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INTRODUCTION |
Intercellular adhesion molecule-1 (ICAM-1,
CD54)1 is a highly
glycosylated 90-110-kDa single chain protein that is up-regulated on
endothelial cells (EC) by inflammatory cytokines such as
interferon-
, interleukin (IL)-1, IL-6, tumor necrosis factor
,
and by phorbol esters. ICAM-1 belongs to a superfamily of
immunoglobulin (Ig)-like proteins and is composed of five extracellular
Ig-like domains, a single transmembrane domain, and a short cytoplasmic
tail. The binding of leukocytes through integrins
L
2 and
M
2
to ICAM-1 on EC results in cellular interactions that regulate immune
and inflammatory reactions (1). Fibrinogen (Fg), a plasma protein, has
been reported to interact with ICAM-1. Fg bound to
M
2 on monocytic cells interact with
ICAM-1 on EC resulting in cellular bridging between monocytic cells and
EC (2, 3). In addition, Fg has been shown to have a vasoconstrictive
effect on the vessel wall through ICAM-1 interaction on EC (4). We have
recently reported that an interaction between Fg and ICAM-1 on
B-lymphoid Raji cells induces a proliferative response in Raji (5).
Amino acid residues 8-22 within the first Ig domain of ICAM-1 and
117-133 within the
chain of Fg have been demonstrated to be
involved in cellular interactions (6, 7), including mitogenesis
(5).
Apart from mitogenesis induced through the ligation of ICAM-1 by Fg,
ICAM-1 ligation with integrins causes oxidative burst in neutrophils
(8), and co-stimulation through ICAM-1 induces the release of IL-2 in
B-cells (9). In addition, ICAM-1 modulates changes in intracellular
Ca2+ in Burkitt's lymphoma (10). These results suggest an
active role for ICAM-1 in signal transduction. However, in recent
reports the occupancy and cross-linking of ICAM-1 by anti-ICAM-1
antibodies has resulted in the phosphorylation of diverse intracellular
proteins (11-13). The activation of certain members of the tyrosine
kinase family of proteins including Lyn and Raf-1 was reported in a
mouse B cell line (11). An increased tyrosine phosphorylation of
cortactin was demonstrated in a rat brain endothelial cell line (12). Also, an increase in tyrosine phosphorylation of cdc2 kinase in peripheral blood T cells resulted in diminished cdc2 kinase activity (13). In these studies the cognitive natural ligands for ICAM-1 were
provided by encephalitogenic T cells (12) and CD3 activated T cells
(11).
As Fg-ICAM-1 interactions mediated through recognition sequences in
this ligand-receptor pair regulate growth in Raji, we sought to
identify signals that may define the unique Fg-dependent proliferative function in this human B-cell line. The proliferative signals generated by activation or ligation of the prototypic growth
factor receptors (platelet-derived growth factor and epidermal growth
factor) results in the activation of the p21ras signaling
pathway. The cytoplasmic tails of the growth factor receptors possess
intrinsic tyrosine kinase activity and become coupled to cytosolic
mitogen-activated protein tyrosine kinases (MAPK) (14, 15). Studies
utilizing either constitutively active or dominant negative mutants of
kinases from the MAP kinase family establish a role for these kinases
in cell proliferation, differentiation, and gene induction (16-18). In
this study using soluble Fg to bind ICAM-1 on Raji, we have observed
that the key proteins that are phosphorylated are pp60Src
and components of the MAP kinase cascade: p44 mitogen-activated protein
kinase (MAPK) and p42 MAPK, also known as ERK-1 and ERK-2 (extracellular signal-regulated protein kinase-1 and -2). In addition, defined regions within Fg and ICAM-1 participate in the signaling process and inhibitors of pp60Src and MAPK kinase (MEK-1)
block Raji mitogenesis induced through the binding of Fg to ICAM-1.
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MATERIALS AND METHODS |
Reagents and Antibodies--
Transferrin, bovine serum albumin
(BSA), and dimethyl sulfoxide were purchased from Sigma.
