Neurodegeneration risk factor, EIF2AK3 (PERK), influences tau protein aggregation.

Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms-unfolded protein response and integrated stress response. Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation, whereas inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the endoplasmic reticulum luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small-molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.

PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

Tauopathy-associated PERK alleles are functional hypomorphs that increase neuronal vulnerability to ER stress.

Tauopathies are neurodegenerative diseases characterized by tau protein pathology in the nervous system. EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), also known as PERK (protein kinase R-like endoplasmic reticulum kinase), was identified by genome-wide association study as a genetic risk factor in several tauopathies. PERK is a key regulator of the Unfolded Protein Response (UPR), an intracellular signal transduction mechanism that protects cells from endoplasmic reticulum (ER) stress. PERK variants had previously been identified in Wolcott-Rallison Syndrome, a rare autosomal recessive metabolic disorder, and these variants completely abrogated the function of PERK's kinase domain or prevented PERK expression. In contrast, the PERK tauopathy risk variants were distinct from the Wolcott-Rallison variants and introduced missense alterations throughout the PERK protein. The function of PERK tauopathy variants and their effects on neurodegeneration are unknown. Here, we discovered that tauopathy-associated PERK alleles showed reduced signaling activity and increased PERK protein turnover compared to protective PERK alleles. We found that iPSC-derived neurons carrying PERK risk alleles were highly vulnerable to ER stress-induced injury with increased tau pathology. We found that chemical inhibition of PERK in human iPSC-derived neurons also increased neuronal cell death in response to ER stress. Our results indicate that tauopathy-associated PERK alleles are functional hypomorphs during the UPR. We propose that reduced PERK function leads to neurodegeneration by increasing neuronal vulnerability to ER stress-associated damage. In this view, therapies to enhance PERK signaling would benefit at-risk carriers of hypomorphic alleles.

Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells.

Increased protein translation in cells and various factors in the tumor microenvironment can induce endoplasmic reticulum (ER) stress, which initiates the unfolded protein response (UPR). We have previously reported that factors released from cancer cells mounting a UPR induce a de novo UPR in bone marrow-derived myeloid cells, macrophages, and dendritic cells that facilitates protumorigenic characteristics in culture and tumor growth in vivo. We investigated whether this intercellular signaling, which we have termed transmissible ER stress (TERS), also operates between cancer cells and what its functional consequences were within the tumor. We found that TERS signaling induced a UPR in recipient human prostate cancer cells that included the cell surface expression of the chaperone GRP78. TERS also activated Wnt signaling in recipient cancer cells and enhanced resistance to nutrient starvation and common chemotherapies such as the proteasome inhibitor bortezomib and the microtubule inhibitor paclitaxel. TERS-induced activation of Wnt signaling required the UPR kinase and endonuclease IRE1. However, TERS-induced enhancement of cell survival was predominantly mediated by the UPR kinase PERK and a reduction in the abundance of the transcription factor ATF4, which prevented the activation of the transcription factor CHOP and, consequently, the induction of apoptosis. When implanted in mice, TERS-primed cancer cells gave rise to faster growing tumors than did vehicle-primed cancer cells. Collectively, our data demonstrate that TERS is a mechanism of intercellular communication through which tumor cells can adapt to stressful environments.

Multiple Mechanisms of Unfolded Protein Response-Induced Cell Death.

Eukaryotic cells fold and assemble membrane and secreted proteins in the endoplasmic reticulum (ER), before delivery to other cellular compartments or the extracellular environment. Correctly folded proteins are released from the ER, and poorly folded proteins are retained until they achieve stable conformations; irreparably misfolded proteins are targeted for degradation. Diverse pathological insults, such as amino acid mutations, hypoxia, or infection, can overwhelm ER protein quality control, leading to misfolded protein buildup, causing ER stress. To cope with ER stress, eukaryotic cells activate the unfolded protein response (UPR) by increasing levels of ER protein-folding enzymes and chaperones, enhancing the degradation of misfolded proteins, and reducing protein translation. In mammalian cells, three ER transmembrane proteins, inositol-requiring enzyme-1 (IRE1; official name ERN1), PKR-like ER kinase (PERK; official name EIF2AK3), and activating transcription factor-6, control the UPR. The UPR signaling triggers a set of prodeath programs when the cells fail to successfully adapt to ER stress or restore homeostasis. ER stress and UPR signaling are implicated in the pathogenesis of diverse diseases, including neurodegeneration, cancer, diabetes, and inflammation. This review discusses the current understanding in both adaptive and apoptotic responses as well as the molecular mechanisms instigating apoptosis via IRE1 and PERK signaling. We also examine how IRE1 and PERK signaling may be differentially used during neurodegeneration arising in retinitis pigmentosa and prion infection.

Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress-induced cell death during the unfolded protein response.

Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress-induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop((-)/(-)) cells are partially resistant to ER stress-induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2α and promotes XIAP degradation through ATF4. Of interest, PERK's down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress-induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a "two-hit" model of ER stress-induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.

Selective activation of ATF6 and PERK endoplasmic reticulum stress signaling pathways prevent mutant rhodopsin accumulation.

PURPOSE: Many rhodopsin mutations that cause retinitis pigmentosa produce misfolded rhodopsin proteins that are retained within the endoplasmic reticulum (ER) and cause photoreceptor cell death. Activating transcription factor 6 (ATF6) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) control intracellular signaling pathways that maintain ER homeostasis. The aim of this study was to investigate how ATF6 and PERK signaling affected misfolded rhodopsin in cells, which could identify new molecular therapies to treat retinal diseases associated with ER protein misfolding. METHODS: To examine the effect of ATF6 on rhodopsin, wild-type (WT) or mutant rhodopsins were expressed in cells expressing inducible human ATF6f, the transcriptional activator domain of ATF6. Induction of ATF6f synthesis rapidly activated downstream genes. To examine PERK's effect on rhodopsin, WT or mutant rhodopsins were expressed in cells expressing a genetically altered PERK protein, Fv2E-PERK. Addition of the dimerizing molecule (AP20187) rapidly activated Fv2E-PERK and downstream genes. By use of these strategies, it was examined how selective ATF6 or PERK signaling affected the fate of WT and mutant rhodopsins. RESULTS: ATF6 significantly reduced T17M, P23H, Y178C, C185R, D190G, K296E, and S334ter rhodopsin protein levels in the cells with minimal effects on monomeric WT rhodopsin protein levels. By contrast, the PERK pathway reduced both levels of WT, mutant rhodopsins, and many other proteins in the cell. CONCLUSIONS: This study indicates that selectively activating ATF6 or PERK prevents mutant rhodopsin from accumulating in cells. ATF6 signaling may be especially useful in treating retinal degenerative diseases arising from rhodopsin misfolding by preferentially clearing mutant rhodopsin and abnormal rhodopsin aggregates.

Acidic stress-ER stress axis for blunted activation of NF-κB in mesothelial cells exposed to peritoneal dialysis fluid.

BACKGROUND: Bacterial peritonitis is a frequent complication in patients on peritoneal dialysis (PD). We previously reported that PD fluid (PDF) suppressed expression of monocyte chemoattractant protein 1 (MCP-1) in mesothelial cells in vitro and in vivo, which was ascribed to the suppression of nuclear factor-κB (NF-κB). To elucidate molecular mechanisms underlying this effect, we tested a role of endoplasmic reticulum (ER) stress. METHODS: Mesothelial cells and other cell types were exposed to acidic stress, and induction of the unfolded protein response was examined. Peritoneal induction of ER stress was also tested in mice exposed to acidic and neutralized PDF. Activation of NF-κB and expression of MCP-1 by tumour necrosis factor-α were evaluated in mesothelial cells under acidic and ER stress conditions. Peritoneal expression of MCP-1 and infiltration of monocytes were compared in lipopolysaccharide (LPS)-treated mice between normal and ER stress conditions. RESULTS: PDF, but not neutralized PDF, caused ER stress in the peritoneum. In vitro, acidic stress, but not metabolic and osmotic stress, induced ER stress in mesothelial cells and other cell types and suppressed activation of NF-κB and NF-κB-dependent MCP-1 induction. This effect was reproducible by other ER stress inducers, and attenuation of ER stress reversed the suppressive effect of low pH on NF-κB. Like PDF, ER stress inducers suppressed expression of MCP-1 and infiltration of mononuclear cells in the peritoneum of LPS-treated mice. CONCLUSION: These results indicate a role for the acidic stress-ER stress pathway in blunted activation of NF-κB, which may cause perturbation of monocyte recruitment by mesothelial cells in PD patients.

