Description
Background: Thapsigargin (TG) mimics the cellular events that occur during pathological stresses, such as ischemia, which dysregulates cellular calcium. By inhibiting SERCA, TG decreases SR/ER calcium in cardiac myocytes, which impairs protein folding in the ER, causing ER stress. Initially, ER stress is adaptive because it activates processes aimed at improving ER protein-folding. However, if ER stress is not resolved, continued accumulation of toxic misfolded proteins in the SR/ER leads to cell death. Initial experiments showed that cells treated with TG in a low media volume were less susceptible to ER stressmediated cell death than cells treated with TG in a high media volume. This did not happen with a different ER stressor, tunicamycin (TM), which does not affect ER calcium. Hypothesis: This observation led to the hypothesis that TG causes the secretion of cytoprotective factors. Specific Aims: This hypothesis was addressed in three specific aims, which were: 1) to characterize the volume-dependent protective effects of TG, and to determine the cause of cell death in high volume TG treatment, 2) to determine if TG-conditioned media (CM) is able to transfer protective factors to naive cells, and 3) to test whether adding recombinant forms of proteins found in proteomic analyses of TG-CM reproduce the protective effects of TG. Approach: These aims were addressed using HeLa and cardiac myocyte cultured cell model systems. Results: (1) The protective effects of TG diminished in direct relation to increases of media volume. (2) CM prepared with TG/low volume treated cells transferred some cytoprotection to naïve cells, although this effect was variable, possibly because the secreted factors are labile. (3) Addition of bacterially-generated, recombinant forms of GRP94, GRP78, Crt and MANF did not consistently confer cytoprotection against TG-mediated cell death. Conclusions: Cytoprotective factors are released from cells upon treatment with TG, but not TM. However, even though some proteins secreted from cells upon treatment with TG have been identified, it remains unclear whether any of these proteins is/are responsible for the observed volume-dependent cytoprotective effects of TG.
