¶
§
* Nora Eccles Harrison Cardiovascular Research and Training Institute, Program in Human Molecular Biology and Genetics,
Eccles Institute of Human Genetics, § Department of Biochemistry,
Department of Internal Medicine, and ¶ Department of
Pathology, University of Utah, Salt Lake City, Utah 84112; and the ** Department of Medicine, Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205
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Abstract |
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Integrins are widely expressed plasma membrane adhesion molecules that tether cells to matrix
proteins and to one another in cell-cell interactions. Integrins also transmit outside-in signals that regulate
functional responses of cells, and are known to influence gene expression by regulating transcription. In
previous studies we found that platelets, which are naturally occurring anucleate cytoplasts, translate preformed mRNA transcripts when they are activated by
outside-in signals. Using strategies that interrupt engagement of integrin IIb
3 by fibrinogen and platelets deficient in this integrin, we found that
IIb
3 regulates
the synthesis of B cell lymphoma 3 (Bcl-3) when platelet aggregation is induced by thrombin. We also found
that synthesis of Bcl-3, which occurs via a specialized
translation control pathway regulated by mammalian
target of rapamycin (mTOR), is induced when platelets adhere to immobilized fibrinogen in the absence of
thrombin and when integrin
IIb
3 is engaged by a conformation-altering antibody against integrin
IIb
3.
Thus, outside-in signals delivered by integrin
IIb
3 are
required for translation of Bcl-3 in thrombin-stimulated aggregated platelets and are sufficient to induce translation of this marker protein in the absence of thrombin. Engagement of integrin
2
1 by collagen also triggered synthesis of Bcl-3. Thus, control of translation
may be a general mechanism by which surface adhesion
molecules regulate gene expression.
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Introduction |
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INTEGRINS are plasma membrane proteins that mediate
adhesion of cells to other cells and to matrix structures (Hynes, 1992). Individual
and
subunits pair
to form heterodimers of characteristic ligand specificity
that interact via their cytoplasmic domains with cytoskeletal and other intracellular proteins (Hynes, 1992
; Shattil
and Ginsberg, 1997
). Interaction of integrin intracellular domains with cytoplasmic proteins confers the ability to
transmit outside-in signals when the extracellular domains
are engaged by specific ligands, in addition to providing a
mechanism by which affinity and avidity of integrins for
their ligands can be regulated (Hynes, 1992
; Clark and
Brugge, 1995
; Schwartz et al., 1995
; Shattil and Ginsberg,
1997
). Outside-in signaling resulting from integrin engagement triggers a variety of responses in cells, including fluxes in intracellular calcium, sodium-proton exchange
and alterations in intracellular pH, phosphatidylinositol
metabolism, activation of calpain, focal adhesion kinase,
mitogen-activated protein kinases and other enzymes,
and induction of nuclear signaling pathways leading to expression of new gene products (Clark and Brugge, 1995
;
Schwartz et al., 1995
; Howe et al., 1998
). These biochemical events lead to both rapid and delayed changes in cellular function and phenotype, including motility, growth,
and differentiation. Outside-in signaling by integrins is
both heterodimer- and cell-specific (Shattil and Ginsberg,
1997
) and has been studied frequently in the context of adhesion of cells to matrix ligands. Although integrins are
also prototypic tethering factors in cell-cell interactions (Zimmerman et al., 1996
), less is known of their outside-in
signaling roles in this context compared with cell-matrix interactions.
The mechanisms by which gene expression is regulated
by cellular adhesion are of considerable interest because
this imposes both spatial and biochemical control on the
process. Studies of fibroblasts and human monocytic cells
demonstrate that engagement of integrins by antibodies
against their extracellular domains or by matrix ligands induces activation and/or nuclear translocation of Rel (NF-B)
and other transcription factors, and consequent transcription of specific mRNAs (Schwartz et al., 1995
; Juliano, 1996
). In some cases, only mRNA transcripts are induced
by integrin engagement alone, and a second signal is required for their translation and expression of the corresponding protein (Juliano, 1996
). Whether integrins directly or indirectly regulate posttranscriptional pathways
is largely unknown, and is a challenging issue to study because of the clear and potent influence of integrin engagement on transcriptional events (Juliano and Haskill, 1993
;
Schwartz et al., 1995
).
