Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2984-2990
Sphingosine 1-Phosphate Stimulates Fibronectin Matrix
Assembly Through a Rho-Dependent Signal Pathway
By
Qinghong Zhang,
Olivier Peyruchaud,
Kelly J. French,
Magnus K. Magnusson, and
Deane F. Mosher
From the Departments of Medicine and Biomolecular Chemistry,
University of Wisconsin-Madison, Madison, WI.
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ABSTRACT |
Fibronectin matrix assembly is a cell-dependent process mediated by
cell surface binding sites for the 70-kD N-terminal portion of
fibronectin. We have shown that Rho-dependent cytoskeleton reorganization induced by lysophosphatidic acid (LPA) or the
microtubule-disrupting agent nocodazole increases fibronectin binding
(Zhang et al, Mol Biol Cell 8:1415, 1997).
Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in
mitogenesis and cytoskeletal remodelling. Both LPA and S1P are present
in increased amounts in serum as compared with plasma as a result of
platelet activation. Addition of S1P to human osteosarcoma MG63 cells
or human foreskin fibroblasts increased cell-mediated binding and
assembly of fibronectin. MG63 cells expressed the Edg-2 and Edg-4
G-protein-coupled receptors for bioactive lipids, whereas foreskin
fibroblasts expressed Edg-2, Edg-3, and Edg-4. The stimulatory effect
of S1P on the binding of fibronectin or the N-terminal 70-kD fragment
of fibronectin was dynamic and due to increases in both the number and
affinity of binding sites. The stimulation of 70-kD fragment binding by nanomolar S1P, like stimulation of binding by LPA or nocodazole, was
blocked by inactivation of Rho with C3 exotoxin but not by pertussis
toxin-mediated inactivation of Gi. These results indicate a common
signal pathway leading to control of cellular fibronectin matrix
assembly by bioactive lipids generated during blood coagulation.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SPHINGOSINE 1-PHOSPHATE (S1P), a
bioactive phospholipid, is present in plasma and released from
platelets after activation.1,2 S1P is the
product of phosphorylation of sphingosine by sphingosine kinase3 and is subsequently cleaved by S1P lyase to a fatty aldehyde and ethanolamine phosphate4 or dephosphorylated by a sphingosine phosphatase.5 S1P, in addition to being an
intermediary catabolite, is a bioactive lipid with interesting
functions, including stimulation of mitogenesis of
fibroblasts,6-8 inhibition of tumor cell
motility,9 platelet activation,10 and neurite
retraction.11
Initial studies focused on S1P as an intracellular second messenger
mediating the proliferative effects of platelet-derived growth factor
(PDGF) and evoking release of Ca2+ from
internal stores by an inositol-phosphate (IP3)-independent mechanism.12-14 Recent studies show that some signaling
responses to S1P are through cell surface G-protein-coupled receptors
(GPCR), of which several candidates have been cloned.15,16
A similar dependence on G proteins has been observed for response to
lysophophatidic acid (LPA),17 a lysoglycerophospholipid
that is structurally similar to S1P. Several GPCRs for LPA have also
recently been cloned.15,18-20 The receptors for S1P and LPA
are called endothelial cell differentiation genes (Edg) in recognition
of the initial identification of Edg-1 in endothelial
cells.21
Fibronectin matrix assembly is a cell-dependent process mediated by
cell surface binding sites for the 70-kD N-terminal portion of
fibronectin. LPA stimulates binding and assembly of fibronectin by a
variety of fibroblastic cells.22,23 Enhanced binding of the
70-kD N-terminal fragment of fibronectin induced by LPA treatment correlates with changes in cell shape and actin-containing
cytoskeleton.23-25 Actin stress fiber formation and cell
contraction are mediated by the small Ras-type GTPase, Rho, and
represent a rapid, labile way that cells can modulate fibronectin
matrix assembly.24
Like LPA, S1P causes stress fiber formation and cell
contraction.11,26 Both LPA and S1P are present in serum as
a result of platelet activation.1,27 Therefore, we tested
whether S1P stimulates cell surface binding and assembly of
fibronectin. S1P (0.1 nmol/L to 10 µmol/L) stimulated 70-kD fragment
binding to MG63 osteosarcoma cells and foreskin fibroblasts. The Edg
receptors common to MG63 osteosarcoma cells and foreskin fibroblasts
were Edg-2 and Edg-4. The stimulatory activity of S1P was inhibited by
C3 exotoxin-mediated inactivation of Rho. These results indicate that a
common signal pathway initiated by bioactive lipids generated during
blood coagulation controls assembly of fibronectin by cells.
