* Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba R3A 1M4, Canada
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Abstract |
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The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with 1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src
family kinases, although caveolin has not been directly
implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with
1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK)
phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes
1
integrin/caveolin complexes. Depletion of caveolin in
uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further,
1
integrin/caveolin complexes could be disassociated by
uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle
cells blocks the association of Src family kinases with
1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling
necessary for normal
1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR
may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting
efficient signal transduction.
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Introduction |
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INTEGRINS are heterodimeric adhesion receptors implicated in many biological processes characterized by
cell movement, including inflammation, tissue remodeling, growth, and tumorigenesis (Hynes, 1992; Ruoslahti
and Reed, 1994
; Schwartz, 1997
). Integrin-dependent cellular adhesiveness depends not only on specific
/
integrin chains expressed by cells but also on ligand-induced
integrin redistribution and signal transduction leading to
cytoskeletal reorganization (Schwartz et al., 1995
; Burridge and Chrzanowska-Wodnicka, 1996
; Parsons, 1996
).
Signaling through the integrin family of adhesion receptors requires their association with cytoplasmic elements
capable of signal transduction because integrins lack intrinsic signaling capacity. In part, these dynamic aspects of integrin function are regulated by the physical association
of integrins with nonintegrin membrane proteins. These
include the tetraspan family of membrane proteins, integrin-associated protein, and CD98 (Lindberg et al., 1993
;
Berditchevski et al., 1996
; Fenczik et al., 1997
). In addition
we have previously reported that
1 integrin function is
regulated by its association with the GPI-linked nonintegrin receptor, the urokinase receptor (uPAR)1 (Wei et
al., 1996
). When human embryonic kidney cells are engineered by transfection to express uPAR, uPAR forms
complexes with
1 integrins and promotes adhesion of
these cells to vitronectin via a vitronectin binding site on
uPAR. Incorporation of
5/
1 integrins into complexes
with uPAR also inhibits the ability of these integrins to
bind fibronectin. Disruption of the uPAR/integrin complexes by uPAR-binding peptides blocks adhesion to vitronectin and restores the normal adhesiveness of 293 cells
for fibronectin. Thus, the ligand specificity and adhesive
function of this class of integrins is altered by the presence
of uPAR.
A second nonintegrin membrane protein, caveolin, was
also found to coimmunoprecipitate with 1 integrins and
uPAR in uPAR-transfected 293 cells (Wei et al., 1996
). Independently, Wary et al. (1996)
observed caveolin to coprecipitate with
1 integrins in detergent-lysed A431 cells,
which express high levels of uPAR and caveolin. Caveolin, discovered as an Src kinase substrate, is an ~21-kD membrane protein which binds cholesterol and a number of signaling molecules potentially linked with integrin function:
Src family kinases, heterotrimeric G proteins, and H-ras
(Li et al., 1995
, 1996a
). Caveolin has an intrinsic propensity to oligomerize and it is currently thought caveolin
oligomers contribute to formation of cholesterol-sphingolipid-rich microdomains within membranes and the distinct membrane structures termed caveolae (Parton, 1996
;
Harder and Simons, 1997
). Caveolae are enriched in a
number of nonreceptor signaling proteins and growth factor
receptor tyrosine kinases, but are not known to contain integrins (Lisanti et al., 1994
; Mineo et al., 1996
). However,
while this work was in progress it was reported that a fraction of cellular caveolin associates with
1 integrins and promotes Fyn-dependent Shc phosphorylation in response
to integrin ligation, leading to mitogen-activated protein
kinase (MAPK) activation and cellular growth (Wary et
al., 1998
). In this report we provide direct evidence that
caveolin is also important to
1 integrin-dependent fibronectin adhesion and focal adhesion kinase (FAK) activation and propose that this operates, at least in part, by
virtue of the ability of caveolin to regulate Src family kinase activity surrounding integrins. Thus, caveolin appears
to be a general regulator of
1 integrin function.
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Materials and Methods |
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Generation and Transfection of Caveolin cDNA and Antisense Constructs
Human caveolin-1 cDNA was generated by reverse transcription and
polymerase chain reaction, subcloned into TA cloning vector (Invitrogen), verified by nucleotide sequencing (Sequenase; United States Biochemical), digested with BamHI and Xhol, and then subcloned into the inducible expression vector pML1 (Lukashev et al., 1994) in an inverted
order. Cells were transfected by electroporation, clones selected in hygromycin, and levels of caveolin-1 assessed by immunoblotting after overnight culture with 3 µM CdCl2. Caveolin cDNA was also subcloned into
the CMV promoter-driven expression vector, pCEP9 (Invitrogen), and
clones selected by growth in G418. Transfected cells were then screened
for protein expression via immunoblot with antibody specific for caveolin-1
(Transduction Labs).
