(Received for publication, February 17, 1995; and in revised form, July 18, 1995)
From the
Thrombospondin is a matrix glycoprotein found in various cells
that can modulate cell attachment, migration, and proliferation. We now
show that intact soluble thrombospondin causes a transient
[Ca]
increase in
IMR-90 fibroblasts. This [Ca
]
increase is mediated partly by the RGD-containing domain of
thrombospondin that binds to the integrin
v
3 as demonstrated
by inhibitor studies using anti-
v
3 antibody and
RGD-containing peptides. A non-RGD and non-
v
3 component of
this [Ca
]
increase is
mediated by the carboxyl-terminal domain of thrombospondin through an
unidentified receptor on fibroblasts as shown by the antibody to the
carboxyl-terminal of thrombospondin, C6.7. In addition, the
carboxyl-terminal derived peptide, RFYVVMWK, also triggers
[Ca
]
increase in
35% of fibroblasts. Both EGTA and Ni
block the
entire [Ca
]
increase
indicating that this is due to an influx of extracellular
Ca
. B6H12, an antibody to the integrin-associated
protein, blocks this [Ca
]
increase by 50%, suggesting that some of the Ca
might be entering through an integrin-associated calcium channel.
The current findings demonstrate that multiple domains on
thrombospondin can trigger signal transduction events by increasing
[Ca
]
through their
interactions with different cell receptors.
Thrombospondin (TSP) ()is a 450-kDa trimeric matrix
glycoprotein. It was first described as an
-granular protein
secreted by platelets upon thrombin activation. TSP is also synthesized
by fibroblasts, endothelial cells, smooth muscle cells, monocytes,
macrophages, osteoblasts, and tumor
cells(1, 2, 3, 4, 5, 6) .
TSP has been shown to modulate cell attachment, migration, and
proliferation and has an important role in wound healing, hemostasis,
and
angiogenesis(2, 3, 5, 7, 8, 9, 10) .
Like other matrix proteins such as fibrinogen and fibronectin, TSP
possesses multiple cell binding domains (6, 11) . Four
known cell binding domains have been described for thrombospondin: (i)
the heparin binding domain at the amino-terminal (12) , (ii)
the type I repeats containing the CSVTCG sequence(13) , (iii)
the Arg-Gly-Asp (RGD) sequence in the type III calcium binding
repeat(7) , and (iv) the carboxyl-terminal cell binding
domain(14, 15) . Each of these domains was found to
bind to distinct cell surface receptors: (i) heparan sulfate
proteoglycans(16) , (ii) CD36 or GPIV (17) , (iii)
integrin
v
3(7, 18) , and (iv) the 105/80-kDa
receptor (4) or the 52-kDa receptor(19) ,
respectively.
Recently there has been major interest in the integrin
v
3 because of its role in angiogenesis and
apoptosis(20, 21) . An initial step in these processes
often involves the binding of a matrix protein to
v
3. There
has been extensive work on the role of TSP on cell attachment involving
v
3; however, little is known about the signal transduction
events(4, 7, 18) . Increasing evidence from
various groups clearly shows that the role of integrins extends beyond
the binding of ligands or matrix
proteins(22, 23, 24) . The binding of various
matrix proteins to their receptors, especially integrins, often
triggers different signals such as tyrosine phosphorylation and changes
in intracellular pH or
Ca
(25, 26, 27) . These
outside-in signaling events serve as important regulatory mechanisms to
mediate subsequent molecular events such as modulation of integrin
affinity (28, 29) or gene transcription(30) .
In this study, we investigated the role of TSP in triggering
Ca mobilization in a lung fibroblast cell line,
IMR-90. In addition, we also used known inhibitors of both the cell
binding domains and the receptors of TSP to elucidate the structural
basis of TSP-induced transmembrane signaling. The findings suggest that
intact TSP can mediate Ca
mobilization via both the
RGD and carboxyl-terminal domains.
R was determined by 2-4 µM ionomycin (Calbiochem) and R
by 10
µM EGTA. S
and S
were determined by procedures published by Grynkiewicz et al.(32) . All single-cell fluorescence recordings were done
at room temperature with background subtracted. Background was
determined using a field on the coverslip with no cells. For each
experiment, the responses of 5-10 individual cells were averaged.
In inhibition experiments, peptides or antibodies were preincubated
with cells for 2-5 min before addition of TSP. Increases in
[Ca
]
due to TSP varied among
different cell preparations in the range of 91-569 nM.
To normalize for this interexperimental difference, % control or %
inhibition was calculated from the TSP response of each experiment.