[methyl-3H]Thymidine was from Amersham Life
Sciences (Arlington Heights, IL) and
-[32P]ATP was
from NEN Life Science Products Inc. Myelin basic protein was from
Upstate Biotechnology (Lake Placid, NY). Human fibronectin purified
according to the method of Vuento and Vaheri (19) was a gift from Dr.
T. Ugarova (Cleveland Clinic Foundation, Cleveland, OH). PD98059, a
specific inhibitor of MEK (20) and geldanamycin and herbimycin A,
inhibitors of pp60Src were purchased from Calbiochem. Each
of these inhibitors was stored at a concentration of 10 mM
in dimethyl sulfoxide at
20 °C. Recombinant protein G-Sepharose
was from Zymed Laboratories Inc. Laboratories (South
San Francisco, CA). Anti-phosphotyrosine antibody PY-20 was purchased
from Upstate Biotechnology (Lake Placid, NY). The polyclonal
antibodies, anti-ERK-1 raised in goat and anti-pp60Src
raised in rabbit, were from Santa Cruz Biotechnology Inc. (Santa Cruz,
CA). Murine anti-ERK-1 antibody was from Zymed Laboratories Inc.
Cell Proliferation Assays--
ICAM-1-expressing lymphoblastoid
Raji cells were obtained from ATCC (Rockville, MD). Raji were grown in
RPMI 1640 (BioWhittaker, Walkersville, MD) containing 7.5% fetal
bovine serum and 1.0 mM glutamine plus penicillin and
streptomycin in incubators maintained at 37 °C and 5%
CO2. Proliferation assays were performed as described previously (5) with minor modifications. Raji were exchanged into
serum-free medium 18 h prior to commencement of an experiment in
order to achieve cellular quiescence. Cells were washed in Iscove's
modified Dulbecco's medium (IMDM) (BioWhittaker) and counted with a
hemocytometer. Aliquots of 0.2 ml of cells (4 × 105
cells/ml) were mixed with 0.2-ml protein solutions diluted with IMDM
and 4 µl of [3H]thymidine (1.0 µCi/µl). Some
protein solutions contained reagents reported to inhibit known cell
proliferation pathways. Cell suspensions (0.1 ml) were aliquoted into
four replicate wells of a 96-well flat-bottomed plate (Becton
Dickinson, Franklin Lakes, NJ) and plates were stored at 37 °C and
5% CO2 for 8 h. To measure uptake of
[3H]thymidine, the contents of each well were transferred
to a 96-well plate with v-shaped wells. Cells were harvested by
centrifugation (200 × g/10 min) and washed twice in
PBS then resuspended in 50 µl of PBS containing 1% (w/v) BSA.
[3H]Thymidine that had been incorporated into cellular
DNA was precipitated by addition of 0.1 ml of 20% (v/v)
trichloroacetic acid for 12 h at 4 °C. Trichloroacetic acid
pellets were solubilized in 0.2 M NaOH containing 2% (v/v)
SDS and assayed for radioactivity in a
-counter.
Preparation of Fg and Fragments of Fg--
Fg was purified from
fresh human plasma by cryoethanol precipitation (21, 22). Using
electrophoretic conditions that allow the separation of fibrin from Fg
monomer and subsequent visualization using Coomassie Blue R-250 (23),
the isolated material was estimated to comprise greater than 95% Fg.
Preparations of Fg were also analyzed for the presence of free
fibrinopeptides A and B by elution on a Sep-Pak C18 high
performance liquid chromatography column using standard preparations of
each of the fibrinopeptides (Sigma). At protein concentrations of at
least 50-fold greater than those used in these experiments, amounts of
fibrinopeptides A and B were below detectable levels.
Synthetic Peptides--
Peptides with amino acid sequences
corresponding to regions of ICAM-1 and Fg (6) were synthesized by the
N-(9-fluorenyl)methoxycarbonyl method on an Applied
Biosystems ABI-66 instrument. Specific sequences were ICAM-1-(8-22),
KVILPRGGSVLVTCS; ICAM-1-(130-145), REPAVGEPAEVTTTV; Fg-
-(117-133),
NNQKIVNLKEKVAQLEA; Fg-
-(117-133), scrambled ALENAEVQNLVKKIQKN; and
Fg-
-(124-133), LKEKVAQLEA. Peptides were cleaved from the resin and
deprotected using crystalline phenol and thioanisole and then purified
by high performance liquid chromatography.
