DIFFERENTIAL UP-REGULATION OF RAP1A AND RAP1B PROTEINS DURING SMOOTH-MUSCLE CELL-CYCLE

The relationship between Rap1 proteins and cell proliferation was asse ssed by investigating the effect of isoforms AA and BE of platelet-der ived growth factor (PDGF) on Rap1 protein and mRNA expression througho ut the smooth muscle cell cycle, Firstly PDGF BB-induced cell cycle tr averse was studied, thus demonstrating entry into S phase at 18 to 20 h, Western blotting carried out on total Rap1 proteins showed that 5 n g/ml of PDGF BE instigated a biphasic induction of total Rap1 proteins during the cell cycle, This involved a 2.1 +/- 0.4-fold increase at 6 h (early G(1)) and a 2.8 +/- 0.6-fold increase at 20 to 24 h (G(1)/S transition), Such an up-regulation was abolished by addition of 1 ng/m l of transforming growth factor-beta 1 (TGF-beta 1), which inhibited u p to 80% of the PDGF BE-induced entry into S phase, Comparative RT-PCR of both rap1a and rap1b mRNAs throughout the cell cycle allowed us to differentiate between the two rap1a and rap1b species, PDGF BE induce d a 1.9 +/- 0.3-fold increase at 4 h and a 2.4 +/- 0.2-fold relative i ncrease at 16 h for rap1b mRNA, whereas a unique 1.9 +/- 0.5-fold incr ease in rap1a mRNA was observed at 14 h. Again, this induction of rap1 a and rap1b mRNAs by PDGF BE was totally abolished by TGF-beta 1, We c onclude that the differential up-regulation of Rap1a and Rap1b protein s during the smooth muscle cell cycle is directly linked to cell proli feration.