(Received for publication, May 24, 1995; and in revised form, September 4, 1995)
From the
We have previously shown that soluble partially degraded
fibrin(ogen) remains in solution after fibrin clot formation and is a
potent fibroblast mitogen (Gray, A. J., Bishop, J. E., Reeves, J. T.,
Mecham, R. P., and Laurent, G. J.(1995) Am. J. Cell Mol. Biol. 12, 684-690). Mitogenic sites within the fibrin(ogen)
molecule are located on the A and B
chains of the protein
(Gray, A. J., Bishop, J. E., Reeves, J. T., and Laurent, G. J.(1993) J. Cell Sci. 104, 409-413). However, receptor pathways
[Abstract]
through which mitogenic effects are mediated are unknown. The present
study sought to determine the nature of fibrin (ogen) receptors
expressed on human fibroblasts which interact with the fibrinogen
B
chain. Receptor complexes were isolated from
I-surface-labeled fibroblasts and purified on a
fibrinogen B
chain affinity column. Subsequent high performance
liquid chromatography and SDS-polyacrylamide gel electrophoresis
analysis indicated fibrinogen B
chain bound specifically to a
60-kDa surface protein. Sequence analysis of the amino terminus of this
protein indicated 100% homology to human calreticulin.
Immunoprecipitation experiments employing a polyclonal
anti-calreticulin antibody provided further evidence that the 60-kDa
protein isolated in this study was calreticulin. Further, polyclonal
antibodies to human calreticulin significantly inhibited the mitogenic
activity of fibrinogen B
chain on human fibroblasts. The present
study has shown that cell surface calreticulin binds to the B
chain of fibrinogen mediating its mitogenic activity.
Blood coagulation at the site of tissue injury is a process
central to wound healing. The deposition of an insoluble fibrin matrix
provides both a hemostatic plug preventing leakage of plasma components
and a matrix over which cells can spread and proliferate(3) .
We have previously shown that the action of thrombin on fibrinogen (in
addition to the formation of insoluble fibrin) generates soluble
partially degraded forms of fibrin(ogen) which are potent fibroblast
mitogens (1, 4) and may further facilitate wound
healing. Mitogenic sites within the fibrinogen molecule have been
investigated, and we have shown both the A and B
chains of
fibrinogen are mitogens for human fibroblasts (2) .
The cell
surface components responsible for binding A and B
chains of
fibrinogen are unknown. The present study sought to determine the
nature of fibroblast receptor(s) involved in binding and subsequently
mediating the mitogenic action of partially degraded fibrin(ogen). Our
approach utilized human fetal fibroblasts which were surface-labeled
with
I. Membrane proteins were extracted with a mild
neutral detergent, and plasma membrane components were purified on a
fibrinogen B
chain affinity column. Protein moieties eluted from
the column were further purified by HPLC and visualized by SDS-PAGE. A
60-kDa surface protein which binds specifically to the B
chain of
fibrinogen was identified. Sequence analysis of the first 13 amino
acids of this protein showed 100% homology with human calreticulin.
Further, polyclonal antibodies to human calreticulin significantly
inhibited the mitogenic activity of the fibrinogen B
chain on
human fetal fibroblasts.
Glass
tubes were coated with IODOGEN, and 1 ml of cell suspension was added
to each tube. 500 µCi of NaI (ICN) was added, and the
mixture was incubated on ice for 12 min with occasional mixing.
Finally, cells were washed several times to remove free
I.
Plasma membrane proteins were extracted from human
fibroblasts which were grown to confluence in roller bottles and
harvested as described above. The same procedure was employed for both Ilabeled cells and unlabeled cells. Cell pellets were
resuspended in the neutral detergent octyl-
-glucopyranoside +
protease inhibitors (Sigma), and membrane-bound proteins were extracted
overnight at 4 °C. The resulting solution was spun at 20,000
g, and the supernatant either was used immediately in
the affinity chromatography assay or stored at -70 °C.
Figure 1:
Fibroblast mitogenic activity in
response to the B chain of fibrinogen. Assays were performed in
serum-free conditions for 48 h. B
chain was serially diluted
across a 96-well tissue culture plate. The starting concentration (1) of B
chain was 1.1
10
M.
Figure 2:
I-surface-labeled 60-kDa
protein eluted from a fibrinogen B
chain affinity column and
further purified by reverse phase HPLC. A, tracks
a-e,
I-surface-labeled proteins constituting
the radioactive peak eluted from a C4 reverse phase HPLC column after
initial purification over a fibrinogen B
chain affinity column.
