In our previous study, we showed that PI 3-kinase was involved in the major signaling pathway in response to FGF-2 stimulation in CECs.
10 We further investigated whether the antiproliferative effect of cAMP is mediated through inhibiting PI 3-kinase/Akt pathways. We first determined the inhibitory action of 8-Br-cAMP on PI 3-kinase activity
(Fig. 2A) . The serum-starved CECs were treated with 8-Br-cAMP for 24 hours in concentrations ranging from 0.1 to 1 mM in the presence of FGF-2. Cell extracts were immune-precipitated with anti-PI 3-kinase (p85 subunit) antibody. They were then assayed for PI 3-kinase activity by measuring the phospholipid product PI-3-P. Cell extracts obtained from FGF-2 treatment contained high levels of PI 3-kinase activity. 8-Br-cAMP inhibited PI 3-kinase enzyme activity in a dose-dependent manner
(Fig. 2A) . Both 8-Br-cAMP at 1 mM and LY294002 at 20 μM again equally inhibited PI 3-kinase enzyme activity. Cells treated with both LY294002 (20 μM) and 8-Br-cAMP (1 mM) showed no synergistic effect on FGF-2–stimulated PI 3-kinase activity.
Figure 2A also demonstrates that 8-Br-cAMP had no effect on the basal PI 3-kinase activity in the absence of FGF-2 stimulation. These data suggest that cAMP is the upstream molecule to PI 3-kinase in the mitogenic pathway of FGF-2. The activation of PI 3-kinase in response to FGF-2 was further confirmed with PI 3-kinase–mediated Akt phosphorylation, using specific antibodies for phosphorylated Akt (Ser473 or Thr308;
Fig. 2B ). Akt phosphorylation at both Thr308 and Ser473 was mediated in response to FGF-2 stimulation, and 8-Br-cAMP markedly reduced Akt phosphorylation at both the Thr308 and Ser 473 sites
(Fig. 2B) . This inhibitory effect of cAMP on Akt activation further confirmed that cAMP is the upstream molecule to PI 3-kinase/Akt pathways. This observation is in agreement with previous reports on the inhibitory action of cAMP on Akt phosphorylation.
26 27 We further investigated whether cAMP regulated Akt expression at the protein level
(Fig. 2C) . When CECs were treated with FGF-2 alone for 1 hour to 24 hours, Akt level was slightly increased as a function of the duration of FGF-2 stimulation. When cells were simultaneously treated with FGF-2 and 8-Br-cAMP, CECs demonstrated a marked time-dependent reduction of Akt expression: an 8-hour treatment of cells with 8-Br-cAMP reduced the protein level by approximately 40%, and longer treatment of cells with 8-Br-cAMP facilitated further reduction of Akt protein level. These findings are different from the previous report
27 in which total Akt protein levels were not altered by 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) treatment, regardless of the treatment duration. We do not know what causes the discrepancy observed in the two studies, except that two different cAMP analogues were used. However, unlike the unstable Akt level, the p85 protein level of PI 3-kinase was not altered by treating cells with 8-Br-cAMP
(Fig. 2C) , suggesting that the reduced levels of Akt after 8-Br-cAMP treatment are not artifacts of the culture system. The reduced levels of Akt mediated by 8-Br-cAMP may be attributable to rapid turnover of the protein. To investigate this possibility, CECs were first stimulated with FGF-2 for 24 hours. Cells were then treated with cycloheximide alone or with 8-Br-cAMP.
Figure 2D shows that, in the cells treated with cycloheximide alone, the Akt level was not altered. However, when cells were simultaneously treated with cycloheximide and 8-Br-cAMP, there was a marked reduction of Akt level within 2 hours of treatment. These data indicate that cAMP regulates the stability of Akt protein, thus leading to a rapid turnover.