From the
Activation of cytoplasmic tyrosine kinases is an important
aspect of signal transduction mediated by integrins. In the human
monocytic cell line THP-1, either integrin-dependent cell adhesion to
fibronectin or ligation of
Members of the integrin family of cell surface receptors are
involved in many key biological processes, including cell to cell and
cell to extracellular matrix adhesion, cell motility, hemostasis,
lymphocyte trafficking, and inflammatory
phenomena(1, 2, 3) . Integrins are comprised of
noncovalently linked
There have been observations in a variety of cell types showing that
interactions with the extracellular matrix (ECM) can modulate gene
expression(14, 15, 16, 17) . During
inflammation, blood monocytes respond to chemotactic factors and
subsequently extravasate into inflamed tissues. Monocytes migrate
through the subendothelial basement membrane and underlying
interstitial structures rich in extracellular matrix proteins and also
interact with vascular endothelial cells and connective tissue cells.
Partly as a consequence of cell-cell and cell-ECM interactions,
monocytes are induced to secrete cytokines and to undergo maturation to
macrophages. Integrins are prominent among the cell surface receptors
that mediate many of the adhesive functions of
monocytes(1, 2) . In human peripheral blood monocytes,
cell adherence to ECM components or ligation of
In this report, we describe a cell culture model using the
human monocytic leukemia cell line, THP-1, which mimics important
aspects of monocyte responses to ECM proteins. THP-1 cell adhesion to
ECM proteins or ligation of
EMSAs were performed by a slight modification
of the method we previously reported(19) . Briefly, the
DNA-protein binding reactions were performed in 10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 0.5 mM EDTA, 1
mM dithiothreitol, 5 mg of BSA, 0.3 µg of poly(dI-dC), and
4% Ficoll in a final volume of 20 µl. Each reaction contained 1
µg of THP-1 nuclear extract and 10-20,000 cpm of 3
Ligation of
The patterns of tyrosine
phosphorylation induced in THP-1 cells by integrin-mediated cell
adhesion or by antibody ligation of
Despite
differences in the overall patterns of tyrosine phosphorylation induced
by cell adhesion or by antibody ligation of integrins, it is clear that
both of these stimuli can activate NF-
We have examined THP-1 lysates for
evidence of integrin-mediated tyrosine phosphorylation of several
proteins previously identified as being involved in signal transduction
cascades or linked to integrin-dependent cytoskeletal reorganization.
In THP-1 cells, the focal contact protein paxillin and the cytoplasmic
tyrosine kinases FAK and Syk became tyrosine phosphorylated in response
to integrin-mediated cell adhesion. The activation of FAK by integrin
ligation, as well as by other stimuli, has been observed in a number of
cell types(35, 39) ; paxillin is a substrate for FAK,
and its tyrosine phosphorylation seems to parallel the activation of
the FAK kinase(23, 41) . That FAK and paxillin are
tyrosine phosphorylated during THP-1 cell adhesion suggests that these
molecules may be involved in cytoskeletal reorganization and formation
of focal adhesive sites in these cells; a similar role for these
proteins has been postulated in fibroblasts(23, 42) .
Since cytochalasin D effectively blocks the tyrosine phosphorylation of
FAK and paxillin, some degree of cytoskeletal organization and cell
spreading seem to be required for these events.
Ligation of
Either integrin-mediated cell adhesion or antibody
ligation of integrins can effectively increase IL-1
Current evidence indicates that
Syk is an integrin-responsive tyrosine kinase. It also suggests the
existence of a signal transduction pathway in monocytic cells that
involves the ligation of integrins, activation of the Syk tyrosine
kinase, the triggering of downstream events, activation of the
NF-
At this point, a number of
issues require further study. It is not certain that activation of the
Syk tyrosine kinase is essential for integrin-mediated gene induction,
as opposed to simply accompanying the induction process, nor can the
participation of additional tyrosine kinases be ruled out. Further
experiments will be required to establish a causal role for Syk in
integrin signaling. In addition, the downstream events linking changes
in tyrosine phosphorylation to alterations of inflammatory gene message
levels remain to be defined. In monocytes, it is clear that integrin
ligation induces transcriptional activation of the IL-1
We thank Drs. A. Baldwin and D. Brenner of University
of North Carolina, Chapel Hill, for providing reporter gene constructs
and advice and Dr. M. Hemler of Harvard for providing the TS2/16
hybridoma. We also thank Dr. J. A. Hughes for help with flow cytometry
analysis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1 integrins with antibodies causes a
rapid and intense tyrosine phosphorylation of two sets of proteins of
about 65-75 and 120-125 kDa. In addition, integrin ligation
leads to nuclear translocation of the p50 and p65 subunits of the
NF-
B transcription factor, to activation of a reporter gene driven
by a promoter containing NF-
B sites, and to increased levels of
mRNAs for immediate-early genes, including the cytokine interleukin
(IL)-1
. The tyrosine kinase inhibitors genistein and herbimycin A
block both integrin-mediated tyrosine phosphorylation and increases in
IL-1
message levels, indicating a causal relationship between the
two events. The components tyrosine phosphorylated subsequent to cell
adhesion include paxillin, pp125
, and the SH2 domain
containing tyrosine kinase Syk. In contrast, integrin ligation with
antibodies induces tyrosine phosphorylation of Syk but not of FAK or
paxillin. In adhering cells, pre-treatment with cytochalasin D
suppresses tyrosine phosphorylation of FAK and paxillin but not of Syk,
while IL-1
message induction is unaffected. These observations
indicate that the Syk tyrosine kinase may be an important component of
an integrin signaling pathway in monocytic cells, leading to activation
of NF-
B and to increased levels of cytokine messages.
