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Biomedical subjects

A Ostman

Publications and source records attributed to A Ostman.

At least 19 recordsLinked to original sources
PubMed1997

Cell scattering and migration induced by autocrine transforming growth factor alpha in human glioma cells in vitro.

In the present investigation, we have transfected a human malignant glioma cell line, U-1242 MG, and derived clones that produce transforming growth factor alpha (TGF-alpha) in an inducible manner using the tetracycline suppressible vector system. TGF-alpha expression was confirmed by Northern analysis, by ELISA, and by immunoprecipitation of metabolically labeled cells. The functional activity of the induced protein was proven by the finding of epidermal growth factor receptor (EGFR) tyrosine phosphorylation on induction of TGF-alpha. A clear effect on cell motility, i.e., cell scattering and an increased phagokinetic track area of individual glioma cells, was demonstrated. The fact that the EGFR tyrosine kinase activation was independent of cell density suggests that autocrine activation of the EGFR kinase occurred at the single-cell level. These findings are of interest, because increased cell motility is most likely a requirement for glioma cell invasion in vivo. The results imply that as a result of coexpression of EGFR and its ligand, individual glioma cells are capable of acting as independent autocrine locomotory units.

Antibodies, Monoclonal↗
PubMed1997

Identification of a 190-kDa vascular endothelial growth factor 165 cell surface binding protein on a human glioma cell line.

Vascular endothelial growth factor (VEGF) is an angiogenesis factor for which two signaling protein tyrosine kinase receptors, Flt1 and KDR, have been identified. We describe here a 190-kDa component present on a human glioma cell line that binds VEGF165 with high affinity. In contrast, VEGF121 is bound only with low affinity, suggesting that the C-terminal part of VEGF165 is important for interaction with the 190-kDa component. No internalization or stimulation of tyrosine phosphorylation was recorded after ligand binding to the 190-kDa component, suggesting that it may not be directly involved in signaling; its function may be to present ligand or stabilize ligand binding to signaling receptors.

Amidohydrolases↗
PubMed1997

Immunoglobulin-like domain 4-mediated receptor-receptor interactions contribute to platelet-derived growth factor-induced receptor dimerization.

Platelet-derived growth factor (PDGF) is a dimeric growth factor that activates its tyrosine kinase receptor by inducing receptor dimerization. In this study, we investigated if receptor-receptor interactions, in addition to ligand-receptor interactions, contribute to the ligand-induced dimerization of the PDGF receptors. Analysis of two deletion mutants of the PDGF alpha-receptor indicated a role for Ig-like domain 4 in ligand-receptor or receptor-receptor interactions. When the fourth Ig-like domain of the PDGF alpha-receptor instead was replaced with the corresponding sequence of the stem cell factor receptor, the binding of PDGF-AA and -BB was not affected, nor was the ability to form homodimeric receptor complexes. This indicates that Ig-like domain 4 does not participate in ligand-receptor interactions. However, the chimeras did not form heterodimers with wild-type PDGF alpha- or beta-receptors. Together, these findings suggest that Ig-like domain 4 mediates specific receptor-receptor interactions. This notion was also supported by the finding that a soluble form of Ig-like domain 4 of the PDGF alpha-receptor acted as a PDGF alpha-receptor antagonist. We conclude that specific receptor-receptor interactions contribute to PDGF receptor dimerization in vivo and that complementary epitopes in Ig-like domain 4 mediate these interactions. Our experiments also identify Ig-like domain 4 as a target for PDGF antagonists.

Animals↗
PubMed1996

Production of cell-associated PDGF-AA by a human sarcoma cell line: evidence for a latent autocrine effect.

