Cell death is regulated mainly through an evolutionarily conserved form of cell suicide termed apoptosis [1]. Deregulation of apoptosis has been associated with cancer, autoimmune diseases and degenerative disorders. Many cells, particularly those of the hematopoietic system, have a default program of cell death and survival that is dependent on the constant supply of survival signals. The Bcl-2 family, which has both pro- and anti-apoptotic members, plays a critical role in regulating cell survival [2]. One family member, the Bcl-2 interacting mediator of cell death (Bim), contains only a protein-interaction motif known as the BH3 domain, allowing it to bind pro-survival Bcl-2 molecules, neutralizing their function [3]. Disruption of the bim gene results in resistance to apoptosis following cytokine withdrawal in leukocytes, indicating that regulation of the pro-apoptotic activity of Bim is critical for maintenance of the default apoptotic program [4]. Here, we report that withdrawal of cytokine results in upregulation of Bim expression concomitant with induction of the apoptotic program in lymphocytes. Activation of the forkhead transcription factor FKHR-L1, previously implicated in regulation of apoptosis in T lymphocytes [5], was sufficient to induce Bim expression. We propose a mechanism by which cytokines promote lymphocyte survival by inhibition of FKHR-L1, preventing Bim expression.

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 p27KIP1 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 KIP1 transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

The cytokine IL-2 plays a very important role in the proliferation and survival of activated T cells. These effects of IL-2 are dependent on signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. We and others have shown that PI3K, through activation of protein kinase B/Akt, inhibits transcriptional activation by a number of forkhead transcription factors (FoxO1, FoxO3, and FoxO4). In this study we have investigated the role of these forkhead transcription factors in the IL-2-induced T cell proliferation and survival. We show that IL-2 regulates phosphorylation of FoxO3 in a PI3K-dependent fashion. Phosphorylation and inactivation of FoxO3 appears to play an important role in IL-2-mediated T cell survival, because mere activation of FoxO3 is sufficient to trigger apoptosis in T cells. Indeed, active FoxO3 can induce expression of IL-2-regulated genes, such as the cdk inhibitor p27Kip1 and the proapoptotic Bcl-2 family member Bim. Furthermore, we show that IL-2 triggers a rapid, PI3K-dependent, phosphorylation of FoxO1a in primary T cells. Thus, we propose that inactivation of FoxO transcription factors by IL-2 plays a critical role in T cell proliferation and survival.