Proteins were eluted with a 20-50% acetonitrile gradient. Tracks a-e eluted from the column with 40% acetonitrile.
Fractions were dried, reconstituted in Laemmli buffer, and run on a
12.5% SDS-PAGE gel.
I signal was visualized after a 48-h
exposure of the dried gel to autoradiography film. B, track a shows a large scale (cold) preparation of protein
visualized in track c of A. Protein moieties eluting
with 40% acetonitrile were collected, dried, and further separated on a
12.5% SDS-PAGE gel. Proteins were blotted onto Problot and visualized
with Coomassie Blue stain. A 60-kDa protein was observed that
corresponded with the 60-kDa protein seen in A, tracks
a-e. Finally, the 60-kDa protein band was cut out, and the
protein sequence was determined employing a ABI protein
Sequencer.
To identify the nature of the 60-kDa protein, a large scale preparation of unlabeled plasma membrane proteins was prepared using the affinity procedure described above. Purification of the 60-kDa protein was confirmed by Coomassie Blue staining of the isolated protein transferred to Problot (Fig. 2B, track a). The stained 60-kDa band was cut out and sequenced using a ABI protein Sequencer. Sequence analysis of the 60-kDa protein indicated an amino-terminal sequence which read: EPAVYFKEQFLDG. These experiments were performed on three separate preparations, and the same sequence was recorded on each occasion. A comparison of the sequence with sequences in the protein data base found 100% homology with human calreticulin.
Further experiments were performed to determine
binding specificity of fibrinogen B chain to the 60-kDa protein,
identified as calreticulin. Fig. 3, track a, shows the
ability of CNBr fragments of fibrinogen B
chain to elute
calreticulin from a B
chain affinity column. For comparison, also
shown in Fig. 3, track b, is the 60-kDa protein eluted
with 40 mM EDTA.
Figure 3:
Elution of I-labeled 60-kDa
protein from a B
chain affinity column with fibrinogen B
chain fragments and EDTA. Track a shows
I-labeled 60-kDa protein eluted from a fibrinogen B
chain affinity column with fibrinogen B
chain fragments generated
by CNBr digest. Track b shows
I-labeled 60-kDa
protein eluted from a fibrinogen B
chain affinity column with 40
mM EDTA.
Calreticulin has previously been reported
as a calcium binding protein found predominantly on the luminal side of
the endoplasmic reticulum. It was therefore important for the present
study to determine that calreticulin was also found on the
extracellular side of the plasma membrane. Fig. 4, track
a, shows an autoradiograph of I-labeled calreticulin
purified from cells scraped from a tissue culture dish in the presence
of protease inhibitors prior to
I labeling; a clear
60-kDa band can be observed. In contrast, Fig. 4, track
b, shows proteins purified in an fashion identical with those
shown in Fig. 4, track a, with the exception that cells
were trypsinized from tissue culture plates prior to
I
labeling.
I-Labeled 60-kDa protein was not isolated from
cells which were harvested with the serine protease trypsin.
Figure 4:
Purification of 60-kDa protein from cells
trypsinized from cell culture dishes prior to I labeling. Track a shows
I-surface-labeled 60-kDa protein
after purification over a fibrinogen B
chain affinity column and a
C4 reverse phase HPLC column. Cells were harvested prior to
I labeling by gently scraping a subconfluent monolayer of
cells from a roller bottle in the presence of protease inhibitors. Track b shows a repetition of the method used to purify the
60-kDa protein visualized in track a, with the exception that
a subconfluent monolayer of cells was trypsinized from a roller bottle
prior to
I surface labeling. Molecular mass markers are
shown along the left-hand side of the
gel.
Additional experiments were performed to confirm the identity of the
60-kDa protein and to further determine its presence as a cell surface
protein capable of binding to fibrinogen B chain. Fig. 5, track a, shows an autoradiograph of
I-surface-labeled proteins immunoprecipitated with a
polyclonal anti-calreticulin antibody. Fig. 5, track b,
shows a repetition of the immunoprecipitation experiment in Fig. 5, track a, with the exception that cells were
trypsinized prior to radiolabeling. The presence of a clear 60-kDa band
(visualized in Fig. 5, track a) confirms that the
protein isolated from plasma membranes and purified on a fibrinogen
B
chain affinity column is a cell surface form of calreticulin.
