|Budget Amount *help
¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1997 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥800,000 (Direct Cost : ¥800,000)
To elucidate the role of protein kinase C (PKC) in vascular cell proliferation, we examined the effects of phorbol-myristate-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) on the cell cycle events in smooth muscle and endothelial cells.
The role of PKC in the G_1/S transition was studied using G_0-synchronized human umbilical artery smooth muscle cells. [^3H] thymidine incorporation started 15 h after stimulation with 20% fetal bovine serum and 10ng/ml basic fibroblast growth factor. PMA inhibited the incorporation over 90% when added earlier than 3 h, but the inhibition was attenuated when PMA was added at 6 h or later. PMA inhibited the phosphorylation of the retinoblastoma rotein (pRb), which normally began at about 9 h. PMA inhibited the activity of Cdk2, which increased from about 9 h, whereas PMA did not inhibit Cdk4/6 activities, which increased from 0-3 h. OAG (10muM) added repeatedly from 3 h also inhivited [^3H] thymidine incorporation, pRb phosphorylation, and Cdk2 act
ivity. PMA did not inhibit the mRNA expression of Cdk2, Cdk3, Cdk4, Cdk5, and cyclins G,C,and D,all of which began at 0-3 h, whereas PMA reduced the mRNA expression of cyclins E and A,which usually began at 3-9 h and about 15 h, respectively. However, PMA did not reduce the protein levels of cyclins E and A.PMA had no influence on the expression of Cdk inhibitors p21 and p27. PMA inhibited the shift of Cdk2 to a slower migrating form in SDS-PAGE that represents Thr160 phosphorylation and Tyr15 dephosphorylation.
The role of PKC in the G_2/M transition was investigated in human umbilical vein endothelial cells released from the G_1/S border. PMA caused G_2 arrest because, firstly, when added to G_2 cells, PMA inhibited subsequent cell division, secondly, these growth-arrested cells did not show morphological features of mitotic cells, and thirdly, PMA did not interrupt mitosis in cells released from nocodazole-induced M phase arrest. OAG also inhibited moitosis. The activation of Cdc2 kinase around the G_2/M transition was suppressed by PMA and OAG.Although Cdc2 was expressed in the presence of PMA,dephosphorylation of its tyrosine residue was inhibited by PMA.In parallel, the expression of Cdc25B was suppressed by PMA.The total and the Cdc2 associated amount of cyclin B were both reduced by PMA.
Therefore the PKC pathway negatively regulates both the G_1/S and G_2/M transitions by inhibiting Cdk2 and Cdc2, respectively. The suppression of Cdk activities may result from inhibited threonine phosphorylation, tyrosine dephosphorylation, and expression of their partner cyclins. Less