Measurement of Protein Tyrosine Phosphorylation in
Raji--
Raji were maintained in serum-free medium for 18 h
prior to the commencement of an experiment. Cells were washed twice in Dulbecco's PBS and once in ion-free Hanks' balanced salt solution containing 25 mM Hepes, pH 7.4, and resuspended in an
incubation medium of Hanks' balanced salt solution containing 1.0 mM CaCl2, 1.0 mM MgCl2,
and 25 mM Hepes, pH 7.4, to an approximate cell concentration of 105-106 cells per ml. Aliquots
of cells (0.1 ml) were mixed with 0.1 ml of incubation medium alone or
containing 200-400 nM amounts of Fg, fibronectin,
transferrin, or BSA. Some cells were incubated for 10 min with either
100-500 nM amounts of synthetic peptides ICAM-1-(8-22) or
ICAM-1-(130-145) or the inhibitory compounds PD98059 or geldanamycin
prior to the addition of proteins. Cell suspensions were maintained at
22 °C for 10-60 min then 20 µl of 0.1 M sodium
orthovanadate was added and cells were separated from medium by
centrifugation (100 × g/30 s). Cells were washed twice
in Dulbecco's PBS containing 10 mM sodium orthovanadate. Cells were then lysed on ice by addition of 50 µl of lysis buffer (10 mM Tris/HCl, pH 7.5, containing 37 mM NaCl, 1%
(v/v) Nonidet P-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 2 mM
EDTA). Levels of protein in each cell lysate were estimated using the
BCA kit (Pierce, Rockford, IL) and portions of each lysate containing
equivalent amounts of protein (50-70 µg) were separated on 12.5%
acrylamide/SDS gels and proteins were transferred to polyvinylidene
difluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes
were soaked in a solution of 0.15 M NaCl, 20 mM
Tris/HCl, pH 7.5, containing 0.05% (v/v) Tween 20 (wash buffer) to
which was added 5% (w/v) BSA for 1 h, rinsed in wash buffer, then
incubated with anti-phosphotyrosine antibody PY20 for 4 h. Membranes were washed and probed with a goat anti-mouse IgG antibody conjugated with alkaline phosphatase. The bound antibody was detected using the Immunostar chemiluminescence kit (Bio-Rad).
ERK-1 or pp60Src were purified from cell lysates by
immunoprecipitation. Cells were lysed as described and precleared by
incubation with 20 µl of protein G-Sepharose. Aliquots containing
equivalent amounts of protein were mixed with 5 µg of an anti-ERK-1
or anti-pp60Src monoclonal antibodies for 4 h at
4 °C, then 30 µl of a slurry of protein G-Sepharose was added to
each immunoprecipitation tube and tubes were mixed for 4 h at
4 °C. Sepharose beads were washed twice in lysis buffer, and once
with Dulbecco's PBS. The precipitated immune complexes were extracted
from the Sepharose beads by boiling in nonreducing SDS gel loading
buffer. Samples were analyzed for phosphorylation of ERK-1 by elution
on 12.5% acrylamide/SDS gels and immunoblotting as described above.
Kinase activity in samples immunoprecipitated with anti-ERK-1
antibodies was measured using a myelin basic protein phosphorylation
assay (24). Sepharose beads containing precipitated immune complexes
were rinsed twice with lysis buffer and twice with kinase assay buffer
(20 mM Hepes, pH 7.4, containing 0.15 M NaCl,
0.2 mM sodium orthovanadate, 0.1% (v/v) Triton X-100, 10%
(v/v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 100 units/ml Trasylol). For the kinase
assay, Sepharose beads containing immune complexes were resuspended in
30 µl of kinase assay buffer then 10 µl of myelin basic protein (2 mg/ml) and 10 µl of a solution of 1 µCi/µl of [
-32P]ATP (50 µM ATP) containing 7.5 mM MgCl2 was added. Tubes were incubated at
30 °C for 30 min and then 25 µl of the reaction mixture was mixed
with SDS gel loading buffer and eluted on 15% acrylamide/SDS gels.
