In the present study, we demonstrate that CA-074Me, a drug described as a potent and specific cathepsin B inhibitor, protects macrophages against LT-dependent cell death without inhibiting LF-mediated translocation or activity (as assessed by MEK cleavage), but instead by blocking some step upstream of caspase-1 activation. LT induced an increase in cytoplasmic cathepsin B activity, indicating a LMP event which occurred earlier than the large-scale lysosomal destabilization coupled to cell lysis (as indicated by a complete loss of LysoTracker Red fluorescence). LMP and cathepsin translocation into the cytosol can begin prior to the loss of lysosomal acidic pH, measurable by LysoTracker fluorescence, sometimes taking 30 to 90 min before dye-based visualization is possible (5
). We hypothesize that there is an initial event triggering LMP that correlates with increased cytoplasmic cathepsin B activity (as measured by our activity assay) and a later, large-scale LMP event immediately preceding cell death (as measured by fluorescence microscopy). It is unclear, however, whether CA-074Me protects against LT-mediated lysis through inhibition of cathepsin B itself or other cellular proteases because a reduction of the enzyme by siRNA in RAW 264.7 cells does not protect against toxin. Furthermore, we cannot state whether CA-074Me protection actually targets protease release associated with LT-induced LMP.
There is an increasing recognition that cathepsin B is critically involved in a number of different functions separate from its established role in lysosomal protein degradation and protein processing (33
). The release of cathepsin B from lysosomes is involved in both apoptosis (typically after partial LMP) and necrosis (after large-scale LMP) (9
). Furthermore, cathepsin B release has been shown to act both upstream and downstream of NLRP3 inflammasome activation (16
). Indeed, lysosomal destabilization and the cytoplasmic activity of lysosomal proteases such as cathepsin B may be a universal mechanism through which a variety of stimuli induce the proinflammatory responses associated with inflammasome activation (reviewed in references 14
Release of cathepsin B into the cytoplasm by LT corresponds closely in time with the activation of caspase-1 by LT. This suggests that a feedback amplification mechanism might be operative, one in which traces of cytosolic proteases associated with the lysosome leakage process might act on the NLRP1b inflammasome or its procaspase-1 component to begin an activation cascade. In this scenario, the CA-074Me inhibitor may actually act on the putative cytoplasmic enzyme required for NLRP1b-mediated caspase-1 activation, preventing further LMP-based enzyme release.
Interestingly, the relationship between NLRP3-activated caspase-1 and cathepsin B is also unclear. Lysosomal destabilization and the release of cathepsin B have been implicated in the activation of the NLRP3 inflammasome/caspase-1 after exposure to nigericin, amyloid β, aluminum salts, silica crystals, microparticle vaccine adjuvants, osmotic lysosomal rupture, and Leu-Leu-OMe (19
). However, in a contrary view, cathepsin B release from lysosomes has been described as resulting from the upstream activation of NLRP3 that occurs upon transfection of disease-associated mutant forms of NLRP3, treatment with certain antiviral drugs, and S. flexneri
infection, although cell death in these scenarios is caspase-1 independent (16
). Thus, LMP has been shown to both cause the release of cathepsin B and be dependent on the activity of cathepsin B, contrasting observations that taken together could support the cathepsin-dependent positive-feedback loop mentioned above (16
Cathepsin B has also been shown to activate caspase-1 and caspase-11 in vitro and in vivo, further suggesting that there could be important interactions between cathepsin B and the inflammasome (4
). In a recent study, both caspase-1 and caspase-11 were found to associate with NLRP1b in response to LT treatment (35
). Caspase-11 has also been reported to be required for caspase-1 activation, and cathepsin B may act on caspase-11 (25
We tried to relate caspase-1 activation to cytoplasmic cathepsin B activity by observing cytoplasmic cathepsin B activity in the presence of a pan-caspase inhibitor. We hypothesized that the inhibition of caspase-1 activity would lead to inhibition of cathepsin B release if caspase-1 was necessary for amplification of the cathepsin B release. These experiments were complicated by the discovery that the potent caspase inhibitor Boc-d
-CMK, previously used to implicate caspase-1 activity in LT-mediated macrophage death (46
), is also a potent cathepsin B inhibitor, much in the manner previously shown for other caspase inhibitors (17
). This inhibitor, a potent protector against LT-mediated lysis (12
), may act through targeting both caspase-1 and cathepsin B. These results underscore the need to interpret data from pharmacological approaches with caution and to use inhibitors in conjunction with other methods to clarify the role of cathepsins and caspases in various cellular pathways. For this very reason, we performed siRNA experiments which suggest that CA-074Me may protect LT by acting on other cellular enzymes in addition to cathepsin B. However, the half-life of previously transcribed and processed mature cathepsin B is around 24 h (6
), and it is likely that some active enzyme remained during toxicity testing. Only a few molecules of cathepsin B could be sufficient for inducing the events that lead to caspase-1 activation, making it difficult to observe a significant change in LT-protection with these knockdown experiments. Furthermore, it has been demonstrated that CA-074Me and CA-074 can inhibit cathepsins L and X (enzymes also released during LMP), and concerns remain that they may also act on other unknown targets (28
). Therefore, it is possible that although cytoplasmic cathepsin B is a clear measure for LT-induced LMP, the enzyme itself is not involved in LT-mediated cell death.
The mechanism(s) by which LT induces LMP remain unknown. One membrane-active enzyme that could play a role is phospholipase A2
is known to be a strong inducer of LMP (61
), and it has previously been implicated in LT-mediated cell death (44
). Various forms of PLA2
are activated by both lysosomal proteases and K+
effluxes, and in turn, PLA2
can regulate caspase-1 activity (3
). Our preliminary data suggest that the iPLA2β
-specific inhibitor (S
)-bromoenol lactone (23
) does, in fact, protect macrophages against LT-dependent cell death (data not shown).
Another organelle affected by LT that may be involved in the regulation of LT-mediated LMP is the mitochondrion. Strong interdependent relationships exist between the state of lysosomes and mitochondria (7
). Mitochondrial membrane potential disruption is an essential, proteasome-dependent step in LT-induced cell death (2
). A key mediator of mitochondrial or lysosomal damage could be LT-induced reactive oxygen species (20
), since reactive oxygen species molecules can easily damage lysosomal membranes (7
). Finally, the channel formed by PA in endosomes could possibly act as a potential small-scale LMP trigger. In cells engineered to overexpress anthrax toxin receptor 1 (TEM8), exposure to PA induces cell death through what was proposed to be an LMP-like event (40
). One can imagine that a membrane-inserted PA channel might traffic to lysosomes and cause small-scale content release, which could act in concert with other LF-dependent events to produce cell death and inflammasome activation.
To summarize, we report that anthrax LT-induced cell death results in LMP events and CA-074Me, an inhibitor of lysosomal proteases, prevents cell death. Although CA-074Me inhibited the NLRP1b inflammasome and caspase-1 activation by LT, it is possible that caspase-1 activation also contributes to LT-induced LMP.