Platelet-derived growth factor. II. Specific binding to cultured cells

We have prepared radioiodinated purified platelet-derived growth factor (125I-PDGF) which retains full mitogenic activity. The binding of 125I-PDGF to Swiss 3T3 cells is saturable and highly competed by whole blood serum, purified unlabeled PDGF, and by material from each stage in the purification of PDGF from platelet-rich plasma. Other purified mitogens and substances tested do not compete. 125I-PDGF binding to fibroblasts, 3T3 cells, and arterial smooth muscle cells shows an apparent dissociation constant of 10(-11) M, comparable to the range in which PDGF is mitogenic. A clone of Swiss 3T3 cells obtained from a population selected repeatedly against mitogenic response to PDGF shows a greatly reduced mitogenic response to PDGF and binds only 5% as much 125I-PDGF/cell. The binding capacity of the different cell types tested ranges from 2,500 binding sites/cell on the poorly responding variant to 390,000 binding sites/cell on one strain of Swiss 3T3 cells. Cell types that do not respond to PDGF do not show specific high affinity binding of 125I-PDGF. At 4 degrees C, 125I-PDGF binding to monolayer cultures is relatively slow. Equilibrium binding of low concentrations of 125I-PDGF is not achieved during 7 h unless the binding medium is constantly mixed. 125I-PDGF binding at 4 degrees C shows a broad pH optimum between 6.3 and 8.0. Binding does not seem to require Ca2+ or Mg2+ but is reduced more than 6-fold if both monovalent and divalent salts are omitted. The initial rate of 125I-PDGF binding is greater at 37 degrees C than at 4 degrees C but cell-associated 125I begins to decline soon after reaching a peak value at 30-60 min. Coincident with this decline, trichloroacetic acid-soluble 125I appears in the medium and the binding capacity of the cells declines. These phenomena suggest that PDGF and its receptor may be internalized and degraded.     

Platelet-derived growth factor receptors form a high affinity state in membrane preparations. Kinetics and affinity cross-linking studies

The specific binding of 125I-PDGF (platelet-derived growth factor) to intact fibroblasts becomes relatively nondissociable during incubation at 37 degrees C. To characterize the interaction of PDGF with its receptors under conditions in which there is no receptor internalization, we have studied the binding of 125I-PDGF to membrane preparations derived from mouse 3T3 cells and rat liver. The binding sites had the affinity and specificity characteristics expected of PDGF receptors. At 37 degrees C (but not at 4 degrees C) the specific binding of 125I-PDGF to membranes gradually became nondissociable as assessed by either dilution or by addition of excess unlabeled PDGF. This tight binding was not due to a covalent interaction since the polyanionic compound suramin readily dissociated specifically bound 125I-PDGF. This property of suramin was used to expose rat liver PDGF receptors which were occupied by endogenous PDGF. Affinity cross-linking studies demonstrated that the formation of the nondissociable state of 125I-PDGF binding was associated with the binding of 125I-PDGF to a 160,000-dalton protein and to a 110,000-dalton species. The cross-linked binding sites could be adsorbed to wheat germ agglutinin and to anion exchange resins. The isoelectric point of both cross-linked species determined by two-dimensional gel electrophoresis was approximately 4.7. These data demonstrate that in membrane preparations, PDGF binds to an anionic 160,000-dalton glycoprotein which is likely to be the receptor. A high affinity state of PDGF binding, which is formed rapidly at 37 degrees C, can be dissociated by suramin.     

Interactions between the receptors for platelet-derived growth factor and epidermal growth factor

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.     

Growth factor modulation of melanoma growth stimulatory activity mRNA expression in human malignant melanoma cells correlates with cell growth

This report demonstrates that the expression of melanoma growth stimulatory activity (MGSA) mRNA can be modulated in a positive fashion in the Hs294T human melanoma cell line by PDGF and MGSA. There is close correlation between MGSA expression and the pattern of cell growth in Hs294T cells.     

