"Translating" tumor hypoxia: unfolded protein response (UPR)-dependent and UPR-independent pathways

Poor oxygenation (hypoxia) is present in the majority of human tumors and is associated with poor prognosis due to the protection it affords to radiotherapy and chemotherapy. Hypoxia also elicits multiple cellular response pathways that alter gene expression and affect tumor progression, including two recently identified separate pathways that strongly suppress the rates of mRNA translation during hypoxia. The first pathway is activated extremely rapidly and is mediated by phosphorylation and inhibition of the eukaryotic initiation factor 2alpha. Phosphorylation of this factor occurs as part of a coordinated endoplasmic reticulum stress response program known as the unfolded protein response and activation of this program is required for hypoxic cell survival and tumor growth. Translation during hypoxia is also inhibited through the inactivation of a second eukaryotic initiation complex, eukaryotic initiation factor 4F. At least part of this inhibition is mediated through a Redd1 and tuberous sclerosis complex 1/2-dependent inhibition of the mammalian target of rapamycin kinase. Inhibition of mRNA translation is hypothesized to affect the cellular tolerance to hypoxia in part by promoting energy homeostasis. However, regulation of translation also results in a specific increase in the synthesis of a subset of hypoxia-induced proteins. Consequently, both arms of translational control during hypoxia influence gene expression and phenotype. These hypoxic response pathways show differential activation requirements that are dependent on the level of oxygenation and duration of hypoxia and are themselves highly dynamic. Thus, the severity and duration of hypoxia can lead to different biological and therapeutic consequences.     

Targeting of the unfolded protein response (UPR) as therapy for Parkinson's disease

Parkinson's disease is the second most common neurodegenerative disorder, leading to the progressive decline of motor control due to the loss of dopaminergic neurons in the substantia nigra pars compacta. At the molecular level, Parkinson's disease share common molecular signatures with most neurodegenerative diseases including the accumulation of misfolded proteins in the brain. Alteration in the buffering capacity of the proteostasis network during aging is proposed as one of the triggering steps leading to abnormal protein aggregation in this disease, highlighting disturbances in the function of the endoplasmic reticulum (ER). The ER is the main subcellular compartment involved in protein folding and quality control. ER stress triggers a signalling reaction known as the unfolded protein response (UPR), which aims restoring proteostasis through the induction of adaptive programs or the activation of cell death pathways when damage is chronic and cannot be repaired. Here, we overview most evidence linking ER stress to Parkinson's disease. Strategies to alleviate ER stress by targeting specific components of the UPR using small molecules and gene therapy are highlighted.     

Tumor necrosis factor alpha (TNFalpha) induces the unfolded protein response (UPR) in a reactive oxygen species (ROS)-dependent fashion, and the UPR counteracts ROS accumulation by TNFalpha

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER overload, resulting in ER stress. To cope with ER stress, mammalian cells trigger a specific response known as the unfolded protein response (UPR). Although recent studies have indicated cross-talk between ER stress and oxidative stress, the mechanistic link is not fully understood. By using murine fibrosarcoma L929 cells, in which tumor necrosis factor (TNF) alpha induces accumulation of reactive oxygen species (ROS) and cell death, we show that TNFalpha induces the UPR in a ROS-dependent fashion. In contrast to TNFalpha, oxidative stresses by H2O2 or arsenite only induce eukaroytic initiation factor 2alpha phosphorylation, but not activation of PERK- or IRE1-dependent pathways, indicating the specificity of downstream signaling induced by various oxidative stresses. Conversely, the UPR induced by tunicamycin substantially suppresses TNFalpha-induced ROS accumulation and cell death by inhibiting reduction of cellular glutathione levels. Collectively, some, but not all, oxidative stresses induce the UPR, and pre-emptive UPR counteracts TNFalpha-induced ROS accumulation.     

Roles of mitochondrial unfolded protein response in mammalian stem cells

The mitochondrial unfolded protein response (UPR^mt) is an evolutionarily conserved adaptive mechanism for improving cell survival under mitochondrial stress. Under physiological and pathological conditions, the UPR^mt is the key to maintaining intracellular homeostasis and proteostasis. Important roles of the UPR^mt have been demonstrated in a variety of cell types and in cell development, metabolism, and immune processes. UPR^mt dysfunction leads to a variety of pathologies, including cancer, inflammation, neurodegenerative disease, metabolic disease, and immune disease. Stem cells have a special ability to self-renew and differentiate into a variety of somatic cells and have been shown to exist in a variety of tissues. These cells are involved in development, tissue renewal, and some disease processes. Although the roles and regulatory mechanisms of the UPR^mt in somatic cells have been widely reported, the roles of the UPR^mt in stem cells are not fully understood. The roles and functions of the UPR^mt depend on stem cell type. Therefore, this paper summarizes the potential significance of the UPR^mt in embryonic stem cells, tissue stem cells, tumor stem cells, and induced pluripotent stem cells. The purpose of this review is to provide new insights into stem cell differentiation and tumor pathogenesis.     

