(Received for publication, November 26, 1996, and in revised form, February 4, 1997)
From the Departments of Surgery, Toronto Hospital-General Division and the University of Toronto, Toronto, Ontario M5G 2C4, Canada
Adhesion molecules such as VLA-4 are important
not only for monocyte adhesion to extracellular matrix proteins, but
also for subsequent cell activation. Monocyte adherence to fibronectin or engagement of VLA-4 has been demonstrated to stimulate production of
potent inflammatory mediators such as tumor necrosis factor-, interleukin-1, and the procoagulant tissue factor protein. However, the
intracellular signaling cascades leading to gene expression have not
been elucidated. Using the human monocytic THP-1 cell line, VLA-4
cross-linking by monoclonal antibodies directed against its
4 and
1 subunits produced a
time-dependent increase in tyrosine phosphorylation of a
broad range of cellular proteins. Using Western blot analysis directed
against the phosphorylated form of the extracellular signal-related
kinase (ERK) mitogen-activated protein (MAP) kinase proteins, as well
as immunoprecipitation and in vitro kinase assays, we found
that VLA-4 cross-linking increased ERK1/ERK2 tyrosine phosphorylation
and activity. In conjunction, integrin cross-linking also increased
NF-
B nuclear translocation and 4-h expression of tissue factor.
Inhibition of tyrosine kinase activity with genistein (10 µg/ml) as
well as selective MAP kinase inhibition with the MEK-1 inhibitor
PD98059 abolished the VLA-4-dependent ERK tyrosine
phosphorylation, inhibited NF
B nuclear binding, and abrogated
tissue factor expression induced by both VLA-4 cross-linking and
adhesion to fibronectin in THP-1 cells and human peripheral blood
monocytes. These studies point to the involvement of the MAP kinase
pathway in the activation of monocytic cells during transmigration to
inflammatory sites.
The integrin family of surface adhesion molecules plays a key role in leukocyte recruitment to areas of extravascular inflammation. These heterodimeric integral membrane proteins are important not only for the adhesion to and transmigration across endothelial barriers, but also for adhesive interactions with extracellular matrix proteins (1-3). While initially considered important only for their adhesive properties, recent studies have suggested that integrin engagement can initiate signal transduction pathways contributing to cellular activation (4-8).
Monocyte recruitment to extravascular sites is an important component of the host response to a variety of stimuli including bacterial infection, tumor deposits, and atherosclerotic plaques. In this location, the surface expression as well as release of a number of macrophage products serve to coordinate the local inflammatory response. Fibrin deposition induced by macrophage tissue factor (TF)1 expression is known to contribute significantly to the development of this response. Within the vascular space, adherence of monocytes to the endothelium stimulates expression of TF on monocytes, a process that likely contributes to local microvascular thrombosis (9, 10). Furthermore, at extravascular sites, products of both the coagulation and fibrinolytic cascades contribute to the generation of the inflammatory response through their interaction with infiltrating cells. Indeed, strategies aimed at reducing fibrin deposition or precluding TF expression have been shown to mitigate the full expression of both the local and systemic inflammatory response depending on the model system studied (9, 11-15).
Recent work has defined a role for integrin engagement in the induction
of monocyte TF expression, as well as other immediate early genes such
as interleukin-1 (16), interleukin-8, and tumor necrosis factor, and
(8) transcription factors I
B (11), c-Jun, and c-Fos (17). While
monocytes are endowed with a variety of surface integrins, engagement
of very late antigen 4 (VLA-4) appears to consistently induce gene
expression (16-23). For example, ligation of VLA-4 by monoclonal
antibody in both human peripheral blood monocytes (PBM) and in the
monocytic THP-1 cell line promotes TF expression, whereas engagement of
2 integrins has little effect (20). The intracellular
signaling mechanisms leading to the VLA-4-mediated induction of TF as
well as other inflammatory genes appears to involve the induction of
tyrosine phosphorylation (16, 18, 24). Recent studies by Lin and
colleagues have implicated a possible signaling role for Syk tyrosine
kinase in this process (18). In human monocytic THP-1 cells, VLA-4
engagement caused prominent tyrosine phosphorylation as well as
activation of Syk tyrosine kinase, an effect that occurred in concert
with the induction of the interleukin-1
gene.
