin or adhesive proteins in response to LPA, whereas beta 1-deficient cells lacked LPA-induced cell migratory ability. Checkerboard analyses indicated that LPA causes both chemotaxis and chemokinesis of beta 1-replete cells. Cells expressing beta 1A with mutations of prolines or tyrosines in conserved cytoplasmic NPXY motifs, threonine in the inter-motif sequence, or a critical aspartic acid in the extracellular domain had low migratory responses to LPA. These findings indicate that active beta 1A integrin is required for cell migration induced by LPA and that the cytoplasmic domain of ligated beta 1A interacts with pathways that are common to both receptor tyrosine kinase and G-protein-linked receptor signaling.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Directed cell migration in a concentration gradient (chemotaxis) is important for a large number of physiological and pathological processes, including development, immunity, wound healing, and cancer metastasis (1-4). Chemotaxis involves the sensing of the gradient of chemoattractant, reorganization of the actin cytoskeleton, and subsequent movement toward the chemoattractant. Cell movement requires the fine control of cellular association with and release from the extracellular matrix (5, 6). Integrins are transmembrane heterodimeric cell adhesion receptors that mediate organization of focal contacts, actin-containing cytoskeleton, and extracellular matrix and may also contribute to cell migration by participating in signal transduction cascades (7-14). It has been suggested that integrin function involves interaction with adhesive ligands ("outside-in" signaling) and cellular control of binding avidity ("inside-out" signaling) (9, 15, 16). In addition, cell migration may be regulated in part by the cycling of integrins between cytoplasmic compartments and the cell surface (17, 18).

Lysophosphatidic acid (LPA)1 is a product of activated platelets and cells and has diverse actions on cells (19). LPA is the serum enhancement factor of fibronectin matrix assembly; enhancement of assembly closely correlates with LPA-induced actin stress fiber formation and cell contraction (20-22). LPA is a mitogen for a number of cells, including endothelial cells (23, 24). LPA induces in vitro invasion across host cell monolayers by several types of tumor cells (25, 26). LPA also stimulates random, nondirectional migration (chemokinesis) of Rat1 fibroblasts (27). Recently, the specific LPA receptor, ventricular zone gene-1 (vzg-1) (28), also known as endothelial differentiation gene-2 (edg-2) (29), has been identified in mouse and human. Related proteins, Edg-1 (30, 31) and Edg-3 (32, 33), have been identified as receptors for sphingosine 1-phosphate. LPA signaling is mediated by the Raf-ERK pathway via Ras activation and through the small GTP-binding protein Rho, leading to activation of mitogen-activated protein kinase (19, 29, 34). Expression of a dominant negative Ras mutant inhibits migration of NIH(M17) cells in response to LPA as well as to other chemoattractants such as platelet-derived growth factor (PDGF) (35).

We recently expressed a set of beta 1A integrin subunits with point mutations of the cytoplasmic domain in fibroblasts derived from beta 1-null stem cells and showed that cells lacking the beta 1 integrin subunit or expressing beta 1A with certain cytoplasmic mutations had impaired ability to migrate toward PDGF or epidermal growth factor (EGF), ligands of receptor tyrosine kinases (36). This system allows the effects of wild type and mutated beta 1 on cell migration to be analyzed without the confounding presence of endogenous beta 1 subunits. In the present study, we demonstrate that restoration of beta 1 integrins is stringently required for LPA-induced cell migration of beta 1-null fibroblasts.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cells-- The GD25 and GD10 fibroblast lines were established after differentiation from beta 1-null stem cells and immortalization with SV40 large T antigen (37, 38). beta 1As with mutations of the cytoplasmic domain were constructed from pBSbeta 1A encoding full-length mouse beta 1A integrin subunit and expressed in GD25 cells as described (36). A point mutation in the extracellular domain of beta 1A (D130A) was introduced as follows. cDNA for beta 1A was excised with XbaI and Acc65I from pBSbeta 1A (38) and cloned into pGEM-7Zf. The mutant was generated by oligonucleotide-primed DNA synthesis using a mutagenesis kit (Pharmacia Biotech, Uppsala, Sweden). The region spanning the HindIII and ClaI sites was analyzed by DNA sequence analysis, and the correctly mutagenized HindIII-ClaI fragment was isolated and ligated into HindIII-ClaI-digested pGEM-7Zf containing the original XbaI-BglII fragment of beta 1A. The XbaI-NcoI fragment was then excised and ligated into XbaI-NcoI-digested pBSbeta 1A, yielding cDNA encoding full-length beta 1A polypeptide containing the point mutation. The plasmid was linearized with XbaI and transfected into GD25 cells by electroporation. Clones stably expressing mutant beta 1A were obtained with the selection by puromycin and analyzed for expression of beta 1A by flow cytometry (36). The population of higher expressing cells in several clones was selected by fluorescence-detected cell sorting and expanded. By flow cytometry, the cells selected for study expressed mutant cell-surface beta 1A at comparable levels with cells expressing wild type beta 1A.

