(Received for publication, October 17, 1995)
From the
Urokinase-type plasminogen activator (u-PA) or its
amino-terminal fragment (ATF) containing the u-PA receptor (u-PAR)
binding domain, is known to promote monocyte adhesion. In the present
study, U937 monocyte adhesion to a plastic surface was used to
investigate the mechanism of its promotion by u-PA and ATF. Adhesion
was found to be inhibited by cycloheximide or actinomycin D,
implicating protein synthesis and gene expression in u-PA-induced
monocyte adhesion. Adhesion was prevented by 2`-deoxyadenosine
3`-monophosphate, indicating that a cAMP-dependent pathway of signal
transduction was involved. This concept was supported by the
complementary finding that u-PA-induced adhesion was greatly promoted
by forskolin, cholera toxin, or 8-bromo-cAMP, which by themselves
induced little adhesion. Furthermore, similar to many other
cAMP-dependent activities, cGMP diminished u-PA-induced adhesion. When
u-PA or ATF was treated with immobilized carboxypeptidase B, its
proadhesive effect was abolished, implicating the involvement of
carboxyl-terminal lysine residues (Lys on u-PA and
Lys
on ATF). Moreover, when a carboxyl-terminal lysine
analog was added, the proadhesive effect of carboxypeptidase B-treated
u-PA or ATF was restored. In conclusion, the present study indicates
that u-PA- or ATF-induced monocyte adhesion involves cAMP-dependent
signal transduction, which is triggered by u-PAR binding. It is also
critically dependent on the presence of a carboxyl-terminal lysine.
Urokinase-type plasminogen activator (u-PA) ()is a
well characterized, highly restricted serine protease that converts
plasminogen to plasmin. It contains three domains, an epidermal growth
factor (EGF)-like domain, a kringle, and a protease domain. The
protease domain catalyzes plasminogen activation and is involved in
many biological processes including cell migration, tissue remodeling,
ovulation, tumor metastasis, and fibrinolysis. In addition, u-PA has
been shown to induce monocyte adhesion(1) , to be involved in
cell focal adhesion (2) , and to promote cell
growth(3, 4) . These latter functions are independent
of the protease domain of u-PA. Instead, they are related to the
occupation of a u-PA receptor (u-PAR) found on the surface of many
cells(1, 3) , which is mediated by the EGF domain of
u-PA(5) .
The u-PAR is a glycosylphosphatidylinositol-anchored protein that does not have a transmembrane domain or intracellular kinase domain(6) . Therefore, a transmembrane protein partner has been implicated in u-PAR-mediated signal transduction (7, 8, 9) but remains to be identified.
It
has been postulated that the physical binding of u-PAR (10, 11) or u-PA (12) to vitronectin accounts
for the proadhesive effect. However, a number of observations are
inconsistent with this hypothesis. First, according to the
u-PA-vitronectin binding mechanism, an excess of free u-PA should
inhibit u-PA-induced cell adhesion(12) , but such an inhibition
was not found(1, 10) . Second, u-PA-induced adhesion
was found to be time-dependent(1) , which is not characteristic
of physical interaction but rather suggests a biochemical process.
Third, when a vitronectin-coated surface instead of plastic was used (1, 10) , the promotion of cell adhesion by u-PA (3
nM) was reduced from 40- to
4-fold(1, 10) . Moreover, the promotion of vitronectin
binding to phorbol myristate acetate-primed U937 cells by u-PA (10
nM) was reported to be only about 2-fold, as was the promotion
of soluble u-PAR binding to immobilized vitronectin(11) . Since
u-PA promotion of monocyte adhesion was more than 50-fold(1) ,
a direct physical interaction between vitronectin and u-PAR/u-PA
appears to be responsible for only a minor part of u-PA-induced
adhesion. Finally, low temperature was found to inhibit u-PA-induced
adhesion, suggesting a metabolic process(10) .
In the present study of u-PA- or ATF-induced monocyte adhesion, evidence for cAMP-dependent signal transduction was found. A carboxyl-terminal lysine residue was also found to be required for this process.
DFP-treated u-PA and the ATF (American Diagnostics, Greenwich, CT) were used in all the experiments, thereby avoiding any complication by the anti-adhesive effect of PAI-1 against enzymatic u-PA(1) .
The cell adhesion assay was performed as
described previously (1) with modifications. U937 cells at
10 cells/ml were labeled with 1 µCi/ml
[
H]thymidine in the culture medium containing
human transforming growth factor-
1 (1 ng/ml) and
1
,25-dihydroxyvitamin D
(52 nM) for 24 h. The
H-labeled cells were collected by centrifugation at 350
g for 4 min at 4 °C. After washing three times
with serum-free RPMI 1640, cells were resuspended in RPMI 1640
containing 10% bovine fetal serum at 10
cells/ml. Test
reagents were added to the cell suspension, and 300 µl of
cells/well were placed in duplicate in 24-well plates (Costar Corp.,
Cambridge, MA). After 17 h of incubation (37 °C) in 5%
CO
, the contents of each well were removed using a Pasteur
pipette, and wells were gently rinsed with phosphate-buffered saline
three times. Adherent cells were lysed in lysis buffer containing 10%
glycerol, 0.2% sodium dodecyl sulfate, and 0.2% Triton X-100 (1
ml/well). Each 1 ml of lysate was added into 10 ml of formula-989 high
flash point mixture and counted in a liquid scintillation counter.
Figure 1:
Inhibition of u-PA-induced U937 cell
adhesion by cycloheximide and actinomycin D. H-Labeled U937
cells at 1
10
cells/ml were incubated with u-PA (1
nM) in the absence and presence of either cycloheximide (1
µg/ml) or actinomycin D (5 µg/ml) in 24-well plates as
described. The quantity of the adherent cells was measured by a liquid
scintillation counter and expressed in counts/min. All the data are
mean values with S.D. of duplicate assays.
