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C H Heldin

Publications and source records attributed to C H Heldin.

At least 19 recordsLinked to original sources
PubMed1992

Platelet-derived endothelial cell growth factor. Pharmacokinetics, organ distribution and degradation after intravenous administration in rats.

Platelet-derived endothelial cell growth factor (PD-ECGF) stimulates chemotaxis of endothelial cells in vitro and has angiogenic activity in vivo. Recently PD-ECGF was shown to have thymidine phosphorylase activity. In order to study possible therapeutic applications of PD-ECGF we used a rat model to determine its pharmacokinetics and tissue distribution after intravenous injection. [125I]PD-ECGF disappeared from the plasma in a biphasic manner, with estimated distribution and elimination half-lives of 17 min and 7 h, respectively. PD-ECGF was metabolized in the liver, excreted via the bile, and not accumulated in any organ system. The stability and long half-life in the circulation, together with the specificity for endothelial cells, suggest that PD-ECGF may be useful as a therapeutic agent to stimulate re-endothelialization in vivo, or, in view of its thymidine phosphorylase activity, in chemotherapy, by decreasing the pool of available thymidine.

Animals↗
PubMed1992

Transforming growth factor-beta 1, -beta 2, and -beta 3 secreted by a human glioblastoma cell line. Identification of small and different forms of large latent complexes.

Transforming growth factor-beta 1 (TGF-beta 1) has been found to occur as latent high molecular weight complexes, with or without an associated component denoted latent TGF-beta 1-binding protein (LTBP). We show here that a human glioblastoma cell line (U-1240 MG) secretes all isoforms of TGF-beta s found in mammalian cells (TGF-beta 1, -beta 2, and -beta 3). Approximately 26% of the secreted TGF-beta is in an active form. Latent TGF-beta s were partially purified from medium conditioned by the U-1240 MG cell line using anion exchange chromatography. Analysis of the different fractions by immunoblotting using antisera against precursor parts of the different TGF-beta isoforms, and against LTBP, revealed that not only TGF-beta 1 but also other isoforms of TGF-beta may occur in high molecular weight forms containing LTBP. In addition, each one of the TGF-beta isoforms occurred in smaller forms not containing LTBP. Interestingly, each of the TGF-beta isoforms was also seen in complexes of about 210 kDa containing associated component(s) distinct from LTBP. These results indicate that each of the different isoforms of TGF-beta is synthesized and secreted by this glioblastoma cell line in several different high molecular weight latent forms; the biological importance of the various latent TGF-beta complexes is discussed.

Carrier Proteins↗
PubMed1992

Identification of a peptide antagonist for platelet-derived growth factor.

A series of peptides derived from the primary sequence of the B-chain of platelet-derived growth factor (PDGF) was analyzed for their ability to inhibit the binding of 125I-PDGF-AA and 125I-PDGF-BB to PDGF alpha-receptors and PDGF beta-receptors, respectively. A 13-amino acid peptide (ANFLVWEIVRKKP), corresponding to amino acids 116-121 and 157-163 in PDGF B-chain, was found to compete with binding to both alpha- and beta-receptors. Modification of this peptide on the tryptophan residue increased its receptor competing activity. The peptide was found to be a receptor antagonist, since it inhibited dimerization and autophosphorylation of PDGF receptors. When analyzed on intact cells, the peptide was found to have, in addition to the specific inhibitory effect at the receptor level, a nonspecific inhibitory effect on [3H]thymidine incorporation. Our study has identified two regions in PDGF that are of importance for receptor interaction.

Amino Acid Sequence↗
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↗
PubMed1992

Platelet-derived growth factor and its receptors in human glioma tissue: expression of messenger RNA and protein suggests the presence of autocrine and paracrine loops.

