Abstract 500: PI 3-Kinase and Phospholipase C-γ Mediate the Platelet-Derived Growth Factor (PDGF)-Induced Cell Cycle Progression by Different Mechanisms
βPDGFR-mediated proliferation of VSMC plays a crucial role in atherogenesis and restenosis.
The βPDGFR activates cytoplasmic signaling molecules such as Src, PI3K, RasGAP, SHP-2 and PLCγ. The βPDGFR-induced signaling pathways leading to cell cycle progression are largely unknown. In order to characterize the signaling molecules implicated in PDGF-induced VSMC proliferation, we used mutated βPDGFR in which the binding sites of each signaling molecule were individually deleted. To bypass endogenous PDGFR, we used chimeric receptors containing the extracellular ligand binding domain of the M-CSFR. BrdU incorporation assays revealed that stimulation of the chimeric wild type receptor (WT) with M-CSF (50 ng/ml) led to a 2.8±0.6-fold increase of DNA-synthesis (p<<med>0.01). Deletion of the binding sites for PI3K or PLCγ diminished DNA-synthesis to 47±6% or 54±5%, respectively, whereas deletion of the binding sites for Src, RasGAP, or SHP-2 had no effect. Accordingly, chimeric mutants which only bind PI3K or PLCγ mediated 43±4% or 52±5% of the WT response. Consequently, we investigated the influence of PI3K and PLCγ on the expression of the keyplayers of cell cycle progression such as immediate early genes (IEGs), cyclins, and inhibitors of cdk. As a first response, IEGs were already upregulated 30 min after stimulation of VSMC with PDGF-BB (c-myc: 14.0±2.1fold, c-fos: 101±24fold and egr-1: 51±14fold, p<<med>0.01) as assessed by real-time PCR. Analysis of the PDGFR mutants demonstrated that up-regulation of IEGs depend mostly on PLCγ. 8 h later, expression levels of cyclins and cdk-inhibitors in VSMC began to change. Westernblot analysis revealed that PI3K induced up-regulation of cyclin D1, whereas PLCγ mediated the down-regulation of the cdk-inhibitor p27kip1. Consistently, PDGF-dependent phosphorylation of the retinoblastoma protein which is important for the G1/S transition was abolished when the binding sites for PI3K or PLCγ were deleted.Our results show that the activated βPDGFR mediates proliferation of VSMC via different signaling pathways, which are activated by PI3K and PLCγ. These signaling molecules mediate the PDGF-induced DNA-synthesis by upregulation of IEGs and cyclins and downregulation of cdk-inhibitors via different signaling pathways.