PubMed Health⌕ Search

Biomedical subjects

B Westermark

Publications and source records attributed to B Westermark.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1992

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protein was found in the malignant tumors, and also in some benign lesions, such as dermatofibroma. In all these cases, benign as well as malignant, the PDGF B-chain mRNA, and less clearly, the PDGF A-chain mRNA, were coexpressed with the beta-receptor. In contrast, high expression of PDGF alpha-receptor mRNA was only found in fully malignant lesions, i.e., malignant fibrous histiocytoma. These data indicate that an autocrine growth stimulation via the PDGF beta-receptor could occur in an early phase of tumorigenesis, and may be a necessary but insufficient event for the progression into fully malignant human connective tissue lesions.

Alcian Blue↗
PubMed1992

Platelet-derived growth factor potentiates phorbol ester-induced neuronal differentiation of human neuroblastoma cells.

Previous studies have shown that platelet-derived growth factor (PDGF) and PDGF receptors are expressed in the mammalian central nervous system and that primary cultured neuroblasts from rat hindbrain have functional PDGF beta-receptors. Here, it is shown that cultured human neuroblastoma cells express PDGF alpha- and beta-receptors, but not PDGF-A and PDGF-B chain mRNA. In contrast to alpha-receptor expression, beta-receptor expression appears to be associated with a mature neuronal phenotype. Under serum-free growth conditions, PDGF-AA and -BB induce a trophic and weak mitogenic response in SH-SY5Y neuroblastoma cells, showing that the PDGF receptors in these cells are functional. In combination with 12-O-tetradecanoylphorbol-13-acetate, all three PDGF isoforms induce sympathetic neuronal differentiation of the SH-SY5Y cells, as shown by morphology and by increased expression of the genes coding for growth-associated protein 43 and neuropeptide tyrosine, respectively. This indicates a potential role for PDGF in the development of sympathetic neurons in particular and of the nervous system in general.

Cell Differentiation↗
PubMed1991

Induction of platelet-derived growth factor alpha- and beta-receptor mRNA and protein by platelet-derived growth factor BB.

We describe that stimulation of human fibroblasts with platelet-derived growth factor BB (PDGF-BB) induces a transient up-regulation of the PDGF alpha- and beta-receptor transcript and protein levels. The effect of PDGF-BB on the receptor transcript levels was more pronounced than those seen when other cytokines were used. Regulation of transcript levels by PDGF-BB was mediated through post-transcriptional mechanisms. No induction could be observed in a nuclear run-on analysis, but cycloheximide treatment attenuated the accumulation of both alpha- and beta-receptor transcripts induced by PDGF-BB. An increase in receptor protein levels was observed using two different experimental approaches. Increased amounts of receptor precursor forms could be immunoprecipitated from metabolically labeled cells, stimulated with PDGF-BB. In a second approach, cells were exposed to different concentrations of PDGF-BB, and, in a subsequent step, ligand binding analysis was performed. In this experiment, an initial down-regulation of receptors was followed by increased levels of the cell surface forms of the receptors. In conclusion, PDGF-BB, but not PDGF-AA, induces increased synthesis of both PDGF alpha- and beta-receptor protein; this constitutes a positive feed-back mechanism, which, for example, could serve to potentiate autocrine stimulation of growth.

Amino Acid Sequence↗
PubMed1991

Neurotrophic activity of platelet-derived growth factor (PDGF): Rat neuronal cells possess functional PDGF beta-type receptors and respond to PDGF.

The different isoforms of PDGF bind with different affinities to two different receptor types. Previously, these receptors have been demonstrated mainly on mesenchymal and glial cells. We show here that PDGF beta-type receptors are present also on rat brain neurons in vivo as well as in vitro. Immunohistochemical analysis of a sequentially sectioned brain of a newborn rat revealed the presence of PDGF beta receptors in neurons throughout the central nervous system. These beta-receptor-bearing cells were shown to contain neurofilaments by double immunofluorescence staining. Furthermore, the PDGF-BB isoform bound to cells in a neuron-enriched culture from newborn rat brains and induced c-Fos protein in these cells. Continuous PDGF-BB treatment of primary rat brain cell cultures resulted in outgrowth of neurites and prolonged survival. These results indicate a neurotrophic effect of PDGF-BB.

Animals↗
PubMed1991

Differential expression of platelet-derived growth factor receptors in human malignant glioma cell lines.