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MATERIALS AND METHODS |
Materials.
LPA was from Avanti Polar Lipids (Birmingham, AL). S1P was obtained
from LC Laboratories (Woburn, MA). Sphingosylphosphorylcholine, sphingomyelin, and fatty acid-free bovine albumin were from Sigma Chemical Co (St Louis, MO). Stock solutions of 2 mmol/L S1P,
sphingosylphosphorylcholine, or sphingomyelin were made in methanol.
Recombinant C3 exotransferase28 was prepared by Dr Tracee
Panetti of our laboratory. Pertussis toxin was purchased from List
Biological Laboratories Inc (Campbell, CA). Recombinant factor XIII was
a gift from Dr Paul Bishop (Zymogenetics, Seattle, WA). Human plasma
fibronectin and the 70-kD N-terminal, gelatin-binding fragment of
fibronectin generated by cathepsin D were isolated, iodinated, and
reisolated as previously described.29,30 The labeled
proteins were stored in portions at 
70°C in TBS containing 0.1% (wt/vol) fatty acid-free bovine albumin. Purity of labeled proteins was assessed by gel electrophoresis with and without reduction
followed by autoradiography. Fibronectin was also labeled with
fluorescein isothiocyanate (FITC).30
Cells.
The MG63 human osteosarcoma cell line was obtained from the American
Type Culture Collection (Rockville, MD). Human foreskin fibroblasts
were a strain derived by Dr Lynn Allen-Hoffmann (University of
Wisconsin-Madison, Madison, WI). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% (for
MG63 cells) or 10% (for foreskin fibroblasts) fetal bovine serum (FBS).
Pertussis toxin and C3 exotransferase treatment.
Cell monolayers were incubated in complete media containing pertussis
toxin (100 µg/L) or C3 exotransferase (50 mg/L) for 24 hours before
fibronectin or 70-kD fragment binding assay. The effects of
pretreatment of activation of MAP kinase was assayed by immunoblotting
with antibodies to dual phosphorylated ERK1, 2 from Promega (Madison, WI).
Binding and cross-linking assays.
Confluent cell layers were usually studied 3 days after sparse seeding
in DMEM plus FBS. For binding studies, the confluent cell layers were
washed with TBS and incubated with 125I-labeled fibronectin
or 70-kD fragment of fibronectin in DMEM containing 0.2% (wt/vol)
fatty acid-free bovine albumin in the absence or presence of different
stimulators for the indicated amount of time. After washing out the
unbound ligand, the cell layers were collected with 1% sodium dodecyl
sulfate (SDS) for counting of the amounts of total cell
layer-associated radioactivity.23 Nonspecific binding in
the presence of 500 mg/L of unlabeled fibronectin or 20 mg/L of
unlabeled 70-kD fragment was determined and subtracted from total
binding to calculate specific binding. Cross-linking with recombinant
factor XIIIa (10 mg/L) was performed after washing out the unbound
ligand and assessed on reducing SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) followed by phosphor-imaging as previously
described.31
Microscopy studies.
Confluent cell layers were washed with TBS and incubated with
FITC-labeled fibronectin in DMEM containing 0.2% (wt/vol) fatty acid-free bovine albumin in the absence or presence of different stimulators for the indicated amount of time. After washing out the
unbound ligand, the cell layers were processed for fluorescent microscopy as previously described.23
RNA isolation, reverse transcription, and polymerase chain reaction
(RT-PCR).
RNA was isolated and RT-PCR were performed using the RNAgents Total RNA
Isolation and Access RT-PCR kit systems (Promega) as
described.32 Primers were selected based on the sequences for human cDNAs: Edg-1, 5'ACGTCAACTATGATATCATCGTCCG3' and
5'CATTTTCAGCATTGTGATATAGCGC3', expected size 391 bp21; Edg-2, 5'AATCGAGAGGCACATTACGG3' and 5'TGTGGACAGCACACGTCTAG3', expected size 432 bp18; Edg-3, 5'CGATCGTTTTATCAATGGTCGC3' and 5'GCGTCGTAAGGCCTCATTTTG3', expected size 631 bp33; and Edg-4, 5'CATCATGCTTCCCGACAACG3' and
5'GGGCTTACCAAGGATACGCAG3', expected size 352 bp
(Genebank entry no. AF011466).