Further, the low intensity of the 60-kDa band visualized in Fig. 5, track b, provides additional evidence that the
60-kDa protein is present on the cell surface and is thus sensitive to
trypsin degradation. Tracks c and d of Fig. 5show IgG controls for both scraped and trypsinized cells,
respectively.
Figure 5:
Immunoprecipitation of surface-labeled
calreticulin. Immunoprecipitation of surface-labeled proteins with a
polyclonal anti-calreticulin antibody. Track a,
immunoprecipitation with calreticulin antibodies of I-surface-labeled proteins isolated from cells scraped
from a tissue culture flask in the presence of protease inhibitors
prior to surface labeling. Track b, immunoprecipitation with
calreticulin antibody of
I-surface-labeled proteins from
cells trypsinized prior to
I surface labeling. Tracks
c and d show normal IgG controls for tracks a and b, respectively.
In addition to surface-labeling cells with I, cells were also surface-biotinylated. Fig. 6, track b, shows biotinylated surface calreticulin
immunoprecipitated with anti-calreticulin antibodies. Finally,
calreticulin was visualized by immunostaining of the cell surface with
polyclonal anti-calreticulin antibodies. Fig. 7A shows human
fetal fibroblasts after incubation with anti-calreticulin antibodies.
In contrast, Fig. 7B shows cells incubated with nonimmune
IgG. Calreticulin is clearly visualized in A, and no signal
was visualized with nonimmune IgG controls (B). Surface
biotinylation and immunostaining provided further evidence, using two
alternative surface-labeling technique, that calreticulin is present on
the surface of the plasma membrane.
Figure 6: Immunoprecipitation of biotinylated calreticulin. Track b, immunoprecipitation with calreticulin antibodies of biotinylated cell surface calreticulin (surface proteins were extracted employing the methods described for Fig. 5, track a). Shown along the left-hand side of the figure are molecular mass markers (track a).
Figure 7: Immunostaining of human fetal fibroblasts for surface calreticulin. Human fetal fibroblasts were incubated with polyclonal anti-calreticulin antibodies or nonimmune IgG controls for 20 min at 37 °C in DMEM. Cells were subsequently fixed and signal-visualized with a fluorescein isothiocyanate-conjugated secondary antibody. A and B show fibroblasts incubated with polyclonal anti-calreticulin antibodies and nonimmune IgG controls, respectively.
Figure 8:
Effects of polyclonal calreticulin
antibody on the ability of fibrinogen B chain to stimulate
fibroblast proliferation. Mitogenic effects on human fetal fibroblasts
of fibrinogen B
chain (B
), B
chain +
anti-calreticulin IgG (c) and B
chain + IgG control (i). Antibody dilutions ranged between 1:500 and 1:5000,
B
chain was assayed at a concentration of 2.5
10
M in each of the three studies.
Mitogenic activity was assessed with a colorimetric assay based on the
uptake and subsequent release of methylene blue (see
``Experimental Procedures''). Inhibition of B
chain
activity by immune IgG raised against human calreticulin was
significant at antibody dilutions of 1:500 and 1:1000 as determined by
Duncan's New Multiple Range Test and Scheffe's S test (*, p < 0.05). Control IgG did not significantly inhibit B
chain activity at any of the dilutions
tested.
It is well established that partially degraded fibrin(ogen)
is a fibroblast mitogen(1) , an action which is mediated in
part by sites within the B chains of the molecule(2) . It
remains to characterize receptors expressed by fibroblasts which
interact with fibrinogen B
chain and possibly mediate its
biological functions. The present study isolated a membrane-bound
protein which specifically bound to the B
chain of fibrinogen. The
purified protein displayed an apparent molecular mass of about 60 kDa
as determined by SDS-PAGE analysis. Sequence analysis of the purified
60-kDa protein has shown it to have an amino-terminal sequence
identical with that of calreticulin. The 60-kDa protein was further
identified through immunoprecipitation experiments and
immunohistochemistry employing polyclonal anti-calreticulin antibodies.
Calreticulin has been described by several independent groups as a
calcium-binding protein with a molecular mass of 46 kDa(6) .
