Pivotal role of ERO1α in the arsenite-dependent regulation of Ca2+ homeostasis and mitochondrial superoxide formation

Arsenic (As) is a chemical element belonging to the group of metalloids. Arsenic compounds, widely diffused in nature and largely used in agrotechnical and industrial processes, represent a serious concern for the ecosystem and human health. Indeed, human exposure to arsenic compounds increases the incidence of a plethora of diseases and various types of cancer. At the molecular level, trivalent arsenic (Na2AsO3, arsenite) causes numerous deleterious effects in target cells, through its binding to protein thiols or via the intermediate formation of reactive oxygen species (ROS), in the mitochondrial respiratory chain and via NADPH oxidase activation. Experimental work performed in our laboratory has focused on the mechanisms regulating mitochondrial ROS formation, an event requiring direct effects of the metalloid in the respiratory chain and, most importantly, a significant increase in the mitochondrial concentration of Ca2+. Studies on the mechanism whereby arsenite affects Ca2+ homeostasis provided evidence for an initial stimulation of the inositol 1,4,5-triphosphate receptor (IP3R), maximally induced at low concentrations, and the subsequent activation of the ryanodine receptor (RyR). Interestingly, the release of the cation from the RyR was critical to promote mitochondrial superoxide (mitoO2•−) formation. It follows that arsenite induces mitoO2•− formation only cells concomitantly expressing the IP3R and the RyR (as undifferentiated-U937 cells or terminally differentiated C2C12 cells). These observations emphasize the relevance of the RyR in events associated with mitochondria ROS formation, possibly due to the close apposition of the RyR with these organelles. Interestingly, we also obtained evidence for the existence of critical regulatory mechanisms for RyR activation, different from conventional Ca2+-induced Ca2+ release mechanisms. We found that low dose arsenite dependent Ca2+ release from the IP3R, was responsible for the triggering of an endoplasmic reticulum (ER) stress response, leading to increased expression of ERO1α, which in turn critically regulated RyR activity. Interestingly, pharmacological inhibition of the activity or expression of ERO1α, or its genetic deletion, was associated with the prevention of Ca2+ release from the RyR and the ensuing mitochondrial accumulation of the cation necessary for the formation of mitoO2•−. Under the same conditions, the above treatments/manipulations prevented the early DNA strand scission or late mitochondrial dysfunction and mitochondrial permeability transition (MPT)-dependent apoptosis. We subsequently identified an additional mechanism leading to increased ERO1α expression, mediated by mitoO2•−-derived H2O2, that however failed to impact on Ca2+ homeostasis. Based on these findings, it appears that only the mechanism driven by IP3R-derived Ca2+ increases the expression of ERO1α in the close vicinity of the RyR, possibly in the mitochondria associated membranes (MAMs). In conclusion, direct stimulation of the IP3R is critical for the triggering of the effects of arsenite on Ca2+ homeostasis. This event was associated with the triggering of an ER stress response leading to increased ERO1α expression, possibly in the MAMs, which mediated RyR activation and the ensuing mitochondrial Ca2+ accumulation, critical for induces mitoO2•− formation. Pharmacological inhibition of ERO1α activity or expression was therefore associated with prevention of induces mitoO2•− formation as well as with prevention of the ensuing geno- and cyto-toxic effects. As a final note, we identified an additional mechanism of ERO1α expression, which however failed to affect Ca2+ homeostasis, possibly due its localization in domains of the ER distal from the RyR and the mitochondria.