Perturbation of the platelet-derived growth factor receptor signaling by dibutyryl-camp in human astrocytoma cells

It has been shown that cAMP may perturb the polypeptide growth factor-induced nuclear events. However, the possible interactions of the cAMP-protein kinase A (cAMP-PKA) and receptor tyrosine kinase pathways in the cytosol have not been fully elucidated. In this study, we use human astrocytoma cells as a model to investigate this issue. The results show that platelet-derived growth factor (PDGF)-induced receptor autophosphorylation in human astrocytoma cells is suppressed by dibutyryl-cAMP pretreatment and such suppression is not due to changes in the ligand-receptor binding properties. Further studies show that PDGF-induced tyrosine phosphorylation of phospholipase C__γ1 (PLC__γ1) and phosphatidylinositol 3-kinase (PI 3-kinase) are also suppressed in dibutyryl-cAMP-pretreated cells. The suppression of PLC__γ1 tyrosine phosphorylation was accompanied by a decreased production of water soluble inositol phosphates. In contrast, similar treatment with normal human astrocytes potentiates the tyrosine phosphorylation of PLC__γ1 and PI 3-kinase. The results indicate that cAMP can either negatively or positively modulate the PDGF receptor tyrosine kinase activity depending on the cell types examined. © 1995 Wiley-Liss, Inc.     

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

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

Ultrastructural localization of platelet-derived growth factor and related factors in normal and transformed cells

Visualization of platelet-derived growth factor (PDGF) and PDGF-like growth factors in cultured cells has been achieved by cryo-ultramicrotomy in combination with immunogold labeling. Immunogold staining of cryosections requires a mild chemical fixation in order to ensure preservation of antigenicity and ultrastructural details. Therefore the effect of several chemical fixatives on the antigenic properties of PDGF and PDGF-like growth factors was studied by indirect immunofluorescence using a polyclonal anti-PDGF antiserum. These studies demonstrated that formaldehyde has no effect on antigenicity, in contrast to glutaraldehyde or acrolein. For this reason formaldehyde was used as the only fixative for the visualization of PDGF in cryosections. PDGF was visualized in cryosections of normal human fibroblasts, preincubated with PDGF under various conditions. Preincubation at 4 degrees C with PDGF resulted in partial internalization of the growth factor. During subsequent warming of the cells to 37 degrees C PDGF was translocated to the nucleus. PDGF was also detected in the cytoplasm of tumor cells producing endogenous PDGF-like growth factors (neuroblastoma and simian sarcoma virus-transformed cells) but in these cases no significant amounts of these growth factors were present in the nucleus or at the extracellular surface of these cells. These results will be discussed in view of the intracellular routing of PDGF in normal responsive cells and of PDGF-like growth factors in factor-producing cells.     

Crystal structure of human platelet-derived growth factor BB.

The crystal structure of the homodimeric BB isoform of human recombinant platelet-derived growth factor (PDGF-BB) has been determined by X-ray analysis to 3.0 A resolution. The polypeptide chain is folded into two highly twisted antiparallel pairs of beta-strands and contains an unusual knotted arrangement of three intramolecular disulfide bonds. Dimerization leads to the clustering of three surface loops at each end of the elongated dimer, which most probably form the receptor recognition sites.     

Transforming growth factor beta 1 partially suppresses the transformed phenotype of ras-transformed hepatocytes.