Recombinant expression, biochemical characterization, and biological activities of the human MGSA/gro protein

Melanoma growth stimulatory activity (MGSA) is a mitogenic protein secreted by Hs294T melanoma cells that corresponds to the polypeptide encoded by the human gro gene. The MGSA/gro cDNA has been expressed in mammalian cells and the secreted recombinant factor has been purified. Biochemical and biological characterization shows that the recombinant protein is identical with the natural protein and is devoid of posttranslational glycosylation, sulfation, and phosphorylation. The two C-terminal amino acids are proteolytically removed from the mature recombinant MGSA, indicating a length of 71 instead of the predicted 73 amino acids. The recombinant MGSA is mitogenically active on the Hs294T melanoma cells. The purified MGSA competes with interleukin 8 for binding to neutrophil receptors and exhibits neutrophil chemotactic activity equivalent to that of interleukin 8.     

Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to beta-thromboglobulin.

Melanoma growth stimulatory activity (MGSA) is a mitogenic polypeptide secreted by Hs294T human melanoma cells. Comparison of the N-terminal sequences of the 13 and 16 kd MGSA species with the cDNA sequence revealed that the mature form of human MGSA is maximally 73 amino acids long. Expression of the cDNA in mammalian cells results in the secretion of this peptide with mitogenic activity. MGSA is structurally related to the platelet-derived beta-thromboglobulin and to several other polypeptides. These factors may constitute a family of growth factors. MGSA mRNA was detected in a variety of cell types. The level of MGSA mRNA in melanoma cells is strongly elevated by treatment with MGSA. MGSA is the gene product of a recently detected gene gro. The gene was mapped to chromosome 4 (region q13----q21). This same region also contains genes for two of the structurally related factors, for c-kit, a receptor for an as yet unidentified ligand, and for 'piebald trait', an inherited skin pigmentation disorder.     

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.     

A significant part of macrophage-derived growth factor consists of at least two forms of PDGF

The macrophage has been suggested to be responsible for the connective tissue cell proliferation that accompanies most chronic inflammatory responses. One of the secretory products of activated macrophages is MDGF, a growth factor (or factors) for fibroblasts, 3T3 cells, smooth muscle, and vascular endothelium. This report demonstrates that a significant portion of the mitogenic activity for 3T3 cells secreted by cultured human alveolar and peritoneal macrophages is due to a molecule (or molecules) similar to platelet-derived growth factor (PDGF). Two size classes (approximately 37,000-39,000 and 12,000-17,000 daltons) of mitogenically active PDGF-like molecules are detected by two criteria--antigenic similarity with PDGF and ability to compete with 125I-PDGF for high-affinity binding to the PDGF receptor. The presence of mRNA for the B chain of PDGF is demonstrated by Northern analysis, and de novo synthesis of these molecules by activated macrophages is shown by immunoprecipitation of 35S-labeled proteins with anti-PDGF IgG.     

Suramin binds to platelet-derived growth factor and inhibits its biological activity

The polyanion suramin was recently found to inhibit binding of 125I-PDGF (platelet-derived growth factor) to Balb/c 3T3 cell membranes. Cultured Swiss 3T3 cells were used to investigate the mode of action of suramin and to monitor its effect on the biological activity of PDGF. Evidence is presented that suramin inhibits cellular binding of PDGF by binding to PDGF itself, thereby preventing it from binding to its cell surface receptor: First, while suramin inhibited 125I-PDGF binding with a half maximum inhibition concentration of approximately 60 microM or 90 micrograms/ml in a simultaneous competition assay, it was inactive in a sequential radioreceptor assay, in which an inhibitor is expected to be active if it interacts with the receptor (even with relatively low affinity) but to be inactive if it interacts with PDGF. Second, suramin prevented immunoprecipitation of 125I-PDGF in a dose-dependent manner, with a half maximum effective concentration of approximately 50 microM. Furthermore, suramin efficiently dissociated 125I-PDGF bound to its cell surface receptor, whereas unlabeled PDGF even in large excess was virtually inactive. This is also in line with the proposed direct interaction between PDGF and suramin, since such an interaction can be envisaged to induce a conformational change in the PDGF-receptor complex, resulting in an increased off-rate of the complex. Reduced 125I-PDGF binding in the presence of suramin correlated directly with a suramin dose-dependent inhibition of PDGF-induced incorporation of 3H-thymidine into quiescent Swiss 3T3 cells and of the proliferation of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytolytic T cells recognize the two amino-terminal domains of H-2 K antigens in tandem in influenza A infected cells

