© 1999 American Heart Association, Inc.
Vascular Biology |
Phosphatidylinositol 3-Kinase and Focal Adhesion Kinase Are Early Signals in the Growth Factor–Like Responses to Thrombospondin-1 Seen in Human Vascular Smooth Muscle
From Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology & Medicine, St. Mary's Hospital, London (J.S.L., S.J.R., G.F.C., K.L.G., A.D.H.), and the Immunology Unit, Glaxo-Wellcome, Medicines Research Centre, Stevenage (C.N., N.T.T.), England.
Correspondence to Joanne S. Lymn, Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology & Medicine, QEQM Wing, St. Mary's Hospital, Paddington, London W2 1NY, England. E-mail j.lymn{at}ic.ac.uk
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—Thrombospondin-1 (TSP-1) is a matricellular protein that is expressed in negligible amounts in normal blood vessels but is markedly upregulated in vascular injury. Although TSP-1 can act as a pleiotropic regulator for human vascular smooth muscle cells (HVSMCs), the intracellular signaling pathways stimulated by this protein remain obscure. In cultured HVSMCs derived from saphenous vein, TSP-1 induces tyrosine phosphorylation of a number of cellular proteins, with a complex temporal pattern of activation. Immunoprecipitation techniques have identified the early tyrosine-phosphorylated signals as being the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-K) and focal adhesion kinase (FAK). Tyrosine phosphorylation of the p85 subunit of PI 3-K showed a biphasic response to TSP-1 stimulation, which corresponded to a biphasic activation of the lipid kinase. Treatment with both wortmannin and LY294002 inhibited PI 3-K activity of HVSMCs but did not affect tyrosine phosphorylation of the p85 regulatory subunit. TSP-1–stimulated FAK phosphorylation, however, was substantially reduced by these inhibitors, as was the TSP-1–induced chemotaxis of these cells. These results suggest that activation of PI 3-K is an early signal induced by TSP-1 and is critical for chemotaxis. Activation of this kinase precedes and may occur upstream from FAK phosphorylation, although the nature of the interaction between these 2 enzymes remains obscure.
Key Words: thrombospondin-1 • focal adhesion kinase • phosphatidylinositol 3-kinase • human vascular smooth muscle
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Thrombospondin-1 (TSP-1), a large, homotrimeric glycoprotein, is a member of the TSP family of matricellular proteins. This family currently consists of 5 isoforms: TSP-1 through TSP-4 and TSP-5/cartilage oligomeric matrix protein,1 which, although implicated in a wide variety of biological processes,2 show differential tissue expression both temporally and spatially.3 4 TSP-1 was originally described as a product of platelet
-granules5
but has since been shown to be synthesized by a number of different
cell types, including vascular endothelial and smooth
muscle cells.6 7 TSP-1 is present in negligible
amounts in normal human blood vessels, but the expression of this
protein is markedly upregulated in injured and diseased
vasculature,8 9 10 suggesting a possible role for TSP-1 in
the abnormal cellular response to vascular injury. Literature evidence would suggest that TSP-1 levels are regulated by growth factors. Both platelet-derived growth factor (PDGF) and transforming growth factor-ß (TGF-ß) have been shown to induce TSP-1 secretion from vascular smooth muscle cells (VSMCs).11 12 TSP-1 mRNA levels have been shown to be similarly regulated. Basic fibroblast growth factor upregulates TSP-1 mRNA in 3T3 cells,13 and PDGF induces upregulation in a manner similar to that of c-myc and c-fos, with TSP-1 being classified as an immediate early response gene.14 These data, in conjunction with the role of TSP-1 in potentiating the chemotactic response of VSMCs to PDGF15 and the proliferative response to epidermal growth factor,16 have led to the belief that TSP-1 plays a modulatory role in the cellular response to vascular injury.
Recent work from our laboratory, however, demonstrates that TSP-1 can in fact act as an independent growth factor for human (H) VSMCs. We have shown that TSP-1 is capable of inducing not only migration but also proliferation of these cells to levels similar to those seen with PDGF, although we have shown that PDGF does not contribute to these mitogenic effects. Indeed, TSP-1 in combination with PDGF induced a synergistic effect on DNA synthesis.17 18 This direct effect of TSP-1 on HVSMCs suggests a growth factor–like role for TSP-1 in the cellular response to injury. Although the functional consequences of TSP-1 stimulation have been demonstrated, the mechanisms by which these signals are transmitted intracellularly remain obscure.
Our previous data have shown that TSP-1 acts as a pleiotropic growth regulator of HVSMCs in much the same way as growth factors like PDGF. In keeping with this observation, recent data from our laboratory have shown that TSP-1–induced DNA synthesis in HVSMCs is completely inhibited by the tyrosine kinase inhibitors genistein and tyrphostin A23. Cellular migration in response to TSP-1 is also substantially reduced by these tyrosine kinase inhibitors.18 This suggests that tyrosine phosphorylation of certain key proteins plays an important role in the intracellular transmission of signals induced by TSP-1. Interestingly TSP-1–enhanced cell spreading of C32 human melanoma cells on sparse vitronectin coatings has been correlated with increased tyrosine phosphorylation of focal adhesion kinase (FAK).19 Although TSP-induced disassembly of focal adhesion plaques in aortic endothelial cells has recently been reported to occur via a pathway involving stimulation of the lipid kinase, phosphatidylinositol 3-kinase (PI 3-K), tyrosine phosphorylation of the p85 regulatory subunit of this enzyme has not been reported.20
We report here that soluble TSP-1 induces tyrosine phosphorylation of a number of cellular proteins in quiescent HVSMCs, with a complex temporal pattern of activation. The primary phosphotyrosine proteins have been identified as PI 3-K (the p85 regulatory subunit) and FAK. PI 3-K is upstream from FAK in the signaling process, in that FAK phosphorylation is dependent on PI 3-K activation.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
HVSMC Culture
HVSM was obtained from saphenous veins from patients undergoing cardiovascular surgery. Tissues were surplus to requirements, and their use conformed to the guidelines of our local ethics committee. VSMCs were cultured by an explant technique as previously described.21 VSMCs were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) buffered with 25 mmol/L HEPES and supplemented with 15% (vol/vol) FCS, 4 mmol/L L-alanyl-L-glutamine (Glutamax-I), penicillin (100 U/mL), streptomycin (100 µg/mL), and gentamicin (25 µg/mL). Cell cultures were maintained in a humidified atmosphere of 5% CO2 (vol/vol) in air at 37°C. VSMCs were routinely used at third passage unless otherwise stated. VSMC purity was routinely checked by immunocytochemical studies for
-actin
staining.
