From the Instituto de Farmacología y
Toxicología CSIC-UCM, Facultad de Medicina, Universidad
Complutense, 28040 Madrid, Spain, the ¶ Servicio de
Inmunología, Hospital de la Princesa, 28006 Madrid, Spain,
** INSERM U268, Hôpital Paul Brousse, Villejuif, 94807 France, the
Departamento de
Bioquímica y Biología Molecular, Facultad de
Ciencias Químicas, Universidad Complutense, 28040 Madrid,
Spain, and the §§ Departamento de
Bioquímica, Facultad de Medicina, Universidad Autónoma de
Madrid, 28029 Madrid, Spain
Received for publication, August 1, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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Tetraspanins associate on the cell membrane with
several transmembrane proteins, including members of the integrin
superfamily. The tetraspanin CD9 has been implicated in cell motility,
metastasis, and sperm-egg fusion. In this study we characterize the
first CD9 conformation-dependent epitope (detected by
monoclonal antibody (mAb) PAINS-13) whose expression depends on changes
in the activation state of associated The tetraspanins (also called TM4SF) are integral membrane
proteins with four transmembrane regions delimiting two extracellular regions of unequal sizes. All tetraspanins share the presence of a CCG
motif and several conserved cysteine residues in the large
extracellular domain. The tetraspanin family currently comprises more
than 25 members, including the differentiation antigens CD9, CD37,
CD53, CD63, CD81, CD82, and CD151. These proteins have been implicated
in different cellular functions, including adhesion, migration,
differentiation, and signal transduction (reviewed in Refs. 1-4).
The tetraspanins act as molecular facilitators or adaptors that
organize a network of interactions among a subset of cell surface
molecules, known as the "tetraspanin web." Among the proteins identified as components of the tetraspanin web are many
immunoglobulin superfamily protein members (including the CD4 and CD8
antigens on T cells), the CD19 molecule on lymphoid cells, the major
histocompatibility complex class II molecules, and several
members of the The tetraspanin CD9 is a 21-24-kDa surface molecule, which was
initially identified as a lymphohemopoietic marker (9), and was later
implicated in cell motility (10), metastasis (11, 12), neurite
outgrowth (13), myotube formation and maintenance (14), and in
sperm-egg fusion (15). CD9 seems to act as a suppressor of metastasis
because its transfection into melanoma cells reduces their metastatic
potential (16) and an inverse correlation between expression of CD9 and
appearance of metastasis in melanoma, colon, lung, and breast cancers
has been described (12, 17-19). The tissue and cellular distribution
of CD9 is very wide, and most immortalized cell lines tested have been
found to express detectable levels of this tetraspanin (20-22).
Notable exceptions are several leukocytic cell lines of both the
lymphocytic (Daudi, Raji, and HSB-2) or the myeloid (U-937, KG1)
lineages (21).
Some members of the In this study we characterize the first CD9
conformation-dependent epitope whose expression depends on
changes in the activation state of associated Cell Culture--
The human microvascular endothelial cell line
HMEC-1 (33) was grown in MCDB-131 medium (Sigma) supplemented with 20%
fetal bovine serum, 2 mM glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, 10 ng/ml recombinant human epidermal growth
factor (Promega), and 1 µg/ml hydrocortisone (Sigma) on
gelatin-coated (0.5%) flasks. Human umbilical vein endothelial cells
(HUVEC) were obtained and cultured as previously described (34) and
used within the first 6 passages. Human T lymphocytes (primary T
lymphoblasts obtained from healthy blood donors as previously described
(35), HSB-2, and Jurkat cell lines), B lymphocytes (Raji, Ramos, and JY
cell lines), K562 erythroleukemic, DX3 and A375 melanoma, NB100
neuroblastoma, Colo320 colocarcinoma, and mouse L929 fibroblast cell
lines were cultured in RPMI 1640 medium supplemented with 10%
fetal bovine serum and antibiotics. K562 cells stably transfected with
Antibodies--
Several anti-integrin monoclonal antibodies were
used in this study, including the anti- Adhesive Proteins--
Matrigel basement membrane matrix was
obtained from the Engelbreth-Holm-Swarm sarcoma cells as previously
described (43). Fibronectin and poly-L-lysine were
purchased from Sigma. Laminin I was either purchased from Sigma or
purified from the Engelbreth-Holm-Swarm sarcoma cells (44).
Generation of PAINS-13 MAb--
Confluent HMEC-1 endothelial
cells were collected using 5 mM EDTA, pH 8, and
intraperitoneally injected into Balb/c mice on days Flow Cytometry Analysis--
Cells were washed twice, incubated
with primary antibodies at 4 °C for 20 min, washed, and incubated
for 30 min on ice with fluorescein isothiocyanate-conjugated anti-mouse
IgG (Sigma). After washing three times, cells were fixed in 2%
formaldehyde in PBS, and fluorescence was measured using a
FACScanTM flow cytometer (BD Biosciences). To study the
effect exerted by temperature on the expression of different cellular
surface molecules, an aliquot of cells was incubated at 4 °C and
another aliquot at 37 °C with the primary antibody for 20 min and
subsequently FACS analysis was performed as described above. The effect
of divalent cations was studied in cells washed with Hepes buffer (20 mM Hepes, 150 mM NaCl, 1 mg/ml glucose)
containing 2 mM EGTA, and then resuspended in Hepes buffer.
The primary antibody was supplemented or not with MnCl2
(1-6 mM depending on the cellular type) during 20 min at
37 °C. For direct activation of Immunofluorescent Staining--
HUVEC endothelial cells were
grown on 12-mm glass coverslips precoated with 1% gelatin. Cells were
incubated with the appropriate primary antibody for 1 h at room
temperature, washed twice, and incubated for 30 min in the presence of
secondary antibody Alexa FluorTM 488 goat anti-mouse IgG
(Molecular Probes, Leiden, Netherlands). After washing with
Tris-buffered saline (TBS) containing 1 mM CaCl2, 1 mM MgCl2, cells were fixed
with 3% formaldehyde in PBS for 10 min at room temperature and mounted
with Mowiol (Calbiochem). In some cases, cellular fixation was
performed before the incubation with the primary antibody and
nonspecific binding sites were blocked with TNB (0.1 M
Tris-HCl, 0.15 M NaCl, 0.5% blocking reagent; Roche
Molecular Biochemicals) for 20 min. Samples were examined with a Nikon
Labophot-2 photomicroscope and images were acquired with a COHU high
performance CCD camera (Cohu, Tokyo, Japan) connected to a LEICA Q550
CW work station (Leica Imaging Systems, Ltd., Cambridge, UK) using the
LEICA QFISH V1.01 Software.
