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FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity - PubMed

  • ️Tue Jan 01 2002

FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal: protein kinase B-enhanced cell survival through maintenance of mitochondrial integrity

Pascale F Dijkers et al. J Cell Biol. 2002.

Abstract

Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, we used a cell line in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induced apoptosis, which was preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlated with the disruption of mitochondrial membrane integrity and cytochrome c release. This was preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduced the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone was sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim-/- mice exhibited reduced levels of apoptosis upon inhibition of PI3K/PKB signaling. These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB--mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death.

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Figures

Figure 1.
Figure 1.

Induction of apoptosis by cytokine withdrawal or FKHR-L1 activity. (A, top) Ba/F3 cells were cultured in the presence or absence of cytokines. FKHR-L1(A3):ER* cells were cultured with IL-3 without or with 4-OHT (100 nM) for 24 h and analyzed for annexin V binding and PI staining as described in the Materials and methods. (Bottom) Ba/F3 cells were IL-3 starved (left) or FKHR-L1(A3):ER*–expressing cells were treated with 4-OHT (100 nM, right) for the times indicated. DNA laddering was analyzed as described in the Materials and methods. (B) Ba/F3 cells were cytokine-starved for the indicated times, lysed, and equal amounts of protein were analyzed for levels of p27KIP1 (top) or Bim (middle). Samples were analyzed with a RACK1 antibody to verify equal protein loading (bottom). (C) Ba/F3 cells stably expressing FKHR-L1(A3):ER* were treated with 4-OHT for the times indicated, lysed, and analyzed as above. Data depicted are representative of at least three independent experiments.

Figure 2.
Figure 2.

Death receptor signaling is not activated in response to cytokine deprivation or FKHR-L1 activation. (A) Caspase-8 cleavage was analyzed in Ba/F3 cells that were cytokine starved for the times indicated. (B) Analysis of caspase-8 cleavage in 4-OHT–treated (100 nM) FKHR-L1(A3):ER* cells. (C) Jurkat cells were treated with or without αFAS18 (1 μg/ml). After 24 h, samples were harvested and protein concentration was measured. An equal amount of protein was analyzed by SDS-PAGE and anti–caspase-8 Western blotting. Data depicted are representative of at least three independent experiments. (D) Ba/F3 cells were electroporated with 1 μg renilla luciferase and either 20 μg of a FasL promoter construct (FasL–LUC) or a construct containing three forkhead binding sites from the FasL promoter (FHRE-LUC). After culture for 18 h with or without IL-3, cells were lysed and analyzed for the amount of produced luciferase protein. Data represent the mean of three independent experi- ments ± SD. (E) Ba/F3 cells or Jurkat cells were treated for 24 h with SUPERFas Ligand (50 ng/ml) and analyzed for annexin V binding and PI staining as described in the Materials and methods.

Figure 3.
Figure 3.

Cell cycle analysis of cytokine-starved cells and cells in which FKHR-L1 activity is induced. (A) Ba/F3 cells were cytokine starved for the times indicated, fixed, stained with PI, and analyzed by FACS®. (B) Ba/F3 cells stably expressing FKHR-L1(A3):ER* were treated with 4-OHT (100 nM) for the times indicated and processed as in A. Data depicted are representative of at least three independent experiments.

Figure 4.
Figure 4.

Analysis of caspase activation and PARP cleavage in cytokine-starved cells and 4-OHT–treated FKHR-L1(A3):ER* cells. (A) Cells were IL-3 starved for the times indicated and caspase-3 and -7 activation was analyzed using cleavage-specific antibodies. Equal protein loading was verified by analyzing samples for RACK1 expression. (B) 4-OHT–treated FKHR-L1(A3):ER* cells were analyzed as in A. (C) PARP cleavage was examined in cytokine-starved cells (left) or 4-OHT–treated FKHR-L1(A3):ER* cells (right). Data depicted are representative of at least three independent experiments.

Figure 5.
Figure 5.

Induction of mitochondrial transmembrane depolarization by cytokine starvation or FKHR-L1 activity. (A) Ba/F3 cells were IL-3 starved for the times indicated and mitochondrial transmembrane depolarization was measured using Rh-123 staining as described in the Materials and methods. (B) 4-OHT–treated FKHR-L1(A3):ER* cells were analyzed as in A. Data depicted are representative of at least three independent experiments.

Figure 6.
Figure 6.

