Several models have been proposed to explain the paradoxical role of the JNK pathway as a pro-survival/proliferative or a pro-death pathway. For example, cross-talk between JNK and other cellular pathways, such as nuclear factor kappa-B, may determine the repertoire of target genes that are induced and the subsequent cellular outcomes (Lee et al., 1997
; Mecurio et al., 1999
). In addition, the duration of JNK activation has been proposed to be an important modulating factor. Transient activation of the pathway, occurring after stimulation by growth factors, has been associated with cell proliferation whereas sustained activation, occurring after exposure to chemical/physical stresses, is associated with cell death (Chen et al., 1996
; Gabai et al., 2000
; Muscarella and Bloom, 2002
; Wicovsky et al., 2007
). However, these observations do not fully explain the complex role of JNK in stress-induced apoptosis. In the present study we show that even when activated by potent, apoptosis–inducing treatments, the requirement for JNK in B-cell apoptosis induction is agent-specific, and depends on its downstream targets, which include either nuclear or cytoplasmic substrates.
Two classes of stress inducing treatments were used in our study: global proteotoxic stress induced by arsenite or lethal heat treatment compared to specific inhibition of microtubule function induced by vincristine. Both activated the JNK pathway in a sustained manner, as analyzed by phosphorylation of the JNK protein and induction of its downstream target c-Jun protein. However, two structurally unrelated JNK inhibitors, SP6 and JNK inhibitor V (Bennet et al., 2001
, Gaillard et al., 2005
) blocked apoptosis induced only by vincristine, not by arsenite. In contrast, an inactive structural analogue of SP6 had no effect, nor did inhibitors of ERK and p38 MAP kinase pathways commonly activated by cellular stress. A major difference found among the treatments was that only vincristine induced the phosphorylation of Bcl-2. Importantly, vincristine-induced apoptosis and Bcl-2 phosphorylation were both blocked by inhibitors of JNK (SP6 and JNK V). Although we cannot formally exclude the potential involvement of some other kinases, the fact that two unrelated JNK inhibitors yielded similar results strongly implicate JNK as an important Bcl-2 – phosphorylating kinase. Furthermore, our results showing that phosphorylation of Bcl-2 is a proapoptotic event is consistent with other published studies (Yamamoto et al., 1999
; Fan et al., 2000a
Several sites in the Bcl-2 protein, including threonine 56, are typically phosphorylated at the G2/M transition (checkpoint) of the cell cycle. It is believed that G2/M-associated Bcl-2 phosphorylation serves to promote the elimination of aberrant cells by sensitizing them to the induction of apoptosis during this critical phase in the cell cycle. The JNK pathway appears to be responsible for this cell-cycle-related phosphorylation of Bcl-2. Therefore, it has been proposed that drug-induced microtubule damage and the G2/M cell cycle checkpoint overlap with respect to activation the JNK pathway, leading to Bcl-2 phosphorylation and that the cell cycle arrest caused by microtubule inhibitors may provide the key cellular signal.
We observed a rapid induction of signaling following vincristine exposure. Bcl-2 phosphorylation occurred concurrently with phosphorylation of JNK and induction of its downstream target, c-Jun, reaching maximal levels within 2 h of drug exposure. Thus, this rapid phosphorylation of Bcl-2 following the induction of microtubule damage precedes cell cycle arrest or accumulation of G2/M cells which occurs gradually over several hours after addition of the microtubule inhibitor (data not shown). This finding is consistent with the hypothesis that signals induced by microtubule damage directly activate pathways that are also involved in the G2/M checkpoint during typical cell cycle progression.
An important issue is why arsenite and heat stress do not induce Bcl-2 phosphorylation despite their potent activation if JNK and ability to induce c-Jun. In order to phosphorylate/induce nuclear targets such as c-Jun, JNK must translocate to the nucleus. This event requires the association of the kinase with the scaffolding protein, JNK-interacting protein (JIP). Genetic disruption of JIP blocks the nuclear translocation of JNK and subsequent induction of c-Jun, but its ability to phosphorylate Bcl-2 is unaffected (Fan et al., 2000b
). Thus, there is at least one major bifurcation point in the JNK pathway that distinguishes its nuclear from cytoplasmic targets () and that may be differentially activated by various agents. The events that lead the differential targeting of downstream JNK-substrates by various chemical stresses are presently unknown although they may be related to the fact that these treatments differentially activate additional signaling pathways outside of JNK. For example, there is evidence that activation of JNK following microtubule damage involves upstream signaling by ASK1 and Ras (Wang et al., 1998
) whereas arsenite exposure results in the activation of several MAP kinase pathways in addition to JNK.
FIG 10 Proposed pathways for JNK-dependent and JNK-independent apoptosis induction by arsenite and vincristine. (A) Vincristine induced microtubule damage leads to phosphorylation of JNK which, in turn, translocates to the nucleus and phosphorylates the transcription (more ...)