Wild type beta 1A was expressed in GD10 cells by transfecting with pBSbeta 1A using LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions.

Cell Migration-- Cell migration assays were performed in modified Boyden chambers containing Nucleopore polycarbonate membranes (5-µm pore size; Costar Corp., Cambridge, MA) as described previously (36). The filters were soaked overnight in a 10 µg/ml solution of vitronectin, fibronectin, or laminin-1 or 100 µg/ml gelatin, briefly rinsed with phosphate-buffered saline containing 0.2% (w/v) bovine serum albumin (BSA), air-dried, and then placed in the chamber. PDGF from porcine platelets (R&D systems, Minneapolis, MN) or 1-oleoyl-LPA (Avanti Polar Lipids, Birmingham, AL) in Dulbecco's modified Eagle's medium containing 0.2% fatty acid-free BSA was added to the lower (and/or upper) compartment of the chambers. Cells suspended in Dulbecco's modified Eagle's medium containing 0.2% fatty acid-free BSA were introduced into the upper compartment. The chambers were then incubated for 6 h at 37 °C. The filters were fixed and stained, and the cells that had migrated to the lower surface were counted at × 400 magnification. In each experiment, two areas from each of two wells were counted. Values are the mean ± S.D. of cells per 0.16-mm2 field.

RNA Isolation and RT-PCR-- Total RNA was isolated using total RNA isolation system (Promega Corp., Madison, WI). RT-PCR was performed on Perkin-Elmer 480 thermal cycler (Perkin-Elmer Corp.) using the Access RT-PCR system (Promega Corp.) as described elsewhere.2 Controls for RT-PCR were studied by omitting the avian myeloblastosis virus reverse transcriptase from the reaction mixture. The specific primers used were based upon human cDNA sequence: 5'-AATCGAGAGGCACATTACGG-3' (nucleotide 429-448) and 5'-TGTGGACAGCACACGTCTAG-3' (nucleotide 857-838) for edg-2 (28, 29); and 5'-ACGTCAACTATGATATCATCGTCCG-3' (nucleotide 306-330) and 5'-CATTTTCAGCATTGTGATATAGCGC-3' (nucleotide 697-673) for edg-1 (30). Molecular size standards (1-kilobase pair DNA ladder) were from Life Technologies, Inc.

Immunoprecipitation and Immunoblotting-- Immunoprecipitation analysis was performed as described previously elsewhere (39), with a slight modification. Briefly, cells grown to 80-90% confluence were starved in medium without serum for 16 h and then stimulated with agonists for 5 min or left unstimulated. The cells were then lysed on ice in buffer containing 1% (v/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, 100 mM sodium fluoride,1 mM sodium orthovanadate, 0.5 mM sodium molybdate, 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 0.1 µg/ml pepstatin A, 0.4 mM pefabloc SC, and 20 mM Tris-HCl, pH 7.4 and centrifuged for 15 min at 12,000 r.p.m. The same amounts of protein from different experimental samples were used for analyses, as determined using a BCA protein assay (Pierce). The supernatants were precleaned with protein A-Sepharose 4 fast-flow (Pharmacia LKB Bio-tech, Uppsala, Sweden) and subsequently incubated with antibody. The complexes were precipitated with protein A-Sepharose 4 fast-flow, and the proteins were eluted from the resins by incubation with SDS-sample buffer. Samples were then subjected to SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes.

For immunoblotting, the blots were probed with the primary antibody, then with a horseradish peroxidase-conjugated secondary antibody (Organon Teknika Corp., Westchester, PA). Immunoreactive bands were developed using the enhanced chemiluminescence (ECL) substrate system (NEN Life Science Products). Rabbit polyclonal antibodies that recognized mouse EGF and PDGF receptors were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody against phosphotyrosine (mAb 4G10) was from Upstate Biotechnology Inc. (Lake Placid, NY). Human recombinant EGF was from Upstate Biotechnology Inc.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Expression of vzg-1/edg-2 and edg-1-- GD25 and GD10 cells were differentiated from beta 1-null embryonic stem cells and are of uncertain lineage. We used RT-PCR to check the expression of vzg-1/edg-2 and edg-1, which have been identified as receptors for LPA and sphingosine 1-phosphate, respectively (28-31). GD25 and GD10 cells lacking beta 1A, and GD25 and GD10 cells expressing wild type beta 1A, were found to express vzg-1/edg-2 LPA-specific receptor (Fig. 1). The related edg-1 receptor was also expressed by these cells (Fig. 1).