The expression of platelet-derived growth factor (PDGF) and its receptors was analyzed in 14 gliomas of various degrees of malignancy and compared with three gliosis cases by in situ hybridization and immunohistochemistry techniques. Expression of both PDGF A- and B-chains was higher in glioblastomas than in astrocytomas. The PDGF A-chain mRNA was predominantly found in cell-rich areas in glioblastomas. The cognate PDGF-alpha receptor (PDGFR-alpha) mRNA was heterogeneously distributed in gliomas of all grades, and PDGFR-alpha expression was higher in gliomas than in gliosis. Within some glioblastomas probed with PDGFR-alpha complementary RNA, cells heavily loaded with grains were intermingled with others containing low or moderate signals. The heavily labeled cells were often found in the vicinity of proliferating capillaries. Immunostaining with an anti-PDGF antibody and an affinity-purified antiserum against the PDGFR-alpha showed strong staining of most tumor cells with both antibodies in glioblastoma. In addition, the PDGFR-alpha antibodies yielded a strong staining of scattered cells, and the anti-PDGF antibody yielded staining of a few cells within the astrocytoma. Furthermore, high levels of the PDGF-beta receptor (PDGFR-beta) and PDGF B-chain mRNA as well as the beta receptor protein were found in hyperplastic capillaries. These results suggest the presence of autocrine and paracrine loops in glioma, activating the PDGFR-alpha in glioma cells and the PDGFR-beta in endothelial cells.

Adolescent↗
PubMed1992

Platelet-derived endothelial cell growth factor has thymidine phosphorylase activity.

Platelet-derived endothelial cell growth factor (PD-ECGF), a protein which stimulates angiogenesis in vivo, is shown to have a 39.2% amino acid sequence similarity over a 439 amino acid region with the thymidine phosphorylase of Escherichia coli (E. coli). Using recombinant human PD-ECGF, we show that PD-ECGF has thymidine phosphorylase activity. Analysis by gel chromatography revealed that recombinant human PD-ECGF occurs as a 90 kDa homodimer, similar to other thymidine phosphorylases. In addition to a possible effect on DNA synthesis, PD-ECGF was shown to affect [3H]thymidine assays in a manner which is not related to cell proliferation. The in vitro and in vivo effects of PD-ECGF may thus occur by an indirect mechanism through its enzymatic activity.

Amino Acid Sequence↗
PubMed1992

Ligand-induced polyubiquitination of the platelet-derived growth factor beta-receptor.

We have analyzed the nature of ligand-induced shift to higher molecular weight forms of the beta-receptor for platelet-derived growth factor expressed in porcine aortic endothelial cells. The modification of the beta-receptor was found to be due to polyubiquitination, as judged by immunoblotting using an anti-ubiquitin antiserum. A mutant beta-receptor made kinase negative by a point mutation (K634A mutant) did not undergo ubiquitination in response to ligand stimulation. A mutant in which carboxyl-terminal 98 amino acids were deleted (CT98 mutant) and which retained kinase activity was likewise not ubiquitinated. These data suggest that the kinase activity, as well as the carboxyl-terminal 98 amino acids, is required for ubiquitination of the beta-receptor. Ligand-induced degradation of the receptor-bound ligand, as well as of the receptor itself, was partially impaired in the CT98-receptor-expressing cells, suggesting that the ubiquitination is of importance for efficient degradation of the ligand-receptor complex.