Transient Transfections--
Mouse fibroblast L929 cells were
trypsinized and resuspended in RPMI 1640, 10% fetal calf serum medium
supplemented with 5 µl of 1.5 M NaCl, 20 µg of
pBlueScript (Stratagene), and 5 µg of the CD9-EGFP or CD151-EGFP
cDNAs. Cells were electroporated at 975 microfarads/200 V in a Gene
Pulser II (Bio-Rad) and used for immunofluorescence experiments 24 h after transfection. The CD9-EGFP fusion protein construct was
obtained by PCR amplification of CD9-cDNA followed by cloning in
the pEGFP-N1 vector (Clontech Laboratories) (45).
For epitope mapping, the CD9 and CD82 negative colocarcinoma Colo320
cells were transiently transfected in 2.5% SBF RPMI 1640 with 20 µg
of different cDNA coding for CD9, CD82, and for the different
chimeric tetraspanin molecules CD9 × 82, CD82 × 9, CD82LEL9, and CD82CCG9 all subcloned in the pcDNA3 vector (Invitrogen). Cells were electroporated at 412.5 V/cm in an
ElectroSquarePorator ECM 830 (BTX) and used for FACS detection 24 h after transfection.
Cell Labeling and Immunoprecipitations--
HMEC-1 confluent
monolayers were labeled using 0.3-0.5 mg/ml biotin
3-sulfo-N-hydroxysuccinimide ester (Sigma) in PBS with 1 mM CaCl2, 1 mM MgCl2,
for 30 min at 4 °C. Cells were lysed in 1% digitonin, 1% Brij 96, or in different concentrations of Triton X-100 buffer (in TBS
supplemented with 1% BSA, 1 mM MgCl2, 1 mM CaCl2, 1 mM phenylmethylsulfonyl
fluoride, 0.2 units/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml
pepstatin A). After 30 min at 4 °C, cellular material was recovered
using a cellular scraper and the insoluble portion was removed by
centrifugation at 14,000 rpm for 15 min. Protein G-Sepharose was
precleared with cellular lysates from unlabeled cells, and coupled with
the specific antibodies for 2-4 h at 4 °C. After removing the
excess antibodies, they were incubated overnight at 4 °C with the
biotinylated lysates. Beads were then rinsed three times with diluted
(1:10) lysis buffer in TBS and immune complexes were eluted by boiling
in Laemmli buffer and resolved in a 12% acrylamide/bisacrylamide
SDS-PAGE gel, followed by transfer to nitrocellulose membranes. Blots
were blocked with 5% BSA in TBS and the chemiluminescence was measured using the Immunopure ABC peroxidase staining kit (Pierce) in TBST (TBS,
0.1% Tween 20) and chemiluminescence (ECL detection kit, Amersham Biosciences).
Immunoblotting--
Immunoprecipitations were performed as
described above from unlabeled cells. After transfer to nitrocellulose
membranes, blots were blocked with 5% BSA in TBS at room temperature
and developed with primary biotinylated antibodies followed by
incubation with avidin peroxidase (Sigma). Chemiluminescence was
measured as described above.
Adhesion and Cellular Spreading Assays--
For cellular
adhesion and spreading assays, 96-microwell plates (Costar) were coated
with different extracellular matrix components (30 µg/ml laminin I, 5 µg/ml fibronectin, or 50 µg/ml poly-L-lysine as a
control) overnight at 4 °C and blocked with 1% heat-denatured BSA
for 2 h at room temperature. The trypsinized HMEC-1 cells were
washed and resuspended in MDCB-131 medium (without serum and growth
factors, and supplemented with 20 mM Hepes and 0.1% BSA)
containing 10 µg/ml of each purified mAb. 1.25 × 105 cells (for adhesion assays) or 1.5 × 104 (for spreading assays) were added to the coated wells
and incubated for 10 min at 4 °C to allow cells to sediment onto the
well bottom, followed by 15 (for laminin-I) or 5 min (for fibronectin)
incubations at 37 °C. In adhesion assays, unbound cells were removed
by filling the wells with PBS, 1 mM CaCl2, 1 mM MgCl2, 20 mM Hepes, 0.1% BSA,
sealing with plastic, and inverting the plates for 30 min at 37 °C.
After fixation of bound cells in 3.7% formaldehyde and treatment with
2% methanol followed by 70% crystal violet solution, adhesion was
analyzed by measuring the absorbance of eluted crystal violet at 540 nm. For cellular spreading assays, the fixed adherent cells were
photographed in a phase-contrast Nikon Diaphot 300 inverted microscope
equipped with a Sony SSC-M350 CE CCD video camera and VCR and the
cellular perimeter was calculated using the image analysis software
Optimas 5.2 (Bioscan, Edmonds, WA).
In Vitro Angiogenesis Assay--
80 µl of Matrigel were added
into flat-bottomed 96-well tissue culture plates at 4 °C and allowed
to gel during 1 h at 37 °C. Then, 25 × 103
HMEC-1 cells were seeded at the top of the gel in a volume of 80 µl
with 20 µg/ml purified mAbs. After 6 h incubation, tubular structures were formed and images were recorded on TMAX 400 film (Kodak) with a phase-contrast microscope (Nikon ELWD 0.3). Tubes were
defined as cellular extensions linking cell masses (46).
Wound Healing Assays--
Wound healing assays were performed as
a modification of the previously described method (42). HMEC-1 cells
were seeded on 0.5% gelatin-coated 24-well plates until confluence.
16-18 h before the assay, complete medium was replaced by basal medium without growth factors. The cellular monolayer was disrupted by a
1200-µm wide wound and immediately the medium was replaced by fresh
medium containing the antibodies (20 µg/ml), and cells were cultured
at 37 °C. Wounds were photographed at the beginning and end of the
assay with a phase-contrast Nikon Diaphot 300 inverted microscope
equipped with a Sony SSC-M350 CE CCD video camera and VCR. The area of
the wound was calculated using the image analysis software Optimas 5.2 (Bioscan).
PI3 Kinase Assay--
PI3K assays were performed as described by
Shaw et al. (47) with some modifications. HMEC-1 cells were
cultured on 0.5% gelatin-coated dishes to confluence and medium
without growth factors was added 16 h before the assay. Antibodies
(20 µg/ml) were added for 20 min at 37 °C. After two washes using
cold PBS, 1 mM CaCl2, 1 mM
MgCl2, an additional cross-linking was performed with a
rabbit anti-mouse IgG (1:200, Sigma) for 10 min at 4 °C, followed by
30 min at 37 °C. Then, chilled lysis buffer (20 mM Tris
buffer, pH 7.4, 140 mM NaCl, 1% Triton X-100, 10%
glycerol, 1 mM sodium orthovanadate, 2 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, pepstatin, and
leupeptin) was added and cells were removed using a cellular scraper.