Glioma cells in culture express platelet-derived growth factor (PDGF) A- and B-chains and secrete PDGF-like activity that is mainly PDGF-AA. In this work, we show that the PDGF alpha- and beta-receptors are independently expressed in human malignant glioma cells. We also define three different receptor phenotypes that are related to the morphology of glioma cells: cells with only alpha-receptors, only beta-receptors, or with both types of receptors. By the help of Northern blot analyses, 125I-PDGF-binding experiments, and immunoprecipitations the receptors are shown to be structurally normal PDGF receptors, except for minor variations in size that probably are due to differences in glycosylation. PDGF-BB induces DNA synthesis in cells of all three receptor phenotypes, whereas PDGF-AA or PDGF-AB has this effect only on cells with alpha- or with alpha- and beta-receptors. 125I-PDGF-AB binds with high affinity and down-regulates beta-receptors only in cells where alpha-receptors are present in addition to beta-receptors. Thus, the different functional capacities of PDGF isoforms on glioma cells fit with their known receptor-binding specificities and are compatible with the hypothesis that the isoforms act by inducing dimeric receptor complexes. When data on PDGF A- and B-chains, as well as alpha- and beta-receptor expression are compiled and the pattern of receptor binding specificity is taken into account, the majority of glioma cell lines are found to have a phenotype that makes autocrine stimulation possible.

DNA↗
PubMed1991

Human glial fibrillary acidic protein: complementary DNA cloning, chromosome localization, and messenger RNA expression in human glioma cell lines of various phenotypes.

Glial fibrillary acidic protein (GFAP) is a constituent of intermediate filaments of glial cells of the astrocyte lineage. We cloned a human GFAP complementary DNA, deduced the amino acid sequence, and established the chromosomal location (17q21) of the GFAP gene by Southern blot hybridization of somatic cell hybrids and by in situ hybridization. The authenticity of the complementary DNA was proven by expressing it in glioma cells lacking endogenous GFAP; after microinjection of the complementary DNA, such cells became positive for staining with GFAP antibodies. The levels of fibronectin (FN) and GFAP mRNA of ten human glioblastoma cell lines, determined by Northern blot hybridization of RNA, were related to other phenotypic characteristics [cell morphology and expression of the genes encoding platelet-derived growth factor (PDGF) receptors]. A high expression of GFAP mRNA was found only in cells lacking fibronectin mRNA and protein. Glioma cells with a fibroblastic phenotype (bipolar, FN+/GFAP-) were found to express both types of PDGF receptors (alpha and beta). Relatively high levels of PDGF alpha-receptor mRNA, in the absence of beta-receptor expression, were found in cell lines that express GFAP and lack detectable levels of fibronectin mRNA. The findings are compatible with the idea that the genes encoding PDGF receptors in glioma cells are regulated in concert with other genes, the expression of which may reflect the developmental program of normal glia cell lineages.

Chromosome Mapping↗
PubMed1991

Binding of epidermal growth factor-dextran conjugates to cultured glioma cells.

Some gliomas, melanomas and squamous carcinomas have large numbers of EGF receptors which could, in these cases, be used for targeting with toxic agents. We investigated whether EGF could be conjugated to dextran, which is a suitable carrier for toxic agents, without losing its ability to bind to the receptor. Dextran of 20 kDa molecular weight was activated with I-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) yielding highly active pyridinium-isourea derivatives. EGF was coupled to the activated dextran through the amino terminus and glycine was added to block residual activity. The EGF-dextran conjugate was, after purification on Sephadex G25 and Sephacryl 200 columns, tested for its receptor binding properties on human malignant glioma, U343MGaC12:6, cells. The conjugate inhibited binding of 125I-EGF in a competitive assay, showing that the binding was receptor-specific. Dextran conjugated with glycine, without EGF, had no inhibitory effect. The conjugate was radio-labelled either on the EGF part with 125I or on the dextran part with 3H-glycine, and the internalization patterns were compared to the internalization of 125I-EGF. The radioactivity of the conjugates remained cell-associated for more than 20 hr, regardless of whether the radioactivity was on the EGF or on the dextran part, while the radioactivity of unconjugated EGF rapidly disappeared from the cells. Most of the cell-associated radioactivity was, at all analysed time intervals, located intracellularly. Thus, it seems promising to use dextran, conjugated with EGF, as a carrier of, for example, toxic radioactive nuclides.

Dextrans↗
PubMed1991

Activation of the human c-kit product by ligand-induced dimerization mediates circular actin reorganization and chemotaxis.

The proto-oncogene c-kit is allelic with the murine white spotting (W) locus and encodes a transmembrane protein tyrosine kinase that is structurally related to the receptors for platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1). Recently the ligand for the c-kit product, stem cell factor (SCF), was identified in both transmembrane and soluble forms. In order to examine the mechanism for receptor activation by SCF and biological properties of the activated c-kit product, we transfected the wild-type human c-kit cDNA into porcine aortic endothelial cells. We found that the receptor was down-regulated and transmitted a mitogenic signal in response to stimulation with soluble SCF. We also demonstrate that SCF induces dimerization of the c-kit product in intact cells, and that dimerization of the receptor is correlated with activation of its kinase. Activation of the c-kit product by SCF was found to induce circular actin reorganization indistinguishable from that mediated by the PDGF beta-receptor in response to PDGF-BB. Furthermore, soluble SCF was a potent chemotactic agent for cells expressing the c-kit product, a property which might be of importance during embryonic development.

