In this report, we analyze the mechanisms of cytokine withdrawal– and FKHR-L1–induced apoptosis, as well as PKB-mediated rescue from apoptosis. Using cells expressing FKHR-L1(A3):ER* allowed us to uniquely analyze the effects of FKHR-L1 activation. Interestingly, no measurable differences between cytokine withdrawal– and FKHR-L1–mediated induction of apoptosis were observed. This suggests that FKHR-L1 alone could account for induction of the apoptotic program triggered by cytokine withdrawal. In both cases, cells were first arrested in G1
and subsequently underwent apoptosis (). This was accompanied by a sequential upregulation of p27KIP1
, which is involved in arresting cells in G1
and the induction of apoptosis (Polyak et al., 1994
; Toyoshima and Hunter, 1994
; Dijkers et al., 2000a
), and Bim, which can induce apoptosis by binding to anti-apoptotic members of the Bcl-2 family (O'Connor et al., 1998
) (). Bim appears to be essential for the induction of apoptosis in lymphocytes, because cytokine-deprived lymphocytes from Bim−/−
mice fail to undergo apoptosis (Bouillet et al., 1999
). Furthermore, lymphocytes derived from p27KIP1−/−
mice undergo apoptosis at a significantly decreased rate compared with those from wild-type mice (Dijkers et al., 2000a
). This suggests that the upregulation of both p27KIP1
and Bim may play critical roles in the induction of the apoptotic program initiated by cytokine withdrawal.
We also analyzed whether Fas/FasL signaling may be involved in the induction of apoptosis upon cytokine withdrawal as previously proposed (Brunet et al., 1999
). Neither cytokine withdrawal nor FKHR-L1 activity resulted in caspase-8 cleavage, an event specific for death receptor signaling (Juo et al., 1998
; Varfolomeev et al., 1998
). In addition, cytokine withdrawal had no effect on FasL promoter activity ( D) or FasL protein expression (unpublished data). Importantly, treatment of Ba/F3 cells with FasL did not induce apoptosis, suggesting a lack of a functional Fas/FasL death receptor signaling pathway. This suggests that apoptosis induced either by cytokine withdrawal, PI3K inhibition, or FKHR-L1 activity is initiated through a death receptor–independent mechanism. In support of this, overexpression of anti-apoptotic Bcl-2 members, which rescue death receptor–independent apoptosis, but not death receptor–dependent apoptosis in lymphocytes (Itoh et al., 1993
; Scaffidi et al., 1998
), are able to rescue both cytokine withdrawal– as well as FKHR-L1–induced apoptosis (Chao et al., 1998
; Dijkers et al., 2000b
). Interestingly, although the FasL promoter was not activated by cytokine withdrawal, a small region of this promoter previously shown to contain three forkhead binding sites was (Brunet et al., 1999
; D). This suggests that in the context of the intact FasL promoter, secondary factors are responsible for mediating promoter activity. Possibly, there are critical forkhead cofactors absent in Ba/F3 cells, or other factors are expressed that actively repress FasL promoter activity. This might explain the differences between our data and that previously described by Brunet et al. (1999)
in T cells.
PKB has been demonstrated to negatively regulate members of a subfamily of forkhead transcription factors: AFX, FKHR, and FKHR-L1 (for review see Datta et al., 1999
). Recently, members of the SGK (serum and glucocorticoid-induced kinases) family, which phosphorylate consensus sequences similar to PKB, were found to be required for full phosphorylation of FKHR-L1 in vivo and in IL-3–mediated survival (Liu et al., 2000
; Brunet et al., 2001
). This suggests that both kinases may be required for phosphorylation-mediated inactivation of FKHR-L1, and may explain why PKB was unable to completely inhibit cytokine withdrawal–induced apoptosis (). However, PKB was capable of significantly abrogating cytokine withdrawal–induced loss of mitochondrial potential ( C). Thus, we can conclude that PKB exerts its anti-apoptotic effect at a premitochondrial level, preventing intracellular release of cytochrome c. A potential role for PKB in rescue from apoptosis and prevention of cytochrome c leakage has also been proposed in apoptosis induced in Rat1 fibroblasts by UV irradiation (Kennedy et al., 1999
), as well as in apoptosis induced in epithelial cells by detachment from the extracellular matrix (Rytomaa et al., 2000
). However, PKB has also been previously shown to inhibit ceramide-induced apoptosis in hybrid neuron motor 1 cells downstream of cytochrome c release (Zhou et al., 2000
). These findings may be explained by differences in apoptotic stimuli in different cell types, and indicates that PKB has the potential to act at multiple levels. Furthermore, difference in species could be an explanation for the differential contribution of PKB in rescue from apoptosis. PKB has been suggested to promote rescue from apoptosis by inhibitory phosphorylation of caspase-9 in human cells (Cardone et al., 1998
), but not in mouse or rat cells because the PKB phosphorylation site in caspase-9 is not present (Fujita et al., 1999
). PKB has also been linked to the upregulation of the anti-apoptotic Bcl-2 member Mcl-1 (Wang et al., 1999
), which is essential in cytokine-mediated rescue from apoptosis (Chao et al., 1998
). This regulation of an anti-apoptotic Bcl-2 member, involved in the maintenance of mitochondrial integrity, also supports a role for PKB upstream of cytochrome c leakage in cytokine-mediated rescue from apoptosis.
