From the Departments of Medicine and Biomolecular
Chemistry, University of Wisconsin, Madison, Wisconsin 53706 and
the § Department of Experimental Pathology, Lund University,
221 85 Lund, Sweden
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ABSTRACT |
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Cells lacking the 1 integrin
subunit or expressing
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
1A integrins on lysophosphatidic acid (LPA)-induced migration of fibroblastic cells derived from
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
1A demonstrated enhanced cell migration across filters
coated with gelatin or adhesive proteins in response to LPA, whereas
1-deficient cells lacked LPA-induced cell migratory
ability. Checkerboard analyses indicated that LPA causes both
chemotaxis and chemokinesis of
1-replete cells. Cells
expressing
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
1A integrin is required for cell migration
induced by LPA and that the cytoplasmic domain of ligated
1A interacts with pathways that are common to both
receptor tyrosine kinase and G-protein-linked receptor signaling.
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INTRODUCTION |
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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 1A integrin subunits with
point mutations of the cytoplasmic domain in fibroblasts derived from
1-null stem cells and showed that cells lacking the
1 integrin subunit or expressing
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
1 on cell migration to be analyzed without the
confounding presence of endogenous
1 subunits. In the
present study, we demonstrate that restoration of
1
integrins is stringently required for LPA-induced cell migration of
1-null fibroblasts.
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EXPERIMENTAL PROCEDURES |
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Cells--
The GD25 and GD10 fibroblast lines were established
after differentiation from 1-null stem cells and
immortalization with SV40 large T antigen (37, 38).
1As
with mutations of the cytoplasmic domain were constructed from
pBS
1A encoding full-length mouse
1A
integrin subunit and expressed in GD25 cells as described (36). A point
mutation in the extracellular domain of
1A (D130A) was
introduced as follows. cDNA for
1A was excised with
XbaI and Acc65I from pBS
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
1A. The XbaI-NcoI fragment was
then excised and ligated into XbaI-NcoI-digested
pBS
1A, yielding cDNA encoding full-length
1A polypeptide containing the point mutation. The
plasmid was linearized with XbaI and transfected into GD25
cells by electroporation. Clones stably expressing mutant
1A were obtained with the selection by puromycin and
analyzed for expression of
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
1A at comparable levels with cells expressing wild type
1A.
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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Expression of vzg-1/edg-2 and edg-1--
GD25 and GD10 cells were
differentiated from 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
1A,
and GD25 and GD10 cells expressing wild type
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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Migration through Gelatin-coated Filters--
The effects of
1A integrin on LPA-induced cell migration through
gelatin-coated filters were analyzed. GD25 cells lacking
1A migrated very little when LPA (500 nM)
was added to the lower compartment (Fig.
2A and Table
I). GD25 cells expressing wild type
1A migrated 10-fold more than nontransfected GD25 cells in response to LPA. GD25 cells and GD25 cells expressing
1A migrated equally well in response to PDGF (Table I).
GD10 cells and GD10 cells expressing wild type
1A
behaved similarly to the GD25 counterparts (Table I). Thus,
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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Migration through Filters Coated with Adhesive
Proteins--
Expression of 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
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
1A migrated 5-fold (on vitronectin) or
6-fold (on fibronectin) more than cells lacking
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
1A integrins
in cell-matrix interactions synergizes with the response to LPA.
LPA-induced migration through laminin-1-coated filters required
expression of
1A (Fig. 3A), as is also the
case for EGF- or PDGF-induced migration (36). Migration of cells expressing wild type
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
1A (Fig. 3B). Y783,795F cells migrated
much less, nearly equivalent to GD25 cells lacking
1A.
Y783F and Y795F cells had migratory behavior that was intermediate between Y783,795F cells and cells expressing wild type
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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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 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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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 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).
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DISCUSSION |
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We previously found that 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
1A to
support migration through filters coated with fibronectin or
vitronectin indicates that
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
1A-expressing GD25 cells is blocked by
anti-
6
1 antibody (36).
GD25 and GD10 cells lacking 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.2, 4 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 1 integrins may account for
the need for
1 integrins in migration of the germ
cell-derived cells: binding of
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
1 integrins (43) both signal by multiple
pathways. Our results indicate that the
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
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
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
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
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.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Julie Nowlen for valuable technical help and Magnus Magnusson, Tracee Panetti, and Qinghong Zhang for advice and encouragement.
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FOOTNOTES |
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* 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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REFERENCES |
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