Recently, we found that stimulated human platelets synthesize proteins from preformed mRNA in an activation-dependent fashion (Weyrich et al., 1998). Because they are
primary anucleate cytoplasts that bear receptors and surface adhesion molecules capable of mediating outside-in
signaling (Shattil et al., 1994
, 1998
), platelets are a unique
system in which to study activation-dependent translational events independent of nuclear influences. In activated platelets, the synthesis of several induced proteins is
inhibited by the immunosuppressant rapamycin in addition to general inhibitors of translation (Weyrich et al.,
1998
), indicating the presence of a specialized pathway of
translational control regulated by mammalian target of rapamycin (mTOR)1 (Brown and Schreiber, 1996
; Thomas
and Hall, 1997
). The transcript for B cell lymphoma protein 3 (Bcl-3), an intracellular regulatory factor, is present
in platelets and is translated via this pathway when platelets are stimulated with thrombin (Weyrich et al., 1998
), making it a useful marker for studies of regulated protein
synthesis in this cell type. Here we show that translation of
Bcl-3 is an adhesion-dependent event that requires engagement of integrin
IIb
3 in platelets activated by thrombin. Integrin
IIb
3 is expressed only by platelets and
megakaryocytes, and transmits outside-in signals in addition to mediating cellular aggregation and adhesion (Phillips et al., 1991
; Hynes, 1992
; Shattil et al., 1998
). We also
show that direct engagement of integrin
IIb
3 induces expression of Bcl-3 in the absence of thrombin or other exogenous agonists, and that synthesis of Bcl-3 is induced
when platelets adhere to collagen via integrin
2
1. These
experiments demonstrate for the first time that integrins
can directly control expression of gene products at translational checkpoints, and that their influence on the flow of
genetic information is not limited to regulation of transcription.
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Materials and Methods |
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Cell Isolation
Platelets were isolated using the methods of Hamburger and McEver
(1990). In brief, human blood was drawn into acid-citrate-dextrose (ACD;
7 ml ACD/42 ml of blood) and was centrifuged (200 g for 20 min) to obtain platelet-rich plasma. Platelet-rich plasma was recentrifuged (500 g for
20 min) in the presence of 100 nM prostaglandin E-1. The supernatant was
discarded and platelet pellet was resuspended in 50 ml of Pipes/saline/glucose (5 mM Pipes, 145 mM NaCl, 4 mM KCl, 50 µM Na2HPO4, 1 mM
MgCl2-6 H2O, and 5.5 mM glucose), containing 100 nM of prostaglandin
E-1 (Sigma Chemical Co.). The platelet suspension was centrifuged (500 g
for 20 min), the supernatant was discarded, and the platelet pellet was resuspended in M199 (phenol red free; Whittaker M.A. Bioproducts). In selected studies, the platelets were suspended in Ca2+ and Mg2+-free HBSS
containing 5 mM EGTA to chelate Ca2+. 2.5 × 108 platelets were used for
each experimental point. Platelets were stimulated with thrombin (Sigma
Chemical Co.), collagen (from human placenta; Sigma Chemical Co.), fibrinogen (Sigma Chemical Co.), or an activating antibody, D3GP3 (provided by Dr. L.K. Jennings, University of Tennessee, Memphis, TN) for 1 h
at 37°C while gently rocking in small volume conical tubes. In selected
studies, platelets were preincubated with
IIb
3 blocking antibodies, 7E3
(obtained commercially and provided by Dr. S. Tam, Centocor, Malvern,
PA), 10E5 (provided by Dr. B.S. Coller, Mount Sinai School of Medicine, New York), G4120, G4709 (provided by Dr. T. Gadek, Genentech, San
Francisco, CA), or integrilin (eptifibatide) (provided by Dr. S. Hollenbach, COR Therapeutics, South San Francisco, CA) before stimulation
with thrombin. Isotype-matched antibodies were used as controls as indicated in the text and figure legends (
-LFA-1 and CD31 from R&D Systems). After 1 h, the platelet pellets were collected and prepared for Western analysis as described below.
Human tissues for examination of platelets in situ were collected at the time of surgical intervention. Surgical tissues were collected according to protocols approved by the University of Utah internal review board after informed consent.
Platelet Adhesion Assay
Platelet adhesion to fibrinogen was studied in 4-well polystyrene chambers (Nunc Inc.) precoated overnight at 4°C with HBSS-human serum albumin (2%), which served as the control, fibrinogen (1 mg/ml; Sigma Chemical Co.), or collagen (50 µg/ml; Sigma Chemical Co.). Plates were washed three times and blocked for 2 h with 1 ml of HBSS-human serum albumin (2%), and washed three times with HBSS followed by three more washes with HBSS containing 0.01% Tween 20. Residual buffer was removed by aspiration and 2.5 × 108 platelets/ml were added to the matrix-coated wells for 1 h at 37°C. After this time, adherent platelets were scraped into Eppendorf tubes and the suspensions were centrifuged for 2 min, 1,000 g at room temperature, and the supernatants were removed. The cell pellets were placed in SDS-PAGE reducing buffer for Western analysis as described below.
