Related Experiment Videos
Analysis of platelet-derived growth factor-induced phospholipase D activation in mouse embryo fibroblasts lacking
J A Hess1, Q S Ji, G Carpenter
1Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295, USA.
Abstract:
Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) in mouse embryo fibroblasts (MEFs). In order to investigate a role for phospholipase C-gamma1 (PLC-gamma1), we used targeted disruption of the Plcg1 gene in the mouse to develop Plcg1(+/+) and Plcg1(-/-) cell lines. Plcg1(+/+) MEFs treated with PDGF showed a time- and dose-dependent increase in the production of total inositol phosphates that was substantially reduced in Plcg1(-/-) cells. Plcg1(+/+) cells also showed a PDGF-induced increase in PLD activity that had a similar dose dependence to the PLC response but was down-regulated after 15 min. Phospholipase D activity, however, was markedly reduced in Plcg1(-/-) cells. The PDGF-induced inositol phosphate formation and the PLD activity that remained in the Plcg1(-/-) cells could be attributed to the presence of phospholipase C-gamma2 (PLC-gamma2) in the Plcg1(-/-) cells. The PLC-gamma2 expressed in the Plcg1(-/-) cells was phosphorylated on tyrosine in response to PDGF treatment, and a small but significant fraction of the Plcg1(-/-) cells showed Ca2+ mobilization in response to PDGF, suggesting that the PLC-gamma2 expressed in the Plcg1(-/-) cells was activated in response to PDGF. The inhibition of PDGF-induced phospholipid hydrolysis in Plcg1(-/-) cells was not due to differences in the level of PDGF receptor or in the ability of PDGF to cause autophosphorylation of the receptor. Upon treatment of the Plcg1(-/-) cells with oleoylacetylglycerol and the Ca2+ ionophore ionomycin to mimic the effect of PLC-gamma1, PLD activity was restored. The targeted disruption of Plcg1 did not result in universal changes in the cell signaling pathways of Plcg1(-/-) cells, because the phosphorylation of mitogen-activated protein kinase was similar in Plcg1(+/+) and Plcg1(-/-) cells. Because increased plasma membrane ruffles occurred in both Plcg1(+/+) and Plcg1(-/-) cells following PDGF treatment, it is possible neither PLC nor PLD are necessary for this growth factor response. In summary, these data indicate that PLC-gamma is required for growth factor-induced activation of PLD in MEFs.
Insights
Platelet-derived growth factor (PDGF) activates phospholipase D (PLD) via phospholipase C-gamma1 (PLC-gamma1) in mouse embryo fibroblasts. Disrupting the Plcg1 gene significantly reduced PDGF-induced PLD activity, indicating PLC-gamma1 is crucial for this signaling pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Platelet-derived growth factor (PDGF) is a key regulator of cellular processes.
- Phospholipase D (PLD) activation is implicated in PDGF signaling pathways.
- The role of phospholipase C-gamma1 (PLC-gamma1) in PDGF-induced PLD activation remains to be fully elucidated.
Purpose of the Study:
- To investigate the necessity of PLC-gamma1 for PDGF-induced PLD activation in mouse embryo fibroblasts (MEFs).
- To characterize the signaling mechanisms downstream of PDGF receptor in relation to PLC-gamma1 and PLD.
Main Methods:
- Development of Plcg1 gene-disrupted (Plcg1(-/-)) and wild-type (Plcg1(+/+)) MEF cell lines.
- Measurement of inositol phosphate production and PLD activity in response to PDGF stimulation.
- Analysis of PDGF receptor phosphorylation and downstream signaling pathways, including mitogen-activated protein kinase (MAPK).
Main Results:
- PDGF treatment led to a time- and dose-dependent increase in inositol phosphates and PLD activity in Plcg1(+/+) MEFs.
- Plcg1(-/-) MEFs exhibited significantly reduced PDGF-induced inositol phosphate production and PLD activity.
- Residual signaling in Plcg1(-/-) cells was attributed to phospholipase C-gamma2 (PLC-gamma2), which showed PDGF-induced phosphorylation and Ca2+ mobilization.
- Restoration of PLD activity in Plcg1(-/-) cells was achieved by mimicking PLC-gamma1 effects.
- MAPK phosphorylation and PDGF-induced membrane ruffling were unaffected in Plcg1(-/-) cells.
Conclusions:
- PLC-gamma1 is essential for the growth factor-induced activation of PLD in MEFs.
- PLC-gamma2 can partially compensate for the loss of PLC-gamma1 in PDGF signaling.
- Specific signaling pathways, like MAPK, are independent of PLC-gamma1 for PDGF-induced activation.