Animals↗
PubMed1992

Retention of the transforming growth factor-beta 1 precursor in the Golgi complex in a latent endoglycosidase H-sensitive form.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized as a latent high molecular weight complex in a human erythroleukemia cell line, HEL, treated with phorbol 12-myristate 13-acetate. The complex is comprised of three components: mature TGF-beta 1, the TGF-beta 1 latency-associated peptide (beta 1-LAP), and the latent TGF-beta 1-binding protein (LTBP). LTBP plays an important role in the assembly and secretion of the latent TGF-beta 1 complex; if the TGF-beta 1 precursor fails to bind to LTBP, much of it remains inside the cells and may contain anomalous disulfide bond(s) between beta 1-LAP and the mature TGF-beta 1 molecule (Miyazono, K., Olofsson, A., Colosetti, P., and Heldin, C.-H. (1991) EMBO J. 10, 1091-1101). In the present work, we have investigated the subcellular localization and properties of the TGF-beta 1 precursor retained intracellularly. When the HEL cells were metabolically labeled and chased for up to 72 h, a considerable part of the TGF-beta 1 precursor was still observed intracellularly in an unprocessed form. The secreted form of the TGF-beta 1 precursor was resistant to endoglycosidase H, whereas the intracellular form of the TGF-beta 1 precursor was sensitive to endoglycosidase H, regardless of the presence or absence of swainsonine, an inhibitor of mannosidase II. Indirect immunofluorescence microscopy revealed that the TGF-beta 1 precursor co-localized with mannosidase II, a marker for the Golgi complex, but not with protein disulfide isomerase, a marker for the endoplasmic reticulum. The intracellular TGF-beta 1 precursor was prepared from phorbol 12-myristate 13-acetate-treated HEL cells and tested for TGF-beta 1 bioactivity. Half-maximal inhibition of the DNA synthesis in mink lung epithelial cells, Mv1Lu, was observed at 80 pM of the acid-treated TGF-beta 1 precursor, whereas nontreated material showed minimal growth inhibitory activity. Taken together, these results indicate that the TGF-beta 1 precursor is retained inside the cells in the Golgi complex, mainly in a latent, immature form.

Animals↗
PubMed1992

Expression of platelet-derived growth factor and its receptors in neuroendocrine tumors of the digestive system.

Carcinoid tumors are slowly growing neuroendocrine neoplasms which often present pronounced fibrosis around the tumor cells. We have previously shown by immunohistochemistry that carcinoid tumors express platelet-derived growth factor (PDGF) beta-receptors on surrounding stromal cells. In this report, 22 midgut carcinoids and 5 endocrine pancreatic tumors were examined for the presence of PDGF with a monoclonal antibody raised against a peptide corresponding to a part of the B-chain of PDGF which reacts strongly with the B-chain and weakly with the A-chain. They were also examined for PDGF alpha-receptors with an affinity-purified polyclonal peptide antibody and for PDGF beta-receptor with the monoclonal antibody PDGFR-B2. PDGF was expressed on tumor cells and on adjacent stroma. PDGF alpha-receptor was seen on clusters of tumor cells and occasionally on adjacent stroma, whereas beta-receptors were seen only in the stroma. Tissue sections from some of these midgut carcinoids were also investigated by in situ hybridization for mRNA of PDGF A- and B-chains as well as alpha- and beta-receptors. By in situ hybridization, abundant expression of mRNA for PDGF beta-receptor and PDGF A-chain was observed in stromal cells adjacent to carcinoid tumor cell clusters, but the mRNA expression in the tumor cells themselves was at a low level. A few clustered tumor cells and stromal cells expressed mRNA for the PDGF alpha-receptor, thus consolidating the immunohistochemical findings. mRNA for the PDGF B-chain was detected in both tumor cells and stroma, but only at low levels. Our data suggest that PDGF is involved in the growth stimulation of the carcinoid tumor cells in an autocrine fashion and in the stimulation of stromal cell growth through paracrine and possibly autocrine mechanisms. Moreover, remarkably strong immunostaining of PDGF and the PDGF alpha-receptor was seen on peripheral nerve fibers.

Adult↗
PubMed1992

Expression of platelet-derived growth factor-beta receptors on human fibroblasts. Regulation by recombinant platelet-derived growth factor-BB, IL-1, and tumor necrosis factor-alpha.