Integrin engagement through interaction with extracellular matrix
proteins has been shown to contribute to the regulation of cellular
growth and differentiation and to modulate tumor behavior (1). In
fibroblasts, this process involves tyrosine phosphorylation and
activation of MAP kinase through a cascade involving Raf-1 and MEK (1,
25-27). While this cascade is known to be activated in macrophages in
response to various proinflammatory stimuli (28-35), its contribution
to the 1 integrin-induced activation of inflammatory
cell gene expression is unknown. In the present studies, engagement of
VLA-4 on the surface of THP-1 monocytic cells and on human monocytes,
both through integrin cross-linking and attachment to a fibronectin
substratum, was shown to induce tyrosine phosphorylation and activation
of the ERK1/ERK2 MAP kinases. This effect occurred in parallel with
nuclear translocation of NF-
B and stimulation of TF on the surface
of these cells. Furthermore, we took advantage of the existence of a
novel selective inhibitor of MEK1 to define the contribution of this
pathway to integrin-induced TF expression. This agent, PD98059, caused
a dose-dependent inhibition of tyrosine phosphorylation and
activation of MAP kinase in response to VLA-4 engagement and
concomitantly prevented NF-
B translocation and TF induction.
Considered together, these studies support a contribution of the MAP
kinase pathway to integrin-induced gene expression in cells of
monocyte/macrophage lineage.
Buffers and Reagents
Genistein was purchased from Calbiochem and prepared in
Me2SO at 10 mg/ml. The selective MEK-1 inhibitor PD98059
was the kind gift of Dr. R. Saltiel, and was prepared in
Me2SO. Escherichia coli O111:B4
lipopolysaccharide (LPS) was purchased from Life Technologies, Inc., as
were endotoxin-free RPMI and HBSS media. Fetal calf serum (FCS) was
from HyClone. The following antibodies were used in the integrin
engagement studies: mouse IgG1 anti-CD49d (mAb HP2.1 (Immunotech) and
mAb 44H6 (Serotec)), CD29 (mAb K20 and mAb Lia1.2 (Immunotech)), goat
F(ab)2 anti-mouse IgG (Immunotech), mouse IgG1 anti-CD45
mAb 1214 (PDI Bioscience), and negative mouse IgG1 (Serotec).
Inhibitory anti-tissue factor antibody (mAb 4509) was obtained from
American Diagnostica.
Cell Preparation
Human monocytic THP-1 cells (ATCC) were propagated in RPMI/10% FCS/penicillin/streptomycin at 37 °C, 5% CO2. Human PBM were isolated from the blood of normal healthy donors by centrifugation over a Ficoll-Hypaque gradient at 400 × g for 20 min. The mononuclear layer was aspirated, washed twice and resuspended in RPMI/2% FCS/L-glutamine. This cell population contained 25-35% monocytes as assessed by Wright's stain and CD14 expression (flow cytometry with fluorescein isothiocyanate-conjugated anti-CD14 Ab; Becton-Dickinson), with >96% viability by trypan blue exclusion and propidium iodide uptake.
Cell Activation
For integrin engagement studies, THP-1 cells were suspended in
RPMI/2% FCS/L-Gln at 5 × 106 cells/ml.
Surface CD29, CD49d, and CD45 antigens were ligated with monoclonal
antibody for 25 min at 15 µg/ml and 4 °C, washed twice in cold
RPMI, and then cross-linked with 5 µg/ml goat anti-mouse F(ab)2 for 25 min at 4 °C. The concentration of primary
antibody was shown to be saturating by flow cytometry (data not shown). Cells were washed twice in cold RPMI, resuspended in RPMI/2%
FCS/L-Gln and incubated at 37 °C, 5% CO2
for times ranging from 1 min to 4 h. Reactions were stopped by
placing the cells on ice. For cell adhesion studies, six-well
polystyrene culture plates were coated either with 1 mg/ml
poly-L-lysine or 0.05 mg/ml fibronectin for 1 h at
25 °C. Poly-L-lysine was cross-linked under UV light for 2 h; all wells were then washed twice in cold RPMI and blocked for
1 h with RPMI/10% FCS at 37 °C. THP-1 (1 × 106) or PBM (0.5 × 106) were layered onto
the coated wells and allowed to settle for 25 min at 4 °C, and then
brought to 37 °C for up to 4 h before being placed on ice. In
inhibition studies, THP-1 cells or human PBM were preincubated in the
presence of 1 and 10 µM PD98059 or 10 µg/ml genistein
for 45 min at 4 °C.