Immunoblotting Procedure
Platelet pellets, collected from activated cells in suspension or those adherent to fibrinogen, were placed in SDS-PAGE reducing buffer, electrophoresed on a 9% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. Western analysis was conducted using affinity-purified, rabbit polyclonal anti-Bcl-3 antibody (Santa Cruz Technology). Immunoreactive protein was detected by affinity-isolated goat anti-rabbit antibody conjugated to peroxidase (Biosource Int.) and an enhanced chemiluminescence detection reagent (Amersham Life Science).
Immunocytochemical and Immunohistochemical Procedures
Immunocytochemical procedures were performed as described previously, with minor modifications (Weyrich et al., 1996, 1998
). In brief,
platelets were spun onto glass slides and immediately fixed with 1%
paraformaldehyde. After a methanol permeabilization step, the cells were
blocked and probed with anti-Bcl-3 (Santa Cruz Technology). Immunoreactive protein for Bcl-3 was detected using an ABC kit from Vectastain
(Vector Laboratories, Inc.) for horseradish peroxidase detection that
yields a brown immunostain product. Control slides included omission of
the primary antibody, omission of the secondary antibody, and/or substitution of nonimmune rabbit IgG. Tissue specimens from abdominal aortic
aneurysms were collected and placed in Histochoice MB fixative (Amresco Inc.). After fixation, the specimens were embedded in paraffin, sectioned into 5-µm slices, and immunoreactivity for Bcl-3 was assayed as described previously (Weyrich et al., 1993
). Sections were viewed and
photographed by Nomarski interference contrast optics using a Zeiss Axioplan light microscope. Tissue collection procedures were approved by
the University of Utah Institutional Review Board.
Aggregometry
0.5-ml aliquots of platelets (2.5 × 108/ml) were preincubated for 5 min at
37°C in the presence of buffer or antibodies before aggregation was initiated by thrombin. Platelets were placed in siliconized cuvettes and aggregation was monitored by a Sienco aggregometer (model DP-247-E) with
constant stirring at 1,000 rpm at a constant temperature of 37°C as described previously (Kouns et al., 1990).
ELISA
Concentrations of RANTES were measured by ELISA as described previously (Weyrich et al., 1996).
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Results |
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The Expression of Bcl-3 Is Enhanced in Aggregated Human Platelets
In previous experiments, we found that isolated human
platelets translate constitutively present mRNA into proteins in an activation-dependent fashion, that this occurs in
platelets stimulated with thrombin, and that Bcl-3 is an informative marker protein to examine in analyses of the
synthetic response in this system (Weyrich et al., 1998). In
addition, we found that when suspensions of thrombin-stimulated platelets were stained using an antibody against
Bcl-3, expression of the protein appeared to be enhanced in aggregated cells compared with single cells. This suggested that signaling of protein synthesis in stimulated
platelets is influenced by adhesion. To further explore this
issue we performed additional immunocytochemical analyses and found that Bcl-3 protein is rapidly expressed in
platelet aggregates after thrombin stimulation, with lesser
amounts in thrombin-stimulated single cells and little or
no protein detectable in platelets in the absence of thrombin (Fig. 1). When the anti-Bcl-3 antibody was deleted or
replaced with a control rabbit immunoglobulin, there was
no staining of Bcl-3 (Weyrich et al., 1998
; data not shown).
We also found that Bcl-3 is present in aggregated platelets
in microvessels of inflamed tissue (Fig. 2), demonstrating
that its synthesis in isolated platelets (Fig. 1) models in
vivo events. The accumulation of Bcl-3 in thrombin-stimulated aggregated platelets examined in vitro was time- and
concentration-dependent (Fig. 3 and data not shown),
consistent with our earlier studies characterizing its synthesis in this cell type (Weyrich et al., 1998
). Because aggregation of human platelets depends on engagement of
integrin
IIb
3 by fibrinogen or von Willebrand factor
(Gawaz et al., 1991
; Williams et al., 1995
), these observations suggested that outside-in signaling via this integrin
heterodimer regulates the synthetic pathway leading to expression of the Bcl-3 protein. Therefore, we characterized the role of integrin
IIb
3 in detail.
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Chelation of Extracellular Calcium Inhibits Platelet-Platelet Clustering and Abolishes Bcl-3 Synthesis
We determined if chelation of extracellular calcium interrupts Bcl-3 synthesis, since it is known to block platelet
aggregation (Fitzgerald et al., 1985; Shattil et al., 1985).
Platelets stimulated with thrombin in calcium-free buffer
containing EGTA (5 mM) did not aggregate in response
to thrombin (Fig. 3) and did not synthesize Bcl-3 (Fig. 3 b).