Gels were fixed and stained with Coomassie Blue R-250, dried, and
exposed to x-ray film overnight. Densitometric quantitation of ECL
exposures and 32P incorporation into myelin basic protein
were performed using a Linotype-Hell Saphir Ultra2 scanner and Adobe
Photoshop software. In each experiment, the final densitometric values
comparing levels of phosphorylation within an experiment were first
corrected to normalize for each amount of relevant proteins immunoprecipitated.
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RESULTS |
Ligation of Raji with Fg Induces Increased Tyrosine Phosphorylation
within Proteins--
Previous results from our laboratory have
identified an increase in cellular proliferation in Raji upon
incubation with nanomolar amounts of Fg (5). This 2-3-fold increase in
proliferation was shown to be dependent in part on the association of
Fg with ICAM-1 on the surface of Raji. As little is known about cell
signaling events associated with occupancy of ICAM-1 by ligands we
investigated the nature of the Fg-induced signal. Since phosphorylation
of tyrosine residues within numerous intracellular proteins is an event
key to the conveyance of a signal from the cell membrane to the
nucleus, we appraised changes in levels of phosphorylation of
intracellular proteins in Raji upon incubation with 200 nM Fg. This concentration of Fg was chosen as it consistently induced cell
proliferation in Raji. Raji were incubated at 37 °C with Fg or
transferrin (as control) for increasing periods of time. Cells were
then rinsed with fresh medium and lysed in TBS containing 1% Nonidet
P-40 and protease inhibitors. Lysates were clarified by centrifugation
and equivalent amounts of protein from cell lysates were separated on
12.5% acrylamide/SDS gels, transferred to PVDF membranes, and probed
with an anti-phosphotyrosine antibody. Fig.
1 shows an increase in phosphorylation of
several proteins in lysates prepared from cells that were treated with
200 nM Fg when compared with control incubations containing
fibronectin or media alone. The major proteins in which a specific
increase in phosphorylation was observed were of approximate molecular masses 32, 40, and 56-65 kDa. The phosphorylation of these proteins was rapid and sustained up to 30 min. The 32-kDa protein became phosphorylated by 5 min and was dephosphorylated by 30 min. The 40-kDa
protein and proteins migrating at 56-65 kDa became phosphorylated at
10 min and were sustained at the 30-min time point (Fig. 1, lane
4).

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Fig. 1.
Increased tyrosine phosphorylation of
intracellular proteins upon incubation of Raji with Fg. Raji were
washed and resuspended in IMDM at a concentration of 5 × 106 cells per ml. Aliquots of cells (0.1 ml) were mixed
with 0.1 ml of incubation medium alone or containing 200 nM
amounts of Fg or fibronectin and incubated at 37 °C for 5-30 min.
Cells were lysed, then equivalent amounts of protein were separated on
12.5% acrylamide/SDS gels, transferred to PVDF membranes, and the
membranes were probed with an anti-phosphotyrosine antibody. Bound
antibody was detected using alkaline phosphatase-conjugated goat
anti-mouse IgG followed by enhanced chemiluminescence.
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Increased Phosphorylation of ERK-1 and pp60Src Induced
by Fg--
In order to identify specific proteins that demonstrated
increased tyrosine phosphorylation as a result of treatment with Fg,
Raji lysates were subjected to immunoprecipitation using antibodies specific for candidate proteins known to be phosphorylated during cellular proliferation events. Intracellular proteins ERK-1 and pp60Src were isolated from cell lysates using monoclonal
antibodies. The resultant immune complexes were captured on protein
G-Sepharose beads. Proteins were eluted from the beads in SDS sample
buffer then separated on SDS-acrylamide gels, transferred onto PVDF
membranes. Membranes were probed with an anti-phosphotyrosine antibody.
