©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Vascular Smooth Muscle Cell-derived, Gla-containing Growth-potentiating Factor for Ca-mobilizing Growth Factors (*)

(Received for publication, November 18, 1994; and in revised form, January 3, 1995)

Toru Nakano (§) Ken-ichi Higashino Norihisa Kikuchi Junji Kishino Koji Nomura Hiroko Fujita Osamu Ohara (¶) Hitoshi Arita

From the Shionogi Research Laboratories, Shionogi & Co., Ltd., 5-12-4 Sagisu, Fukushima-ku, Osaka 553, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Proliferation of vascular smooth muscle cells (VSMC) (^1)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.


MATERIALS AND METHODS

Time Course of DNA Synthesis in Thrombin- or EGF-stimulated VSMCs

Rat VSMCs were plated in 24-well tissue culture plates and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum. At confluence, the cells were made quiescent by incubation for 48 h in DMEM containing 0.1% bovine serum albumin (BSA). Next, with or without the culture medium replaced with fresh DMEM containing 0.1% BSA, the cells were stimulated with 0.1 unit/ml thrombin or 0.1 nM EGF. The cells were labeled with [^3H]thymidine (0.5 µCi/well) for 2 h at the times indicated in Fig. 1. Incorporation of [^3H]thymidine was measured as trichloroacetic acid-insoluble radioactivity in the cells.


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 (bullet), 0.1 nM EGF (box), or vehicle (DMEM containing 0.1% BSA) (up triangle). The cells were labeled for 2 h with [^3H]thymidine at the indicated times.



Assay for GPF Activity

Confluent rat VSMCs in 24-well tissue culture plates were cultured for 48 h in DMEM containing 0.1% BSA. Prior to the assay, the culture medium was replaced with a fresh one to remove GPF from the medium. The cells were then stimulated with 0.1 unit/ml thrombin in the presence of the samples. Eighteen hours later, the cells were labeled with [^3H]thymidine (0.5 µCi/well) for 2 h, and the incorporation of [^3H]thymidine was measured as the trichloroacetic acid-insoluble radioactivity in the cells.

Purification of GPF

Confluent rat VSMCs in 15-cm tissue culture dishes were cultured for 2 days in 25 ml of DMEM with 0.1% BSA. Conditioned medium was filtered with a 0.2-µm filter and concentrated by ultrafiltration using a P0200 ultrafilter membrane (ADVANTEC, Japan). Approximately 1.5 liters of the conditioned medium was concentrated to 20 ml and separated with a Sephacryl S-300 gel filtration column (2.5 times 110 cm, Pharmacia Biotech Inc.) (Fig. 1). The column was equilibrated with 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl and eluted at a flow rate of 10 ml/h. Each 5-ml fraction was collected, and 30-µl aliquots of each fraction were assayed for GPF activity. Active fractions (Fig. 2A, peaka) from four runs of Sephacryl S-300 chromatography were pooled, dialyzed against 10 mM Tris-HCl, pH 8.3, 0.01% Tween 20 and applied to a column of Q-Sepharose HP (25 ml, Pharmacia) equilibrated with the same buffer. The column was eluted with a gradient of 0-0.5 M NaCl in the same buffer (600 ml) at a flow rate of 180 ml/h. GPF activity was eluted as a single peak at 0.32 M NaCl (data not shown). The active fractions were dialyzed against 10 mM sodium phosphate, pH 6.8, 0.01% Tween 20 and applied to a hydroxyapatite column (Econo-Pac HTP cartridge, 5 ml; Bio-Rad) equilibrated with the same buffer. The column was eluted with a gradient of 10-400 mM sodium phosphate, pH 6.8, 0.01% Tween 20 (80 ml) at a flow rate of 0.5 ml/min. A single peak of GPF activity was eluted at 150 mM sodium phosphate (data not shown). The active fractions were concentrated with a Centricon-50 concentrator (Amicon) to 0.1 ml. The samples were then applied to TSK gel G3,000SW columns ((7.8 mm times 30 cm) times 2, linked in tandem, Tosoh, Japan). The columns were equilibrated with 10 mM sodium phosphate, pH 6.8, 0.4 M NaCl, 0.01% n-octyl-beta-D-thioglucoside and eluted at a flow rate of 0.5 ml/min. Each 0.25-ml fraction was collected, and 1-µl aliquots of each fraction were tested for GPF activity.


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 (bullet) or without (circle) 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 (bullet) 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.



Cloning of Rat GPF cDNA

Purified GPF was subjected to NH(2)-terminal analysis with a protein sequencer (Applied Biosystems 477A). For determination of the internal amino acid sequence of GPF, the purified GPF was digested with lysyl endopeptidase (Wako Chemicals, Japan), and the fragments were separated by HPLC on a C(8) column. Each isolated fragment was analyzed with the protein sequencer. The partial amino acid sequences suggested that GPF might be related to potential products of a murine and human growth arrest-specific gene (gas6)(8) . Therefore, we designed two oligonucleotide primers corresponding to nucleotide positions 1276-1295 (5`-CCATCAACCACGGCATGTGG-3`) and 1845-1864 (5`-TCGCACACCTTGATTTCCAT-3`) of mouse gas6 cDNA(8) . Polymerase chain reaction was performed using these primers and rat VSMC cDNA, and an amplified product of the expected size was purified on an agarose gel. Using the cDNA fragments thus obtained as a probe, the cDNA library constructed from rat VSMC was screened by colony hybridization. Positive clones containing cDNA encoding GPF were isolated and sequenced in both strands. The nucleotide sequence data for rat GPF can be found in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data bases with accession number D42148.

Binding Assays

Purified GPF was iodinated by the chloramine-T method(9) . Membrane fractions of cultured rat VSMC were prepared as described elsewhere(10) . The binding experiments in the membrane fractions were carried out at 4 °C in a total volume of 0.2 ml of the incubation buffer (10 mM Tris-HCl, pH 7.4, 10 mM CaCl(2), and 0.25% BSA) with I-GPF (5,500 cpm/fmol) at the indicated concentration. After a 2-h incubation period, ice-cold phosphate-buffered saline was added, and the reaction mixture was immediately filtered by suction through a Whatman GF/C glass filter. The filter was then washed three times with ice-cold phosphate-buffered saline and analyzed for radioactivity. The specific binding is defined as the difference between binding in the presence and absence of the unlabeled GPF (20-fold amount of added I-GPF in each tube).


RESULTS AND DISCUSSION

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 [^3H]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 [^3H]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 (box) 0.1 unit/ml thrombin. B, proliferation of VSMC. Rat VSMCs were plated (2 times 10^4 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(2)-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(2)-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(d) 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 nMI-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(d) 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(max) 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. (^2)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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-6-458-5861; Fax: 81-6-458-0987.

Present address: Laboratory of DNA Technology, Kazusa DNA Research Inst., 1532-3 Yana-Uchino, Kisarazu-shi, Chiba 292, Japan.

(^1)
The abbreviations used are: VSMC, vascular smooth muscle cell; EGF, epidermal growth factor; bFGF, basic fibroblast growth factor; G-protein, heterotrimeric guanine nucleotide binding protein; GPF, growth-potentiating factor; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis.

(^2)
H. Fujita and T. Nakano, unpublished observations.


ACKNOWLEDGEMENTS

We thank Ayako Terawaki and Miho Okuyama for excellent technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.