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C D Stiles

Publications and source records attributed to C D Stiles.

At least 19 recordsLinked to original sources
PubMed1999

Platelet-derived growth factor stimulation of monocyte chemoattractant protein-1 gene expression is mediated by transient activation of the phosphoinositide 3-kinase signal transduction pathway.

Platelet-derived growth factor (PDGF) stimulates transcription of an immediate-early gene set in Balb/c 3T3 cells. One cohort of these genes, typified by c-fos, is induced within minutes following activation of PDGF receptors. A second cohort responds to PDGF only after a significant time delay, although induction is still a primary response to receptor activation as shown by "superinduction" in the presence of the protein synthesis inhibitor cycloheximide. PDGF-receptor activated signaling pathways for the "slow" immediate-early genes are poorly resolved. Using gain-of-function mutations together with small molecule inhibitors of kinase activity, we show that activation of PI 3-kinase is both necessary and sufficient for the induction of the prototype slow immediate-early gene, monocyte chemoattractant-1 (MCP-1). Following activation of PDGF receptors, MCP-1 mRNA does not begin to accumulate for at least 90 min. However, only a brief (10 min) interval of PI 3-kinase activity is required to trigger this delayed response. The serine/threonine protein kinase, Akt/PKB, likely functions as a downstream affector of PI 3-kinase for this induction.

3T3 Cells↗
PubMed1999

Activation of neurotrophin-3 receptor TrkC induces apoptosis in medulloblastomas.

Elevated expression of the neurotrophin-3 (NT-3) receptor TrkC by childhood medulloblastomas is associated with favorable clinical outcome. Here, we provide evidence that TrkC is more than simply a passive marker of prognosis. We demonstrate that: (a) medulloblastomas undergo apoptosis in vitro when grown in the presence of NT-3; (b) overexpression of TrkC inhibits the growth of intracerebral xenografts of a medulloblastoma cell line in nude mice; and (c) trkC expression by individual tumor cells is highly correlated with apoptosis within primary medulloblastoma biopsy specimens. TrkC-mediated NT-3 signaling promotes apoptosis by activating multiple parallel signaling pathways and by inducing immediate-early gene expression of both c-jun and c-fos. Considered collectively, these results support the conclusion that the biological actions of TrkC activation affect medulloblastoma outcome by inhibiting tumor growth through the promotion of apoptosis.

Animals↗
PubMed1999

TrkA glycosylation regulates receptor localization and activity.

The human nerve growth factor receptor (TrkA) contains four potential N-glycosylation sites that are highly conserved within the Trk family of neurotrophin receptors, and nine additional sites that are less well conserved. Using a microscale deglycosylation assay, we show here that both conserved and variable N-glycosylation sites are used during maturation of TrkA. Glycosylation at these sites serves two distinct functions. First, glycosylation is necessary to prevent ligand-independent activation of TrkA. Unglycosylated TrkA core protein is phosphorylated even in the absence of ligand stimulation and displays constitutive kinase activity as well as constitutive interaction with the signaling molecules Shc and PLC-gamma. Second, glycosylation is required to localize TrkA to the cell surface, where it can trigger the Ras/Raf/MAP kinase cascade. Using confocal microscopy, we show that unglycosylated active Trk receptors are trapped intracellularly. Furthermore, the unglycosylated active TrkA receptors are unable to activate kinases in the Ras-MAP kinase pathway, MEK and Erk. Consistent with these biochemical observations, unglycosylated TrkA core protein does not promote neuronal differentiation in Trk PC12 cells even at high levels of constitutive catalytic activity.

Animals↗
PubMed1997

Activation of ErbB2 during wallerian degeneration of sciatic nerve.

We used anti-phosphopeptide-immunodetecting antibodies as immunohistochemical reagents to define the location and activity state of p185(erbB2) during Wallerian degeneration. Nerve damage induces a phosphorylation event at Y1248, a site that couples p185(erbB2) to the Ras-Raf-MAP kinase signal transduction pathway. Phosphorylation of p185(erbB2) occurs within Schwann cells and coincides in time and space with Schwann cell mitotic activity, as measured by bromodeoxyuridine uptake. These visual images of receptor autophosphorylation link activation of p185(erbB2) to the Schwann cell proliferation that accompanies nerve regeneration.