Fig. 2 shows an increased tyrosine
phosphorylation of pp60Src (panel A) and ERK-1
(panel B) in samples isolated from cell preparations that
had been treated with 200 nM Fg but not control proteins. The lower portion of each panel demonstrate that equivalent amounts of
each protein were isolated from the cell lysates. Under the assay
conditions, we observed a marked increase in the phosphorylation of pp60Src when cells were incubated in the presence of Fg
compared with cells incubated in media lacking Fg (Fig. 2A, upper
panel). The extent of ERK-1 phosphorylation was modest when Raji
were incubated in the presence of Fg (Fig. 2B). The extent
of phosphorylation in these two proteins could be a reflection of their
relative abundance in Raji. More importantly, the inclusion of 20 µM PD98059 (a cell permeable inhibitor of MEK-1, the
enzyme responsible for phosphorylating ERK-1) to Raji cultures blocked
the increased phosphorylation of ERK-1 induced by 200 nM
Fg. This result validates the specificity of ERK-1 activation in cells
incubated with Fg.

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Fig. 2.
Increased phosphorylation of tyrosine
residues within pp60Src and ERK-1 and a corresponding
increase in ERK-1 kinase activity in Raji treated with fibrinogen.
5 × 105 Raji were incubated at 37 °C in IMDM alone
or containing 200 nM Fg or fibronectin for 10 min. The
MEK-1 inhibitor PD98059 (20 µM) was included in some
cultures containing Fg. Cells were lysed and monoclonal antibodies
against either pp60Src (panel A) or ERK-1
(panel B) were used to purify these proteins from lysates
containing equivalent amounts of protein. Immunocomplexes were captured
using protein G-Sepharose and eluted with 3 × SDS buffer
(upper panels A and B) then captured proteins
were separated on SDS gels, transferred to PVDF membranes, and probed
using an anti-phosphotyrosine antibody. Membranes were then stripped
and reprobed with anti-pp60Src antibody or a rabbit
anti-ERK-1 antibody to determine equal loading of pp60Src
and ERK-1 (lower panels A and B). Levels of
kinase activity were estimated in samples of protein G-captured
immunocomplexes formed using the ERK-1 antibody. Immunoprecipitated
ERK-1 was mixed with 10 µg of the ERK-1 substrate myelin basic
protein and 20 µM [ -32P]ATP (10 µCi)
and samples were incubated at 30 °C for 20 min. The reaction was
stopped by addition of 3 × SDS buffer and proteins were separated
on SDS gels, dried, and subjected to autoradiography (upper panel
C). Portions of immunoprecipitated ERK-1 were subjected to
immunoblotting using a rabbit anti-ERK-1 antibody to estimate amounts
of ERK-1 immunoprecipitated (lower panel C). Relative band
densities were measured as described under "Materials and Methods"
and are indicated.
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The increased ERK-1 activity in cultures treated with Fg was further
confirmed using a kinase assay. Samples of ERK-1 that had been purified
from control or Fg-treated Raji lysates by immunoprecipitation were
measured in an assay using [
-32P]ATP as a phosphate
donor and myelin basic protein as a kinase substrate. Assay tubes were
incubated at 30 °C for 20 min before the addition of 3 × SDS
gel loading buffer. Tubes were boiled and the contents were separated
on 15% acrylamide/SDS gels and levels of incorporation of
32P were estimated by autoradiography and densitometry.
Fig. 2 (panel C) illustrates an increase in levels of
32P associated with material corresponding to the molecular
mass of myelin basic protein (18 kDa) in samples isolated from
Fg-treated Raji lysates. These results indicate that phosphorylation of
ERK-1 renders it enzymatically active in a manner that is dependent on
the concentration of Fg. Densitometric measurements of the 32P-labeled bands revealed a 2.29-fold increase in
incorporation of radioactivity into myelin basic protein by ERK-1
isolated from cells that had been treated with 20 µg/ml Fg, as
compared with control cells treated with 20 µg/ml BSA. This fold
increase in ERK-1 activity (Fig. 2C) parallels the extent of
phosphorylation observed in Fig. 2B. An increased
phosphorylation of tyrosine residues within both of these proteins is
known to correspond to increased kinase activity of these proteins and
correlates with known roles for these proteins in intracellular
signaling cascades resulting in increased cellular proliferation.
