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(KIP1) transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Apoptosis / drug effects
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Apoptosis / genetics
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B-Lymphocytes / cytology
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B-Lymphocytes / metabolism
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Cell Cycle Proteins*
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Cell Line
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Cells, Cultured
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Cyclin-Dependent Kinase Inhibitor p27
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Cyclin-Dependent Kinases / antagonists & inhibitors
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Cytokines / metabolism*
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DNA-Binding Proteins / antagonists & inhibitors
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Enzyme Inhibitors / metabolism*
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Enzyme Inhibitors / pharmacology
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Eosinophils / drug effects
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Eosinophils / metabolism
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Forkhead Box Protein O1
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Forkhead Transcription Factors
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Gene Expression Regulation
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Genes, Reporter / genetics
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Hematopoietic Stem Cells / cytology
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Hematopoietic Stem Cells / metabolism
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Humans
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Hydroxytestosterones / pharmacology
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Interleukin-3 / genetics
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Interleukin-3 / metabolism*
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Mice
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Microtubule-Associated Proteins / genetics*
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Microtubule-Associated Proteins / metabolism
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Mutation
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PTEN Phosphohydrolase
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Phosphatidylinositol 3-Kinases / metabolism*
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Phosphoric Monoester Hydrolases / genetics
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Phosphoric Monoester Hydrolases / metabolism
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Promoter Regions, Genetic / drug effects
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RNA / metabolism
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Signal Transduction
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Sirolimus / pharmacology
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Tamoxifen / pharmacology
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Transcription Factors / antagonists & inhibitors
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Transcription, Genetic / genetics*
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Tumor Suppressor Proteins*
Substances
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Cdkn1b protein, mouse
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Cell Cycle Proteins
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Cytokines
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DNA-Binding Proteins
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Enzyme Inhibitors
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FOXO1 protein, human
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Forkhead Box Protein O1
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Forkhead Transcription Factors
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Foxo1 protein, mouse
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Hydroxytestosterones
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Interleukin-3
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Microtubule-Associated Proteins
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Transcription Factors
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Tumor Suppressor Proteins
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Tamoxifen
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Cyclin-Dependent Kinase Inhibitor p27
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4,17 beta-dihydroxy-4-androstene-3-one
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RNA
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Phosphatidylinositol 3-Kinases
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Cyclin-Dependent Kinases
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Phosphoric Monoester Hydrolases
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PTEN Phosphohydrolase
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PTEN protein, human
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Sirolimus