Activation Of Cellular Stress Response Research Articles

DDX41 haploinsufficiency causes inefficient hematopoiesis under stress and cooperates with p53 mutations to cause hematologic malignancy

Germline heterozygous mutations in DDX41 predispose individuals to hematologic malignancies in adulthood. Most of these DDX41 mutations result in a truncated protein, leading to loss of protein function. To investigate the impact of these mutations on hematopoiesis, we generated mice with hematopoietic-specific knockout of one Ddx41 allele. Under normal steady-state conditions, there was minimal effect on lifelong hematopoiesis, resulting in a mild yet persistent reduction in red blood cell counts. However, stress induced by transplantation of the Ddx41+/− BM resulted in hematopoietic stem/progenitor cell (HSPC) defects and onset of hematopoietic failure upon aging. Transcriptomic analysis of HSPC subsets from the transplanted BM revealed activation of cellular stress responses, including upregulation of p53 target genes in erythroid progenitors. To understand how the loss of p53 affects the phenotype of Ddx41+/− HSPCs, we generated mice with combined Ddx41 and Trp53 heterozygous deletions. The reduction in p53 expression rescued the fitness defects in HSPC caused by Ddx41 heterozygosity. However, the combined Ddx41 and Trp53 mutant mice were prone to developing hematologic malignancies that resemble human myelodysplastic syndrome and acute myeloid leukemia. In conclusion, DDX41 heterozygosity causes dysregulation of the response to hematopoietic stress, which increases the risk of transformation with a p53 mutation.

Exploiting inter-tissue stress signaling mechanisms to preserve organismal proteostasis during aging.

Aging results in a decline of cellular proteostasis capacity which culminates in the accumulation of phototoxic material, causing the onset of age-related maladies and ultimately cell death. Mechanisms that regulate proteostasis such as cellular stress response pathways sense disturbances in the proteome. They are activated to increase the expression of protein quality control components that counteract cellular damage. Utilizing invertebrate model organisms such as Caenorhabditis elegans, it has become increasingly evident that the regulation of proteostasis and the activation of cellular stress responses is not a cell autonomous process. In animals, stress responses are orchestrated by signals coming from other tissues, including the nervous system, the intestine and the germline that have a profound impact on determining the aging process. Genetic pathways discovered in C. elegans that facilitate cell nonautonomous regulation of stress responses are providing an exciting feeding ground for new interventions. In this review I will discuss cell nonautonomous proteostasis mechanisms and their impact on aging as well as ongoing research and clinical trials that can increase organismal proteostasis to lengthen health- and lifespan.

DOES THE ENERGETIC COST OF STRESS ACCELERATE BIOLOGICAL AGING AND SHORTEN LIFESPAN?

Abstract In humans, chronic activation of cellular stress responses predict functional decline, accelerate aging, and increase mortality, but the cellular basis for the stress-aging cascade remains unclear. Here we induced chronic stress in primary human fibroblasts from multiple donors with constant i) ATP-synthase inhibition (Oligomycin-1nM) or i) glucocorticoid stimulation (Dexamethasone-100nM) cultured for up to 10 months. Stressors triggered mtDNA instability and activated integrated stress responses resulting in both transcriptional activation and secretion of cytokines and metabokines. In parallel, chronic stress increased cellular energy expenditure or the “cost of living” by 62–108% (Ps<0.001). Thus, chronically stressed cells considerably expend more energy to undergo each cell division. This severe state of hypermetabolism led to faster rates of telomere shortening and of genome-wide DNA methylation-based epigenetic aging monitored across the cellular lifespan, reflecting mito-nuclear signaling. This accelerated aging phenotype culminated in 20–40% fewer maximal cell divisions (i.e., Hayflick limit). Based on findings that hypermetabolism and increased energy flux through mitochondria may shorten lifespan, we pharmacologically inhibited carbon entry (glutamine, pyruvate, long-chain fatty acids) into the Krebs cycle across the entire cellular lifespan. While this manipulation successfully decreased OxPhos activity, it increased glycolysis-derived ATP synthesis and total energy expenditure, exacerbating the accelerated aging phenotype. Combined, our longitudinal bioenergetic and multi-omic profiling of primary human cells show that chronic heterotypic stressors converge on an acceleration of metabolism (i.e., hypermetabolism), and commensurately accelerate the progression of multiple aging hallmarks. These findings also implicate long-term mito-nuclear signaling in the stress-aging cascade in a human model.

