scholarly journals Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells

1999 ◽  
Vol 339 (3) ◽  
pp. 759 ◽  
Author(s):  
Claudia GUTACKER ◽  
Gerd KLOCK ◽  
Patrick DIEL ◽  
Claudia KOCH-BRANDT
Keyword(s):  
Gene Expression ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Pc12 Cells ◽  
Nerve Growth ◽  
Epidermal Growth

scholarly journals Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells

1999 ◽  
Vol 339 (3) ◽  
pp. 759-766 ◽  
Author(s):  
Claudia GUTACKER ◽  
Gerd KLOCK ◽  
Patrick DIEL ◽  
Claudia KOCH-BRANDT
Keyword(s):  
Gene Expression ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Neuronal Differentiation ◽  
Pc12 Cells ◽  
Binding Studies ◽  
Nerve Growth ◽  
Epidermal Growth ◽  
Differentiation And Proliferation

Clusterin (apolipoprotein J) is an extracellular glycoprotein that might exert functions in development, cell death and lipid transport. Clusterin gene expression is elevated at sites of tissue remodelling, such as differentiation and apoptosis; however, the signals responsible for this regulation have not been identified. We use here the clusterin gene as a model system to examine expression in PC12 cells under the control of differentiation and proliferation signals produced by nerve growth factor (NGF) and by epidermal growth factor (EGF) respectively. NGF induced clusterin mRNA, which preceded neurite outgrowth typical of neuronal differentiation. EGF also activated the clusterin mRNA, demonstrating that both proliferation and differentiation signals regulate the gene. To localize NGF- and EGF-responsive elements we isolated the clusterin promoter and tested it in PC12 cell transfections. A 2.5 kb promoter fragment and two 1.5 and 0.3 kb deletion mutants were inducible by NGF and EGF. The contribution to this response of a conserved activator protein 1 (AP-1) motif located in the 0.3 kb fragment was analysed by mutagenesis. The mutant promoter was not inducible by NGF or EGF, which identifies the AP-1 motif as an element responding to both factors. Binding studies with PC12 nuclear extracts showed that AP-1 binds to this sequence in the clusterin promoter. These findings suggest that NGF and EGF, which give differential gene regulation in PC12 cells, resulting in neuronal differentiation and proliferation respectively, use the common Ras/extracellular signal-regulated kinase/AP-1 signalling pathway to activate clusterin expression.

scholarly journals Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells.

1983 ◽  
Vol 97 (1) ◽  
pp. 92-98 ◽  
Author(s):  
J Boonstra ◽  
W H Moolenaar ◽  
P H Harrison ◽  
P Moed ◽  
P T van der Saag ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Growth Stimulation ◽  
Initial Growth ◽  
Nerve Growth ◽  
Pump Activity ◽  
Epidermal Growth

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.

Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors

1993 ◽  
Vol 13 (9) ◽  
pp. 5500-5512
Author(s):  
K L Suen ◽  
X R Bustelo ◽  
T Pawson ◽  
M Barbacid
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Pc12 Cells ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Binding Studies ◽  
Trk Receptors ◽  
Nerve Growth ◽  
Epidermal Growth

We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein.

scholarly journals Nerve growth factor- and epidermal growth factor-stimulated phosphorylation of a PC12 cytoskeletally associated protein in situ.

1985 ◽  
Vol 100 (3) ◽  
pp. 677-683 ◽  
Author(s):  
G E Landreth ◽  
G D Rieser
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Pc12 Cells ◽  
Receptor Occupancy ◽  
Hormone Action ◽  
Intact Cells ◽  
Nerve Growth ◽  
Epidermal Growth

Nerve growth factor (NGF) and epidermal growth factor (EGF) produce stable alterations in PC12 cells that persist in the detergent-insoluble cytoskeleton, resulting in the phosphorylation of a 250,000-mol-wt cytoskeletally associated protein in situ. Treatment of PC12 cells with NGF or EGF, followed by detergent lysis of the cells and incubation of the resulting cytoskeletons with gamma-32P-ATP, permitted detection of hormonally stimulated, energy-dependent events, which result in the enhanced phosphorylation of a cytoskeletally associated protein as an immediate consequence of receptor occupancy. These events were elicited only upon treatment of intact cells at physiological temperatures. The NGF- and EGF-stimulated events occurred rapidly; however, they were a transient effect of hormone action. NGF and EGF were found to act through independent mechanisms to stimulate the in situ phosphorylation of the 250,000-mol-wt protein, as the effects of NGF, but not EGF, were blocked by methyltransferase inhibitors. The 250,000-mol-wt protein was phosphorylated on serine and threonine residues in response to both NGF and EGF although in somewhat different proportions. The data suggest that the hormone-stimulated labeling of the 250,000-mol-wt protein may be the result of either the direct activation of a protein kinase, the redistribution of the kinase relative to its substrates as a consequence of hormone action, or the coincident occurrence of these events.

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