Adhesion and Binding Assays
Cells (5 × 104) were seeded in fibronectin (5 µg/ml; Sigma Chemical Co.)
or vitronectin (1 µg/ml; Becton Dickinson) coated 96-well tissue culture
plates, incubated at 37°C for 1 h, and adherent cells quantified as described previously (Wei et al., 1996). Before the adhesion assay, antisense
caveolin 293 or uPAR/293 cells were induced with 3 µM CdCl2 at 37°C
for 8 h.
Fibronectin binding was assessed on cells seeded in polylysine-coated
96-well tissue culture plates, cultured overnight, induced with 3 µM CdCl2
at 37°C for 8 h, and then washed with binding buffer (DMEM, 1 mg/ml
BSA). Half of the cells were treated with 1 integrin activating antibody
TS2/16 (3 µg/ml; Endogen) at 4°C for 30 min, and then all cells were incubated with buffer containing 125I-fibronectin (1, 5, and 10 nM) at 4°C for
1.5 h. After washing, cells were lysed in 0.2% SDS, 0.2% Triton X-100,
10% glycerol in PBS for 30 min, and radioactivity was measured. Nonspecific binding was determined by inclusion of 0.5 mM RGDS (American
Peptide Co.) in the reaction mixture.
Flow Cytometry
Cells transfected with caveolin antisense constructs were induced with 3 µM
CdCl2 at 37°C for 8-18 h. Cells were then detached and incubated with
PBS containing 0.1% BSA and primary antibodies to uPAR (R2) or integrins 1 (JB1A) (Wilkins et al., 1996
),
5
1 (Chemicon), and
v (L230)
(American Type Culture Collection) on ice for 30 min. After washing,
cells were incubated with FITC-conjugated goat anti-mouse IgG (Sigma
Chemical Co.) and analyzed on a flow cytometer (FACScan®; Becton
Dickinson).
Immunofluorescence and Confocal Fluorescence Microscopy
To assess the distribution of caveolin and 1 integrins, human saphenous
vein smooth muscle cells (SMC) and 293 or uPAR/293 cells coexpressing
caveolin were cultured overnight on glass coverslips in 10% FBS. After
washing, cells were incubated for 30 min with JB1A and/or K20 (Immunotec) antibodies to
1 or isotype controls at 4°C and fixed for 20 min in
3.7% paraformaldehyde. Fixed cells were permeabilized and incubated
with rabbit polyclonal antibody to caveolin (Transduction Labs) for 30 min at room temperature, then incubated with FITC- or Rhodamine red-
conjugated secondary antibodies and coverslips mounted in Prolong (Molecular Probes). Fluorescence staining was analyzed by confocal laser
(model MRC1024; Bio-Rad Laboratories) attached to a Zeiss microscope
(model Axiovert S100) using separate filters for each fluorochrome. Optical planes were imported into Adobe Photoshop and processed. To visualize integrin and caveolin clustering, SMC in suspension were incubated with FITC-K20
1 antibodies without or with goat anti-mouse secondary antibodies for 1 h at 37°C, immobilized on 50 µg/ml polylysine-coated coverslips for 10 min, and then fixed and permeabilized as above. Cells were
stained for caveolin and analyzed by confocal laser microscopy.
Immunoprecipitation and Blotting
Control or antisense caveolin clones were cultured for 8 h in serum-free
DMEM containing 3 µM CdCl2 on either fibronectin (5 µg/ml), vitronectin (1 µg/ml), or polylysine (50 µg/ml) and lysed on ice for 30 min in buffer
containing 50 mM Hepes (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM
sodium orthovanadate, 1 mM phenylmethyl-sulfonylfluoride, and leupeptin (10 µg/ml). After preclearing with protein A-agarose, lysates were incubated with antibodies to 1 integrin (JB1A) or cortactin (Upstate Biotechnology Inc.) at 4°C overnight. In some experiments, the lysates were made 10 µM in the caveolin-related peptide, cav-1 (DGIWKASFTTFTVTKYWFYR), or the control cav-x peptide (WGIDKAFFTTSTVTYKWFRY), in 0.25% DMSO (Michel et al., 1997
). The cav-x peptide
has an identical amino acid composition and 60% identical sequence to
the cav-1 peptide. The immunoprecipitates were blotted with caveolin antibody (Transduction Labs), stripped, and reblotted for Src family kinases (Santa Cruz Biotech), cortactin, or 4G10 (Upstate Biotechnology Inc.).