The effect of transforming growth factor beta type 1 (TGF-beta 1) on DNA synthesis, anchorage-dependent and anchorage-independent proliferation, cytoskeletal organization, and gene expression in ras-transformed simian virus 40 (SV40)-immortalized hepatocyte cell lines was measured. An SV40-immortalized cell line (CWSV1), a control neo-transfected and selected cell line (N1), and neo+ras-transfected and selected cell lines (NR3 and NR4) were used for this study. CWSV1 and N1 cells do not grow in soft agarose and are not tumorigenic. The ras-transformed hepatocytes NR3 and NR4 grow in soft agar and are tumorigenic. TGF-beta 1 treatment did not inhibit DNA synthesis or anchorage-dependent growth in the SV40-immortalized hepatocyte cell line CWSV1 or in the ras-transformed hepatocytes. TGF-beta 1 treatment inhibited anchorage-independent growth, increased actin cytoskeleton organization, and altered the morphology of ras-transformed hepatocytes; that is, with regard to all three of these properties, TGF-beta 1-treated ras-transformed hepatocytes more closely resembled the immortalized parent cell line. c-Ha-ras and c-myc RNA levels were not altered in TGF-beta 1-treated NR4 cells. TGF-beta 1 treatment did alter expression of some genes in NR4 cells. The level of expression of alpha 1 integrin RNA was higher in CWSV1 cells than in NR4 cells and increased in NR4 cells when they were treated with TGF-beta 1. Similarly, the levels and profiles of integrins on the cell surface of CWSV1 cells compared to NR4 cells, as determined by cell surface protein iodination, differed and in TGF-beta 1-treated NR4 cells more closely resembled the surface integrin profile for CWSV1 cells.     

Identification of the type-B receptor for platelet-derived growth factor in human embryonal carcinoma cells

The human embryonal carcinoma (EC) cell line Tera 2 clone 13 (T2/13) can be induced to differentiate in vitro into neuroectodermal cell types with retinoic acid. Undifferentiated cells are characterized by rapid proliferation, whereas differentiated cells show a prolonged generation time, have a limited life span, and possess new cell-surface markers. In the present study we establish that both differentiated and undifferentiated T2/13 cells express the type-B platelet-derived growth factor (PDGF) receptor mRNA and bind PDGF-BB with high affinity. Differentiation causes a three-fold increase in receptor number per cell and leaves the affinity of the receptors unaffected. These data are the first to describe expression of this receptor in EC cells. The biosynthesis and degradation of this receptor were studied in undifferentiated as well as in differentiated T2/13 cells using an anti-type-B receptor antibody. These experiments revealed that high concentrations of recombinant PDGF-AA did not accelerate receptor metabolism in both cell types. In contrast, human PDGF or recombinant PDGF-BB added to the culture dishes readily increased receptor degradation. These results demonstrate that T2/13 cells express functional type-B PDGF receptors and suggest that cells responsive to PDGF might be present during mammalian development before the onset of mesoderm formation.     

Progression of the phenotype of transformed cells after growth stimulation of cells by a human papillomavirus type 16 gene function.

Alteration of the growth properties of the established murine fibroblast cell lines NIH 3T3 and 3Y1 was studied in monolayer cultures and in cells suspended in semisolid medium after introduction of a cloned human papillomavirus type 16 (HPV16) DNA. HPV 16 DNA stimulated both cell lines to grow beyond their saturation densities in monolayer cultures without any apparent morphological changes or tendency to pile up. These cells were also stimulated to grow in soft agar. Since essentially all the cells that received the viral gene were stimulated to grow, the growth-stimulatory activity of HPV16 appeared to be due to the direct effect of a viral gene function. The NIH 3T3 cells showed an additional change in growth properties upon prolonged incubation of dense monolayers of cells containing the HPV16 DNA; morphologically recognizable dense foci appeared at a frequency of about 10(-3). These cells, when cloned from the foci, grew more rapidly in soft agar than the parental cells and were morphologically transformed. In other words, there were two sequential steps in cell transformation induced by HPV16. Practically all the viral DNAs were present in the cells as large rearranged multimers and were integrated into host chromosomal DNA. There was no obvious difference in the state of viral DNA in the cells of the original clone or the three subclones derived from it as dense foci. There was no difference in the amount or the number of viral RNA species expressed in the cells at these two stages. The secondary changes in the growth properties of NIH 3T3 cells appear to be due to some cellular alterations.     

