XBP1U inhibits the XBP1S-mediated upregulation of the iNOS gene expression in mammalian ER stress response

Upregulation of the inducible nitric oxide synthase (iNOS) gene is associated with many pathological conditions such as endoplasmic reticulum (ER) stress, and X-box binding protein 1 (XBP1) is critical in mediating ER-stress responsive genes, including iNOS. Nonetheless, the mechanism by which XBP1 regulates iNOS during ER stress remains unexplored. Here we show that the active/spliced form of XBP1 protein, XBP1S, directly binds to the AABS (A-activator-binding site) in the iNOS promoter in vitro and in living cells. XBP1S exhibits dose-dependent activation of iNOS-specific reporter gene activity and endogenous iNOS expression. XBP1S is elevated whereas the unspliced form of XBP1, XBP1U, reduced in ER stress in HepG2 cells. In addition, XBP1U binds to XBP1S and this complex is associated with the iNOS promoter in response to ER stress. Furthermore, XBP1U acts as a negative mediator and suppresses XBP1S-mediated induction of iNOS. Collectively, we present the first evidence demonstrating the regulation of iNOS gene induction by the interaction between the spliced and unspliced forms of XBP1 in response to ER stress.     

MicroRNA-382 silencing induces a mitonuclear protein imbalance and activates the mitochondrial unfolded protein response in muscle cells

Proper mitochondrial function plays a central role in cellular metabolism. Various diseases as well as aging are associated with diminished mitochondrial function. Previously, we identified 19 miRNAs putatively involved in the regulation of mitochondrial metabolism in skeletal muscle, a highly metabolically active tissue. In the current study, these 19 miRNAs were individually silenced in C2C12 myotubes using antisense oligonucleotides, followed by measurement of the expression of 27 genes known to play a major role in regulating mitochondrial metabolism. Based on the outcomes, we then focused on miR-382-5p and identified pathways affected by its silencing using microarrays, investigated protein expression, and studied cellular respiration. Silencing of miRNA-382-5p significantly increased the expression of several genes involved in mitochondrial dynamics and biogenesis. Conventional microarray analysis in C2C12 myotubes silenced for miRNA-382-5p revealed a collective downregulation of mitochondrial ribosomal proteins and respiratory chain proteins. This effect was accompanied by an imbalance between mitochondrial proteins encoded by the nuclear and mitochondrial DNA (1.35-fold, p < 0.01) and an induction of HSP60 protein (1.31-fold, p < 0.05), indicating activation of the mitochondrial unfolded protein response (mtUPR). Furthermore, silencing of miR-382-5p reduced basal oxygen consumption rate by 14% ( p < 0.05) without affecting mitochondrial content, pointing towards a more efficient mitochondrial function as a result of improved mitochondrial quality control. Taken together, silencing of miR-382-5p induces a mitonuclear protein imbalance and activates the mtUPR in skeletal muscle, a phenomenon that was previously associated with improved longevity.     

Ubiquitin-like protein 5 positively regulates chaperone gene expression in the mitochondrial unfolded protein response

Perturbation of the protein-folding environment in the mitochondrial matrix selectively upregulates the expression of nuclear genes encoding mitochondrial chaperones. To identify components of the signal transduction pathway(s) mediating this mitochondrial unfolded protein response (UPR(mt)), we first isolated a temperature-sensitive mutation (zc32) that conditionally activates the UPR(mt) in C. elegans and subsequently searched for suppressors by systematic inactivation of genes. RNAi of ubl-5, a gene encoding a ubiquitin-like protein, suppresses activation of the UPR(mt) markers hsp-60::gfp and hsp-6::gfp by the zc32 mutation and by other manipulations that promote mitochondrial protein misfolding. ubl-5 (RNAi) inhibits the induction of endogenous mitochondrial chaperone encoding genes hsp-60 and hsp-6 and compromises the ability of animals to cope with mitochondrial stress. Mitochondrial morphology and assembly of multi-subunit mitochondrial complexes of biotinylated proteins are also perturbed in ubl-5(RNAi) worms, indicating that UBL-5 also counteracts physiological levels of mitochondrial stress. Induction of mitochondrial stress promotes accumulation of GFP-tagged UBL-5 in nuclei of transgenic worms, suggesting that UBL-5 effects a nuclear step required for mounting a response to the threat of mitochondrial protein misfolding.     

Sphingolipids and the unfolded protein response

The unfolded protein response (UPR) is a response by the endoplasmic reticulum to stress, classically caused by any disruption to cell homeostasis that results in an accumulation in unfolded proteins. However, there is an increasing body of research demonstrating that the UPR can also be activated by changes in lipid homeostasis, including changes in sphingolipid metabolism. Sphingolipids are a family of bioactive lipids with important roles in both the formation and integrity of cellular membranes, and regulation of key cellular processes, including cell proliferation and apoptosis. Bi-directional interactions between sphingolipids and the UPR have now been observed in a range of diseases, including cancer, diabetes and liver disease. Determining how these two key cellular components influence each other could play an important role in deciphering the causes of these diseases and potentially reveal new therapeutic approaches.