Measurement of Procoagulant Activity
At 4 h, THP-1 cells (0.5 × 106) were
sedimented at 1000 × g for 3 min. The cell pellet was
resuspended at 106 cells/ml RPMI, freeze-thawed at
70 °C, and procoagulant activity (PCA) measured by single-stage
recalcification clotting assay (36). PCA was expressed as
milliunits/106 cells by comparison to rabbit brain
thromboplastin. In a typical experiment,
1 integrin
cross-linking shortened clotting times from 70 s to 56 s,
representing an increase in PCA from 74 milliunits/106
cells to 376 milliunits/106 cells. In the cell adhesion
studies, THP-1 or PBM were harvested using a rubber policeman over ice
after a 4 h incubation, washed, and resuspended at 106
cells/ml RPMI for PCA assessment. PCA was attributed to TF expression on the basis of a complete reversal of PCA with inhibitory anti-TF antibody (see "Results") and failure of FVII-deficient serum to clot (data not shown).
Western Blot Analysis
Following integrin cross-linking, THP-1 cells were lysed in ice-cold cell lysis buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 2 mM sodium orthovanadate, 10 µg/ml leupeptin, 50 mM NaF, 5 mM EDTA, 1 mM EGTA, and 1 mM PMSF. Postnuclear supernatants were collected following centrifugation at 10,000 × g for 5 min and diluted with 2 × Laemmli buffer, 0.1 M dithiothreitol (DTT). Following adhesion to poly-L-lysine or fibronectin substrata, PBM were lysed in the culture wells with ice-cold lysis buffer and prepared in a similar fashion. Lysates prepared from 100,000 cells were separated on 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Mobilion). Blots were then probed with polyclonal rabbit (Transduction Laboratories) anti-phosphotyrosine antibody, rabbit anti-phospho-ERK antibody (New England Biolabs), rabbit anti-ERK1 or -ERK2 antibody (Santa Cruz Biotechnologies). Following incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (Sigma), blots were developed using an ECL-based system (Amersham).
Immunoprecipitations and Immune Complex Kinase Assays
Phosphotyrosine and ERK ImmunoprecipitationsCells (3 × 106) were lysed as above and the postnuclear supernatant precleared with Protein G-Sepharose (Pharmacia Biotech Inc.). Cellular proteins were immunocomplexed using polyclonal anti-phosphotyrosine antibody (Transduction) or anti-ERK-1 and -ERK-2 antibody (SCB) for 1 h at 4 °C. Protein G-Sepharose was added and incubated at 4 °C for 1 h. The resulting immune complexes were washed five times with cold phosphate-buffered saline/0.01% Tween 20, and then separated from beads by 2 × Laemmli buffer, 0.1 M DTT and boiling at 100 °C for 5 min. Beads were then sedimented by ultracentrifugation and the supernatant collected for Western blot analysis.
ERK-2 Immune Complex Kinase AssaysERK-2 immunocomplexes
were washed with five changes of cold phosphate-buffered saline/0.01%
Tween 20, and then incubated for 30 min at 30 °C with 20 µg of
ultra-pure myelin basic protein (MBP, Upstate Biotechnology, Inc.) in
kinase assay buffer composed of 0.4 mM cold and 0.4 mM [-32P]ATP (DuPont NEN), 50 mM Tris-HCl (pH 7.4), and 10 mM
MgCl2. Reactions were stopped with the addition of 2 × Laemmli buffer, 0.1 M DTT and boiling at 100 °C for 5 min. Equal volumes were loaded and run on 10% SDS-PAGE. The
radioactivity of the phosphorylated MBP band running at 20 kDa was
quantified on a Molecular Dynamics SI PhosphorImager.