Simian sarcoma virus-transformed NIH 3T3 (SSV-NIH 3T3) and SSV-NRK cells secrete a potent growth-promoting activity identical with the platelet-derived growth factor (PDGF) in mitogenic assays. The secreted activity is blocked by anti-PDGF antisera and competes with 125I-PDGF for receptor binding, suggesting that the secreted protein is the transforming protein of SSV, p28v-sis, or its processed product. Secreted p28v-sis appears to stimulate autocrine cell growth of SSV-transformed cells because anti-PDGF antisera block 3H-thymidine incorporation into growing SSV-NIH 3T3 and SSV-NRK cells. SSV-transformed cells have reduced numbers of high-affinity 125I-PDGF receptors; PDGF/p28v-sis receptor was purified from SSV-NIH 3T3 cells and retained active protein tyrosine kinase activity stimulated by PDGF. The rate of tumor growth in athymic nude mice injected with SSV-transformed cells was compared with levels of secreted growth factor activity. The rate of tumor growth in nude mice correlated directly with levels of p28v-sis secreted by SSV-transformed cells.     

Monoclonal antibody C3.1 is a platelet derived growth factor (PDGF) antagonist

A monoclonal antibody C3.1 raised against NR6 cells and partially purified PDGF receptor blocked PDGF stimulated [3H] thymidine incorporation in Swiss mouse 3T3 cells and immunoprecipitated a 180 kDa phosphoprotein from NR6 cells. The phosphoprotein bound to a C3.1 sepharose 4B affinity matrix and could be specifically eluted with PDGF but not by EGF or basic FGF. These preliminary results suggests that the ability of C3.1 to inhibit PDGF stimulated mitogenesis may be due to its direct or allosteric interaction at the PDGF receptor binding site.     

Cultured endothelial cells do not respond to a platelet-derived growth-factor-like protein in an autocrine manner

Cultured endothelial cells produce a growth factor similar or identical to platelet-derived growth factor (PDGF). Endothelial cells are able to proliferate in plasma-supplemented medium, while most nontransformed cells require serum-supplemented medium. Since PDGF is a major serum mitogen, we have tested the possibility that endothelial cells interact with and respond to the autologously produced PDGF-like (PDGF-c) protein. We have found that bovine aortic and rat heart endothelial cells express little or no cell surface PDGF receptors as determined by binding of pure 125I-PDGF. Treating these cells under acidic conditions, which release receptor-bound PDGF in control cells without affecting receptor function, did not reveal a population of cryptic receptors. In addition, when rat heart endothelial cells were grown in the presence of an antibody to PDGF, proliferation was unimpaired, though no detectable free PDGF was present in the medium. An equivalent amount of antibody completely blocked the mitogenic response of human fibroblasts that had been preincubated for 1 h at 37 degrees C with an equivalent dose of PDGF. Thus, endothelial cells do not respond mitogenically in a manner that would be expected from the interaction of autologously produced PDGF with its cell surface receptor. Endothelial cells were detergent-solubilized and immobilized on nitrocellulose in an attempt to detect the presence of intracellular PDGF receptors. Specific binding of 125I-PDGF to adsorbed, solubilized bovine aortic or rat heart endothelial cells was undetectable, though significant binding to adsorbed, solubilized fibroblasts, used as a positive control, was observed. We conclude that endothelial cells do not have detectable intracellular PDGF receptors.     

Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor

Two monoclonal antibodies against the receptor for platelet-derived growth factor (PDGF) were obtained by immunizing mice with pure PDGF receptor preparations derived from porcine uterus. The antibodies, denoted PDGFR-B1 and PDGFR-B2, both bound to the external domain of the receptor, as demonstrated by indirect immunofluorescence and binding of 125I-labeled antibodies to intact human fibroblasts. Both antibodies precipitated pure 175-kDa 32P-labeled autophosphorylated porcine PDGF receptor as well as a Mr 175,000 glycoprotein from metabolically labeled cells. The monoclonal antibodies did not inhibit binding of 125I-PDGF to human fibroblasts and did not stimulate these cells to undergo mitosis. Both antibodies induced clustering and down-regulation of their antigen. However, this resulted in only a partial loss of cell surface binding sites for PDGF itself, consistent with the conclusion that the monoclonals recognized only one of two or several receptors for PDGF. Clustering and down-regulation were not seen when the cells were incubated with monovalent Fab' fragments of the PDGFR-B2 antibody. The antibodies also stimulated autophosphorylation of pure PDGF receptor, and PDGFR-B2 was shown to stimulate phosphorylation of phosphofructokinase, an exogenous substrate for the PDGF receptor kinase. High concentrations of PDGFR-B2 antibody, or Fab' fragments thereof, failed to enhance the PDGF receptor kinase activity, compatible with the possibility that dimerization was of importance in the antibody-stimulated kinase activity of purified PDGF receptors.     

