While exploring the sequence of protein-protein interactions initiated by the death receptor Fas (CD95), we discovered an adapter protein, FADD/Mort1. This protein associates with death receptors and recruits MACH/caspase-8, a member of the caspase cysteine protease family that plays a crucial role in all apoptotic processes. We also discovered the protein cFLIP/CASH, a Caspase-8 homologue that serves as a biological inhibitor of death induction and initiator of non-apoptotic effects of the receptors. Both genetic screens and protein-purification approaches are applied to isolate regulatory proteins that associate with Caspase-8 and cFLIP/CASH.
Our analysis of the in vivo role of Caspase-8 by targeted disruption of its gene in mice, and by its conditional knockout using the Cre/loxP recombination system, has confirmed that the enzyme plays a pivotal role in death induction. Deletion of Caspase-8 in hepatocytes, for example, protected them from Fas-induced hepatocyte death and from fatal liver damage similar to that which occurs in acute hepatitis.
In addition, this analysis revealed that Caspase-8 also serves cellular functions that are non-apoptotic. Caspase-8 deletion in endothelial cells resulted in degeneration of the yolk sac vasculature and embryonic death due to circulatory failure. Its deletion in bone-marrow cells resulted in arrest of hematopoietic progenitor functioning, and, in cells of the myelomonocytic lineage - in arrest of differentiation into macrophages and to death.
An alternative form of programmed cell death termed programmed necrosis or necroptosis has emerged recently. The induction of this death form does not require caspase activity, but is rather inhibited by it. It turns out that, contrary to the prior connotation of “necrosis” as a haphazard phenomenon, specific triggers can induce necrosis in a highly regulated manner. One of the key proteins in this death pathway, a pseudokinase MLKL, mediates cell death by binding to cellular membranes, thereby triggering ion fluxes. We revealed further complexity in this process: the association of MLKL with the cell membrane in necroptotic death is preceded by the translocation of activated MLKL to the nucleus. We are characterizing the molecular and functional consequences of this translocation.
There are indications that MLKL, as well as other core components of the necroptotic machinery, also signal for some non-deadly functions. In our hands, mice with specific deletion of caspase-8 in dendritic cells developed an aggressive systemic inflammatory disease and were highly susceptible to the lethal effect of LPS. LPS stimulation of caspase-8 deficient dendritic cells led to MLKL-dependent enhanced activation of the NLRP3/ASC1/caspase-1 inflammasome and IL-1β expression not accompanied by cell death.
In another study, we demonstrated that mice lacking Caspase-8 in the skin epidermis develop fatal acute perinatal cutaneous inflammation accompanied by marked hyperactivation of the type-I interferon pathway. In this model, Caspase-8–deficient keratinocytes exhibited enhanced gene activation by transfected DNA. We proposed that Caspase-8 acts to restrain excessive activation of pathways of response to endogenous intracellular nucleic acids, wherefore its deficiency can directly trigger sterile inflammation.
We are now analyzing a series of additional model systems and procedures for modification of Caspase-8 action both in cell culture and in vivo, aiming at identification of novel proinflammatory signaling regulators downstream of Caspase-8.