(Received for publication, November 18, 1994; and in revised form, January 3, 1995)
From the
Proliferation of vascular smooth muscle cells (VSMC) is
triggered by two types of growth factors. One activates tyrosine
kinase-type receptors and the other activates G-protein-coupled
receptors. We found that a conditioned medium of rat VSMC contained a
growth-potentiating activity for the latter type of growth factor, and
we purified a 70-kDa growth-potentiating factor (GPF) from the
conditioned medium. Analyses of GPF and its cDNA revealed GPF to be a
-carboxyglutamic acid-containing protein encoded by a growth
arrest-specific gene, gas6, which related to protein S. GPF
specifically potentiated cell proliferation mediated by
Ca
-mobilizing receptors. The presence of a specific
binding site suggests that the effect of GPF is mediated by a receptor.
Thus, GPF may be a new type of extracellular factor regulating VSMC
proliferation.
Proliferation of vascular smooth muscle cells (VSMC) ()is one of the critical events in intimal thickening of the
vascular wall accompanying atherosclerosis or restenosis. However,
there are still many questions regarding the growth factors involved in
intimal thickening(1) . Two distinct intracellular signaling
pathways induce cell proliferation(2, 3, 4) .
One pathway is activated by receptors that have intrinsic
protein-tyrosine kinases. This type of receptor is activated by
``classical'' growth factors such as epidermal growth factor
(EGF), platelet-derived growth factor, and basic fibroblast growth
factor (bFGF). The other signaling pathway is stimulated by a receptor
group that interacts with heterotrimeric guanine nucleotide binding
proteins (G-proteins). Activated G-protein then stimulates
phospholipase C, resulting in intracellular Ca
mobilization and activation of protein kinase C. This type of
receptor is activated by several factors such as thrombin,
angiotensin-II, or lysophosphatidic acid, which are also candidates for
intimal thickening of the vascular
wall(5, 6, 7) . However, it is not clear why
these two distinct signaling mechanisms lead to the same output, i.e. mitogenesis.
In order to try to answer this question, we compared the characteristics of VSMC proliferation stimulated by these different types of growth factors, using EGF and thrombin as their representatives, and found that VSMC-conditioned medium displayed a growth-potentiating activity that enhanced thrombin-stimulated mitogenesis but not the EGF-stimulated one. Here, we describe the purification, cloning, and characteristics of the growth-potentiating factor (GPF) and also demonstrate the presence of a specific binding site for GPF on the VSMC membrane.
Figure 1:
Time course of EGF- or thrombin-induced
DNA synthesis in VSMC and effect of replacement of culture medium.
Confluent rat VSMCs were cultured for 48 h in DMEM containing 0.1% BSA.
Next, with (brokenlines) or without (solidlines) replacement of the medium with fresh DMEM
containing 0.1% BSA, the cells were stimulated with 0.1 unit/ml
thrombin (), 0.1 nM EGF (
), or vehicle (DMEM
containing 0.1% BSA) (
). The cells were labeled for 2 h with
[
H]thymidine at the indicated
times.
Figure 2:
Purification of GPF. A,
separation of VSMC-conditioned medium by gel filtration chromatography.
Conditioned medium of VSMCs (1.5 liters) was concentrated to 20 ml by
ultrafiltration and separated on a column of Sephacryl S-300. The GPF
activity of each fraction was assayed as described under
``Materials and Methods.'' For the assay, VSMCs were
stimulated with () or without (
) 0.1 unit/ml thrombin in the
presence of 30 µl of each fraction. B,
purification of GPF with gel filtration HPLC. The active peak from
hydroxyapatite chromatography (see ``Materials and Methods'')
was separated on a TSKgel G3,000SW
column, and GPF
activity (
) was assayed as described under ``Materials and
Methods.'' For the assay, VSMCs were stimulated with 0.1 unit/ml
thrombin in the presence of 1 µl of each fraction. C,
purified GPF (0.5 µg) was analyzed by SDS-PAGE in the presence or
absence of 5% 2-mercaptoethanol (2ME). The gel was stained
with Coomassie Brilliant Blue.
One hypothesis for the mechanism of mitogenesis induced by
Ca-mobilizing receptors is that the mitogenesis is
due to secondarily produced tyrosine kinase-activating growth factors.