Preparation of Nuclear Extracts
Following cell activation by VLA-4 cross-linking, 5 million THP-1 cells were washed twice in cold HBSS and lysed in 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, and 0.1% Nonidet P-40. Following centrifugation at 13,000 rpm (4 °C) for 10 min, the nuclear pellet was resuspended in 15 µl/107 cells extract buffer containing 20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 0.5 mM spermidine, 0.15 mM spermine, and 5 µg/ml each of leupeptin, pepstatin, and aprotinin. Supernatants were collected after a 15-min centrifugation at 14,000 rpm (4 °C) and diluted with 75 µl of buffer containing 20 mM HEPES (pH 7.9), 20% glycerol, 0.2 mM EDTA, 50 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF and immediately frozen on dry ice. Protein concentrations were determined using the Bradford protein assay (Bio-Rad).
Electrophoretic Mobility Shift Assay
5 µg of nuclear extract protein were preincubated with the
nonspecific DNA competitor poly(dI-dC) (5 µg, Pharmacia) for 10 min
at room temperature. 32P-Radiolabeled probe containing 2 NF-B sites derived from the human immunodeficiency virus-1 enhancer
(HIV-ENH), or containing the TF-specific NF-
B site (49), was
incubated for an additional 20 min at room temperature. DNA-protein
complexes were resolved on a 5% non-denaturing polyacrylamide (60:1
cross-link)/Tris glycine gel and autoradiographs prepared by exposure
at
70 °C using a Kodak X-Omat film. To demonstrate specificity of
the protein-DNA complex, 125 M excess of unlabeled probe or
mutated TF
B probe was added to the nuclear extract before adding
the radiolabeled probe. The sequences of the plus strands of the
oligonucletides used were as follows.
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Statistical Analysis
The data are represented as the mean and standard error of the indicated number of experiments. Where representative studies are shown, these are indicative of at least three equivalent studies. Statistical comparisons were made for continuous data using one-way ANOVA with post hoc Tukey. For comparisons of experiments involving two treatment groups only, the two-tailed Student's t test was employed.
Fig. 1A demonstrates that
cross-linking of either the 1 or
4
subunits of VLA-4 induces a time-dependent increase in
phosphotyrosine accumulation in THP-1 cells. The increase occurred
within 1 min, reached a maximum at 5-10 min, and persisted for ~30
min. While receptor ligation alone caused a small increase in
phosphotyrosine residues, cross-linking markedly amplified this effect
(Fig. 1B). This effect was not due to the addition of the
secondary antibody per se, since this did not induce
phosphotyrosine accumulation (Fig. 1C). We also compared the
effect of cross-linking using antibodies directed against different
epitopes of the
4
1 integrin. As shown in
Fig. 1D, cross-linking of
4
1
with the inhibitory antibody Lial 1.2 directed against the
1 subunit caused a similar pattern of phosphotyrosine
accumulation compared with the non-inhibitory antibody K20, although
the magnitude was somewhat less. HP2/1, an inhibitory antibody directed
against the
4 subunit caused similar accumulation of
phosphotyrosine residues, while use of 44H6, also directed against
4, failed to induce tyrosine phosphorylation despite
surface binding equivalent to mAb HP2/1. (data not shown, as assessed
by flow cytometry, Coulter Epics MCL). Finally, the effect was not due
to the process of cross-linking of surface antigen per se,
since neither cross-linking with mAb 1214 (Fig. 1C) nor 44H6
induced an increase in tyrosine-phosphorylated proteins (Fig.
1D). As for 44H6, mAb 1214 had surface binding
characteristics equivalent to K20 and HP2/1 (data not shown, as
assessed by flow cytometry).
