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C H Heldin

Publications and source records attributed to C H Heldin.

At least 109 records · Page 6Linked to original sources
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

Mutation of a Src phosphorylation site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis.

Ligand induced activation of the beta-receptor for platelet-derived growth factor (PDGF) leads to activation of Src family tyrosine kinases. We have explored the possibility that the receptor itself is a substrate for Src. We show that Tyr934 in the kinase domain of the PDGF receptor is phosphorylated by Src. Cell lines expressing a beta-receptor mutant, in which Tyr934 was replaced with a phenyalanine residue, showed reduced mitogenic signaling in response to PDGF-BB. In contrast, the mutant receptor mediated increased signals for chemotaxis and actin reorganization. Whereas the motility responses of cells expressing wild-type beta-receptors were attenuated by inhibition of phosphatidylinositol 3'-kinase, those of cells expressing the mutant receptor were only slightly influenced. In contrast, PDGF-BB-induced chemotaxis of the cells with the mutant receptor was attenuated by inhibition of protein kinase C, whereas the chemotaxis of cells expressing the wild-type beta-receptor was less affected. Moreover, the PDGF-BB-stimulated tyrosine phosphorylation of phospholipase C-gamma was increased in the mutant receptor cells compared with wild-type receptor cells. In conclusion, the characteristics of the Y934F mutant suggest that the phosphorylation of Tyr934 by Src negatively modulates a signal transduction pathway leading to motility responses which involves phospholipase C-gamma, and shifts the response to increased mitogenicity.

Actins↗
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

Structural determinants in the platelet-derived growth factor alpha-receptor implicated in modulation of chemotaxis.

Activation of the platelet-derived growth factor (PDGF) beta-receptor leads to cell growth and chemotaxis. The PDGF alpha-receptor also mediates a mitogenic signal, but fails to induce cell migration in certain cell types. To examine this difference in signal transduction, a series of point-mutated PDGF alpha-receptors were analyzed. Porcine aortic endothelial cells expressing mutant PDGF alpha-receptors, in which tyrosine residues 768, 993, or 1018 were changed to phenylalanine residues migrated toward PDGF, whereas wild-type alpha-receptors and mutant alpha-receptors changed at tyrosine residues 720, 944, or 988 failed to migrate. All mutant receptors were mitogenically active and their capacity to activate phosphatidylinositol 3'-kinase and phospholipase C-gamma was not different from that of the wild-type receptor. Tyr-768 was found to be phosphorylated in PDGF-stimulated cells; in the Y768F mutant, there was a considerable increase in phosphorylation of Ser-767. Tyr-993 was not phosphorylated, but mutation of this tyrosine residue to a phenylalanine residue resulted in increased efficiency of phosphorylation on Tyr-988. Tyr-1018 is known to be an autophosphorylation site. Phosphorylated Tyr-768 and Tyr-1018 may bind signal transduction molecules involved in negative modulation of the chemotactic signaling capacity, whereas phosphorylated Tyr-988 may mediate increased chemotaxis. Thus our data indicate that the PDGF alpha-receptor has an intrinsic ability to transduce a chemotactic signal, and that this signal is counteracted by overriding negative signals.

Amino Acid Sequence↗
PubMed1996

Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain.

A detailed comparison in the developing rat central nervous system between the distribution of the NG2 proteoglycan and the alpha-receptor for platelet-derived growth factor (PDGF) shows that these two molecules are co-expressed by glial progenitor cells of the O2A lineage and can serve as reliable markers for identification of O2A cells in vivo. Our mapping experiments indicate that NG2-positive, PDGF alpha-receptor positive O2A cells are abundant throughout the developing central nervous system in both white and gray matter. The earliest cells immunoreactive for either of the two markers are found adjacent to the central canal of the embryonic day 15 (E15) spinal cord. These cells express only PDGF alpha-receptor and not NG2. By E17, process-bearing cells expressing both NG2 and PDGF alpha-receptor in a highly co-localized fashion are found throughout the central nervous system. The first postnatal week marks the peak in the number of NG2 and PDGF alpha-receptor immunoreactive cells, as well as the peak in the level of expression and the extent of co-localization of the two molecules. After the first week, the level of expression of both NG2 and PDGF alpha-receptor declines, although both molecules continue to be expressed in the adult brain. On O2A cells in the mature brain, NG2 and PDGF alpha-receptor are not as well co-localized at the subcellular level as they are on O2A cells in the younger brain. The functional consequences of co-localization and subsequent dissociation of NG2 and PDGF alpha-receptor on maturing O2A progenitors are investigated in the accompanying paper (Nishiyama et al.: J Neurosci Res 43:315-330, 1996).