To analyze FAK and EGF receptor activation, clones were induced with
CdCl2 and plated on fibronectin- or polylysine-coated 100-mm dishes or
their
1 integrins clustered with antibodies. Cells were lysed or stimulated
with EGF (10 ng/ml; Upstate Biotechnology Inc.) for 5 min before lysing
in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% deoxycholate, 0.1% SDS, 1% Triton X-100) supplemented with protease inhibitors.
Lysates were immunoblotted for ErbB-2, -3, and -4 (Santa Cruz Biotech)
and phosphotyrosine (Upstate Biotechnology Inc.).
1 immunoprecipitates from m1-m5 clones were immunoblotted with uPAR antibody 399R
(American Diagnostica).
Kinase Assay
To measure total enzymatic activity of 1-associated Src kinases, induced
cells were lysed in Triton X-100 buffer and immunoprecipitated with
1
antibody (JB1A). The immunocomplexes were suspended in 24 µl of reaction solution containing 50 mM Pipes, pH 7.0, 10 mM MnCl2, 5 µg of
acid-denatured enolase, 10 µM ATP, 10 µCi of [
-32P]ATP, and reacted at
room temperature for 10 min. The kinase reaction was stopped by sample
buffer, and samples were resolved on 10% SDS-PAGE and visualized by autoradiography.
For determination of basal activity of Src, induced cells were lysed in RIPA buffer and ~500 µg of lysate immunoprecipitated with Src mAb (Calbiochem) and procedures described above followed.
Phospho-MAPK Assay
SMC were plated onto fibronectin- (5 µg/ml) or polylysine-coated (50 µg/ ml) dishes at 37°C for 20 min in serum-free DMEM/BSA with or without peptide 25 or 36 (75 µM). Cells were lysed in RIPA buffer and immunoblotted with phospho-specific MAPK antibody which detects p42 and p44 MAPK catalytically activated by phosphorylation at Tyr204/Thr202 (New England Biolabs). The membrane was stripped and reprobed for total MAPK protein as control.
Migration Assay
Passage 1-3 SMC (105) were seeded into Transwell (Costar; Becton Dickinson) inserts containing 8-µm polycarbonate filters precoated on the bottom with fibronectin, and then cultured overnight in serum-free DMEM containing BSA (5 mg/ml) with or without peptide 25 or 36 (50-100 µM). Cells on both sides of the filter were detached by trypsin-EDTA and counted. All assays were performed in triplicate and the data expressed as percent inhibition by the peptides.
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Results |
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1 Integrins and Caveolin Partially Colocalize
Because caveolin oligomers are relatively insoluble in nonionic detergents, the reported association between 1 integrins and caveolin could possibly be an artifact of cellular
disruption by detergents. Therefore, the distribution of
1
integrins and caveolin in cells in situ was assessed by immunostaining. Primary cultures of human vascular SMC
(Fig. 1, A and B) were examined because these cells express relatively high amounts of
1 integrins (red) and caveolin-1 (green). Confocal microscopic analysis indicated these two proteins had marked spotty or array-like colocalizations over the cell body of SMC grown on glass
coverslips (Fig. 1 C). Substitution of isotype Ig controls
for primary
1 and caveolin antibodies resulted in much
weaker or no staining (Fig. 1 D) verifying the specificity of
the immunostaining. Because
1 integrins and caveolin-1
only partially colocalize, we determined if
1 integrins and
caveolin would cocluster. Clustering of SMC
1 integrins
in suspension at 37°C resulted in the emergence of bright
fluorescent signals of integrins (green) (Fig. 1 F) and caveolin (red) (Fig. 1 E) indicating clusters. Superimposition of
the signals (Fig. 1 G) confirmed coclustering. No caveolin
clustering was observed when the integrins were nonclustered (Fig. 1 H) or nonimmune Ig was substituted for the
primary caveolin antibodies (Fig. 1 I). These images indicate that fractions of
1 integrins and caveolin colocalize
and are physically linked in cells before cellular disruption
with detergents and are consistent with the hypothesis that
caveolin has a role in integrin function.
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Caveolin Depletion in 293 Cells Blocks 1
Integrin Function
To explore more directly the importance of caveolin to 1
integrin function, parental 293 cells were transfected with
antisense full-length mRNA for caveolin-1 under the influence of an inducible murine metallothionein promoter.
Several clones with inducible suppression of caveolin protein expression were identified and examined. After 12-18 h
of induction in the presence of 3 µM cadmium, two clones
with normal caveolin levels, 6 and 9 (Fig. 2 A), displayed strong adhesion to fibronectin (Fig. 2 B). By contrast, two
representative antisense clones, 31 and 32, which had 15-
50% the level of caveolin in parental 293 cells (Fig. 2 A),
displayed markedly impaired adhesion to fibronectin (Fig.