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RESULTS |
S1P stimulates binding of fibronectin or the N-terminal 70-kD fragment
to cells: Comparison to LPA and nocodazole.
LPA and the microtubule-disrupting agent nocodazole are potent
stimulators of fibronectin matrix assembly.22-24 The
addition of S1P (200 nmol/L) to cell layers of strain of human foreskin fibroblasts or MG63 osteosarcoma cells increased the initial binding to
cells of the N-terminal 70-kD matrix assembly domain of fibronectin (Fig 1A) to levels stimulated by LPA (200 nmol/L) or nocodazole (10 µmol/L). This effect was specific for S1P,
inasmuch as the same dose of sphingosylphosphorylcholine or
sphingomyelin, two related sphingolipids,34 did not
increase the binding of 70-kD fragment to MG63 cells (data not shown).
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| Fig 1.
Binding of 70-kD N-terminal fragment of fibronectin (A)
and FITC-fibronectin (B) to confluent cells. (A) Binding of
125I-70-kD fragment of fibronectin to MG53 cells (MG63) or
human foreskin fibroblasts (AH-1F) was determined in the absence
(control) or presence of 200 nmol/L LPA (LPA), 10 µmol/L nocodazole
(NOC), or 200 nmol/L S1P (S1P) as described in Materials and Methods.
Specific binding was calculated by subtraction of nonspecific binding
(in the presence of 500 mg/L unlabeled fibronectin) from total binding
and expressed as nanograms of 70-kD fragment bound per milligram of
cellular protein. Data represent the mean ± SD (n = 3). (B)
Deposition of FITC-fibronectin by confluent MG53 cells was determined
after incubation for 1 hour in the absence (control) or presence of 200 nmol/L LPA (LPA), 10 µmol/L nocodazole (NOC), or 200 nmol/L S1P
(S1P). Bar = 35 µm.
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Fluorescence microscopy of MG63 cells incubated with FITC-labeled
fibronectin showed that S1P enhanced deposition of fibronectin into
extracellular matrix (Fig 1B). When MG63 cells were treated with S1P,
LPA, or nocodazole, fluorescent fibers were more numerous and coarser.
Starting at 0.1 nmol/L, S1P's effect on 70-kD fragment binding to MG63
cells was dose-dependent over 6 orders of magnitude (Fig 2). Unlike the results with
LPA,23 S1P caused increasing stimulation of 70-kD fragment
binding at concentrations as high as 10 µmol/L, and a plateau was not
achieved.
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| Fig 2.
Dose response to S1P of binding of 70-kD N-terminal
fragment of fibronectin to confluent MG63 cells. Binding of
125I-70-kD fragment of fibronectin was determined in the
presence of increasing concentrations of S1P. Specific binding was
calculated by subtraction of nonspecific binding (in the presence of
500 mg/L unlabeled fibronectin) from total binding and expressed as
nanograms of 70-kD fragment bound per milligram of cellular protein.
Data represent the average of duplicates that varied by less than
10%.
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Long-term incubation of LPA with cells has been shown to result in loss
of its activity to stimulate fibronectin binding to cells, whereas the
effect of microtubule-disrupting agent nocodazole is
sustained.23,24 When 100 nmol/L S1P was included in
long-term binding assays, it lost the stimulatory activity by 6 hours
(Fig 3A). S1P- or LPA-containing medium
conditioned by MG63 cells for 20 hours both lost the ability to enhance
binding of the 70-kD fragment to fresh cells, whereas S1P- or
LPA-containing medium incubated at 37°C but not incubated with
cells retained the ability to enhance binding when subsequently added
to fresh cells (Fig 3B). In experiments not shown, S1P-containing
medium conditioned by cells for 2 to 6 hours showed gradual loss of
enhancing activity when added to fresh cells. These results indicate
that both LPA and S1P are degraded or cleared from the medium by
cell-based mechanisms.
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| Fig 3.