Aging↗
PubMed1996

Interaction between NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells is required for optimal response to PDGF.

Previous studies on the NG2 chondroitin sulfate proteoglycan have shown that NG2 is expressed on A2B5-positive O2A progenitor cells, which are known to respond to platelet-derived growth factor (PDGF). In the accompanying paper (Nishiyama et al.; J Neurosci Res 43:299-314, 1996) we show that on O2A progenitors in the embryonic and newborn rat brain, NG2 and PDGF alpha-receptor display an extensive co-localization which becomes less pronounced as the brain matures past the first postnatal week. The present communication describes the relationship between NG2 and PDGF alpha-receptor in vitro. NG2 and PDGF alpha-receptor are highly co-localized on A2B5-positive O2A cells isolated from neonatal rat cerebrum. Mimicking the situation in vivo, the level of expression of the two molecules and the extent of co-localization decline as these cells differentiate into O4-positive pre-oligodendrocytes. However, maintenance of the cells in a progenitor state by treatment with bFGF results in increased levels of both NG2 and PDGF alpha-receptor on the cell surface, suggesting that expression of the two molecules may be coordinately regulated. Furthermore, NG2 can be co-immunoprecipitated from radiolabeled O2A extracts with a rabbit antibody to PDGF alpha-receptor, indicating the presence of a molecular complex that includes NG2 and the receptor. Finally, antibody-patching and subsequent down-regulation of NG2 results in reduced expression of PDGF alpha-receptor and diminishes the proliferative response of the cells to PDGF. These findings suggest that correct co-expression of the NG2 proteoglycan and PDGF alpha-receptor on the surface of O2A progenitor cells is important for the cells' ability to respond effectively to the mitogenic stimulus of PDGF.

Animals↗
PubMed1996

Association of loss of heterozygosity on chromosome 17p with high platelet-derived growth factor alpha receptor expression in human malignant gliomas.

The aim of this study was to examine platelet-derived growth factor alpha receptor (PDGFR-alpha) expression in gliomas of various degrees of malignancy and to correlate the findings with genetic alterations present in the same tumor samples. We analyzed 83 tumors by in situ hybridization using a PDGFR-alpha cRNA probe. Increased PDGFR-alpha mRNA expression was observed in astrocytic tumors of all stages of malignancy, although the highest levels were found in glioblastoma multiforme. To evaluate the frequency of PDGFR-alpha gene amplification, differential PCR requiring less DNA than Southern analysis was used with fluorescence-labeled primers corresponding to the kinase insert region of the PDGFR-alpha. Only 7 of 43 glioblastomas and none of the other tumors tested showed amplification of the PDGFR-alpha gene, suggesting that a mechanism other than gene amplification is responsible for the overexpression of PDGFR-alpha in glial brain tumors. Comparison of the in situ hybridization data with genetic alterations in the same tumor material showed a significant correlation of loss of heterozygosity on chromosome 17p (Fisher's exact, P < 0.0002) with high expression levels of PDGFR-alpha. Because that was the case in both low- and high-grade astrocytomas, our data imply that PDGFR-alpha is actively involved in tumor cell proliferation in early and late stages of glioma development. The association of PDGFR-alpha expression with a distinct subset of glioblastomas characterized by loss of heterozygosity 17p further supports the differentiation of these tumors into molecular variants.