Specific Inhibitors of Intracellular Kinases Block Proliferation of
Raji induced by Fg--
Geldanamycin (25, 26) and herbimycin A (27)
are inhibitors of pp60Src. PD98059 (20), a specific
inhibitor of the upstream regulator of ERK-1 (MEK-1), has been
previously used to block cell proliferation mediated through the MAP
kinase pathway (17, 28). The above reagents were used to assess the
role of these kinases in Fg-induced Raji proliferation. Fig.
3 shows that the inclusion of 62 nM geldanamycin (panel A), 2.5 µM
herbimycin A (panel B), or 25 µM PD98059
(panel C) resulted in greater than 50% blockade of
proliferation of Raji induced by 200 nM Fg. Higher
concentrations of geldanamycin (1.0 µM), herbimycin A
(>5.0 µM), or PD98059 (100 µM) completely
abrogate the proliferation in Raji. Equivalent amounts of vehicle
(dimethyl sulfoxide) alone did not influence the proliferation. These
results indicate that the induction of proliferation in Raji generated by Fg requires the activation of the Src family and MAP kinase family
of proteins, in a manner reminiscent of proliferation induced by growth
hormones receptors.

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Fig. 3.
Fg-induced proliferation in Raji is blocked
by inhibitors of pp60Src and MEK-1. Serum-depleted
Raji (104 cells/well) were mixed with IMDM containing 1.0 µCi of [3H]thymidine per well, 200 nM Fg,
and increasing concentrations of inhibitors of pp60Src,
geldanamycin (panel A), or herbimycin (panel B)
or the MEK-1 inhibitor PD98059 (panel C). Cells were
incubated at 37 °C for 8 h, then washed. Levels of
[3H]thymidine incorporated into macromolecules were
assessed by radioactive assay of trichloroacetic acid precipitates.
Results are the average of 4 replicate wells for each incubation and
are representative of three experiments. Control incubations containing
200 nM Fg and equivalent amounts of dimethyl sulfoxide gave
results similar to incubations containing 200 nM Fg
alone.
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A Peptide Corresponding to ICAM-1 Fragment-(8-22) Blocks
Fg-induced Phosphorylation of ERK-1--
The region ICAM-1-(8-22) has
been shown to mediate Fg-binding (6) and block Fg-induced proliferation
in Raji (5). To confirm a requirement for occupancy of ICAM-1 by Fg to
generate increased phosphorylation of ERK-1, Fg was preincubated with
peptides corresponding to ICAM-1 amino acid fragment-(8-22) or
fragment-(130-145). Raji were incubated with 200 nM
Fg/peptide mixtures at 37 °C for 10 min then cells were rinsed,
lysed, and ERK-1 was isolated by immunoprecipitation as described.
Immune complexes were eluted from protein G beads and separated on
12.5% acrylamide/SDS gels, transferred to PVDF membranes, and probed
with an anti-phosphotyrosine antibody. Fig.
4 demonstrates that inclusion of 50 µM amounts of ICAM-1-(8-22) but not ICAM-1-(130-145)
caused a reduction of the increased phosphorylation of ERK-1 in Raji,
induced by 200 nM Fg. An analysis of ICAM-1-(8-22) and
ICAM-1-(130-145) indicates that these two peptides are structurally
comparable (5). Densitometric scanning of the upper panel of
Fig. 4 indicated a 26% reduction in the band intensity in incubations
containing ICAM-1-(8-22) after correction for gel loading, compared
with ERK-1 isolated from incubations treated with Fg alone. Inclusion
of either ICAM-1-(8-22) or ICAM-1-(130-145) alone in incubations did
not influence phosphorylation of ERK-1 (data not shown). In our
proliferation assays we observe that 50-100 µM
ICAM-1-(8-22) causes up to 60% reduction in Fg-induced proliferation
of Raji (5). This indicates that association of Fg with ICAM-1 via the
region spanning amino acids 8-22 is important for the generation of
intracellular signals and increased tyrosine phosphorylation observed
in ERK-1 upon treatment of Raji with Fg.

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Fig. 4.
