Forkhead Transcription Factor FKHR-L1 Modulates Cytokine-Dependent Transcriptional Regulation of p27^KIP1

Abstract

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27^KIP1 through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27^KIP1 promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27^KIP1 appears to be critical in the regulation of cell survival since mere ectopic expression of p27^KIP1 was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27^KIP1 null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27^KIP1transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

ACKNOWLEDGMENTS

We thank Tom O'Toole for technical help with the fetal liver cultures, Kris Reedquist for critically reading the manuscript, and Geert Kops for helpful discussions. Thanks also to Ivo Touw for helpful discussions and providing the p27^KIP1 luciferase construct, Anke Klippel for providing the myrPKB:ER* construct and M. E. Greenberg for the FKHR-L1 and FKHR-L1(A3) constructs.

Eric W.-F. Lam is supported by the Leukemia Research Fund of Great Britain.