Animals↗
PubMed1997

Trk receptors function as rapid retrograde signal carriers in the adult nervous system.

During development target-derived neurotrophins promote the survival of neurons. However, mature neurons no longer depend on the target for survival. Do target-derived neurotrophins retain retrograde signaling functions in mature neurons, and, if so, how are they executed? We addressed this question by using a phosphotyrosine-directed antibody to locate activated Trk receptors in adult rat sciatic nerve. We show that catalytically active Trk receptors are located within the axon of adult rat sciatic nerve and that they are distributed throughout the length of the axons. These catalytically active receptors are phosphorylated on tyrosine at a position that couples them to the signal-generating proteins Ras and PI3 kinase. Neurotrophin applied at sciatic nerve terminals increases both catalytic activity and phosphorylation state of Trk receptors at distant points within the axons. Trk activation initiated at the nerve terminals propagates through the axon toward the nerve cell body at an initial rate that exceeds that of conventional vesicular transport. However, our data suggest that this rapid signal is nevertheless vesicle-associated. Thus, in mature nerves, activated Trk receptors function as rapid retrograde signal carriers to execute remote responses to target-derived neurotrophins.

3T3 Cells↗
PubMed1997

Detection of activated platelet-derived growth factor receptors in human meningioma.

The beta receptor subunit of platelet-derived growth factor (PDGF) and its corresponding ligand (PDGF-BB) are coordinately expressed in fresh surgical isolates of human meningioma. These observations imply that PDGF autocrine loops are engaged in human meningioma and suggest that activated PDGF-beta receptors might contribute to the pathology of this common brain neoplasm. The study of PDGF autocrine loops and human meningioma has been slowed by the scarcity of meningioma cell culture model systems. Furthermore, in meningioma tumor tissue, the activation state of PDGF receptors is difficult to assess with conventional reagents, because the tumor is intermixed with normal stroma. In fact, there is no evidence that PDGF receptors within the tumor are activated by ligand. We used a synthetic tyrosine phosphopeptide to raise an antibody that reports the phosphorylation state of tyrosine 751 in the human PDGF-beta receptor. Phosphorylated tyrosine 751 is a recognition site for phosphatidylinositol 3'-kinase, a cytoplasmic effector of PDGF-induced mitogenesis, chemotaxis, and membrane ruffling. Immunoblotting and immunostaining analyses with this antibody show that the PDGF-beta receptor is constitutively phosphorylated at tyrosine 751 within multiple fresh surgical isolates of human meningioma. These findings are consistent with a role for activated PDGF receptors in the proliferation of human meningiomas.

3T3 Cells↗
PubMed1997

A PDGF-regulated immediate early gene response initiates neuronal differentiation in ventricular zone progenitor cells.

When exposed to platelet-derived growth factor (PDGF), uncommitted neuroepithelial cells from the developing cortex of embryonic day 14 (E14) rats develop into neurons. Outward signs of the neuronal phenotype are not observed for 4 days following exposure to PDGF. However, only a brief (2-3 hr) period of PDGF receptor activation is required to initiate neuronal development. During the window of receptor activation, RNA synthesis is essential, but protein synthesis is not. These observations indicate that specification of neuronal fate is mediated by an immediate early gene response.

Animals↗
PubMed1997

Phosphorylation-directed antibodies in high-flux screens for compounds that modulate signal transduction.

Synthetic tyrosine phosphopeptides were used to generate antibodies that recognize phosphotyrosine in the context of a defined sequence of flanking amino acids. Using phosphopeptide immunogens derived from regulatory or signal-generating motifs, "phosphorylation-directed antibodies" can be targeted to specific growth factor receptors or signal-generating proteins. In his paper, we show how phosphorylation-directed antibodies can be used in a colorimetric, high-throughput screen for drugs that modulate the function of specific growth factor receptors or signal-generating proteins.

Amino Acid Sequence↗
PubMed1997

Temporal and spatial specificity of PDGF alpha receptor promoter in transgenic mice.

