IFN-α has been employed for treatment of tumors and viral diseases (hepatitis C) [1
]. Type I IFN-α exerts multiple functions including anti-viral action, immune modulation, cell proliferation, and cell death [3
]. IFN-α actions against tumors involve induction of apoptosis as well as indirect effects through induction of cytotoxic cells [7
]. We have previously demonstrated that IFN-α induced a sustained activation of pro-apoptotic molecule JNKs [12
], concomitant with upregulation of anti-apoptotic molecule c-Flip through NF-κB activation [30
]. However, the molecular mechanisms of IFN-α-induced apoptotic cell death are not completely understood. In the present study, we explored the upstream component of IFN-α-induced JNK activation and found that PKC-δ activation occurs upstream of JNK and Stat1 activation.
Several studies have demonstrated that PKC-δ has multiple targets in response to apoptotic stimuli, including IFN-α [15
]. For example, it has been shown that PKC-δ mediates JNK activation in response to DNA damage [18
]. Consistent with this observation, genetic silencing of PKC-δ using dnPKC-δ reduced the IFN-α-induced JNK activation (Figure ), supporting the notion that PKC-δ signaling participates in the IFN-α-induced JNK activation. Because PKC-δ did not translate to the nucleus in response to IFN-α (Additional file 4
), it is unlikely that PKC-δ affects gene expression via transcription factor. Rather, downstream component of PKC-δ signaling pathway including JNK may participate in the upregulation of TRAIL promoter activity [12
The interaction of TRAIL with its receptors TRAIL-Rs leads to caspase-8 activation through recruitment of adaptor protein Fas-associated death domain (FADD) [31
]. The caspase-8 activation in most cell types is amplified in the mitochondria. The IFN-α-induced loss of ΔΨm and increase in sub-G1 fraction was reduced in the dnPKC-δ-overexpressing cells, while the mitochondrial dissipation was enhanced in the caPKC-δ-overexpressing cells. PKC-δ inhibitor rottlerin was not suitable for analysis of IFN-α-induced loss of ΔΨm in Daudi cells, because rottlerin alone induced mitochondrial dissipation (Additional file 1
]). Such rottlerin-induced mitochondrial damage may lead to a small level of PKC-δ activation. Thus, the following pathway is proposed for the IFN-α-induced apoptosis: IFN-α induces JNK1 activation via PKC-δ → upregulation of TRAIL → interaction of TRAIL with its TRAIL-Rs. → loss of ΔΨm → apoptosis. Interestingly, a recent report clearly demonstrated that TRAIL induces PKC-δ activation through diacylglycerol in Jurkat cells [33
], which can form a positive feedback loop involving PKC-δ and TRAIL. Alternatively, it is also possible that the activated JNK induces migration of p21Bax-α to the mitochondria from cytosol, which is cleaved into p18Bax-α, a more potent form [34
]. These changes lead to loss of ΔΨm, especially in the later phases of apoptotic processes, which is TRAIL-independent [36
The TRAIL promoter contains a possible IFN-stimulated response element (ISRE) and AP-1 sites [37
], which might be involved in TRAIL promoter activation. Originally, JNK was found to induce phosphorylation of c-Jun, which combines with c-Fos, creating a heterodimer of c-Jun/c-Fos (AP-1) [10
]. Indeed, IFN-α caused AP-1 activation in several cell lines including Daudi lymphoma cells, probably through JNK activation (Yanase et al. ms. in preparation, [12
]). The ISRE binds transcription factor IFN-stimulated gene factor-3 (ISGF3) comprising Stat1, Stat2, and IRF-9 [7
]. IFN-α caused phosphorylation of Stat1 on Ser727, at least through PKC-δ signaling [19
]. We also confirmed the phosphorylation of Stat1 (Ser727) in the IFN-α-stimulated Daudi cells (Figure ). The IFN-α-stimulated phosphorylation of Stat1 on Ser727, but not Tyr701, was markedly abrogated in the dnPKC-δ-expressing Daudi cells, confirming that PKC-δ signaling plays a crucial role in the Ser727-PKC-δ phosphorylation. Stat1 activation favours induction of apoptosis in some settings, whereas Ser727-Stat1 promotes survival in others probably through gene induction [40
]. Experiments are required to address whether IFN-α-induced Ser727-Stat1 functions as proapoptotic or anti-apoptotic molecule.
Several kinases phosphorylate and activate PKC-δ following stimulation with IFN-α. For example, tyrosine phosphorylation of PKC-δ on Tyr 311 was induced by Src-tyrosine kinase [43
]. Interestingly, PI3-K and phosphoinositide-dependent kinase (PDK)1 [45
] phosphorylated PKC-δ on the Thr505 residue that is stimulated by IFN-α (Figure ). It remains unclear what kinases are involved in IFN-α-stimulated phosphorylation of PKC-δ in Daudi B lymphoma cells.
Stimulation of IFN-α together with chemotherapeutic agents appears to be effective for treatment of cancer patients [46
]. PKC-δ-JNK signaling pathway is involved in induction of apoptosis in response to chemotherapeutic agents [18
]. Here, we demonstrated that the IFN-α-induced apoptosis also involves PKC-δ-JNK signaling pathway in human Daudi B lymphoma cells. Concurrent with the activation of pro-apoptotic pathway, we recently demonstrated that IFN-α-induced-PKC-α activation caused upregulation of anti-apoptotic c-Flip through NF-κB activation [30
]. Thus, the balance between IFN-α-induced pro-apoptotic (such as PKC-δ-JNK) and anti-apoptotic (such as PKC-α-NF-κB) signaling pathways might determine the cell phenotype. These findings would be valuable for the design of treatment modalities of cancer patients with IFN-α together with or without chemotherapeutic agents.