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Introduction
Integrin is a family of cell adhesion molecules, which are
heterodimeric transmembrane receptors composed of
various α and β subunits[1]. The primary function of integrins is
the mechanical connection of cells to the extracellular matrix
or to other cells by binding to specific
ligands[1,2]. Ligand binding to integrins leads to the generation of intracellular
signals, which in concert with other signals, coordinate cell
adhesion with cell migration, growth, and
differentiation[3,4].
The αvβ3 integrin is found in many cell types and
influences cell adhesion and migration with effects on angiogenesis,
restenosis, tumor cell invasion, and
atherosclerosis. It was shown to be critical in angiogenesis induced by both basic
fibroblast growth factor and tumor necrosis factor
(TNF)[5_7], and blocking of this integrin prevents angiogenesis in
several models. Basic fibroblast growth factor and TNF
costimulate αvβ3 expression on developing blood vessels
in the chick chorioallantoic membrane and on the rabbit
cornea[6,8]. Integrin αIIbβ3 shares the common
β3 subunit with αvβ3, is a receptor for fibrinogen, von Willebrand factor
(vWF), fibronectin, and vitronectin (VN), and is essential for
platelet aggregation[9,10].
Integrins have been implicated in a wide variety of
post-receptor occupancy events that occur as a result of ligand
binding. These include the activation of cytoplasmic
protein tyrosine kinases, increased intracellular pH, and gene
induction[3,11]. Additionally, intracellular signaling events
can modulate integrin ligand-binding affinity, a process
termed "activation" or "inside-out"
signaling[4,12]. This has been best characterized in the platelet fibrinogen receptor
integrin αIIbβIIbb3[13_15] . A critical feature of the function of
αIIbβ3 is that it is modulated by platelet agonists. As a
consequence of platelet activation triggered by
platelet agonists, such as thrombin, produced from the activated
coagulation cascade upon vascular injury, integrin
αIIbβ3 rapidly (<1 s) switches from a low-affinity to a
high-affinity ligand-binding conformation of its ectodomains through
conformational changes initiated by intracellular events
referred to as affinity modulation, priming, or inside-out
signaling[16_18], which converge on the C-terminal cytoplasmic
tails of the integrin subunits. However, the affinity
regulation of αvβ3, the VN receptor, is not well
understood[19,20].
The integrin α and β cytoplasmic domains plays key roles
in integrin signaling[21_24]. It has been reported that the
COOH-terminal Arg-Gly-Thr (RGT) sequence of β3 is important for
outside-in signaling; the
T755NITY759 sequence of β3
containing an NXXY motif is critical to inside-out
signaling[25,26]. However, the molecular mechanisms of integrin
αvβ3 affinity regulation have been hampered by the lack of a suitable
model in cultured cells convenient for adhesion assay. It
has been reported that the binding of αvβ3 to VN is
regulated[27,28]; however, the measurement of the alternation of
the adhesion affinity of the αvβ3 expressing cells to VN
has proven to be difficult.
To address the role of the α and β cytoplasmic domains
in the affinity regulation of αvβ3, we constructed a series of
chimeric αIIb/αv and truncated β3 molecules. The
extracellular and transmembrane domains of the αIIb subunit
was fused to the cytoplasmic domain of the wild-type
av subunit, and the chimeric gene was stably coexpressed in
Chinese hamster ovary (CHO) cells with wild-type β3 and
the β3 truncations at sites COOH terminal to
T741, Y747, and
F754, which have been shown to occur after hydrolysis by
calpain[29]. These cells were used to test the cytoplasmic
domains of α and β on receptor affinity regulation and
post-receptor signal transduction through binding to soluble or
immobilized fibrinogen.
Materials and methods
Construction of chimeric a integrin The recombinant
αIIb/αv gene, in which the αIIb cytoplasmic sequence has
been replaced by the corresponding αv sequence, were
prepared as follows: human wild-type av cDNA was cloned from
MDA-MB435 cells (a cell line isolated from the pleural
effusion of a patient with breast carcinoma). The
αIIb extracellular and transmembrane PCR products were generated under the
αIIb cDNA template pcDNA3-IIb (a gift from the Shanghai
Institute of Hematology, Ruijin Hospital, Shanghai, China) with
primers 5´ GCTCTA-GAAGATTGGCCAGAGC-TTTGTGT 3´
and 5´ CCATCC-TCCACATGGCCAGGACC 3´ and αv
cytoplasmic PCR products with primers 5´
GGCCATGTGGAG-GATGGGCTTTTTTAAAC 3´ and 5´
GGGGTACCTCAG-GCACTACCT GTCTTAT 3´. Using αIIb extracellular and
transmembrane PCR products and αv cytoplasmic PCR products as templates, and with Ex
Taq polymerase (Takara, Tokyo, Japan), dNTP Mix, and Ex
Taq buffer in the first 3 rounds of PCR, we obtained PCR products that were then
used as templates for PCR with primers 5´
GCTCTAGAA-GATTGGCCAGAGCTTTGTGT 3´ and 5´
GGGGTACCTCA-GGCACTACCTGTCTTAT 3´. The final products were
digested with XbaI and KpnI and inserted into pcDNA3.1 zeo
(-)digested with the same enzymes, creating a pcDNA3.1 zeo
(-)αIIb/Cαv construct.