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. I. Evidence for reversible binding and inactivation of the platelet-derived growth factor by heparin

Summary We have investigated the effects of interactions between growth factors and heparin-like glycosaminoglycans on untransformed human arterial smooth muscle cells (hASMC) in vitro. The results indicate that heparin in the presence of serum mitogens prevents the cells from entering the S phase of the cell cycle by binding and inactivating reversibly some serum mitogen(s). Our results suggest that platelet-derived growth factor (PDGF) is one of them and that it is the most potent stimulator of hASMC growth in vitro. Thymidine incorporation as well as increase in DNA content was inhibited not only by the presence of heparin in serum-containing medium but also when serum was chromatographed on Heparin-Sepharose at physiologic salt concentrations before exposure to the cells. The mitogenic activity of the unretained serum fraction was restored by the addition of PDGF AA, AB, or BB dimers or of a fraction (RF I) that dissociated from Heparin-Sepharose at 0.2 to 0.6M NaCl. Radiolabeled recombinant PDGF (c-sis) dissociated from Heparin-Sepharose within a concentration range of NaCl similar to that of RF I. Neither the unretained material nor the RF I or PDGF dimers were effective alone. The effect of RF I was significantly decreased by the addition of an anti-PDGF IgG that is known to neutralize the PDGF mitogenic activity partially. Addition of heparin abolished DNA-synthesis when the PDGF dimers or RF I were combined with the unretained fraction. A second fraction (RF II) bound strongly to Heparin-Sepharose and eluted between 1.1 and 1.6M NaCl. The RF II also induced DNA synthesis but was neither as efficient as RF I nor depending on other serum fractions for growth promotion and it was not inhibited by anti-PDGF IgG. A similar strong affinity for Heparin-Sepharose was found for labeled basic fibroblast growth factor and we cannot exclude the possibility that RF II represent fibroblast growth factor. Under these culture conditions, inhibition of hASMC proliferation was directly correlated with the expression of smooth muscle specific alpha actin isoforms in stress fibers and the suppression of a proliferating cell-specific nuclear antigen. Conversely, stimulation of hASMC proliferation was associated with the opposite phenomenon. We conclude that heparin-like glycosaminoglycans influence growth and phenotype of hASMCs in vitro by binding and inactivating PDGF. Inasmuch as heparin-like substances constitute a significant proportion of the proteoglycan-associated glycosaminoglycans of the arterial wall, such mechanisms might be important for the development of atherosclerotic lesions.     

Suppression of the chemically transformed phenotype of BHK cells by a human cDNA.

Transformation of the baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis (A. Stoler and N. P. Bouck, Proc. Natl. Acad. Sci. USA 82:570-574, 1985). A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent normal cellular phenotype after transfection with human cDNA. The human cDNA capable of inducing stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.     

Actions of platelet-derived growth factor isoforms in mesangial cells

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

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. II. The platelet-derived growth factor A-chain contains a sequence that specifically binds heparin

Summary Synthetic oligopeptides were used to study the specificity of the interaction between heparin and platelet-derived growth factor (PDGF) in competition experiments. DNA synthesis in PDGF-dependent human arterial smooth muscle cell (hASMC) cultures was used as a biological tracer of PDGF activity. Oligo-108-124 (corresponding to amino acid residues 108-124 of the long PDGF A-chain isoform) had no effect on DNA synthesis in itself but competed at 10⁻¹⁰ M concentration effectively with PDGF for binding to heparin and released the block on thymidine incorporation induced by heparin. Poly-lysine-serine (lysine:serine ratio 3:1) was also effective but at a considerably higher concentration (10⁻⁶ M). Poly-arginine-serine did not compete with PDGF for heparin as deduced from the cell assay. This suggested that among basic amino acids, lysine was more important than arginine for heparin binding. Deletion of lysine residues 115 and 116 in Oligo-108-124 abolished its effect on the interaction between PDGF and heparin in the cell assay. Likewise, Oligo-69-84 (corresponding to the PDGF A-chain residues 69–84), with three lysine residues interrupted by a proline, was ineffective. In Oligo-108-124, the lysine residues are interrupted by an arginine. Our results suggested that the binding between PDGF and heparin is specific and that the amino acid sequence [-Lys¹¹⁵-Lys¹¹⁶-Arg¹¹⁷-Lys¹¹⁸-Arg¹¹⁹-] is of major importance. They do not however, exclude other domains of the PDGF A or B chains as additional binding sites for heparin nor do they exclude the possibility that heparin and the PDGF receptor share a common binding site.     

Effects of platelet-derived growth factor on human and mouse osteoblastic cells isolated from the trabecular bone surface.

