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Microinjection of a p21ras antibody into PC12 cells inhibits neurite outgrowth induced by nerve growth factor and
J G Altin1, R Wetts, R A Bradshaw
1Department of Biological Chemistry, California College of Medicine, Irvine.
Abstract:
The role of p21ras in signal transduction in PC12 cells was studied using an antibody that blocks its function. Native cells were microinjected with either a control solution or a solution containing the monoclonal antibody Y13-259. Treatment of the cells with growth factors appeared to enhance the ability of the cells to survive the microinjection procedure. Of the cells microinjected with the control solution 66-69% of those treated with either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) were still present 24 h post-injection, compared with only 57% for those not treated with growth factor after microinjection. This effect of the growth factors was inhibited by introduction of the Y13-259 antibody, suggesting that it occurs through a pathway that involves p21ras. Similarly, introduction of the Y13-259 antibody into cells also resulted in a statistically significant decrease in the percentage of neurite-bearing cells; 25-36% of the cells microinjected with the control solution had neurites, whereas 12-14% of the cells microinjected with the antibody solution had neurites. This decrease suggests that the induction of neurite outgrowth and the maintenance of established neurites by these growth factors is dependent on a functional p21ras pathway. As well as complementing the finding that p21ras is apparently involved in the mechanism of action of NGF in PC12 cells, these results further establish (1) that p21ras is also involved in the mechanism of action of bFGF, and (2) that the effect of NGF and bFGF on the number of labeled cells still present 24 h postinjection requires a functional p21ras protein.
Insights
The p21ras protein is crucial for PC12 cell survival and neurite outgrowth, mediating the effects of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF). Blocking p21ras function with an antibody inhibits these growth factor-induced responses.
Area of Science:
- Cell Biology
- Molecular Biology
- Neuroscience
Background:
- PC12 cells are a model system for studying neuronal differentiation.
- p21ras is a key signaling protein involved in cell growth and differentiation.
- Nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) are known to promote survival and neurite outgrowth in PC12 cells.
Purpose of the Study:
- To investigate the role of p21ras in signal transduction pathways activated by NGF and bFGF in PC12 cells.
- To determine if p21ras mediates the survival effects of NGF and bFGF in PC12 cells.
- To assess the involvement of p21ras in NGF- and bFGF-induced neurite outgrowth.
Main Methods:
- Microinjection of PC12 cells with a monoclonal antibody (Y13-259) that inhibits p21ras function or a control solution.
- Treatment of cells with NGF or bFGF.
- Assessment of cell survival 24 hours post-injection.
- Quantification of neurite-bearing cells.
Main Results:
- Growth factors (NGF and bFGF) enhanced PC12 cell survival after microinjection, an effect blocked by the Y13-259 antibody.
- The Y13-259 antibody significantly reduced the percentage of neurite-bearing cells induced by growth factors.
- These findings indicate that p21ras is essential for mediating the survival and neurite outgrowth effects of NGF and bFGF.
Conclusions:
- p21ras plays a critical role in the signal transduction pathways of both NGF and bFGF in PC12 cells.
- Functional p21ras protein is required for the survival-promoting effects of NGF and bFGF.
- p21ras is essential for NGF- and bFGF-induced neurite outgrowth and maintenance in PC12 cells.