Plasmids with β3 cDNA wild type and 3 kinds of
plasmids with muant β3 cDNA bearing truncations
at sites T741,
Y747, and F754 of the COOH-terminal, respectively, were kindly
provided by the Shanghai Institute of Hematology, Ruijin
Hospital. The mutations were confirmed by analysis of the
recombinant cDNA in automated DNA sequencing analysis
(Invitrogen, America).
Cell culture and transfection The CHO cells were grown
in F12 medium supplemented with 10% fetal bovine serum,
glutamine, and non-essential amino acid. Transfection was
performed using Lipofectamine 2000 (Invitrogen, America).
Each mutant β3 cDNA was cotransfected with αIIb/αv at a
ratio of 7:1. Forty-eight hours after transfection, the cells
were collected and diluted into fresh medium containing
Zeocin (Invitrogen, America) at 0.2 mg/mL. The cells were
cultured with selective medium every 3 to 4 d until cell
foci were clearly visible. The cell colonies were then
collected and transferred into 24-well plates. The cells were
cultured to subconfluence before expanding to larger plates.
Stable cell lines expressing proteins were maintained in
the culture in 0.1 mg/mL Zeocin.
Flow cytometric analysis of chimeric integrin
expression The expression of chimeric integrins was monitored by
flow cytometry using CD41a (BD Pharmingen, San Diego,
California, America). The transfected cells were harvested
using 0.5 mmol/L EDTA in phosphate-buffered saline (PBS),
washed with PBS, resuspended at a density of
1×106 cells/100 µL in modified Tyrode's solution [2.5 mmol/L
N-2-hydroxyethylpiperazine-N-2-ethanesulphonic
acid (HEPES), 150 mmol/L NaCl, 2.5 mmol/L KCl, 12 mmol/L
NaHCO3, 5.5 mmol/L D-glucose, 1 mmol/L
CaCl2, 1 mmol/L MgCl2, and 0.1%
bovine serum albumin (BSA), pH 7.4], and incubated for 30
min at 37 °C with monoclonal antibodies specific to the
extracellular domain of human αIIbβ3. Next, the cells were washed
and exposed to the fluorescein-isothiocyanate (FITC)-F(ab)
fragment of rabbit antimouse immunoglobulin G (Santa Cruz,
CA, USA) at 37 °C for 30 min, and the intensity of fluorescence
was quantified in a Coulter flow cytometer
(FACSCalibur, Becton Dickinson, San Jose, CA, USA).
Binding of soluble fibrinogen to transfected CHO cells
The transfected CHO cells were resuspended at
1×106 cells/100 µL in modified Tyrode's solution with 15
µg/mL Alexa Fluor 488-conjugated fibrinogen (Invitrogen,
America) for 30 min at room temperature. After washing,
the cells were resuspended and analyzed by flow cytometry.
Adhesion of CHO cell lines to immobilized fibrinogen
In total, 96-well plates were coated overnight at 4 °C with 25
µg/mL fibrinogen in 0.5 mmol/L
NaHCO3 (pH 8.3). The wells then were blocked with 2% BSA-PBS at 37 °C for 2 h. Cell
suspension (3×104 cells/well in F12 with 1% BSA) was added
to the ligand-coated microtiter wells and incubated for 90
min at 37 °C in a CO2 incubator. After 3 washes, cell spreading
was examined under an inverted microscope (40×objective lens).
In the quantitative assays, 50 µL of 0.3%
p-nitrophenyl phosphate in 1% Triton X-100 and 50 mmol/L sodium acetate (pH
5.0) were added to 96 wells and incubated at 37 °C for 1 h.
The reaction was stopped by adding 50 µL of 1 mol/L NaOH.
The results were determined by reading the optical density
at a 405 nm wavelength.
Antibodies, proteins, and reagents Monoclonal
antibodies against the integrin αIIbβ3 complex (CD41a) were
purchased from BD Pharmingen (America). Alexa Fluor
488-conjugated human fibrinogen were purchased from
Invitrogen (America). The integrin αIIb cDNA clone in
pCDNA3 and β3 in the pCDM8 vector were provided by the
Shanghai Institute of Hematology, Ruijin Hospital.
