Sustained MAP kinase activation is required for the expression of cyclin D1, p21(Cip1) and a subset of AP-1 proteins in CCL39 cells

In CCL39 cells thrombin is a potent growth factor which requires sustained activation of mitogen activated protein kinases (MAPKs) to promote DNA synt hesis. Some of the effects of thrombin can be mimicked by peptides based on the new amino terminus of the cleaved receptor; however, these thrombin re ceptor peptides (TRPs) fail to induce sustained activation of MAPK or DNA s ynthesis, We have used thrombin, TRP-7 and other agonists which elicit sust ained or transient MAPK activation to identify immediate-early and delayed- early genes which are only expressed under conditions of sustained MAPK act ivation focusing on cyclin D1, p21(Cip1) and the AP-1 transcription factor, Of the stimuli tested only FBS and thrombin were able to stimulate a susta ined activation of MAPK, expression of cyclin D1, p21(Cip1) and cell cycle re-entry, The expression of cyclin D1 was strongly, though not completely, inhibited by the MEK1 inhibitor PD098059. Thrombin stimulated a rapid but t ransient accumulation of c-Fos whereas the expression of Fra-1, Fra-2, c-Ju n and JunB was sustained throughout the G1 phase of the cell cycle. We focu ssed our analysis on c-Fos (typical of AP-1 genes which are expressed rapid ly and transiently) and Fra-1 and JunB (typical of AP-1 genes expressed aft er a delay but in a sustained manner). The expression of c-Fos, Fra-1 and J unB was dependent upon the activation of MAPK since these responses were in hibited by PD098059, However, a comparison of responses to FBS, thrombin, T RPs, LPA and EGF revealed that Fra-1 and JunB expression required sustained activation of MAPK whereas c-Fos expression was strongly induced even by n on-mitogenic stimuli which elicited only transient MAPK activation, The exp ression of c-Fos (in response to thrombin; TRP or LPA) or Fra-1, JunB and c yclin D1 (thrombin only) was also inhibited by pertussis toxin, This sugges ts that both early and late AP-1 gene expression is regulated by the same G i-mediated, MEK-dependent MAPK signalling pathway but that expression of la te AP-1 genes and cyclin D1 requires that this pathway be persistently acti vated. The results suggest that the duration of receptor signalling and the refore MAPK activation is a key determinant of qualitative changes in gene expression during cell cycle re-entry.