Effects of Sphingosylphosphorylcholine on Vascular Smooth Muscle Cell Growth and on the Delayed Growth Response Cell Cycle Proteins Cdk2 and Cdc2
Abnormal vascular smooth muscle cell (VSMC) proliferation plays an important pathophysiological role in the development of vascular disease. We examined the effect of Sphingosylphosphorylcholine (SPC) on DNA synthesis ([3H]thymidine-incorporation method), cell number, cell volume, and cell diameter (CASY-1 coulter counter) as well as on the delayed response cell cycle proteins cyclin-dependent kinase2 (Cdk2) and Cdc2 (a serine/threonineprotein kinase) after stimulation of quiescent VSMCs with 5 μg/mL SPC for 2, 4, 6, 8, 12, 14, 16, 18, 20, 24, and 36 h. Cdk2 protein level is reduced when cells are in the G0-phase and its level is increased by the progression from G1- to S-phase upon addition of growth factors. Progression of the cells from the G2-phase (high level of tyrosine15-phosphorylated form of Cdc2) into M-phase is regulated by dephosphorylation of the Tyr15 residue of Cdc2 (34 kDa). Cdc2 phosphorylation status at Tyr15 and Cdk2 protein level were examined by chemiluminescence western blotting analysis using primary antibodies which recognizes Tyr15-phosphorylated Cdc2 and Cdk2 protein, respectively. Stimulation of VSMCs for 12, 14, 16, 18, 20, 24, and 32 h caused a 50±6, 50±11, 143±3, 192±15, 304±32, 221±29 and 245±26% increase in [3H]thymidine incorporation. Stimulation of VSMCs with SPC for 24, and 32 hour resulted in an 8 and 32% increase in cell number. Cell volume and radius was increased by 40 and 11%, respectively. Stimulation of quiescent VSMCs with SPC for 12, 16, 20, 24 and 32 h caused an 7, 51, 50, 76, and 32% increase of Cdk2 level. After stimulation of VSMCs for 20 and 24 h a 91 and 103% increase of Tyr15-phosphorylation, respectively, occurred. An almost complete Tyr15-dephoshorylation occurred after 32 h. We might conclude that SPC is a potent mitogenic and hyperthrophic factor for VSMCs acting through stimulation of Cdk2 and Cdc2.