A genetic screen for proteins involved in Non‐Stop mRNA decay in C. elegans

Non‐stop mRNAs, which lack a stop codon due to mutation or errors in mRNA transcription or processing, serve as templates for production of aberrant and potentially toxic protein products. Ultimately, translation of non‐stop mRNAs leads to a failure in normal translation termination and ribosome collisions, which in eukaryotes initiates ubiquitin‐dependent degradation of the protein product and Non‐Stop Decay (NSD) of the problematic mRNA. Ribosome collisions in yeast are linked to activation of cellular stress responses and failure to resolve stalled ribosomes leads to neurodegeneration in mice, highlighting the biological importance of mRNA quality control.In the nematode Caenorhabditis elegans, loss of the core NSD components pelo‐1 and skih‐2 leads to temperature‐sensitive sterility, as does the loss of endogenous siRNAs that are triggered by NSD at some loci. To further probe the link between NSD and the biology of germ cells, we have used an RNAi approach to screen for temperature‐sensitive reduction in brood size for the C. elegans orthologs of several proteins known to be part of the NSD machinery in other organisms. Preliminary results have identified 2 NSD‐associated genes with conditionally reduced brood sizes. We will present progress on examining the requirement of these genes for Non‐Stop decay in C. elegans, as well as investigation of their specific contributions to the biology of the germ line.

The melatonin receptor antagonist luzindole induces the activation of cellular stress responses and decreases viability of rat pancreatic stellate cells.

In this study, we have examined the effects of luzindole, a melatonin receptor-antagonist, on cultured pancreatic stellate cells. Intracellular free-Ca2+ concentration, production of reactive oxygen species (ROS), activation of mitogen-activated protein kinases (MAPK), endoplasmic reticulum stress and cell viability were analyzed. Stimulation of cells with the luzindole (1, 5, 10 and 50 μm) evoked a slow and progressive increase in intracellular free Ca2+ ([Ca2+ ]i ) towards a plateau. The effect of the compound on Ca2+ mobilization depended on the concentration used. Incubation of cells with the sarcoendoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin (1 μm), in the absence of Ca2+ in the extracellular medium, induced a transient increase in [Ca2+ ]i . In the presence of thapsigargin, the addition of luzindole to the cells failed to induce further mobilization of Ca2+ . Luzindole induced a concentration-dependent increase in ROS generation, both in the cytosol and in the mitochondria. This effect was smaller in the absence of extracellular Ca2+ . In the presence of luzindole the phosphorylation of p44/42 and p38 MAPKs was increased, whereas no changes in the phosphorylation of JNK could be noted. Moreover, the detection of the endoplasmic reticulum stress-sensor BiP was increased in the presence of luzindole. Finally, viability was decreased in cells treated with luzindole. Because cellular membrane receptors for melatonin have not been detected in pancreatic stellate cells, we conclude that luzindole could exert direct effects that are not mediated through its action on melatonin membrane receptors.