Base Sequence↗
PubMed1996

Effect of transforming growth factor-beta and platelet-derived growth factor-BB on articular cartilage in rats.

The short-term and long-term effects on the growth zone in articular cartilage of transforming growth factor-beta 1 and platelet-derived growth factor-BB injected intraarticularly into the knee joint of growing rats were investigated. The changes induced by five injections of 0.5 micrograms of transforming growth factor-beta 1 included a rapid decrease in the size and number of hypertrophic cells and an enhanced subchondral bone formation. The changes were most marked in the patella but were also apparent in the tibia and femur. The proliferating cells became swollen and lost their normal organization. From the seventh day of the experiment to about 3 weeks, the matrix stained intensely with safranin O for proteoglycans. The alterations induced by transforming growth factor-beta also included synovial fibroplasia and synovitis, consisting predominantly of mononuclear cells. Localised necroses in the cartilage sometimes appeared after 21 days. In long-term studies, destroyed cartilage was found in three of six rats and partial ossification of the joint cartilage was found in two after 90 and 180 days. Ossicles developed in the tendons in all six patellae. Injection of platelet-derived growth factor-BB resulted in an early and transitory minor increase in the osteogenic activity in the zone between cartilage and red bone marrow and later produced an ossicle in one of four tendons. None of the other changes noted after injection of transforming growth factor was observed.

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↗
PubMed1996

Signaling via hetero-oligomeric complexes of type I and type II serine/threonine kinase receptors.

Members of the transforming growth factor-beta (TGF-beta) superfamily have been found to signal by inducing the formation of hetero-oligomeric complexes of different type I and type II serine/threonine kinase receptors. Recent data indicate that binding of TGF-beta to its constitutively active type II receptor recruits the type I receptor into the complex; the type I receptor is thereafter phosphorylated and activated, processes which are necessary and sufficient for most TGF-beta mediated responses. Recent genetic analyses of Drosophila also indicate a strict requirement for both type I and type II receptors in decapentaplegic signaling in vivo.

Activin Receptors, Type I↗
PubMed1996

Bone morphogenetic protein type IB receptor is progressively expressed in malignant glioma tumours.

The distribution of bone morphogenetic protein (BMP) type I receptors and the activin type I receptor (ActR-I) was investigated in 16 cases of human glioma and five cases of non-tumourous gliosis tissue by immunohistochemical technique. Both BMP type IA (BMPR-IA) and the type IB (BMPR-IB) receptors were detected in human glioma cells. A significant increase in BMPR-IB in tumour cells was observed in malignant glioma compared with both low-grade astrocytomas (n=16, P<0.005) and gliosis (n=13, P<0.001). However, enhancement of BMPR-IA staining was moderate and ActR-I staining was only weakly expressed in the malignant glioma tumours. Osteogenic protein (OP)-1/BMP-7, which is known to bind BMPR-IA, BMPR-IB and ActR-I, was expressed in nervous tissue and was also detected in anaplastic areas of malignant glioma. In contrast to the tissue materials, BMPR-IA was expressed to a stronger degree than BMPR-IB in human glioma cell lines; the growth of these cells was suppressed by OP-1. These results suggest the presence of BMP receptors and a functional role for BMPs in malignant glioma.

Activin Receptors↗
PubMed1996

Protein tyrosine kinase receptors.

More than 50 PTK receptors are known to be involved in regulation of cell growth, differentiation, chemotaxis and actin reorganization. PTK receptors can be classified into subfamilies according to their structural features. PTK receptors are activated by ligand induced homo- or heterodimerization, which leads to receptor autophosphorylation on tyrosine residues. In certain receptors, the autophosphorylation regulates the catalytic activity of the kinase. Moreover, autophosphorylated tyrosine residues bind signal transduction molecules with SH2 or PTP domains. Such molecules are activated by the actual binding to the receptors or by phosphorylation on tyrosine residues by the receptor kinase. There are also examples of constitutively active signal transduction molecules that are translocated to act at the cell membrane by binding to autophosphorylated PTK receptors. In this way, specific intracellular signal transduction pathways are initiated. After ligand binding and activation, PTK receptors are internalized and deactivated by dephosphorylation as well as by degradation in the cytoplasm or in the lysosomes.