The apparent anomaly between the molecular size determined by SDS-PAGE
analysis and molecular size determined from cDNA is thought to be a
consequence of the low isoelectric point of calreticulin which may
result in its slow migration through SDS-PAGE gels(7) . In
addition to the well characterized role of calreticulin as a major
calcium storage protein of the endoplasmic reticulum(8) ,
calreticulin also displays a number of diverse activities which have
direct effects on cell function. For example, a recent study by Burns et al.(9) showed that calreticulin binds to the
DNA-binding domain of the glucocorticoid receptor; an event that
prevented receptor-ligand interaction. These results suggest that
calreticulin may play a direct role in gene transcription by regulating
receptor activity. Additionally, calreticulin binds the highly
conserved KXGFFKR sequence found in the cytoplasmic domain of
all -integrin subunits(10) , thus mediating cell
attachment to the extra cellular matrix(11) . The
KXGFFKR sequence found as a component structure of
-integrin subunits is similar to the sequence found in the
DNA-binding domain of the glucocorticoid receptor. It has been
speculated that these two binding events are coordinated and that
calreticulin may mediate the transduction of signals from integrins to
the nucleus(11) .
Calreticulin was originally thought to be confined to the endoplasmic reticulum (ER) on account of its carboxyl-terminal KDEL sequence; a sequence known to play a role in the retention of proteins within the ER(12) . It has become apparent over the last few years that, although a large proportion of intercellular calreticulin is retained within the ER, it is also found in several other locations. For example, calreticulin is found as a component of the nuclear envelope(13) , it is also associated with component proteins on the cytosolic side of the plasma membrane. Additionally, calreticulin has been identified on the cell surface of human leukocytes, platelets, and endothelial cells(14, 15) . Release of cell surface calreticulin from stimulated neutrophils is thought to play a role in some autoimmune disorders(16) .
Although there have been reports
of extracellular calreticulin (14, 15, 16, 17) , it was considered
important to determine that the calreticulin described in the present
study was expressed on the extracellular side of the plasma membrane.
The first question considered was as follows. Did the I-cell surface-labeling procedure label only surface
proteins? The efficiency of cell surface labeling was verified using
the protease trypsin.
I-labeled 60-kDa protein was not
eluted from a fibrinogen B
chain affinity column when cells had
been incubated with trypsin prior to
I labeling. This
observation suggested that iodination experiments performed in the
present study labeled exclusively proteins expressed on the cell
surface. Additionally,
I-labeled and biotinylated surface
calreticulin was immunoprecipitated with a polyclonal antibody, and a
clear 60-kDa band was observed. However, immunoprecipitation of
calreticulin from cells trypsinized prior to
I-labeling
yielded only a very small quantity of
I-labeled
calreticulin.
Immunostaining of cultured fibroblasts for calreticulin provided further evidence that calreticulin was present as a surface component. In this series of experiments, antibodies were incubated with live cells to ensure that cell membranes were intact and, thus, that only surface calreticulin was labeled.
Finally,
polyclonal antibodies to calreticulin inhibited the mitogenic activity
of fibrinogen B chain on human fetal fibroblasts. These results
suggest the antibody interacts with an epitope on the cell surface,
thus inhibiting cellular proliferation.
Binding specificity of the
B chain of fibrinogen to calreticulin was also considered in the
present study. Calreticulin was eluted from the B
chain affinity
column with EDTA, suggesting a cation-dependent binding event. A
similar requirement for divalent cations has also been described for
the interaction of calreticulin with the
-integrin
subunit(11) . In the present study, we found that calreticulin
was also eluted from a fibrinogen B
chain affinity column with
soluble B
chain fragments indicating a competitive interaction
between bound and free ligand.
The function of fibrinogen B
chain/calreticulin interactions were investigated with purified IgG
raised against human calreticulin. Incorporation of anti-calreticulin
antibodies into the methylene blue cell replication assay significantly
inhibited the mitogenic activity of the fibrinogen B
chain.
Further, IgG controls neither stimulated nor inhibited the mitogenic
effects of the B
chain.
It is becoming apparent that the role of calreticulin is more than simply one of calcium storage. The observation that a form of calreticulin is expressed on the cell surface and has the ability to trigger cell replication is in its self interesting. In the light of the present findings, it is also of interest to note that calreticulin contains a sequence which has the potential for phosphorylation by a number of kinases(18) . It has recently been observed that a simian homologue of human calreticulin is phosphorylated, a function which facilitates its binding to viral RNA(19) . It is now important to determine whether the cell surface form of calreticulin described in this study is in fact phosphorylated or is capable of phosphorylating other protein components.