Nonlinear relationship between ER Ca2+ depletion versus induction of the unfolded protein response, autophagy inhibition, and cell death

Endoplasmic reticulum (ER) Ca2+ depletion activates the unfolded protein response (UPR), inhibits bulk autophagy
and eventually induces cell death in mammalian cells. However, the extent and duration of ER Ca2+
depletion required is unknown. We instigated a detailed study in two different cell lines, using sarco/endoplasmic
reticulum Ca2+-ATPase (SERCA) inhibitors to gradually reduce ER Ca2+ levels in a controlled
manner. Remarkably, UPR induction (as assessed by expression analyses of UPR-regulated proteins) and autophagy
inhibition (as assessed by analyses of effects on starvation-induced bulk autophagy) required substantially
higher drug concentrations than those needed to strongly decrease total ER Ca2+ levels. In fact, even
when ER Ca2+ levels were so low that we could hardly detect any release of Ca2+ upon challenge with ER Ca2+
purging agents, UPR was not induced, and starvation-induced bulk autophagy was still fully supported.
Moreover, although we observed reduced cell proliferation at this very low level of ER Ca2+, cells could tolerate
prolonged periods (days) without succumbing to cell death. Addition of increasing concentrations of extracellular
EGTA also gradually depleted the ER of Ca2+, and, as with the SERCA inhibitors, EGTA-induced activation
of UPR and cell death required higher EGTA concentrations than those needed to strongly reduce ER Ca2+
levels. We conclude that ER Ca2+ depletion-induced effects on UPR, autophagy and cell death require either an
extreme general depletion of ER Ca2+ levels, or Ca2+ depletion in areas of the ER that have a higher resistance to
Ca2+ drainage than the bulk of the ER.