2 B). Although 293 cells have only weak vitronectin adhesion, mediated by
v
5 and
v
1, this adhesion was also
abrogated in caveolin-deficient cells (not shown). As is
also indicated in Fig. 2 A, there was substantially less caveolin in these clones even without induction, indicating leakage of antisense expression, which has been reported for
this expression vector in previous studies (Lukashev et al.,
1994
). Adhesion assays mirrored the extent of caveolin depletion: although they were capable of spreading on fibronectin, clones 31 and 32 were less adhesive even in the absence of induction (Fig. 2 B). Nonetheless, clones 31 and
32 grew nearly as well as clones 6 and 9 over many months
of passage. Flow cytometric analyses revealed no differences in surface level of
1 or
v or
5
1 heterodimers among
these clones (Fig. 2 C), indicating that depletion of caveolin expression affected the function rather than expression
of
1 or its dimerization with
5 or other integrins.
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Caveolin antisense clones also exhibited time-dependent changes in cell shape with decreasing caveolin expression. Clones 6 and 31 were allowed to spread onto fibronectin for 1 h at 37°C, and then antisense caveolin mRNA induced. Within 8 h, clone 31 cells, but not clone 6 cells, became less spread. Within 18 h, clone 31 cells became round (Fig. 2 D) and numerous cells actually detached from the matrix. The shape changes paralleled loss of focal adhesion sites as indicated by loss of focal paxillin accumulation on ventral surfaces (Fig. 2 D). In spite of the marked changes in adhesiveness and shape, most cells were found to respread and adhere better within hours of removing cadmium.
Caveolin Depletion in 293 Cells Does Not Impair
Integrin Ligand Binding or Lateral Movement but
Blocks Signaling through 1 Integrins
We next considered mechanisms by which caveolin could
influence integrin function. Because integrin expression
per se was not changed in caveolin-depleted cells (Fig. 2
C), the capacity of the various clones to bind 125I-fibronectin in the absence and presence of a stimulating antibody (TS2/16) known to promote fibronectin binding by the
major fibronectin receptor, 5
1, was determined (Masumoto and Hemler, 1993
). After induction, clones 6 and 31 were found to bind fibronectin equally at 4°C and exposure of the cells to TS2/16 enhanced fibronectin binding
to a similar degree (Fig. 3 A), indicating that the affinity states of
5
1 in control and caveolin-depleted clones are
similar. Further, when surface
1 integrins were cross-linked by antibodies, the
1 integrins of normal (clone 6)
and caveolin-depleted cells (clones 31 and 32) clustered
equally well as judged by confocal microscopic analysis
(not shown). In spite of the capacity of
1 integrins on caveolin-depleted clones to engage ligand, the changes in
cell shape and adhesiveness provoked by caveolin depletion (Fig. 2) could not be reversed by the stimulatory
1
integrin antibody (TS2/16). These data imply that a postreceptor binding event(s) important to adhesion, rather than
binding per se, is most likely impaired in caveolin-depleted
cells.
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Src family tyrosine kinases are required for fibronectin
receptor 5
1 and vitronectin receptor
v
5-mediated
cell adhesion (Bergman et al., 1995
; Kaplan et al., 1995
).
Moreover, tyrosine phosphorylation of FAK and other
signaling molecules important to cytoskeletal reorganization and cell spreading after ligand engagement of
1 integrins may depend on interactions between FAK and
Src family kinases (Parsons, 1996
; Schlaepfer and Hunter, 1997
). Because caveolin has been shown previously to interact with c-Src (Li et al., 1996 a,b), it is possible that integrin-mediated tyrosine phosphorylation depends on caveolin. To test this hypothesis, we initially treated 293 cells
with the tyrosine kinase inhibitor genestein (100 µM) and
noted that 293 cells failed to attach and spread on fibronectin (data not shown). When
1 integrins were clustered with antibodies or 293 cells were allowed to adhere to fibronectin, increased FAK phosphorylation was apparent (Fig. 3, B and C). In contrast, 293 cells with suppressed
caveolin expression failed to exhibit FAK phosphorylation
whether in response to antibody-induced clustering or
ligand binding. This defect was not a general defect in tyrosine phosphorylation as autophosphorylation of ErbB-2, an EGF receptor-like tyrosine kinase, after binding of
EGF, was not different between control and caveolin-deficient clones (Fig. 3 D).
