Nobuhiko Hiramatsu

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.

Chronic ER stress down-regulates XIAP by activating the PERK branch of the UPR. PERK attenuates Xiap translation via eIF2α phosphorylation. PERK promotes XIAP degradation via ATF4. CHOP induction and XIAP suppression act in parallel to sensitize cells to ER stress–induced apoptosis.

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 PERKs 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.

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.

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 UPRs 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.

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

Prostate cancer cells release an ER stress response signal that enhances tumor growth and drug resistance. Stress signals improve tumor fitness Mechanisms that promote the survival of healthy cells are often exploited by tumor cells. Tumors experience increased cellular stress, and targeting the endoplasmic reticulum (ER) stress response, an adaptive response to increased protein translation, has been proposed as an anticancer therapy. Rodvold et al. found that prostate cancer cells undergoing an ER stress response transmit some signal to cocultured, naïve cancer cells that then also launch an ER stress response. This phenomenon, which the authors call “transmissible ER stress” (TERS), promoted faster tumor growth and resistance to common anticancer drugs in xenograft mouse models. The findings show that tumor cells leverage this intrinsically adaptive stress response to enhance the fitness of the overall tumor. Inhibiting this signal (once identified) or the pathways induced in the recipient cells might avert drug resistance in prostate cancer patients. 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.

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.

Tauopathy-Associated PERK Alleles are Functional Hypomorphs that Increase Neuronal Vulnerability to ER Stress

&NA; 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 PERKs 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.

Abstract B51: Tumor ER stress transmission to infiltrating myeloid cells in vivo require IRE1alpha and TLR4

The unfolded protein response (UPR) facilitates tumor outgrowth through evoking a variety of cell intrinsic processes that promote cancer survival, angiogenesis, and chemoresistance. The UPR can be transmitted from stressed cancer cells to receiver myeloid cells in vitro through a process termed transmissible endoplasmic reticulum stress (TERS) in which receiver cells undergo a UPR and acquire tumor promoting pro-inflammatory (IL-6, TNF-alpha, IL-23) and immune-suppressive (Arg1) characteristics. Here, we confirmed that CD11b+ myeloid cells isolated from subcutaneous murine melanoma tumors in C57BL/6 mice display the TERS signature. In light of the fact that TERS signaling in myeloid receiver cells is partly TLR4-dependent, we interrogated subcutaneous tumors in TLR4 mice. We found that these tumors had delayed onset, reduced growth kinetics, and improved survival over tumors growing in wildtype mice. Most significantly, isolated CD11b+ cells from TLR4-/- tumors did not undergo a UPR nor upregulate inflammatory genes when compared to splenic controls, though, interestingly, still upregulated Arg1. Next, we probed individual signaling arms of the UPR in relation to TERS production. We utilized a panel of novel transgenic cell lines in which specific arms of the UPR can be conditionally activated and found that activated inositol requiring enzyme-1alpha (IRE1alpha) transmitted stress to myeloid cells. The remaining two UPR arms (PERK and ATF6) had little transmitting potential, if any. Bone marrow derived myeloid cells treated with TERS generated from IRE1alpha KO mouse embryonic fibroblasts (MEFs) had a markedly reduced UPR than those treated to wildtype MEF TERS. Subcutaneous tumors consisting of IRE1alpha KO TERS-conditioned myeloid dendritic cells and melanoma cancer cells had delayed onset and grew at reduced rate compared to tumor mixtures in which wildtype MEF TERS was used. Based on these data, we probed a patient melanoma for its co-localization of stress, myeloid infiltrate, and inflammation. Together, these data identify a novel signaling mechanism dependent on IRE1alpha and TLR4 used by stressed tumor cells to co-opt infiltrating myeloid cells to become tumor promoting. Citation Format: Jeffrey J. Rodvold, Nobuhiko Hiramatsu, Kevin T. Chiu, Navin R. Mahadevan, Jonathan Lin, Maurizio Zanetti. Tumor ER stress transmission to infiltrating myeloid cells in vivo require IRE1alpha and TLR4. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B51.