/
heterodimers(4) . Different
and
subunits can associate in various combinations, which
then determine the ligand-binding specificities of the intact integrin
heterodimer complexes(2, 4) . Recently, it has become
apparent that integrins function not only as adhesive proteins but can
also transduce biochemical signals into the interior of the
cell(5) . One mode of integrin signal transduction involves the
activation of cytoplasmic tyrosine kinases. In fibroblasts, platelets,
endothelial cells, and cultured tumor cells, integrin-induced tyrosine
phosphorylation involves a novel cytoplasmic tyrosine kinase termed
pp125 focal adhesion kinase
(pp125
)
(
)(6, 7, 8) .
However, other aspects of integrin signaling have been observed,
including calcium transients(9) , changes in cytoplasmic
pH(10) , modulation of ion channels(11) , activation of
protein kinase C(12) , as well as numerous other
effects(13) . The relationship between these later events and
integrin-mediated tyrosine phosphorylation is currently unclear.
1 integrins with
antibodies results in the rapid induction of multiple inflammatory
mediator genes including several
cytokines(18, 19, 20) . The 5`-regulatory
regions of many of the genes induced by integrin ligation contain
binding motifs for the NF-
B transcription factor, suggesting a
role for this factor in the gene induction process(5) . In
parallel to gene induction, a rapid and profound increase in protein
tyrosine phosphorylation is observed, with the predominant
phosphorylated component(s) having a molecular mass of about 76
kDa(21) . Both of these responses are blocked by tyrosine kinase
inhibitors, suggesting an important role for protein tyrosine
phosphorylation in integrin signaling pathways, leading to inflammatory
mediator gene induction. Aside from enhanced tyrosine phosphorylation,
little is known of integrin-mediated signal transduction in monocytic
cells. A significant reason for this is the difficulty involved in
performing biochemical or molecular studies on peripheral blood
monocytes as well as the donor to donor variability observed with these
cells.
1 integrins with antibody induces
protein tyrosine phosphorylation, increases inflammatory mediator gene
message levels, and activates the NF-
B transcription factor. These
responses are blocked by tyrosine kinase inhibitors such as herbimycin
A and genistein. Among the proteins tyrosine phosphorylated in THP-1
cells in response to cell adhesion to ECM components are the
pp125
(6, 22) , the focal contact protein
paxillin(23) , and the nonreceptor tyrosine kinase
Syk(24, 25) . Ligation of
1 integrins with intact
antibodies or with F(ab`)
fragments results in the tyrosine
phosphorylation of Syk but not of FAK or paxillin. The tyrosine
phosphorylation of Syk is accompanied by an increase in its kinase
activity. These results indicate that, like FAK, the Syk kinase is an
integrin-responsive non-receptor tyrosine kinase. They also suggest
that activation of Syk is closely correlated with the induction of
inflammatory mediator gene messages, while there is no such correlation
for FAK. Thus, Syk may be a vital part of an integrin signaling pathway
in monocytic cells, leading to transcription factor activation, and to
increased levels of cytokine messages.
Materials
Monoclonal antibodies
reactive with FAK, Raf-1, PTP1D (Syp), and paxillin, as well as goat
anti-mouse IgG-peroxidase and goat anti-rabbit IgG-peroxidase
conjugates, were purchased from Transduction Laboratories (Lexington,
KY). The anti-1 integrin antibody P4C10 was obtained from Life
Technologies, Inc. Anti-Syk kinase polyclonal antibodies were raised in
rabbits using fusion proteins as previously described(26) . The
mouse hybridoma TS2/16 (anti-
1 integrin subunit) was a generous
gift of Dr. M. Hemler (Dana Farber Cancer Research Institute, Boston,
MA). TS2/16 F(ab`)
fragment was prepared by proteolytic
digestion using a kit from Pierce following the manufacturer's
directions. Removal of intact antibody or Fc fragments was accomplished
using a protein G affinity column. The purity of the F(ab`)
was evaluated by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) prior to use in experiments. Herbimycin A, genistein, and
calyculin A were purchased from Calbiochem. Protein G-Sepharose was
from Pharmacia Biotech Inc. Human fibronectin, collagen type I,
collagen type IV, and laminin, and the tissue culture reagents were
from Life Technologies, Inc. Other reagents and chemicals were from
Sigma.
Cell Culture
THP-1 cells were maintained
in RPMI 1640 medium containing 10% fetal calf serum (heat inactivated),
50 µM 2-mercaptoethanol, 50 µg/ml streptomycin, and 50
units/ml penicillin. Substratum-coated dishes were prepared by
incubating 10 µg/ml fibronectin, collagen, or laminin in
phosphate-buffered saline (PBS) in tissue culture dishes at 4 °C
overnight. The dishes were blocked with 0.1% bovine serum albumin (BSA)
and washed with PBS prior to use.
Adhesion Assay
Adhesion studies were
performed using a modification of a previously described
assay(27) . Briefly, 48-well tissue culture plates were coated
overnight at 4 °C with 0.2 ml of PBS containing 10 µg/ml ECM
proteins. The wells were blocked with 0.1% BSA in PBS for 2 h at 4
°C and washed with PBS prior to use. THP-1 cells were washed,
resuspended in RPMI 1640 medium, and added to substratum-coated wells
(1 10
cells/well) for 30 min at 37 °C. The
wells were washed two times with warm PBS and stained with a solution
containing 0.1% crystal violet and 10% methanol in PBS for 15 min.
Following three washes with PBS, the crystal violet stain was
solubilized with 1% SDS and quantitated by measuring the absorbance at
540 nm. The absorbance from 1
10
stained cells was
used to calculated the number of adhesive cells per well. Each assay
was performed in triplicate.