Inhibition of PI3K or direct activation of FKHR-L1 results in cytochrome c release from mitochondria. Ba/F3 cells were IL-3 starved or treated with LY294002, and FKHR-L1(A3):ER cells were treated with 4-OHT for the times indicated (in the presence of IL-3). Mitochondrial and cytosolic fractions were prepared as described in the Materials and methods. Protein concentrations were measured, and an equal amount of protein from the different fractions was analyzed by SDS-PAGE and cytochrome c Western blotting.

Figure 7.
Figure 7.

Activation of PKB rescues cells from apoptosis and maintains mitochondrial transmembrane potential. (A) Ba/F3 cells stably expressing myrPKB:ER* were left untreated or were treated with 4-OHT (100 nM) for 36 h. PKB phosphorylation was measured using a PKB phospho ser-473–specific antibody. (B) myrPKB:ER* cells were cytokine starved in the absence (left) or presence (right) of 4-OHT (100 nM) for 36 h, and the percentage of annexin V-FITC–positive cells was determined as described in the Materials and methods. (C) Ba/F3 cells or Ba/F3 cells stably expressing myrPKB:ER* were cytokine starved in the absence or presence of 4-OHT (100 nM) or cultured with IL-3 for 36 h. Mitochondrial integrity was examined using Rh-123 as described in the Materials and methods. (D) Caspase-3 (left) and -7 (right) activation was measured in myrPKB:ER* cells that were cytokine starved for 36 h in the absence or presence of 4-OHT (100 nM) using activation-specific antibodies. These cells were then reprobed with RACK1 antibody to verify equal protein loading. All data depicted are representative of at least three independent experiments.

Figure 8.
Figure 8.

Inhibition of FKHR-L1 transcriptional activity protects cells from apoptosis induced by cytokine withdrawal. (A) COS cells (6-cm wells) were transfected with 50 ng of a renilla luciferase construct together with 2 μg of the pGL2-p27kip luciferase promoter construct (top) or 2 μg of a pGL2–6xDBE luciferase construct (bottom). Cells were cotransfected either with an empty vector (lane 1) with 1, 2, or 4 μg of pSG5–mycFKHRL1-DBD (lanes 2–4, respectively), or with 2 μg pECE-HA-FKHR-L1(A3) (lane 5) together with 1, 2, or 4 μg of pSG5–mycFKHRL1- DBD (lanes 6–8, respectively). After 24 h, cells were lysed and analyzed for luciferase activity. Data represent the mean of three independent experiments ± SEM. (B) Ba/F3 cells (107) were electroporated with 10 μg pGL2-p27kip luciferase promoter construct together with 10 μg of either a control plasmid (pSG5) or pSG5–mycFKHRL1-DBD. After 24 h, cells were lysed and analyzed for luciferase activity. Data represent the mean of three independent experiments ± SD. (C) Ba/F3 cells (107) were electroporated with 2 μg spectrin–GFP and either 18 μg control vector (top), FKHRL1-DBD (middle), or gagPKB (bottom). After electroporation, dead cells were removed as described in the Materials and methods. 48 h later GFP-positive (right panels) and GFP-negative (left panels) cells were analyzed by FACS® for apoptosis by annexin V–phycoerythrin binding. The percentage of annexin V–positive cells is shown. Data depicted are representative of at least three independent experiments.

Figure 9.
Figure 9.

Regulation of Bim expression is critical for PI3K-mediated rescue from apoptosis. Hematopoietic stem cells were isolated from either wild-type mice or mice lacking both (−/−) alleles of the BIM gene as described in the Materials and methods. Cells were treated with cytokines, with or without incubation with 10 μM LY294002, or were cytokine starved. (A) Annexin V binding was analyzed after 24 h. (B) The integrity of the mitochondrial transmembrane potential was measured by FACS® analysis of Rh-123 binding after 48 h. Data represent the mean of four independent experiments ± SEM.

Figure 10.
Figure 10.

A model for cytokine withdrawal–induced apoptosis. In the absence of cytokines, PKB and SGK are inactive, preventing elevation of the anti-apoptotic Bcl-2 member Mcl-1, and resulting in dephosphorylation and subsequent activation of the pro-apoptotic Bcl-2 member Bad and the FKHR-L1 transcription factor. Transcriptional activity of FKHR-L1 elevates levels of Bim and p27KIP1. p27KIP1 inhibits cell cycle progression and helps to promote apoptosis in an as yet unidentified manner. Bim, possibly together with Bad, promotes leakage of cytochrome c and subsequent loss of mitochondrial integrity. This triggers the activation of caspases and subsequent cleavage of downstream targets, resulting in apoptosis.

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