Endoplasmic reticulum (ER) stress is observed in many human diseases, often associated with inflammation. ER stress can trigger inflammation through nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammasome, which might stimulate inflammasome formation by association with damaged mitochondria. How ER stress triggers mitochondrial dysfunction and inflammasome activation is ill defined. Here we have used an infection model to show that the IRE1alpha ER stress sensor regulates regulated mitochondrial dysfunction through an NLRP3-mediated feed-forward loop, independently of ASC. IRE1alpha activation increased mitochondrial reactive oxygen species, promoting NLRP3 association with mitochondria. NLRP3 was required for ER stress-induced cleavage of caspase-2 and the pro-apoptotic factor, Bid, leading to subsequent release of mitochondrial contents. Caspase-2 and Bid were necessary for activation of the canonical inflammasome by infection-associated or general ER stress. These data identify an NLRP3-caspase-2-dependent mechanism that relays ER stress to the mitochondria to promote inflammation, integrating cellular stress and innate immunity.

Exposure to the toxic metalloid arsenic is associated with diabetes and cancer and causes proteotoxicity and endoplasmic reticulum (ER) stress at the cellular level. Adaptive responses to ER stress are implicated in cancer and diabetes; thus, understanding mechanisms of arsenic-induced ER stress may offer insights into pathogenesis. Here, we identify genes required for arsenite-induced ER stress response in a genome-wide RNAi screen. Using an shRNA library targeting ∼20,000 human genes, together with an ER stress cell model, we performed flow cytometry-based cell sorting to isolate cells with defective response to arsenite. Our screen discovered several genes modulating arsenite-induced ER stress, including sodium-dependent neutral amino acid transporter, SNAT2. SNAT2 expression and activity are up-regulated by arsenite, in a manner dependent on activating transcription factor 4 (ATF4), an important mediator of the integrated stress response. Inhibition of SNAT2 expression or activity or deprivation of its primary substrate, glutamine, specifically suppressed ER stress induced by arsenite but not tunicamycin. Induction of SNAT2 is coincident with the activation of the nutrient-sensing mammalian target of rapamycin (mTOR) pathway, which is at least partially required for arsenite-induced ER stress. Importantly, inhibition of the SNAT2 or the System L transporter, LAT1, suppressed mTOR activation by arsenite, supporting a role for these transporters in modulating amino acid signaling. These findings reveal SNAT2 as an important and specific mediator of arsenic-induced ER stress, and suggest a role for aberrant mTOR activation in arsenic-related human diseases. Furthermore, this study demonstrates the utility of RNAi screens in elucidating cellular mechanisms of environmental toxins.

Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia. The pathogenesis of these diseases involves β-cell dysfunction and death. The primary function of β-cells is to tightly regulate the secretion, production, and storage of insulin in response to blood glucose levels. In order to manage insulin biosynthesis, β-cells have an elaborate endoplasmic reticulum (ER). The ER is an essential organelle for the proper processing and folding of proteins such as proinsulin. Proteins fold properly when the ER protein load balances with the ER folding capacity that handles this load. Disruption of this ER homeostasis by genetic and environmental stimuli leads to an accumulation of misfolded and unfolded proteins, a condition known as ER stress. Upon ER stress, the unfolded protein response (UPR) is activated. The UPR is a signaling network that aims to alleviate ER stress and restore ER homeostasis promoting cell survival. Hence, the UPR allows β-cells to handle the physiological fluctuations of insulin demand. However upon severe unresolvable ER stress conditions such as during diabetes progression, the UPR switches to pathological outputs leading to β-cell dysfunction and apoptosis. Severe ER stress may also trigger inflammation and accumulating evidence suggests that inflammation also contributes to β-cell failure, but the mechanisms remain elusive. In this dissertation, we demonstrate that thioredoxin interacting protein (TXNIP) mediates ER stress induced β-cell inflammation and apoptosis. During a DNA microarray analysis to identify novel survival and death components of the UPR, we identified TXNIP as an interesting proapoptotic candidate as it has been linked to glucotoxicity in β-cells. During our detailed investigation, we discovered that TXNIP is selectively expressed in β-cells of the pancreas and is strongly induced by ER stress through the IRE1α and PERK-eIF2α arms of the UPR and specifically its transcription is regulated by activating transcription factor 5 (ATF5) and carbohydrate response element binding protein (ChREBP) transcription factors. As TXNIP has been shown to activate the Nod-like receptor protein 3 (NLRP3) inflammasome leading to the production of the inflammatory cytokine interleukin-1β (IL- 1β), we hypothesized that perhaps TXNIP has a role in IL-1β production under ER stress. We show that ER stress can induce IL-1β production and that IL-1β is capable of binding to IL-1 type 1 receptor (IL-1R1) on the surface of β-cells stimulating its own expression. More importantly, we demonstrate that TXNIP does indeed play a role in ER stress mediated IL-1β production through the NLRP3 inflammasome. Furthermore, we also confirmed that TXNIP is a mediator of β-cell apoptosis under ER stress partially through IL-1β signaling. Collectively, we provide significant novel findings that TXNIP is a component of the UPR, mediates IL-1β production and autostimulation, and induces cell death under ER stress in β-cells. It is becoming clear that TXNIP has a role in the pathogenesis of diabetes and is a link between ER stress, oxidative stress and inflammation. Understanding the molecular mechanisms involved in TXNIP expression, activity, and function as we do here will shed light on potential therapeutic strategies to tackle diabetes.