Stability of S1P. (A) Time course of effects of LPA,
nocodazole, and S1P on binding of 70-kD fragment to MG63 cells. Binding
of 125I-70-kD fragment of fibronectin in the absence ( )
or presence of 200 nmol/L LPA ( ), 10 µmol/L nocodazole ( ), or
100 nmol/L S1P ( ) was determined at different time points. Specific
binding was calculated by subtraction of nonspecific binding (in the
presence of 500 mg/L unlabeled fibronectin) from total binding and
expressed as nanograms of 70-kD fragment bound per milligram of
cellular protein. Data represent the average of duplicates that varied
by less than 10%. (B) 200 nmol/L LPA (LPA) or S1P (S1P) was
preincubated with MG63 cells for 20 hours at 37°C ( ) or left at
37°C for 20 hours () and was transferred to naive cells for a
45-minute 125I-70-kD fragment binding assay. Specific
binding was calculated by subtraction of nonspecific binding (in the
presence of 500 mg/L unlabeled fibronectin) from total binding and
expressed as nanograms of 70-kD fragment bound per milligram of
cellular protein. Data represent the mean ± SD (n = 3).
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The enhancement of 70-kD fragment binding to MG63 cells by LPA or
nocodazole is due to changes in both the kd and the cell surface
binding sites for the N-terminal matrix assembly
domain.23,24 Binding isotherms for binding of the 70-kD
fragment to MG63 cells in the presence or absence of 200 nmol/L S1P
were similarly determined (Fig 4A).
Scatchard plotting of the result indicated that for S1P-treated MG63
cells, the kd was 3.1 nmol/L versus 6.4 nmol/L for control cells, and
there were 218,000 binding sites per S1P-treated cell versus 92,000 sites for control cells. To compare S1P further with other modulators
of fibronectin matrix assembly, we cross-linked bound
125I-70-kD fragment with factor XIIIa. Cross-linking of the
cell surface-bound 70-kD fragment to MG63 cells treated
with S1P was to molecules of large apparent molecular mass of 3 MD and >>3 MD (Fig 4B), as is also the case with LPA
or nocodazole treatment.24
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| Fig 4.
Binding isotherm (A) and cross-linking (B) of
125I-70-kD fragment to cells. (A) Increasing amounts of
125I-70-kD fragment were incubated with MG63 cells in the
absence (control) or presence of 200 nmol/L S1P (S1P) for 45 minutes.
To achieve concentrations less than 0.5 mg/L, ligand with a specific
activity of 1,000 cpm/ng was added. To achieve concentrations greater
than 0.5 mg/L, unlabeled 70-kD fragment was added to
125I-70-kD fragment to yield mixtures with progressively
lower specific activity. Specific binding was calculated by subtraction
of nonspecific binding (in the presence of 500 mg/L unlabeled
fibronectin) from total binding and expressed as nanograms of 70-kD
fragment bound per well. Data represent the average of duplicate
values. (B) Confluent MG63 cells were incubated for 60 minutes with
125I-70-kD fragment (0.5 mg/L) in medium containing
unlabeled 70-kD fragment (20 mg/L; NSB), control with nothing
additional (CON), 200 nmol/L LPA (LPA), 10 µmol/L nocodazole (NOC),
or 100 nmol/L S1P (S1P). Cell layers were then washed and incubated for
an addition of 5 minutes with factor XIIIa (10 mg/L). Cell lysates were
analyzed by reducing SDS-PAGE and phosphorimaging. Top, top of the
stacking gel; Int, interface of the 3% stacking and 8% separating
polyacrylamide gels; and 70-kD, un-cross-linked 125I-70-kD
fragment. Quantitation of bands at the top, the interface, and the
position of the un-cross-linked probe yielded the following ratios of
density of the three bands in treated cultures compared with control:
LPA/CON: 2.0, 2.2, and 1.9; NOC/CON: 2.1, 2.5, and 2.3; and S1P/CON:
2.1, 1.9, and 1.7.
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Signaling mechanism: Involvement of Rho in enhancement of fibronectin
assembly.
