A glioma-derived PDGF A chain homodimer has different functional activities from a PDGF AB heterodimer purified from human platelets

Glioma-derived growth factor I (GDGF-I) is structurally similar to a platelet-derived growth factor (PDGF) A chain homodimer, whereas PDGF purified from human platelets is a heterodimer of one A and one B chain. Binding experiments revealed that GDGF-I and PDGF bound to a common receptor on human fibroblasts, but also suggested the presence of a second receptor type recognizing only PDGF. In contrast to PDGF, GDGF-I had only a limited mitogenic activity, a low ability to stimulate receptor autophosphorylation and actin reorganization, and no chemotactic activity. GDGF-I did, however, cause transmodulation of EGF receptors, suggesting that it, like PDGF, activates protein kinase C in fibroblasts. These data indicate that different PDGF-like growth factors have different functional activities, which are possibly mediated via different receptors.     

A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain

Platelet-derived growth factor, PDGF, purified from human platelets is a disulfide-bonded dimer consisting of two homologous polypeptide chains denoted A and B; it has not been known whether it is a heterodimer or a mixture of homodimers. We present here evidence that a major part of PDGF has a heterodimer structure. A highly homogeneous, 31-kDa PDGF was purified in the presence of protease inhibitors and shown to contain both chains by means of immunoprecipitations with peptide antisera specific for the A and B chains, respectively. The susceptibility of PDGF to mild acid treatment and its chromatographic behavior in reversed-phase high performance liquid chromatography and immobilized metal ion affinity chromatography, as compared to A and B chain homodimers, is consistent with a heterodimer structure. Analysis of PDGF purified according to our routine, large scale procedure revealed the major part to have a heterodimer structure. In addition, B chain homodimers were also found. With the demonstration that a major part of PDGF purified from human platelets occurs as a heterodimer, all three dimeric forms of PDGF have been identified. The following nomenclature to distinguish the various forms is suggested: PDGF-AA, a homodimer of A chains; PDGF-AB, a heterodimer; PDGF-BB, a homodimer of B chains; PDGF, any dimeric form of A or B chains.     

The B chain of PDGF alone is sufficient for mitogenesis.

The platelet-derived growth factor (PDGF) is a mitogen derived from human platelets consisting of two related polypeptides termed A and B chains. The entire B chain of PDGF is highly (96%) homologous to a portion of p28sis, the transforming protein of simian sarcoma virus. It has been suggested that p28sis exerts its transforming potential by mimicking the growth promoting activity of PDGF and stimulating the cell in an autocrine manner. We have directly examined the mitogenic potential of p28sis and the B chain homologous region by expressing these heterologous sequences in the yeast Saccharomyces cerevisiae. In our constructions, these proteins are encoded by portions of the v-sis gene. Expression and secretion from the yeast cell is achieved by using a yeast promoter and the alpha-factor pheromone secretory leader. The sis proteins thus expressed and secreted are immunoreactive with anti-PDGF antisera and are mitogenic for cultured fibroblasts. Furthermore, they mediate this mitogenic activity by specific binding to the PDGF cell surface receptor. Gel electrophoresis and cell binding analysis indicates that the mitogenic species is primarily a disulphide-bonded dimer. We are able to conclude that p28sis is a mitogen and that a polypeptide corresponding to the B chain alone is sufficient to account for the mitogenic activity attributed to PDGF.     

The A-type receptor for platelet-derived growth factor mediates protein tyrosine phosphorylation, receptor transmodulation and a mitogenic response.

A clonal human glioma cell line, U-343 MGa 31L, which expresses the A-type but not the B-type receptor for platelet-derived growth factor (PDGF), was used in a functional study of the A-type receptor. PDGF-AA induced, in a dose- and time-dependent manner, phosphorylation on tyrosine residues of the receptor in metabolically labelled cells. The optimal dose was around 30 ng/ml; at 100 ng/ml, phosphorylation was maximal at 15 min and had almost returned to the control level after 60 min. The phosphorylation on tyrosine residues of the PDGF A-type receptor was stimulated by PDGF-AA, PDGF-AB and PDGF-BB; these isoforms also stimulated [3H]thymidine incorporation into U-343 MGa 31L cells. In addition, activation of the A-type PDGF receptor induced transmodulation of the epidermal growth factor receptor.     

Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10

Human umbilical vein endothelial (HUVE) cells have been previously reported to express the genes for the A and B chains of PDGF and to secrete PDGF-related factors into culture media. Antihuman PDGF IgG affinity chromatography was used to purify PDGF-related activity from HUVE cell-conditioned media. Immunoblot analysis of the affinity-purified proteins with anti-PDGF IgG and antibodies specific for the A or B chain peptides of PDGF combined with chemotactic and mitogenic assays revealed that the major PDGF immunorelated molecule secreted by HUVE cells is a monomer of approximately 36-38 kD and that less than 10% of the purified biologically active molecules are PDGF A or B chain peptides. Screening of an HUVE cell cDNA library in the expression vector lambda gtl 1 with the anti-PDGF antibody resulted in the cloning and sequencing of a cDNA with an open reading frame encoding a 38-kD cysteine-rich secreted protein which we show to be the major PDGF-related mitogen secreted by human vascular endothelial cells. The protein has a 45% overall homology to the translation product of the v-src-induced CEF-10 mRNA from chick embryo fibroblasts. We have termed this new mitogen connective tissue growth factor.     

Affinity purification and characterization of CIF, an insect immunoresponsive factor with NF-kappa B-like properties.

1. A rapid DNA affinity purification procedure was worked out for the purification of the Cecropia Immunoresponsive Factor (CIF) from the pupae of Hyalophora cecropia. 2. CIF consists of a single polypeptide chain of 65 kDa and is present as a homodimer under native conditions. 3. CIF binds to the kappa B-like sequences upstream of the H. cecropia immune genes with the following order of affinity: attacin kappa B greater than lysozyme kappa B greater than cecropin A kappa B greater than cecropin B kappa B. 4. The purified CIF also strongly binds to the kappa B sequences from both the immunoglobulin kappa light chain gene and the MHC class I gene. 5. The DNA binding of CIF can be inhibited by antisera directed against NF-kappa B-related proteins. 6. The cytoplasmic factor Cl, co-purified from the affinity column, contains two polypeptide chains, one of which has the same molecular weight as CIF.     

Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli

Preparations of the mitogen platelet-derived growth factor (PDGF) from human platelets contain two related polypeptides termed A chain and B chain. PDGF-B is highly homologous to a portion of p28v-sis, the transforming protein of simian sarcoma virus. We have studied the mitogenic potential of a PDGF-BB-like homodimer by expressing the sequence coding for the mature part of PDGF-B in Escherichia coli. Expression was achieved as cro-beta-gal-PDGF-B fusion protein which was exclusively found in the "inclusion bodies". A monomeric PDGF-B fragment shortened by 12 amino acid residues from the NH2 terminus was excised from the fusion protein by CNBr cleavage. After protection of thiols by S-sulfonation, this fragment was purified by gel permeation chromatography and reversed-phase high-performance liquid chromatography. This monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active rPDGF-BB with an overall yield of approximately 0.7 mg of rPDGF-BB/L of culture. Escherichia coli rPDGF-BB stimulated [3H]thymidine incorporation into AKR2B fibroblast at concentrations of about 1 ng/mL.     