If this is the case, the mitogenesis induced by
Ca
-mobilizing growth factor should be delayed for
several hours compared with that induced by tyrosine kinase-activating
growth factors(11, 12) . However, as shown by the solidlines in Fig. 1, thymidine incorporation
reached a maximum at 16-18 h with both thrombin stimulation and
EGF stimulation, indicating that thrombin-induced proliferation is not
mediated by secondarily produced ``growth factors'' but by a
direct effect of thrombin stimulation.
In the above experiment, the
cells were serum-starved for 2 days in DMEM, 0.1% BSA and then
stimulated with EGF or thrombin without a change of medium. However,
when the medium was replaced with a fresh one prior to the stimulation,
thrombin-induced [H]thymidine incorporation was
markedly attenuated while the EGF-induced response was not (Fig. 1, brokenlines). This result suggested
that the VSMC-conditioned medium contained a factor that potentiated
thrombin-induced VSMC proliferation but not the EGF-induced response.
A preliminary study using ultrafiltration suggested that a factor
potentiating thrombin-induced proliferation could be recovered in a
fraction larger than 50 kDa (not shown). We thus concentrated the
VSMC-conditioned medium by ultrafiltration and separated it with a gel
filtration column (Sephacryl S-300) (Fig. 2A). The
fractions were assayed for enhancement of
[H]thymidine incorporation in VSMC, in the
presence or absence of thrombin. Two peaks of proliferation-stimulating
activities with distinct characteristics were separated. One of them
was eluted along with a major peak of protein (Fig. 2A, peakb), which stimulated thymidine incorporation by
itself. This peak may be due to VSMC-derived growth factors such as
platelet-derived growth factor, bFGF, or heparin-binding EGF-like
growth factor(13, 14, 15) . The other peak (Fig. 2A, peaka) was eluted earlier
than peakb. It did not stimulate thymidine
incorporation by itself but enhanced the thrombin-induced response.
Thus, peaka was pooled and sequentially
chromatographed on an anion exchange column (Q-Sepharose) and
hydroxyapatite column (see ``Materials and Methods'').
Finally, GPF was purified to homogeneity with a gel filtration column
by HPLC (Fig. 2B). Upon SDS-PAGE, GPF migrated as a
band of about 70 kDa under non-reducing conditions and 90 kDa under
reducing conditions (Fig. 2C). Therefore, GPF appears
to be a monomeric polypeptide containing intramolecular disulfide
bonds. Purified GPF was completely inactivated by incubation with 5
mM dithiothreitol for 2 h, indicating that the intramolecular
disulfide bonds were necessary for its activity. Approximately 100
µg of GPF was purified from 7.5 liters of VSMC-conditioned medium.
Purified GPF enhanced thrombin-induced thymidine incorporation in a
dose-dependent manner with half-maximal stimulation at approximately
0.4 nM (Fig. 3A). Fig. 3B shows that GPF significantly enhanced the thrombin-induced
increase of the cell numbers of VSMC. GPF also enhanced
lysophosphatidic acid- or angiotensin II-induced DNA synthesis but not
the EGF- or bFGF-induced response (Fig. 3C). Thus, GPF
appears to specifically enhance cell proliferation, which is induced by
growth factors activating Ca-mobilizing receptors.
Figure 3:
Characterization of GPF. A,
growth-potentiating activity of purified GPF. The assay for GPF
activity was performed as described under ``Materials and
Methods.'' Various concentrations of purified GPF were added to
VSMCs with () or without (
) 0.1 unit/ml thrombin. B, proliferation of VSMC. Rat VSMCs were plated (2
10
cells/well, 24-well plate) in DMEM, 10% calf serum.
After 4 h, the medium was replaced with DMEM, 0.5% calf serum,
containing vehicle (DMEM, 0.1% BSA), 3 nM GPF, 0.1 unit/ml
thrombin, or GPF plus thrombin. The media were changed every day. Cell
number was counted after 5 days. Data points are mean ± S.D. (n = 4). C, specificity of GPF. VSMCs were
serum-starved for 48 h, and the medium was replaced with a fresh one.
The cells were stimulated with 0.1 unit/ml thrombin, 1 µM angiotensin II (Ang-II), 10 µM lysophosphatidic acid (LPA), 0.1 nM EGF, or 1
nM bFGF in the presence (solidbars) or
absence (openbars) of 3 nM GPF.