The alternative splicing of platelet-derived growth factor A-chain is known to result in 2 different protein products. One variant is encoded by transcripts containing the 69 nts representing exon 6 (PDGF-AA(L)), and one variant is encoded by transcripts in which exon 6 is excluded (PDGF-AA(S). Transfection assays have suggested that the long splice variant of the A-chain is mainly associated with membrane- and matrix-associated heparan sulphate proteoglycans, whereas the shorter variant is soluble. We describe a human sarcoma cell line (U-2197) that expresses a high level of PDGF-A transcripts. Immunoprecipitations revealed cell-associated protein products of mainly 24, 28 and 33 kDa and less abundant forms of 40-45 kDa, while no PDGF was found in the medium. Analysis of extracellular medium in a radioreceptor assay confirmed that PDGF was not secreted by the U-2197 cells. The addition to U-2197 cultures of a carboxy terminal peptide that specifically competes with the binding of the long splice variant of PDGF-AA to extracellular matrix and cell membranes resulted in the release of 3 PDGF-AA-specific dimeric proteins with molecular masses of 33, 37 and 45 kDa. Furthermore, polymerase chain reaction studies discriminating between the long and the short splice variants of the PDGF-A transcripts revealed that U-2197 expressed relatively higher amounts of the long splice variant compared with U-343 MGa Cl 2:6, which is known to secrete PDGF-AA. These cell-associated forms of PDGF, released to the medium by adding carboxy terminal peptide, increased the tyrosine kinase activity of the endogenous PDGF alpha-receptor.

Aged↗
PubMed1996

Inhibitory DNA ligands to platelet-derived growth factor B-chain.

We have identified a group of DNA molecules that bind to platelet-derived growth factor (PDGF)-AB with subnanomolar affinity from a randomized DNA library using in vitro selection. Individual ligands cloned from the affinity-enriched pool bind to PDGF-AB and PDGF-BB with comparably high affinity (Kd approximately 10(-10) M) and to PDGF-AA with lower affinity (> 10(-8) M), indicating specific recognition of the PDGF B-chain in the context of the hetero- or homodimer. The consensus secondary structure motif for most of the high-affinity ligands is a three-way helix junction with a three-nucleotide loop at the branch point. Photo-cross-linking experiments with 5-iodo-2'-deoxyuridine-substituted ligands establish a point contact between a thymidine nucleotide in the helix junction loop region and phenylalanine 84 of the PDGF-B chain. Representative minimal DNA ligands inhibit the binding of 125I-PDGF-BB but not of 125I-PDGF-AA to PDGF alpha- or beta-receptors expressed in porcine aortic endothelial (PAE) cells in a concentration-dependent manner with half-maximal effects of approximately 1 nM. The same ligands also exhibit a similar inhibitory effect on PDGF-BB-dependent [3H]thymidine incorporation in PAE cells expressing the PDGF beta-receptors. These DNA ligands represent a novel class of specific and potent antagonists of PDGF-BB and, by inference, PDGF-AB.

Binding, Competitive↗
PubMed1996

Identification of three amino acid residues in the B-chain of platelet-derived growth factor with different importance for binding to PDGF alpha- and beta-receptors.

The B-chain homodimer isoform of platelet-derived growth factor (PDGF) binds with high affinity both to alpha- and to beta-receptors. In order to localize amino acid residues in PDGF-BB of differential importance for the binding to the two receptors, PDGF-BB mutants were analyzed in which single amino acid residues were changed to alanine residues. We found that Phe-118 in loop 1 of the PDGF B-chain is crucial for binding to both receptors, and that the surrounding amino acids, Asn-117 and Leu-119, appear to be important primarily for binding to the beta-receptor. In contrast, Lys-161 in loop 3 was found to be more important for binding to alpha-receptors than beta-receptors. Previous studies have shown that the receptor binding epitope of PDGF-BB is composed mainly of loops 1 and 3; the findings of the present study show that the alpha- and beta-receptors interact with different amino acid residues in these regions.

Amino Acid Sequence↗
PubMed1996

Protein-tyrosine phosphatase-mediated decrease of epidermal growth factor and platelet-derived growth factor receptor tyrosine phosphorylation in high cell density cultures.