Endotoxin is known to cause tyrosine phosphorylation in monocytic cells (31, 34). The observed effect of cross-linking was unlikely to be due to endotoxin contamination of the system. Polymyxin B did not reverse the stimulation of phosphotyrosine accumulation, and heating of the primary antibody prior to incubation with the cells completely prevented induction of tyrosine phosphorylation. Furthermore, as indicated above, neither primary nor secondary antibody alone mimicked the stimulatory effect.
Tyrosine Phosphorylation and Activation of ERK1/ERK2 following VLA-4 Cross-linkingWhile cross-linking induced a pattern of tyrosine phosphorylation encompassing a broad range of proteins, cross-linking with K20 caused some degree of persistent phosphorylation in the 42-50-kDa range. To determine whether p44 ERK1/p42 ERK2 MAP kinases might be among the candidate substrate proteins, cell lysates were probed first for phosphotyrosine residues, and the blots stripped and reprobed for p42 ERK2 and p44 ERK1. Both ERK proteins comigrated with an area of VLA-4-induced persistent tyrosine phosphorylation (data not shown). Importantly, the absolute amount of ERK protein in the cell lysates did not change over time following integrin engagement.
To conclusively identify the ERK proteins as targets of the
VLA-4-induced tyrosine phosphorylation, two approaches were used. First, immunoprecipitation studies were carried out at 5 min after cell
stimulation. The upper panel of Fig.
2A shows a blot of immunoprecipitated tyrosine phosphoproteins probed with anti-ERK2 Ab under each of the
treatment conditions. Integrin cross-linking with K20 markedly enhances
the amount of tyrosine-phosphorylated ERK2 protein compared with
control cells, while engagement alone causes a slight increase. A
similar effect is observed when cell lysates are sedimented with either
anti-ERK1 Ab or anti-ERK2 Ab and then probed with anti-phosphotyrosine
Ab (Fig. 2A, middle and lower panels,
respectively). Similar results were obtained when cell surface
integrins were cross-linked with mAb against the 4
subunit (data not shown). ERK phosphorylation induced by VLA-4
cross-linking was also evaluated by Western blot studies using an
antibody specific to ERK phosphorylated on tyrosine residues.
Cross-linking of VLA-4 by mAb against the
1 subunit
induced a time-dependent increase in ERK tyrosine
phosphorylation, particularly of the p42 ERK2 (Fig. 2B).
This effect occurred early, peaked at 30 min, and persisted for at
least 60 min. This was accompanied by a slight retardation in the
electrophoretic mobility, consistent with phosphorylation of ERK2.
Integrin ligation alone caused a somewhat delayed rise in phospho-ERK
accumulation, although cross-linking induced more phosphorylation at
any given time point.
Having demonstrated tyrosine phosphorylation of ERK1/ERK2 proteins,
studies were performed to evaluate their level of activation. Using MBP
as a substrate, Fig. 3 (A and B)
shows that cross-linking of 4 and
1
subunits of VLA-4, respectively, cause a time-dependent increase in immunoprecipitated ERK2 activity, the magnitude of which is
similar to that seen with 1 µg/ml LPS. Similar results were seen with
immunoprecipitated ERK-1, although the increase in activity stimulated
by VLA-4 engagement was less, approximately 2-3-fold (data not shown).
As expected from the pattern of induction of phosphotyrosine
accumulation, cross-linking mAb Lia1.2 markedly increased
immunoprecipitated ERK2 activity, while cross-linking 44H6 or mAb 1214 had little to no effect (data not shown).
Adherence to Fibronectin Stimulates Tyrosine Phosphorylation of ERK
To determine whether adhesion to a physiological substratum
might induce a similar activation of ERK, human cells were plated onto
fibronectin or poly-L-lysine substrata. Fig.
4A shows the pattern of accumulation of
tyrosine-phosphorylated proteins in response to adhesion to the
indicated substratum. In particular, proteins with molecular masses
corresponding to 42, 72, 85, and 95 kDa were tyrosine-phosphorylated in
fibronectin-exposed cells but not in cells adhered to
poly-L-lysine. This pattern generally corresponded to that
observed for VLA-4 cross-linking by antibodies in suspended, although
the pattern was less complex (compare with Fig. 1). Tyrosine
phosphorylation of ERK following adhesion was also evaluated by
immunoprecipitation. Cell lysates were recovered at varying time points
after adhesion to fibronectin or poly-L-lysine, and ERK2
was immunoprecipitated and probed with anti-phosphotyrosine antibody.