Aberrant expression of the platelet-derived growth factor alpha receptor (PDGF alpha R) has been linked to developmental abnormalities in vertebrate models, and has been implicated in multiple disease states in humans. To identify cis-acting regulatory elements that dictate expression of this receptor, we generated transgenic mice bearing the reporter gene beta-galactosidase (lacZ) under the control of a 6-kb promoter sequence. Expression of lacZ was monitored throughout embryonic development, with special focus on nervous tissue, skeleton, and several organ systems wherein PDGF alpha R expression is thought to play a pivotal role. In several independent transgenic mouse strains, lacZ expression recapitulated predominant features of PDGF alpha R gene expression during mouse development. These results demonstrate that critical tissue-specific regulatory elements for PDGF alpha R expression are located within a 6-kb upstream region of the PDGF alpha R gene.

Animals↗
PubMed1996

Differential utilization of Trk autophosphorylation sites.

Tyrosine autophosphorylation controls the catalytic and signaling activities of the neurotrophin receptors, the Trks. To analyze the regulation of distinct tyrosine sites, we generated a panel of antibodies that report the phosphorylation state of individual tyrosines within the Trk cytoplasmic domain. Using pheochromocytoma-derived cell lines, we show that individual tyrosines within the nerve growth factor receptor TrkA are phosphorylated in a non-coordinate fashion following receptor activation. The non-coordinate response of these tyrosines reflects their separate functions in regulating the catalytic and signaling activities of Trk receptors. The differential utilization of distinct sites on Trk receptor tyrosine kinases suggests that the receptor can specify both the timing and the nature of neurotrophin-stimulated signal transduction pathways. Moreover, we show that these Trk autophosphorylation sites, which have hitherto been mapped and characterized only in non-neuronal cell lines, are activated in normal neurons in response to ligand stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Platelet-derived growth factor induction of the immediate-early gene MCP-1 is mediated by NF-kappaB and a 90-kDa phosphoprotein coactivator.

A broad panel of agents including serum, interleukin-1, double-stranded RNA, and platelet-derived growth factor (PDGF) stimulate transcription of the "slow" immediate-early gene MCP-1. These disparate inducers act through a tight cluster of regulatory elements in the distal 5'-flanking sequences of the MCP-1 gene. We describe a 22-base element in this cluster which, in single copy, confers PDGF-inducibility to a tagged MCP-1 reporter gene. In mobility shift assays, the element binds a PDGF-activated form of NF-kappaB, and a 90-kDa protein (p90) which binds constitutively. Antibody supershift and UV cross-linking experiments indicate that the PDGF-activated NF-kappaB species is a Rel A homodimer. The DNA binding form of p90 is a nuclear-restricted serine/threonine phosphoprotein. Mutagenesis of the 22-base element shows that the NF-kappaB and p90 binding sites overlap, but binding of the two species is mutually independent. Both sites, however, are required for optimum PDGF induction of MCP-1. Therefore, p90 appears to be a coactivator with NF-kappaB in PDGF-mediated induction of MCP-1.

3T3 Cells↗
PubMed1995

Expression of PDGF and PDGF receptors in human astrocytoma operation specimens supports the existence of an autocrine loop.

Established cell lines derived from human malignant astrocytomas typically express a combination of platelet-derived growth factor (PDGF) and PDGF receptor which could form an autocrine loop. In this study, we screened for the essential components of a PDGF autocrine loop in fresh surgical isolates of human astrocytomas, using in situ hybridization and immunohistochemical techniques. Eight malignant astrocytomas (6 glioblastomas and 2 anaplastic astrocytomas), 5 low-grade astrocytomas and 4 non-neoplastic glial specimens (mesial temporal sclerosis) were evaluated. Malignant astrocytomas, and to a lesser extent low-grade astrocytomas, expressed more PDGF-A and PDGF-B than non-neoplastic glia. PDGF-alpha-receptor expression was elevated both in malignant and in low-grade astrocytomas. These data support the argument that PDGF autocrine loops contribute to the unregulated growth of human astrocytomas. Expression of PDGF and PDGF receptor in low-grade astrocytomas suggests that activation of PDGF autocrine loops may be an early event in the pathogenesis of malignant astrocytomas.

