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

Abstract

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.

Keywords: Autophagy; Cell death; ER Ca(2+)depletion; SERCA; Thapsigargin; Unfolded protein response.