Actins↗
PubMed1991

Effect of receptor kinase inactivation on the rate of internalization and degradation of PDGF and the PDGF beta-receptor.

The complementary DNAs for wildtype and tyrosine kinase-inactivated (K634A) forms of the PDGF beta-receptor were expressed in porcine aortic endothelial cells. We examined the internalization and degradation of ligands and receptors after exposure of receptor expressing cells to PDGF-BB, which binds to the beta-receptor with high affinity, and PDGF-AB, which binds with lower affinity. Cells expressing wildtype beta-receptors were able to internalize and degrade the receptor, as well as the ligand, after exposure to PDGF-BB or -AB. Cells expressing the kinase-inactivated mutant receptor also internalized and degraded both receptor and ligand, but with lower efficiency compared with the wildtype receptor cells. The degradation of either form of receptor was inhibited by treatment of the cells with the lysosomotropic drug chloroquine. Exposure of wildtype and K634A receptor expressing cells to PDGF-AB resulted in a twofold slower rate of internalization of this ligand as compared with PDGF-BB, whereas the relative rate of degradation was similar for the two ligands. Our data indicate that tyrosine kinase activity promotes, but is not a prerequisite for, ligand-induced internalization and degradation of the ligand-receptor complex.

Animals↗
PubMed1991

Identification of a cell retention signal in the B-chain of platelet-derived growth factor and in the long splice version of the A-chain.

The B-chain homodimer of platelet-derived growth factor (PDGF) is only very inefficiently secreted and remains largely associated with the producer cell; in contrast, the dimer of the short, and most common, splice variant of the A-chain is secreted. To identify the structural background to the differences in the secretory pattern between the different isoforms of PDGF, a set of chimeric PDGF A/B cDNAs was generated and expressed in COS cells. Analyses of the biosynthesis and processing of the corresponding products led to the identification of a determinant for cell association in the carboxy-terminal third of the PDGF B-chain precursor. Introduction of stop codons at various positions in the carboxy-terminal prosequence of the PDGF B-chain localized this determinant to an 11-amino-acid-long region (amino acids 219-229). This region contains an 8-amino-acid-long basic sequence that is homologous to a sequence present in an alternatively spliced longer version of the PDGF A-chain. In contrast to the short splice variant, the long splice A-chain version, like the B-chain, was found to remain predominantly cell associated. Thus, we have identified a conserved sequence that inhibits the secretion of some of the PDGF isoforms. Our data also suggest that switching of splicing patterns can be a mechanism to regulate the formation of secreted or cell-associated forms of PDGF-AA and possibly other growth factors.

Amino Acid Sequence↗
PubMed1991

Down-regulation of cellular platelet-derived growth factor receptors induced by an activated neu receptor tyrosine kinase.

The functional integration of growth factor signaling occurs at several levels in target cells. One of the most proximal mechanisms is receptor transmodulation, by which one activated receptor can regulate the expression of other receptors in the same cells. Well-established transregulatory loops involve platelet-derived growth factor (PDGF) down-regulation of epidermal growth factor (EGF) receptors and beta-type transforming growth factors modulation of PDGF receptors. We have studied the relationship between neu tyrosine kinase activation and the expression of the PDGF receptors in transfected NIH/3T3 cells. Expression of the neu oncogene, but not of the neu proto-oncogene, was associated with a decrease of PDGF alpha- and beta-receptors on the cell surface, as measured by [125-I]PDGF-AA and -BB binding. These results were corroborated by metabolic labeling and immunoprecipitation of the PDGF beta-receptors. PDGF alpha- and beta-receptor mRNAs were strongly decreased in the neu oncogene-transformed cells in comparison with control cells expressing the neu proto-oncogene. Down-regulation of the PDGF receptors and their mRNAs was also observed after EGF treatment of cells expressing a chimeric EGF receptor/neu receptor, where the neu tyrosine kinase is activated by EGF binding. These results show that the neu tyrosine kinase can down-modulate PDGF receptor expression, and the effect is mediated via decreased PDGF receptor mRNA levels.

3T3 Cells↗
PubMed1991

Inhibition of human natural killer cell activity by platelet-derived growth factor (PDGF). III. Membrane binding studies and differential biological effect of recombinant PDGF isoforms.

