Expression of mRNAs of multiple growth factors and receptors by neuronal cell lines: Detection with RT-PCR (original) (raw)
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Cellular and Molecular Neurobiology, 1995
1. Although glial cells in culture are known to secrete growth factors and are also known to be responsive to some of them, detailed comparisons are difficult because the bulk of information was based on various animals of origin, developmental stages, growth properties, culture age, and culture conditions. 2. To present a unified picture of the growth factors and their receptors found in glial cells, we surveyed the expression of messenger RNAs of a panel of growth factors and receptors, using reverse transcription-polymerase chain reaction (RT-PCR), in three common glial cell types: rat astrocytes in primary culture, rat glioma line C6, and human glioma line A172. 3. We observed that normal and neoplastic glial cells in culture express multiple growth factors and also possess most of the receptors to these factors, suggesting multiple autocrine functions. In addition, glia produce growth factors known to be capable of acting on neurons, implicating paracrine function involving gila-neuron interaction. Glial cells also produce growth factors and receptors that are capable of communicating with hematopoietic cells, suggesting
Identification and Characterization of Glial Growth Factor
2019
A combination of biochemical, cell biological and immunological techniques have been employed to identify a novel and potent polypeptide mitogen of the brain and pituitary. This molecule, named glial growth factor (GGF), stimulates DNA synthesis and cell division in cultured rat Schwann cells, astrocytes, and fibroblasts. Three independent lines of evidence indicate that GGF activity resides in a basic protein of molecular weight 3.1 x 104. (a) When partially purified preparations are analyzed by native gel electrophoresis at pH 4.5, mitogenic activity migrates with a protein of this molecular weight, as revealed by bioassay coupled with a second dimension of SDS gel electrophoresis. (b) A set of monoclonal antibodies which deplete growth factor activity from heterogeneous solutions specifically recognize a 31,000 dalton protein antigen, as determined by gel immunoautoradiography. (c) GGF activity is recovered at a molecular weight of 3.1 x 104 after denaturing polyacrylamide gel el...
Growth factors, glia and gliomas
Journal of Neuro-oncology, 1997
The abilities of growth factors to cause normal cells to express theproperties associated with transformed cells is discussed in specificreference to the oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell. Inthe O-2A lineage, it has been possible to use growth factors and otherdefined molecules to induce or promote in normal cells all of the mainproperties of tumor cells, these being continued cell division in theabsence of differentiation, more subtle modulations of self-renewalprobabilities, promotion of cell migration and inhibition of programmed celldeath. In addition to our studies on primary cells, our application to thegrowth of human tumor specimens of techniques utilized to study primaryglial progenitor cells has allowed us to isolate a human glioblastomamultiforme (GBM)-derived population that expresses many properties otherwiseuniquely expressed by oligodendrocyte-type-2 astrocyte (O-2A) progenitorcells. Hu-O-2A/Gb1 (for Human O-2A lineage Glioblastoma number 1) cellsresponded to similar mitogens and differentiation modulators as rodent O-2Aprogenitors, and generated cells with features of precursor cells,oligodendrocytes and astrocytes. Moreover, 1H-NMR analysis ofamino acid composition demonstrated a striking conservation of types andquantities of free amino acids between the human tumour cells and the rodentprimary cells. Hu-O-2A/Gb1 cells represent the first human glioma-derivedpopulation for which unambiguous lineage assignment has been possible. Ourresults thus demonstrate that the human O-2A lineage can contribute to oneof the most malignant of glial tumours. Our analyses further indicate thatat least two distinct glial lineages can generate glioblastomas. Inaddition, the highly diagnostic 1H-NMR spectrum expressed byHu-O-2A/Gb1 cells raises the possibility of eventual non-invasiveidentification of tumors of this lineage.
Molecular Brain Research, 2002
Nerve growth factor (NGF) influences neuronal development, function, and response to injury. Using reverse transcriptase polymerase chain reaction, we find that mouse and rat cortex and spinal cord, and both neurons and glia in culture, express NGF mRNA. In the mouse, NGF is regulated by at least two promoters that govern synthesis of four different transcripts, A through D, that are all expressed in the mouse tissues and cells examined. In contrast, rat NGF expression varies with tissue and with cell type: transcript C is expressed strongly in brain but weakly in spinal cord, and transcript D is undetectable in rat central nervous system (CNS). In addition to speciesand tissue-specific expression, NGF transcripts also exhibit cell type-specific expression: transcripts B, C and D are expressed in rat astrocytes but poorly or not at all in rat neurons, identifying glia as an important source of NGF in rat. NGF increases sharply after injury. TGF-b1, which also increases immediately after injury, induces NGF mRNA and protein in rat and mouse glia but not in neurons. Furthermore, transcripts A, B and D, but not C, are upregulated by TGF-b1 in mouse glia, whereas in rat glia, the major responsive transcript is C. Thus, there may be multiple TGF-b1-responsive elements in the NGF promoters located upstream of exons 1 and 3 that may differ between mouse and rat. Moreover, NGF transcripts are differentially expressed in a species-, cell type-, and inducer-specific manner. These results have implications for the use of mice versus rats as models for the study of NGF regulation following CNS injury. (M. Fahnestock).