Cell Stimulation
Passage 2 HVSMCs were subcultured and plated onto Petri dishes
at a known density (106 cells per dish) and
allowed to attach for 24 hours in DMEM supplemented with 15% (vol/vol)
FCS. Cells were then washed twice with PBS and maintained in serum-free
DMEM for a period of 7 days. Quiescent cells were then washed in
serum-free DMEM and stimulated with 1 µg/mL TSP-1 in DMEM for a
period of 30 minutes at 37°C, unless otherwise stated. Cells were
then washed twice with ice-cold PBS, scraped into 1 mL of ice-cold
lysis buffer (50 mmol/L Tris [pH 7.4]; 150 mmol/L NaCl;
1 mmol/L EGTA; 1% [vol/vol] NP-40; 0.25% sodium deoxycholate,
1 mmol/L NaF, sodium orthovanadate, and PMSF; and 1 µg/mL
aprotinin, pepstatin, and leupeptin), and allowed to stand on ice for
10 minutes before centrifugation (14 000 rpm, 15
minutes, 4°C). The resulting cell lysates were used for protein
measurement.
Immunoprecipitation
A known concentration of cellular protein (
250 µg) was
removed from all samples and precleared for 1 hour with
albumin-agarose before incubation with primary antibody (1 µg
of antibody per 100 µg of protein) for 3 hours at 4°C.
Immunoprecipitates were captured on protein A–agarose in the manner
described.22
Immunoblotting
Proteins were separated on 10% SDS-polyacrylamide gels
before being transferred to nitrocellulose by using a Bio-Rad wet gel
transfer system. After successful transfer, the nitrocellulose blots
were blocked in 5% BSA for 1 hour and washed 3 times in Tris-buffered
saline containing 0.05% Tween-20 before being probed with the primary
antibody for 1 hour. Blots were washed well in Tris-buffered
saline/Tween-20 before being probed with the appropriate secondary
antibody and developed by enhanced chemiluminescence.
PI 3-K Activity Assay
Passage 4 cells were washed twice with PBS and maintained in
serum-free DMEM for a period of 7 days before stimulation with TSP-1.
Cells were stimulated and scraped into lysis buffer as described above
and then incubated with anti–rat PI 3-K, rabbit whole antiserum, and
protein A–agarose overnight at 4°C. The immunoprecipitate was washed
twice with 0.1 mol/L Tris-HCl, 0.5 mol/L LiCl (pH 7.5) and twice with
kinase assay buffer (20 mmol/L HEPES, 10 mmol/L
MgCl2, and 25 µmol/L ATP, pH 7.4). Samples
were resuspended in 40 µL of kinase buffer with 10 µL of
[33P]ATP and 10 µL of PI and incubated at
room temperature for 15 minutes. The reaction was stopped by the
addition of 50 µL of 1 mol/L HCl and 100 µL of chloroform/methanol
(1:1, vol/vol), and the samples were centrifuged at 14 000 rpm
for 5 minutes to give 2 separate phases. The chloroform layer was
spotted onto potassium oxalate–coated thin-layer
chromatography plates and developed in a mixture of
chloroform/acetone/methanol/acetic acid/water (40:15:13:12:7,
vol/vol/vol/vol/vol) with a running time of 2 hours. Plates were
dried thoroughly, wrapped in Clingo-rap, and exposed to a
PhosphorImager cassette for 2 days.
Cell Migration Assays
These assays were performed with blind-well chemotaxis chambers
as previously described.23 In brief, the upper and lower
compartments of the blind-well chambers were separated by
gelatin-coated polycarbonate filters. TSP-1 (10 µg/mL) was added to
the lower chamber and acted as the chemoattractant. The cell suspension
(2.25x105 cells/mL), with or without PI 3-K
inhibitors, was added to the upper chamber, and migration
was allowed to proceed for 5 hours. The filters were then removed,
fixed in ethanol, and stained in toluidine blue (1%, vol/vol). The
migrated cells were counted under a light microscope.
Materials
Cell-culture plasticware, media, supplements, and TSP-1
(purified from human platelets) were obtained from Life
Technologies. FCS was obtained from M.B. Meldrum Ltd. BSA fraction V
was purchased from Boehringer Mannheim. The bicinchoninic acid
protein assay kits were obtained from Pierce Warriner. Hybond C
nitrocellulose, enhanced chemiluminescence reagents, and streptavidin
complex were obtained from Amersham. Protein A–agarose was obtained
from Cambridge Bioscience. Anti-phosphotyrosine antibody PY20 and
anti-rabbit IgG (horseradish peroxidase linked) were obtained from
Affiniti. All other antibodies were obtained from TCS, and all other
reagents were obtained from Sigma.