Stimulation of human fibroblasts by platelet-derived growth factor (PDGF)-BB leads to a down-regulation of PDGF beta-receptors and a concomitant appearance of intracellular granular accumulations of receptors, as determined by stainings with the mAb PDGFR-B2. The granules contained both the ligand and PDGF beta-receptors, as revealed by double-immunofluorescence staining, and were formed in response to PDGF-BB but not in response to other cytokines tested. The formation of intracellular PDGF beta-receptor granules was dependent on PDGF-BB concentration and time of stimulation. The granular PDGF beta-receptor staining on cells treated with PDGF-BB for 1 h at 37 degrees C was used to investigate the effects of macrophage-derived cytokines on PDGF beta-receptor expression. The number of PDGF beta-receptor granules was found to be reduced in fibroblasts grown for 48 h in the presence of PDGF-BB, TNF-alpha, or IL-1; PDGF-AA under the same conditions had no effect. The reduction observed was paralleled by a decrease in cell surface expression of PDGF beta-receptors, measured as binding of 125I-PDGF-BB and of the PDGFR-B2 antibody. Furthermore, both TNF-alpha and IL-1 decreased the detergent-extractable pool of PDGF-beta receptors in the fibroblasts, as revealed by immunoblotting of detergent cell extracts. Finally, the decrease in PDGF beta-receptors after culturing of the cells in the presence of TNF-alpha and IL-1 was accompanied by a decreased incorporation of [3H]thymidine in response to PDGF-BB stimulation. In conclusion, our data suggest that certain macrophage-derived cytokines can modulate the expression of PDGF beta-receptors by cultured fibroblasts, which may contribute in part to their reduced responsiveness to PDGF.

Cells, Cultured↗
PubMed1992

PDGF alpha- and beta-receptors activate unique and common signal transduction pathways.

We have examined the signal transduction pathways of the PDGF alpha- and beta-receptors, in order to characterize the specificity of each receptor type in the signaling. Porcine aortic endothelial cell lines expressing equal levels of either PDGF alpha- or beta-receptors were established. The alpha- and beta-receptor cells responded mitogenically to stimulation with the proper PDGF isoforms. Three aspects of actin reorganization were examined after ligand stimulation: loss of stress fibres, appearance of edge ruffles and formation of circular membrane ruffles. The beta-receptor cells showed a response to ligand stimulation which included all three features. The alpha-receptor cells exhibited edge ruffles and loss of stress fibres, but circular ruffles could not be found in several independent alpha-receptor cell lines. The beta-receptor cells, but not the alpha-receptor cells, were able to migrate chemotactically towards a concentration gradient of ligand. The molecular basis for the differences in signalling were explored by comparing the pattern of increased phosphorylation of potential substrates for the alpha- and beta-receptors in [32P]orthophosphate labelled intact cells and using an in vitro kinase assay. Certain of the observed substrates were common for the two receptors, whereas others were specific for either one. We conclude that certain of the known PDGF induced cellular effects are transduced only by the beta-receptor; the presence of alpha-receptor-specific substrates suggests that there are also alpha-receptor-specific signals, which have yet to be identified.

Actins↗
PubMed1992

Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma.

Two novel sites of autophosphorylation were localized to the C-terminal tail of the PDGF beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) compared to the wild type PDGF beta-receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC-gamma could be detected. These data show that tyrosine phosphorylation of PLC-gamma is dependent on autophosphorylation of the PDGF beta-receptor at Tyr1009 and Tyr1021.

Amino Acid Sequence↗
PubMed1992

Effects of homodimeric isoforms of platelet-derived growth factor (PDGF-AA and PDGF-BB) on wound healing in rat.