Cell morphology was monitored by phase contrast microscopy. None of the
antagonists or antibodies used in these experiments significantly
affected the morphology of these cells within the time period of the
experiment.
Addition of TSP to IMR-90 cells caused a
concentration-dependent transient rise in
[Ca]
ranging from 55-
1000
nM in >90% of the cells (Fig. 1). This
[Ca
]
increase peaked within
10-30 s and returned to resting levels after 60-180 s
depending on the concentration of TSP (Fig. 1). With 0.08
µM TSP, the mean increase in
[Ca
]
was 220 ± 34
nM (n = 20) above the resting level of 93
± 8 nM (n = 18) (Fig. 2A). This increase in
[Ca
]
was eliminated completely
in the absence of extracellular Ca
by the addition of
5 mM EGTA (Fig. 2B). It is possible that the
blocking of the [Ca
]
increase
is due to EGTA's effect on Ca
-dependent ligand
binding to TSP receptors rather than depletion of the source of
Ca
available for transmembrane
fluxes(4, 33) . Chelation of Ca
has
been shown to dissociate the integrin heterodimer and alter the
tertiary structure of
TSP(7, 18, 34, 35) . This, in turn,
could indirectly affect ligand binding and thus the ability to trigger
an increase in [Ca
]
. To verify
that the attenuated Ca
was due to inhibition of
Ca
influx, cells were pretreated with
Ni
, which is known to block Ca
fluxes through many types of Ca
channels.
Ni
at 10 mM abolished the rise in
[Ca
]
induced by TSP similar to
that of EGTA (Fig. 2C). Neither EGTA nor Ni
affected the baseline [Ca
]
under these conditions. These results indicate that the increase
in [Ca
]
from TSP is dependent
on extracellular Ca
fluxes into the cells.
Figure 1:
Thrombospondin-induced
transient increase in [Ca]
in IMR-90 lung fibroblasts. TSP-induced increase in
[Ca
]
in a
concentration-dependent manner. Representative
[Ca
]
tracings of
single cell recordings from IMR-90.
Figure 2:
Thrombospondin-induced transient increase
in [Ca]
in IMR-90 is
dependent on extracellular Ca
. Representative
[Ca
]
tracings of
recordings from cells stimulated with 0.08 µM TSP (A) and pretreatment with 5 mM EGTA (B) or
10 mM Ni
(C) before TSP addition.
Note the inhibition of this increase in
[Ca
]
by EGTA or
Ni
pretreatment. Representative tracings of single
cell recordings from at least four separate experiments. Assay was
carried out as described under ``Experimental
Procedures.''
Figure 3:
Cell surface expression of thrombospondin
receptors in IMR-90 fibroblasts by flow cytometry. Cells were gated by
forward and side light scatter, and 5,000 gated events were collected.
IMR-90 expressed both v
3 and
v
5 but not CD36 on
their cell surface. Assay was carried out as described under
``Experimental Procedures.''
Figure 4:
[Ca]
tracings showing the effect of various anti-
v
antibodies on the thrombospondin-induced rise in
[Ca
]
. Cells were
pretreated with 4 µg/ml LM609 (anti-
v
3) (A), 4
µg/ml B6H12 (anti-IAP) (B), or 10 µg/ml P1F6
(anti-
v
5) (C) before addition of 0.08 µM TSP. Representative tracings of single cell recordings from at
least four separate experiments. Note the partial inhibition of
TSP-induced increases in [Ca
]
with LM609 and B6H12 but not with P1F6. Assay was carried
out as described under ``Experimental
Procedures.''
Figure 5:
Effect of anti-v antibodies on the
thrombospondin-induced rise in
[Ca
]
. Antibodies LM609
and B6H12 (4 and 10 µg/ml) partially inhibited TSP-induced
increases in [Ca
]
.
P1F6 at a similar concentration has minimal effect. Mean ± S.E. (n = 4). Assay was carried out as described under
``Experimental Procedures.''
Since this Ca increase was
apparently due to a calcium influx, we investigated further whether it
was via the integrin-associated protein (IAP) which has been shown to
mediate integrin-regulated Ca
flux into
cells(39) . We tested the effect of a recently described
antibody to IAP, B6H12(31) , on TSP-induced increases in
[Ca
]
. B6H12, at 4 and 10
µg/ml, inhibited TSP-induced increases in
[Ca
]
by 42% and 50%,
respectively (Fig. 4B and Fig. 5). On the other
hand, the TSP-induced increase in
[Ca
]
was not inhibited by the
control antibody to
v
5 (Fig. 4C and Fig. 5). In addition, LM609 and B6H12 do not block non-TSP-, i.e. thrombin, mediated increases in
[Ca
]
, which indicates they
specifically block
v
3 integrin-mediated Ca
flux. (
)None of these monoclonal antibodies raised the
baseline [Ca
]
at the
concentrations tested.