Lysis was performed during 30 min at 4 °C and the nonsoluble
material was removed by centrifugation at 12,000 rpm for 10 min.
Cellular lysates were incubated overnight at 4 °C with an anti-p85
subunit of PI3K antibody bound to Protein G-Sepharose. The
immunoprecipitates were washed three times with diluted lysis buffer,
and twice in kinase buffer (20 mM Tris, pH 7.5, 75 mM NaCl, 20 mM Hepes, 10 mM
MgCl2, and 200 µM adenosine). Beads were then
resuspended in 40 µl of kinase buffer containing 10 µg of
L- MAb PAINS-13 Is Specific for Human CD9 Tetraspanin--
The
monoclonal antibody PAINS-13 was selected as a CD9-specific antibody
based on the following criteria: (i) its high reactivity and specific
pattern of immunofluorescence staining of monolayers of primary HUVEC
endothelial cells (Fig. 1A);
(ii) PAINS-13 immunoprecipitates from Brij 96 lysates of human
microvasculature HMEC-1 cells were identical to immunoprecipitates
obtained with other characterized anti-CD9 mAbs (Fig. 1B);
(iii) the anti-CD9 specific mAb VJ1/20 recognizes a band of 24 kDa
corresponding to CD9 in Western blots of PAINS-13 and VJ1/20
immunoprecipitates from HMEC-1 lysates (Fig. 1C); (iv) mAb
PAINS-13 specifically recognizes mouse fibroblast L929 cells
transfected with a human CD9-EGFP cDNA construct (Fig. 1D) but was unreactive with mock-transfected cells or cells
transfected with other tetraspanin-coding cDNA constructs
(CD151-EGFP, data not shown), and (v) mAb cross-competition binding
assays on HMEC-1 cells in which cell preincubation with anti-CD9 mAb
VJ1/20 completely abolished subsequent reactivity of mAb PAINS-13, but
preincubation with different antibodies to CD31, VE-cadherin,
The Epitope Recognized by MAb PAINS-13 on the CD9 Tetraspanin
Is Dependent on the Activation State of Associated
These striking differences in cellular reactivity between mAb PAINS-13
and other "classical" anti-CD9 antibodies led us to consider the
possibility that PAINS-13 might be recognizing a subpopulation of CD9
molecules displaying a specific conformation. To test this hypothesis,
we compared the PAINS-13 immunoprecipitates from HMEC-1 cells
solubilized in increasing stringency conditions (i.e. 0.1%
Triton X-100, 0.5% Triton X-100, 1% Triton X-100) under which the
interactions formed among TM4SF and associated membrane proteins are
disrupted. As Fig. 2b shows, a 24-kDa band corresponding to
CD9 is clearly visible in the mAb PAINS-13 precipitates from lysates
obtained with 0.1% Triton X-100 (panel A). In contrast, mAb
PAINS-13 was no longer able to precipitate the CD9 molecule under more
stringent conditions such as 0.5% Triton X-100 (panel B) or
1% Triton X-100 (panel C) or under 1% digitonin lysis
conditions (data not shown). In addition, the fact that PAINS-13 is
unreactive in Western blotting (not shown) also suggests that PAINS-13
could be specific for a CD9 conformational epitope.
As the motion in the plane of membrane, the level of association and
changes in conformation of many cell surface molecules are all
processes characterized by their temperature dependence, we decided to
analyze whether the expression of the PAINS-13 epitope on the CD9
molecules was also affected by this parameter. For this purpose, we
compared the expression of the PAINS-13 epitope on different cell types
at 4 and 37 °C. Fig. 3A
shows that in DX3 or NB100 cells, which display a high expression of
the CD9 molecule and moderate basal expression of PAINS-13, this
epitope was up-regulated at 37 °C compared with 4 °C. In
contrast, the expression of the CD9 epitopes detected by two classical
anti-CD9 mAbs, VJ1/20 and VJ1/10 (not shown), was not affected. As
expected, 37 °C was also ineffective at inducing any expression of
PAINS-13 epitope in all the CD9-negative cell lines tested, including
HSB2, Ramos, and Raji (not shown). Most importantly, the PAINS-13
epitope was not inducible on the CD9-positive B lymphocytic cell line JY, which lacks
The presence of
Although changes in the temperature and divalent cations regulate the
activation state of
At this point we decided to answer whether the CD9 conformation
detected by mAb PAINS-13 was preferentially dependent on a particular
integrin Location of the PAINS-13 Epitope on the CD9 Molecule--
To
identify the CD9 region that is detected by mAb PAINS-13 we used the
following CD9 × CD82 chimeric molecules (depicted in Fig.
5): CD9 × 82 (comprising the
NH2 portion of the CD9 molecule up to the beginning of the
large extracellular loop (LEL)); CD82 × 9 (comprising the
NH2 portion of CD82 molecule up to the beginning of the
LEL; CD82LEL9 (comprising the large extracellular loop of CD9) and
CD82CCG9 (comprising the NH2 portion of CD82 molecule up to
the CCG motif of LEL). Transient expression of full-length CD9,
full-length CD82, and of each of these cDNA chimeric constructions in the human CD9 Relationship between CD9 Conformation and
To probe the functional consequences of CD9 engagement with mAb
PAINS-13, we analyzed the effect of this antibody in in
vitro wound healing assays of HMEC-1 endothelial cell monolayers.
As shown in Fig. 6A, both the
inhibitory anti-
Although the wound healing assay is dependent on
As the functional effects of PAINS-13 epitope engagement were very
similar to those exerted upon activation of
As integrin-mediated adhesion in adherent cells leads to reorganization
of cytoskeleton and cellular spreading, we also quantitated changes in
the spreading of HMEC-1 endothelial cells adhering to laminin-1 and to
fibronectin substrates. As shown in Fig. 7B, the stimulatory
mAb TS2/16 induced a 149% increase in cellular spreading on laminin-1
over control conditions (100%), and mAb PAINS-13 similarly induced a
169% increase, reflecting the activation of the
Engagement of the PAINS-13 Epitope Activates PI3K--
Activation
of PI3K is a hallmark of The tetraspanin CD9 is a widely distributed cell surface molecule
found in numerous tumor cells, as well as in neural, vascular, and
leukocytic cells. CD9 expression in several types of cancer is
inversely correlated with their metastatic potential and patient survival (12). Through the use of transfection and monoclonal antibodies specific for the CD9 molecule, this tetraspanin has been
implicated in cell motility and migration (10), in vitro wound healing repair and angiogenesis (50), metastasis (17), neurite
outgrowth (13), myotube formation and maintenance (14), and in
sperm-egg fusion (15). Recently, some of us have described the
implication of CD9 in transendothelial migration of melanoma cells
through a mechanism involving strengthening of melanoma-endothelial cell heterotypic interactions (45). The involvement of CD9 in all these
functions seems to depend on its association with other proteins, and
particularly those belonging to the integrin family, in molecular
complexes known as the tetraspanin web.
