Tumor Necrosis Factor-related Apoptosis-inducing Ligand Activates a Lysosomal Pathway of Apoptosis That Is Regulated by Bcl-2 Proteins

Abstract

The present studies were performed to determine whether lysosomal permeabilization contributes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity and to reconcile a role for lysosomes with prior observations that Bcl-2 family members regulate TRAIL-induced apoptosis. In KMCH cholangiocarcinoma cells stably expressing Mcl-1 small interference RNA (siRNA), treatment with TRAIL induced a redistribution of the cathepsin B from lysosomes to the cytosol. Pharmacological and small hairpin RNA-targeted inhibition of cathepsin B attenuated TRAIL-mediated apoptosis as assessed by morphological, biochemical, and clonogenic assays. Neither Bid siRNA nor Bak siRNA prevented cathepsin B release. In contrast, treatment of the cells with Bim siRNA or the JNK inhibitor SP600125 attenuated lysosomal permeabilization and cell death. Moreover, Bim and active Bax co-localized to lysosomes in TRAIL-treated cells in a JNK-dependent manner, and Bax siRNA reduced TRAIL-induced lysosomal permeabilization and cell death. Finally, BH3 domain peptides permeabilized isolated lysosomes in the presence of Bax. Collectively, these data suggest that TRAIL can trigger an apoptotic pathway that involves JNK-dependent activation of Bim, which in turn induces Bax-mediated permeabilization of lysosomes. Tumor necrosis factor (TNF)2 -related apoptosis-inducing ligand (TRAIL), a death ligand that preferentially induces apoptosis in transformed and virally infected cells, is under evaluation as an anti-cancer agent in humans (1-3). TRAIL induces apoptosis by binding to death receptors 4 and 5 (DR4/ TRAIL-R1 and DR5/TRAIL-R2), type I transmembrane proteins that contain death domains in their cytosolic tails. TRAIL binding results in receptor oligomerization (4) followed by recruitment of Fas-associated protein with death domain. This polypeptide in turn recruits procaspases-8 and -10 to the receptor complex through homotypic interactions involving death effecter domains in Fas-associated protein with death domain and these two apical caspases (4). The resulting activation of the bound caspases is essential for TRAIL-induced apoptosis (5).The cytotoxicity of TRAIL is also regulated by members of the Bcl-2 family of polypeptides (6). In particular, the multidomain proapoptotic Bcl-2 family member Bax is essential for TRAIL-induced killing of a colon cancer cell line that has low level expression of Bak (7). Other Bcl-2 family members also affect TRAIL action. We and others have demonstrated that the multidomain antiapoptotic protein Mcl-1 blocks TRAILmediated apoptosis in various cell lines (8 -10). The related proteins Bcl-2 and Bcl-X L have also been reported to inhibit TRAIL-induced killing (11,12). Conversely, certain proapoptotic BH3-only family members, which function as sensors of various cellular stresses (13), participate in TRAIL-induced killing. Specifically, Bid and Bim have both been implicated in TRAIL cytotoxicity (10,14). How these Bcl-2 family members integrate into a composite signaling pathway involving apica...

JNK mediates UVB-induced apoptosis upstream lysosomal membrane permeabilization and Bcl-2 family proteins

“…However, the phosphorylation target for JNK-dependent regulation of LMP remains unidentified. Interestingly, a recent report demonstrates that Bim is translocated to lysosomes in a JNK-dependent manner during TRAIL-induced apoptosis [15]. However, we found no colocalization of Bim and lysosomes in human melanocytes after UVB irradiation.…”

Measurement of BH3-only protein tolerance

“…Thus, although BH3 profiling is being explored as a strategy to predict sensitivity or resistance of particular cell lines or cancers to specific treatments, 50 measurement of BH3-only tolerance has the potential to provide insight into endogenous signaling networks within intact cells that include BCL2 family members bound to surfaces other than the MOM. 44,51 Despite its potential usefulness in understanding the action of drugs that upregulate BH3-only proteins, measurement of BH3-only tolerance also has some limitations. First, because it measures cell death at a fixed point in time, it might underestimate the number of cells ultimately killed by overexpressed BH3-only proteins.…”

Diverse regulatory roles for lysosomal proteases in the immune response

“…What seems to be clear is that the precise mechanism will depend on the initial triggers of LMP. Of interest are reports indicating that, in addition to their role in MOMP, Bcl2 family members such as Bim and Bax, also appear to be involved in LMP [90]. Caspases and even cathepsins themselves may be involved in amplifying the LMP pathway of apoptosis.…”