Earlier studies have reported that integrin
clustering is very important in signal
transduction(25, 40, 41) . Here we
investigated the effect of TSP and inhibitors of the TSP-v
3
interaction on integrin clustering. As shown in Fig. 6, TSP
induces significant integrin clustering in IMR-90. This is not seen in
cells that were not exposed to TSP or those that were pretreated with
LM609, suggesting a specific interaction between the integrin and the
matrix protein.
Figure 6:
Immunolocalization of v
3
integrin on IMR-90 using indirect fluorescence. Fluorescence images
showing very diffuse distribution of
v
3 integrin on the
surface of IMR-90 (A). With pretreatment of 0.17
µM TSP for 2 min (B), cells showed significant
integrin clustering as indicated by the white arrows. A 2-min
preincubation with 20 µg/ml LM609, which prevents the interaction
of
v
3 with TSP, showed significantly less clustering (C). Cells incubated with isotype match antibody did not stain
(not shown). The distribution of
v
3 was observed using LM609
followed by Cy3-conjugated goat anti-mouse IgG as described under
``Experimental Procedures'' (magnification,
1000).
Figure 7:
Effect of RGD-containing peptides on the
thrombospondin-induced rise in
[Ca]
. Cells were
pretreated with 0.8 mM GRGDSP (A), 0.8 mM GRGESP (B), or 0.8 µM echistatin (C) before the addition of 0.08 µM TSP. Note the
partial inhibition of TSP-induced increases in
[Ca
]
with GRGDSP and
echistatin but not with the control inactive peptide GRGESP. However,
GRGDSP at this concentration also increases
[Ca
]
. Representative
tracings of single cell recordings from at least four separate
experiments. Assay was carried out as described under
``Experimental Procedures.''
Figure 8:
Dose-response relationship of the
inhibitory effect of RGD-containing peptides. The effect of echistatin
(), GRGDSP (
), or GRGESP (
) on TSP-induced increases
in [Ca
]
. Echistatin
showed more potent inhibition of TSP-induced increases in
[Ca
]
than GRGDSP with
a shift of 3-4 log concentrations to the left. Each data
point is the mean of at least four experiments with S.E.
10%.
Assay was carried out as described under ``Experimental
Procedures.''
In addition to
blocking the TSP-induced increase in
[Ca]
, GRGDSP by itself, at
concentration >0.8 mM, caused a transient increase in
[Ca
]
in >90% of the cells.
It is possible that the depletion of available Ca
due
to this GRGDSP-induced increase in
[Ca
]
might be responsible for
the attenuation of [Ca
]
signal
when TSP is added. However, this is unlikely since subsequent
stimulation with other agonists, such as thrombin, was able to elicit
another transient increase in
[Ca
]
.
This response
to GRGDSP was RGD-specific as the control peptide, GRGESP, did not
induce an increase in [Ca
]
.
Also, echistatin, by itself, demonstrated a similar increase in
[Ca
]
in 50% of the cells at 5
µM, but no effect was seen at 1 µM.
One of the potential candidates is CD36, which
is the receptor for the type I repeat of TSP(17) . This surface
glycoprotein CD36 has been shown to mediate signal transduction in
platelets by increasing
[Ca]
(43) . However, in
this system, it is unlikely that CD36 mediates the increase in
[Ca
]
as IMR-90 do not express
CD36 on their cell surface (Fig. 3). In addition, the peptide
CSVTCG, which corresponds to the CD36 binding motif on TSP and has been
shown to inhibit cell adhesion(13) , did not have any effect on
TSP-induced rises in [Ca
]
(Fig. 9). CSVTCG by itself had no effect on baseline
[Ca
]
. Likewise, the heparin
binding domain of TSP also did not seem to play a role in generating
this [Ca
]
increase because
heparin, at a concentration (100 µg/ml) that had been shown to
effectively block cell adhesion(15) , did not inhibit
TSP-induced rises in [Ca
]
(Fig. 9).