As a control to test whether signaling via the thrombin receptor was still intact under these conditions, we measured
the secretion of RANTES, a preformed chemokine that is stored in alpha granules (Kameyoshi et al., 1992
; Weyrich
et al., 1996
), and found that it was released in response to
stimulation (Fig. 3 c). These findings suggested that disruption of adhesive interactions between the aggregating
platelets by removal of extracellular cations resulted in
impaired synthesis of Bcl-3. Alterations in cation concentrations also have the potential to inhibit intracellular enzymes and other components required for protein synthesis, however, so we pursued this issue further using additional experimental strategies.
mAbs Directed Against Integrin IIb
3 Inhibit Platelet
Aggregation and Bcl-3 Synthesis
We next determined if engagement of the IIb
3 integrin is
required for Bcl-3 synthesis in thrombin-stimulated platelets. Platelet aggregation occurs when
IIb
3 heterodimers
on adjacent activated platelets are engaged by fibrinogen
released from alpha granules or added exogenously (Gawaz
et al., 1991
; reviewed in Williams et al., 1995
). mAb 10E5
blocks binding of fibrinogen to
IIb
3 and prevents platelet
aggregation (Coller et al., 1983
; Coller, 1985
). Therefore,
we determined if mAb 10E5 inhibits Bcl-3 accumulation in
stimulated human platelets. As expected, preincubation of
platelets with mAb 10E5 markedly attenuated thrombin-induced platelet aggregation compared with control cells
in the absence of antibody (Figs. 1 and 4). In addition, Bcl-3
accumulation was inhibited by pretreatment of platelets
with mAb 10E5 when examined by immunocytochemistry
(Fig. 1) and Western analysis (Fig. 4 b). In some incubations, there were scattered small residual aggregates that
were visible by microscopy in suspensions pretreated with mAb 10E5 and then stimulated with thrombin, although
the majority of cells did not aggregate; these residual aggregates contained Bcl-3 when examined by immunocytochemical analysis (Fig. 1 c). An isotype-matched mAb
against
L
2 integrin (
-LFA-1; IgG2a) did not inhibit
Bcl-3 accumulation in thrombin-stimulated platelets or
block platelet aggregation (Fig. 4).
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We performed similar experiments with the Fab fragment of the chimeric human-murine mAb 7E3. This antibody recognizes integrins IIb
3 and
v
3, inhibits fibrinogen binding to platelets, and has been used to block
platelet aggregation as a clinical antithrombotic agent (Coller, 1985
; Reverter et al., 1996
; Coller, 1997
). mAb 7E3 attenuated Bcl-3 accumulation in thrombin-stimulated platelets and also inhibited aggregation in parallel incubations
(not shown). In contrast, a mAb against
v
3 did not block
Bcl-3 expression.
Peptides That Block Engagement of Integrin IIb
3
Inhibit Synthesis of Bcl-3 in Stimulated
Human Platelets
Binding of fibrinogen to integrin IIb
3 on activated platelets requires engagement of a dodecapeptide sequence in
the
chain of fibrinogen by the integrin heterodimer, an
event that can be blocked by peptides that contain arginine-glycine-asparagine (RGD) sequences (Du et al., 1991
;
Phillips et al., 1991
). Substitution of lysine (K) for arginine
(R) in the RGD sequence confers specificity for integrin
IIb
3 compared with other integrins, and cyclic peptides
containing the KGD sequence that are based on the snake venom disintegrin, barbourin, potently inhibit fibrinogen
binding to
IIb
3 and platelet aggregation (Scarborough et
al., 1991
; Scarborough et al., 1993
). We first examined linear RGD peptides as antagonists of aggregation-dependent Bcl-3 synthesis in human platelets and found that under some conditions the peptides themselves had weak
agonist effect (not shown), consistent with previous observations (Du et al., 1991
). Then, we examined a cyclic KGD heptapeptide that specifically binds to integrin
IIb
3 (Schulman et al., 1996
; Pursuit Trial Investigators, 1998). The cyclic peptide antagonist inhibited both thrombin-induced
platelet aggregation (Fig. 5 a) and Bcl-3 accumulation (Fig.
5 b). The inhibition was concentration-dependent (range
1-10 µg/ml) with maximal inhibition at 10 µg/ml (Fig. 5 b).
A control peptide had no effect. A second blocking cyclic
peptide, G4120 (Barker et al., 1992
), also inhibited Bcl-3
synthesis in a concentration-dependent fashion, whereas a
control peptide did not (not shown). Thus, inhibition of
engagement of
IIb
3 integrin with competitive peptides
attenuates accumulation of Bcl-3 in platelets stimulated
with thrombin (Fig. 5), as does treatment of the platelets
with blocking antibodies (Fig. 4).
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Bcl-3 Synthesis Is Absent or Reduced When Platelets
Deficient in Integrin IIb
3 Are Stimulated
with Thrombin
Platelets from patients with Glanzmann thrombasthenia
have significant reductions or absence of IIb
3 on their
surfaces (George et al., 1990
; Newman and Poncz, 1995
).
The deficiency in integrin
IIb
3 prevents normal binding
of ligands and consequent platelet aggregation, accounting
for the hemostatic defect that characterizes these subjects.