Statistical Analysis of Data
Control data were defined as 100%, and experimental data were
expressed as mean±SEM in relation to the control. Statistical
differences between means in terms of analysis of temporal
effects were determined with a Freidman nonparametric ANOVA
for multiple repeated measures. Other data were compared by a
Wilcoxon matched-pairs signed rank test. A value of
P
0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
TSP-1 Stimulates Tyrosine Phosphorylation of Novel Protein Bands
HVSMCs were incubated with or without TSP-1 (1 µg/mL) for 60 minutes, and the tyrosine phosphorylation profiles of crude cell lysates were examined and compared. Control cells demonstrated a small number of constitutively phosphorylated protein bands, 3 of which (approximate molecular weights of 97, 75, and 63 kDa) exhibited significantly enhanced tyrosine phosphorylation after stimulation with TSP-1. More important, TSP-1 stimulation of HVSMCs resulted in the appearance of a number of tyrosine-phosphorylated protein bands that were not observed in control, unstimulated cells. Densitometric profile analysis of the TSP-1–stimulated cell lysates compared with biotinylated molecular weight markers attributed molecular weights of
170, 118, and 88 kDa to these novel
tyrosine-phosphorylated bands (data not shown). The
stimulation of novel tyrosine-phosphorylated proteins
by TSP-1 correlates well with previous data from our laboratory that
suggest that TSP-1 acts as an independent growth factor for
HVSMCs.18
Temporal Nature of TSP-1–Induced Tyrosine Phosphorylation
Further investigation of the effect of TSP-1 stimulation on the
tyrosine phosphorylation profile of cell lysates
demonstrated a complex temporal pattern of tyrosine
phosphorylation in response to cell stimulation.
Protein bands that showed some constitutive tyrosine
phosphorylation appeared to demonstrate increasing
tyrosine phosphorylation in response to TSP-1 in a
time-dependent manner. The peak tyrosine
phosphorylation levels of these particular protein
bands were generally observed between 60 and 90 minutes after
initiation of TSP-1 stimulation. Other proteins, however, appeared to
be tyrosine phosphorylated early in response to TSP-1,
between 20 and 30 minutes. These proteins then appeared to undergo a
certain amount of subsequent dephosphorylation on
prolonged exposure to TSP-1, although they remained
phosphorylated above control levels at 60 minutes.
Protein bands falling into this category of rapidly
phosphorylated signals included
tyrosine-phosphorylated proteins of 118 and 88 kDa
(Figure 1
).
|
Identification of Early Signals in Response to TSP-1
Owing to the complex temporal nature of the phosphotyrosyl protein
profile and because the nature of the primary signaling process in
response to TSP-1 in HVSMCs is obscure, we concentrated on identifying
and understanding the early phosphotyrosine signals. The growth
factor–like nature of TSP-1 and the estimated molecular weights of the
early tyrosine-phosphorylated proteins led us to
investigate the possibility that these protein bands might
represent FAK and the p85 regulatory subunit of PI 3-K.
Quiescent cells were exposed to TSP-1 (1 µg/mL), and the temporal
pattern of tyrosine phosphorylation of p85 PI 3-K and
FAK was identified by immunoprecipitation followed by Western blotting
techniques. Initially, phosphotyrosine immunoprecipitates were
collected from 200 µg of crude cell lysate and probed with antibodies
to specific cellular proteins. Parallel studies in which the specific
protein was immunoprecipitated followed by
immunoblotting with an anti-phosphotyrosine antibody
yielded results similar to those in which phosphotyrosine proteins were
immunoprecipitated. These results demonstrated a single peak of FAK
phosphorylation in response to TSP-1 stimulation at
20 to 30 minutes (Figure 2A and 2B).
In contrast, the p85 regulatory subunit of PI 3-K exhibited a biphasic
tyrosine phosphorylation in response to TSP-1
stimulation. Tyrosine phosphorylation levels were
markedly increased within the first 10 minutes of TSP-1 stimulation,
decreased transiently, and then exhibited a second peak in tyrosine
phosphorylation at 20 to 30 minutes (Figure 2C).
Prolonged exposure to TSP-1 was generally associated with
reduced phosphorylation of the p85 regulatory subunit
of PI 3-K to near control levels. Intriguingly, the peak in FAK
phosphorylation coincided exactly with the later peak
in the biphasic PI 3-K response. This behavior was a consistent
trend in all of the human cell strains tested, although the time of
peak phosphorylation and the degree of
phosphorylation above control levels varied from strain
to strain (Figure 3A).
|
|
PI 3-K Activity
Although FAK phosphorylation can be taken as an
indicator of activation,24 the same is not necessarily
true of PI 3-K. With PI 3-K, phosphorylation occurs in
the p85 regulatory subunit whereas catalytic activity is located in the
p110 subunit, and although phosphorylation and activity
are generally linked, activation of PI 3-K is not concomitant with
tyrosine phosphorylation of the p85 subunit.
Consequently, modulation of PI 3-K activity in response to TSP-1 was
measured and compared with the p85 tyrosine
phosphorylation results. Temporal analysis of
the PI 3-K activity in immunoprecipitates from stimulated cells (5- to
90-minute time course) demonstrated that this lipid kinase activity was
indeed stimulated by TSP-1 in a biphasic manner (Figure 3B
).
This increase in activity, which was statistically significant
(P<0.05) at 5 minutes, correlates well with the temporal
nature of the increased tyrosine phosphorylation of the
p85 regulatory subunit of PI 3-K in response to TSP-1, which was
statistically significant at both 5 and 20 minutes (Figure 3A).