In the present study we have characterized the first CD9 epitope,
recognized by mAb PAINS-13, whose expression depends on conformational
changes transmitted upon activation of the associated In a recent paper by Geary et al. (51) several monoclonal
antibodies to the tetraspanin CD151 have been characterized and found
to react differentially with this molecule based on tissue staining and
binding to hemopoietic cells. These authors demonstrate that these
differences in mAb reactivity are because of masking/unfolding of the
conformational epitopes recognized in the integrin-CD151 protein
complexes. Interestingly, in this study it is shown that transfection
of K562 cells with either Through the use of different CD9 × CD82 intertetraspanin chimeric
constructions we have demonstrated that the reactivity of mAb PAINS-13
requires the integrity of the CD9 region comprising the first
NH2-half LEL, which encompasses residues 112-154. The regions of the CD9 molecule involved in the association of this tetraspanin with integrins have not been characterized yet, but interestingly, Berditchevski and co-workers (49, 50) have recently
described that the region of tetraspanin CD151 necessary and sufficient
for stable association with integrin Several tetraspanins, including CD9, have been proposed to mediate some
of the functional activities of associated cell surface molecules,
including regulation of cell migration and
adhesion-dependent signaling through In the wound healing assays, it is known that endothelial cells produce
as they move a deposition of different extracellular matrix components
including laminin, type IV collagen, and thrombospondin (54). In this
type of assay endothelial cell motility is inhibited by mAbs that
affect the activity of Increased adhesion and subsequent spreading onto specific ligands for
The formation of cord-like structures by endothelial cells plated on
basement membrane matrix (Matrigel) depends on Signal transduction through integrins in many different cellular
systems involves the activation of PI3K, an event which in turn
regulates many other important cell functions such as cell survival and
apoptosis and the activity of Rho family small GTPases, which control
cell morphology, polarization, and migration (26, 47, 55, 56). So far,
however, very few reports have described the implication of
tetraspanins in integrin-mediated signal transduction. Sugiura and
Berditchevski (24) have reported that different Taken together, our results obtained in these four different functional
assays clearly show that the functional biological effects after
engagement of the CD9 conformation-dependent epitope PAINS-13 are similar to those exerted by the stimulatory
anti-1 integrins.
MAb PAINS-13 precipitates CD9 under conditions that preserve the
association of this tetraspanin with integrins, but not under
conditions that disrupt these interactions. Induction of activation of
1 integrins by temperature, divalent cation
Mn2+, or mAb TS2/16 correlated with enhanced expression of
the PAINS-13 epitope on a variety of cells. Through the use of
different K562 myeloid leukemia transfectant cells expressing specific
members of the
1 integrin subfamily we show that the
expression of the PAINS-13 epitope depends on CD9 association with
6
1 integrin. The mAb PAINS-13 reactivity
has been mapped to the CD9 region comprising residues 112-154 in the
NH2 half of the large extracellular loop. Also, we
show that the CD9 conformation recognized by mAb PAINS-13 is
functionally relevant in
1 integrin-mediated cellular processes including wound healing migration, tubular morphogenesis, cell adhesion and spreading and in signal transduction involving phosphatidylinositol 3-kinase activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 subfamily of integrins on many cell
types (Refs. 1-4, and references therein). The organization of the
tetraspanin web is based on several levels of interactions (2, 5-7).
The first level relates to primary interactions between a particular
tetraspanin and specific proteins known as tetraspanin partners. These
first level interactions are direct, resistant to detergents such as
digitonin and Triton X-100, and of high stoichiometry (i.e.
>50% in the complex). A second level of interactions are indirect,
more numerous and resistant to Brij 96 detergent extraction, and arise
as tetraspanins associate to each other linking multiple primary
complexes. A third level of tetraspanin organization relates to the
large and light complexes that are resistant to detergents such as Brij
99 or CHAPS1 and display
partial lipid rafts properties (8).
1 integrin subfamily, particularly
3
1 (VLA-3),
4
1 (VLA-4), and
6
1 (VLA-6), are among the
tetraspanin-associated cell surface proteins found in the tetraspanin
web in most cellular systems. The contribution of tetraspanins to
adhesion-dependent signaling seems to be linked to their
ability to recruit signaling components such as protein kinase
C, PI3K, or PI4K into the integrin complexes (23, 24). The
1 integrin subfamily comprises heterodimeric transmembrane proteins that mediate cell adhesion to extracellular matrix proteins, including laminin (
3
1
and
6
1), collagens (
1
1,
2
1,
and
3
1), fibronectin
(
4
1 and
5
1), and also cell-cell interactions
(
4
1/VCAM-1) (25). Integrins not only mediate cell adhesion but work as effective bidirectional signal transducers (25, 26). The adhesive and signaling capacities of
integrins depend on transitions between conformational changes in these
receptors that reflect distinct "activation states" defined by
their increased or decreased ability to mediate ligand interactions or
signal transduction (27-29). Ligand binding, physiologic temperature (37 °C), the presence of divalent cations Mg2+ and
Mn2+ in the extracellular medium, and several specific
"stimulatory" monoclonal antibodies (mAbs) that provoke a
conformational change upon binding to a particular integrin, are among
the factors known to induce the activation of integrins. The activation
state of many integrins can be monitored with the use of a different
type of specific mAbs ("activation reporters") that recognize
conformation-dependent integrin epitopes whose expression
reflect changes in the integrin avidity or/and affinity (29-32).
1
integrins. Through the use of different K562 transfectant cells
expressing specific members of the
1 integrin subfamily
we have determined that the expression of the PAINS-13 epitope depends
preferentially on the presence of
6
1 integrin. These interactions are functionally relevant in
1 integrin-dependent cellular processes
including wound healing, migration, tubular morphogenesis, cell
adhesion and spreading, and signal transduction involving PI3K
activation and may therefore play a regulatory role in cell migration
during angiogenesis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 and
4 integrin subunits were kindly
provided by Dr. J. Teixidó (Centro de Investigaciones
Biológicas, CSIC, Madrid, Spain) (36).
3-K562 transfectants were a gift from Dr. M. Hemler (Dana-Farber Cancer Institute, Boston, MA) (37) and
6-K562 transfectants
were a gift from A. Sonnenberg (Netherlands Cancer Institute,
Amsterdam, The Netherlands) (38). Stable K562 transfectants were
maintained in 0.2 mg/ml G418 as selection agent.