Exogenous S1P has been shown to influence cellular functions through
GPCRs on the cell surface.1,7,10,11,16,35,36 RT-PCR
indicated that MG-63 cells express Edg-2 and Edg-4 and that foreskin
fibroblasts express Edg-2, Edg-3, and Edg-4 (data not shown). S1P's
effects on mitogenesis,7 MAP kinase
activation,37 intracellular Ca2+
mobilization,36 and activation of
IK(Ach) in atrial myocytes35 are
abolished by pertussis toxin. In contrast, the effects of S1P on
neurite retraction11 and cell aggregation16
depend on Rho. Pretreatment of MG63 cells with Clostridium
botulinum C3 exoenzyme, which ADP-ribosylates and thereby
inactivates Rho,28 caused loss of enhancement of 70-kD
fragment binding to cells in response to 200 nmol/L S1P
(Fig 5). Enhanced deposition of FITC-fibronectin into extracellular matrix in response to S1P was also
lost after treatment with C3 exo-enzyme (data not shown). S1P's
stimulation of 70-kD fragment binding was not affected by pretreatment
of MG63 cells with pertussis toxin (Fig 5), even though the activation
of MAP kinase in MG63 cells by S1P was totally abolished by the
pertussis toxin pretreatment (data not shown). These results indicate
that a nanomolar dose of S1P stimulates initial fibronectin binding to
cells through a Rho-dependent signaling pathway.
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| Fig 5.
Effects of C3 exotransferase or pertussis toxin on
enhanced binding of 70-kD N-terminal fragment of fibronectin to MG63
cells by LPA, nocodazole, or S1P. Confluent MG63 cells were pretreated
with complete medium (None), 50 mg/L C3 transferase (C3), or 100 µg/L
pertussis toxin (PTX) for 24 hours and then incubated for 60 minutes
with 125I-70-kD fragment in medium with 200 nmol/L LPA
(), 10 µmol/L nocodazole ( ), or 100 nmol/L S1P ( ). Data are
presented as specific binding in the presence of additive divided by
specific binding in the absence of any additives as a percentage of
control. Data represent the mean ± SD (n = 4).
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Differentiation of the effects of S1P from the stimulatory effects of
LPA and microtubule-disrupting agent.
To check whether S1P's effect on fibronectin binding to cells is
secondary to production of LPA, phospholipase B, which cleaves the
remaining fatty acid chain from the glycerol backbone and destroys
LPA,23 was included in the binding mix with the stimulator. Phospholipase B (1,000 U/L) totally eliminated the stimulation of 70-kD
fragment binding to MG63 cells by LPA, but did not affect the response
to S1P (Fig 6). Furthermore, when the cells
were preincubated for 2 hours with 20 µmol/L taxol, a
microtubule-stabilizing agent,38 enhancement of 70-kD
fragment binding by nocodazole was blocked, whereas MG63 cells
pretreated with taxol responded to S1P just as well as the cells not
treated with taxol (Fig 6). These results indicate that the stimulatory
activity on fibronectin binding by S1P is neither secondary to
extracellular LPA production nor due to the disruption of microtubules.
When S1P and LPA at concentrations of 1, 10, or 100 mmol/L were
combined, the enhancement of 70-kD fragment binding was 20% to 60%
greater than if each lipid alone at the same concentration was
incubated with cells (data not shown). This result indicates that the
effects of the two lipids on cells are additive.
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| Fig 6.
Effects of phospholipase B or taxol on enhanced binding
of 70-kD N-terminal fragment of fibronectin to MG63 cells by LPA,
nocodazole, or S1P. Confluent MG63 cells were pretreated with complete
medium (None), 1,000 U/L phospholipase B (PLB), or 20 µmol/L taxol
(Taxol) for 2 hours and were then incubated for 60 minutes with
125I-70-kD fragment in medium with 200 nmol/L LPA (), 10 µmol/L nocodazole (), or 100 nmol/L S1P (). Data are presented
as the specific binding in the presence of additive divided by specific
binding in the absence of any additives as a percentage of control.
Data represent the mean ± SD (n = 4).