ICAM-1-(8-22) blocks Fg-induced tyrosine
phosphorylation of ERK-1 in Raji. Aliquots of 200 nM
Fg were incubated at 37 °C with 50 µM amounts of
peptides with sequences corresponding to ICAM-1-(8-22) or
ICAM-1-(130-145) for 1 h. Fg/peptide mixtures were then incubated
with 106 Raji at 37 °C for 10 min then cells were lysed
and ERK-1 protein was isolated from cell lysates containing equivalent
amounts of protein. Levels of tyrosine phosphorylation in
immunoprecipitated ERK-1 was estimated by immunoblotting using an
anti-phosphotyrosine antibody (upper panel) and measurement
of relative band densities are indicated. Equivalent amounts of ERK-1
were confirmed by stripping immunoblots and reprobing with a rabbit
anti-ERK-1 antibody (lower panel).
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A Region of Fg Induces Increased Activity in ERK-1--
A discrete
region of Fg known to bind to ICAM-1 (7) is able to induce a
proliferative response in Raji (5). We investigated whether this
portion of Fg encompassing amino acids 117-133 of the
chain could
induce increased phosphorylation of ERK-1 in Raji. Raji were incubated
with 100 µM amounts of either Fg peptides
-(117-133)
or
-(117-133 scrambled) for 10 min at 37 °C, then cells were
washed and lysed as described under "Materials and Methods." ERK-1
was immunoprecipitated from equivalent amounts of protein from each
lysate, and levels of tyrosine phosphorylation were analyzed by
immunoblotting as described under "Materials and Methods." Fig.
5 shows increased tyrosine
phosphorylation of ERK-1 extracted from cell preparations treated with
100 µM amounts of Fg-
-(117-133) but not
Fg-
-(117-133 scrambled), suggesting the interaction of this
discrete region of Fg with ICAM-1 was sufficient to increase activity
of ERK-1 in Raji. This result is in good agreement with previous data
which demonstrated an increased cell proliferation in Raji that were
incubated with 100 µM amounts of Fg-
-(117-133) (5).
Raji binding studies using soluble 125I-labeled
Fg-
-(117-133) produce a binding curve which suggests binding of
Fg-
-(117-133) to a single class of proteins on Raji (data not
shown). Comparison of the primary structure of this peptide with amino
acid sequences of other known peptide hormones reveal no homology. It
remains to be determined whether the tertiary structure of this peptide
resembles the structure of other peptides known to have a role in cell
proliferation.

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Fig. 5.
A peptide from within the chain of Fg induces increased tyrosine phosphorylation of
ERK-1. 5 × 105 Raji were incubated at 37 °C
in IMDM containing either 200 nM Fg or 100 µM
amounts of Fg peptides -(117-133) or -(117-133) scrambled for
10 min. Cells were lysed and a monoclonal antibody against ERK-1 was
used to purify ERK-1 from lysates containing equivalent amounts of
protein. Immunocomplexes were captured using protein G-Sepharose,
washed, and eluted with 3 × SDS buffer then proteins were
separated on SDS gels, transferred to PVDF membranes, and probed using
an anti-phosphotyrosine antibody (upper panel). Bound
antibodies were visualized using alkaline phosphatase-conjugated
secondary antibody and enhanced chemiluminescence. Membranes were then
stripped and reprobed with a rabbit anti-ERK-1 antibody to determine
equal loading of ERK-1 (lower panel). Relative band
densities are indicated.
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|
 |
DISCUSSION |
We have previously reported that Fg binding to ICAM-1 expressing
B-lymphoid Raji cells results in the proliferation of these cells (5).
In the present report, our results indicate that ligation of ICAM-1 by
Fg, a dimeric molecule, induces the rapid activation of
pp60Src and ERK-1. These kinases become specifically
tyrosine phosphorylated within Raji in the presence of soluble 200 nM Fg. The immediate upstream regulator of ERK-1 is MAPK
kinase also known as MEK-1. A specific inhibitor of MEK-1, PD98059,
blocked Fg induced cell proliferation in Raji and blocked the ERK-1
activation in Raji lysates (Figs. 2B and 3C). At
similar concentrations PD98059 blocked growth activity induced by
platelet-derived growth factor and epidermal growth factor (17).