The 1 integrins were next examined for interaction
with nonreceptor tyrosine kinases by coprecipitation. Caveolin depletion of 293 cells (clones 31 and 32) resulted in
loss of caveolin and Src family kinases in the
1 immunoprecipitates (Fig. 4 A). The antibody used in this experiment was a pan-Src antibody recognizing multiple members of the src gene family. In additional experiments using specific antibodies we found c-Src, Fyn, and Yes in
the
1 immunoprecipitates, all of which were almost completely lost in the
1 immunoprecipitates of caveolin-depleted cells (not shown). A major substrate for Src
kinase, cortactin, implicated in adhesion and migration
(Vuori and Ruoslahti, 1995
), was also markedly depleted in these precipitates. In situ kinase assays of
1 immunoprecipitates confirmed markedly reduced activity in cells
(clones 31 and 32) depleted of caveolin (Fig. 4 B).
Although little or no Src family kinase or cortactin associates with 1 integrins in caveolin-depleted cells (Fig. 4
A), this was not because tyrosine kinases could not be activated in these cells. Rather, kinase assays of immunoprecipitated c-Src confirmed a two- to fourfold increase in
total c-Src activity in the caveolin-deficient clones (not
shown). Previously, caveolin levels have been linked inversely to cellular Src kinase activity (Li et al., 1996a
). Enhanced total kinase activity correlated with a striking hyperphosphorylation of cortactin in the caveolin-depleted
clones (31 and 32) compared with the control clone 6 (Fig.
4 A). Immunoblots of the cortactin immunoprecipitates
revealed increased Src family kinases associated with tyrosine-phosphorylated cortactin, possibly through Src SH2 domain (Thomas and Brugge, 1997
). These data indicate
that caveolin-deficient 293 cells have dysregulated Src
family kinase activity and little or no tyrosine kinase activity associated with their
1 integrins.
Caveolin Depletion Disrupts uPAR/Integrin Complexes and Abrogates uPAR-mediated Adhesion to Vitronectin
uPAR forms complexes with 1 and
2 integrins and such
complexes are important to uPAR-dependent adhesion to
matrix vitronectin via a vitronectin binding site on uPAR
(Wei et al., 1996
). Caveolin is also found in uPAR/
1 integrin complexes. To explore the functional importance of
caveolin to these complexes, we created stable cotransfectants of 293 cells expressing both uPAR and the antisense
full-length mRNA for caveolin-1. Several clones with inducible suppression of caveolin expression were again
identified. Control clones expressing uPAR but without
reduction in caveolin levels adhered avidly to vitronectin
(Fig. 5 A). Clones with <50% the normal level of caveolin protein (clones m2-m5) adhered weakly or not at all to
vitronectin (Fig. 5 A) even though uPAR and
1 integrin
surface expression was not changed as judged by FACS®
analysis (data not shown). Immunoprecipitation of
1 integrins coprecipitated uPAR in proportion to caveolin levels and the ability of the cells to adhere to vitronectin (Fig.
5 A), indicating that caveolin is required for stable uPAR/
integrin complexes and vitronectin adhesion. As with
nontransfected 293 cells (Fig. 4), depletion of caveolin in
uPAR/293 cells resulted in almost complete loss of Src
family kinases from the
1 integrin immunoprecipitates (Fig. 5 B). As expected, the caveolin-depleted uPAR/293
cells also failed to adhere to fibronectin (not shown).
In prior work, we observed that incorporation of 5/
1
integrins into complexes with uPAR, while stabilizing caveolin/integrin interactions, inhibited the ability of these
integrins to bind fibronectin. Indeed, uPAR-transfected
293 cells had impaired fibronectin adhesion (Wei et al.,
1996
). In light of the critical role for caveolin in 293 cell adhesion to fibronectin (Fig. 2) and the fact that 293 cells
have relatively low levels of caveolin, we considered the
possibility that uPAR expression also inhibited
1 integrin
function indirectly by sequestering caveolin into uPAR/
integrin complexes. Therefore, we transfected uPAR/293
cells with wild-type caveolin-1 and assessed the capacity of
these transfectants to adhere to vitronectin and fibronectin. Concurrent caveolin overexpression in uPAR/293 cells
had no effect on uPAR-dependent adhesion to vitronectin
but completely restored the capacity of these cells to adhere to fibronectin (Fig. 5 C). These data suggest competition for limited caveolin between unbound
1 integrin and uPAR/
1 integrin complexes may largely explain the impaired fibronectin adhesion of uPAR-transfected 293 cells.