The unfolded protein response is a major mechanism by which LRP1 regulates Schwann cell survival after injury.

In peripheral nerve injury, Schwann cells (SCs) must survive to exert a continuing and essential role in successful nerve regeneration. Herein, we show that peripheral nerve injury is associated with activation of endoplasmic reticulum (ER) stress and the adaptive unfolded protein response (UPR). The UPR culminates in expression of C/EBP homology protein (CHOP), a proapoptotic transcription factor in SCs, unless counteracted by LDL receptor-related protein-1 (LRP1), which serves as a major activator of phosphatidylinositol 3-kinase (PI3K). Sciatic nerve crush injury in rats induced expression of the ER chaperone GRP78/BIP, reflecting an early, corrective phase of the UPR. However, when LRP1 signaling was inhibited with receptor-associated protein, PI3K activity was decreased and CHOP protein expression increased, particularly in myelinating SCs. In cultured SCs, the PKR-like ER kinase target eIF2α was phosphorylated and CHOP was induced by (1) inhibiting PI3K, (2) treating the cells with tumor necrosis factor-α (TNF-α), or (3) genetic silencing of LRP1. CHOP gene deletion in SCs decreased cell death in response to TNF-α. Furthermore, the effects of TNF-α on phosphorylated eIF2α, CHOP, and SC death were blocked by adding LRP1 ligands that augment LRP1-dependent cell signaling to PI3K. Collectively, our results support a model in which UPR-activated signaling pathways represent a major challenge to SC survival in nerve injury. LRP1 functions as a potent activator of PI3K in SCs and, by this mechanism, limits SC apoptosis resulting from increased CHOP expression in nerve injury.

Induction of CCAAT/enhancer-binding protein-homologous protein by cigarette smoke through the superoxide anion-triggered PERK-eIF2α pathway.

Cigarette smoke triggers apoptosis through oxidative stress- and endoplasmic reticulum (ER) stress-dependent induction of CCAAT/enhancer-binding protein-homologous protein (CHOP) (Tagawa et al., 2008. Free Radic. Biol. Med. 45, 50-59). We investigated roles of individual reactive oxygen/nitrogen species in the transcriptional induction of CHOP by cigarette smoke. Exposure of bronchial epithelial cells to O(2)(-), ONOO(-) or H(2)O(2) induced expression of CHOP, whereas NO alone did not. Induction of CHOP mRNA by cigarette smoke extract (CSE) was attenuated by scavengers for O(2)(-), ONOO(-) or NO, whereas scavenging H(2)O(2) did not affect the induction of CHOP. Like CSE, O(2)(-) and ONOO(-) caused activation of the CHOP gene promoter. Scavengers for O(2)(-), ONOO(-) or NO attenuated CSE-triggered activation of the CHOP gene promoter. CSE, O(2)(-) and ONOO(-) induced phosphorylation of protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) and caused induction of downstream activating transcription factor 4 (ATF4). Scavengers for O(2)(-), ONOO(-) or NO attenuated induction of ATF4 by CSE. Furthermore, dominant-negative inhibition of the PERK-eIF2α pathway exclusively suppressed CSE-triggered induction of CHOP and consequent apoptosis. These results suggest that O(2)(-) and ONOO(-) are selectively involved in CSE-triggered induction of CHOP and that the PERK-eIF2α pathway plays a crucial role in the induction of CHOP and apoptosis downstream of the particular reactive oxygen species.

Selective abrogation of BiP/GRP78 blunts activation of NF-κB through the ATF6 branch of the UPR: involvement of C/EBPß and mTOR-dependent dephosphorylation of Akt.