Platelet-derived growth factor (PDGF) has been suggested to have a significant role in wound healing. The present work was aimed at studying the effects of PDGF-AA and PDGF-BB homodimers on developing granulation tissue in rats. Subcutaneously implanted hollow cylindrical cellulose sponges were used as an inductive matrix for the ingrowth of granulation tissue. Fifty microliters of solutions containing 0, 5, 50, or 500 ng of PDGF-AA or PDGF-BB homodimers was injected daily into the sponges; 7 days after implantation the granulation tissue in the sponge cylinders was analyzed. Five hundred nanograms of PDGF-BB stimulated significantly the accumulation of collagen, indicated by the elevated hydroxyproline content of the sponge (+34%, P < 0.001). Similarly, the amounts of RNA-ribose, nitrogen, hexosamines, and uronic acids were significantly higher, reflecting a PDGF-BB-induced increase in the accumulation of RNA, protein, and glycosaminoglycans. Analyses of wound fluid showed no essential changes in the composition of different cell types after PDGF-BB-treatment. The PDGF-AA-treatment increased significantly the mean amount of RNA but there were no significant changes in other parameters. In vitro both PDGF-AA and PDGF-BB increased significantly the number of rat granulation tissue derived fibroblasts in culture at concentrations of 10 and 30 ng/ml. This proliferative effect resulted in a lowered level of protein synthesis per cell. To conclude, PDGF-BB accelerates granulation tissue formation both in vitro and in vivo, whereas PDGF-AA is effective in vitro but it is clearly less effective in vivo.

Animals↗
PubMed1992

Hydrocortisone-induced increase of PDGF beta-receptor expression in a human malignant mesothelioma cell line.

The effect of hydrocortisone (HC) on PDGF beta-receptor expression was studied in the human malignant mesothelioma cell line Mero-14. HC was found to induce a time- and dose-dependent increase of PDGF beta-receptor mRNA. Nuclear run off analysis revealed that HC induced increased transcription of the PDGF beta-receptor gene. The expression of PDGF beta-receptor protein was also elevated by HC as demonstrated with an immunoblotting assay. However, the number of PDGF-BB binding sites on the cell surface of Mero-14 remained unchanged upon HC treatment. These results suggest that steroid hormones can regulate PDGF receptor expression in vivo.

Binding Sites↗
PubMed1992

PDGF-AA and PDGF-BB biosynthesis: proprotein processing in the Golgi complex and lysosomal degradation of PDGF-BB retained intracellularly.

Platelet-derived growth factor is a potent mitogen for cells of mesenchymal origin. It is made up of two polypeptide chains (A and B) combined in three disulfide-linked dimeric forms (AA, AB, and BB). Here, the biosynthesis and proteolytic processing of the two homodimeric forms of PDGF (AA and BB) were studied in CHO cells stably transfected with A-chain (short splice version) or B-chain cDNA. PDGF-AA was processed to a 30-kD molecule which was secreted from the cells. In contrast, PDGF-BB formed two structurally distinct end products; a minor secreted 30-kD form and a major cell-associated 24-kD form. Immunocytochemical studies at light- and electron-microscopical levels revealed presence of PDGF in the Golgi complex, in lysosomes, and to a smaller extent in the ER. From analysis of cells treated with brefeldin A, an inhibitor of ER to Golgi transport, it was concluded that dimerization occurs in the ER, whereas the proteolytic processing of PDGF-AA and PDGF-BB precursors normally occurs in a compartment distal to the ER. Exposure of the cultures to the lysosomal inhibitor chloroquine led to an increased cellular accumulation of PDGF-BB, as determined both by metabolic labeling experiments and immunocytochemical methods, indicating that the retained form of PDGF-BB is normally degraded in lysosomes. Structural analysis of the two end products of PDGF-BB revealed that the secreted 30-kD form is a dimer of peptides processed as the B-chain of PDGF purified from human platelets, and that the retained 24-kD form is made up of subunits additionally processed in the NH2-terminus. Also, the 24-kD form was shown to be composed of proteolytic fragments held together by disulfide bridges. Taken together these findings suggest that the newly synthesized PDGF A- and B-chains are dimerized in the ER and thereafter transferred to the Golgi complex for proteolytic processing. From there, PDGF-AA is carried in vesicles to the cell surface for release extracellularly by exocytosis. A smaller part of PDGF-BB (the 30-kD form) is handled in a similar way, whereas the major part (the 24-kD form) is generated by additional proteolysis in the Golgi complex, from which it is slowly carried over to lysosomes for degradation.

Animals↗