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Fig. 1.   Expression of mRNAs for the vzg-1(edg-2) LPA receptor and the related sphingosine 1-phosphate receptor edg-1 detected by RT-PCR. Amplification products were resolved on 2% agarose gel. Lane 2, control (no addition of the avian myeloblastosis virus reverse transcriptase); lane 3, beta 1-deficient GD25 cells; lane 4, GD25 cells expressing wild type beta 1A; lane 5, beta 1-deficient GD10 cells; lane 6, GD10 cells expressing wild type beta 1A. Lane 1 contained the size standards (1-kilobase pair DNA ladder) with positions (base pairs (bp)) indicated to the left. The specific amplification products for edg-1 and vzg-1(edg-2) are 391 and 409 base pairs, respectively.

Migration through Gelatin-coated Filters-- The effects of beta 1A integrin on LPA-induced cell migration through gelatin-coated filters were analyzed. GD25 cells lacking beta 1A migrated very little when LPA (500 nM) was added to the lower compartment (Fig. 2A and Table I). GD25 cells expressing wild type beta 1A migrated 10-fold more than nontransfected GD25 cells in response to LPA. GD25 cells and GD25 cells expressing beta 1A migrated equally well in response to PDGF (Table I). GD10 cells and GD10 cells expressing wild type beta 1A behaved similarly to the GD25 counterparts (Table I). Thus, beta 1-null fibroblasts demonstrate a profound defect in migration through gelatin-coated filters in response to LPA that is not because of an intrinsic inability to migrate through the gelatin-coated pores.


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Fig. 2.   Cell migration through gelatin-coated filters in response to LPA of beta 1-deficient GD25 cells and GD25 cells expressing wild type beta 1A (A) and GD25 cells expressing beta 1A with mutations (B). LPA (500 nM) was in the lower chamber. Bars represent the means of cell number/0.16-mm2 field. Error bars indicate ± S.D. of quadruplicate determinations. GD25, beta 1-deficient cells; beta 1GD25, GD25 cells expressing wild type beta 1A; other cells are designated by mutation(s). In the absence of LPA, less than 10 cells/0.16-mm2 moved across filters coated with gelatin.

                              
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Table I
Effect of LPA or PDGF on cell migration through gelatin-coated filters
Cells and chemotactic agents were added to the upper and lower compartments, respectively. Numbers represent the mean ± S.D. of at least four determinations.

Cells expressing mutant beta 1As were tested for an ability to migrate in response to LPA. The mutations in the cytoplasmic domain fell into two groups, active (D759A; Y783F; Y795F; Y783,795F) and inactive (T788P; P781,793A), based upon the reactivity of expressing cells with anti-beta 1 (9EG7) antibody, alpha 6beta 1-dependent adhesion to laminin-1, and ability to support fibronectin assembly (36). T788P or P781,793A cells, which express inactive beta 1As, migrated much less in response to LPA through gelatin-coated filters than cells expressing wild type beta 1A (Fig. 2B). D130A cells, carrying a point mutation of extracellular domain of beta 1A known to impair ligand binding (40), also showed less ability to migrate. In contrast, cells expressing beta 1A with an activating D759A mutation of conserved aspartate in the membrane-proximal region of the cytoplasmic domain migrated 9-fold more than GD25 cells lacking beta 1A and equivalently to the cells expressing wild type beta 1A. Although mutation of one or both of the tyrosines in the two NPXY motifs (Tyr783, Tyr795) to phenylalanines results in active beta 1A as assessed by adhesion to laminin-1 and reactivity with the 9EG7 antibody (36), Y795F or Y783,785F cells migrated much less than cells expressing wild type beta 1A and no more than GD25 cells lacking beta 1A (Fig. 2B). Y783F cells showed intermediate migratory behavior between GD25 cells lacking beta 1A and expressing wild type beta 1A.

