Abstract 10623: Phosphorylation of Forkhead Transcription Factors Regulate Thrombin-Induced Proliferation of Human Vascular Smooth Muscle Cells
Forkhead box-O (FoxO) transcription factors regulate cellular processes including mitogenesis and cell cycle arrest. Phosphorylation of FoxO controls these cellular responses via the PI3K/AKT kinase pathway. Thrombin activates mitogenic signaling in vascular smooth muscle cells (VSMCs) via stimulation of the G-protein-coupled protease activated receptors (PAR-1, -3, and -4). Thereby thrombin elicits cellular responses that contribute to the pathogenesis of vascular disorders such as atherosclerosis. We investigated that FoxO proteins are downstream effectors of thrombin signaling in human VSMCs in vitro. Consequently, we aimed to determine whether phosphorylation of FoxO proteins upon stimulation of PARs is involved in thrombin-mediated proliferative responses in human VSMCs. The VSMCs were stimulated by thrombin (3U/ml) or PAR-1, -3 and -4-selective peptides. Protein expression was determined by immunoblotting; gene silencing was carried out using specific FoxO siRNAs. Mitogenesis and proliferation were performed by DNA synthesis via [3H]-thymidine incorporation and cell counting, respectively. FoxO dependent expression of p21CIP1 and p27kip1 was measured by qRT-PCR, target genes involved in cell cycle regulation. Phosphorylation of FoxO1 and FoxO3 occurred in a time-dependent manner after stimulation with thrombin and PAR-1 activating peptide, while activation of PAR-3 and PAR-4 by selective activating peptides had no effect (n=3). In parallel, phosphorylation of AKT, ERK1/2 and p38 was also observed (n=3). These effects were prevented by the selective inhibitor of PI3K, LY-294002, but not by inhibition of the ERK1/2 or p38 pathways (n=3). Thrombin-enhanced mitogenesis and proliferation were attenuated by siRNA-mediated silencing of the FoxO1 and FoxO3 genes (n=6). This suggested that thrombin caused deactivation of FoxO factors in the nucleus, which subsequently down-regulated the mRNA and protein levels of p21CIP1 and p27kip1 (n=5). Our results suggest that thrombin-induced VSMC proliferation is dependent on phosphorylation and subsequent deactivation of FoxO factors via PAR-1 in a PI3K/AKT-dependent manner. This conclusion is further supported by the observed downregulation of the p21CIP1 and p27kip1 target genes.
- © 2011 by American Heart Association, Inc.