AIP1: a new player in TNF signaling.

Apoptosis signal-regulating kinase 1 (ASK1) is an upstream activator of JNK and p38 MAPK signaling cascades. Evidence now shows that the ASK1-interacting protein, AIP1, plays an important role in TNF-alpha-induced ASK1 activation by facilitating dissociation from its inhibitor.

Cell stress gives a red light to the mitochondrial cell death pathway.

Although the ultimate outcome of prolonged exposure of cells to stress is often death, the early response appears to be the activation of survival pathways that are likely to give the cell an opportunity to repair low-level damage. How these stress-initiated survival pathways influence B cell lymphoma/leukemia 2 (Bcl-2) proteins, the core cell death machinery, has remained unclear; however, two papers now provide insight into stress-mediated survival mechanisms. The liver is unusually resistant to p53-mediated apoptosis. It appears that p53-mediated induction of the gene that encodes insulin-like growth factor-binding protein-1 (IGFBP1) attenuates the cell death response in hepatocytes by preventing the formation of a complex between p53 and the proapoptotic protein BAK. This is especially interesting as IGFBP1 is not a member of the Bcl-2 family, yet it inhibited BAK. In three unrelated cell lines, another regulatory interaction that influences cell survival occurs at the mitochondria. In this case, protein phosphatase 1gamma (PP1gamma) regulated the phosphorylation status of the Bcl-2/Bcl-X(L)-associated death promoter (BAD). The prefoldin family member URI is normally phosphorylated by S6 kinase 1, which liberates PP1gamma from a URI-PP1gamma complex. However, the withdrawal of growth factors or nutrients stabilizes this complex, which renders PP1gamma inactive. The net response of this stress stimulus is an increased abundance of phosphorylated BAD, which raises the threshold required to trigger cell death. These two studies have identified new players and mechanisms that integrate stress responses and cell death.

Tumor necrosis factor-related apoptosis-inducing ligand activates a lysosomal pathway of apoptosis that is regulated by Bcl-2 proteins.

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.

Bid is upstream of lysosome-mediated caspase 2 activation in tumor necrosis factor alpha-induced hepatocyte apoptosis.

BACKGROUND & AIMS: During tumor necrosis factor alpha-mediated hepatocyte cytotoxicity, cathepsin B is released from lysosomes and contributes to apoptosis by indirectly promoting mitochondrial dysfunction. How this lysosomal pathway mediates mitochondrial dysfunction is unclear. Because Bcl-2 family proteins and caspase 2 have been implicated in proximal apoptosis-signaling pathways, we examined the role of these proteins in tumor necrosis factor alpha-induced lysosomal permeabilization and cathepsin B-mediated mitochondrial dysfunction. METHODS: Studies were performed in primary hepatocytes from wild-type cathepsin B knockout, Bid knockout, and caspase 2 knockout mice and in the rat hepatoma cell line McArdle7777 by using tumor necrosis factor alpha/actinomycin D. RESULTS: Studies in wild-type and Bid knockout hepatocytes showed that tumor necrosis factor alpha-mediated lysosomal permeabilization is Bid dependent. After tumor necrosis factor alpha/actinomycin D treatment, caspase 2 activity increased severalfold in wild-type hepatocytes, whereas minimal activity was observed in hepatocytes from cathepsin B knockout mice or in hepatoma cells treated with a cathepsin B inhibitor. In contrast, Bax was activated independently of cathepsin B. Pharmacological, genetic, or small interfering RNA-mediated inhibition of caspase 2 attenuated tumor necrosis factor alpha-mediated mitochondrial dysfunction, downstream caspase activation, and hepatocyte apoptosis. CONCLUSIONS: These data suggest that tumor necrosis factor alpha triggers Bid-dependent lysosomal permeabilization, followed by release of cathepsin B into the cytosol and activation of caspase 2. Caspase 2 then facilitates efficient mitochondrial cytochrome c release and apoptosis.

TNF-alpha-mediated lysosomal permeabilization is FAN and caspase 8/Bid dependent.

TNF-alpha cytotoxic signaling involves lysosomal permeabilization with release of the lysosomal protease cathepsin B (ctsb) into the cytosol. However, the mechanisms mediating lysosomal breakdown remain unclear. Because caspase-8 and factor associated with neutral sphingomyelinase activation (FAN) have been implicated as proximal mediators of TNF-alpha-associated apoptosis, their role in lysosomal permeabilization was examined. Cellular distribution of ctsb-green fluorescent protein (ctsb-GFP) in a rat hepatoma cell line was imaged by confocal microscopy. ctsb-GFP fluorescence was punctate under basal conditions but became diffuse after treatment with TNF-alpha/actinomycin D. This cellular redistribution of ctsb-GFP was blocked by transfection with a vector expressing a dominant-negative Fas-associated protein with death domain (DeltaFADD), cytokine response modifier A, or a pharmacological caspase-8 inhibitor, IETD-fmk. Consistent with the concept that caspase 8-mediated apoptosis is also Bid-dependent in hepatocytes, ctsb-GFP release from lysosomes was reduced in hepatocytes from Bid(-/-) mice. Interestingly, transfection with a vector expressing a dominant-negative FAN (DeltaFAN) also blocked ctsb-GFP release and caspase-8 activation. Paradigms that inhibited ctsb-GFP release from lysosomes also reduced apoptosis as assessed by morphology and biochemical criteria. In conclusion, these studies suggest FAN is upstream of caspase-8/Bid in a signaling cascade culminating in lysosomal permeabilization.

Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent.

Cathepsin B (Cat B) is released from lysososomes during tumor necrosis factor-alpha (TNF-alpha) cytotoxic signaling in hepatocytes and contributes to cell death. Sphingosine has recently been implicated in lysosomal permeabilization and is increased in the liver by TNF-alpha. Thus the aims of this study were to examine the mechanisms involved in TNF-alpha-associated lysosomal permeabilization, especially the role of sphingosine. Confocal microscopy demonstrated Cat B-green fluorescent protein and LysoTracker Red were both released from lysosomes after treatment of McNtcp.24 cells with TNF-alpha/actinomycin D, a finding compatible with lysosomal destabilization. In contrast, endosomes labeled with Texas Red dextran remained intact, suggesting lysosomes were specifically targeted for permeabilization. LysoTracker Red was released from lysosomes in hepatocytes treated with TNF-alpha or sphingosine in Cat B(+/+) but not Cat B(-/-) hepatocytes, as assessed by a fluorescence-based assay. With the use of a calcein release assay in isolated lysosomes, sphingosine permeabilized liver lysosomes isolated from Cat B(+/+) but not Cat B(-/-) liver. C(6) ceramide did not permeabilize lysosomes. In conclusion, these data implicate a sphingosine-Cat B interaction inducing lysosomal destabilization during TNF-alpha cytotoxic signaling.