Subtilase cytotoxin (SubAB) that selectively cleaves BiP/GRP78 triggers the unfolded protein response (UPR) and protects mice from endotoxic lethality and collagen arthritis. We found that pretreatment of cells with SubAB suppressed tumor necrosis alpha (TNF-α)-induced activation of NF-κB and NF-κB-dependent chemokine expression. To elucidate underlying mechanisms, the involvement of C/EBP and Akt, putative regulators of NF-κB, was investigated. Among members of the C/EBP family, SubAB preferentially induced C/EBPß. Overexpression of C/EBPß suppressed TNF-α-induced NF-κB activation, and knockdown of C/EBPß attenuated the suppressive effect of SubAB on NF-κB. We identified that the ATF6 branch of the UPR plays a crucial role in the induction of C/EBPß. In addition to this effect, SubAB depressed basal and TNF-α-induced phosphorylation of Akt via the UPR. It was mediated by the induction of ATF6 and consequent activation of mTOR that dephosphorylated Akt. Inhibition of Akt attenuated activation of NF-κB by TNF-α, suggesting that the mTOR-Akt pathway is another target for SubAB-initiated, UPR-mediated NF-κB suppression. These results elucidated that SubAB blunts activation of NF-κB through ATF6-dependent mechanisms, i.e., preferential induction of C/EBPß and mTOR-dependent dephosphorylation of Akt.

Monitoring and manipulating mammalian unfolded protein response.

The unfolded protein response (UPR) is a conserved, intracellular signaling pathway activated by endoplasmic reticulum (ER) stress. In mammalian cells, the UPR is controlled by three ER-resident transmembrane proteins: inositol-requiring enyzme-1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor-6 (ATF6), by which cytoprotective mechanisms are initiated to restore ER functions. However, if cellular homeostasis is not restored by the UPR's initial events, UPR signaling triggers apoptotic cell death, which correlates with the pathogenesis of a wide range of human diseases. The intrinsic function of the UPR in regulating cell survival and death suggests its importance as a mechanistic link between ER stress and disease pathogenesis. Understanding UPR regulatory molecules or signaling pathways involved in disease pathogenesis is critical to establishing therapeutic strategies. For this purpose, several experimental tools have been developed to evaluate individual UPR components. In this chapter, we present methods to monitor and quantify activation of individual UPR signaling pathways in mammalian cells and tissues, and we review strategies to artificially and selectively activate individual UPR signaling pathways using chemical-genetic approaches.

Real-time monitoring of ER stress in living cells and animals using ESTRAP assay.

Endoplasmic reticulum (ER) stress is involved in a wide range of pathologies. Detection and monitoring of the unfolded protein response are required to disclose the link between ER stress and diseases. Assessment of ER stress is also essential for evaluation of therapeutic drugs in vitro and in vivo; that is, their therapeutic utility as well as adverse effects. For detection and monitoring of ER stress in living cells and animals, ER stress-responsive alkaline phosphatase (ESTRAP), also called secreted alkaline phosphatase (SEAP), serves as a useful indicator. In cells genetically engineered to express SEAP, secretion of SEAP is quickly downregulated in response to ER stress. This phenomenon is observed in a wide range of cell types triggered by various ER stress inducers. The magnitude of the decrease in extracellular SEAP is proportional to the intensity of ER stress, which is inversely correlated with the induction of endogenous ER stress markers. In contrast to SEAP, the activity of intracellular luciferase is not affected by ER stress. ER stress causes a decrease in SEAP activity not via transcriptional suppression but via abnormal posttranslational modification, accelerated degradation, and reduced secretion of SEAP protein. In mice constitutively producing SEAP, in vivo induction of ER stress similarly causes rapid reduction in serum SEAP activity. Using SEAP as an indicator, real-time monitoring of ER stress in living cells and animals is feasible. The ESTRAP method provides a powerful tool to investigate the pathogenesis of ER stress-associated diseases, to assess toxicity and the adverse effects of drugs, and to develop therapeutic agents for the treatment of ER stress-related disorders.

Impairment of MCP-1 expression in mesothelial cells exposed to peritoneal dialysis fluid by osmotic stress and acidic stress.