GPF was digested with lysyl endopeptidase, and the amino acid
sequences of the isolated peptides were analyzed. Computer-based
comparison (16) of the relatedness of partial amino acid
sequences and the NH-terminal amino acid sequence of rat
GPF suggested that GPF is related to potential products of a murine and
human growth arrest-specific gene (gas6)(8) . This led
us to clone a cDNA encoding rat GPF (Fig. 4). The residue
identities between rat GPF and potential products of human and murine gas6 were 82 and 94%, respectively. As described for gas6 products, GPF shows homology to protein S, with 43% identity
between 674 residues of rat GPF and 676 residues of human protein S
(see (8) for detailed discussion about structure comparison),
which is a negatively regulating factor of blood coagulation and a
vitamin K-dependent protein containing 10
-carboxyglutamic acid
residues/mol of protein(17) . Next, we analyzed the amino acid
composition of GPF after alkaline hydrolysis and detected 11.7 Gla
residues/mol of protein(18) , which indicated that GPF is also
a vitamin K-dependent protein.
Figure 4:
Comparison of sequences of GPF, murine and
human gas6 products and protein S. The predicted amino acid
sequence of GPF (A) contains the NH-terminal
sequence of mature GPF (dotted) and all the amino acid
sequences of peptides obtained (doubleunderlined).
Sequence identities are represented by hyphens, while
different amino acids (a.a.) in murine gas6 product (B), human gas6 product (C), and protein S (D) are shown. Dots represent
deletions.
In order to assess the action
mechanism of GPF, binding assay was performed. Specific binding of I-GPF was detected in both intact VSMC and VSMC membrane
(not shown). Scatchard analysis of the specific binding of
I-GPF to VSMC membrane revealed that the K
value was 0.3 nM and the binding capacity was
approximately 170 fmol/mg protein (Fig. 5). Displacement study
showed that GPF inhibited the binding of 0.3 nM
I-GPF with IC
values of 1.2 nM (not shown). Despite the structural homology, human protein S did
not inhibit the binding of
I-GPF even at 30 nM.
It also did not have growth-potentiating activity. The binding was not
inhibited by EGF, PDGF, or bFGF at 30 nM, but VSMC
proliferation was induced by them. Therefore, the binding appears to be
specific to GPF. Although there is no direct evidence, good
correspondence between the K
value of the binding
and the ED
value of biological function suggests that the
biological action of GPF is mediated by a receptor.
Figure 5:
Scatchard plot of specific I-GPF binding. The membrane fractions of rat VSMC were
incubated with various concentrations of
I-GPF at 4
°C. The specific binding was calculated by subtracting the
nonspecific binding obtained with a 20-fold amount of unlabeled GPF.
Under the postulation that the membrane fraction had a single class of
binding site for GPF, the K
value and B
were 0.3 nM and 170 fmol/mg protein,
respectively.
The gas6 gene was originally cloned as one of the genes whose expression
was up-regulated during serum starvation and down-regulated during
growth induction(8) . Expression of GPF mRNA was regulated in
the same manner in rat VSMC. ()Based on the gene expression
profile, some mitogenesis-related functions were suggested as possible
biological activities of gas6 products. However, there had
been no clear evidence for the biological function of the gas6 product until our present finding of GPF having a
growth-potentiating activity for VSMC. Unlike other growth factors, GPF
only potentiates cell proliferation stimulated by growth factors that
provoke intracellular Ca
mobilization. Since this
type of growth factor was recently hypothesized as being involved in
intimal thickening of the vascular
wall(5, 6, 7) , we speculate that GPF plays a
critical role in the pathogenesis of vascular diseases. Therefore,
inhibition of GPF production or action may be a new way to treat
restenosis or atherosclerosis.
Moreover, since GPF action is
specific to Ca-mobilizing growth factors in VSMC,
intracellular signaling responses induced by GPF, which have not been
clarified yet, may be responses induced only by tyrosine
kinase-activating growth factors and may be essential for initiating
VSMC proliferation induced by Ca
-mobilizing growth
factors. Further analysis of the intracellular signaling process should
provide the key to clarifying the difference between the signaling
mechanisms of these two types of growth factors.