Migration through Filters Coated with Adhesive Proteins-- Expression of beta 1A in GD25 cells enhances haptotactic migration through filters coated with vitronectin, fibronectin, or laminin-1 as well as migration through such filters in response to PDGF or EGF (36). Migration of cells expressing wild type beta 1A through vitronectin-, fibronectin-, or laminin-1-coated filters in response to LPA was greater than migration through gelatin-coated filters (compare Table I and Fig. 3A). In response to LPA, GD25 cells expressing beta 1A migrated 5-fold (on vitronectin) or 6-fold (on fibronectin) more than cells lacking beta 1A and 10-fold (on vitronectin) or 3.5-fold (on fibronectin) more than the haptotactic responses in the absence of LPA (Fig. 3A). These results suggest that the participation of beta 1A integrins in cell-matrix interactions synergizes with the response to LPA. LPA-induced migration through laminin-1-coated filters required expression of beta 1A (Fig. 3A), as is also the case for EGF- or PDGF-induced migration (36). Migration of cells expressing wild type beta 1A was half-maximal in response to approximately 50 nM LPA through all the adhesive substrates (Fig. 3A). Migration of cells expressing the cytoplasmic D759A mutation was somewhat less than that of cells expressing wild type beta 1A (Fig. 3B). Y783,795F cells migrated much less, nearly equivalent to GD25 cells lacking beta 1A. Y783F and Y795F cells had migratory behavior that was intermediate between Y783,795F cells and cells expressing wild type beta 1A (Fig. 3B). D130A cells also had an impaired migratory response through filters coated with fibronectin, vitronectin, or laminin-1 (not shown).


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Fig. 3.   Cell migration through vitronectin-, fibronectin-, or laminin-coated filters in response to LPA. Migration was quantified for beta 1A-deficient GD25 cells (open squares) and cells expressing wild type beta 1A (filled squares) (A) and GD25 cells expressing D759A (open circles), double Y783,795F (closed circles), Y783F (open triangles), and Y795F (asterisks) mutants (B). LPA in the concentrations of 50, 150, 500, and 1,500 nM was added in the lower chamber, and the dose dependence was analyzed. Each symbol represents the mean of cell number per 0.16-mm2 field. Error bars indicate ± S.D. of quadruplicate determinations.

Checkerboard Analysis-- Previous studies suggested that LPA causes random, nondirectional migration (chemokinesis) rather than chemotaxis of Rat1 fibroblasts (27). Checkerboard analysis in which different concentrations of chemoattractant were added to the upper and lower chamber of the apparatus was therefore carried out to characterize the effect of LPA on cell migration by derivatives of GD25 cells (Fig. 4). Such an analysis differentiates directed migration across the filter in response to the gradient of chemoattractant (chemotaxis) from increased random motility because of the presence of the chemoattractant per se (chemokinesis). Cells expressing wild type beta 1A, the D759A mutant, or the Y783F mutant displayed directional motility in a LPA gradient, although there were substantial increases in random motility when LPA was present at equal concentrations in both chambers: the chemotactic responses were 1.2-1.5-fold greater than the chemokinetic responses. Migration of Y795F cells was greater when LPA was present in both chambers than when there was a gradient of LPA, indicating that the main effect was a chemokinetic response. Migration of Y783,795F cells or nontransfected GD25 cells was too low to classify responses as chemotaxis or chemokinesis.


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Fig. 4.   Checkerboard analyses of LPA-induced cell migration of beta 1-deficient GD25 cells and GD25 cells expressing wild type or mutant beta 1A. Cell migration assays through vitronectin (A)- or fibronectin (B)-coated filters were performed as described under "Experimental Procedures" with variable concentrations of LPA being added to the upper as well as the lower chamber. Results are expressed as the mean of duplicate experiments with duplicate determinations in each experiment (n = 4). S.D. values were <10% of the means in all cases. GD25, beta 1-deficient cells; beta 1GD25, GD25 cells expressing wild type beta 1A; other cells are designated by mutation(s).

Relation to EGF and PDGF Signaling-- Signaling pathways initiated by high concentration of LPA (10-25 µM) are known to "cross-talk" with EGF signaling pathways to cause phosphorylation of the EGF receptors (41, 42). Because GD25 cells are also deficient in migration in response to EGF or PDGF (36), we checked the presence of EGF and PDGF receptors in GD25 cells and GD25 cells expressing beta 1A. Lysates of both cells contained comparable amounts of both receptors when analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting (not shown). As assessed by anti-phosphotyrosine immunoblotting of immunoprecipitated receptors, both receptors in both cells were activated appropriately by concentrations of 3 ng/ml EGF or PDGF but not by 500 nM LPA that caused migration (not shown).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We previously found that beta 1A with an intact cytoplasmic tail, including intact NPXY motifs, is important for optimal chemotaxis of GD25 fibroblasts through filters coated with vitronectin, fibronectin, or laminin-1 in response to PDGF or EGF (36). The purposes of the present studies were to learn if GD25 cells or its derivatives migrate in response to LPA and then to compare and contrast LPA-induced migration to that induced by PDGF or EGF. The fact that the extracellular D130A mutation ablated ability of beta 1A to support migration through filters coated with fibronectin or vitronectin indicates that beta 1 integrins must be able to interact with extracellular ligands. However, which integrins are responsible for migration on the various coated filters is open to question, with the exception of laminin-1, to which adhesion of beta 1A-expressing GD25 cells is blocked by anti-alpha 6beta 1 antibody (36).