BACKGROUND: Bacterial peritonitis is one of the most frequent complications in patients on peritoneal dialysis. In the present study, we investigated effects of peritoneal dialysis fluid (PDF) on mesothelial cell recruitment of macrophages, focusing especially on unphysiological properties of PDF. METHODS: Human and murine mesothelial cells were exposed to PDF or individual properties of PDF (low pH, high glucose concentration, hyperosmolality, high lactate concentration) in vitro and in vivo, treated with inflammatory stimuli, and subjected to analyses of monocyte chemoattractant protein-1 (MCP-1), nuclear factor-κB (NF-κB), mitogen-activated protein (MAP) kinases, and MAP kinase phosphatase-1 (MKP-1). RESULTS: We found that intraperitoneal administration of PDF suppressed expression of MCP-1 and infiltration of mononuclear cells in the peritoneum of mice following injection with lipopolysaccharide. Among the unphysiological properties of PDF, low pH and hyperosmolality caused blunted induction of MCP-1 in cytokine-stimulated mesothelial cells. The attenuated response was ascribed to suppression of NF-κB by low pH and inhibition of p38 MAP kinase by hyperosmolality. Furthermore, the attenuated phosphorylation of p38 MAP kinase by osmotic stress was associated with induction of MKP-1. CONCLUSION: These results suggest a possibility that mesothelial cells exposed to PDF exhibit attenuated MCP-1 expression and consequent impairment of macrophage recruitment through dual mechanisms, that is, inhibition of NF-κB by acidic stress and blunted activation of p38 MAP kinase by osmotic stress. In patients on peritoneal dialysis, blunted expression of chemokines may lead to perturbation of bacterial clearance by macrophages in the peritoneal cavity.

Selective deletion of adipocytes, but not preadipocytes, by TNF-alpha through C/EBP- and PPARgamma-mediated suppression of NF-kappaB.

Tumor necrosis factor-alpha (TNF-alpha) is a key regulator of adipose tissue mass, but mechanisms underlying this effect have not been fully elucidated. We found that exposure to TNF-alpha caused a significant decrease in the number of adipocytes, but not preadipocytes. Subsequent experiments revealed that TNF-alpha selectively deleted adipocytes through induction of apoptosis. Following exposure to TNF-alpha, rapid activation of nuclear factor-kappaB (NF-kappaB) was observed only in preadipocytes, but not in adipocytes. Inhibition of NF-kappaB rendered preadipocytes susceptible to TNF-alpha-induced apoptosis, suggesting that different activity of NF-kappaB is the determinant for the distinct apoptotic responses. During adipocyte differentiation, expression and activity of peroxisome proliferator-activated receptor-gamma (PPARgamma) were upregulated. Treatment of preadipocytes with a PPARgamma agonist attenuated NF-kappaB activation and rendered the cells vulnerable to TNF-alpha-induced apoptosis. Conversely, treatment of adipocytes with a PPARgamma antagonist enhanced NF-kappaB activation and conferred resistance to TNF-alpha-induced apoptosis, suggesting involvement of PPARgamma in the suppression of NF-kappaB in adipocytes. We also found that, following differentiation, expression and activity of CCAAT/enhancer binding protein (C/EBP), especially C/EBPalpha and C/EBPbeta, were upregulated in adipocytes. Overexpression of individual C/EBPs significantly inhibited activation of NF-kappaB in preadipocytes. Furthermore, transfection with siRNA for C/EBPalpha or C/EBPbeta enhanced activity of NF-kappaB in adipocytes, suggesting that C/EBP is also involved in the repression of NF-kappaB in adipocytes. These results suggested novel mechanisms by which TNF-alpha selectively deletes adipocytes in the adipose tissue. The C/EBP- and PPARgamma-mediated suppression of NF-kappaB may contribute to TNF-alpha-related loss of adipose tissue mass under certain pathological situations, such as cachexia.

Anti-inflammatory subtilase cytotoxin up-regulates A20 through the unfolded protein response.

We recently reported that subtilase cytotoxin (SubAB) has the potential to attenuate experimental models of inflammatory diseases [3]. Currently, little is known about underlying mechanisms involved in this therapeutic effect. In the present report, we show that SubAB induces A20, the endogenous negative regulator of NF-kappaB, in vitro and in vivo. This stimulatory effect occurred at the transcriptional level, and SubAB induced activation of the A20 promoter. We found that, in the early phase, SubAB triggered activation of NF-kappaB in a dose-dependent manner. Blockade of NF-kappaB abrogated expression of A20 by SubAB. SubAB rapidly triggered the unfolded protein response (UPR), and induction of the UPR by other agents (thapsigargin and A23187) mimicked the stimulatory effects of SubAB, both on NF-kappaB and on A20. The induction of A20 by thapsigargin was correlated with activation of the A20 promoter, which was not observed in the kappaB-mutated A20 promoter. Furthermore, induction of A20 by SubAB was substantially attenuated by treatment with different chemical chaperones. These results elucidated for the first time that the anti-inflammatory SubAB has the potential to induce A20 through the UPR-NF-kappaB-dependent pathway.