This finding suggests that TSP-1 stimulates a biphasic increase in PI
3-K enzyme activity, with the primary, transient stimulation occurring
within 5 to 10 minutes after exposure to TSP-1, and the second
prolonged peak of activity occurring at 30 minutes. These peaks in
PI 3-K activity appear to be coupled to increases in the tyrosine
phosphorylation levels of the p85 regulatory subunit of
this enzyme.
Effect of PI 3-K Inhibition on FAK Phosphorylation
The biphasic nature of the PI 3-K response to TSP-1 compared with
that of FAK led us to investigate possible interactions between PI 3-K
and FAK in the signaling pathway. Wortmannin at nanomolar
concentrations has been shown to be a highly selective
inhibitor of class 1 PI3-K,25 as has LY294002,
a structurally dissimilar but equally effective PI 3-K
inhibitor.26 Pretreatment of HVSMCs with
either of these membrane-permeable inhibitors before a
30-minute exposure to TSP-1 resulted in a substantial decrease in the
level of TSP-1–stimulated tyrosine phosphorylation of
FAK. This inhibition was apparent in samples wherein immunoprecipitated
FAK was probed with an anti-phosphotyrosine antibody (Figure 4A) and in phosphotyrosine
immunoprecipitates probed with anti-FAK (Figure 4B). These data
suggest that not only is PI 3-K activity stimulated by TSP-1 but also
that this activation occurs upstream from FAK activation in the
signaling cascade and that FAK activation is dependent on PI 3-K
activity.
|
Neither wortmannin nor LY294002 had a significant effect on the ability of the p85 regulatory subunit to associate with tyrosine-phosphorylated proteins or on tyrosine phosphorylation of the subunit itself (densitometric analysis of p85 tyrosine phosphorylation in arbitrary units: TSP-1, -4.5±1.5, n=8; wortmannin, -3.8±1.5, n=8; and LY294002, -2.4±1.3, n=6). Both wortmannin and LY294002 interact with the p110 catalytic subunit of PI 3-K and would not necessarily be expected to affect tyrosine phosphorylation of the p85 subunit.
The effect of wortmannin and LY294002 on cell viability was determined by measuring lactic dehydrogenase release from the cells by using a nonradioactive cytotoxicity assay. Lactic dehydrogenase released from control cells was expressed as 100%, and all other data were compared with this value (lactic dehydrogenase positive control for these cells was 3136±1380%; TSP-1, 105±27%; wortmannin, 296±150%; and LY294002, 102±15%). These data indicated that PI 3-K inhibitor treatment did not significantly affect cell viability.
Nature of the FAK–PI 3-K Interaction
Western blotting techniques were used to determine the nature of
the interaction between FAK and PI 3-K and the temporal effect of TSP-1
stimulation on this interaction. Lysates from agonist-stimulated cells
were immunoprecipitated with FAK, and these immunoprecipitates were
immunoblotted for PI 3-K. At no stage after TSP-1
stimulation was the p85 regulatory subunit of PI 3-K detected in these
immunoprecipitates. Conversely, FAK protein expression was not detected
in p85 PI3-K immunoprecipitates from agonist-stimulated cells (data not
shown). These data suggest that the TSP-1–stimulated interaction
between FAK and PI 3-K may not constitute a direct protein-protein
interaction between the cytosolic tyrosine kinase and the p85
regulatory subunit of the lipid kinase.
TSP-1–Induced Cellular Migration
TSP-1 (10 µg/mL) has been shown to induce levels of HVSMC
chemotaxis similar to those seen with PDGF-BB. These are significantly
reduced by tyrosine kinase inhibitors, suggesting that
tyrosine phosphorylation of cell proteins plays a key
role in this process.18 FAK has long been recognized as a
constituent of focal adhesions,27 and the assembly of
focal adhesions accompanies cell migration.28 Activation
of FAK in focal adhesions appears to occur downstream from their
assembly and to be a key factor in the regulation of cell
motility.29 30 31 32 If FAK is associated with the ability of
cells to migrate, then inhibition of signals upstream from FAK in the
signaling process should also have the ability to inhibit cell
migration. Consequently, we set out to examine the effect of inhibiting
PI 3-K activity on cell migration in response to TSP-1.
The addition of both PI 3-K inhibitors, wortmannin and
LY294002, to cells before their exposure to a directed gradient of
TSP-1 resulted in a concentration dependent decrease in the ability of
these cells to migrate in response to TSP-1 (Figure 5). The PI 3-K inhibitor
wortmannin has been shown in some cell lines to affect phospholipase
A2 (PLA2), which may in
turn affect cellular responses such as chemotaxis. Consequently, the
effect of the PLA2 inhibitor
7,7-dimethyleicosadienoic acid on TSP-1–induced chemotaxis was
investigated. PLA2 inhibition had no effect on
TSP-1–induced chemotaxis (TSP-1–induced chemotaxis was taken to
represent 100%; the TSP-1+100 µmol/L
7,7-dimethyleicosadienoic acid value was -114±22%, n=3), suggesting
that inhibition of PLA2 did not contribute to the
action of wortmannin. This result suggests that PI 3-K plays a key role
in the regulation of cellular migration. Wortmannin (25 to 100 nmol/L)
inhibited the ability of cells to migrate to TSP-1 in a
concentration-dependent manner. LY294002 (0.1 to 10 µmol/L)
similarly inhibited TSP-1–induced migration.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Previous data from our laboratory have shown that TSP-1 may act as an independent growth factor for HVSMCs.18 We have previously shown that TSP-1–induced DNA synthesis in these HVSMCs is unaffected by the presence of neutralizing antibodies to both PDGF and TGF-ß1, although the response to these factors is completely inhibited in the presence of the appropriate antibody. Similarly, we have been unable to detect the presence of either PDGF or TGF-ß1 in the commercially available TSP-1 used for these experiments. Indeed, we have demonstrated both DNA synthesis and migration of HVSMCs in response to recombinant TSP-1.18 These results suggest that the responses seen to TSP-1 are unlikely to be due to contamination of this protein with either PDGF or TGF-ß1.