Results
Gene expression of αIIb/αv and β3 in CHO cells
To study the structure-functional relationship of
β3 integrins, we generated 1 wild-type and 3 C-terminal
truncated β3 genes and cotransfected these genes with
the chimeric αIIb/αv gene in Chinese hamster ovary (CHO) cells. The CHO cells
cotransfected with wild-type αIIb and β3 genes were used
as controls. Accordingly, 5 CHO cell lines were established.
In order to assess the cell surface expression of
recombinant integrins transfected in CHO cells, we used flow
cytometry to detect the reactivity of monoclonal antibodies specific to
the extracellular domain of the human β3 chain (CD61). As
shown in Figure 1, all the mutants used in the present study
exhibited similar levels of cell surface expression of the
β3 subunit. Since all these cell lines express the
αIIbβ3 complex in the outer side of cell surfaces, we then used a flow
cytometer to evaluate their expression with the monoclonal
antibodies specific to the extracellular complex of human
αIIbβ3 (CD41a). As shown in Figure 2, all the cell lines expressed
similar levels of the CD41a antigen, indicating similar levels
of the αIIbβ3 complex on these cells.
Function of chimeric αIIb/αvβ3 integrins as a
fibrinogen receptor It is known that αIIbβ3 is a specific receptor for
fibrinogen[9,30,31]. Thus, we first examined fibrinogen
binding to the cell surface αIIbβ3 complex after the replacement
of the αIIb cytoplasmic domain by the αv cytoplasmic
domain. This was assessed by measuring the amount of
fluorescence-labeled soluble fibrinogen bound to the
resuspended cells without any treatment in a flow cytometer. As
shown in Figure 3, the cells expressing αIIb/αvβ3 bound
soluble fibrinogen. This binding was inhibited by
Arg-Gly-Asp-Ser (RGDS peptide), but the cells expressing wild-type
αIIbβ3 could not bind soluble fibrinogen. Thus, unlike
natural αIIbβ3 integrin, activation was not required for the cells
bearing chimeric genes to bind soluble fibrinogen. This also
indicated that the cells bearing the chimeric
aIIb/av gene formed the αIIbβ3 complex in a correct conformation on the
cell surfaces, because RGD-dependent fibrinogen binding is
a restricted functional marker highly specific for
αIIbβ3[32,33]. Then we examined the soluble fibrinogen binding of the cells
with the C-terminal truncated β3 at the intracellular
compartment. As demonstrated in Figure 4, soluble
fibrinogen bound to αIIb/αvβ3/741, β3/747, and β3/754 cells at
comparably high levels as to aIIb/αvβ3/762 (WT) cells, showing
that aIIb/αvβ3 was in a constant activation state regardless
of the truncation of β3 within the intracellular tail.
Integrins mediate cell adhesion and spreading on the
fibrinogen matrix Another important function of
β3 integrins is cell adhesion to the extracellular matrix. The cytoplasmic
chain of β3, especially its C-terminal, has been proven to
play an important role in integrin signaling and to regulate
its function. We further examined the behavior of these cell
lines in adhesion and spreading on the fibrinogen matrix.
The adhesive properties of the cells expressing
αIIb/αvβ3 containing full-length or truncated β3 subunit proteins were
studied. The cell lines β3/741, β3/747, β/754, and
b3/762, the cell line αIIbβ3, as well as the non-transfected control cell
lines, were added to fibrinogen-coated polystyrene wells.
As shown in Figure 5, non-transfected CHO cells lacking
αIIb/αvβ3 were incapable of adhering to the fibrinogen matrix.
CHO cells expressing αIIbβ3 and the cells expressing
aIIb/αvβ3/762 (WT) adhered to and spread well on the
fibrinogen matrix. In contrast, all CHO cells displaying truncated
β3 in the αIIb/αvβ3 complex, including β3/741, β3/747, and
β3/754, failed to adhere to the fibrinogen matrix. These
results showed that the full length of β3 was required for the
cell adhesion to immobilize fibrinogen; any deletion of the
C-terminal sequence of β3 would abolish aIIb/αvβ3-mediated
adhesion and spreading.
Discussion
In this study, we generated the chimeric
αIIb/αvβ3 integrin protein, which fused the extracellular and
transmembrane sequences of αIIb to the intracellular sequences of
αv and successfully expressed this chimeric integrin on the
surface of CHO cells. This model was designed to test whether
such a chimeric molecule is functionally active and if any
signal transduction can be observed by the ligand binding
assay and cell adhesion assay. Since αIIbβ3 binding to
fibrinogen is extremely well documented and easy to test, this
chimeric integrin was expected to provide a useful model for
the study of signal transduction mediated by the
αvβ3 intracellular domains.