MP54-12 IMPAIRMENT OF AMPK-α2 BY ISCHEMIA PROVOKES DETRUSOR OVERACTIVITY VIA CELLULAR STRESS SENSORS

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology (MP54)1 Apr 2020MP54-12 IMPAIRMENT OF AMPK-α2 BY ISCHEMIA PROVOKES DETRUSOR OVERACTIVITY VIA CELLULAR STRESS SENSORS Jinghua Yang, Yedan Li, Michelle Azad, and Kazem Azadzoi* Jinghua YangJinghua Yang More articles by this author , Yedan LiYedan Li More articles by this author , Michelle AzadMichelle Azad More articles by this author , and Kazem Azadzoi*Kazem Azadzoi* More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000916.012AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Adenosine monophosphate-activated protein kinase alpha 2 (AMPK-α2) is a subunit of AMPK with catalytic kinase domain that senses cellular energy deprivation and signals cellular nutrients deficiency. Our goal was to examine functional consequences of AMPK-α2 responses in bladder ischemia. METHODS: A rat model of bladder ischemia was developed by engendering aorto-iliac arterial atherosclerosis. After 8 weeks, ischemic and sham control bladder tissues were processed for analysis of AMPK-α2 expression and AMPK-α2 translocation and activity using western blotting and ELISA. Ischemia-mediated cellular stress was examined by real-time PCR analysis of the cellular stress sensor upstream stimulatory factor 1 (USF1). USF1-mediated smooth muscle contractile activity was examined in the organ bath. In follow up experiments, rats with bladder ischemia were treated with the AMPK-α2 activator 5-aminoimidazole-4-carboxamide-1-beta-D ribofuranoside (AICAR) or placebo in daily basis for 4 weeks then conscious cystometry was performed and bladder tissues were processed for analysis of AMPK-α2, USF1, and smooth muscle contractile activity, as described earlier. RESULTS: Ischemia upregulated AMPK-α2 expression while blocking AMPK-α2 translocation from cytoplasm to the nucleus. Blockade of AMPK-α2 translocation was associated with significant decrease in AMPK-α2 catalytic activity, suggesting loss of cellular energy deprivation sensing via AMPK-α2 in bladder ischemia. Impairment of AMPK-α2 activity upregulated the expression of cellular stress sensor USF1, implying cellular stress responses via AMPK-α2/USF1 pathway in bladder ischemia. These changes shifted contractile reactivity of ischemic bladder tissues to electrical field stimulation (EFS) to the left and produced cystometric changes consistent with detrusor overactivity. Treatment of bladder tissues with the USF1 inhibitor C527 reversed hyperreactivity to EFS. Treatment of rats with the AMPK-α2 activator AICAR significantly downregulated USF1 expression and diminished cystometric markers of detrusor overactivity in bladder ischemia. CONCLUSIONS: Impairment of AMPK-α2 by ischemia upregulated USF1 and provoked detrusor overactivity by activation of cellular stress responses. The mechanism appeared to involve USF1-directed smooth muscle hyperreactivity via cellular stress signaling pathway. Our data suggest that activators of AMPK-α2 and inhibitors of USF1 may have valuable therapeutic potentials against overactive bladder. Source of Funding: Supported by Merit Review Award Number I01 BX004372 from the United States (U.S.) Department of Veterans Affairs Biomedical Laboratory R&D (BLRD) Service. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e795-e796 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jinghua Yang More articles by this author Yedan Li More articles by this author Michelle Azad More articles by this author Kazem Azadzoi* More articles by this author Expand All Advertisement PDF downloadLoading ...

Activating the unfolded protein response in osteocytes causes hyperostosis consistent with craniodiaphyseal dysplasia.

Bone remodeling is a balanced process between bone synthesis and degradation, maintaining homeostasis and a constant bone mass in adult life. Imbalance will lead to conditions such as osteoporosis or hyperostosis. Osteoblasts build bone, becoming embedded in bone matrix as mature osteocytes. Osteocytes have a role in sensing and translating mechanical loads into biochemical signals, regulating the differentiation and activity of osteoblasts residing at the bone surface through the secretion of Sclerostin (SOST), an inhibitor of WNT signaling. Excessive mechanical load can lead to activation of cellular stress responses altering cell behavior and differentiation. The unfolded protein response (UPR) is a shared pathway utilized by cells to cope with stress stimuli. We showed that in a transgenic mouse model, activation of the UPR in early differentiating osteocytes delays maturation, maintaining active bone synthesis. In addition, expression of SOST is delayed or suppressed; resulting in active WNT signaling and enhanced periosteal bone formation, and the combined outcome is generalized hyperostosis. A clear relationship between the activation of the unfolded protein response was established and the onset of hyperostosis that can be suppressed with a chemical chaperone, sodium 4-phenobutyrate (4-PBA). As the phenotype is highly consistent with craniodiaphyseal dysplasia (CDD; OMIM 122860), we propose activation of the UPR could be part of the disease mechanism for CDD patients as these patients are heterozygous for SOST mutations that impair protein folding and secretion. Thus, therapeutic agents ameliorating protein folding or the UPR can be considered as a potential therapeutic treatment.