1 mAb 5E8D9 (39),
the stimulatory anti-
1 integrin mAb TS2/16 (40), the
"blocking" anti-
1 integrin mAb Lia1/2 (41), and
the "activation reporter" mAb HUTS-21 that recognizes the
activated conformation of
1 integrin (30). The anti-CD9
antibodies used in this study were VJ1/20 and VJ1/10 (42), and mAb
10B1, which detects an epitope located in the second (COO2)
half of the large extracellular loop of
CD9.2 The anti-CD82 mAb TS82
has been previously described (21). Other antibodies used were mAb Tea
1/31 (anti-VE-cadherin) and mAb TP1/15 (anti-CD31) (42). The monoclonal
Ig P3X63 myeloma protein (IgG1,
) was used as a negative control
(41). The anti-p85 subunit of PI3K antibody was purchased from
Transduction Laboratories (Lexington, KY). When necessary, purified
antibodies were biotinylated with
3-sulfo-N-hydroxysuccinimide ester (Sigma) as previously described (30).
22 and
12 and
intravenously on day
3. Spleen cells were fused on day 0 with SP2
mouse myeloma cells at a 4:1 ratio and hybridomas were distributed in
96-well culture plates. After 7 days the hybridoma supernatants were
harvested and their reactivity against the HMEC-1 used in the
immunization was assayed by flow cytometry. Positive clones obtained by
the limiting dilution method were selected and inoculated into Balb/c
mice to generate ascitic fluid. Monoclonal antibodies were purified
from ascitic fluid and concentrated by affinity chromatography using a
column of protein G-Sepharose and eluted using sodium citrate 0.1 M, pH 3.0. MAb PAINS-13 is an IgG3.
1 integrins, cells
were incubated for 20 min at 37 °C with the stimulatory mAb TS2/16
(10 µg/ml) in Hepes buffer containing 1 mM
CaCl2, 1 mM MgCl2, and biotinylated
PAINS-13 or HUTS-21 (20 µg/ml) mAbs were then added and incubated at
37 °C for an additional period of 20 min and finally incubated with
avidin-fluorescein isothiocyanate (Sigma) and processed for FACS
analysis. To increase the HUTS-21 epitope expression on the K562
transfectants, N-acetylcysteine (50 mM) was
used, and the FACS assay was performed after eliminating this agent
before the primary antibody was added to the cells.
-phosphatidylinositol (Sigma), the reaction was
started by addition of 5 µCi of [
-32P]ATP, carried
out for 15 min at room temperature, and stopped on ice. Phospholipids
were extracted in chloroform/methanol/HCl, desiccated under
N2 flow, and dissolved in
chloroform/methanol/H2O (75:25:2 in volume) prior to being
resolved by thin layer chromatography on Silica Gel 60 plates (Merck)
and analyzed by autoradiography. Total precipitated enzyme was
quantified by Western blot using the same anti-p85 subunit antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,
2,
3,
4,
5,
6, or
1 integrin had no
blocking effect (data not shown).
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Fig. 1.
mAb PAINS-13 is specific for CD9.
A, immunofluorescence staining of HUVEC cells. HUVEC
cells were plated on glass coverslips coated with 1% gelatin and
cultured to confluence and stained with the specific mAbs TS2/16
( 1 integrin), Tea 1/31 (VE-cadherin), VJ1/20 (CD9), and
PAINS-13. B, immunoprecipitation analysis with mAb
PAINS-13 from HMEC-1 lysates. HMEC-1 cells were surface-biotinylated
and lysed in 1% Brij 96 detergent. Immunoprecipitations were carried
out using mAbs TS2/16 (
1 integrin), VJ1/20 (CD9),
PAINS-13, and the ×63 negative control mAb (
). The immune complexes
were resolved by reducing and nonreducing 12% SDS-PAGE, and
biotinylated proteins were detected using the ABC avidin peroxidase
system. Double volume of cellular lysate was used for precipitation
with mAb PAINS-13 compared with the other antibodies.
C, Western blot (WB) analysis of PAINS-13
immunoprecipitates (IP). HMEC-1 cells were lysed with 1%
Brij 96 and precipitated with either PAINS-13 or VJ1/20 (CD9). After
migration under nonreduced conditions, proteins were transferred to a
nitrocellulose membrane and probed with the biotinylated-VJ1/20 mAb
followed by avidin peroxidase and ECL chemiluminescence.
D, PAINS-13 recognizes CD9-transfected cells. Mouse
fibroblasts L929 were transiently transfected with a CD9-EGFP cDNA
construct and plated in glass coverslips. After 24 h of culture,
cells were fixed with 3% formaldehyde and stained with the mAb
PAINS-13 followed by the Rhodamine RedTM-X goat anti-mouse
IgG secondary antibody. hCD9-EGFP expressing cells are shown in the
left panel and PAINS-13 staining in the right
panel.
1
Integrins--
When the cellular reactivity of anti-CD9 mAb PAINS-13
was compared with that of the previously described anti-CD9 antibody VJ1/20 in a variety of cells from different lineages (Fig.
2a), striking
differences were observed: whereas the reactivity of mAb VJ1/20 was
strong for all the CD9-positive cell types tested, PAINS-13 only
yielded a high reactivity in HUVEC and HMEC-1 endothelial cells but a
weak to moderate staining in NB100, DX3, A375, and Jurkat cell
lines.
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Fig. 2.
The CD9 epitope recognized by mAb PAINS-13
differs from other CD9 epitopes. a, analysis of the
expression of the epitope recognized by mAb PAINS-13 on different cell
types. Flow cytrometric analysis of the reactivity of mAb PAINS-13 with
a variety of human cells, including endothelial HUVEC and HMEC-1 cells,
melanoma (DX3 and A375), neuroblastoma (NB100), erythroblastoma (K562),
primary interleukin 2-activated T lymphoblasts (T blasts), T
lymphocytic (Jurkat and Ramos), and B lymphocytic (Raji and JY) cell
lines. Cells were stained with mAb PAINS-13 at 4 °C followed by a
fluorescein isothiocyanate-conjugated secondary anti-mouse antibody and
analyzed on a FACScan. The fluorescence profiles of stained cells
(black) are compared with ×63 control mAb
(white). b, CD9 recognition by PAINS-13 in cell lysates is lost under increasing detergent stringency
conditions. Surface-biotinylated HMEC-1 cells were lysed using 0.1%
Triton X-100 (A), 0.5% Triton X-100 (B), and 1%
Triton X-100 (C). After overnight immunoprecipitations using
mAbs TS2/16 ( 1 integrin), VJ1/20 (CD9), PAINS-13, and
the ×63 control mAb (
), the immune complexes were resolved by
nonreducing SDS-PAGE, and the total surface-biotinylated proteins were
revealed by blotting using the ABC avidin peroxidase system.
1 integrin expression (Fig.
3A).
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Fig. 3.
Regulation of PAINS-13 expression.