Melanoma growth stimulatory activity: isolation from human melanoma tumors and characterization of tissue distribution

Melanoma growth stimulatory activity (MGSA) is an acid and heat stable, auto-stimulatory growth factor which was first isolated from culture medium conditioned by the Hs294T human melanoma cell line. In this report, we describe the purification of MGSA from acid ethanol extracts of Hs294T tumors grown in nude mice using a series of Bio-Gel P30, reverse phase-high performance liquid chromatography and heparin-sepharose steps. This modified procedure provides a 10-fold improved yield of MGSA over previously published procedures. Purified MGSA-stimulated melanoma cell growth in both 3H-thymidine and cell number assays over a concentration range of 0.06 to 6 ng/ml. The MGSA bioactivity was primarily associated with fractions which exhibited molecular weights of 16 and 13-14 Kd based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), transforming growth factor-beta (TGF beta), and epidermal growth factor (EGF) in combination with TGF beta did not stimulate 3H-thymidine incorporation in Hs294T cells under the conditions used for MGSA bioassay. Monoclonal antibody to MGSA was used to screen melanoma and benign nevus cultures as well as fixed sectioned tissue for MGSA. The majority of the melanoma cultures were MGSA positive, while most nevus cultures were MGSA negative. However, when fixed sectioned tissue was screened for MGSA immunoreactivity, melanoma tissue was MGSA positive and three-fourths of the benign nevi were MGSA positive. In addition, epidermal keratinocytes and several tissues exhibiting proliferative disorders contained immunoreactive MGSA. These data suggest that MGSA may be a normal regulator of growth and that the microenvironment of the cell may regulate both production of MGSA and response to MGSA.     

DNA synthesis in U-2 OS human osteosarcoma cells is independent of PDGF binding to functional cell surface receptors

Previous studies have shown that suramin reveals specific PDGF binding sites on U-2 OS human osteosarcoma cells. Studies presented here indicate that U-2 OS cells pretreated with suramin internalize and degrade 125I-PDGF and respond to PDGF by increased tyrosine kinase activity and amino acid transport. However, DNA synthesis in these cells is not reduced by incubation with the PDGF blocking agent suramin and is not stimulated by exogenous PDGF. These data indicate that U-2 OS cells possess functional PDGF receptors but that high levels of DNA synthesis in these cells is unrelated to the binding of secreted PDGF to these cell surface receptors. Thus, it is unlikely that the PDGF mitogen produced by U-2 OS cells stimulates proliferation through an autocrine mechanism involving secretion and subsequent binding to PDGF receptors.     

Basic fibroblast growth factor modulates the mitogenic potency of the platelet-derived growth factor (PDGF) isoforms by specific upregulation of the PDGF alpha receptor in vascular smooth muscle cells.

Platelet-derived growth factor AA (PDGF AA), in contrast to PDGF AB and BB, is a poor mitogen for smooth muscle cells (SMC). However, together with basic fibroblast growth factor (bFGF) it acts synergistically on DNA synthesis of these cells. Northern blot analysis revealed that bFGF selectively increases the PDGF-receptor alpha subtype (PDGF-R alpha) mRNA level without a significant effect on the PDGF-R beta mRNA level. The amount of PDGF-R alpha protein is also selectively increased after stimulating SMC with bFGF as shown by immunoprecipitation of lysates from SMC with anti-PDGF-R alpha antibodies. The number of binding sites for 125I-PDGF AA is more than doubled after bFGF-treatment, whereas the specific binding for PDGF AB and BB increased only by approximately 30 and 20%, respectively. The increase in the number of PDGF-R alpha renders the SMC responsive for PDGF AA as demonstrated by the induction of the proto-oncogene c-fos as well as by an increased cell proliferation. The enhanced PDGF binding after bFGF treatment may in fact explain the observed synergistic behavior. These data are discussed with regard to a possible role of growth factor-induced transmodulation of receptor expression during atherogenesis.     