Disruption of 1 Integrin/Caveolin Complexes with
Caveolin and uPAR-Binding Peptides
To explore further the functional connection between caveolin and uPAR in assembly of integrin-associated signaling molecules, lysates of uPAR/293 cells were immunoprecipitated for 1 integrins in the presence of either a
peptide (peptide 25) (Wei et al., 1996
) which disrupts
uPAR/integrin associations or a peptide derived from the
"scaffolding domain" of caveolin-1 (cav-1). In experiments using purified proteins, cav-1 disrupts self-oligomerization
of caveolin and blocks direct interactions between caveolin and other proteins, including c-Src (Li et al., 1996a
;
Michel et al., 1997
). Both peptide 25 and cav-1 blocked coprecipitation of caveolin with
1 integrins in lysates prepared from either uPAR/293 cells or primary human vascular SMC (Fig. 6 A). Both peptides, but not inactive
control peptides, also blocked coprecipitation of Src family kinases and cortactin with
1 integrins (Fig. 6 A), verifying that the stable association of these cytoplasmic proteins with integrins is dependent upon the membrane
proteins caveolin and uPAR in these cells. Neither control
peptide (peptide 36 and cav-x) had any effect on caveolin,
cortactin, or Src coprecipitation with
1 integrins when
compared with a no peptide control (Fig. 6 A). Further,
peptide 25 had no effect on coprecipitation of caveolin (or
Src kinases) with
1 integrins in nontransfected 293 cells
(not shown), confirming the specificity of this peptide for uPAR.
Vascular SMC are known to express uPAR and 1 integrins (Clyman et al., 1992
; Okada et al., 1995
) and we confirmed uPAR expression by immunostaining. Clustering
of
1 integrins resulted in uPAR coclustering, though no
uPAR was detectable in these
1 immunoprecipitates by
immunoblotting (data not shown). Although complex formation of
1 integrins with uPAR could be expected to inhibit fibronectin adhesion (Wei et al., 1996
), SMC attach
and spread well on fibronectin, mostly via
5
1. As discussed above, this may be explained by the relatively high
levels of caveolin in SMC as compared with 293 cells.
Nonetheless, peptide 25 but not a control peptide, was
found to disrupt caveolin and Src kinase associations with
1 integrins almost completely in lysates of SMC (Fig. 6
A). Therefore, we used this peptide to explore the role of
caveolin in the integrin function of intact SMC. Incubation
of SMC adherent to fibronectin with peptide 25 before lysis resulted in loss of Src kinases (as well as caveolin) in
the
1 immunoprecipitates (Fig. 6 B). Consistent with the
loss of signaling molecules, integrin-dependent MAPK activation was also blocked by peptide 25 (Fig. 6 B). It
should be noted that the pathway of integrin and uPAR-
dependent MAPK activation in these cells is not yet defined. Although MAPK is reproducibly phosphorylated
within 30 min of fibronectin attachment, we can detect
no Shc or FAK phosphorylation (not shown). The inhibitory effects of the peptide on association of Src family kinases with integrins was correlated with loss of integrin
function. Although neither peptide blocked adhesion,
peptide 25 delayed SMC spreading on fibronectin and
blocked migration of human SMC across microbore filters
to fibronectin-coated surfaces (Fig. 6 C), consistent with
its effect on integrin signaling.
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Discussion |
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Expression of the capacity of integrins to mediate cellular
adhesion has several distinct facets: (a) the conformational
state of integrins, affecting the affinity of ligand binding; (b)
ligand-induced integrin clustering, enhancing the strength
of attachment and promoting interactions between kinases
and their substrates important to signal transduction; and
(c) signaling itself, activating a cascade of events leading
to organization of the cytoskeleton and cell spreading
(Schwartz et al., 1995; Burridge and Chrzanowska-Wodnicka, 1996
; Parsons, 1996
). Numerous in vitro studies and
recent evaluations of mice with targeted deletions in nonreceptor tyrosine kinases implicate FAK and Src family kinases as key mediators of integrin signaling (Furata et al.,
1995; Ilic et al., 1997
; Meng and Lowell, 1998
). Thus, the
molecular events leading to assembly and activation of
these kinases surrounding integrins are important determinants of integrin function. This conclusion has focused
much attention on nonintegrin membrane proteins which
might regulate this process and our data identify one such
protein as caveolin. While caveolin does not affect integrin
expression or the intrinsic capacity of
1 integrins to bind
fibronectin (Figs. 2 and 3), caveolin is required for the normal assembly of adhesion plaques which develop in response to ligand engagement and integrin clustering. In
the absence of sufficient
1 integrin-associated caveolin, there is loss of integrin-associated Src kinase activity, little or no FAK activation after ligand binding, and impaired
association of integrins with structural proteins such as tyrosine-phosphorylated cortactin important to
1 integrin-
dependent adhesion (Fig. 4). As a result, the characteristic
accumulation of enzymes and structural proteins which
comprise integrin-dependent adhesion sites fails to develop (Fig. 2).
