Detection Of Endoplasmic Reticulum Stress Research Articles

IRE1 signaling increases PERK expression during chronic ER stress

The Unfolded Protein Response (UPR) is an essential cellular process activated by the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), a condition referred to as ER stress. Three ER anchored receptors, IRE1, PERK and ATF6 act as ER stress sensors monitoring the health of the ER. Upon detection of ER stress, IRE1, PERK and ATF6 initiate downstream signaling pathways collectively referred to as the UPR. The overarching aim of the UPR is to restore ER homeostasis by reducing ER stress, however if that is not possible, the UPR transitions from a pro-survival to a pro-death response. While our understanding of the key signaling pathways central to the UPR is well defined, the same is not true of the subtle signaling events that help fine tune the UPR, supporting its ability to adapt to varying amplitudes or durations of ER stress. In this study, we demonstrate cross talk between the IRE1 and PERK branches of the UPR, wherein IRE1 via XBP1s signaling helps to sustain PERK expression during prolonged ER stress. Our findings suggest cross talk between UPR branches aids adaptiveness thereby helping to support the plasticity of UPR signaling responses.

The copper (II) complex of salicylate phenanthroline induces immunogenic cell death of colorectal cancer cells through inducing endoplasmic reticulum stress

BackgroundIn our previous study, Cu(sal)phen was found to have anti-tumor effects, yet its precise mechanism remains unknown. Research has shown that dying tumor cells release damage-associated molecular patterns (DAMPs) to promote anti-tumor immune response. Therefore, we have further explored the effects and potential molecular mechanisms of Cu(sal)phen-induced immunogenic cell death (ICD) in colorectal cancer (CRC). MethodsELISA and flow cytometry were used to detect the effects of Cu(sal)phen treatment on ICD markers. The molecular mechanisms of Cu(sal)phen-induced ICD were investigated through the detection of endoplasmic reticulum stress (ERS) and reactive oxygen species (ROS) in vitro using Western blot and flow cytometry. Additionally, a mouse model was constructed to study the effects of Cu(sal)phen on immune cells and anti-tumor-related cytokines in vivo. ResultsCu(sal)phen induced the release of calreticulin (CRT), adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1), the main molecular markers of ICD, by promoting the accumulation of ROS and inducing ERS. Furthermore, Cu(sal)phen promoted the maturation of dendritic cells (DCs) and activation of CD8+T cells, as well as the secretion of interleukin-12 (IL-12) and interferon-γ (IFN-γ), while downregulating transforming growth factor-β (TGF-β) levels, thereby activating the anti-tumor immune response. ConclusionCu(sal)phen has the potential to induce ICD in tumors and activate the adaptive immune response to achieve anti-tumor effects. This makes Cu(sal)phen a promising candidate for the treatment of CRC.

Detection of ER Stress in iPSC-Derived Neurons Carrying the p.N370S Mutation in the GBA1 Gene.

Endoplasmic reticulum (ER) stress is involved in the pathogenesis of many human diseases, such as cancer, type 2 diabetes, kidney disease, atherosclerosis and neurodegenerative diseases, in particular Parkinson's disease (PD). Since there is currently no treatment for PD, a better understanding of the molecular mechanisms underlying its pathogenesis, including the mechanisms of the switch from adaptation in the form of unfolded protein response (UPR) to apoptosis under ER stress conditions, may help in the search for treatment methods. Genetically encoded biosensors based on fluorescent proteins are suitable tools that facilitate the study of living cells and visualization of molecular events in real time. The combination of technologies to generate patient-specific iPSC lines and genetically encoded biosensors allows the creation of cell models with new properties. Using CRISPR-Cas9-mediated homologous recombination at the AAVS1 locus of iPSC with the genetic variant p.N370S (rs76763715) in the GBA1 gene, we created a cell model designed to study the activation conditions of the IRE1-XBP1 cascade of the UPR system. The cell lines obtained have a doxycycline-dependent expression of the genetically encoded biosensor XBP1-TagRFP, possess all the properties of human pluripotent cells, and can be used to test physical conditions and chemical compounds that affect the development of ER stress, the functioning of the UPR system, and in particular, the IRE1-XBP1 cascade.