Identification and purification of glial growth factor
The Journal of Neuroscience, 1984
Cultured rat Schwann cells are stimulated to divide by a protein growth factor, present in extracts of bovine brain and pituitary, which we have named glial growth factor (GGF). Two lines of evidence indicate that GGF activity in both brain and pituitary resides in a protein of Mr = 31,000. (1) Four independently isolated monoclonal antibodies that immunoprecipitate the activity react with an antigen of this molecular weight in sodium dodecyl sulfate (SDS)-polyacrylamide gels. (2) After SDS-polyacrylamide gel electrophoresis of partially purified preparations, mitogenic activity on Schwann cells is recovered at this molecular weight. GGF has been purified approximately 10(5)-fold to apparent homogeneity from bovine pituitary anterior lobes by a combination of column chromatography steps and preparative SDS gel electrophoresis. Purified human platelet-derived growth factor, a molecule with properties similar to those of GGF, is inactive on Schwann cells and therefore appears to be di...
Journal of Neuroscience Research, 1992
The neurotrophic proteins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are related in their primary amino acid structures. In this study we investigated the extent to which the low-affinity NGF receptor (LNGFR) in C6 glioma cells can discriminate between the neurotrophins NGF and BDNF. LNGFR-immunoreactivity (IR) was studied in C6 cells treated for 16 hr with NGF (50 ng/ml) or BDNF (10 ng/ml), using immunogold labeling and electron microscopic morphometric analysis. The cells were exposed to the anti-NGFR antibody 192-IgG, followed by immunoglobulin conjugated with colloidal gold. Untreated C6 cells exhibited some surface gold label (positive LNGFR-IR). Cells treated with NGF or BDNF displayed significantly increased LNGFR-IR on all surfaces in terms of gold labeling, which was more pronounced in NGF-treated cells. LNGFR-IR was also localized in coated endocytotic vesicles, in smooth endoplasmic reticulum, and in secondary multivesicular lysosomes in neurotrophin-treated and untreated cells. The increase in LNGFR protein was further substantiated by a correspondingly higher content of LNGFR mRNA detected after 15 hr of either NGF or BDNF treatment. These results suggest that the LNGFR in glial cells can be upregulated by the structurally related neurotrophins NGF and BDNF.
Neuro-glia interaction effects on GFAP gene: a novel role for transforming growth factor-β1
European Journal of Neuroscience, 2002
Central nervous system (CNS) development is highly guided by microenvironment cues specially provided by neuron±glia interactions. By using a transgenic mouse bearing part of the gene promoter of the astrocytic maturation marker GFAP (glial ®brillary acidic protein) linked to the b-galactosidase (b-Gal) reporter gene, we previously demonstrated that cerebral cortical neurons increase transgenic b-Gal astrocyte number and activate GFAP gene promoter by secretion of soluble factors in vitro. Here, we identi®ed TGF-b1 as the major mediator of this event. Identi®cation of TGF-b1 in neuronal and astrocyte extracts revealed that both cell types might synthesize this factor, however, addition of neurons to astrocyte monolayers greatly increased TGF-b1 synthesis and secretion by astrocytes. Further, by exploiting the advantages of cell culture system we investigated the in¯uence of neuron and astrocyte developmental stage on such interaction. We demonstrated that younger neurons derived from 14 embryonic days wild-type mice were more ef®cient in promoting astrocyte differentiation than those derived from 18 embryonic days mice. Similarly, astrocytes also exhibited timed-schedule developed responsiveness to neuronal in¯uence with embryonic astrocytes being more responsive to neurons than newborn and late postnatal astrocytes. RT-PCR assays identi®ed TGF-b1 transcripts in young but not in old neurons, suggesting that inability to induce astrocyte differentiation is related to TGF-b1 synthesis and secretion. Our work reveals an important role for neuron±glia interactions in astrocyte development and strongly implicates the involvement of TGF-b1 in this event.
Neuroscience Letters, 1999
Glia maturation factor (GMF) is a 17-kDa protein unique to the nervous system. Although GMF was initially characterized as a growth/differentiation factor, the absence of a leader sequence and its intracellular localization in normal brain suggest an intracellular function as well. In this paper we transfected the C6 glioma cells with GMF cDNA by infecting the cells with a GMF/adenovirus construct. The transfected cells overexpressed GMF but did not secret the protein into the culture medium. However, the transfected cells showed an increased expression of the neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The increase in neurotrophic activity of the C6 cell conditioned medium was demonstrable by its ability to promote neurite outgrowth in PC12 cells. q