Cell Adherence and Integrin
Ligation
THP-1 cells were harvested from suspension
culture, washed extensively with cold RPMI 1640 medium, and suspended
in medium with 0.1% BSA. Cell adherence was initiated by adding the
cells to 100-mm tissue culture dishes coated with ECM proteins. Cells
were incubated at 37 °C for the times indicated in the figure
legends. For integrin ligation, THP-1 cells were incubated for 45 min
at 4 °C in RPMI 1640 medium or medium containing intact anti-1
IgG, or F(ab`)
fragment, washed twice with cold medium, and
then incubated at 37 °C in RPMI 1640 medium.
Preparation of Cell Lysates
Cells were
lysed in a buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 1 mM sodium vanadate, 0.2 µM calyculin A, 5 mM NaF, 5 mM sodium pyrophosphate, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 0.5% Triton
X-100, and 0.1% sodium deoxycholate, and the lysates were cleared by
centrifugation at 30,000 g for 30 min at 4 °C.
Protein concentration in the lysates was determined using the
bicinchonic acid assay (Pierce).
Immunoblotting
Total cell lysates from
equivalent cell numbers or immunoprecipitated proteins were separated
by SDS-PAGE (8%) under reducing conditions. The proteins were
transferred eletrophoretically onto polyvinylidene fluoride membranes
(Immobilon P, Millipore Corp.). The membranes were blocked with 1% BSA
and 0.1% Tween 20 in PBS. The membranes were subsequently probed with
primary antibody (1 µg/ml) in PBS containing 1% BSA and 0.1% Tween
20. The antibody-antigen complexes were detected by using goat
anti-mouse IgG or goat anti-rabbit IgG peroxidase conjugates, followed
by use of an enhanced chemiluminescence kit (Amersham Corp.) according
to the manufacturer's instruction. In some cases, the blots were
stripped of bound antibodies by incubating the membranes with stripping
buffer containing 62.5 mM Tris, pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS for 30 min at 50 °C. The
stripped blots were reprobed with other antibodies.
Immunoprecipitation and Syk Kinase
Autophosphorylation Assay
Cell lysates were precleared by
incubation with protein G-Sepharose. The cleared lysates were first
incubated with anti-FAK, anti-Raf-1, anti-paxillin, anti-PTP1D, or
anti-Syk kinase antibody for 3 h at 4 °C, followed by the addition
of protein G-Sepharose, and then incubated for additional 3 h at 4
°C. The precipitates were washed extensively with lysis buffer. For
Western analysis, the precipitates were boiled with SDS-PAGE sample
buffer to dissociate the proteins. For Syk kinase autophosphorylation
assay, the anti-Syk immunocomplexes were further washed twice with
kinase assay buffer (50 mM HEPES, pH 7.6, 10 mM MnCl, 2 mM MgCl
, and 1
mMp-nitrophenyl phosphate) and resuspended in 50
µl of the same buffer containing 10 µCi of
[
-
P]ATP (3000 Ci/mmol) and 2 µM ATP. After 10 min at 30 °C, the reactions were stopped by
addition of 20 µl SDS-PAGE sample buffer (4
) and boiling for
3 min.
Reporter Gene Assays
3XMHCwt56CAT, a
construct containing NF-
B-responsive elements upstream of the
chloramphenicol acetyltransferase (CAT) reporter gene, has been
previously described(28) . h-fos-luc, a construct containing the
5`-regulatory region of the c-fos gene upstream of the
luciferase reporter gene has also been
described(29, 30) . These constructs were transfected
into THP-1 cells using the DEAE-dextran method(50) . Transfected
cells were manipulated in various ways, as indicated in the legends,
and CAT or luciferase enzyme activities measured using established
protocols(51) .
Electrophoretic Mobility Shift Assay
(EMSA)
For EMSAs, THP-1 nuclear and cytoplasmic extracts
were made using a modification of the procedure described by Cordle and
co-workers(31) . Each treatment group utilized 5-10
10
cells. Following incubation, THP-1 cells were
collected by centrifugation, washed with cold PBS, and suspended in
ice-cold cytoplasmic extraction buffer (CEB) (10 mM Tris-HCl,
pH 7.9, 60 mM KCl, 1 mM EDTA, and 1 mM dithiothreitol). After equilibration in CEB for 5 min, the cells
were collected by centrifugation and lysed on ice in 50
the
packed cell volume of Nonidet P-40/CEB/PI (CEB containing 0.1% Nonidet
P-40, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml
antipain, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 40 µg/ml
bestatin, 3 µg/ml E64, 1 mM 1,10-phenanthroline, and 100
µg/ml chymostatin). Adherent cells were equilibrated with 2 ml of
ice-cold CEB buffer and subsequently recovered from the tissue culture
dish by gently scraping the cells into 500 µl of Nonidet
P-40/CEB/PI. Nuclei were pelleted and washed in cold CEB containing
protease inhibitors but no detergent and then suspended and mixed in 25
µl of nuclear extraction buffer (NEB) (20 mM Tris-HCl, pH
8.0, 0.4 M NaCl, 1.5 mM MgCl
, 1.5 mM EDTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 50 µg/ml antipain, 1 µg/ml
leupeptin, 1 µg/ml pepstatin, 40 µg/ml bestatin, 3 µg/ml
E64, 1 mM 1,10-phenanthroline, 100 µg/ml chymostatin, and
25% glycerol). After 10 min of incubation on ice, the samples were
clarified by centrifugation, and the supernatants (nuclear extracts)
were collected and snap-frozen on dry ice before storing at -70
°C. Protein concentrations were determined using the bicinchoninic
acid method (Pierce).