Contact-induced growth inhibition is a characteristic feature of normal cells grown in monolayer. The importance of reversible tyrosine phosphorylation in mitogenic signaling, together with earlier reports of increased levels of protein-tyrosine phosphatases (PTPs) in densely cultured cells, has led to the proposal that PTPs may be involved in mediating contact inhibition of cell growth. We have compared net levels of ligand-induced tyrosine phosphorylation of the epidermal growth factor (EGF) receptor in mink lung epithelial cells cultured under sparse or dense conditions. The levels of net tyrosine phosphorylation of the stimulated EGF receptor was found to be more than 4-fold higher in sparse cultures. This difference was greatly reduced when cells were pretreated with the PTP inhibitor phenyl arsine oxide. Monitoring of dephosphorylation rates in vivo of the stimulated EGF receptors revealed increased EGF receptor-directed PTP activity in dense cultures. The platelet-derived growth factor beta-receptor, expressed in stably transfected porcine aortic endothelial cells, also displayed lower levels of ligand induced net tyrosine phosphorylation in cells from dense cultures. This density-dependent difference in tyrosine phosphorylation was reduced by pretreatment of cultures with the PTP inhibitor orthovanadate. A PTP-mediated decrease of the in vivo net levels of ligand induced tyrosine phosphorylation of EGF and platelet-derived growth factor receptors in cells at high density have thus been demonstrated. Loss of this previously unnoticed regulatory pathway may be involved in cellular transformation.

Animals↗
PubMed1996

Ligand-induced dimerization of growth factor receptors: variations on the theme.

Growth, differentiation, apoptosis and movement of cells are regulated, in part, by polypeptide growth factors, or cytokines, which exert their effects by binding to cell surface receptors on the target cells. Recent observations have indicated that a common mechanism of activation of several classes of such receptors is ligand-induced dimerization or oligomerization of the receptors.

Dimerization↗
PubMed1995

Involvement of loop 2 of platelet-derived growth factor-AA and -BB in receptor binding.

Platelet-derived growth factor (PDGF) is a disulfide-bonded antiparallel dimer of A- and B-polypeptide chains. Each subunit contains two loops (loops 1 and 3) which point in the same direction, and which are located close to a region (loop 2) from the other subunit of the dimer. Previous studies have shown that epitopes in loops 1 and 3 are important for binding to PDGF alpha- and beta-receptors. The aim of the present investigation was to determine the importance of loop 2 for receptor interactions. PDGF A- and B-chain cDNA:s were mutated in the loop 2 regions and transfected into COS cells. Analyses of conditioned media of such cell cultures revealed that PDGF B-chain mutated in the loop 2 region lost its ability to compete with 125I-PDGF for binding to PDGF beta-receptors, but retained 2-5% of its binding of alpha-receptors. The A-chain binds only to alpha-receptors; 2-5% of this binding was also retained after mutation of the loop 2 region. In conclusion, the loop 2 region of PDGF is important for receptor binding, but appears to be more important for binding to the PDGF beta-receptors than to the alpha-receptors.

Amino Acid Sequence↗
PubMed1994

Expression of DEP-1, a receptor-like protein-tyrosine-phosphatase, is enhanced with increasing cell density.

cDNA encoding a receptor-like protein-tyrosine-phosphatase (PTP) termed DEP-1 was isolated from a HeLa cell library. The cDNA predicts an enzyme consisting of an extracellular segment containing eight fibronectin type III repeats, a single transmembrane segment, and a single intracellular PTP domain. Following expression of DEP-1 cDNA in COS cells a glycoprotein of 180 kDa was detected and PTP activity was demonstrated in immunocomplexes with a C-terminal peptide antiserum. Endogenous DEP-1 was detected in WI-38 human embryonic lung fibroblasts by immunoblotting and immunocomplex PTP assays. Immunoblot analysis of DEP-1 expression in WI-38 cells revealed dramatically increased levels and activity of the PTP in dense cultures relative to sparse cultures. Also, DEP-1 activity, detected in PTP assays of immunocomplexes, was increased in dense cell cultures. In contrast, the expression levels of PTP-1B did not change with cell density. This enhancement of DEP-1 expression with increasing cell density was also observed in another fibroblast cell line, AG1518. The increase in DEP-1 occurs gradually with increasing cell contact and is initiated before saturation cell density is reached. These observations suggest that DEP-1 may contribute to the mechanism of contact inhibition of cell growth.

Amino Acid Sequence↗
PubMed1994

Characterization of the retention motif in the C-terminal part of the long splice form of platelet-derived growth factor A-chain.