Platelets from patients with Glanzmann thrombasthenia also have impaired outside-in signaling (Wang et al.,
1997
). We studied platelets from a patient with type I
Glanzmann thrombasthenia that do not aggregate when
stimulated by thrombin or a variety of other agonists (Jin
et al., 1996
). When compared with platelets from a normal
control subject isolated in parallel, these mutant platelets
exhibited a dramatic defect in accumulation of Bcl-3 in response to thrombin stimulation (Fig. 6). There was no accumulation of Bcl-3 in the integrin
IIb
3-deficient platelets at concentrations of thrombin (0.05, 0.1 U/ml) that
induced synthesis of the protein marker in the simultaneously assayed control platelets (Fig. 6) or in platelets
from other control subjects (Figs. 1 and 3-5). At higher
concentrations of thrombin, accumulation of Bcl-3 in the
IIb
3-deficient platelets was attenuated but not absent
(Fig. 6).
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Direct Activation of Integrin IIb
3 Induces
Bcl-3 Synthesis
We showed previously that adhesion of platelets to purified immobilized fibrinogen induces the synthesis of multiple proteins in the absence of thrombin or another agonist
(Weyrich et al., 1998). This is consistent with earlier studies indicating that integrin
IIb
3 on the platelet surface
can engage immobilized fibrinogen and transmit outside-in signals without requiring an exogenous agonist, whereas
engagement by soluble fibrinogen requires agonist-stimulated cellular activation (Savage and Ruggeri, 1991
; Haimovich et al., 1993
; Shattil et al., 1994
). To determine if engagement of integrin
IIb
3 is sufficient to induce translation of the marker protein Bcl-3, we first used this immobilized fibrinogen system. We found that platelets adherent to a fibrinogen matrix accumulated Bcl-3 (Fig. 7 a). In
contrast, there was little or no Bcl-3 in platelets incubated
in suspension (not shown) or on immobilized albumin in
parallel. mAbs 10E5 and 7E3 (see above) inhibited Bcl-3
accumulation in platelets adherent to immobilized fibrinogen. In addition, LY 294002 and Wortmannin, which inhibit phosphatidylinositol-3-kinase (PI3K), blocked Bcl-3
expression in platelets adherent to immobilized fibrinogen
(not shown), as they do in thrombin-stimulated aggregated
platelets (Weyrich et al., 1998
).
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We then used an alternative strategy to ask if engagement of integrin IIb
3 delivers outside-in signals to the
translation pathway that regulates synthesis of Bcl-3. mAb
D3GP3 is directed against the
3 chain of the integrin
IIb
3 heterodimer and induces a conformational change
that makes the integrin competent to bind fibrinogen and
mediate aggregation in the absence of thrombin or another stimulus (Kouns et al., 1990
; Kouns and Jennings,
1991
). We found that incubation of platelets with mAb
D3GP3 resulted in their aggregation (not shown), as previously reported, and also triggered synthesis of Bcl-3 (Fig. 7
b). An isotype-matched antibody against another protein
on the platelet plasma membrane, PECAM-1 (CD31), did
not induce Bcl-3 synthesis (not shown). When examined
by immunocytochemistry, Bcl-3 was predominantly located in aggregated platelets in suspensions treated with
D3GP3, with few single platelets showing staining (not
shown). The accumulation of Bcl-3 in platelets incubated
with mAb D3GP3 was not as great as that in platelets
stimulated with thrombin in parallel (Fig. 7 b), consistent
with the fact that the antibody induces submaximal aggregation under these conditions (Kouns et al., 1990
) (our experiments not shown). Platelets treated with mAb D3GP3
bind exogenously added fibrinogen in an enhanced fashion (Kouns et al., 1990
). When we added soluble fibrinogen to the incubation, the accumulation of Bcl-3 was enhanced in platelets incubated with D3GP3 (Fig. 7 b).
Exogenous soluble fibrinogen did not induce Bcl-3 synthesis in platelets incubated with the control mAb against
PECAM-1 (not shown).
Collagen Triggers Bcl-3 Synthesis by Human Platelets
Collagen is recognized by integrin 2
1 as well as by other
adhesion molecules on platelets, and treatment of isolated
platelets with collagen in solution induces activation of intracellular kinases and aggregation equivalent in magnitude to that triggered by thrombin (Lipfert et al., 1992
;
Shattil et al., 1994
). We found that collagen in solution
induced synthesis of Bcl-3 in a concentration-dependent fashion (Fig. 8 a). In addition, we found that platelets adherent to immobilized collagen synthesized Bcl-3 (Fig. 8
b). The accumulation of Bcl-3 in platelets adherent to immobilized collagen was inhibited by a blocking antibody
against the integrin
2 subunit (not shown) and also by
mAb 7E3 (see above) (Fig. 8 b) but not by a control mAb
against
v
3 (not shown). The latter findings are consistent with a previous report that mAb 7E3 and an antibody
against
2
1 integrin each inhibited outside-in signaling of
platelets when the cells adhered to an immobilized collagen matrix (Haimovich et al., 1993
). mAb 10E5 also
blocked synthesis of Bcl-3 under similar conditions (Fig. 8
b). Thus, engagement of integrin
2
1 and/or other surface
receptors for collagen together with integrin
IIb
3 may
mediate signaling of translational events leading to Bcl-3
synthesis in a costimulatory fashion.