Figure 9:
Effect of inhibitors against the non-RGD
domains of thrombospondin on thrombospondin-induced rise in
[Ca]
. Antibodies to
TSP cell binding domains, C6.7 (1:100), A4.1 (1:100), heparin (100
µg/ml), and control mouse IgG (50 µg/ml) were preincubated with
TSP for 30 min before addition to cells. Peptide CSVTCG (0.1
mM) was added directly to the IMR-90 cells. Of these
inhibitors only the antibody to the carboxyl-terminal domain of TSP,
C6.7, is effective in inhibiting TSP-induced increase in
[Ca
]
. Assay was
carried out as described under ``Experimental
Procedures.''
Another possibility is the carboxyl-terminal
cell binding domain of TSP which has been described to support
non-RGD-dependent cell adhesion in melanoma cell
lines(14, 15) . When TSP was preincubated with C6.7,
an antibody against this domain, the TSP-induced increase in
[Ca]
is inhibited by 50%. This
suggests that part of the Ca
signal generated by TSP
may be due to the binding of the carboxyl-terminal domain to its
receptor (Fig. 9). This effect is apparently specific to the
carboxyl-terminal binding domain since antibody
A4.1(10, 44) , directed against the amino-terminal
half of the central stalk region of TSP, did not block the
[Ca
]
increase. Nonspecific
antibody did not have any significant effect on
[Ca
]
increase. Interestingly,
when both LM609 (10 µg/ml) and C6.7 (1:100) were used together, no
synergistic or additive effect was seen. (
)It is unclear, at
this point, if this is due to a lack of cooperativity of the two sites.
Nevertheless, more direct evidence of the role of the carboxyl-terminal
of TSP came from studies with the carboxyl-terminal domain peptide
RFYVVMWK(45) . This peptide at 100 µM triggered a
transient increase in [Ca
]
that
reached
2 µM in 35% of the cells (Fig. 10).
This suggests that the carboxyl-terminal cell binding domain may
mediate part of the TSP-induced rise in
[Ca
]
. Altogether, these data
indicate that intact TSP can mediate an increase in
[Ca
]
in IMR-90 fibroblasts via
at least two pathways: the RGD domain binding to the
v
3
integrin and the carboxyl-terminal domain possibly binding to an
unidentified receptor.
Figure 10:
[Ca]
tracing showing the effect of thrombospondin
carboxyl-terminal peptide on [Ca
]
in IMR-90 cells. The carboxyl-terminal peptide, RFYVVMWK
(100 µM), increases
[Ca
]
in IMR-90.
Representative tracings of single cell recordings from at least four
separate experiments. Assay was carried out as described under
``Experimental Procedures.''
In this study we have shown that TSP, a matrix protein which
binds to multiple cell surface receptors, increases
[Ca]
in IMR-90 fibroblasts.
This increase, which is partially mediated by the RGD domain of TSP
binding to the integrin
v
3, is not limited to IMR-90, as we
have also observed this in endothelial cells.
We have also
shown that known inhibitors of
v
3, GRGDSP peptide and
echistatin, in addition to blocking TSP-induced signaling, are capable
of triggering an increase in [Ca
]
by themselves. This suggests that RGD-containing peptides can
function as both competitive antagonists and partial agonists for their
receptors. This had been reported previously by several other groups;
however, our threshold concentrations are much higher than those
reported elsewhere(36, 37) . One possible explanation
is that high concentrations of peptides or antibodies could induce
clustering of integrins, which could activate the integrins and trigger
transmembrane Ca
flux not normally seen at the lower
concentrations(26, 46) . This clustering is seen in
the present study when
v
3 integrins interact with TSP. These
findings illustrate that integrin clustering, presumably caused by
multimeric interactions between the ligand and its receptors, is
important in transmembrane signaling(41) . This is further
supported by the observation that RGD peptide can produce an increase
in [Ca
]
(>1 mM) in
kidney epithelial cells when coupled to beads but not when it is in the
soluble monomeric form(29) . In addition, earlier studies with
2 integrin (CD11b/CD18) demonstrate that antibodies to the
and
can induce Ca
signal only if they are
cross-linked(40) .
Another confounding factor is that this
ligand-induced integrin signaling may be dependent on the type of cell
preparation being used. Sjaastad et al.(29) have
reported that soluble RGD (0.3 mM) was not able to
produce an increase in [Ca
]
in
kidney epithelial cells, whereas two other groups both observed an
increase in osteoclasts at a similar
concentration(36, 37) . Even within the same cell type
the data are not consistent. In that regard, echistatin was shown to
induce increases in [Ca
]
in rat
osteoclasts by one group but not by the other
group(36, 37) . This discrepancy indicates the
complexity of ligand/integrin-mediated signaling.