As shown in Fig. 4B, there was there was a
time-dependent rise in phosphorylated ERK in
fibronectin-exposed cells, but not in those adhered to
poly-L-lysine. Total ERK protein after immunoprecipitation did not differ between substrata (data not shown).
PD98059 Inhibits Both ERK Activation and Tyrosine Phosphorylation
Recent studies have reported the development of a
synthetic inhibitor of the MAP kinase pathway. This compound, PD98059,
was shown to specifically block the activation of MEK1, the upstream activator of ERK1/ERK2, without an effect on other protein kinases including the stress activated protein kinases and p38 (37, 38). Having
demonstrated that VLA-4 cross-linking was able to induce activation of
ERK1/ERK2, we used this agent to discern the role of this pathway in
stimulating gene expression in macrophages in response to integrin
engagement. First, the effect of PD98059 on VLA-4-stimulated ERK
activation was studied. PD98059 prevented ERK-2 activation following
VLA-4 cross-linking in a dose-dependent manner, with
complete inhibition at 10 µM (Fig. 5,
A and B). Binding studies with the flow cytometer
and fluorescein isothiocyanate-labeled secondary Ab demonstrated that
PD98059 did not influence overall binding of antibody to integrin (data
not shown). Consistent with its known mechanism of action, PD98059 also
inhibited the increase in phospho-ERK which occurs in response to VLA-4
cross-linking (Fig. 5C). In addition, this agent prevented
the slight mobility shift observed with activation of ERK. The
inhibitory effect of PD98059 occurred without altering cell viability,
as assessed by propidium iodide uptake, and also had no effect on total
cellular levels of the predominant isoform ERK2 (data not shown). In a similar fashion, the tyrosine kinase inhibitor genistein (10 µg/ml) also decreased ERK2 activation (Fig. 5, A and B).
Fig. 5D demonstrates that genistein causes a global
reduction in the level of tyrosine phosphoproteins following
cross-linking of 1, while PD98059 has little effect on
the overall pattern of phosphorylation. Considered together, these
studies demonstrate that both tyrosine kinase inhibition as well as
inhibition of MEK-1 activation are able to prevent ERK activation in
response to VLA-4 cross-linking.
PD98059 and Genistein Inhibit VLA-4-stimulated Monocytic Tissue Factor Expression
Consistent with previous reports (20, 23),
VLA-4 engagement by antibody cross-linking or adhesion to fibronectin
caused marked increases in monocyte PCA (Fig. 6,
A and B, respectively). The ability to stimulate
PCA correlated with the increase in phosphotyrosine accumulation in
response to treatment. In Fig. 6A, PCA was increased following cross-linking with K20, HP2/1, and Lia1.2, but not 44H6 or
CD45 (compare with Fig. 1). Furthermore, the increase did not occur
following engagement with primary Ab alone or secondary antibody alone.
The induction of PCA was comparable to that observed following
stimulation with LPS (1 µg/ml). The effect of adhesion to fibronectin
was observed both in freshly plated human blood monocytes as well in
THP-1 cells (Fig. 6B). PCA was attributed to tissue factor
induction on the basis of inhibitory studies with anti-TF Ab. In a
typical experiment, control cells had clotting times of 67 ± 5 s (112 ± 58 PCA milliunits), while cells cross-linked with
mAb K20 exhibited induction of PCA (56 ± 1 s, 393 ± 50 milliunits). The ability of cross-linked cells to shorten clotting time
was completely eliminated by inhibitory anti-TF Ab (to 66 ± 1 s, 118 ± 1 milliunits), but not by mouse anti-human IgA Ab
(Jackson). Furthermore, this effect was not due to LPS contamination,
since heated mAb did not induce PCA and polymyxin B (50 µg/ml) did
not reverse the effect of cross-linking.
