Inositol-requiring enzyme 1 alpha is essential for dentinogenesis

The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.

Inflamed cystic fibrosis (CF) human bronchial epithelia (HBE), or normal HBE exposed to supernatant from mucopurulent material (SMM) from CF airways, exhibit endoplasmic reticulum (ER)/Ca(2+) store expansion and amplified Ca(2+)-mediated inflammation. HBE inflammation triggers an unfolded protein response (UPR) coupled to mRNA splicing of X-box binding protein-1 (XBP-1). Because spliced XBP-1 (XBP-1s) promotes ER expansion in other cellular models, we hypothesized that XBP-1s is responsible for the ER/Ca(2+) store expansion in inflamed HBE. XBP-1s was increased in freshly isolated infected/inflamed CF in comparison with normal HBE. The link between airway epithelial inflammation, XBP-1s, and ER/Ca(2+) store expansion was then addressed in murine airways challenged with phosphate-buffered saline or Pseudomonas aeruginosa. P. aeruginosa-challenged mice exhibited airway epithelial ER/Ca(2+) store expansion, which correlated with airway inflammation. P. aeruginosa-induced airway inflammation triggered XBP-1s in ER stress-activated indicator (ERAI) mice. To evaluate the functional role of XBP-1s in airway inflammation linked to ER/Ca(2+) store expansion, control, XBP-1s, or dominant negative XBP-1 (DN-XBP-1) stably expressing 16HBE14o(-) cell lines were used. Studies with cells transfected with an unfolded protein response element (UPRE) luciferase reporter plasmid confirmed that the UPRE was activated or inhibited by expression of XBP-1s or DN-XBP-1, respectively. Expression of XBP-1s induced ER/Ca(2+) store expansion and potentiated bradykinin-increased interleukin (IL)-8 secretion, whereas expression of DN-XBP-1 inhibited bradykinin-dependent IL-8 secretion. In addition, expression of DN-XBP-1 blunted SMM-induced ER/Ca(2+) store expansion and SMM-induced IL-8 secretion. These findings suggest that, in inflamed HBE, XBP-1s is responsible for the ER/Ca(2+) store expansion that confers amplification of Ca(2+)-dependent inflammatory responses.

Domain interaction, a structural property of apolipoprotein E4 (apoE4), is predicted to contribute to the association of apoE4 with Alzheimer disease. Arg-61 apoE mice, a gene-targeted mouse model specific for domain interaction, have lower brain apoE levels and synaptic, functional, and cognitive deficits. We hypothesized that domain interaction elicits an endoplasmic reticulum (ER) stress in astrocytes and an unfolded protein response that targets Arg-61 apoE for degradation. Primary Arg-61 apoE astrocytes had less intracellular apoE than wild-type astrocytes, and unfolded protein response markers OASIS (old astrocyte specifically induced substance), ATF4, and XBP-1 and downstream effectors were up-regulated. ER stress appears to cause global astrocyte dysfunction as glucose uptake was decreased in Arg-61 apoE astrocytes, and astrocyte-conditioned medium promoted neurite outgrowth less efficiently than wild-type medium in Neuro-2a cell cultures. We showed age-dependent up-regulation of brain OASIS levels and processing in Arg-61 apoE mice. ER stress and astrocyte dysfunction represent a new paradigm underlying the association of apoE4 with neurodegeneration.

The flavonol quercetin stabilizes the dimeric active form of the intracellular stress sensor IRE1.

Upon detecting endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) orchestrates adaptive cellular changes to reestablish homeostasis. If stress resolution fails, the UPR commits the cell to apoptotic death. Here we show that in hematopoietic cells, including multiple myeloma (MM), lymphoma, and leukemia cell lines, ER stress leads to caspase-mediated cleavage of the key UPR sensor IRE1 within its cytoplasmic linker region, generating a stable IRE1 fragment comprising the ER-lumenal domain and transmembrane segment (LDTM). This cleavage uncouples the stress-sensing and signaling domains of IRE1, attenuating its activation upon ER perturbation. Surprisingly, LDTM exerts negative feedback over apoptotic signaling by inhibiting recruitment of the key proapoptotic protein BAX to mitochondria. Furthermore, ectopic LDTM expression enhances xenograft growth of MM tumors in mice. These results uncover an unexpected mechanism of cross-regulation between the apoptotic caspase machinery and the UPR, which has biologically significant consequences for cell survival under ER stress.

Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated, which subsequently converts an unspliced X-box binding protein 1 (XBP1U) mRNA to a spliced mRNA that encodes a potent XBP1S transcription factor. XBP1S is essential for relieving ER stress and secretory cell differentiation. We previously established Twist2-Cre;Xbp1 CS/+ mice that constitutively expressed XBP1S in the Twist2-expressing cells as well as in the cells derived from the Twist2-expressing cells. In this study, we analyzed the dental phenotype of Twist2-Cre;Xbp1 CS/+ mice. We first generated a mutant Xbp1s minigene that corresponds to the recombinant Xbp1 Δ26 allele (the Xbp1 CS allele that has undergone Cre-mediated recombination) and confirmed that the Xbp1s minigene expressed XBP1S that does not require IRE1α activation in vitro. Consistently, immunohistochemistry showed that XBP1S was constitutively expressed in the odontoblasts and other dental pulp cells in Twist2-Cre;Xbp1 CS/+ mice. Plain X-ray radiography and µCT analysis revealed that constitutive expression of XBP1S altered the dental pulp chamber roof- and floor-dentin formation, resulting in a significant reduction in dentin/cementum formation in Twist2-Cre;Xbp1 CS/+ mice, compared to age-matched Xbp1 CS/+ control mice. However, there is no significant difference in the density of dentin/cementum between these two groups of mice. Histologically, persistent expression of XBP1S caused a morphological change in odontoblasts in Twist2-Cre;Xbp1 CS/+ mice. Nevertheless, in situ hybridization and immunohistochemistry analyses showed that continuous expression of XBP1S had no apparent effects on the expression of the Dspp and Dmp1 genes. In conclusion, these results support that sustained production of XBP1S adversely affected odontoblast function and dentin formation.

Saturated fatty acids (SFAs) have been shown to induce endoplasmic reticulum (ER) stress and chronic inflammatory responses, as well as alter sphingolipid metabolism. Disruptions in ER stress and sphingolipid metabolism have also been implicated in intestinal inflammation. Therefore, to elucidate the roles of SFAs in ER stress and inflammation in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, induced ER stress signaling, including activation of inositol-requiring enzyme 1 (IRE1) and X-box binding protein 1 (XBP1) signaling. Myristate significantly increased C14-ceramide levels, whereas palmitate increased several long-chain ceramides. To define the role of ceramide synthases (CerSs) in myristate-induced ER stress, we used the pharmacologic inhibitor, fumonisin B1 (FB1), and small interfering RNA (siRNA) for CerS5 and 6, the primary isoforms that are involved in C14-ceramide generation. FB1 and siRNA for CerS5 or 6 suppressed myristate-induced C14-ceramide generation and XBP1 splicing (XBP1s). Moreover, increased XBP1s induced the downstream expression of IL-6 in a CerS5/6-dependent manner. In addition, a myristate-enriched milk fat-based diet, but not a lard-based diet, increased C14-ceramide, XBP1s, and IL-6 expression in vivo. Taken together, our data suggest that myristate modulates ER stress and cytokine production in the intestinal epithelium via CerS5/6 and C14-ceramide generation.-Choi, S., Snider, J. M., Olakkengil, N., Lambert, J. M., Anderson, A. K., Ross-Evans, J. S., Cowart, L. A., Snider, A. J. Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells.

The non-steroidal anti-inflammatory drug aspirin and its metabolite, sodium salicylate, have profound effects on cellular protein synthesis and cell physiology. However, the underlying mechanism by which they cause these responses remains unclear. We show here that salicylates induce phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2alpha), resulting in the inhibition of mRNA translation in cells. Exposure of cells to acetyl salicylic acid resulted in strong activation of eIF2alpha stress-activated protein kinase R-like endoplasmic reticulum kinase (PERK). Analysis of fibroblasts with a targeted deletion of the perk gene revealed that PERK is indispensable for triggering the phosphorylation of eIF2alpha as well as the inhibition of protein synthesis induced by salicylates. Although salicylate treatment did not trigger activation of inositol-requiring enzyme 1, there was an increased expression of the pro-apoptotic transcription factor CHOP-(gadd153), a downstream event to eIF2alpha phosphorylation known to mediate endoplasmic reticulum stress-mediated responses. Thus, salicylates selectively trigger an endoplasmic reticulum stress-responsive signaling pathway initiated through activation of PERK to induce their cellular effects.