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![]() |
Discussion |
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The flow of genetic information is regulated at sequential checkpoints that, together, provide precise control of
the expression of protein products (Darnell, 1982; Kozak,
1991
). Here we show that integrin
IIb
3 regulates translation of a marker protein, Bcl-3, in thrombin-stimulated aggregated platelets. Integrin
IIb
3 is the principle integrin
of human platelets and is a herald member of the integrin
family that has yielded many insights into the structure
and function of these adhesion proteins (Phillips et al.,
1991
; Hynes, 1992
; Shattil et al., 1994
; Shattil et al., 1998
).
We also found that outside-in signals delivered via integrin
IIb
3 trigger Bcl-3 synthesis when the integrin heterodimer is engaged by immobilized ligand or a function-perturbing antibody. Our findings raise the possibility that a
general mechanism by which integrins regulate gene expression is by interacting at posttranscriptional checkpoints, in addition to mediating nuclear signaling and transcriptional events. It has been suggested previously that
1
integrins on leukocytes may influence posttranscriptional steps (Mondal et al., 1995
). Also, mechanical signaling via
engaged
1 integrins may orchestrate local accumulation
of mRNA and ribosomes in the region of focal adhesion
complexes (Chicurel et al., 1998
). However, our findings
provide the first evidence that outside-in signaling via a
specific integrin heterodimer regulates expression of protein products in the absence of nuclear effects.
Human platelets may be a particularly informative system in which to study adhesion-dependent signaling of
translation in a primary cell type, and mechanisms elucidated in platelets may also reflect similar processes at the
earlier, nucleated, megakaryocyte stage (Weyrich et al.,
1998). Platelets have ribosomes and other components required for protein synthesis and carry stable mRNA transcripts (Warshaw et al., 1967
; Morgenstern, 1980
; Belloc
et al., 1982
; Kieffer et al., 1987
; Newman et al., 1988
; Roth
et al., 1989
; Power et al., 1995
). Multiple new proteins are
synthesized in thrombin-stimulated platelets and in platelets adherent to immobilized fibrinogen; this synthesis is
interrupted by puromycin and cycloheximide and, for a
subset of these proteins, by rapamycin (Weyrich, 1998; and
our unpublished observations). To date, we have identified Bcl-3 as one of the newly synthesized products and
five others as proteins that regulate or are involved in cytoskeletal interactions (Weyrich A.S., N.D. Tolley, M.L.
Wade, T.M. McIntyre, S.M. Prescott, Z. Wu, and G.A.
Zimmerman, manuscript in preparation). Although mitochondrial transcription occurs in platelets (Agam et al.,
1976
), Bcl-3 is translated from preformed mRNA and the transcriptional inhibitor actinomycin D does not prevent
synthesis (Weyrich et al., 1998
). Platelets contain key enzymes in the specialized mTOR pathway that controls
translation of a subset of mRNAs with specific structural
features (Brown and Schreiber, 1996
; Thomas and Hall,
1997
), including the mRNA for Bcl-3 (Weyrich et al., 1998
). Current evidence from cell lines and transfected cell
models indicate that activity in this pathway is initiated by
a signal at the plasma membrane followed by a cascade involving PI3K and 3-phosphoinositide-dependent protein
kinase 1 (PDK1) and culminating in phosphorylation of
the translation repressor eIF4E-binding protein 1 (4EBP-1),
causing it to dissociate from eukaryotic translation initiation factor 4E (eIF4E) and allowing cap-dependent translation to proceed (Sonenberg and Gingras, 1998
). Phosphorylation and activation of the ribosomal S6 kinase, p70
S6 kinase (p70S6K), also occurs. In previous studies of lymphocytic cell lines and fibroblasts, this pathway was shown
to be triggered by growth factors and mitogens (reviewed
in Brown and Schreiber, 1996
; Thomas and Hall, 1997
;
Peterson and Schreiber, 1998
; Sonenberg and Gingras, 1998
). In aggregating platelets, PI3K is triggered in an
adhesion-dependent fashion and PDK1 is also present
(Clark and Brugge, 1995
; Banfic et al., 1998a
,b). Inhibitors
of PI3K block synthesis of Bcl-3 in thrombin-stimulated
aggregated platelets (Weyrich et al., 1998
) and in platelets
adherent to immobilized fibrinogen (this study). In addition, p70S6K is present in human platelets (Papkoff et al.,
1994
) and is activated when they aggregate in response
to thrombin (Weyrich, A.S., unpublished experiments).
