FKHR-L1 can act as a critical effector of cell death induced by cytokine withdrawal

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.

Prolactin induces c-Myc expression and cell survival through activation of Src/Akt pathway in lymphoid cells

“…To determine the possible role of FKHRL1 on c-Myc expression in W53 cells, we used an active mutant of FKHRL1, in which the three inhibitory phosphorylation sites were mutated to alanines, FKHRL1-A3 (Dijkers et al, 2002). Cells were transiently transfected with the c-Myc promoter luciferase reporter construct (Lee and Ziff, 1999) together with pSG5-FKHRL1-A3, or the empty plasmid as control.…”

“…In concordance with this result, inducible expression of Akt-CAAX Box blocked PRL stimulation of FKHRL1. This transcription factor controls the expression of p27 KIP1 , Bim, and cyclins D, promoting cell proliferation and survival (Dijkers et al, 2000;Dijkers et al, 2002;Schmidt et al, 2002). Inactivation of forkhead transcription factors by Akt could be related to the observed c-Myc upregulation.…”

“…This cell line has been always maintained in culture using Tet-free foetal bovine serum (Tet System Approved FBS, BD Biosciences Clontech, Palo Alto, CA, USA) to avoid possible contamination of normal foetal bovine serum with tetracycline. For transient transfections, W53 cells were electroporated (960 mF and 300 V) with 10 mg of the c-Myc promoter luciferase reporter construct (Lee and Ziff, 1999) together with pSG5-FKHRL1-A3 (provided by Boudewijn (University Medical Center, Utrecht, Nederlands) (Dijkers et al, 2002), or the empty plasmid as control. After transfection, cells were cultured in the absence or presence of PRL (100 ng/ml) for 24 h. Cells were harvested, lysed with the commercially luciferase lysis buffer (Promega, Madison, USA), the cell extracts were normalized for the protein concentration, and the luciferase activity was measured.…”

FOXO3a Induces Differentiation of Bcr-Abl-transformed Cells through Transcriptional Down-regulation of Id1

“…Cell Culture-Ba/F3 cells were cultured in RPMI 1640 medium supplemented with 8% HyClone serum (Invitrogen) and recombinant mouse IL-3 produced in COS cells (29). BaF3-FOXO3a(A3):ER* cells were previously described (29).…”

“…Cells were co-transfected with 50 ng of a Renilla luciferase plasmid (pRL-TK, Promega, Madison, WI) to normalize for transfection efficiency. After transfection cells were cultured with or without IL-3 or in the presence of IL-3 with or without 4-hydroxytamoxifen (100 nM) for 24 h. Cells were then harvested and lysed in commercially available luciferase lysis buffer, and luciferase activity was determined as in a previous study (29).…”