Role of Endoplasmic Reticulum Calcium Disequilibria in the Mechanism of Homocysteine-Induced ER Stress

Abstract

Our laboratory demonstrated that hyperhomocysteinemia accelerates atherosclerosis in mouse models through ER stress and activation of the unfolded protein response (UPR). In this study, we tested the hypothesis that homocysteine-induced ER stress may arise from ER-Ca²⁺ disequilibria. We found that homocysteine-induced cytosolic Ca²⁺ transients in T24/83 cells and human aortic smooth muscle cells (HASMCs). These calcium effects occurred at concentrations of homocysteine in the external medium (1–5 mM) that increase intracellular homocysteine in these cell types. Prolonged homocysteine treatment (5 h) at these exogenous concentrations reduced ER-Ca²⁺ emptying evoked by thapsigargin. However, these homocysteine-induced effects on ER-Ca²⁺ emptying were of a much smaller magnitude than those evoked by A23187 or thapsigargin (ER stressors known to induce ER stress through ER-Ca²⁺ depletion). T24/83 cells stably overexpressing the Ca²⁺-binding ER chaperone GRP78 showed diminished cytosolic Ca²⁺ transients induced by homocysteine and reduced ER-Ca²⁺ emptying evoked by thapsigargin. Prevention of the homocysteine-induced UPR by cycloheximide pretreatment normalized GRP78 expression and ER-Ca²⁺ emptying evoked by thapsigargin. These results are inconsistent with a mechanism of ER stress induction by homocysteine through ER-Ca²⁺ depletion.