Inhibition of the mitochondrial unfolded protein response by acetylcholine alleviated hypoxia/reoxygenation-induced apoptosis of endothelial cells

The mitochondrial unfolded protein response (UPR^mt) is involved in numerous diseases that have the common feature of mitochondrial dysfunction. However, its pathophysiological relevance in the context of hypoxia/reoxygenation (H/R) in endothelial cells remains elusive. Previous studies have demonstrated that acetylcholine (ACh) protects against cardiomyocyte injury by suppressing generation of mitochondrial reactive oxygen species (mtROS). This study aimed to explore the role of UPR^mt in endothelial cells during H/R and to clarify the beneficial effects of ACh. Our results demonstrated that H/R triggered UPR^mt in endothelial cells, as evidenced by the elevation of heat shock protein 60 and LON protease 1 protein levels, and resulted in release of mitochondrial pro-apoptotic proteins, including cytochrome C, Omi/high temperature requirement protein A 2 and second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low PI, from the mitochondria to cytosol. ACh administration markedly decreased UPR^mt by inhibiting mtROS and alleviating the mitonuclear protein imbalance. Consequently, ACh alleviated the release of pro-apoptotic proteins and restored mitochondrial ultrastructure and function, thereby reducing the number of terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL)-positive cells. Intriguingly, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3AChR) inhibitor, abolished the ACh-elicited attenuation of UPR^mt and TUNEL positive cells, indicating that the salutary effects of ACh were likely mediated by M3AChR in endothelial cells. In conclusion, our studies demonstrated that UPR^mt might be essential for triggering the mitochondrion-associated apoptotic pathway during H/R. ACh markedly suppressed UPR^mt by inhibiting mtROS and alleviating the mitonuclear protein imbalance, presumably through M3AChR.     

The stress rheostat: an interplay between the unfolded protein response (UPR) and autophagy in neurodegeneration

The unfolded protein response (UPR) is a conserved adaptive reaction that increases cell survival under conditions of endoplasmic reticulum (ER) stress. The UPR controls diverse processes such as protein folding, secretion, ER biogenesis, protein quality control and macroautophagy. Occurrence of chronic ER stress has been extensively described in neurodegenerative conditions linked to protein misfolding and aggregation, including Amyotrophic lateral sclerosis, Prion-related disorders, and conditions such as Parkinson's, Huntington's, and Alzheimer's disease. Strong correlations are observed between disease progression, accumulation of protein aggregates, and induction of the UPR in animal and in vitro models of neurodegeneration. In addition, the first reports are available describing the engagement of ER stress responses in brain post-mortem samples from human patients. Despite such findings, the role of the UPR in the central nervous system has not been addressed directly and its contribution to neurodegeneration remains speculative. Recently, however, pharmacological manipulation of ER stress and autophagy - a stress pathway modulated by the UPR - using chemical chaperones and autophagy activators has shown therapeutic benefits by attenuating protein misfolding in models of neurodegenerative disease. The most recent evidence addressing the role of the UPR and ER stress in neurodegenerative disorders is reviewed here, along with therapeutic strategies to alleviate ER stress in a disease context.     

Effect of the mitochondrial unfolded protein response on hypoxic death and mitochondrial protein aggregation

Mitochondria are the main oxygen consumers in cells and as such are the primary organelle affected by hypoxia. All hypoxia pathology presumably derives from the initial mitochondrial dysfunction. An early event in hypoxic pathology in C. elegans is disruption of mitochondrial proteostasis with induction of the mitochondrial unfolded protein response (UPR^mt) and mitochondrial protein aggregation. Here in C. elegans, we screen through RNAis and mutants that confer either strong resistance to hypoxic cell death or strong induction of the UPR^mt to determine the relationship between hypoxic cell death, UPR^mt activation, and hypoxia-induced mitochondrial protein aggregation (HIMPA). We find that resistance to hypoxic cell death invariantly mitigated HIMPA. We also find that UPR^mt activation invariantly mitigated HIMPA. However, UPR^mt activation was neither necessary nor sufficient for resistance to hypoxic death and vice versa. We conclude that UPR^mt is not necessarily hypoxia protective against cell death but does protect from mitochondrial protein aggregation, one of the early hypoxic pathologies in C. elegans.Subject terms: Necroptosis, Energy metabolism     

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.     