4EBP-1 is also phosphorylated in thrombin-stimulated platelets and this event and the synthesis of Bcl-3 are
blocked by inhibitors of PI3K and by rapamycin (Weyrich
et al., 1998
). Thus, platelets have critical enzymatic and
regulatory molecules that are required for translation control, including components of the mTOR pathway, and the
activities of these systems are influenced by outside-in signals.
Using blocking antibodies, competitive peptides, and
deficient platelets from a subject with Glanzmann thrombasthenia, we found that engagement of integrin IIb
3
regulates Bcl-3 synthesis in aggregating platelets stimulated by thrombin. Thus, signals transmitted by integrin
IIb
3 are linked to translation control pathways. We also
found that ligation of integrin
IIb
3 by immobilized fibrinogen or binding of a conformation-altering antibody induces synthesis of Bcl-3 in the absence of thrombin stimulation. These experiments and our previous observations
(Weyrich et al., 1998
) indicate that engagement of integrin
IIb
3 is sufficient to signal activation of translational pathways and synthesis of a variety of proteins. Binding of fibrinogen to integrin
IIb
3 in the presence of an activating
"LIBS" anti-
3 antibody triggers PI3K and apparent PDK1
activities, responses that require platelet-platelet contact and aggregation to be maximal (Banfic et al., 1998a
,b).
Thus, engagement of integrin
IIb
3 can activate key enzymes in the transduction cascade that relay signals from
the plasma membrane to translational pathways (see above).
Whether there are intracellular signaling cascades that are
specific to integrin
IIb
3 (Banfic et al., 1998a
) is unknown.
In other systems, integrins and growth factors or mitogens
use common, rather than unique, intracellular mechanisms
to trigger gene expression (Juliano, 1996
; Howe et al.,
1998
). How integrin
IIb
3 interfaces with downstream
components of the translation control pathway that regulate phosphorylation of 4EBP1 and p70S6K activation is
also currently unknown. In a previous study, adhesion of a
cell line to immobilized fibronectin, laminin, or vitronectin activated p70S6K, implying that regulation of this enzyme is
linked to engagement of integrins of both the
1 and
3
classes (Malik and Parsons, 1996
). Additional experiments
indicated that focal adhesion kinase was partially required
for p70S6K activation. Whether focal adhesion kinase, which
is signaled by integrin
IIb
3 engagement (Lipfert et al.,
1992
; reviewed in Shattil et al., 1994
; reviewed in Clark
and Brugge, 1995
; Lyman et al., 1997
; and reviewed in
Shattil et al., 1998
), is involved in p70S6K activation and
translational regulation in platelets remains to be explored.
In cell-cell interactions, signals delivered through adhesion molecules are integrated with signals from surface receptors, such as those for growth factors or chemokines, to
yield qualitatively distinct responses (Weyrich et al., 1996;
Zimmerman et al., 1996
). Signals delivered by integrins
and growth factors converge and are integrated in this
fashion (reviewed in Schwartz et al., 1995
; Juliano, 1996
;
Sastry and Horwitz, 1996
). Our finding that Bcl-3 expression in platelets is induced by thrombin stimulation raises the possibility that outside-in signals transmitted by engagement of integrin
IIb
3 interface and are integrated
with those generated by ligation of the thrombin receptor
resulting in translation of mRNAs. The thrombin receptor
and integrin
IIb
3 transmit convergent signals to other response pathways in human platelets (Ferrell and Martin,
1989
; Golden et al., 1990
; Clark et al., 1994
; Shattil et al.,
1994
; Cichowski et al., 1996
). In our experiments, expression of Bcl-3 in thrombin-activated platelets required engagement of integrin
IIb
3 at concentrations of thrombin
(0.01-0.1 U/ml) that triggered maximal or near-maximal
platelet aggregation, and was blocked by inhibitory antibodies or peptides against
IIb
3 (see Results). The results
argue that at these concentrations ligation of the thrombin
receptor is not sufficient to induce translation. At higher concentrations of thrombin, there appeared to be an
IIb
3-independent mechanism of signaling when platelets from
a subject with Glanzmann thrombasthenia were studied
(Fig. 6). This is potentially due to differential signaling
through other receptors on platelets that recognize thrombin (Schmidt et al., 1998
). Alternatively, this may represent the activity of a small number of residual copies of integrin
IIb
3 on the platelets that we used for this study
(Jin et al., 1996
).