Fig. 7 shows the effect of PD98059 and genistein on
VLA-4-induced PCA. At concentrations of PD98059 that preclude ERK
activation in response to integrin cross-linking (10 µM
for HP 2/1 cross-linking and 1 µM for K20 cross-linking),
this inhibitor prevented induction of PCA (Fig. 7A).
Similarly, genistein prevented the rise in PCA following cross-linking
of VLA-4 (Fig. 7A), at a concentration that caused near
complete inhibition of MBP phosphorylation. Fig. 7B
demonstrates the ability of PD98059 to abrogate the rise in PCA that
occurs in response to attachment to a fibronectin substratum in both
THP-1 cells and human monocytes.
VLA-4-induced NF-
Previous studies have reported that VLA-4 engagement
dramatically increases NF-B nuclear translocation and specific
binding in THP-1 cells (20). Since NF-
B binding to the promoter of the tissue factor gene is required for induction of gene transcription, we examined whether PD98059 might exert its effect through inhibition of NF-
B translocation. Fig. 8 illustrates two
representative studies. Cross-linking VLA-4 induces a dramatic increase
in both HIV-ENH and TF-specific NF-
B translocation. Preincubation of the cells with 10 µM PD98059 largely abolished the
HIV-ENH shift, and consistently effected a partial inhibition of the TF
B shift.
Macrophage-mediated fibrin deposition via the surface expression of the procoagulant molecule tissue factor contributes significantly to the pathogenesis of both intravascular and extravascular inflammation. Induction of tissue factor occurs in response to a variety of proinflammatory stimuli including tumor necrosis factor, C3a, formyl peptides, as well as various bacterial species and their surface components (9, 11). Recent studies including those reported here demonstrate that engagement of monocyte surface VLA-4 by specific antibody is able to induce tissue factor expression (20). The present results clearly demonstrate that tyrosine phosphorylation and activation of ERK1/ERK2 MAP kinase are involved in integrin-induced signaling pathway leading to tissue factor expression. Several lines of evidence support this conclusion. First, integrin aggregation through cross-linking causes tyrosine phosphorylation of these proteins. This was definitively shown by immunoprecipitation studies, as well as by experiments using an antibody directed against the phosphorylated form of ERK. Furthermore, adhesion of both THP-1 and human monocytes to fibronectin caused phosphorylation of ERK. Second, VLA-4 cross-linking caused a time-dependent activation of ERK1/ERK2 MAP kinase as assessed by its ability to phosphorylate its substrate protein myelin basic protein. Finally, two strategies shown to prevent phosphorylation and activation of ERK precluded the induction of tissue factor, not only in response to cross-linking, but also following adhesion to fibronectin. These included the use of the tyrosine kinase inhibitor genistein as well as the novel specific inhibitor of the upstream activator of ERK, PD98059. Considered together, these findings invoke a role for the MEK-1/MAP kinase cascade in the integrin-induced activation of monocyte coagulation molecules.
The data presented here are consistent with those recently reported by
Lin and colleagues showing that adhesion of THP-1 monocytes to
fibronectin lead to the tyrosine phosphorylation of
pp125FAK, paxillin, and the nonreceptor tyrosine kinase Syk
(18, 39). Syk phosphorylation was also associated with its activation.
At least two pathways leading to the activation of the Ras signaling cascade may link these effects to the activation of MAP kinase demonstrated in this study. First, phosphorylation of FAK creates an
SH2-binding site for Grb2, resulting in localization of Grb2·SOS complexes and subsequent activation of Ras (40). In addition, in
cultured mast cells, activated Syk has been shown to cause tyrosine
phosphorylation of Shc in response to engagement of the Fc RI with
antigen (41). Consequent association with Grb2 may lead to Ras
activation. The precise signaling pathways leading to MAP kinase
activation following integrin engagement, however, require further
definition. While the activation of MEK-1/MAP kinase following integrin
engagement in monocytic cells suggests the involvement of the Ras as
well as the downstream kinase Raf in the signaling pathway, two lines
of evidence suggest that the pathway does not align precisely along the
lines suggested for growth factor-induced cell signaling. For example,
a recent study using NIH 3T3 fibroblasts demonstrated activation of
Raf-1, MEK-1, and MAP kinase following adhesion to fibronectin in a
manner that was independent of Ras (42). Second, the studies reported
by Lin and colleagues demonstrated that neither phosphorylation nor activation of Raf-1 occurred following
1 engagement in
monocytes (18). Recent studies have suggested the existence of
unidentified MEK activators that may not have been detected in these
studies (43). Further studies are required to evaluate the upstream activation pathway leading to MAP kinase activation following
1 integrin engagement in cells of monocytic lineage.