Dimerization↗
PubMed1995

Efficient association of an amino-terminally extended form of human latent transforming growth factor-beta binding protein with the extracellular matrix.

Latent transforming growth factor-beta (TGF-beta) binding protein-1 (LTBP-1) is a component of the high molecular weight latent TGF-beta complex found in various cells, including human platelets. LTBP-1 is observed as different molecular sizes in different cell types, probably due to proteolytic processing and alternative splicing. We here report a novel form of human LTBP-1, which is longer in its NH2-terminal part (LTBP-1L). Northern hybridization analysis revealed that the LTBP-1L is derived from a 7.0-kilobase mRNA, whereas the originally reported shorter form (LTBP-1S) is derived from a 5.2-kilobase mRNA. Transfection of cDNA for LTBP-1L and -1S in COS cells revealed that LTBP-1L bound more efficiently to the extracellular matrix than did LTBP-1S. These results suggest that the different splice forms of LTBP-1 mediate different localization patterns of the latent TGF-beta complexes in vivo.

Amino Acid Sequence↗
PubMed1995

Cloning and characterization of a human type II receptor for bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily. Several members of this family have been shown to transduce their signals through binding to type I and type II serine-(threonine) kinase receptors. Here we report the cDNA cloning and characterization of a human type II receptor for BMPs (BMPR-II), which is distantly related to DAF-4, a BMP type II receptor from Caenorhabditis elegans. In transfected COS-1 cells, osteogenic protein (OP)-1/BMP-7, and less efficiently BMP-4, bound to BMPR-II. BMPR-II bound ligands only weakly alone, but the binding was facilitated by the presence of previously identified type I receptors for BMPs. Binding of OP-1/BMP-7 to BMPR-II was also observed in nontransfected cell lines. Moreover, a transcriptional activation signal was transduced by BMPR-II in the presence of type I receptors after stimulation by OP-1/BMP-7.

Amino Acid Sequence↗
PubMed1995

Enhanced expression of transforming growth factor-beta and its type-I and type-II receptors in human glioblastoma.

Immunohistochemical studies of transforming growth factor-beta (TGF-beta) and its receptors have been carried out on 16 glioma tissues and compared with 5 cases of gliosis. Significantly higher expressions of TGF-beta I, as well as type-I and type-II TGF-beta receptors (T beta R-I and T beta R-II, respectively), were observed in advanced-malignant-glioma tissues when compared with non-tumorous gliosis. Immunoreactivities of TGF-beta and T beta R-I were localized in the cytoplasm of spindle-shaped tumor cells surrounding proliferating vessels or around areas of necrosis. The advancing edge of the tumor clusters frequently stained positive. Similar expression patterns were found for TGF-beta 2 and TGF-beta 3, whereas only weak or no expression was found for endoglin. In low-grade astrocytomas and in gliosis cases, the expression was moderate for T beta R-I and weak for TGF-beta and T beta R-II. In 3 examined human malignant glioma cell lines, clear immunostainings were detected for TGF-beta and its receptors. Ligand-induced heteromeric complexes of the receptors were formed in these cell lines, but the amount of the receptors was less than that of mink lung epithelial cells, which are sensitive target cells for TGF-beta. TGF-beta I showed no growth-inhibitory activity on any of these glioma cell lines. These results suggest that malignant gliomas produce TGF-beta and receptors, but are refractory to TGF-beta, implying dysregulation in the signalling pathway in the tumor cells. It is possible that the released TGF-beta acts on neighboring cells and affects stromal growth, angiogenesis, metastasis or immune surveillance in human glioma.

Brain Neoplasms↗