The p21ras-activated protein kinase MEK has also been proposed to rescue cells from apoptosis (Perkins et al., 1996
; Shimamura et al., 2000
), potentially through activation of downstream targets that phosphorylate Bad (Shimamura et al., 2000
). Furthermore, MEK-initiated signals can result in the phosphorylation of anti-apoptotic members of the Bcl-2 family, thereby enhancing their stability (Breitschopf et al., 2000
). However, using the myrPKB:ER* cell line, we have demonstrated that PKB alone is sufficient to protect cells from apoptosis (). Our data do not, however, rule out the possibility that MEK plays a role in these events.
Two critical experiments have demonstrated that FKHR-L1 is a critical effector of cell death induced by cytokine withdrawal, and that Bim is an important downstream target of PI3K/PKB action. First, in we demonstrate that ectopic expression of an inhibitory FKHR-L1 significantly reduces the levels of apoptosis observed after cytokine withdrawal. This is similar to the effect of expressing a constitutively active mutant of PKB. Although inhibition of apoptosis is dramatic, it is not complete. This could either be due to additional pro-apoptotic pathways, or simply that the levels of expression of these transfected proteins is relatively low in Ba/F3 cells. Using Bim knockout mice, we demonstrate that hematopoietic stem cells isolated from these animals have much reduced levels of apoptosis compared with wild-type mice upon cytokine withdrawal. This is in agreement with observations previously made in leukocytes isolated from these mice (Bouillet et al., 1999
). If inhibition of Bim transcriptional levels is indeed critical for PI3K/PKB–mediated cell survival, it follows that inhibition of PI3K activity would have a significantly reduced effect on apoptosis in cells isolated from Bim-deficient mice. Indeed our data demonstrate this to be the case (). Thus, it appears that in cytokine-dependent cells, repression of Bim expression may be one of the major mechanisms by which PI3K/PKB signaling results in enhanced cell survival in vivo. It should, however, be noted that the cytoprotective effects of FKHRL1-DBD and Bim knockout are not complete. This suggests the possibility that there are other targets and mechanisms contributing to the induction of apoptosis upon cytokine withdrawal. Similarly, it has not been shown that FKHR-L1 is critical for the induction of Bim upon cytokine withdrawal.
Taken together, our data suggest that cytokine-induced signaling can inhibit cells from apoptosis through activation of PKB (or possibly SGK), which inhibits FKHR-L1 and Bad through phosphorylation and transcriptionally upregulates Mcl-1. In the absence of cytokines, PKB is inactive, resulting in dephosphorylation and activation of Bad and transcription of FKHR-L1 targets p27KIP1 and Bim. This results in induction of the apoptotic program through loss of mitochondrial integrity, leakage of cytochrome c, subsequent activation of caspases, and cleavage of substrates. The events mediating cytokine withdrawal–induced apoptosis, as well as, cytokine-mediated rescue from apoptosis are summarized in .
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 (more ...)
Our findings provide greater insight into the mechanisms regulating induction of apoptosis in lymphocytes, and probably other hematopoietic lineages, upon cytokine withdrawal. PKB alone is sufficient to inhibit apoptosis through the maintenance of mitochondrial transmembrane potential. This is likely due to the inhibition of FKHR-L1, which prevents transcription of the pro-apoptotic Bcl-2 family member Bim. A greater understanding of the mechanisms by which cytokines regulate cellular survival will help toward the design of novel pharmacological agents for therapeutic intervention in a variety of proliferative and degenerative disorders of the immune system.