In addition to integrating of signals delivered via receptors for mitogens and growth factors, integrins of different
classes may also signal cooperatively (Schwartz et al.,
1995; Juliano, 1996
). Our experiments in which adhesion
of platelets to immobilized collagen induced synthesis of
Bcl-3 (Results and Fig. 8) indicate that integrin
IIb
3 and
integrin
2
1 cooperatively signal translation events. We
found that an antibody against the
2 subunit of
2
1 integrin, which recognizes collagen, and mAb 7E3 and 10E5,
which block ligand binding by integrin
IIb
3, each inhibited Bcl-3 synthesis in platelets that adhered to immobilized collagen matrices (Results). One explanation for this
experimental outcome is that adhesion of individual platelets to immobilized collagen caused outside-in signaling
via integrin
2
1 and triggered degranulation and local secretion of fibrinogen, with secondary formation of microaggregates caused by binding of fibrinogen to integrin
IIb
3 on adjacent platelets. Haimovich et al. (1993)
reported that microaggregate formation occurs when platelet suspensions are incubated on immobilized collagen and
that this is blocked by mAb 7E3. Thus, it is possible that
engagement of integrin
IIb
3 by endogenously released
fibrinogen alone signals expression of Bcl-3 under these
conditions. Alternatively, engagement of integrin
2
1
may signal directly to the mTOR pathway when platelets
bind to immobilized collagen and additionally triggers local secretion of fibrinogen and engagement of integrin
IIb
3, concomitantly inducing translation of Bcl-3. Although these two possibilities cannot be resolved yet, our
results are consistent with cooperative interaction of the
two platelet integrins in regulating translational events.
Additional evidence for cooperative interaction between
integrins
IIb
3 and
2
1 has also been reported (Coller et
al., 1989
; Lipfert et al., 1992
; Savage et al., 1998
). Whether
other receptors for collagen (Shattil et al., 1994
; Savage et al.,
1998
; Watson and Gibbins, 1998
) also signal translation in
adherent platelets is unknown at this time.
Our finding that engagement of integrins regulates synthesis of proteins in platelets (Results and Weyrich et al.,
1998) suggests that control of translation is a general
mechanism by which integrins and other classes of adhesion molecules influence gene expression. Adhesion-dependent signaling, a process by which integrins and other
adhesion molecules can specifically modulate or induce
synthesis of particular gene products, adds spatial regulation to this process (Juliano, 1996
; Schwartz et al., 1996
;
Zimmerman et al., 1996
). In addition to spatial regulation,
activation of translation pathways by outside-in signals delivered through integrins or other adhesion molecules can
rapidly induce synthesis of proteins from preformed mRNA,
influencing the temporal sequence of expression of gene products. Specific modulation by integrins of translation
checkpoints, which are downstream of transcription, mRNA
processing, nuclear export, mRNA degradation, also adds
precision and variety in signaling of gene expression that
would not be available if transcription were the only point
of influence (Darnell, 1982
). Our ongoing studies indicate
that translation control occurs when other adhesion molecules besides integrins are engaged (Mahoney, T.S., A.S.
Weyrich, G.A. Zimmerman, T.M. McIntyre, S.M. Prescott, manuscript in preparation), suggesting that signaling
to translational control pathways is a general mechanism
of adhesion-dependent regulation of gene expression.
![]() |
Footnotes |
---|
Address correspondence to Andrew S. Weyrich, Ph.D., or Guy A. Zimmerman, M.D., University of Utah, CVRTI, 95 South 2000 East, Salt Lake City, UT 84112-5000. Tel.: (801) 581-8183. Fax: (801) 581-3128. E-mail: andrew_weyrich{at}gatormail.cvrti.utah.edu or guy_zimmerman{at}gatormail.cvrti.utah.edu
Received for publication 12 August 1998 and in revised form 24 November 1998.
We thank Jeanne Falk, Donnie Benson, and Wenhua Li for excellent technical assistance. We also thank Barry S. Coller, Lisa Jennings, Tom Gadek, Stanley Hollenbach, and Susan Tam for the gifts of important reagents. We are grateful to our colleagues at the CVRTI for their helpful comments and critical reading of the manuscript, to Cletus D'Souza for performing protein determinations, and Diana Lim and Richard Kuenzler for preparation of figures. We appreciate the help of Leona Montoya and Michelle Bills in preparation of the manuscript.
This work was supported by the Nora Eccles Treadwell Foundation, and the Richard A. and Nora Eccles Harrison Fund for Cardiovascular Research, the National Institutes of Health (HL44525), and the Wellcome Trust (UK). Dr. Ravinder Pabla is a Wellcome International Traveling Fellow (grant 046937/Z/96/Z/).
![]() |
Abbreviations used in this paper |
---|
Bcl-3, B cell lymphoma 3; mTOR, mammalian target of rapamycin; PDK1, 3-phosphoinositide-dependent protein kinase 1; PI3K, phosphatidylinositol-3-kinase.
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