A novel function of FoxO transcription factors in thrombin-stimulated vascular smooth muscle cell proliferation.

Thrombin exerts coagulation-independent effects on the proliferation and migration of vascular smooth muscle cells (SMC). Forkhead box-O (FoxO) transcription factors regulate cell proliferation, apoptosis and cell cycle arrest, but a possible functional interaction between thrombin and FoxO factors has not been identified to date. In human cultured vascular SMC, thrombin induced a time-dependent phosphorylation of FoxO1 and FoxO3 but not FoxO4. This effect was mimicked by an activating-peptide (AP) for protease-activated receptor (PAR)-1, and abolished by a PAR-1 antagonist (SCH79797). APs for other PARs were without effect. FoxO1 and FoxO3 phosphorylation were prevented by the PI3 kinase (PI3K) inhibitor LY294002 while inhibitors of ERK1/2 (PD98059) or p38MAPK (SB203580) were ineffective. LY294002 moreover prevented thrombin-stimulated SMC mitogenesis and proliferation. FoxO1 and FoxO3 siRNA augmented basal DNA synthesis and proliferation of SMC. Nuclear content of FoxO proteins decreased time-dependently in response to thrombin, coincided with suppressed expression of the cell cycle regulating genes p21CIP1 and p27kip1 by thrombin. FoxO1 siRNA reduced basal p21CIP1 while FoxO3 siRNA attenuated p27kip1 expression; thrombin did not show additive effects. LY294002 restored p21CIP1 and p27kip1 protein expression. Immunohistochemistry revealed that human native and failed saphenous vein grafts were characterised by the cytosolic presence of p-FoxO factors in co-localisation of p21CIP1 and p27kip1 with SMC. In conclusion, thrombin and FoxO factors functionally interact through PI3K/Akt-dependent FoxO phosphorylation leading to expression of cell cycle regulating genes and ultimately SMC proliferation. This may contribute to remodelling and failure of saphenous vein bypass grafts.