Gradual ER calcium depletion induces a progressive and reversible UPR signaling

The unfolded protein response (UPR) is a widespread signal transduction pathway triggered by endoplasmic reticulum (ER) stress. Because calcium (Ca²⁺) is a key factor in the maintenance of ER homeostasis, massive Ca²⁺ depletion of the ER is a potent inducer of ER stress. Although moderate changes in ER Ca²⁺ drive the ubiquitous Ca²⁺ signaling pathways, a possible incremental relationship between UPR activation and Ca²⁺ changes has yet to be described. Here, we determine the sensitivity and time-dependency of activation of the three ER stress sensors, inositol-requiring protein 1 alpha (IRE1α), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 alpha (ATF6α) in response to controlled changes in the concentration of ER Ca²⁺ in human cultured cells. Combining Ca²⁺ imaging, fluorescence recovery after photobleaching experiments, biochemical analyses, and mathematical modeling, we uncover a nonlinear rate of activation of the IRE1α branch of UPR, as compared to the PERK and ATF6α branches that become activated gradually with time and are sensitive to more important ER Ca²⁺ depletions. However, the three arms are all activated within a 1 h timescale. The model predicted the deactivation of PERK and IRE1α upon refilling the ER with Ca²⁺. Accordingly, we showed that ER Ca²⁺ replenishment leads to the complete reversion of IRE1α and PERK phosphorylation in less than 15 min, thus revealing the highly plastic character of the activation of the upstream UPR sensors. In conclusion, our results reveal a dynamic and dose-sensitive Ca²⁺-dependent activation/deactivation cycle of UPR induction, which could tightly control cell fate upon acute and/or chronic stress.