MHC enhancer probe(28) . In parallel EMSAs, specific NF-
B
subunits were identified in shifted complexes using supershifting or
blocking antibodies against the p50, p65, and Rel components of
NF-
B(32, 33) .
RNA Isolation and Northern Analysis
Total
cellular RNA was isolated by the guanidium
isothiocyanate-phenol-chloroform extraction method(34) .
Northern analysis was performed according to a published
procedure(21) . Briefly, total RNA was electrophoresed on 1.2%
agarose-formaldehyde gels, transferred onto polyvinylidene fluoride
membranes (Immobilon N, Millipore), and fixed to the membrane by UV
irradiation. Human cDNA probes for IL-1 (20) and
-actin (Clontech) were labeled using a random primer kit (U.S.
Biochemical Corp.) with [
-
P]dCTP (Amersham
Corp.). The membranes were hybridized with the denatured probe at 60
°C overnight in hybridization buffer (0.5 M sodium
phosphate, pH 7.0, 1% BSA, 1 mM EDTA, and 7% SDS) and were
washed at 60 °C three times with washing buffer (40 mM sodium phosphate, pH 6.8, 1 mM EDTA, and 1% SDS). The
blots were visualized by exposing the membranes to films at -80
°C.
THP-1 Cells Adhere to ECM Components via
Integrins
We surveyed the expression of integrins on the
THP-1 cell surface by flow cytometry. THP-1 cells maintained in
suspension culture expressed several integrin subunits including
1,
2,
2,
3,
4, and
5 (data not shown). We
also examined whether these cells could adhere to ECM component-coated
substrata. As shown in Fig. 1A, THP-1 cells adhered to
tissue culture plates coated with fibronectin, laminin, collagen type
I, or collagen type IV. THP-1 cells exhibited the highest level of cell
adhesion to the fibronectin substratum, a moderate level of adherence
to laminin, and lower levels of cell adhesion to collagen type I or
collagen type IV, while only a few cells adhered to albumin-coated
control wells. To test whether cell adhesion to fibronectin was
specifically mediated by integrins, THP-1 cells were treated with RGDS
peptide or with a mouse antibody recognizing the
1 integrin
subunit. Fig. 1B shows that both RGDS peptide and
anti-
1 integrin antibody inhibited cell adhesion to fibronectin,
whereas treatment with RGES peptide or normal mouse IgG had no effect.
These results indicate that THP-1 cells use
1 integrins to
interact with and adhere to ECM components such as fibronectin.
Figure 1:
THP-1 cells
adhere to ECM components via integrins. A, THP-1 cells
suspended in RPMI 1640 medium were plated on to tissue culture plates
(48-well, 1 10
cells/well) coated with fibronectin (Fn), laminin (Ln), collagen type I (CnI), or collagen type IV (CnIV). Cells
were incubated for 30 min at 37 °C for cell adhesion assay as
described under ``Experimental Procedures.'' Greater than 95%
of cells adhered to fibronectin-coated wells, while less than 3% of
cells adhered to control wells (BSA blocked). The values shown are the
mean ± S.E. of three independent determinations. B,
THP-1 cells were incubated with RPMI 1640 medium or medium containing
RGDS peptide (10 mM), RGES peptide (10 mM),
anti-
1 integrin antibody (ascites of clone P4C10, 1:10 dilution),
or normal mouse IgG (0.5 mg/ml) for 15 min on ice. Cells were then
plated on to fibronectin-coated 48-well plates for 30 min at 37 °C
for the cell adhesion assay. The values represent the mean ±
S.E. of three assays.
Engagement of THP-1 Cell Integrins Increases Protein
Tyrosine Phosphorylation and IL-1
THP-1
cells were plated on fibronectin-coated dishes for different periods of
time (7.5-60 min). Lysates from adherent cells were examined for
protein phosphorylation on tyrosine residues by using
anti-phosphotyrosine immunoblotting. As shown in Fig. 2A (toppanel), cell adhesion to fibronectin gave
rise to a marked increase in tyrosine phosphorylation of several
proteins, including three broad bands centered around 67, 71, and 120
kDa. The increase in tyrosine phosphorylation was readily detected 7.5
min after cells were plated on to the fibronectin substratum, reached a
maximal level after 15 min, and then declined, but was still
significantly elevated after 1 h. Cell adhesion to fibronectin also
induced increased message levels of the inflammatory mediator gene
IL-1 Message Levels
. Fig. 2A (middlepanel)
shows that maximal induction of IL-1
message was reached 1 h after
cell adhesion to fibronectin began. By comparing the time courses, it
can be seen that the induction of tyrosine phosphorylation occurred
prior to IL-1
message expression. The adhesion-induced increase in
IL-1
message over basal levels was approximately 4-5-fold.
We have observed that certain other cytokine messages (e.g. IL-8) are also increased upon THP-1 adhesion to fibronectin (data
not shown).
Figure 2:
Induction of tyrosine phosphorylation and
IL-1 message expression by cell adhesion to fibronectin-coated
dishes or
1 integrin ligation. A, THP-1 cells were plated
on fibronectin-coated dishes and incubated at 37 °C for the times
indicated in the figure. B, THP-1 cells were incubated
nonadherently (NAD), plated on fibronectin-coated dishes (Fn), or treated with the anti-
1 integrin antibody TS2/16
at 4 °C and then incubated nonadherently at 37 °C (
1). C, THP-1 cells were incubated in RPMI 1640 medium or medium
containing anti-
1 antibody TS2/16 or its F(ab`)
fragment (both 2 µg/ml) for 45 min at 4 °C. The cells
were washed and incubated with RPMI 1640 medium at 37 °C. For
Western blotting (toppanels), cell lysates harvested
after 30 min of incubation or at the times as indicated in A were probed with anti-phosphotyrosine antibody (100 µg of
protein/lane). For Northern blotting (middle and bottompanels), total cellular RNA isolated after 1 h of
incubation or at the times as indicated in A was probed with
IL-1
or
-actin cDNA probes (6 µg total
RNA/lane).