A, modulation by temperature. Cells expressing CD9
(DX3 and NB100) or not (HSB2), or without
expression of 1 integrin (JY) were
resuspended in Hepes/NaCl, 1 mM CaCl2, 1 mM MgCl2, and incubated with VJ1/20 or PAINS-13
at 4 °C (white histogram) or at 37 °C (black
histogram) followed by fluorescein isothiocyanate-conjugated
anti-mouse IgG. B, modulation by divalent cations.
Cells previously washed with Hepes/NaCl-EGTA buffer were incubated at
37 °C with the TS2/16 (
1 integrin), HUTS-21
(activated
1 integrin), VJ1/20 (CD9), or PAINS-13
antibody in the absence (white histogram) or presence of 1 mM MgCl2 (black filled histogram).
C, modulation by activation of
1
integrin. DX3 cells were stimulated using the TS2/16 activatory
antibody (left panels), a combination of TS2/16 mAb and
divalent cations (central panels), or with a nonstimulatory
5E8D9 mAb (
1 integrin) as a control (right
panels). The
1 integrin activation was measured by
the HUTS-21 expression (upper panel) without stimuli
(white histogram) or in its presence (black filled
histogram) compared with the modulation on the PAINS-13 epitope
expression (lower panel).
1 integrins in PAINS-13
immunoprecipitates together with the modulation by temperature of the
expression of this epitope on some cell lines but not on the
1-negative JY cells, prompted us to investigate whether
the expression of the CD9 conformation detected by mAb PAINS-13 was
dependent on changes in conformation of
1 integrins. In
this regard, we first studied the effects of the divalent cation
Mn2+ (a well characterized stimulus for the activation of
the adhesive and signaling properties of
1 integrins) on
the PAINS-13 expression. As shown in Fig. 3B, an important
increase in the expression of the PAINS-13 epitope on DX3 and NB100
cells occurred upon replacement of extracellular divalent cations
Ca2+ and Mg2+ by a concentration of 1 mM Mn2+, which correlated with a parallel
increase in the expression of the activation-reporter epitope HUTS-21
on the
1 integrin molecules. As with temperature,
treatment with Mn2+ did not induce any detectable increase
in the expression of PAINS-13 either on the
CD9-positive/
1-negative cell line JY nor on the CD9-negative cell lines HSB2 (Fig. 3B), Ramos, or Raji (not
shown), further pointing out the
1 integrin dependence
for expression of the PAINS-13 epitope. The Mn2+-induced
up-regulation of PAINS-13 expression was specific, as the total
expression of CD9 (detected by mAb VJ1/20) or
1 integrin (detected by mAb TS2/16) remained unaltered after Mn2+
treatment (Fig. 3B).
1 integrins, these conditions could also be affecting a variety of other cellular targets. To address more
specifically the issue of the dependence of the PAINS-13 epitope
expression on
1 integrin activation, we directly induced the activation of
1 integrins by using the stimulatory
mAb TS2/16. As shown in Fig. 3C, pretreatment of DX3 cells
with mAb TS2/16 induced an important increase in expression of the
PAINS-13 epitope, which correlated with the enhanced expression of the
1 integrin activation epitope HUTS-21. Taken together,
our results clearly demonstrate that mAb PAINS-13 recognizes an epitope
on the CD9 molecule whose expression is dependent on conformational
changes conveyed through members of the
1 integrin subfamily.
subunit. For this purpose, we employed the erythroblastoid
K562 cells (wt-K562), which displays moderate levels of the CD9
molecule and only express the
5 integrin subunit associated to
1 (
5-K562), and K562
transfectant cells expressing
2 (
2-K562),
3 (
3-K562),
4
(
4-K562), or
6 (
6-K562)
integrin subunits. All these cells were treated with 5 mM
Mn2+ to induce activation of the
1
integrins. We also used N-acetylcysteine, an antioxidant
agent that induces important changes in the conformation of
1 integrins (presumably by reduction of disulfide bonds
in the cysteine-rich region of the
1 integrin
subunit),3 as revealed by the
strong expression of the HUTS-21 epitope. As shown in Fig.
4, Mn2+ or
N-acetylcysteine only induced a detectable increase of
PAINS-13 expression in the
6-K562 cells, clearly
implying that the
6
1 integrin molecules
in the tetraspanin web are preferentially responsible for transmitting
the conformational changes to CD9 molecules resulting in expression of
PAINS-13 epitope.
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Fig. 4.
Modulation of PAINS-13 expression upon
activation of different 1 integrin
heterodimers in K562 transfectants.
1 integrin
activation measured by changes in HUTS-21 epitope (upper
panels) and modulation of PAINS-13 epitope (lower
panels) were assayed in K562 cells expressing only
5
1 integrin and in the stable
transfectant cells of each one of the integrin subunits
(
2,
3,
4, and
6) as described under "Experimental Procedures."
Basal levels of each epitope are shown by gray filled
histograms. Expression after treatment with 5 mM
Mn2+ is shown by thick line histograms and
expression after treatment with 50 mM
N-acetylcysteine (NAC) is shown by thin
line histograms.
/CD82
Colo320 cell line and
subsequent FACS analysis clearly showed that mAb PAINS-13 reacts with
the region comprising the NH2 half of CD9 LEL, which
encompasses CD9 residues 112-154.
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Fig. 5.
Mapping of the PAINS-13 epitope on the CD9
molecule. The CD9 /CD82
Colo320
colocarcinoma cells were transiently transfected with the cDNA
coding for full-length CD9 (solid thick line) and CD82
(dotted line) tetraspanins and with cDNA constructs
coding for the following chimeric proteins: CD9 × 82 (comprising
the NH2 portion of CD9 molecule up to the beginning of the
LEL); CD82 × 9 (comprising the NH2 portion of CD82
molecule up to the beginning of the LEL; CD82LEL9 (comprising the LEL
of CD9) and CD82CCG9 (comprising the NH2 portion of CD82
molecule up to the CCG motif of LEL). The precise amino acidic sequence
in junctions of each chimeric molecule is shown under each
schematic construct. FACS analysis of transfected cells was performed
using the indicated mAbs. Data are expressed as mean fluorescence
intensity (M.I.F.) and percent of positive cells. Positive
expression of the PAINS-13 epitope is highlighted in gray
boxes.
1 Integrin
Function--
The tetraspanin CD9 has been proposed to mediate some of
the functional activities of associated cell surface molecules,
including regulation of cell migration and
adhesion-dependent signaling through
1
integrins (23, 24, 48, 49).
1 integrin mAb Lia1/2 and the
stimulatory
1 integrin-specific mAb TS2/16 caused an important (43.6 and 50.7%, respectively) inhibition in the repaired area of the wounds over control conditions, clearly indicating the
dependence on a balanced level of
1 integrin adhesion
for endothelial motility in this assay. The mAb PAINS-13 caused a 64.2% inhibition of endothelial cell motility in this type of assay.
