P125Endoplasmic reticulum stress responses to disrupted endoplasmic reticulum ca2+ homeostasis

Abstract

The disruption of the energy or nutrient balance is the fundamental cause of endoplasmic reticulum (ER) stress, a process that mobilizes corrective strategies to reestablish ER and cellular homeostasis known as the unfolded protein response (UPR). In cardiac tissue, numerous conditions can disrupt this balance, resulting in ER stress and disease states. In this study, we focused on identifying ER stress coping responses (pathways) induced by disruption of the ER homeostasis by ER Ca2+ stores depletion. We employed a small interfering RNA (siRNA) library screen combined with deep sequencing analysis of micro RNA (miRNA) to identify factors that mediate UPR modulation. We utilized the XBP1 splicing luciferase reporter, a target of IRE1α endoribonuclease activity, to screen for modulators of the IRE1α branch of the UPR. We uncovered an ER oxidoreductase (PDIA6) and miR-322-dependent mechanism that regulates the IRE1α branch of UPR. PDIA6 is a recently discovered ER stress response protein and is involved in the protection of cardiomyocytes from ischemia/reperfusion-mediated cell death. We show that PDIA6 interacts with the luminal domain of IRE1α via cysteine residues. PDIA6 is required to maintain IRE1α activity as monitored by IRE1α phosphorylation and XBP1 mRNA splicing. Deep sequencing analysis identified miR-322 as one of the miRNAs that was down-regulated after ER Ca2+ store depletion-induced ER stress. The PDIA6 gene is a target of miR-322, and miR-322 abundance is sensitive to changes in ER and cytosolic Ca2+ concentrations. ER Ca2+ depletion and activation of store operated Ca2+ entry, critical during cardiac function, reduces the abundance of different miRNAs including miR-322, which increases PDIA6 mRNA stability and consequently stabilizes IRE1α activity during the ER stress response. We discovered that silencing of the gene encoding PDIA6 affected ER Ca2+ depletion-dependent activation of the IRE1α signaling branch. This work identifies PDIA6 as a unique component of the UPR and demonstrates interplay between ER and cytosolic Ca2+, PDIA6, IRE1α and miR-322 as a part of a novel pathway (coping mechanisms) activated by disrupted ER Ca2+ homeostasis and activation of SOCE as an adaptive response to cope with ER stress during cardiac function.