Platelet-derived growth factor (PDGF) A-chain is found in two different splice variants; the long A-chain variant differs from the short one in that it contains a stretch of basic amino acid residues in the C-terminal part that mediates retention of the growth factor inside the producer cell and to the cell matrix. By analyzing mutants in which different amino acid residues in the retention motif had been changed to alanine residues, we found that the total positive charge of the sequence is of importance for the function of the retention motif. Moreover, we showed that retention also occurs if only one of the polypeptides in the PDGF dimer carries the retention motif. Surface iodination and competition with a peptide having the sequence of the retention motif revealed that the long A-chain variant, in contrast to the short A-chain variant, is localized on the outside of the cells and is also associated to the cell matrix. The association is likely to be mediated partially through heparan sulfate proteoglycans since treatment of matrix with heparitinase released the long A-chain variant.

Alternative Splicing↗
PubMed1994

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 beta-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 (U-343MGa 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 transformed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists.

Antibodies, Monoclonal↗
PubMed1994

Differences in binding to the solid substratum and extracellular matrix may explain isoform-specific paracrine effects of platelet-derived growth factor.

We have studied the paracrine response of fibroblasts to the two homodimeric isoforms of platelet-derived growth factor (PDGF-AA and -BB). CHO-cells stably transfected with a B-chain cDNA expression vector (CHO-PDGF-B cells), were found to elicit a marked paracrine response when seeded at clonal density on preformed monolayers of human or murine fibroblasts; no such response was elicited by CHO-PDGF-A cells. Immunofluorescence microscopy of CHO-PDGF-B cell cultures showed the presence of a pericellular deposit of material reacting with antibodies against PDGF-BB; no corresponding PDGF-AA immunoreactive material was found in the CHO-PDGF-A cultures. The pericellular material, deposited by CHO-PDGF-B cells, was shown to have a growth promoting effect on target cells. Furthermore, we could show that 125I-PDGF-BB binds more efficiently than 125I-PDGF-AA to extracellular matrix prepared from foreskin fibroblast cultures, as well as to defined extracellular matrix components (fibronectin, laminin and collagen III). The results reveal a marked difference in the paracrine activity of PDGF-AA and PDGF-BB; the latter has a strong local growth enhancing effect, that is mostly likely to be ascribed to its association with components of the extracellular matrix.

3T3 Cells↗
PubMed1993

Assignment of intrachain disulfide bonds in platelet-derived growth factor B-chain.

Platelet-derived growth factor (PDGF)-BB is a dimeric protein held together by two disulfide bonds involving the 2nd and 4th cysteine residues from the NH2 terminus. To localize the three intrachain disulfide bonds in PDGF, a method was devised that made it possible to cleave PDGF at specific sites. A set of PDGF derivatives in which specific amino acids were mutated to methionine residues was generated. The recombinant proteins, immunoprecipitated from metabolically labeled transfected COS cells, were then subjected to CNBr cleavage and analyzed by SDS-gel electrophoresis under nonreducing conditions. Based on whether the mutated proteins remained in one piece or fell apart after CNBr cleavage, it was possible to deduce the disulfide bond arrangement in the PDGF B-chain; one bond involves the 1st and 6th cysteine residues, another the 3rd and 7th, and the last the 5th and 8th. The latter disulfide bond was found to be dispensable for receptor binding, whereas the former two were found to be essential for the correct folding or stability of the PDGF B-chain.

Animals↗
PubMed1993

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.

3T3 Cells↗
PubMed1992

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Aminoglycosides↗
PubMed1992

Assignment of interchain disulfide bonds in platelet-derived growth factor (PDGF) and evidence for agonist activity of monomeric PDGF.

Platelet-derived growth factor (PDGF) is a dimeric factor stabilized by disulfide bonds. Using an approach involving partial reduction of PDGF, we have identified the 2nd and 4th cysteine residues in the PDGF chains as the cysteine residues forming interchain disulfide bonds. Analysis of PDGF mutants in which the 2nd and 4th cysteine residues were mutated to serine residues revealed that the disulfide bonds are arranged in a cross-wise manner, with the 2nd cysteine residue in one chain being linked to the 4th cysteine residue in the other. A PDGF B-chain mutant, in which both the 2nd and 4th cysteine residues were substituted with serine residues, migrated as a monomer in sodium dodecyl sulfate gel electrophoresis and retained receptor binding activity. When analyzed in receptor dimerization and autophosphorylation assays, this mutant showed agonistic activity. Thus, structural information has been obtained that will allow the large scale production of properly folded monomeric PDGF, as well as design of specific PDGF heterodimers.

Amino Acid Sequence↗