Introduction: the unfolded protein response’s role in disease pathophysiology

To study the crosstalk between the activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1)-X-box binding protein 1 (XBP1) pathway in oxygen-glucose deprivation/reoxygenation (OGD/R)-injured mouse hippocampal neuronal cell line HT22. The OGD/R-injured HT22 cell model was used to observe the changes of the indicators of endoplasmic reticulum stress (ERS), cell viability, and apoptosis at different OGD/R time points (0, 3, 6, 12, and 24 hours). HT22 cells in the logarithmic growth phase were randomized into blank control group, control+ATF6 activator (AA147) group, control+IRE1 inhibitor (4μ8c) group, OGD/R model group, OGD/R+AA147 group and OGD/R+4μ8c group (10 μmol/L AA147 or 16 μmol/L 4μ8c was given during the whole process in the AA147 group and 4μ8c group). Western blotting was used to detect the expression of ERS-related proteins [glucose-regulated protein 78 (GRP78), phosphorylated-inositol-requiring enzyme 1 (p-IRE1), and phosphorylated-eukaryotic translation initiation factor-2α (p-eIF2α)], and apoptosis-related proteins (Bcl-2, Bax, caspase-3, and cleaved caspase-3). The mRNA of ERS-related genes, and ATF6 [homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (Herpud1), protein disulfide isomerase associated 4 (Pdia4) and Sel-1 suppressor of lin-12-like (Sel1L)] and spliced XBP1 [XBP1s, include DnaJ heat shock protein family member B9 (Erdj4), Sec24 related gene family, member D (Sec24d) and signal sequence receptor, gamma (Ssr3)] induced transcriptional response-related genes were measured by real-time quantitative polymerase chain reaction (RT-qPCR). A cell counting kit-8 (CCK-8) assay was used to detect the viability of HT22 cells. Immunofluorescence was utilized to test the expression of cleaved caspase-3. Compared with the blank control group, the expression of ERS-related proteins p-IRE1 and p-eIF2α were significantly increased at 12 hours and 3 hours following OGD/R, respectively (p-IRE1/β-actin: 2.09±0.10 vs. 1.00±0.00, p-eIF2α/β-actin: 1.39±0.11 vs. 1.00±0.00, both P < 0.01). The mRNA expressions of ERS-related genes [ATF6, XBP1s, unspliced XBP1 (XBP1u), activating transcription factor 4 (ATF4), CCAAT/EBP homologous protein (CHOP)] were also upregulated in different OGD/R timepoint in HT22 cells, which indicated ERS was activated in OGD/R-stimulated HT22 cells. Compared with the OGD/R model group, the expression of protein p-IRE1 was not changed, but the mRNA of XBP1s and XBP1u were obviously downregulated in the OGD/R+AA147 group [XBP1s (2-ΔΔCt): 0.76 (0.71, 0.92) vs. 1.13 (1.03, 1.29), XBP1u (2-ΔΔCt): 0.29±0.05 vs. 0.52±0.04, both P < 0.01], whereas the expressions of XBP1s-induced transcriptional response downstream genes did not change significantly. Compared with the OGD/R model group, the protein of short-form ATF6 (sATF6) and GRP78 were not changed after administration of 4μ8c, neither was the mRNA expression of ATF6-induced transcriptional response-related genes. These results showed that the mRNA expression of XBP1s and XBP1u were inhibited by AA147-induced activation of ATF6, but no crosstalk was observed between the transcriptional response induced by ATF6 and XBP1s. Compared with the blank control group, the cell viability decreased significantly at OGD/R 3 hours [(44.64±5.12) % vs. (99.13±5.76) %, P < 0.01], the ratios of apoptosis-related proteins Bax/Bcl-2 and cleaved caspase-3/caspase-3 were significantly increased at OGD/R 3 hours and OGD 0 hour, respectively (Bax/Bcl-2: 6.15±1.65 vs. 1.00±0.00, cleaved caspase-3/caspase-3: 17.48±2.75 vs. 1.00±0.00, both P < 0.01), which indicated that apoptosis was activated in OGD/R-treated HT22 cells. Compared with the OGD/R model group, the cell viability decreased significantly [(36.52±17.78)% vs. (69.90±9.43)%, P < 0.01], and the ratios of Bax/Bcl-2 and cleaved caspase-3/caspase-3 were significantly upregulated in the OGD/R+AA147 group in HT22 cells (Bax/Bcl-2: 2.06±0.31 vs. 1.10±0.25, cleaved caspase-3/caspase-3: 3.35±0.59 vs. 0.55±0.09, both P < 0.01). Under our experimental conditions, no obvious crosstalk between the transcriptional response induced by ATF6 and XBP1s was observed, while ATF6 activation induced by AA147 suppressed mRNA expression of XBP1s and XBP1u and promoted cell death in OGD/R-treated HT22 cells.