The data presented here argue strongly in favor of TSP-1 inducing its functional effects through the temporally regulated tyrosine phosphorylation of a series of cellular proteins. The early tyrosine-phosphorylated signals seen in response to TSP-1 include FAK and PI 3-K, the activity of both enzymes being significantly enhanced by a 20- to 30-minute stimulation with 1 µg/mL TSP-1. Indeed, preliminary data from this laboratory have recently demonstrated tyrosine phosphorylation of both FAK and the PI 3-K p85 regulatory subunit by baculovirus-derived recombinant TSP-1 (a generous gift from Dr J. Adams, University College, London). Although stimulation of FAK activity by TSP-1 has been previously shown in human C32 melanoma cells,19 our data represent the first direct demonstration that soluble TSP-1 induces tyrosine phosphorylation of FAK in VSMCs. Similarly, these data represent the first demonstration of PI 3-K activation, accompanied by a corresponding tyrosine phosphorylation of the p85 regulatory subunit, induced by TSP-1 in any cells to date.
TSP belongs to a family of matricellular proteins that bind to other
extracellular matrix proteins as well as to cell-surface receptors and
other molecules such as serine proteases. Although signaling through
TSP-1 is obscure, there is evidence that another member of this
matricellular family, osteopontin, produces rapid activation of PI
3-K.33 Recent evidence from Greenwood and
colleagues20 have demonstrated that TSP-1 signals for
focal adhesion disassembly in cultured bovine aortic
endothelial cells operate through a pathway involving
stimulation of PI 3-K. These authors showed a 2.5-fold increase in PI
3-K activity with Hep-I (a synthetic peptide containing amino acids 17
to 35 of TSP-1), although these authors failed to detect a
corresponding increase in tyrosine phosphorylation of
the p85 regulatory subunit of the lipid kinase. This
endothelial cell activation data correlate well with
the HVSMC data reported here, which show a similar increase in PI 3-K
activity (2-fold) at 20 to 30 minutes after TSP-1 stimulation.
Although both FAK and PI 3-K are early tyrosine-phosphorylated signals in response to TSP-1, immunoprecipitation data show a biphasic tyrosine phosphorylation response for PI 3-K that is correlated with a biphasic activation of the lipid kinase. This biphasic phosphorylation was not detected initially when we examined tyrosine phosphorylation profiles, primarily because of the time course used for assessment and the somewhat crude picture that cell lysate analysis gives. The single peak of FAK phosphorylation correlates well with the latter peak of PI 3-K phosphorylation and activation, suggesting that PI 3-K activation may precede FAK activation. In fact, both of the PI 3-K inhibitors, wortmannin and LY294002, though structurally different, substantially reduced the level of TSP-1–stimulated FAK phosphorylation, suggesting that FAK phosphorylation occurs downstream from PI 3-K and that activation of the former is dependent on the activation of PI 3-K.
There is evidence to suggest that the p110 subunit of PI 3-K functions
as a dual kinase. Aside from its ability to phosphorylate
inositol lipids at the D3 position of the inositol ring, PI 3-K has
been implicated as a serine/threonine protein kinase. Although no
protein kinase substrates have yet been identified in
vivo,34 a number of serine/threonine kinases have been
placed downstream from PI 3-K in receptor-stimulated signaling. These
downstream targets of PI 3-K include protein kinase B,35
protein kinase C,36 and
p70s6K.37 Data presented
here, however, suggest that the cytosolic tyrosine kinase FAK can also
be placed downstream from PI 3-K in TSP-1–stimulated cells. Such a
link has previously been suggested by other groups with regard to
PDGF-stimulated phosphorylation of
FAK.38 39 The mechanism by which PI 3-K exerts its effects
on FAK phosphorylation remains obscure, although a
direct and stable association between FAK and the p85 subunit of PI 3-K
has previously been reported in vitro.40 41 42 Data
presented here, however, provide no evidence for such a direct
protein-protein association between FAK and PI 3-K in HVSMCs. FAK was
consistently absent from our immunoprecipitates of PI 3-K, and
similarly, PI 3-K was consistently absent from
immunoprecipitates of FAK, suggesting an indirect mechanism of FAK
activation by PI 3-K. In this respect, the p85 subunit of PI 3-K has
been demonstrated to contain a breakpoint cluster region–homology
domain, which has been suggested to contain a G protein–binding site
and may function as a GTPase-activating protein for monomeric
GTP-binding proteins.43 Similarly, both rac-mediated
lamellipodia and rho-mediated stress fiber and focal adhesion formation
can be induced by membrane targeting of the p110 catalytic subunit of
PI 3-K,44 suggesting that PI 3-K can induce small
GTPase-mediated cellular responses. Interestingly, an increasing number
of reports now link activated rho to the tyrosine
phosphorylation of a number of cellular proteins,
including FAK.45 A possibility then is that tyrosine
phosphorylation and the concomitant activation of FAK
by PI 3-K presented in this report may occur indirectly through
the activation of a small GTPase of the rho family. In keeping with
this concept, the lipid products of PI 3-K have recently been shown
to bind selectively to a number of small G protein exchange
factors.46 Alternatively, there is evidence to suggest
that the products of PI 3-K activity initiate downstream signaling
by recruiting and/or activating target proteins. PI
3,4,5-trisphosphate, a product of PI 3-K activity, has been
demonstrated to compete with tyrosine-phosphorylated
proteins for binding to SH2 domains. Thus, PI 3,4,5-trisphosphate can
act to recruit SH2 domain–containing proteins to the cell membrane and
has been shown to bind to src47 and to activate
phospholipase C
.48 It is possible then that PI 3-K
exerts its effects on FAK indirectly through the cytosolic tyrosine
kinase src. Such an interaction between src and FAK has been seen in a
number of cell types.33 49
TSP-1 can induce levels of chemotaxis similar to those seen with PDGF-BB in HVSMCs.18 This suggests that the upregulation in TSP-1 levels noted after platelet degranulation or in injured and diseased blood vessels may act as a powerful chemoattractant, inducing cells to migrate into the intima of the vessel. In atherosclerosis and saphenous vein graft occlusion, lesions are found in the vascular intima, and the SMCs that compose these lesions are clonal. This suggests that migration of SMCs from the media is a key event in the early stages of atherosclerosis and restenosis.50 51 Understanding the cellular signals involved in the TSP-1–induced migration of HVSMCs may therefore enable us to target specific signaling molecules in a therapeutic approach for prevention of these vascular occlusions.