We have previously reported that platelet-derived growth factor (PDGF) substantially inhibits human natural killer (NK) cell cytotoxicity, and that NK cells possess high-affinity surface binding sites for the PDGF-AB isoform. In this communication, we present direct evidence for the presence of A-type (alpha) PDGF receptors on human NK cells by demonstrating that human NK cells have approximately 150,000 high-affinity, surface binding sites for recombinant (r)PDGF-AA and approximately 300,000 high-affinity, surface binding sites for rPDGF-BB. This was determined by the competitive binding of 125I-labelled rPDGF-AA or 125I-labelled rPDGF-BB and homologous unlabelled rPDGF-AA or rPDGF-BB to FACS-sorted, CD16+ lymphoid (NK) cells, and Scatchard analysis of these data. In addition, we also demonstrate that the various isoforms of PDGF have differential effects on NK-cell cytotoxicity. Physiological quantities (100 ng/ml) of rPDGF-BB homodimers, highly purified PDGF-AB heterodimers from outdated platelets, and rPDGF-AB heterodimers substantially inhibited NK-cell cytotoxicity in both a dose- and time-dependent manner. In contrast, pretreatment of NK cells with equivalent nanogram amounts of rPDGF-AA homodimers resulted in a significantly weaker inhibitory effect on NK-cell cytotoxicity as compared with the PDGF-BB and PDGF-AB isoforms. The implications of these findings are discussed.

Adult↗
PubMed1991

Thyrocyte migration and histiotypic follicle regeneration are promoted by epidermal growth factor in primary culture of thyroid follicles in collagen gel.

The effect of epidermal growth factor (EGF) on the morphology of porcine thyroid follicles cultured in collagen gel was studied by light and electron microscopy. Treatment with EGF (10 ng/ml) for 1-4 days induced a progressive migration of thyrocytes radiating from the mother follicles into the collagen lattice. Migrating cells were often connected with junctional complexes, thus forming small follicles containing microlumina, in which the limiting portion of the plasma membrane expressed microvilli. Autoradiography of [3H]thymidine incorporation in EGF-treated cultures showed that more than 80% of nuclei in mother follicles and migrating cells were labeled after 4 days. TSH (2 mU/ml) given simultaneously with EGF did not influence the effect of EGF on multiplication, migration, and formation of microfollicles. TSH given for 12 h after 4 days of exposure to EGF was able to induce luminal dilation of mother follicles, but caused no change in the appearance of microlumina. It is concluded that thyrocytes in collagen gel retain epithelial characteristics during prolonged stimulation with EGF, in spite of a migrating response. The whole sequence of events, starting with the intact follicle, i.e. multiplication, migration of thyrocytes, and formation of new follicles, could, thus, be visualized to occur in response to a single mitogen, EGF. EGF might be involved in the generation of new follicles in the intact gland.

Animals↗
PubMed1991

Coexpression of functionally active receptors for thyrotropin and platelet-derived growth factor in human thyroid carcinoma cells.

In the present study we show the simultaneous expression of functionally active receptors for TSH and platelet-derived growth factor (PDGF) in a newly established human anaplastic thyroid carcinoma cell line, HTh 74. In Northern blot analysis of RNA extracted from HTh 74 cells a low expression of both TSH and PDGF receptor messenger RNA was found. These observations in conjunction with the fact that the cells contain cytokeratin clearly demonstrate that the cells are bona fide epithelial thyroid cells. Stimulation of HTh 74 cells with TSH led to a concentration-dependent increase in cAMP formation, showing a functional activity of the TSH receptors. Northern blot analysis, immunoprecipitation, immunofluorescence staining, and binding experiments showed the presence of both alpha- and beta-type PDGF receptors in the HTh 74 cells. The functional activity of the PDGF receptors was demonstrated by ligand-induced internalization of the receptors and PDGF-induced growth of the HTh 74 cells. The significance of the expression of PDGF receptors on thyroid epithelial cells is not clear. However, it might reflect the gain of a new growth stimulatory pathway participating in the transformation of the epithelial thyroid cells. Alternatively, the PDGF receptors may be remnants from an immature progenitor cell from which the undifferentiated carcinoma has evolved.

Carcinoma↗
PubMed1991

A somatic point mutation in a putative ligand binding domain of the TSH receptor in a patient with autoimmune hyperthyroidism.

Nucleotide sequence analysis of PCR fragments of TSH receptor cDNA synthesized from thyroid RNA of a patient with autoimmune hyperthyroidism, revealed two different sequences in the first position of codon 36. In one of the sequences, there was a C for G substitution leading to the D36----H substitution in the predicted peptide. Both variants were also found in genomic DNA of thyroid tissue. However, only the germ line sequence was found in other tissues representing all three different germ layers. The novel sequence is therefore likely to represent a somatic mutation in the thyroid tissue, of possible relevance for the pathogenesis of the patient's thyroid disorder.

Adult↗