Actions of platelet-derived growth factor isoforms in mesangial cells

Platelet-derived growth factor (PDGF) occurs as homodimers or heterodimers of related polypeptide chains PDGF-BB, -AA, and -AB. There are two receptors that bind PDGF, termed alpha and beta. The beta receptor recognizes PDGF B chain and is dimerized in response to PDGF BB. The alpha receptor recognizes PDGF B as well as A chains and can be dimerized by the three dimeric forms of PDGF AA, AB, and BB. To characterize PDGF receptor signaling mechanisms and biologic activities in human mesangial cells (MC), we explored the effects of the three PDGF isoforms on DNA synthesis, phospholipase C activation, and PDGF protooncogene induction. PDGF-BB homodimer and AB heterodimer induced a marked increase in DNA synthesis, activation of phsopholipase C, and autoinduction of PDGF A and B chain mRNAs, whereas PDGF-AA homodimer was without effect. The lack of response to PDGF AA could be accounted for by down regulation of the PDGF-alpha receptor since preincubation of MC with suramin restored PDGF AA-induced DNA synthesis. Ligand binding studies demonstrate specific binding of labeled PDGF BB and AB and to a lower extent PDGF AA isoforms to mesangial cells. These results are consistent with predominant expression of PDGF beta receptor in MC, which is linked to phospholipase-C activation. The potent biologic effects of PDGF-AB heterodimer in cells that express very few alpha receptors and do not respond to PDGF AA are somewhat inconsistent with the currently accepted model of PDGF receptor interaction and suggest the presence of additional mechanisms for PDGF isoform binding and activation. © 1994 Wiley-Liss, Inc.     

Purification of a monocyte chemotactic factor secreted by nonhuman primate vascular cells in culture

A protein chemotactic for peripheral blood monocytes (SMC-CF) of potential importance in their recruitment to the arterial intima in atherogenesis was purified from serum-free medium conditioned by cultured baboon aortic medial smooth muscle cells. The purification of SMC-CF was monitored by a filter assay using human peripheral blood mononuclear cells and was achieved by batch separation on a cation-exchange gel followed by gel permeation chromatography, ion-exchange high-performance liquid chromatography (HPLC), and reversed-phase HPLC. The overall recovery was approximately 10% of the initial activity and yielded 0.5-1 microgram of SMC-CF/L of conditioned medium. On analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SMC-CF migrated as a monomeric protein with an apparent molecular weight of 14,500. A dose-dependent relationship was observed between SMC-CF concentration and monocyte chemotactic activity, with maximal and half-maximal biologic activity being observed at approximately 5 and 0.1 nM, respectively. Cultured baboon aortic smooth muscle cells also express the genes for both the A and B polypeptide chains of platelet-derived growth factor, which has been reported to be chemotactic for blood monocytes and neutrophils [Deuel, T. F., Senior, R. M., Huang, J. S., & Griffin, G. L. (1982) J. Clin. Invest. 69, 1046-1049]. Amino acid composition analyses indicate that SMC-CF is not derived either from polypeptide chain of this growth factor or from certain potentially chemotactic connective tissue proteins.     

Effects of platelet-derived growth factor on bone formation in vitro

Platelet-derived growth factor (PDGF) is a polypeptide found in a variety of tissues, including bone, where it could act as an autologous regulator of skeletal remodeling. Therefore, a recombinant B chain homodimer of human PDGF was studied for its effects on bone formation in cultured rat calvariae. PDGF at 10-100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to sixfold and increased the DNA content and the number of colcemid-induced metaphase arrested cells. This effect was observed in the fibroblast and precursor cell-rich periosteum. As a result of its mitogenic actions, PDGF enhanced [3H]proline incorporation into collagen, an effect that was observed primarily in the osteoblast-rich central bone. The effect of PDGF was not specific for collagen since it also increased noncollagen protein synthesis. In addition, PDGF increased bone collagen degradation. PDGF and insulin-like growth factor (IGF) I had additive effects on calvarial DNA synthesis, but PDGF opposed the stimulatory effect of IGF I on collagen synthesis and IGF I prevented the PDGF effect on collagen degradation. In conclusion, PDGF stimulates calvarial DNA synthesis which causes an increased number of collagen-synthesizing cells, but PDGF also enhances bone collagen degradation.     

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.     