In conclusion, TSP-1 stimulates the phosphorylation and activation of both PI 3-K and FAK. PI 3-K lies upstream from FAK in the signaling cascade that leads to cell chemotaxis. Inhibition of PI 3-K activity leads to both a decrease in FAK phosphorylation and a decrease in the ability of cells to migrate in response to a directed gradient of TSP-1. The mechanism by which PI 3-K exerts its effects on FAK, however, remains to be defined.
|
Acknowledgments |
|---|
This work was supported by the British Heart Foundation (PG/98141) (to J.S.L. and A.D.H.). We thank the surgeons and theater staff of St Mary's Hospital for supplying samples of saphenous vein.
Received August 7, 1998; accepted January 25, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Bornstein P. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin-1. J Cell Biol. 1995;130:503–506.
2. Frazier WA. Thrombospondins. Curr Opin Cell Biol. 1991;3:792–799.[Medline] [Order article via Infotrieve]
3.
O'Shea KS, Dixit VM. Unique distribution of the
extracellular matrix component thrombospondin in the developing mouse
embryo. J Cell Biol. 1988;107:2737–2748.
4. Ireula Arispe ML, Liska DJ, Sage EH, Bornstein P. Differential expression of thrombospondin 1, 2 and 3 during murine development. Dev Dyn. 1993;197:40–56.[Medline] [Order article via Infotrieve]
5.
Lawler JW, Slayter HS, Coligan JE. Isolation and
characterization of a high molecular weight glycoprotein
from human blood platelets. J Biol Chem. 1978;253:8609–8616.
6.
Mosher DF, Doyle MJ, Jaffe EA. Synthesis and secretion
of thrombospondin by cultured human endothelial cells.
J Cell Biol. 1982;93:343–348.
7.
Raugi GJ, Mumby SM, Abbott-Brown D, Bornstein P.
Thrombospondin: synthesis and secretion by cells in culture.
J Cell Biol. 1982;95:351–354.
8. Wight TN, Raugi GJ, Mumby SM, Bornstein P. Light microscopic immunolocation of thrombospondin in human tissues. J Histochem Cytochem. 1985;33:295–302.[Abstract]
9.
Brody JI., Pickering NJ, Fink GB. Immunocytochemical
features of obstructed saphenous vein coronary artery bypass
grafts. J Clin Pathol. 1989;42:477–482.
10. Botney MD, Kaiser LR, Cooper JD, Mecham RP, Parghi D, Roby J, Parks WC. Extracellular matrix protein gene expression in atherosclerotic hypertensive pulmonary arteries. Am J Pathol. 1992;140:357–364.[Abstract]
11.
Majack RA, Coates Cook S, Bornstein P.
Platelet-derived growth factor and heparin-like
glycosaminoglycans regulate thrombospondin
synthesis and deposition in the matrix by smooth muscle cells.
J Cell Biol. 1985;101:1059–1070.
12.
Majack RA, Majesky MW, Goodman LV. Role of PDGF-A
expression in the control of vascular smooth muscle cell growth by
transforming growth factor-ß. J Cell Biol. 1990;111:239–247.
13. Donoviel DB, Amacher SL, Judge KW, Bornstein P. Thrombospondin gene expression is associated with mitogenesis in 3T3 cells: induction by basic fibroblast growth factor. J Cell Physiol. 1990;145:16–23.[Medline] [Order article via Infotrieve]
14.
Majack RA, Mildbrandt J, Dixit VM. Induction of
thrombospondin messenger RNA levels occurs as an immediate primary
response to platelet-derived growth factor. J Biol
Chem. 1987;262:8821–8825.
15. Yabkowitz R, Mansfield PJ, Ryan US, Suchard SJ. Thrombospondin mediates migration and potentiates platelet-derived growth factor-dependent migration of calf pulmonary artery smooth muscle cells. J Cell Physiol. 1993;157:24–32.[Medline] [Order article via Infotrieve]
16.
Majack RA, Coates Cook S, Bornstein P. Control of
smooth muscle cell growth by components of the extracellular matrix:
autocrine role for thrombospondin. Proc Natl Acad Sci
U S A. 1986;83:9050–9054.