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DISCUSSION |
Organization of soluble fibronectin into extracellular matrix is a
tightly regulated process, mediated by initial reversible binding by
the 70-kD N-terminal region of fibronectin to specific cell surface
binding sites, followed by insolubilization into fibrils.39
The adhesive information present after insolubilization of fibronectin
is postulated to play a central role in various physiological and
pathophysiologic processes, including embryogenesis, wound healing,
inflammation, and degenerative disease processes such as
atherosclerosis and fibrosis.40,41
S1P is abundantly stored in platelets and rapidly released into the
extracellular environment upon stimulation with physiologic agonists,1,2 whereas LPA is quickly generated upon platelet activation.27 S1P is a normal constituent of human plasma
as well, and the mean S1P levels in plasma and serum are 190 and 480 nmol/L, respectively.1 LPA is generated by activated
platelets during blood clotting process, at least partially through
secretion of phospholipase A2 from platelet granules that then acts on
shedded microvesicles.42 Whole blood serum is estimated to
contain 1 to 5 µmol/L LPA, whereas no LPA was detected in plasma
prepared by methods that minimized platelet activation.27
LPA has been identified as the major enhancer of fibronectin matrix
assembly in 0.1% to 2% serum based on the ability of phospholipase B
to destroy activity.23 Another component, viz, S1P, derived
from platelet activation could play an additional role at higher serum concentrations. We found that S1P in the 100 to 500 nmol/L
concentration range present in plasma and serum enhances fibronectin
binding considerably and that LPA and S1P added together at a
concentration of 100 nmol/L enhance binding more than each lipid by
itself. Thus, S1P and LPA may function additively and/or redundantly
during early wound healing. Redundancy may be important because of the fact that the pathways for generation of S1P and LPA are different: platelet secretion in the case of S1P and action of secretory phospholipase on microvesicles in the case of LPA. In addition, the
pathways for destruction of S1P and LPA may be different. Finally, S1P,
for which there is a higher plasma/serum concentration ratio than LPA,
has the potential to act tonically on fibronectin assembly in tissues
in which there is high permeability to plasma proteins.
S1P's effect on 70-kD fragment binding to MG63 cells was
dose-dependent over 6 orders of magnitude (Fig 2). Unlike the results with LPA,23 S1P caused increasing stimulation of 70-kD
fragment binding at concentrations as high as 10 µmol/L, and a
plateau was not achieved. The complex dose response is compatible with the suggestions that S1P's effect on binding of 70-kD fragment to MG63
cells may involve both a receptor-mediated pathway at lower doses and
second messenger functions at higher doses.34
S1P regulates intracellular second messengers and membrane channels
through activation of specific receptors present in many different cell
types. A family of seven transmembrane spanning domain receptor encoded
by endothelial differentiation genes (Edg) have recently
been recognized as receptors for S1P and LPA. Of this family, Edg-2 and
Edg-4 are expressed by MG63 cells and Edg-2, Edg-3, and Edg-4 are
expressed by foreskin fibroblasts. Edg-2 isolated from both
mouse20,43 and human18 has been shown to be
stimulated by LPA. S1P has been described as a ligand for Edg-315 as well as for Edg-1.16 LPA has been
described as a ligand for Edg-4.44 We have developed
evidence from transfection of MG63 cells and HT1080 fibrosarcoma cells
that both LPA and S1P are able to signal through Edg-2 to cause
increased stress fiber formation and shape change (Peyruchaud et al,
unpublished observation). Thus, Edg-2, which is shared by
MG63 cells and foreskin fibroblasts, likely plays a major role in the
effect of S1P on matrix assembly with an additional unknown
contribution from Edg-4 and perhaps still to be described Edg family receptors.
S1P-induced neurite retraction,11,45 cell
aggregation,16 and 3T3 cell shape change have been shown to
be mediated by Rho based on sensitivity to C3
exotransferase.26 S1P-stimulated fibronectin binding was
blocked by the exotransferase, as in the case with LPA or
nocodazole.24 These results indicate that a common
Rho-dependent cytoplasmic mechanism similar to that described for other
effects of S1P on cells regulates the display of cell surface sites for
assembly of fibronectin. The stimulatory activity of S1P or LPA on
fibronectin binding was not sensitive to pertussis toxin, indicating
lack of the participation of Gi in this signaling. The G12/G13 family
of heterotrimeric G protein has been shown to activate Rho
specifically.46 Thus, a G12 or G13 complex likely links the
S1P receptor(s) to the Rho-dependent machinery.
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ACKNOWLEDGMENT |
The authors thank Julie Nowlen and Renee Schultz for excellent
technical support.
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FOOTNOTES |
Submitted May 11, 1998; accepted December 22, 1998.
Supported by National Institutes of Health Grant No. HL21644. O.P. was
a postdoctoral fellow of the Sanofi Association for Thrombosis Research.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Deane F. Mosher, MD, Departments of
Medicine and Biomolecular Chemistry, University of Wisconsin-Madison,
1300 University Ave, Madison, WI 53706; e-mail:
dfmosher{at}facstaff.wisc.edu.
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ABSTRACT
INTRODUCTION