Figure 2:
Effect of cAMP elevation on u-PA-induced
U937 cell adhesion. A,H-labeled U937 cells at 1
10
cells/ml were incubated with forskolin
(0-150 µM) or 2`-deoxyadenosine 3`-monophosphate
(0-300 µM) in the absence or presence of u-PA (1
nM), and cell adhesion was measured (mean and S.D.). B, U937 cells as above were incubated with cholera toxin (16.7 ng/ml)
or u-PA (0.5 nM) alone, or cholera toxin (16.7 ng/ml) plus
u-PA (0.5 nM). C, U937 cells as above were incubated
with 8-Br-cAMP or 8-Br-cGMP (0-5 mM) in the absence or
presence of u-PA (1 nM).
The findings indicate that the signal of u-PA binding is amplified by an increase in cAMP by adenylyl cyclase, which triggers the other events involved in monocyte adhesion such as RNA and protein synthesis. The activity of adenylyl cyclase is known to be usually regulated by G-protein. This would suggest that the transmembrane partner protein of u-PAR may interact with G-protein. Similar to many other cAMP-dependent activities, cGMP was found to be an antagonist of cAMP in these experiments, diminishing u-PA-induced adhesion (Fig. 2C), suggesting that u-PA-induced monocyte adhesion was regulated by the opposing effects of cAMP and cGMP.
It is noteworthy that cAMP elevation by u-PA was previously reported to inhibit adhesion in cultured mesangial cells(16, 17) . This suggests that the function of cAMP-dependent signal transduction by u-PA may be cell type-specific. Furthermore, in the case of mesangial cells in contrast to U937 cells, adhesion was dependent on the enzymatic activity of u-PA(17) .
A tyrosine kinase (7) and a serine kinase (8, 9) have been reported to be activated by u-PA occupation of u-PAR. However, using their corresponding inhibitors, herbimycin A (2 µM) for protein tyrosine kinases and H7 (10 µM) for serine kinases, no significant effects were seen in the present study, even at the highest concentrations of the inhibitors possible without interfering with cell growth. Therefore, u-PA-induced monocyte adhesion appears to be independent of these protein kinases.
Increased protein expression of u-PAR by 2-fold and u-PA by 5-fold in U937 monocytes was observed to be induced by u-PA or ATF (10 nM) (data not shown). This finding was consistent with previous reports that u-PAR expression was up-regulated in endothelial cells by activation of adenylyl cyclase (18) and in LLC-PK1 kidney cells by cAMP elevation(19) . The increase in u-PAR or u-PA expression was probably insignificant (maximally 2-fold) as an explanation for the induced adhesion based on an increase in physical binding sites. However, it could have a significant effect if it represents a positive feedback mechanism in u-PA(R)-induced signaling. In the presence of 1 nM exogenous u-PA or ATF, there was little increase of u-PAR and u-PA expression (data not shown), while a >10-fold increase in adhesion was seen (Fig. 1). This finding suggests that other cell surface proteins (probably some adhesive integrins) were responsible for the induced adhesion and that they were synthesized or activated as a consequence of u-PA(R) signaling.
Figure 3:
Effect of CPB treatment of u-PA or ATF on
U937 cell adhesion. A,H-labeled U937 cells at 1
10
cells/ml were incubated with u-PA- or
CPB-pretreated u-PA (0.5-5 nM), and cell adhesion was
measured as described. B, U937 cells as above were incubated
with ATF or CPB-pretreated ATF (0.5-4
nM).
Figure 4:
Inhibition of I-u-PA binding
to receptor by ATF, CPB-pretreated ATF, u-PA, and CPB-pretreated u-PA.
U937 cells at 2
10
cells/ml were incubated in
medium containing: 1, 8 nM
I-u-PA; 2, 8 nM
I-u-PA plus 100 nM ATF; 3, 8 nM
I-u-PA plus 100
nM CPB-pretreated ATF; 4, 8 nM
I-u-PA plus 100 nM u-PA; or 5, 8
nM
I-u-PA plus CPB-pretreated u-PA. The
cell-bound radioactivity was measured and expressed in counts/min. Mean
values ± S.D. are shown.
Figure 5:
Restoration of the proadhesive effect of
CPB-pretreated u-PA by EACA. H-Labeled U937 cells at 1
10
cells/ml were cultured in medium containing: 1, no additions; 2, 0.4 mM EACA; 3,
0.5 nM u-PA; 4, 0.5 nM CPB-pretreated u-PA;
or 5, 0.5 nM CPB-pretreated u-PA plus 0.4 mM EACA. Cell adhesion was measured as
described.
In conclusion, the present study indicates that u-PA-induced monocyte adhesion involves cAMP-dependent signal transduction and new protein synthesis, which are triggered by u-PAR occupancy. The presence of a carboxyl-terminal lysine residue was found to be additionally required. Therefore, it is postulated that when u-PA or ATF binds to u-PAR, its transmembrane protein partner is activated, which may interact with G-protein-activating adenylyl cyclase, initiating cAMP-dependent signal transduction and new protein synthesis. The resulting cell adhesion probably occurs mainly due to expression of adhesive integrins. The role of a carboxyl-terminal lysine in this sequence of reactions remains to be determined. However, since kringle domains are well known to interact with carboxyl-terminal lysines in the fibrinolysis-related proteins, like plasminogen and plasminogen activators, it may be speculated that the carboxyl-terminal lysine involved in cell adhesion also interacts with a kringle-carrying protein. Two families of transmembrane proteins that are tyrosine kinases have been found to have one or more kringle domains(21, 22) . Kringle domains are not common among transmembrane proteins.