17. Patel MK, Clunn GF, Lymn JS, Hughes AD Thrombospondin-1 induces DNA synthesis and migration in human vascular smooth muscle cells. [Abstract]. Biochem Soc Trans.. 1996;24:446S.[Medline] [Order article via Infotrieve]
18.
Patel MK, Lymn JS, Clunn GF, Hughes AD.
Thrombospondin-1 is a potent mitogen and chemoattractant for human
vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1997;17:2107–2114.
19.
Gao A-G, Lindberg FP, Dimitry JM, Brown EJ, Frazier WA.
Thrombospondin modulates
vß3 function through
integrin-associated protein. J Cell Biol. 1996;135:533–544.
20.
Greenwood JA, Pallero MA, Theibert AB, Murphy-Ullrich
JE. Thrombospondin signaling of focal adhesion disassembly requires
activation of phosphoinositide 3-kinase. J
Biol Chem. 1998;273:1755–1763.
21. Chan P, Munro E, Patel M, Betteridge L, Schachter M, Sever P, Wolfe J. Cellular biology of human intimal hyperplastic stenosis. Eur J Vasc Surg. 1993;7:129–135.[Medline] [Order article via Infotrieve]
22.
Abedi H, Dawes KE, Zachary I. Differential
effects of platelet-derived growth factor BB on p125 focal adhesion
kinase and paxillin tyrosine phosphorylation and on
cell migration in rabbit aortic vascular smooth muscle cells and Swiss
3T3 fibroblasts. J Biol Chem. 1995;270:11367–11376.
23.
Clunn GF, Refson JS, Lymn JS, Hughes AD.
Platelet-derived growth factor ß-receptors can both promote
and inhibit chemotaxis in human vascular smooth muscle cells.
Arterioscler Thromb Vasc Biol. 1997;17:2622–2629.
24. Zachary I, Rozengurt E. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell. 1992;71:891–894.[Medline] [Order article via Infotrieve]
25. Ui M, Okada T, Hazeki K, Hazeki O. Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase. Trends Biol Sci. 1995;20:303–307.
26.
Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific
inhibitor of phosphatidylinositol 3-kinase,
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).
J Biol Chem. 1994;269:5241–5248.
27.
Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds
AB, Parsons JT. pp125FAK, a structurally distinctive protein-tyrosine
kinase associated with focal adhesions. Proc Natl Acad Sci
U S A. 1992;89:5192–5196.
28.
Nukolls GH, Romer LH, Burridge K. Microinjection of
antibodies against talin inhibits the spread and migration of
fibroblasts. J Cell Sci. 1992;102:753–762.
29. Wilson L, Carrier MJ, Kellie S. pp125FAK tyrosine kinase activity is not required for the assembly of F-actin stress fibres and focal adhesions in cultured mouse aortic smooth muscle cells. J Cell Sci. 1995;108:2381–2391.[Abstract]
30. Gilmore AP, Romer LH. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Mol Biol Cell. 1996;7:1209–1224.[Abstract]
31. Ilic D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M, Yamamoto T, Aizawa S. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature. 1995;377:539–544.[Medline] [Order article via Infotrieve]
32. Cary LA, Chang JF, Guan JL. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci. 1996;109:1787–1794.[Abstract]
33.
Hruska KA, Rolinck F, Huskey M, Alvarez U, Cheresh D.
Engagement of the osteoclast integrin
vß3 by osteopontin
stimulates phosphatidylinositol 3-hydroxyl kinase activity.
Endocrinology. 1995;136:2984–2992.[Abstract]
34. Vanhaesebroeck B, Leevers SJ, Panayotou G, Waterfield MD. Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biol Sci. 1997;22:267–272.
35. Burgering BMTh, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995;376:599–602.[Medline] [Order article via Infotrieve]
36. Ishizuka T, Nagashima T, Yamamoto M, Kajita K, Yamada K, Wada H, Itaya S, Yasuda K, Nozawa Y. Effects of wortmannin on glucose uptake and protein kinase C activity in rat adipocytes. Diabetes Res Clin Pract. 1995;29:143–152.[Medline] [Order article via Infotrieve]
37. Scott PH, Belham CM, Al-Hafidh J, Chivers ER, Peacock AJ, Gould GW, Plevin R. A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth muscle cells. Biochem J. 1996;318:965–971.
38. Saito Y, Seijiro M, Koutaro Y, Tetsuto K, Yasushi S, Nobuhiro M. Phosphatidylinositol 3-kinase activity is required for the activation process of focal adhesion kinase by platelet-derived growth factor. Biochem Biophys Res Commun. 1996;224:23–26.[Medline] [Order article via Infotrieve]
39.
Rankin S, Hooshmand-Rad R, Claesson-Welsh L, Rozengurt
E. Requirement for phosphatidylinositol 3'-kinase activity in
platelet-derived growth factor-stimulated tyrosine
phosphorylation of p125 focal adhesion kinase and
paxillin. J Biol Chem. 1996;271:7829–7834.
40.
Chen H-C, Guan J-L. Association of focal adhesion
kinase with its potential substrate phosphatidylinositol 3-kinase.
Proc Natl Acad Sci U S A. 1994;91:10148–10152.
41.
Chen H-C, Guan J-L. Stimulation of phosphatidylinositol
3'-kinase association with focal adhesion kinase by
platelet-derived growth factor. J Biol Chem. 1994;269:31229–31233.
42.