Interestingly, two conformation-independent mAbs specific for CD9,
VJ1/10 and VJ1/20, exerted very different effects on endothelial cell
motility, with mAb VJ1/20 causing a 81.2% inhibition, whereas mAb
VJ1/10 caused only a marginal 19.6% inhibition. These results show
that the functional consequences after ligation of the tetraspanin CD9
clearly depend on the specific antibody engagement of distinct epitopes
on CD9 molecules.
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Fig. 6.
Functional effects of mAb PAINS-13 in wound
healing and Matrigel morphogenesis. A, PAINS-13
inhibits in vitro wound healing. A 1200-µm wide wound was
inflicted to monolayers of HMEC-1 endothelial cells and cultured at
37 °C in the absence (CONTROL) of any mAb or in the
presence of 20 µg/ml purified mAbs TS2/16 (stimulatory
1 integrin), PAINS-13, VJ1/20, or VJ1/10 (CD9), TP1/15
(CD31), or Lia1/2 (inhibitory
1 integrin). Area wounds
for each case were measured before and 9 h after the treatment,
and the repaired area was calculated. Experiments were performed in
duplicate and data represent the mean ± S.E. from five
independent experiments. B, effect of PAINS-13 in
Matrigel in vitro angiogenesis assay. HMEC-1 were seeded on
Matrigel either in the absence (CONTROL) or presence of 20 µg/ml Lia1/2 (anti-
1 integrin), TS2/16 (anti
1 integrin), VJ1/20 (anti-CD9), or PAINS-13 mAbs during
6 h while tubular structures were formed. Images are
representative of three independent experiments, each done in
triplicate. The number of tubes in each condition was quantitated and
is shown in the bar graph.
1
integrin adhesion, it does not permit discrimination between agents
that act through augmentation or inhibition of adhesive activity of these integrins. We therefore analyzed the formation of anastomosing angiotubular cord-like cellular structures on Matrigel basement membranes, which has been used as an in vitro model for cell
morphogenesis and angiogenesis. As shown in Fig. 6B, while
the inhibitory anti-
1 mAb Lia1/2 completely blocked the formation of
angiotubes, the stimulatory anti-
1 mAb TS2/16 and the
anti-CD9 mAbs VJ1/20 and PAINS-13 all induced an important enhancement
in the number of these cellular cord-like structures.
1 integrin with the stimulatory mAb TS2/16 in both functional cellular assays tested (inhibition of in vitro wound healing and enhancement
of formation of angiotubular cellular structures), we decided to probe
the direct effects of PAINS-13 engagement on
1 integrin activation. For this aim, we first quantitated the effects of mAb
PAINS-13 on the adhesion of HMEC-1 endothelial cells to laminin-1 (a
ligand for
6
1 integrin) and fibronectin
(a ligand for
4
1 and
5
1 integrins). Interestingly, PAINS-13
engagement only induced an increase in cell adhesion to laminin-1 but
had no effect on cell adhesion to fibronectin (Fig.
7A), again supporting the
specific dependence on
6
1 integrin for
PAINS-13 functional effects. As expected, the stimulatory mAb TS2/16
induced an increase in cell adhesion to both ligands.
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Fig. 7.
mAb PAINS-13 induces increased cell adhesion
and spreading of HMEC-1 cells specifically on laminin-1.
A, effect of PAINS-13 on adhesion of HMEC-1 cells.
Adhesion assays were performed in the presence of the indicated mAbs as
described under "Experimental Procedures." Data represent the
mean ± S.E. of percentages of adherent cells from three
independent experiments. Adhesion under control conditions was
considered as 100%. B, effect of PAINS-13 on spreading
of HMEC-1 cells. Spreading assays were performed in the presence of the
indicated mAbs as described under "Experimental Procedures."
Inset shows the spreading of several representative cells on
laminin-1 in the absence or presence of mAb PAINS-13. Data represent
the mean ± S.E. of cellular perimeter (µm) of 100 cells in each
condition from four independent experiments. Quantitation of cellular
spreading under control conditions was considered as 100%.
PLL, poly-L-lysine.
6
1 integrin. As expected, mAb TS2/16 also
increased the spreading of HMEC-1 cells on fibronectin but, in
contrast, mAb PAINS-13 had no effect on cells spreading on this
substrate, clearly indicating that the observed effects are
specifically mediated by
6
1 integrin.
1-mediated integrin signaling.
Because mAb PAINS-13 recognizes a CD9 epitope whose expression depends
on
1 integrin activation, we used this antibody to probe the functional implication of the specific subpopulation of CD9 molecules expressing this epitope. As shown in Fig.
8, cross-linking of CD9 molecules with
mAb PAINS-13 induced a strong activation of PI3K in endothelial HMEC-1
cells. This activation was similar to that induced after direct
activation of
1 integrin with mAb TS2/16. Cross-linking
of other conformation independent anti-CD9 mAbs (VJ1/10 and VJ1/20) or
cross-linking of other abundantly expressed molecules (anti-CD31 mAb
TP1/15) on HMEC-1 cells only exerted a minimal effect on activation of
PI3K. Taken together, these results show that the functional
effects after engagement of the PAINS-13 epitope are very similar to
those obtained after ligation of
1 integrin with the
stimulatory antibody TS2/16.
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Fig. 8.
PAINS-13 induces the PI 3-kinase activation
in HMEC-1 cells. Monolayers of HMEC-1 cells were untreated ( ) or
treated with 20 µg/ml Lia1/2 or TS2/16 (
1 integrin),
VJ1/10 or VJ1/20 (CD9), TP1/15 (CD31), or PAINS-13 mAbs for 20 min at
37 °C, followed by cross-linking with a rabbit anti-mouse IgG. 1%
Triton X-100 lysates were immunoprecipitated using an anti-PI3K-p85
subunit antibody and assayed for PI3K activity. The
32P-labeled lipid products (PtdIns-3-P) were
separated by TLC. Total precipitated enzyme was quantified by Western
blot (WB) using the same anti-p85 subunit antibody. The
results shown are representative for three similar experiments.
Bar graph represents the increment in PI3K activity relative
to control basal conditions (
).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
integrins, and particularly of the
6
1
integrin heterodimer. This has been demonstrated by immunoprecipitation
analysis under different detergent stringency conditions, direct
induction of
1 integrin activation, and the use of K562
transfectants expressing specific
1 integrin members.
3 or
6
integrins results in increased expression of a subset of conformational
epitopes and reduced expression of others. One of the anti-CD151 mAbs
included in this report is TS151r, originally described by Rubinstein
and co-workers (5), which detects a CD151 conformational epitope that
is lost upon interaction with the
3
1
integrin. The expression of the CD9 epitope recognized by mAb PAINS-13
as described in this paper shows the opposite behavior, as it depends
on the presence of
1 integrins and particularly
6
1 on the cell surface, and is increased
upon integrin activation.
