Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP

cAMPhas been found to play a role in mediating the negative regulation of c ell motility, although its underlying molecular mechanism remains poorly understood. By using CHO (Chinese hamster ovary) cells that express the EP2 subtype of PGE(2) (prostaglandin E,) receptors, we provide evidence that an increase in cellular cAMP content leads to inhibition of cellular Rac activity, which. serves as a mechanism for this negative regulation. In CHO cells expressing EP2, but not in vector control cells, PGE, dose-dependently inhibited chernotaxis towards IGF-I (insulin like growth factor-I). which is a Rac-dependent process, with the maximal 75 % inhibition observed at 10(-8) M PGE(2). EP2 stimulation failed to inhibit tyrosine phosphorylation either of IGF-I receptor or IRS-1 (insulin receptor substrate-1), or activation of phosphoinositicle 3-kinase or Akt in response to IGF-I, but potently and dose -dependently inhibited IGF-I-induced activation of cellular Rae activity and membrane ruffling. However, PGE(2) failed to inhibit Val(12) -Rac-induced membrane ruffling. Similar to the case of CHO cells, PGE, inhibited PDGF (platelet-derived growth factor)-induced Rae activation and chernotaxis in vascular smooth muscle cells endogenously expressing EP2. The inhibitory effects of PGE, on IGF-I-induced chernotaxis, membrane ruffling and Rac activation were faithfully reproduced by a low concentration of forskolin, which induced a comparable extent of cAMP elevation as with 10(-8) M PGE(2), and were potentiated by isobutylmethylxanthine. The protein kinase A inhibitor Rp isomer of adenosine 3',5'-cyclic monophosphorothioate reduced PGE(2) inhibition of Rac activation and chernotaxis. These results indicate that EP2 mediates Rae inhibition through a mechanism involving cAMP and protein kinase A, thereby inhibiting membrane ruffling and chernotaxis.