Antibody-mediated ligation of integrins has been
previously used to mimic the integrin clustering process that occurs
during formation of adhesive contacts(35) . Nonadherent THP-1
cells were treated with the anti-1 integrin antibody TS2/16 and
then analyzed for tyrosine phosphorylation and message levels. As shown
in Fig. 2B (toppanel), ligation of
1 integrins resulted in increased tyrosine phosphorylation (lane3), although the pattern was somewhat different
from that induced by cell adhesion to fibronectin (lane2). With antibody ligation, bands at about 115 and 67 kDa
were most prominent. Ligation of
1 integrins with antibody could
also increase IL-1
message levels (Fig. 2B, middlepanel, lane3). The observed
induction of tyrosine phosphorylation and message expression by
anti-
1 antibody was not due to engagement of Fc receptors, since
TS2/16 F(ab`)
induced tyrosine phosphorylation and
IL-1
message as effectively as intact antibody (Fig. 2C).
Integrin Ligation Leads to Activation of
NF-
To determine whether integrin ligation might lead
to activation of transcription factors and the initiation of
transcription, we employed gel mobility shift assays, as well as
reporter gene assays. Since the IL-1B
promoter, as well as the
promoters for several other monocyte immediate-early genes, contains
NF-
B motifs(5) , we decided to focus on activation of the
NF-
B transcription complex. THP-1 cells were transfected with a
plasmid containing several copies of the NF-
B motif driving a CAT
reporter gene, by a similar plasmid with mutated NF-
B sites, or by
a luciferase reporter construct driven by the c-fos promoter.
As seen in Fig. 3A, THP-1 cell adhesion to fibronectin
resulted in a substantial induction of CAT activity in cells
transfected with the construct containing NF-
B motifs but not in
cells transfected with the mutated NF-
B construct. Expression of
luciferase driven by the c-fos promoter was not affected by
cell adhesion, indicating that the results observed with the
NF-
B-driven vector were not due to generalized increases in
transcriptional activity. As seen in Fig. 3B, activation
of the NF-
B-driven reporter could be triggered by integrin
ligation with intact anti-
1 antibody or with F(ab`)
fragments of the antibody, suggesting that integrin ligation,
rather than cell adhesion, was sufficient for reporter activation.
Figure 3:
Activation of NF-B by integrin
ligation. THP-1 cells were transfected with an NF-
B-driven CAT
reporter plasmid (panelsA and B) or a
c-fos-driven luciferase reporter plasmid (panelA). 24 h after transfection, cells (3
10
) were resuspended in 1% BSA in RPMI medium and left in
suspension or plated on fibronectin-coated 60-mm dishes.
NF-
B-transfected cells were also stimulated with 5 µg/ml
anti-
1 integrin TS2/16 mAb IgG or TS2/16 F(ab`)
fragments followed by F(ab`)
goat anti-mouse Ab for
60 min at 4 °C and then incubated at 37 °C (panelB). Cells were collected 24 h later and lysed to measure
CAT activity or 6 h later for luciferase activity.
c-fos-transfected cells were also stimulated with 20 ng/ml
phorbol 12-myristate 13-acetate, and luciferase activity was measured 6
h later. In A, solid bars represent non-adherent
cells, hatched bars represent cells adhered to Fn, stippled bars represent cells treated with phorbol ester. Data
are ±S.E. PanelC depicts the results of gel
shift assays using a probe containing NF-
B motifs from the class I
MHC enhancer; the experimental conditions are the same as PanelsA and B. The identification of the NF-
B
p50/p65 heterodimer was confirmed by supershifting the p65 band and
blocking the p50 band with specific antibodies (32, 33). The faster
moving band was similarly identified as p50/p50 homodimer. Cells were
analyzed after 1 h (lanes1-5) or after
overnight stimulation (lanes6-10) under the
following conditions. Lanes1 and 6,
adherence to fibronectin; lanes 2 and 7, no
stimulation (controls); lanes 3 and 8, TS2/16
antibody; lanes 4 and 9, TS2/16 F(ab`)
fragments; lanes 5 and 10, TS2/16 F(ab`)
followed by goat anti-mouse second antibody. Individual
components were identified by interference with the mobility of p50 and
p65; lane11 is the same as lane10 but is pretreated with anti-p65; lane12,
pretreated with anti-p50; lane13, pretreated with
anti-c-rel.
Integrin ligation by antibodies or integrin-mediated adhesion to
fibronectin also caused activation and nuclear translocation of the p50
and p65 subunits of the NF-B complex. As seen in Fig. 3C, using a probe containing the MHC class I
enhancer NF-
B site, within 1 h there was a strong increase in
bands representing p50 and p65 complexes; an even more robust effect
was observed after overnight stimulation. Use of a probe containing the
IL-1
NF-
B motif gave similar results (data not shown). These
observations, along with the reporter gene assays, suggest that
integrin ligation can activate NF-
B, allowing this factor to
contribute to the stimulation of transcription of certain genes.