Importantly, our data indicate that global protein damage alone may not be the critical signal initiating JNK-pathway activation by arsenite. Hsp70 induction, a known response to general protein damage, was induced at low concentrations of arsenite and by non-lethal heat stress – treatments that did not induce JNK phosphorylation or apoptosis. However, arsenite and lethal heat stress have the ability to directly affect mitochondrial function and also increase the generation of reactive oxygen species – events that strongly induce JNK/c-Jun. In addition, although classically induced by DNA-strand breaks, the p53 pathway has also been shown to be activated by stresses such as arsenite and heat stress most likely by a mechanism involving oxidative damage (Seo et al., 1999
; Yih and Lee, 2000
; Miyakoda et al., 2002
The p53 protein is expressed constitutively at low levels under normal conditions due to its short half-life. Wild-type p53 binds to MDM2, which facilitates its ubiquitinization and subsequent proteosomal degradation. Phosphorylation of p53 protein blocks its binding to MDM2, thus preventing its degradation. This stabilized p53 protein directs the transcription of several apoptosis-associated genes, including those encoding several pro-apoptotic members of the Bcl-2 family. Alternatively, a direct interaction of p53 with Bcl-2 has recently been reported (Marchenko et al., 2000
; Mihara et al., 2003
; Deng et al., 2006
). These studies showed that p53 protein is able to bind to Bcl-2 via the flexible loop region thereby inactivating the protein and promoting the release of cytochrome c from mitochondria. Although we have yet to determine the precise mechanism, our results showing the robust activation of the p53 pathway following treatment with arsenite or lethal heat stress suggest that the activation of the p53 pathway may overcome Bcl-2 mediated resistance in EW36 B-cells in a manner that is independent of the inactivation of Bcl-2 by phosphorylation.
In the current study, we found that the interaction between heat stress and vincristine and subsequent apoptosis induction depended on the specific conditions of heat exposure. Pre-treatment of EW36 B-cells with either limited or prolonged heat stress blocked vincristine-induced Bcl-2 phosphorylation and induced Hsp70. However, pre-treatment of cells with limited, non-lethal, heat stress, attenuated vincristine-induced JNK and Bcl-2 phosphorylation and inhibited apoptosis.
Studies have shown that pre-treatment with limited heat stress protects cells against apoptosis induced by subsequent, more extensive, heat stress as well as by certain classes of drugs (Lee et al., 1992
; Mosser et al., 1997
; Samali and Cotter, 1996
). There are several possible mechanisms by which non-lethal heat stress may protect cells against drug-induced apoptosis. As molecular chaperones, heat-stress proteins function to interact with and stabilize proteins, thus protecting them from damage and promoting their re-folding after deaturation. Several apoptosis-regulating proteins have been identified that may be preferentially targeted by Hsp70. For example, Hsp70 has been reported to interact with and block the activity of caspases, thus protecting cells against apoptosis (Mosser et al., 1997
). Alternatively, Hsp70 has also been shown to interact directly with microtubules. Expression of Hsp70 protects 9L rat brain cells from vincristine-induced apoptosis (Lee et al., 1992
). At present we do not know the critical targets by which non-lethal heat stress protects EW36 B-cells, although the reduction of Bcl-2 phosphorylation suggests that protection occurs at a point relatively early in vincristine-induced signaling. Although Hsp70 induction and/or inhibition of Bcl-2 phosphoryaltion may be required for this protection, these signals alone are not sufficient – lethal heat stress induced apoptosis even in the presence of high levels of the stress protein and inhibition of Bcl-2 phosphorylation. Thus, the activation of other cell death pathways, such as p53, may override the effects of these cytoprotective events.
The findings from our study, summarized in , contribute to an understanding of the agent-specific outcomes of JNK activation – a pathway that plays a central role in stress-signaling. In addition, we have identified two mechanisms, one JNK-dependent and the other JNK-independent, facilitating bypass of resistance conferred by Bcl-2 over-expression: first, by JNK-dependent Bcl-2 phosphorylation (following microtubule damage) and, second, by JNK-independent p53 activation (following general protein damage induced by lethal heat stress or arsenite exposure). This study also identified important interactions between the two classes of apoptosis-inducing treatments. The interaction of heat stress with vincristine depended on the specific conditions of heat treatment, with short versus prolonged treatment having essentially opposing effects on cell survival following vincristine treatment. This is significant because the use of heat stress, alone or in conjunction with chemotherapeutic drugs, is currently being explored for the treatment of malignancies (Ohnishi and Ohnishi, 2001
). Furthermore, like EW36, lymphomas and other cancers over-express Bcl-2 but retain wild-type p53. Thus, it has been proposed that targeting the p53 pathway with newer therapies may be an important treatment strategy (Martins et al., 2006
). Clearly, the precise conditions of p53 activation and its interaction with other pathways activated by different classes of drugs will profoundly influence the efficacy of this approach and requires detailed molecular information on the modes of action of each agent.