We show here that purified platelet derived growth factor (PDGF) stimulates DNA synthesis in normal endosteal mouse and human osteoblastic cells isolated by selective migration from the trabecular bone surface. Maximum DNA synthesis as measured by (3H)-thymidine incorporation into DNA was increased at 50 ng/ml PDGF (48-72 hours). In both species, the effect of PDGF (25 ng/ml) was lower than the mitogenic effect of 10% FCS. We found that the mitogenic effect of PDGF on human trabecular cells decreased with the number of cell passages. DNA synthesis was increased about 4-fold by PDGF (25 ng/ml) in early passaged cells that expressed low basal growth rate and high osteocalcin production in basal conditions and in response to 1,25(OH)2 vitamin D, whereas DNA synthesis was increased 1.2 fold by PDGF in late passaged cells that showed high basal growth rate and low osteocalcin release in absence or presence of 1,25(OH)2D. PDGF alone had no effect on osteocalcin production. These results indicate that PDGF has mitogenic effect on normal mouse and human osteoblastic cells lining the trabecular bone surface and that the responsiveness to PDGF of human trabecular cells varies with the stage of differentiation.     

Somatomedin-C and platelet-derived growth factor stimulate human fibroblast replication

Previous studies have demonstrated that serum contains mitogens, such as platelet-derived growth factor (PDGF), which may alter fibroblast responsiveness to growth factors contained in plasma. Somatomedin-C (SM-C) has been identified as one of the plasma growth factors required for mouse Balb/c 3T3 fibroblasts to initiate DNA synthesis. The present experiments were undertaken to explore the interaction between PDGF, human growth hormone (hGH), SM-C, and other growth-promoting agents in stimulating the growth of human fibroblasts. Proliferation of human dermal fibroblasts plated at low density (3,000 cells/cm²) was found to be equally stimulated by continuous exposure to either normal or somatomedin-C-deficient serum. In contrast, when confluent monolayers were sequentially exposed to PDGF, followed either by normal platelet poor plasma (PPP) or hypopituitary PPP, the cells exposed to normal PPP entered the “S” phase of the cell cycle 50% faster. This difference could be abolished by a 6-hour incubation with growth hormone (10 ng/ml) or somatomedin-C (5 ng/ml) preceding the addition of plasma. When medium containing either hGH or Sm-C was changed frequently so as to remove factors secreted by fibroblasts, only those cells exposed to exogenous somatomedin-C entered DNA synthesis. This finding is in agreement with previous findings that human fibroblasts are capable of making Sm-C in response to hGH. These findings support the hypothesis that somatomedin is required for fibroblast replication in vitro, and that growth hormone appears to stimulate replication indirectly through somatomedin production.     

Shear-induced platelet-derived growth factor gene expression in human endothelial cells is mediated by protein kinase C.

Our previous studies have shown that steady shear stress causes a transient increase of platelet-derived growth factor (PDGF) A and B chain mRNA levels in human umbilical vein endothelial cells (HUVEC). In the present study, we elucidated the signaling pathway of shear stress in HUVEC by examining the roles of protein kineses, intracellular calcium, cyclooxygenase, and guanine nucleotide-binding proteins (G proteins) in the PDGF gene induction by shear. The protein kinase C inhibitors, H7 and staurosporine, strongly inhibited the shear-induced PDGF gene expression in HUVEC. In contrast, HA1004, a cAMP- and cGMP-dependent protein kinases inhibitor, was only slightly inhibitory. BAPTA/AM, an intracellular calcium chelator, partially (50%) inhibited the shear-induced PDGF gene expression. The cyclooxygenase inhibitors, ibuprofen and indomethacin, were slightly inhibitory. A 35-50% inhibition of shear-induced PDGF gene expression was found with GDP-beta-S, an inhibitor of G proteins. These results suggest that shear-induced PDGF gene expression in HUVEC is mainly mediated by protein kinase C activation and requires intracellular calcium. Furthermore, G proteins seem to be involved in this process, whereas prostaglandin synthesis via cyclooxygenase pathway is not. We propose a mechanism of shear-induced PDGF gene expression in HUVEC: Shear stress, either directly or indirectly (G protein-mediated), enhances the membrane phosphoinositide turnover via phospholipase C, producing diacylglycerol, an activator of protein kinase C. The activated protein kinase C then triggers the subsequent PDGF gene expression.