Herbimycin A and Genistein Inhibit Tyrosine
Phosphorylation and IL-1
To evaluate
whether tyrosine phosphorylation plays a critical role in the signaling
pathways leading to increased message levels, THP-1 cells were treated
with protein-tyrosine kinase inhibitors and examined for effects on
tyrosine phosphorylation and IL-1 Message Expression Induced by Cell
Adhesion or by Ligation of
1 Integrins
message induction. Herbimycin A
and genistein are selective inhibitors of tyrosine kinases with
distinct mechanisms of action; herbimycin A blocks tyrosine kinases by
attacking critical sulfhydryl groups(36) , while genistein
inhibits these enzymes by binding to the ATP binding sites(37) . Fig. 4A (toppanel) demonstrates that
treatment of THP-1 cells with herbimycin A resulted in an inhibition of
the tyrosine phosphorylation induced by cell adhesion to fibronectin.
The inhibition was dose dependent; 10 µM herbimycin A
strongly suppressed the induction of tyrosine phosphorylation, while 2
µM was less effective. Expression of IL-1
mRNA was
inhibited by herbimycin A in a manner similar to the effect on tyrosine
phosphorylation (Fig. 4A, middlepanel). Another tyrosine kinase inhibitor, genistein,
also exhibited similar inhibitory effects on tyrosine phosphorylation
and IL-1
expression (Fig. 4B); the inhibitory
effects of genistein were noticeable at 20 µM and
virtually complete at 100 µM. As in the case of
adherence-induced events, the responses induced by anti-
1 antibody
could be blocked with herbimycin A or genistein. As shown in Fig. 4C, TS2/16-induced tyrosine phosphorylation and
IL-1
expression were also inhibited by herbimycin A or genistein
in a dose-dependent manner. Thus, the responses induced by integrin
ligation or by adhesion could be inhibited by the tyrosine kinase
inhibitors herbimycin A or genistein, suggesting that integrin-mediated
increases in IL-1
message levels require protein-tyrosine kinase
activity.
Figure 4:
Inhibition of adhesion- and integrin
ligation-induced tyrosine phosphorylation and IL-1 mRNA expression
by herbimycin A or genistein. THP-1 cells were pretreated at 37 °C
with the concentrations (indicated in the figure) of herbimycin A for 4
h or of genistein for 1 h. The cells were then incubated nonadherently (NAD) or plated on fibronectin-coated dishes (ADH) (panelsA and B), or they were ligated with
anti-
1 integrin antibody at 4 °C (
1), washed, and
incubated nonadherently at 37 °C (panelC). For
Western blotting (toppanels), cell lysates harvested
after 30 min of incubation were probed with anti-phosphotyrosine
antibody (100 µg of protein/lane). For Northern blotting (middle and bottompanels), total cellular
RNA isolated after 1 h of incubation was probed with IL-1
or
-actin probe (6 µg total RNA/lane).
Identification of Proteins Tyrosine Phosphorylated in
Response to Cell Adhesion to a Fibronectin Substratum or to Ligation of
We sought to identify
proteins that were tyrosine phosphorylated in THP-1 cells subsequent to
engagement of integrins, which might be important in integrin-mediated
message induction. We obtained antibodies to several proteins known to
be involved in signal transduction cascades or in cytoskeletal
organization and examined their tyrosine phosphorylation status. We
examined FAK (125 kDa) as a possible component of the 120-kDa complex
and Raf-1 (74 kDa), PTP1D (72 kDa), paxillin (68 kDa), and Syk kinase
(72 kDa) as possible components of the 65 kDa-75-kDa complexes.
Specific antibodies were used to immunoprecipitate these potential
substrates, followed by immunoblotting with anti-phosphotyrosine
antibody. As shown in Fig. 5A, it is clear that FAK,
paxillin, and Syk kinase were tyrosine phosphorylated upon cell
adhesion to fibronectin; there was no evidence for tyrosine
phosphorylation of PTP1D or Raf. Only Syk kinase, but not FAK or
paxillin, was found to be tyrosine phosphorylated when cells were
stimulated by treatment with anti-1 Integrins with Antibodies
1 integrin antibody. The
induction tyrosine phosphorylation of Syk kinase was not due to
engagement of Fc receptors, since the F(ab`)
fragment of
TS2/16 was also effective (Fig. 5B). Thus, increased
tyrosine phosphorylation of Syk occurs during integrin-mediated cell
adhesion or subsequent to integrin ligation by antibodies.
Immunodepletion experiments revealed that Syk, paxillin, and FAK
contribute to the overall patterns of tyrosine phosphorylation observed
in response to adhesion or integrin ligation with antibody, but they do
not fully account for the patterns (data not shown); thus, additional
unidentified proteins are also tyrosine phosphorylated in response to
integrin ligation.
Figure 5:
Tyrosine phosphorylation of FAK,
paxillin, and/or Syk kinase by cell adhesion to fibronectin or by
1 integrin ligation. THP-1 cells were plated on fibronectin-coated
dishes for 30 min at 37 °C or ligated at 4 °C with the
anti-
1 integrin antibody TS2/16 or its F(ab`)
fragment, washed, and then incubated in suspension for 30 min at
37 °C. Following the incubations, cell lysates were
immunoprecipitated with antibodies to PTP1D, Raf-1, FAK, Syk, or
paxillin as indicated in the figure. The precipitated immunocomplexes
were analyzed for phosphotyrosyl-containing proteins by
anti-phosphotyrosine immunoblotting. The blots were stripped and
reprobed with the respective antibody used for immunoprecipitation. NAD, nonadherent; Fn, adherent to fibronectin;
1, ligated with TS2/16; Ig, ligated with intact
TS2/16; F(ab`), ligated with this fragment of
TS2/16.