Rat epidermal growth factor: complete amino acid sequence. Homology with the corresponding murine and human proteins; isolation of a form truncated at both ends with full in vitro biological activity

Epidermal growth factor (EGF) isolated from the submaxillary gland of the rat (rEGF) is missing the COOH-terminal five residues present in both mouse and human EGF. rEGF competes for the binding of 125I-labelled mEGF to human carcinoma cells with the same affinity as mEGF. rEGF and mEGF have identical mitogenic activities on mouse 3T3 fibroblasts, thus the C-terminal region of the sequence is not necessary for the in vitro activity of EGF. Using reversed-phase high-performance liquid chromatography, four molecular forms of EGF have been extracted from rat submaxillary glands. These forms represent rEGF, rEGF(2-48), rEGF(3-48) and rEGF(4-48); all forms appear to be equipotent in both the receptor binding and mitogenic assays. The isoelectric points of these rEGFs are in the range of pH 5.1 to 5.2. The primary structure of rEGF was determined from approximately 10 micrograms protein by sequence analysis of the intact molecule and fragments obtained from the reduced and alkylated protein by chemical cleavage with CNBr and enzymic cleavage with chymotrypsin and a proline-specific endopeptidase. Subnanomole amounts of generated peptides were purified to homogeneity by reversed-phase microbore high-performance liquid chromatography and analysed by automated Edman degradation in a gas-phase sequencer. There are 48 amino acid residues in the complete polypeptide chain which lacks alanine, phenylalanine, lysine and tryptophan. The amino acid sequence of rat epidermal growth factor is: Asn-Ser-Asn-Thr-Gly-Cys-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-Cys-Leu-Asn- Gly-Gly-Val-Cys-Met-Tyr-Val-Glu-Ser-Val-Asp-Arg-Tyr-Val-Cys-Asn-Cys -Val-Ile-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Gln-His-Arg-Asp-Leu-Arg. The calculated relative molecular mass from the sequence analysis is 5377.     

Extracellular matrix is a source of mitogenically active platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a chemotactic and mitogenic agent for fibroblasts and smooth muscle cells and plays a key role in the development of atherosclerotic lesions. PDGF is produced by a number of normal and transformed cell types and occurs as homo- or heterodimers of A and B polypeptide chains. Using Chinese hamster ovary (CHO) cells transfected with various forms of PDGF, we have previously shown that PDGF A_s (short splice version) is secreted, PDGF A_L (long splice version) predominantly extracellular matrix-associated, and PDGF B divided between medium, cells, and matrix. In the present study we have demonstrated the mitogenic activity of matrix-localized PDGF in artificial and more physiologically relevant models by culturing Balb/c-3T3 cells (3T3), human foreskin fibroblasts (HFF), and rabbit aortic smooth muscle cells (SMC) on extracellular matrix (ECM) laid down by PDGF-expressing CHO cells and human umbilical vein endothelial cells (HUVEC). These cells responded to the local growth stimulus of PDGF-containing CHO ECM and HUVEC ECM. We showed that 3T3 cells required proteolytic activity to utilize matrix-localized PDGF, as aprotinin and η-ACA inhibited growth and 3T3 cells were shown to possess plasminogen activator activity. HFF and SMC did not appear to require proteolytic activity (including metalloproteinase and serine protease activity) as a prerequisite for mitogenesis but were able to access immobilized PDGF by contact with the matrix. An understanding of the mechanisms whereby the utilization of stored PDGF is controlled in situations of excessive cellular proliferation will aid in the development of therapy for these conditions. © 1996 Wiley-Liss, Inc.     

A small v-sis/platelet-derived growth factor (PDGF) B-protein domain in which subtle conformational changes abrogate PDGF receptor interaction and transforming activity.