The oxidative stress: endoplasmic reticulum stress axis in cadmium toxicity.

Cadmium preferentially accumulates in the kidney, the major target for cadmium-related toxicity. Several underlying mechanisms are postulated, and reactive oxygen species (ROS) have been considered as crucial mediators for tissue injuries. In addition to oxidative stress, we recently disclosed that endoplasmic reticulum (ER) stress also plays a critical role. Cadmium causes ER stress in vitro and in vivo and mediates induction of apoptosis in target tissues. In this article, we describe a role for ER stress and involvement of particular branches of the unfolded protein response (UPR) in cadmium-triggered tissue injury, especially nephrotoxicity. We also discuss relationship between oxidative stress and ER stress, and involvement of selective ROS in the induction of pro-apoptotic branches of the UPR.

ER stress depresses NF-kappaB activation in mesangial cells through preferential induction of C/EBP beta.

Modest induction of endoplasmic reticulum (ER) stress confers resistance to inflammation in glomeruli. Recently, we found that ER stress leads to mesangial insensitivity to cytokine-induced activation of NF-kappaB, but the underlying mechanisms are incompletely understood. ER stress can trigger expression of CCAAT/enhancer-binding proteins (C/EBPs), which interact with transcription factors including NF-kappaB. Here, we investigated a role for C/EBPs in the ER stress-induced resistance to cytokines. Mesangial cells preferentially induced C/EBPbeta after exposure to thapsigargin or tunicamycin; induction of C/EBPdelta was modest and transient, and expression of C/EBPalpha was absent. The induction of C/EBPbeta correlated with accumulation of C/EBPbeta protein and enhanced transcriptional activity of C/EBP. Overexpression of C/EBPbeta markedly suppressed TNF-alpha-induced activation of NF-kappaB, independent of its transacting potential. Knockdown of C/EBPbeta by small interfering RNA reversed the suppressive effect of ER stress on NF-kappaB. In vivo, preconditioning of mice with ER stress induced renal C/EBPbeta and suppressed NF-kappaB-dependent gene expression in response to LPS. Using dominant negative mutants and null mutants for individual branches of the unfolded protein response, we identified the RNA-dependent protein kinase-like ER kinase (PERK) and the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1) pathways as the unfolded protein response responsible for ER stress-induced C/EBPbeta. These results suggest that ER stress blunts cytokine-triggered activation of NF-kappaB, in part through PERK- and IRE1-mediated preferential induction of C/EBPbeta.

Activation of the Akt-NF-kappaB pathway by subtilase cytotoxin through the ATF6 branch of the unfolded protein response.

Shiga toxin has the potential to induce expression of inflammation-associated genes, although the underlying mechanisms are not well understood. We examined the effects of subtilase cytotoxin (SubAB), an AB(5) toxin produced by some Shiga toxigenic Escherichia coli, on the activation of NF-kappaB. SubAB is known to be a protease which selectively degrades GRP78/Bip. Treatment of NRK-52E cells with SubAB caused rapid cleavage of GRP78. Following the degradation of GRP78, transient activation of NF-kappaB was observed with a peak at 6-12 h; the activation subsided within 24 h despite the continuous absence of intact GRP78. The activation of NF-kappaB was preceded by transient phosphorylation of Akt. Treatment of the cells with a selective inhibitor of Akt1/2 or an inhibitor of PI3K attenuated SubAB-induced NF-kappaB activation, suggesting that activation of Akt is an event upstream of NF-kappaB. Degradation of GRP78 caused the unfolded protein response (UPR), and inducers of the UPR mimicked the stimulatory effects of SubAB on Akt and NF-kappaB. SubAB triggered the three major branches of the UPR including the IRE1-XBP1, PERK, and ATF6 pathways. Dominant-negative inhibition of IRE1alpha, XBP1, or PERK did not attenuate activation of NF-kappaB by SubAB. In contrast, genetic and pharmacological inhibition of ATF6 significantly suppressed SubAB-triggered Akt phosphorylation and NF-kappaB activation. These results suggested that loss of GRP78 by SubAB leads to transient phosphorylation of Akt and consequent activation of NF-kappaB through the ATF6 branch of the UPR.