Activation of Syk Kinase by Engagement of
Integrins
Following stimulation of the cells through
integrins, there were significant increases in the activity of the Syk
kinase, as indicated by immunocomplex kinase assays (Fig. 6). In
nonadherent THP-1 cells, there was detectable kinase activity, but the
activity increased 4-, 3.5-, and 3-fold, respectively, upon cell
adhesion to a fibronectin substratum, ligation of 1 integrins with
TS2/16, or with TS2/16 F(ab`)
; the radioactivity in each
band was quantitated using a PhosphorImager. As shown in Fig. 7,
herbimycin A and genistein suppressed the kinase activity of Syk. The
concentrations of inhibitors required to block Syk activation were
similar to those required to inhibit the overall pattern of tyrosine
phosphorylation and the induction of IL-1
message. Thus, in
monocytic cells, Syk is an integrin-responsive tyrosine kinase whose
activation parallels, and may impinge on, the induction of cytokine
messages.
Figure 6:
Induction of Syk kinase activity by cell
adhesion to fibronectin or by 1 integrin ligation. Cell lysates
obtained as described in Fig. 5 were immunoprecipitated with anti-Syk
antibody. The precipitated Syk kinase was analyzed for its
autophosphorylation activity as described under ``Experimental
Procedures'' (toppanel). In parallel with the
kinase assay, the amount of Syk kinase in the precipitated
immunocomplexes was examined by Western blot (bottompanel). NAD, nonadherent; Fn, adherent
to fibronectin; Ig, ligated with intact TS2/16; F(ab`), ligated with this fragment of
TS2/16.
Figure 7:
Inhibition of adhesion- and integrin
ligation-induced Syk kinase activity by herbimycin A or genistein.
THP-1 cells were treated with herbimycin A or genistein as described in
Fig. 4. Syk kinase was immunoprecipitated from cell lysates and
analyzed for its enzyme activity as described under ``Experimental
Procedures'' (toppanel). In parallel with the
kinase assay, the amount of Syk kinase in the precipitated
immunocomplexes was examined by Western blot (bottompanel). Her., 10 µM herbimycin A; Gen., 100 µM genistein.
Role of the Cytoskeleton
We have explored
the role of the cytoskeleton in the induction of tyrosine
phosphorylation and IL-1 message expression by use of the drug
cytochalasin D, an inhibitor of actin filament assembly(38) . As
seen in Fig. 8A, treatment with cytochalasin D inhibits
spreading of THP-1 cells on fibronectin but does not affect adhesion.
The tyrosine phosphorylation of FAK, and particularly of paxillin, that
is induced in THP-1 cells by interaction with a fibronectin substratum
is strongly inhibited by cytochalasin D (Fig. 8B). This
suggests that some degree of microfilament assembly is required for
these phosphorylation events. By contrast, adhesion-induced tyrosine
phosphorylation of Syk is not blocked by cytochalasin D treatment;
likewise, IL-1
message induction is not affected. These
observations clearly distinguish between presumptive integrin signaling
events that require actin filament assembly and events such as Syk
activation that do not have this requirement. They also indicate that
extensive cytoskeletal reorganization is not required for
integrin-mediated increases in IL-1
message levels.
Figure 8:
Effect of cytochalasin D on tyrosine
phosphorylation and IL-1 induction. A, THP-1 cells were
allowed to adhere and spread on fibronectin-coated dishes in the
presence (+) or absence (-) of cytochalasin D (0.5
µM) at 37 °C for 30 min before being photographed at
300
magnification. B, cells were incubated
nonadherently or plated on to fibronectin-coated dishes in the presence
(+) or absence (-) of 0.5 µM cytochalasin D for
30 min at 37 °C. Following the incubations, cell lysates were
immunoprecipitated (IP) with antibodies as indicated in the
figure. The precipitated immunocomplexes were analyzed for
phosphotyrosyl-containing proteins by anti-phosphotyrosine (anti-PY) immunoblotting. The blots were stripped and reprobed
with the respective antibody used for immunoprecipitation. C,
cells were treated as described in panelB for 1 h.
Total cellular RNA was probed with IL-1
or
-actin probes (6
µg total RNA/lane). NAD, nonadherent; ADH,
adherent to fibronectin.
1 integrins in peripheral blood monocytes
results in a prompt and robust induction of a number of inflammatory
mediator genes including IL-1
, IL-8, IL-6, and tumor necrosis
factor, as well as genes for a number of transcription factors
(18-20). Recently, we have demonstrated that
1 integrin
ligation in monocytes also results in a burst of tyrosine
phosphorylation and that this is necessary for subsequent
integrin-mediated gene induction(21) . Here, we have shown that
similar events, including integrin-mediated tyrosine phosphorylation
and increases in inflammatory gene message levels, also occur in THP-1
cells, a monocytic cell line that is more amenable to study of
biochemical and molecular events than are primary monocytes. We also
show that the transcription factor NF-
B is activated in response
to integrin ligation. Further, we have identified some of the proteins
that are tyrosine phosphorylated in THP-1 cells subsequent to integrin
ligation; these include the focal contact protein paxillin and the FAK
and Syk non-receptor tyrosine kinases.
1 integrins are similar but
distinct. Both types of stimuli result in prominent tyrosine
phosphorylation of several components in the 65-75- and the
115-125-kDa ranges. However, as observed in whole cell lysates,
integrin-mediated adhesion to fibronectin produces a strong doublet at
65-75 kDa, while antibody ligation results primarily in
phosphorylation of the lower molecular mass component of the doublet;
likewise, among the tyrosine-phosphorylated proteins at 115-125
kDa, adhesion results in strong phosphorylation of a slowly migrating
component (120 kDa), while antibody ligation primarily affects a more
rapidly migrating component (115 kDa). The basis for the differences in
tyrosine phosphorylation patterns in THP-1 cells stimulated by
anti-integrin antibodies and those stimulated by integrin-mediated cell
adhesion is unclear at this time. One reasonable possibility is that
cell adhesion results in a more complete engagement of the cytoskeleton
than does the formation of integrin dimers or multimers triggered by
antibodies and the degree of cytoskeletal organization can affect the
recruitment of proteins subject to tyrosine phosphorylation.