Although integrin ligation by antibody without aggregation induces
phosphorylation and activation of p42 ERK-2, cross-linking consistently
induces the most phosphorylation, ERK activation, and procoagulant
response (Figs. 1 and 3). In addition, clustering by either
adhesion-inhibiting antibodies as well as non-inhibitory antibodies
induced comparable effects. These findings are consistent with previous
reports demonstrating the need to aggregate integrin receptors in
monocytes as well as in other cell types to achieve maximal tyrosine
phosphorylation and gene expression (16, 44, 45). Considered together,
the data suggest that the clustering of integrins plays a central role
in initiating the signaling cascade leading to inflammatory gene
induction. However, while integrin aggregation is necessary, it does
not appear to be sufficient. The antibody 44H6 bound to THP-1 cells
with equivalent affinity, yet was unable to induce either tyrosine
phosphorylation or tissue factor expression when cross-linked.
Particular VLA-4 epitopes as defined by mAb clones have already been
suggested to play selective roles in adhesive functions (46). For
example, Sato et al. demonstrated that mAb 8F2 (directed
against the 4 VLA-4 subunit epitope "C") induced
little phosphotyrosine accumulation in Jurkat T-cells, while two other
"C"-specific clones did so strongly (24). From a physiological
standpoint, the process of integrin aggregation using mAb is a rather
artificial one. Monocyte adhesion to endothelial cells or extracellular
matrix proteins in vivo would presumably result in both
engagement of the integrin ligand as well as aggregation of integrin
receptors. In this regard, adhesion of monocytes to fibronectin (Fig.
6) as well as interaction with endothelial cells has been shown to
induce monocytic tissue factor expression (10, 47). It should be noted
that the increase in TF induced by THP-1 or human PBM adherence to
culture plates coated with 50 µg/ml fibronectin is only a moderate
stimulus for TF expression when compared with that obtained with 1 µg/ml LPS. However, the observed increase is consistent with the
ability of fibronectin to induce phosphotyrosine accumulation and
stimulation of immediate early genes, as reported by Lin et
al. (18).
The promoter region of the tissue factor gene has a cis-acting integrin
response element containing two AP-1 sites and a single B-like
sequence (20, 48, 49). Mutation of either site resulted in reduced
integrin responsiveness (20). In the present studies, induction of
tissue factor expression following integrin cross-linking and TF
B
nuclear translocation were fully or partially precluded, respectively,
by treatment with the MEK-1 inhibitor PD98059. HIV-ENH binding was
abrogated by PD98059. Taken together, these data suggest an association
between MAP kinase activation and NF-
B translocation leading to gene
activation. Interestingly, in response to LPS, macrophages similarly
exhibit both ERK activation and NF-
B translocation to the nucleus.
However, these two events do not appear to be causally related,
although both contribute to gene activation (50, 51). Similar
dissociation has been demonstrated in response to cellular hypoxia
(52). By contrast, overexpression of ERK-1 in Jurkat cells caused a
marked increase in the DNA binding activity of NF-
B (53). Together,
these data suggest possible interaction between these two events, which
may be cell- and stimulus-specific.
The present study is the first to describe a contributory role for the ERK pathway in the induction of adhesion-dependent inflammatory response in cells of monocyte/macrophage lineage. Since endothelial cell adhesion of monocytes via engagement of surface integrins is an early event in the mobilization of cells to sites of inflammation, it will be of interest to discern how inflammatory and antiinflammatory mediator molecules acting via this or other signaling cascades might interact with this activated pathway to modulate the inflammatory response.