Sustained MAP kinase activation is required for the expression of cyclin D1, p21Cip1 and a subset of AP-1 proteins in CCL39 cells

Oncogene. 1999 May 20;18(20):3085-97. doi: 10.1038/sj.onc.1202647.


In CCL39 cells thrombin is a potent growth factor which requires sustained activation of mitogen activated protein kinases (MAPKs) to promote DNA synthesis. Some of the effects of thrombin can be mimicked by peptides based on the new amino terminus of the cleaved receptor; however, these thrombin receptor peptides (TRPs) fail to induce sustained activation of MAPK or DNA synthesis. We have used thrombin, TRP-7 and other agonists which elicit sustained or transient MAPK activation to identify immediate-early and delayed-early genes which are only expressed under conditions of sustained MAPK activation focusing on cyclin D1, p21CiP1 and the AP-1 transcription factor. Of the stimuli tested only FBS and thrombin were able to stimulate a sustained activation of MAPK, expression of cyclin D1, p21Cip1 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 transient accumulation of c-Fos whereas the expression of Fra-1, Fra-2, c-Jun and JunB was sustained throughout the G1 phase of the cell cycle. We focussed our analysis on c-Fos (typical of AP-1 genes which are expressed rapidly and transiently) and Fra-1 and JunB (typical of AP-1 genes expressed after a delay but in a sustained manner). The expression of c-Fos, Fra-1 and JunB was dependent upon the activation of MAPK since these responses were inhibited by PD098059. However, a comparison of responses to FBS, thrombin, TRPs, LPA and EGF revealed that Fra-1 and JunB expression required sustained activation of MAPK whereas c-Fos expression was strongly induced even by non-mitogenic stimuli which elicited only transient MAPK activation. The expression of c-Fos (in response to thrombin, TRP or LPA) or Fra-1, JunB and cyclin D1 (thrombin only) was also inhibited by pertussis toxin. This suggests that both early and late AP-1 gene expression is regulated by the same Gi-mediated, MEK-dependent MAPK signalling pathway but that expression of late AP-1 genes and cyclin D1 requires that this pathway be persistently activated. The results suggest that the duration of receptor signalling and therefore MAPK activation is a key determinant of qualitative changes in gene expression during cell cycle re-entry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium-Calmodulin-Dependent Protein Kinases / antagonists & inhibitors
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Cell Cycle
  • Cell Division
  • Cell Line
  • Cricetinae
  • Cricetulus
  • Cyclin D1 / metabolism*
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins / metabolism*
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Flavonoids / pharmacology
  • Gene Expression Regulation / drug effects
  • Kinetics
  • Lysophospholipids / pharmacology
  • Pertussis Toxin
  • Thrombin / pharmacology
  • Transcription Factor AP-1 / antagonists & inhibitors
  • Transcription Factor AP-1 / metabolism*
  • Virulence Factors, Bordetella / pharmacology


  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins
  • Enzyme Inhibitors
  • Flavonoids
  • Lysophospholipids
  • Transcription Factor AP-1
  • Virulence Factors, Bordetella
  • Cyclin D1
  • Pertussis Toxin
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Thrombin
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one