Adolescent↗
PubMed1995

Applications of electroporation of adherent cells in situ, on a partly conductive slide.

Nontraumatic, simple, and reproducible procedures for the introduction of nonpermeant molecules into adherent mammalian cells by in situ electroporation are described. Cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse is applied in the presence of the molecules to be introduced, and their effect on the cellular phenotype can be observed. The cells growing on the nonconductive side of the slide do not receive any pulse and serve as controls. Careful adjustment of electric field strength can achieve the introduction of the molecules into essentially 100% of the cells, and this treatment causes no detectable disruption to cellular metabolism. This is applied in the presence of the fluorescent dye, Lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide. The migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination.

Animals↗
PubMed1995

A new platelet-derived growth factor-regulated genomic element which binds a serine/threonine phosphoprotein mediates induction of the slow immediate-early gene MCP-1.

The MCP-1 chemokine gene belongs to a cohort of immediate-early genes that are induced with slower kinetics than c-fos. In this study, we identified a cluster of four platelet-derived growth factor (PDGF)-responsive elements within a 240-bp enhancer found in the distal 5' flanking MCP-1 sequences. Two of the elements bind one or more forms of the transcription factor NF-kappa B. We focused on the other two elements which are hitherto unreported, PDGF-regulated genomic motifs. One of these novel elements, detected as a 28-mer by DNase I footprinting, restores PDGF inducibility when added in two copies to a 5' truncated MCP-1 gene. A single copy of the second novel element, a 27-mer, restores PDGF inducibility to a 5' truncated MCP-1 gene. The 27-base element interacts with a PDGF-activated serine/threonine phosphoprotein that is detected only within the nucleus of PDGF-treated 3T3 cells. DNA binding of this phosphoprotein is activated by PDGF treatment with slow kinetics that match the time course of MCP-1 gene expression, and activation is not inhibited by cycloheximide. PDGF-activated binding to the 27-mer is shown to involve a single 30-kDa protein by UV-cross-linking analysis.

3T3 Cells↗
PubMed1995

Expression of platelet derived growth factor and platelet derived growth factor receptor mRNA in a glioblastoma from a patient with Li-Fraumeni syndrome.

Expression of platelet derived growth factor (PDGF) and PDGF-receptor mRNA was examined from a glioblastoma taken from a patient with Li-Fraumeni syndrome. Northern blot analysis and in situ hybridisation showed very high concentrations of both PDGF-A and PDGF alpha-receptor mRNA in the tumour. The overall pattern of PDGF expression was similar to those found in sporadic glioblastomas. Mutations in p53 has been implicated as an early pathogenic event leading to sporadic low grade astrocytomas, and is the third most common tumour type in patients with Li-Fraumeni syndrome, where they are predisposed due to a germline mutation in the p53 tumour suppressor gene. This study suggests that progression towards a glioblastoma in both the general population and in patients with Li-Fraumeni syndrome may involve potential autocrine and paracrine stimulation by growth factors such as PDGF.

Adult↗
PubMed1995

Axonal growth and fasciculation linked to differential expression of BDNF and NT3 receptors in developing cerebellar granule cells.

In the developing cerebellum, young granule neurons in the external germinal layer respond preferentially to BDNF, while mature neurons within the inner portion of the cerebellum respond preferentially to NT3. Here we show that this anatomic distinction reflects a developmentally regulated switch at the level of neurotrophin receptor gene expression. The salient feature of the developmental switch is a change in the ration of mRNA transcripts encoding functional BDNF and NT3 receptor tyrosine kinases. The ratio of the BDNF receptor trkB to the NT3 receptor trkC reverses from 5:1 in neonatal cerebellum to 1:3 in adult cerebellum. TrkB and TrkC are closely related transmembrane tyrosine protein kinases. However, activation of BDNF and NT3 receptors in cerebellar granule neurons do not give equivalent biological responses. In aggregate cell culture and single cell assays, BDNF enhances axonal outgrowth of early granule cells by influencing neurite elongation. In contrast, NT3 alters the morphology of outgrowth. Collectively, these findings suggest that regulation of neurotrophin receptors during cerebellar development is important for the timing and morphology of axonal growth.

Animals↗