The activation of αIIbβ3 has been well
documented[12_14]. The natural αIIbβ3 on the platelets was inactive and turns
into an active form once the cells are stimulated by agonists,
such as thrombin, through inside-out
signaling[30,34,35]. Our results are in agreement with this notion since fibrinogen
did not bind to the CHO cells expressing αIIbβ3, while these
cells expressed high levels of the β3 chain (CD61) and
αIIbβ3 complex (CD41a). There are also some reports
about the constitutive activation of αIIbβ3 by
demonstrating that the chimeric α subunits with the extracellular and
transmembrane of αIIb joined to the cytoplasmic domains of
α5, α6A, or α6B conferred a high affinity state to the
recombinant integrins[36]. In the present study, we showed
that once the αIIb cytoplasmic tail was substituted by the
αv tail, the extracellular αIIbβ3 receptors were constitutively active for
their ligands in the absence of the inside-out signals. The
activated state of the receptors remained unimpaired with
the truncations of the cytoplasmic sequences of β3, as we
could see the significant amount of the soluble fibrinogen
bound to the cells expressing these truncated versions of
integrin β3. This binding was specifically mediated by
αIIb/αvβ3 because it was completely inhibited by the RGD peptide.
Based upon our data and those of others, we propose a
model for integrin activation, in which the αIIb and
β3 chains within extracellular domains tend to expose
fibrinogen-binding sites. This tendency is however blocked by the
interaction of the intracellular part of the αIIb and
β3 subunits. Accordingly, any structural alternation that impairs the
interaction of the αIIb and β3 subunits within the intracellular
domains will activate the integrin. We believe that the
proposal of this model would be beneficial in understanding the
mechanisms of integrin activation which is thought to be
regulated by inside-out
signaling[12_14]. It is tempting to
speculate that the disturbance of the interaction between
the intracellular and transmembrane domains of the
αIIb and β3 subunits, in the case of the chimeric
α subunit as in this study or of talin interaction, causes a "deblockade" of the
suppressed receptor activity and this will be responsible for
integrin activation[37,38].
We showed that both the cells expressing αIIbβ3 and
the cells expressing chimeric αIIb/αvβ3 molecules adhered
to and spread well on immobilized fibrinogen in a similar
manner. In contrast, the 3 cell lines expressing truncated
β3 at the C-terminal failed to adhere firmly and spread. Our data
showed that the truncations of β3 did not alter the soluble
fibrinogen binding capacity of αIIb/αvβ3, but it did alter the
cell adhesion on immobilized fibrinogen, indicating that
fibrinogen binding capacity is not sufficient to support
fibrinogen-mediated cell adhesion and spreading. In
fact, it showed that the cell spreading on immobilized
fibrinogen was a more complicated phenomenon and depended on the
integrity of intracellular β3, which has been reported to play
a key role in signal
transduction[4,25,26]. It is known that
binding of ligands to αIIbβ3 not only forms adhesive bonds
between platelets, but also transmits outside-in signals to
induce a series of cellular responses, such as protein
phosphorylation[39,40], elevation of intracellular
Ca2+ [41], and cytoskeleton
reorganization[42], leading to cell spreading and
the stabilization of cell
adhesion[43_45]. In particular, the hydrolysis of short peptides at the C-terminal of
the β3 chain has been shown to be involved in this signal
transmission[25,29]. Therefore, the failure of the cells with truncated
β3 to adhere to and spread on immobilized fibrinogen could be due
to the disruption of the outside-in signals that occurred in
natural αIIbβ3. This interpretation underlines once again
the importance of the regulatory role of β3 within the
intracellular αvβ3 complex in outside-in signal transmission.
However, further study is needed to elucidate the detailed
mechanisms.
Our data indicate that the intracellular interactions within
the αvβ3 cytoplasmic tail of the cell line CHO
αIIb/αvβ3 regulates the adhesion function of the extracellular
αIIbβ3 domain to immobilize fibrinogen and consequent spreading.
Therefore, the cell line CHO αIIb/αvβ3 will be a useful model
for studying the intracellular protein-protein interaction in
which αvβ3 intracellular domains are involved. Furthermore,
this model is potentially useful for the drug screening of
active substances interfering in αvβ3-mediated signaling.
In addition, as we showed, the chimeric integrin
αIIb/αvβ3 was constitutively activated for ligand binding with no need
of agonist stimulation, which suggests that the cell model
would be also a useful tool in drug screening of new
substances interfering with fibrinogen binding to
αIIbβ3, a critical step during platelet aggregation and blood clot formation.
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