Detection of endoplasmic reticulum stress and the unfolded protein response in naturally-occurring endocrinopathic equine laminitis

BackgroundLaminitis is often associated with endocrinopathies that cause hyperinsulinemia and is also induced experimentally by hyperinsulinemia, suggesting that insulin initiates laminitis pathogenesis. Hyperinsulinemia is expected to activate pro-growth and anabolic signaling pathways. We hypothesize that chronic over-stimulation of these pathways in lamellar tissue results in endoplasmic reticulum stress, contributing to tissue pathology, as it does in human metabolic diseases. We tested this hypothesis by asking whether lamellar tissue from horses with naturally-occurring endocrinopathic laminitis showed expression of protein markers of endoplasmic reticulum stress.ResultsThree markers of endoplasmic reticulum stress, spliced XBP1, Grp78/BiP and Grp94, were upregulated 2.5–9.5 fold in lamellar tissues of moderately to severely laminitic front limbs (n = 12) compared to levels in controls (n = 6–7) measured by immunoblotting and densitometry. Comparing expression levels between laminitic front limbs and less affected hind limbs from the same horses (paired samples from 7 to 8 individual horses) demonstrated significantly higher expression for both spliced XBP1 and Grp78/BiP in the laminitic front limbs, and a similar trend for Grp94. Expression levels of the 3 markers were minimal in all samples of the control (n = 6–7) or hind limb groups (n = 7–8). Immunofluorescent localizations were used to identify cell types expressing high levels of Grp78/BiP, as an indicator of endoplasmic reticulum stress. Grp78/BiP expression was highly elevated in suprabasal epidermal keratinocytes and only observed in laminitic front limbs (10/12 laminitic samples, compared to 0/7 in sections from the hind limbs and 0/5 of controls).ConclusionsThese data demonstrate that the endoplasmic reticulum stress pathway is active in naturally occurring cases of laminitis and is most active within a subset of epidermal keratinocytes. These data provide the rationale for further study of endoplasmic reticulum stress in experimental models of laminitis and the links between laminitis and human diseases sharing activation of this stress pathway. Pharmacological options to manipulate the endoplasmic reticulum stress pathway under investigation for human disease could be applicable to laminitis treatment and prevention should this pathway prove to be a driver of disease progression.

Real-Time and Non-invasive Monitoring of the Activation of the IRE1α-XBP1 Pathway in Individuals with Hemodynamic Impairment

Many stressors that are encountered upon kidney injury are likely to trigger endoplasmic reticulum (ER) stress, subsequently activating transcriptional, translational and metabolic reprogramming. Monitoring early cellular adaptive responses engaged after hemodynamic impairment yields may represent a clinically relevant approach. However, a non-invasive method for detecting the ER stress response has not been developed. We combined a metabolomic approach with genetic marker analyses using urine from individuals undergoing scheduled cardiac surgery under cardiopulmonary bypass to investigate the feasibility and significance of monitoring the ER stress response in the kidney. We developed an original method based on fragment analysis that measures urinary levels of the spliced X-box binding protein 1 (sXBP1) mRNA as a proxy of inositol-requiring enzyme 1α (IRE1α) activity because sXBP1 is absolutely sensitive and specific for ER stress. The early engagement of the ER stress response after ischemic stress is critical for protecting against tissue damage, and individuals who mount a robust adaptive response are protected against AKI. The clinical consequences of our findings are of considerable importance because ER stress is involved in numerous conditions that lead to AKI and chronic kidney disease; in addition, the detection of ER stress is straightforward and immediately available in routine practice.

Detection of ER stress in vivo by Raman spectroscopy

The endoplasmic reticulum (ER) is an organelle in which most membrane and secretory proteins are synthesized. If these proteins are not folded correctly, unfolded proteins accumulate in the ER lumen, causing a cellular situation known as ER stress. Recently, many studies on the relationship between ER stress and diseases have been reported. Thus, studies of ER stress in vivo should yield information that is useful in pathology. Model mice have been developed as a powerful tool to visualize ER stress in vivo, but this approach depends on transgenic technology. Here, we report on a method of detecting ER stress in vivo by Raman spectroscopy. Our experiments revealed that two specific Raman bands were reduced in both cultured cells and animal tissues in an ER stress dependent manner. This suggests that Raman spectroscopy could be a useful tool to detect ER stress in vivo without transgenic technology.