Ligand-induced
signaling through integrins does not follow the paradigm for
traditional signaling receptors. In general, integrins differ from the
known signaling receptors in their short cytoplasmic domain, lack of
kinase activity, and lack of direct interaction with G-proteins (23) . In this study we have shown that the
[Ca]
signal triggered by the
soluble matrix protein, TSP, via the integrin
v
3, is
dependent on extracellular calcium. The present data using EGTA or
Ni
indicates that the increase in
[Ca
]
is due primarily to an
influx of extracellular Ca
, possibly through some
type of Ca
channel. Additional evidence supporting
this hypothesis was demonstrated by our data with the antibody to IAP,
B6H12, which was shown to block
50% of
[Ca
]
increase. IAP, which has
been shown to be closely associated with
v
3(31) , was
recently shown by Schwartz et al.(39) to be required
specifically for integrin-regulated Ca
influx. This
suggests that IAP may function as an integrin-associated calcium
channel. Moreover, purified integrin
IIb
3 was reported to
function as a calcium channel when reconstituted into
liposomes(47) . This suggests that the integrin itself can act
as a conduit for transmembrane Ca
flux that is
distinct from the classical Ca
channels. Taken
together, it is possible that the
v
3 integrin or its
associated proteins, upon ligand binding, can be activated and function
as a Ca
channel for signal transduction.
The
present data also show that there are other components of the
transmembrane Ca flux that are non-RGD and
non-
v
3-dependent. This notion comes from the inability of the
synthetic RGD-peptide and LM609 to completely inhibit TSP-induced rises
in [Ca
]
and is verified by the
observation that an antibody to carboxyl-terminal domain, C6.7, is also
able to inhibit 50% of the Ca
signal. The
carboxyl-terminal domain of TSP has been reported to support chemotaxis
and cell migration(48, 49) . Further studies using the
carboxyl-terminal domain peptide, RFYVVMWK, indicate that the
carboxyl-terminal domain of TSP is also capable of triggering
Ca
increases by itself. Since this Ca
increase is observed only in a subpopulation of cells, this
receptor might not be present on all cells. Two possible receptors for
the carboxyl-terminal binding domain have so far been identified, an
80/105-kDa complex on carcinoma cells (4) and a 52-kDa domain
on K-562 cells(19) . Even if either of these receptors exists
in the IMR-90, it is unclear if they are responsible for the binding of
the carboxyl-terminal TSP fragment and the subsequent transduction of
the signal across the cell membrane. The carboxyl-terminal peptide and
GRGDSP trigger increases in [Ca
]
in the millimolar range at peptide concentrations of 0.1 and 0.8
mM, respectively. With intact TSP, only 1 µM is
required to trigger similar increases in
[Ca
]
. The main reason for this
disparity in threshold concentrations is the multivalent and higher
affinity binding of the multimeric matrix protein TSP versus the monovalent and lower affinity binding of the peptide
fragments(41, 50) . In addition to having high
affinity receptors, multimeric ligands are more effective in inducing
receptor clustering as shown in the present study; together, these have
been shown to work synergistically in transmembrane
signaling(41) .
In addition, TSP might potentially mediate
signaling via other receptors. One such TSP receptor is CD36 which was
not studied here due to the lack of expression on IMR-90 fibroblasts.
There is evidence from the literature that CD36 is capable of mediating
signal transduction by increasing
[Ca]
in platelets and
triggering oxidative bursts in monocytes(43, 51) .
However, these two studies utilized monoclonal antibodies against CD36
to trigger the signaling events, and, therefore, it is uncertain if
intact TSP or its fragments will produce similar results via CD36.
In summary, we have demonstrated that TSP can trigger transient
increases in [Ca]
in IMR-90
fibroblasts by increasing Ca
influx. The response is
inhibitable by specific TSP receptor antibodies indicating that this is
a receptor-mediated effect. While we have shown this to occur primarily
via the RGD and carboxyl-terminal domains of TSP and their
corresponding receptors, we cannot rule out the possibility of the
other TSP receptors as signaling molecules in other cellular systems.
The data from the present study, together with previous reports,
indicate that TSP can support cell attachment and signaling through its
different cell binding
domains(4, 7, 13, 14, 18, 45) .
These observations confirm that matrix proteins, with their multiple
cell binding domains, can interact with corresponding cell surface
receptors to transduce signals across the cell membrane. These
outside-in signaling events will be important in understanding the role
TSP plays in cell attachment, migration, and proliferation.