Suppression of the proinflammatory response of metastatic melanoma cells increases TRAIL-induced apoptosis

Melanoma is the most lethal form of human skin cancer. However, only limited chemotherapy is currently available for the metastatic stage of the disease. Since chemotherapy, radiation and sodium arsenite treatment operate mainly through induction of the intrinsic mitochondrial pathway, a strongly decreased mitochondrial function in metastatic melanoma cells, could be responsible for low efficacy of the conventional therapy of melanoma. Another feature of metastatic melanoma cells is their proinflammatory phenotype, linked to endogenous expression of the inflammatory cytokines, such as TNFα IL6 and IL8, their receptors, and constitutive NF-κB- and STAT3-dependent gene expression, including cyclooxygenase-2 (PTGS2/COX2). In the present study, we treated melanoma cells with immunological (monoclonal antibody against TNFα or IL6), pharmacological (small molecular inhibitors of IKKβ-NF-κB and JAK2-STAT3) or genetic (specific RNAi for COX-2) agents that suppressed the inflammatory response in combination with induction of apoptosis via TRAIL. As a result of these combined treatments, exogenous TRAIL via interactions with TRAIL-R2/R1 strongly increased levels of apoptosis in resistant melanoma cells. The present study provides new understanding of the regulation of TRAIL-mediated apoptosis in melanoma and will serve as the foundation for the potential development of a novel approach for a therapy of resistant melanomas.     

Depletion of TMEM65 leads to oxidative stress,apoptosis, induction of mitochondrial unfolded protein response,and upregulation of mitochondrial protein import receptor TOMM22

Mutation in the transmembrane protein 65 gene (TMEM65) results in mitochondrial dysfunction and a severe mitochondrial encephalomyopathy phenotype. However, neither the function of TMEM65 nor the cellular responses to its depletion have been fully elucidated. Hence, we knocked down TMEM65 in human cultured cells and analyzed the resulting cellular responses. Depletion of TMEM65 led to a mild increase in ROS generation and upregulation of the mRNA levels of oxidative stress suppressors, such as NFE2L2 and SESN3, indicating that TMEM65 knockdown induced an oxidative stress response. A mild induction of apoptosis was also observed upon depletion of TMEM65. Depletion of TMEM65 upregulated protein levels of the mitochondrial chaperone HSPD1 and mitochondrial protease LONP1, indicating that mitochondrial unfolded protein response (UPR^mt) was induced in response to TMEM65 depletion. Additionally, we found that the mitochondrial protein import receptor TOMM22 and HSPA9 (mitochondrial Hsp70), were also upregulated in TMEM65-depleted cells. Notably, the depletion of TMEM65 did not lead to upregulation of TOMM22 in an ATF5-dependent manner, although upregulation of LONP1 reportedly occurs in an ATF5-dependent manner. Taken together, our findings suggest that depletion of TMEM65 causes mild oxidative stress and apoptosis, induces UPR^mt, and upregulates protein expression of mitochondrial protein import receptor TOMM22 in an ATF5-independent manner.     

p53 triggers apoptosis in oncogene-expressing fibroblasts by the induction of Noxa and mitochondrial Bax translocation

The mechanism of p53-dependent apoptosis is still only partly defined. Using early-passage embryonic fibroblasts (MEF) from wild-type (wt), p53(-/-) and bax(-/-) mice, we observe a p53-dependent translocation of Bax to the mitochondria and a release of mitochondrial Cytochrome c during stress-induced apoptosis. These events proceed independent of zVAD-inhibitable caspase activation, are not prevented by dominant negative FADD (DN-FADD), but are negatively regulated by Mdm-2. Bcl-x(L) expression prevents the release of mitochondrial Cytochrome c and apoptosis, but not Bax translocation. At a single-cell level, enforced expression of p53 is sufficient to induce Bax translocation and Cytochrome c release. Real-time RT-PCR analysis reveals a significant induction of RNA expression of Noxa and Bax in p53(+/+), but not in p53(-/-) MEF. Noxa protein expression becomes detectable prior to Bax translocation, and downregulation of endogenous Noxa by RNA interference protects wt MEF against p53-dependent apoptosis. Hence, in oncogene-expressing MEF p53 induces apoptosis by BH3 protein-dependent caspase activation.