Deletion scanning mutagenesis within the transforming region of the v-sis oncogene was used to dissect structure-function relationships. Mutations affecting codons within a domain encoding amino acids 136 through 148 had no effect upon homodimer formation or recognition by antisera which detect determinants dependent upon native intrachain disulfide linkages, yet the same mutations completely abolished transforming activity. A platelet-derived growth factor B (PDGF B) monoclonal antibody that prevents its interaction with PDGF receptors recognized v-sis, delta 142 (deletion of codon 142), and delta 148 but not delta 136, delta 137, or delta 139 mutants. These findings mapped the epitope recognized by this monoclonal antibody to include amino acid residues 136 to 139. Furthermore, mutations in the codon 136 to 148 domain caused markedly impaired ability to induce PDGF receptor tyrosine phosphorylation. Thus, subtle conformational alterations in this small domain critically affect PDGF receptor recognition and/or functional activation.     

Cultured endothelial cells produce multiple growth factors for connective tissue cells

Cultured bovine aortic endothelial cells (BAEC) secrete into their medium a growth-promoting factor that stimulates many connective tissue cells in culture. We now report that this growth-promoting activity is due to at least two different proteins which are biochemically separable and immunologically distinct. Cation exchange chromatography (Carboxymethyl-Sephadex) of concentrated BAEC-conditioned medium yields two major peaks of growth-promoting activity which adsorb at pH 8 and elute with a salt gradient. One of these peaks contains as well a protein that inhibits the binding of radioiodinated platelet-derived growth factor (PDGF) to its receptor on target cells. The PDGF-like mitogen is purified approx. 25-fold by this chromatographic step. A second peak of mitogenic activity exhibits no binding to the PDGF receptor. Both the PDGF-like mitogenic activity and the PDGF-distinct mitogenic activity are highly cationic, stable to boiling, sensitive to beta-mercaptoethanol, and between 30 and 50 kD in molecular weight. Complementary studies with human umbilical vein endothelial cells in culture were performed. These human cells also produce both growth-promoting activity and a protein that binds to the PDGF receptor. The latter activity is greatly inhibited by a specific antiserum to human PDGF, whereas the growth-promoting activity of the conditioned medium is minimally affected. The degree of inhibition of the two activities is, however, quantitatively consistent: 3.5 ng of PDGF-like activity in the radioreceptor assay is inhibited, while 5 ng of PDGF-like activity in the DNA synthesis assay is inhibited. The data from the two species are consistent with the proposal that cultured endothelial cells produce at least two distinct mitogens, one of which is biochemically and immunologically related to PDGF.     

PDGF A chain homodimers drive proliferation of bipotential (O-2A) glial progenitor cells in the developing rat optic nerve.

The bipotential glial progenitor cells (O-2A progenitors), which during development of the rat optic nerve give rise to oligodendrocytes and type 2 astrocytes, are stimulated to divide in culture by platelet-derived growth factor (PDGF), and there is evidence that PDGF is important for development of the O-2A cell lineage in vivo. We have visualized PDGF mRNA in the rat optic nerve by in situ hybridization, and its spatial distribution is compatible with the idea that type 1 astrocytes are the major source of PDGF in the nerve. We can detect mRNA encoding the A chain, but not the B chain of PDGF in the brain and optic nerve, suggesting that the major form of PDGF in the central nervous system is a homodimer of A chains (PDGF-AA). PDGF-AA is a more potent mitogen for O-2A progenitor cells than is PDGF-BB, while the reverse is true for human or rat fibroblasts. Fibroblasts display two types of PDGF receptors, type A receptors which bind to all three dimeric isoforms of PDGF, and type B receptors which bind PDGF-BB and PDGF-AB, but have low affinity for PDGF-AA. Our results suggest that O-2A progenitor cells possess predominantly type A receptors, and proliferate during development in response to PDGF-AA secreted by type 1 astrocytes.     

Activated platelet-derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line

Human glioblastoma cells (A172) were found to concomitantly express PDGF-BB and PDGF β-receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up-regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U343MGa 31L) or on Ki-ras-transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transtormed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists. © 1994 Wiley-Liss, Inc.