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

Oncogene volume 18, pages 3085–3097 (1999)Cite this article

Abstract

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.

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Acknowledgements

We would like to thank members of the Babraham Inositide laboratory for stimulating discussions and Drs Michelle Garret and Nancy Pryer for advice on antibodies for cyclin D1 and p21Cip1. We are grateful to Dr Fergus McKenzie and Prof Jacques Pouysségur for providing CCL39 cells and Dr David Gillespie for providing c-Jun antisera. In addition we would like to thank members of the Babraham Microchemical Facility for production of reagents, particularly Mark Quigley for synthesis of the TRP-7 peptide and synthesis and conjugation of the p44MAPK/ERK1 C-terminal peptide and Arthur Davis for innoculation of rabbits and collection of serum. This work was supported by a competitive strategic grant from the BBSRC.

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  1. Signalling Programme, The Babraham Institute, Babraham Hall, Cambridge, CB2 4AT, UK

    Kathryn Balmanno & Simon J Cook

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Balmanno, K., Cook, S. Sustained MAP kinase activation is required for the expression of cyclin D1, p21Cip1 and a subset of AP-1 proteins in CCL39 cells. Oncogene 18, 3085–3097 (1999). https://doi.org/10.1038/sj.onc.1202647

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