Kinetics of 125I-PDGF binding and down-regulation of PDGF receptor in human arterial smooth muscle cell strains during cellular senescence in vitro

Platelet-derived growth factor (PDGF) is one of the major mitogens in serum to stimulate replication of human smooth muscle cells (SMCs) in culture. Previous studies using human fibroblasts failed to demonstrate changes in the receptor systems for growth factors during cellular senescence. We investigated the kinetics of ¹²⁵I-PDGF(-BB) binding and down-regulation of the PDGF receptor in three human arterial SMC strains during cellular aging. The number of specific ¹²⁵I-PDGF binding sites per cell increased slightly at a population doubling level (PDL) of 60%–80% of life span and then decreased at the PDL above 90%. The number of receptors per cell-surface area decreased with increasing in vitro age. The apparent Kd for the ¹²⁵I-PDGF binding decreased with in vitro senescence. The internalization and degradation of ¹²⁵I-PDGF per receptor were significantly reduced in senescent SMCs than young cells. Furthermore, down-regulation of the PDGF receptor was significantly greater in sensescent SMCs than young cells. Immunoblot studies demonstrated that changes in b?-subunit of the PDGF receptor accounted for those in the studies using ¹²⁵I-PDGF and that tyrosine phosphorylation of the PDGF receptor was significantly greater in young SMCs than aged cells. Our results suggest that age-related changes in the receptor systems for PDGF may be important contributors to the failure of DNA synthesis in senescent SMCs. © 1995 Wiley-Liss, Inc.     

Recombinant interferon-gamma inhibits the mitogenic effect of platelet-derived growth factor at a level distal to the growth factor receptor

Highly purified preparations of recombinant human interferons (rIFNs)-alpha A, -beta, and -gamma all inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis in normal human dermal fibroblasts, as monitored by incorporation of [3H]-thymidine into trichloroacetic acid (TCA)-insoluble material. rIFN-gamma was the most potent, since it blocked the PDGF response by 50% at about 10 U/ml or 0.3 ng/ml, whereas with rIFN-alpha A and rIFN-beta 4000 U/ml and 600 U/ml, respectively (10 ng/ml in both cases), were required to achieve the same effect. There was a close parallelism between the ability of these rIFNs to inhibit PDGF mitogenic activity and their capacity to inhibit cell proliferation in serum-containing medium. None of the rIFNs inhibited specific binding of 125I-PDGF to fibroblasts, and none interfered with receptor internalization. The mechanism of action of rIFN-gamma was analyzed further. rIFN-gamma did not inhibit uptake of [3H]-thymidine into these cells. However, it shifted if the time point of initiation of DNA synthesis from about 14 h after stimulation with PDGF to about 18 to 21 h and decreased significantly the rate of the DNA synthesis. rIFN-gamma could be added up to 6 h following stimulation with PDGF with no loss of its inhibitory effect. rIFN-gamma also blocked the mitogenic activity of epidermal growth factor and basic fibroblast growth factor. Taken together these results implicate that rIFN-gamma exerts its antimitogenic effect by inhibiting a process that occurs late in the PDGF signaling pathway and onto which the activity pathways of other mitogens converge. In view of the important role PDGF may play in wound-healing and in the pathogenesis of the proliferative lesions of arteriosclerosis, these data point to a possible role IFN-gamma may play as a regulator of these processes in vivo.     

A naturally occurring secreted form of fibroblast growth factor (FGF) receptor 1 binds basic FGF in preference over acidic FGF.

Alternatively spliced variants of fibroblast growth factor receptor 1 mRNA are predicted to encode secreted forms and membrane-bound forms of receptors. The predicted amino acid sequences of these receptor variants differ in a portion of the extracellular region. In this study, we characterized the function of one of these splice variants which was predicted by its cDNA to be a secreted FGF receptor. We expressed this secreted form of the human FGFR1 (sFGFR1) in Chinese hamster ovary cells. The sFGFR1 protein oligomerized upon ligand binding. Surprisingly, the sFGFR1 preferentially bound basic FGF over acidic FGF. In cross-linking experiments, the sFGFR1 showed higher binding affinity for basic FGF (Kd approximately 30 nM) than for acidic FGF (Kd greater than 300 nM). These results suggest that this secreted form of FGF receptor has an unusual ligand binding specificity that may be important for its biological role in vivo.