Concentrations of geldanamycin and herbimycin A similar to those used
in other studies to specifically inhibit pp60Src (25, 29,
30) were sufficient to block Raji proliferation. In our previous
report, Fg recognition peptide ICAM-1-(8-22) blocked mitogenesis,
while the ICAM-1 recognition peptide Fg-
-(117-133) induced
proliferation of Raji (5). In concordance with the above results, 50 µM ICAM-1-(8-22) blocked the hyperphosphorylation of
ERK-1 induced through the interaction of Fg with Raji (Fig. 4).
Fg-
-(117-133) on the other hand through its interaction with ICAM-1
induced ERK-1 activation. The peptide results are an independent verification and extend the findings that suggest that the activation of the MAP kinase pathway is involved in B-cell proliferation induced
through Fg interaction with ICAM-1. Therefore, Fg and its derivative
Fg-
peptide-(117-133), utilize and transduce growth signal similar
to that of bona fide growth factors (platelet-derived growth factor and
epidermal growth factor), phorbol esters, and cytokines. In this
context it is tempting to speculate that Fg binding to ICAM-1 may cause
ICAM-1 dimerization or clustering (31), as is the case with
platelet-derived growth factor and epidermal growth factor receptor
upon ligand interaction. Nevertheless, this is the first report of
Fg-induced ICAM-1 signaling, and more interestingly through a small
discrete region Fg-
-(117-133) that ligates ICAM-1.
The structure of ICAM-1 lacks the common tyrosine-containing motif
required for the recognition of Src family kinases that has been
identified in other receptors (14, 15). However, Lyn has been
demonstrated to bind IL-3 and IL-5 receptors that lack the consensus
Src family binding motifs (32). It is likely that ICAM-1 may transduce
signals by association with adaptor proteins such as Grb 2, SOS, and
Shc; some of these proteins are known to participate in signaling
through integrins (33). Nevertheless, recently the Src-related protein
tyrosine kinase Lyn, has been shown to be phosphorylated upon ICAM-1
ligation with monoclonal antibodies in murine B-cells (11). However, in
this report a role for Lyn in mitogenesis was not investigated.
Interestingly, in the rat brain endothelial cell line (RBE4), ligation
of ICAM-1 with syngeneic encephalitogenic T cells induces tyrosine
phosphorylation of pp60Src and its substrate cortactin, an
85-kDa actin-binding protein (12). Such cellular and cytoskeletal
changes may facilitate the transmigration of lymphocytes in the brain.
The physical association of ICAM-1 cytoplasmic tail with another
actin-binding protein,
-actinin, has also been documented (34). The
interaction of Fg with integrin
M
2 on
monocytic cells augments the ability of these cells to adhere and
transmigrate through the EC (2, 35). In this context, future
experiments will verify whether pp60Src and cortactin
become phosphorylated, in contrast to ERK-1, in a distinct ICAM-1
ligation process.
There is now overwhelming evidence that the tyrosine phosphorylation of
ERK-1 is involved in cell proliferation. However, recent reports now
indicate that ERK-1/2 could also act as negative regulators of cell
activation, in particular the integrin adhesion receptors (36, 37). ERK
is also implicated in smooth muscle cell contraction (38). In a recent
report (4), Fg induced contraction of saphenous vein endothelium in an
ICAM-1-dependent manner. If the activation of ERK could be
demonstrated in these cells through Fg-ICAM-1 interaction, the above
findings may have a mechanistic explanation.
Fibrin and derivatives of Fg have been implicated in the mitogenesis of
endothelial cells (39, 40). Our results have now implicated intact Fg
and a peptide within the
chain of Fg (Fg-
-(117-133)) in the
activation of a classical MAP kinase pathway in proliferation of
B-lymphoid cells through an adhesive receptor, ICAM-1. The Fg-ICAM-1
pathway has been proposed to play a role in inflammatory processes and
in malignant cell growth (2, 5). Since compounds such as PD98059,
herbimycin A, and geldanamycin, block proliferative signals and cell
growth through Fg-ICAM-1 interaction, these reagents may help
understand vascular diseases such as restenosis after angioplasty
(17).