Our results confirm and extend the recent findings of
Wary and colleagues (1998) that caveolin expression is required for the association of Fyn kinase with 1 integrins
and ligand-dependent Shc phosphorylation. These investigators began with cells (Fisher rat thyroid cells) expressing
little or no caveolin or
5 integrin and transfected both
proteins. They found that physical association of the Src
family kinase Fyn with
5/
1 required concurrent coexpression of caveolin-1. Fyn but not Src was required for
5/
1-dependent Shc phosphorylation. Beginning with cells
having functional
1 integrins and expressing caveolin-1,
we suppressed caveolin expression and found
1 integrin
association with several Src family kinases to be completely dependent on caveolin. In both cases, deficient
1
integrin-associated Src family kinase activity led to correspondingly marked changes in integrin function. Together,
these findings indicate caveolin is a general regulator of
1
integrin function.
How does caveolin promote integrin signaling? Although our understanding is incomplete, we favor a model
(Fig. 7) in which caveolin functions primarily to sequester
Src family kinases in an inactive configuration at sites
proximate to integrins, promoting their presentation to integrins and activation during ligand-induced integrin clustering. Caveolin has been shown previously to interact predominantly with the inactive form of Src and to reduce
Src activity when overexpressed in 293 cells, perhaps by
sequestering Src kinases from activating phosphatases and
substrates (Li et al., 1996b; Rodgers and Rose, 1996
). Consistent with this model, caveolin-deficient 293 cells were
found to have two- to fourfold higher levels of basal c-Src
kinase activity (not shown) and to exhibit striking hyperphosphorylation of a major Src substrate, cortactin (Fig.
4). If one function of caveolin is to suppress basal Src kinase activity, the model in Fig. 7 also offers a possible molecular explanation of how caveolin contributes to Src
family kinase activation as a consequence of integrin clustering. Caveolin interactions with Src family kinases are
thought to occur through the same membrane proximal
sites on caveolin as those involved in formation of caveolin
homo-oligomers (Okamoto et al., 1998
). Because coclustering of caveolin with integrins (Fig. 1) could be expected
to favor caveolin homo-oligomer formation, integrin clustering is likely to modify the physical interaction of caveolin with Src kinases. This may lead to outright release of
these kinases from caveolin (as shown in Fig. 7) and/or an
altered, more accessible position of these kinases in the
cluster favoring their activation through contact with substrate or through a phosphatase. In either case, these studies directly implicate caveolin function in the membrane
proximate events of ligand-induced,
1 integrin-dependent tyrosine kinase activation. As caveolin levels are reportedly low in a number of transformed cells (Koleske et al.,
1995
; Okamoto et al., 1998
), our data raise the possibility
that low caveolin levels contribute to the defective fibronectin adhesion and matrix assembly seen in many cancer cells and thought to be important to their metastatic
potential (Hynes, 1992
; Schwartz, 1997
).
|
A further implication of the studies reported here and
the recent studies of Wary and colleagues (1998) is that
Fyn and Src may have overlapping but distinct roles in 1
integrin signaling: Fyn being primarily required for Shc
phosphorylation and activation of an MAPK-dependent
growth pathway and Src being primarily required for FAK
and cytoskeletal phosphorylation and assembly of focal adhesion sites. This concept is supported by results of
prior studies examining mice deficient in the negative regulator of Src family kinase activity, Csk (Thomas et al.,
1995
). Fibroblasts from Csk
embryos exhibit hyperphosphorylation of adhesion sites and striking hyperphosphorylation of cortactin, reminiscent of that seen in caveolin-deficient cells (Fig. 4). The hyperphosphorylation of
adhesion sites and cortactin was largely corrected by crossing the Csk
mice with Src
mice but not by crossing with
Fyn
mice, again implicating Src as primarily being involved in regulation of integrin-dependent adhesion. Clearly
this is an oversimplification since we also observe Yes as
well as Fyn and Src in
1 integrin/caveolin complexes and
even kinase-inactive Src can promote focal contact assembly in Src
cells (Kaplan et al., 1995
). Still, these recent studies support the possibility that different members of
the src gene family have favored roles in integrin signaling
even in cells expressing multiple family members.
