Characterization of the signaling pathways required to initiate apoptosis from the endoplasmic reticulum

Apoptosis, or programmed cell death, is an evolutionarily conserved mechanism essential for development and homeostasis in multicellular organisms. Dysregulation of the cell death process is associated with neurodegenerative diseases, autoimmunity and cancer. The second most common cancer of the hematologic system is multiple myeloma (MM), an incurable plasma cell malignancy. Plasma cells are professional secretory cells with a highly developed endoplasmic reticulum (ER) that is specialized for the production of thousands of antibody molecules per second. Bortezomib (Velcade(TM), PS-341), a specific inhibitor of the 26S proteasome, has shown significant anti-tumor activity against MM with a favorable toxicity profile and was recently approved by the United States Food and Drug Administration for the treatment of relapsed or refractory MM. However, the 26S proteasome is a multisubunit complex present in the nucleus and the cytoplasm of all eukaryotic cells and is responsible for the degradation of over 80% of all cellular proteins. Thus the nature of the selectivity of proteasome inhibitors (PIs) for MM cells has not been completely understood. As proteasome inhibition has been shown to inhibit both the removal of misfolded proteins from the ER lumen and their subsequent degradation, we hypothesized that the nature of the sensitivity of MM cells to PIs was related to their function as professional secretory cells. Consistent with reports that physiological unfolded protein response (UPR) components are required for plasma cell differentiation, we have demonstrated that the ER chaperones GRP78 and GRP94 are constitutively expressed in MM cells and that the levels of these proteins do not change in response to various apoptotic stimuli. However, PIs and classical ER stress-inducing agents activate the ER stress-specific eIF-2alpha kinase, PERK, within 30 minutes of treatment leading to the rapid induction of its downstream targets: ATF4 and the proapoptotic transcription factor, GADD153. These results suggest that in secretory cells that cannot further induce the expression of pro-survival UPR components, PIs initiate a terminal UPR that is associated with the induction of apoptosis. Interestingly, we also determined that ER-associated initiator caspase (murine caspase-12 or human caspase-4) expression is not required for PI-induced apoptosis. As the proteins that communicate a death signal from the ER to the cellular apoptotic machinery have yet to be conclusively identified, we examined the role of caspase-12/-4 in the initiation of ER stress-induced apoptosis. (Abstract shortened by UMI.