B and can give rise to
increased levels of cytokine messages. Furthermore, as we have
demonstrated here for THP-1 cells and previously for
monocytes(21) , inhibitors of tyrosine kinases can block message
induction mediated by cell adhesion or by ligation of
1 integrins.
These inhibitors also blocked activation of an NF-
B driven
promoter-reporter construct in transient transfection assays (data not
shown). Thus, it seems highly probable that integrin engagement can
trigger activation of tyrosine kinases and protein tyrosine
phosphorylation events that are critical for the control of
inflammatory mediator genes in monocytic cells. Therefore, it is
important to carefully analyze integrin-mediated tyrosine
phosphorylation to try to define those events most closely related to
the message induction process.
1
integrins with antibodies did not result in tyrosine phosphorylation of
FAK or paxillin but did cause increased tyrosine phosphorylation of
Syk, a cytoplasmic tyrosine kinase that contains SH2
domains(24, 25) . The Syk kinase has been reported to
associate with several receptors, including the antigen receptors on
B-cells(43) , the Fc receptors for IgE (Fc
RI) (44) and IgG (Fc
RI, Fc
RIII)(45, 46) ,
and granulocyte colony-stimulating factor receptor(26) . Syk
kinase becomes activated and tyrosine phosphorylated when those
receptors bind their respective ligands. A consensus motif has been
defined in the cytoplasmic domains of receptors that bind Syk or the
related kinase ZAP70(26) . Impaired signal transduction
processes have been found in Syk kinase negative cells or in cells that
express a Syk mutant lacking kinase activity(43) . Since Syk can
be activated via Fc receptors, we used F(ab`)
fragments of
anti-integrin antibodies to demonstrate that integrin ligation, rather
than Fc receptor engagement, was responsible for the increase in Syk
enzymatic activity and tyrosine phosphorylation that we observed. In
addition, the fact that Syk can be activated by integrin-mediated
adhesion to fibronectin substrata also indicates that integrins rather
than Fc receptors are involved. Since integrin cytoplasmic domains do
not contain any obvious homologies to the Syk binding motif found in
other cognate receptors(26) , it seems likely that the
interaction between integrins and Syk is indirect. In support of this,
we have not been able to co-immunoprecipitate Syk and integrins from
cell lysates (data not shown). Recent studies in platelets have shown
that the Syk kinase can be activated via engagement of the
IIb/
3 integrin by fibrinogen(48) . Thus, in both
monocytic cells and platelets, Syk is an integrin-responsive tyrosine
kinase. The activation of Syk, in contrast to the activation of FAK, is
not completely dependent on cytoskeletal assembly as observed here as
well as in previous studies on platelets (48). Interestingly, recent
investigations in rat basophilic leukemia cells have shown that
cross-linking of the Fc
RI receptor can trigger tyrosine
phosphorylation of paxillin, while receptor cross-linking in adherent
cells can induce tyrosine phosphorylation of both FAK and a stronger
phosphorylation of paxillin(47, 49) . These results may
have similar underlying mechanisms to the results presented here, since
multi-valent cross-linking of Fc
RI receptors may cause engagement
of the cytoskeleton, setting the stage for FAK and paxillin
phosphorylation.
message
levels, as well as causing changes in protein tyrosine phosphorylation.
However, antibody ligation affected the Syk tyrosine kinase but not FAK
or its substrate paxillin. Furthermore, treatment with cytochalasin D,
which effectively blocked cell spreading and FAK and paxillin tyrosine
phosphorylation, failed to block Syk tyrosine phosphorylation or
increases in IL-1
message levels. Thus, it seems that FAK is not
required for integrin-mediated IL-1
message induction in THP-1
cells, consistent with our previous observations on primary
monocytes(21) . Conversely, our results suggest that Syk may
play an important role in the signal transduction process leading from
integrin engagement to the induction of inflammatory mediator genes.
Thus, the activation of Syk uniformly precedes and accompanies
integrin-mediated gene induction, while similar concentrations of
tyrosine kinase inhibitors block both Syk autophosphorylation and
IL-1
message induction. The integrin-mediated changes in tyrosine
phosphorylation and Syk activation are transient even during continued
cell adhesion or antibody ligation. This suggests that these processes
are regulated, perhaps through the agency of protein phosphatases that
are induced or activated subsequent to integrin stimulation. Similar
events have been associated with the regulation of kinase activities
stimulated by mitogens(40) .
B transcription complex, and the eventual induction of messages
for inflammatory mediator genes. Extensive cytoskeletal reorganization
does not seem to be required in this pathway, since cytochalasin D
treatment had little effect on IL-1
message levels. In those types
of monocytic cells that do express FAK, such as THP-1, there is a
second integrin-mediated signaling pathway that involves FAK
activation, and that seems to impact primarily on cytoskeletal
organization rather than gene induction.
gene but
does not affect message stability.
(
)In the THP-1
cell system, integrin ligation leads to activation of NF-
B,
increased transcription from NF-
B-driven reporter constructs, and
increases in IL-1
message levels. However, it is not clear at
present if integrin-mediated changes in IL-1
message levels in
THP-1 cells are primarily controlled at the transcriptional level or by
other means, such as changes in message stability or RNA splicing. If
transcriptional activation does occur, the precise role of NF-
B
and of other transcription factors remains to be defined. Despite these
questions, current observations provide important initial insights into
integrin signal transduction processes in monocytic cells and emphasize
a key role for specific tyrosine phosphorylation events.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.