The structural basis for the association between caveolin
and 1 integrins is uncertain. First, most if not all cellular
caveolin is reportedly found in purified caveolae (Lisanti
et al., 1994
; Schnitzer et al., 1995
). Although integrins have
not been observed in purified caveolae, when we subject
293 cells to mechanical lysis, sonicate, and centrifuge the
postnuclear supernatant through a sucrose gradient, caveolin in 293 cells appears at the 5/35% sucrose interface,
typical for that reported for caveolae preparations in 293 and other cells (Song et al., 1996
; Ikezu et al., 1998
). Almost all of the
1 integrins appear in the fractions containing caveolin (Yang, X., unpublished observation). These
findings are consistent with the scenario that
1 integrin/
caveolin/Src family kinase complexes (encircled in Fig. 7)
exist as signaling units in caveolae and separate from caveolae in response to ligand-induced clustering and cytoskeletal reorganization. However, the exact composition of
caveolae remains controversial and appears strongly dependent on the method of purification (Lisanti et al., 1994
; Schnitzer et al., 1995
; Mineo et al., 1996
; Stan et al., 1998
). An alternative possibility is that some fraction of caveolin
traffics outside caveolae, as has been argued by Wary and
colleagues (1998). This will be an important issue for future experiments. In either case, the fraction of
1 integrins which colocalize and cocluster with caveolin (Fig. 1)
appears to be critical to
1 integrin signaling.
Whether 1 integrins directly bind caveolin is also uncertain. Wary and colleagues (1998) reported that the
5
transmembrane domain was required for coprecipitation
of
5/
1 and caveolin. However, no direct binding has
been demonstrated. Most if not all other proteins that are
reported to bind caveolin bind sequences of caveolin that
are membrane proximal but in the cytoplasmic compartment (Okamoto et al., 1998
). Indeed, in SMC we observed a peptide comprised of a membrane proximal region of caveolin (amino acids 82-101) blocking coprecipitation of
caveolin with
1 integrins (Fig. 5). Thus, it is uncertain
whether
1 integrins and caveolin directly bind or associate indirectly through a common affinity for certain membrane lipids or a third protein such as uPAR which may
directly interact both with integrins and cholesterol-rich lipid domains enriched in caveolin. The physical basis for
the association of
1 integrins and caveolin also requires
further investigation.
The marked inhibition of 1 integrin function by uPAR-binding peptides in human SMC (Fig. 6) is remarkable.
Although we could detect uPAR in SMC by immunostaining and could cocluster uPAR along with
1 integrins, no
uPAR was detected in the
1 immunoprecipitations of
SMC. This suggests the association of uPAR with
1 integrins and caveolin in these cells is relatively weak. There
also appears to be a stoichiometric excess of
1 integrins
compared with uPAR in SMC. Thus, it is surprising that
the coprecipitation of most caveolin- and integrin-associated Src kinases with
1 integrins would be blocked by
peptide 25, raising the possibility of an in vitro artifact.
This is an unlikely explanation for the findings because the
uPAR-binding peptide (peptide 25), but not two control peptides (peptide 36 and a scrambled version of peptide
25), also had clear biochemical and functional effects on
intact SMC. Prior treatment of SMC with the active peptide blocked Src kinase association with
1 integrins and
inhibited both signaling and migration of the cells on fibronectin (Fig. 6). These results indicate that, although not
intrinsically required for the function of integrin/caveolin (Fig. 2), the presence of uPAR organizes caveolin and its
associated signaling molecules in an interdependent manner such that integrins, caveolin, and uPAR form a unit
promoting integrin function. Perhaps this is possible because only a fraction of
1 integrins associates with caveolin (Fig. 1) and uPAR associates mainly with activated integrins (Wei et al., 1996
). As indicated in Fig. 7, integrin activation initiated by ligand-induced integrin clustering
may promote formation of these signaling complexes. Similar mechanisms may account for observations that uPAR
is required for normal function of the
2 integrins, CD11/
CD18 (Mac-1; Simon et al., 1996
; Sitrin et al., 1996
; May et
al., 1998
), and for migration on vitronectin mediated by
v
5 (Yebra et al., 1996
), suggesting a promoting effect of
uPAR on integrin function may be a widespread phenomenon.
![]() |
Footnotes |
---|
Address correspondence to Harold A. Chapman, M.D., Brigham and Women's Hospital, Thorn Research Building, Rm. 803, 20 Shattuck St., Boston, MA 02115. Tel.: (617) 732-6074. Fax: (617) 232-4623. E-mail: hchapman{at}rics.bwh.harvard.edu
Received for publication 19 August 1998 and in revised form 2 February 1999.
Y. Wei and X. Yang contributed equally to this work.
The authors thank Martin Hemler for helpful discussions and Thomas Michel for providing cav-1 and cav-x peptides.
This work was supported by a grant from the National Institutes of Health (HL44712) to H.A. Chapman.
![]() |
Abbreviations used in this paper |
---|
FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase; SMC, smooth muscle cells; uPAR, urokinase receptor.
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