3
1
is the second COO2-half of CD151 LEL. As it is likely that
equivalent regions in different tetraspanins are responsible for their
molecular association with
1 integrins, we could infer
that the tetraspanin CD9 associates with integrin
6
1 through its LEL COO2-half
and upon integrin activation a change in conformation is conveyed to
CD9 that is reported by enhanced expression of PAINS-13 epitope. Interestingly, two other anti-CD9 mAbs (VJ1/10 and VJ1/20) that recognize constitutively expressed/conformation-independent epitopes (see Figs. 2a and 3, A and B) also
recognize sequences located in the same first NH2-half of
CD9 LEL (see Fig. 5). These results suggest that this CD9 region must
be highly immunogenic and contain both conformation-independent type
(VJ1/10 and VJ20) and conformational-type (PAINS-13) epitopes.
1
integrins (23, 24, 48, 49, 53). We have addressed in this study the
functional consequences of CD9 engagement with mAb PAINS-13 in four
different types of functional assays in which the implication of
1 integrins is well established: (i) in vitro
wound healing assays in monolayers of endothelial cells, (ii) formation
of angiotubular endothelial cell structures on Matrigel, (iii) cell
adhesion to and spreading on
1 integrin-specific ligands
laminin-1 and fibronectin, and (iv) signal transduction and activation
of PI3K.
1 integrins, either through a
blockade or stimulation of their adhesive function (50). Engagement of
PAINS-13 epitope in these assays resulted in a strong inhibition of
endothelial cell motility, which might be reflecting an effect on the
associated
1 integrins. We have confirmed this
possibility by observing that mAb PAINS-13 actually induces an increase
in endothelial cell adhesion and spreading onto laminin-1, a ligand for
integrin
6
1, but not on the
4
1 and
5
1
ligand fibronectin. These results underline the specific dependence on
6
1 integrin for PAINS-13 functional effects.
1 integrins reflect changes in the activation state of
these adhesion receptors that depend on an alteration of their affinity
or/and avidity properties. Through detection of changes in expression
of the HUTS-21 epitope we have tested the possibility that PAINS-13
engagement might change the affinity of associated
1
integrins. In this regard, we have not been able to detect such
1 conformational changes (not shown) suggesting that the observed increase in cell adhesion and spreading onto laminin-1 are
mainly dependent on changes in integrin avidity. Because cell motility
requires subtle and dynamic changes in the activity of
1
integrins, another attractive possibility to explain the effect of
PAINS-13 on cell motility is that upon engagement of this CD9 epitope
the conformation of associated integrins might be kept in a more rigid
or fixed conformation that would be incompatible with motility. The
fact that two anti-CD9 mAbs (VJ1/10 and VJ1/20) exerted different
effects on endothelial cell motility clearly reflects that depending on
the CD9 epitope engaged the functional effects on associated integrins
can be very diverse.
1
integrin-dependent cell adhesion and migration. Matrigel
contains abundant laminin-1 and type IV collagen, which are ligands for
distinct members of the
1 integrin subfamily, including
3
1 and
6
1,
and indeed, cellular morphogenesis characterized by formation of these
structures depends on a correct balance of the adhesive capacities of
this subfamily of integrins on the endothelial cells. In this regard, our results have confirmed the previous results of Sincock et al. (46), by showing that a blocking anti-
1
integrin antibody (Lia1/2) completely inhibits the formation of
angiotubular structures and by demonstrating that a stimulatory
anti-
1 integrin mAb (TS2/16) exerts the opposite effect,
i.e. augments the formation of these structures. In a recent
paper by Zhang et al. (53) it has been established that
endothelial cell morphogenesis in Matrigel is inhibited by blocking
antibodies directed to either
6
1 integrin or the tetraspanin CD151, demonstrating the relevance of the
6
1-CD151 complexes in angiogenesis. Our
results showing in this case the augmentation of endothelial
morphogenesis by mAb PAINS-13, clearly establish the relevance of the
6
1-CD9 complexes in these phenomena.
3
1-tetraspanin protein complexes control
invasive migration of tumor cells through two distinct
PI3K-dependent mechanisms. The levels of activation of PI3K
on serum-starved human breast cancer MDA-MB-231 cells after antibody
engagement of
3 integrin or of tetraspanins CD9, CD63,
and CD81, remained, however, nearly unaltered. In this paper, we have
confirmed that, in agreement with previous reports, engagement of
1 integrins with stimulatory mAb TS2/16 induces
activation of PI3K in human endothelial HMEC-1 cells. We report here
that engagement of the CD9 tetraspanin on HMEC-1 endothelial cells with
mAb PAINS-13 directed to a conformation-dependent epitope
induced an important increase in the activity of PI3K. Interestingly,
engagement of the CD9 molecule with two other anti-CD9 mAbs did not
induce activation of this lipid-phosphorylating enzyme.
1 integrin antibody TS2/16 and underline the
functional relevance of the
1 integrin-CD9 protein
complexes in the regulation of cellular morphogenesis, migration, and
signal transduction. A general conclusion from our studies is that
different conformational states exist for the CD9 tetraspanin that are
likely dependent on multiple molecular associations, which can be
monitored adequately by specific mAbs and whose functional relevance is
just beginning to be unraveled.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Lorena Sánchez-Martín, Dr. José Luis Rodríguez-Fernández, and Dr. Luis del Peso for critical reading of the manuscript and helpful comments.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from Ministerio de Ciencia y Tecnología CICYT SAF 2001-2807 and a grant from Ministerio de Sanidad y Consumo FIS-01/1367 (to C. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by Comunidad de Madrid Grant 8.3/0010/1999.
Recipient of a Formación de Profesorado
Universitario predoctoral fellowship from Ministerio de
Educación, Cultura y Deporte.
¶¶ To whom correspondence should be addressed: Instituto de Farmacología y Toxicología (CSIC-UCM), Facultad de Medicina UCM, Pabellón III, 28040 Madrid, Spain. Tel.: 34-91-3941444; Fax: 34-91-3941470; E-mail: cacabagu@med.ucm.es.
Published, JBC Papers in Press, October 30, 2002, DOI 10.1074/jbc.M207805200
2 E. Rubinstein, unpublished results.
3 A. Luque and C. Cabañas, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PI3K, phosphatidylinositol 3-kinase; PI4K, phosphatidylinositol 4-kinase; mAb, monoclonal antibody; EGFP, epidermal growth factor protein; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; TBS, Tris-buffered saline; BSA, bovine serum albumin; LEL, large extracellular loop.
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REFERENCES |
---|
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