Guinebault C, Payrastre B, Racaud-Sultan C, Mazarguil
H, Breton M, Mauco G, Plantavid M, Chap H. Integrin-dependent
translocation of phosphoinositide 3-kinase to the
cytoskeleton of thrombin-activated platelets involves
specific interactions of p85 with actin filaments and focal
adhesion kinase. J Cell Biol. 1995;129:831–842.
43.
Musacchio A, Cantley LC, Harrison SC. Crystal structure
of the breakpoint cluster region-homology domain from
phosphoinositide 3-kinase p85 -subunit.
Proc Natl Acad Sci U S A. 1996;93: 14373–14378.
44. Reif K, Nobes CD, Thomas G, Hall A, Cantrell DA. Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho-dependent effector pathways. Curr Biol. 1996;6:1445–1455.[Medline] [Order article via Infotrieve]
45. Flinn HM, Ridley AJ. Rho stimulates tyrosine phosphorylation of focal adhesion kinase, p130 and paxillin. J Cell Sci. 1996;109:1133–1141.[Abstract]
46.
Rameh LE, Arvidsson A-K, Carraway KL III, Couvillon AD,
Rathbun G, Crompton A, VanRenterghem B, Czech MP, Ravichandran KS,
Burakoff SJ, Wang D-S, Chen C-S, Cantley LC. A comparative
analysis of the phosphoinositide binding
specificity of pleckstrin homology domains. J Biol
Chem. 1997;272:22059–22066.
47. Rameh LE, Chen C-S, Cantley LC. Phosphatidylinositol (3,4,5)P3 interacts with SH2 domains and modulates PI 3-kinase association with tyrosine-phosphorylated proteins. Cell.. 1995;83:821–830.[Medline] [Order article via Infotrieve]
48.
Bae YS, Cantley LG, Chen C-S, Kim S-R, Kwon K-S, Rhee
SG. Activation of phospholipase C- by phosphatidylinositol
3,4,5-trisphosphate. J Biol Chem. 1998;273:4465–4469.
49.
Thomas JW, Ellis B, Boerner RJ, Knight WB, White GC
II, Schaller MD. SH2- and SH3-mediated interactions between focal
adhesion kinase and Src. J Biol Chem. 1998;273:577–583.
50. Schwartz SM. Smooth muscle migration in atherosclerosis and restenosis. J Clin Invest. 1997;99:2814–2816.[Medline] [Order article via Infotrieve]
51. Newby AC, George SJ. Proliferation, migration, matrix turnover, and death of smooth muscle cells in native coronary and vein graft atherosclerosis. Curr Opin Cardiol. 1996;11:574–582.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  X.-J. Wang, K. Maier, S. Fuse, A. I. Willis, E. Olson, S. Nesselroth, B. E. Sumpio, and V. Gahtan Thrombospondin-1-Induced Migration Is Functionally Dependent Upon Focal Adhesion Kinase Vascular and Endovascular Surgery, June 1, 2008; 42(3): 256 - 262. [Abstract] [PDF]
|
|
|
|
 |
|
 X. Cai, D. Lietha, D. F. Ceccarelli, A. V. Karginov, Z. Rajfur, K. Jacobson, K. M. Hahn, M. J. Eck, and M. D. Schaller Spatial and Temporal Regulation of Focal Adhesion Kinase Activity in Living Cells Mol. Cell. Biol., January 1, 2008; 28(1): 201 - 214. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Thamilselvan, D. H. Craig, and M. D. Basson FAK association with multiple signal proteins mediates pressure-induced colon cancer cell adhesion via a Src-dependent PI3K/Akt pathway FASEB J, June 1, 2007; 21(8): 1730 - 1741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. W. Orr, M. A. Pallero, W.-C. Xiong, and J. E. Murphy-Ullrich Thrombospondin Induces RhoA Inactivation through FAK-dependent Signaling to Stimulate Focal Adhesion Disassembly J. Biol. Chem., November 19, 2004; 279(47): 48983 - 48992. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. G. Spinale Cell-Matrix Signaling and Thrombospondin: Another Link to Myocardial Matrix Remodeling Circ. Res., September 3, 2004; 95(5): 446 - 448. [Full Text] [PDF]
|
|
|
|
|
|
M. Campbell, W. E. Allen, C. Sawyer, B. Vanhaesebroeck, and E. R. Trimble Glucose-Potentiated Chemotaxis in Human Vascular Smooth Muscle Is Dependent on Cross-Talk Between the PI3K and MAPK Signaling Pathways Circ. Res., August 20, 2004; 95(4): 380 - 388. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. I. Willis, D. Pierre-Paul, B. E. Sumpio, and V. Gahtan Vascular Smooth Muscle Cell Migration: Current Research and Clinical Implications Vascular and Endovascular Surgery, January 1, 2004; 38(1): 11 - 23. [Abstract] [PDF]
|
|
|
|
 |
|
 E. A. Papakonstanti, M. Kampa, E. Castanas, and C. Stournaras A Rapid, Nongenomic, Signaling Pathway Regulates the Actin Reorganization Induced by Activation of Membrane Testosterone Receptors Mol. Endocrinol., May 1, 2003; 17(5): 870 - 881. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Lymn, M. K. Patel, G. F. Clunn, S. J. Rao, K. L. Gallagher, and A. D. Hughes Thrombospondin-1 differentially induces chemotaxis and DNA synthesis of human venous smooth muscle cells at the receptor-binding level J. Cell Sci., November 15, 2002; 115(22): 4353 - 4360. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Huang and C. D. Kontos Inhibition of Vascular Smooth Muscle Cell Proliferation, Migration, and Survival by the Tumor Suppressor Protein PTEN Arterioscler Thromb Vasc Biol, May 1, 2002; 22(5): 745 - 751. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||