GD25 and GD10 cells lacking beta 1A expressed the vzg-1/edg-2 LPA receptor and also the related edg-1 receptor. The generality of the findings for better defined cell types remains to be elucidated. Bovine heart or aortic endothelial cells expressing edg-1 respond to LPA with enhanced cell migration.3 In contrast, human MG63 osteosarcoma cells expressing vzg-1/edg-2 respond to LPA with inhibited migration.24 Thus, the germ cell-derived lines should be considered as models of cellular behavior rather than of any one cell type.

At least two functions of beta 1 integrins may account for the need for beta 1 integrins in migration of the germ cell-derived cells: binding of beta 1 integrins by insolubilized ligands to allow haptotaxis and synergism between integrin-initiated and LPA-initiated signaling pathways to drive chemotaxis and chemokinesis. Although high concentrations of LPA (10-25 µM) have been shown to induce tyrosine phosphorylation of EGF receptors in a variety of cell types (41, 42), we could not show such cross-talk for EGF or PDGF receptors of GD25 cells with a concentration of LPA (500 nM) that induced optimal chemotaxis, suggesting that the initial steps in LPA-induced signaling are independent of tyrosine kinase receptors. LPA (19) and ligated beta 1 integrins (43) both signal by multiple pathways. Our results indicate that the beta 1A integrin must not only be active but have intact NPXY motifs in the cytoplasmic domain to support LPA-induced migration. The latter requirement differentiates the effect of LPA on migration from the effect of LPA on fibronectin matrix assembly, which is up-regulated by LPA in GD25 cells expressing beta 1A with conservative Tyr to Phe substitutions (36). We previously hypothesized that conversion of both tyrosines between nonphosphorylated and phosphorylated states was critical for directed movement, based upon the fact that the Y783F and Y795F mutations caused loss of migration ability to PDGF or EGF (36). One scenario is that the conversion between phosphorylated and dephosphorylated states is important for cycling of integrins to facilitate migration in response to a variety of agonist-receptor systems. Phosphorylated integrins may initiate a pathway leading to changes in F-actin containing cytoskeleton and thus to generation of the cellular polarity required for directional movement. In addition, NPXY motifs may regulate cycling of beta 1 integrins. Upon phosphorylation of the NPXY motifs, the integrin may lose its affinity for both extracellular ligand and cytoplasmic components of the focal contacts and exit the focal contact. Dephosphorylation of the motifs would allow the integrin to participate in a new round of ligation and focal contact formation. The phosphorylation-dephosphorylation cycle may also allow polarization of receptors for chemotactic agents. An alternative scenario is that the enzymatic cascade initiated by ligation of integrins and phosphorylation of beta 1A is linked with a common pathway initiated by ligated LPA receptor or tyrosine kinase receptors. Expression of a dominant negative Ras inhibits migration in response to both LPA and PDGF but not to soluble fibronectin (35). Ligated LPA receptors (19), ligated beta 1 integrins (43), and ligated EGF or PDGF receptors (41, 42) all can work through Ras. Thus, downstream targets of Ras are the most likely common pathway.

    ACKNOWLEDGEMENTS

We gratefully acknowledge Julie Nowlen for valuable technical help and Magnus Magnusson, Tracee Panetti, and Qinghong Zhang for advice and encouragement.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL21644 and HL54462, fellowship funds from the Cell Science Research Foundation and Marion Merrill Dow (to T. S.), a postdoctoral grant from Sanofi Association for Thrombosis Research (to O. P.), and a grant from the Swedish National Research Foundation.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: Depts. of Medicine and Biomolecular Chemistry, University of Wisconsin-Madison, 4285B, Medical Science Center, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-1576; Fax: 608-263-4969; E-mail: dfmosher{at}facstaff.wisc.edu.

1 The abbreviations used are: LPA, lysophosphatidic acid; edg, endothelial differentiation gene; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; vzg-1, ventricular zone gene-1; BSA, bovine serum albumin; RT-PCR, reverse transcription-polymerase chain reaction.

2 O. Peyruchaud and D. F. Mosher, submitted for publication.

3 T. Panetti, O. Peyruchaud, and D. F. Mosher, manuscript in preparation.

4 M. K. Magnusson and D. F. Mosher, submitted for publication.

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Discussion
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