Stress management: How the unfolded protein response impacts fatty liver disease

The unfolded protein response (UPR) is an adaptive signaling pathway activated in response to endoplasmic reticulum (ER) stress. The effectors of the UPR are potent transcription activators; however, some genes are suppressed by ER stress at the mRNA level. The mechanisms underlying UPR-mediated gene suppression are less known. Exploration of the effect of UPR on NK cells ligand expression found that the transcription of NK group 2 member D (NKG2D) ligand major histocompatibility complex class I polypeptide-related sequence A/B (MICA/B) is suppressed by the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) pathway of the UPR. Deletion of IRE1 or XBP1 was sufficient to promote mRNA and surface levels of MICA. Accordingly, NKG2D played a greater role in the killing of IRE1/XBP1 knockout target cells. Analysis of effectors downstream to XBP1s identified E2F transcription factor 1 (E2F1) as linking UPR and MICA transcription. The inverse correlation between XBP1 and E2F1 or MICA expression was corroborated in RNA-Seq analysis of 470 primary melanoma tumors. While mechanisms that connect XBP1 to E2F1 are not fully understood, we implicate a few microRNA molecules that are modulated by ER stress and possess dual suppression of E2F1 and MICA. Because of the importance of E2F1 and MICA in cancer progression and recognition, these observations could be exploited for cancer therapy by manipulating the UPR in tumor cells.-Obiedat, A., Seidel, E., Mahameed, M., Berhani, O., Tsukerman, P., Voutetakis, K., Chatziioannou, A., McMahon, M., Avril, T., Chevet, E., Mandelboim, O., Tirosh, B. Transcription of the NKG2D ligand MICA is suppressed by the IRE1/XBP1 pathway of the unfolded protein response through the regulation of E2F1.

Cigarette Smoke Extract Promotes Human Lung Myofibroblast Differentiation by the Induction of Endoplasmic Reticulum Stress

Methylenetetrahydrofolate reductase (MTHFR), an enzyme in folate and homocysteine metabolism, influences many cellular processes including methionine and nucleotide synthesis, methylation reactions, and maintenance of homocysteine at nontoxic levels. Mild deficiency of MTHFR is common in many populations and modifies risk for several complex traits including vascular disease, birth defects, and cancer. We recently demonstrated that MTHFR can be up-regulated by NF-kappaB, an important mediator of cell survival that is activated by endoplasmic reticulum (ER) stress. This observation, coupled with the reports that homocysteine can induce ER stress, prompted us to examine the possible regulation of MTHFR by ER stress. We found that several well characterized stress inducers (tunicamycin, thapsigargin, and A23187) as well as homocysteine could increase Mthfr mRNA and protein in Neuro-2a cells. The induction of MTHFR was also observed after overexpression of inositol-requiring enzyme-1 (IRE1) and was inhibited by a dominant-negative mutant of IRE1. Because IRE1 triggers c-Jun signaling, we examined the possible involvement of c-Jun in up-regulation of MTHFR. Transfection of c-Jun and two activators of c-Jun (LiCl and sodium valproate) increased MTHFR expression, whereas a reported inhibitor of c-Jun (SP600125) and a dominant-negative derivative of c-Jun N-terminal kinase-1 reduced MTHFR activation. We conclude that ER stress increases MTHFR expression and that IRE1 and c-Jun mediate this activation. These findings provide a novel mechanism by which the ER can regulate homeostasis and allude to an important role for MTHFR in cell survival.

Inositol-requiring enzyme 1 (IRE1) is a kinase and ribonuclease that executes the splicing of X box binding protein 1 (XBP-1) mRNA in response to the accumulation of unfolded protein in the ER, a signal cascade termed the unfolded protein response. Recently, IRE1 has been implicated in mRNA and miRNA cleavage and degradation, a pathway termed regulated IRE1-dependent decay (RIDD). Deletion of XBP-1 in the liver and pancreas strongly enhances RIDD by upregulating IRE1 protein levels and enhancing its ribo-nuclease activity. Because XBP-1 is essential for generating plasma cells with developed secretory capacity, we sought to evaluate the contribution of RIDD to this regulation. Mice were conditionally deleted for XBP-1 and/or IRE1 in their B-cell lineage. Similarly to the liver, deletion of XBP-1 induces IRE1 expression in LPS-treated B cells. In vitro, IRE1 cleaves the mRNA of secretory μ chains, which explains the reduction in secretory μ mRNA and its synthesis in XBP-1 KO plasma cells. In accordance, the IgM response is partially restored in XBP-1/IRE1 double KO mice relative to XBP-1 KO mice. Interestingly, the IgG1 response is reduced to a similar level in XBP-1 KO, IRE1 KO, and their double knockout animals. Our data demonstrate a specific contribution by RIDD in curtailing immunoglobulin synthesis and secretion.