OP-24 - Novel Dysregulation of Mitochondria Impacts Brain Insulin Signaling

Obesity-related disorders, such as type 2 diabetes, are characterized by insulin resistance not only in peripheral organs but also in the brain. Dysregulation of brain insulin signaling causes obesity and neurological alterations, such as depressive-like behavior. Causal factors of neuronal insulin resistance are multifactorial including exposure to elevated glucose and fatty acid levels, activation of cellular stress responses as well as neuroinflammation. While the effect of the endoplasmatic unfolded protein response (ER stress response) on insulin signaling has been described in detail, the effect of mitochondrial stress is less well understood. We observed that type 2 diabetic mice exhibit hypothalamic insulin resistance and mitochondrial dysfunction due to decreased expression of mitochondrial proteins involved in the mitochondrial unfolded protein response. Reduction of mitochondrial chaperones in neurons is sufficient to cause severe mitochondrial dysfunction with aberrant mitochondrial protein homeostasis leading to oxidative stress and insulin resistance. We further observe that mitochondrial dysfunction is both a causal factor of brain insulin resistance and a consequence of altered brain insulin signaling causing neurological abnormalities.

BPDE-induced genotoxicity: relationship between DNA adducts, mutagenicity in the in vitro PIG-A assay, and the transcriptional response to DNA damage in TK6 cells

Benzo[a]pyrene is a known human carcinogen. As underlying mechanism, the induction of stable DNA adducts and mutations have been repeatedly demonstrated. Also, the activation of cellular stress response on the transcriptional level has been described. Nevertheless, the interrelationship between these different events is less well understood, especially at low, for human exposure relevant concentrations. Within the present study, we applied the reactive metabolite benzo[a]pyrene diolepoxide (BPDE) in the nanomolar, non-cytotoxic concentration range in human TK6 cells and quantified the induction and repair of stable DNA adducts at the N2-position of guanine by HPLC with fluorescence detection. Significant levels of DNA lesions were detected even at the lowest concentration of 10 nM BPDE, with a linear increase up to 50 nM. Relative repair was similar at all damage levels, reaching about 30% after 8 h and 60% after 24 h. Mutation frequencies were quantified as GPI-deficient cells by the recently established in vitro PIG-A mutagenicity assay. Again, a linear dose–response-relationship in the before-mentioned concentration range was observed, also when plotting the number of GPI-deficient cells against the number of DNA adducts. Furthermore, we explored the time- and concentration-dependent DNA damage response on the transcriptional level via a high-throughput RT-qPCR technique by quantifying the impact of BPDE on the transcription of 95 genes comprising DNA damage response, DNA repair factors, oxidative stress response, cell cycle arrest, cell proliferation, and apoptosis. As expected, BPDE activated DNA damage signaling, p53 and AP-1 dependent signaling, oxidative stress response, and apoptosis. However, in contrast to DNA adducts and mutations, the onset of the transcriptional DNA damage response was restricted to higher concentrations, indicating that its respective activations require a certain level of DNA lesions. Altogether, the results indicate that in case of BPDE, DNA lesions and mutations were correlated at all concentrations, suggesting that repair is not complete even at low levels of DNA damage. Considering the ongoing discussion on potential thresholds also for genotoxic carcinogens, the results are of major relevance, both with respect to basic research as well as to risk assessment of chemical carcinogens.

P89 - Assessment of an in vitro model of lung epithelial cell stress responses

The ability of cells to adapt and survive environmental and physiological stress relies on activation of cellular stress responses. These include anti-oxidant, inflammatory and apoptotic responses. These cellular responses are mediated by cell signalling pathways, including those controlled by the transcription factors Nrf2 (antioxidant response) and NF-?ß (inflammatory response). As part of a suite of in vitro models for the comparison of different nicotine delivery products, an in vitro lung cell stress response model has been developed. Human bronchial epithelial cells were exposed to positive controls or cigarette smoke aqueous extracts (CSEaq) from 3R4F and 1R5F reference cigarettes for 4hours. Cellular responses were then measured as oxidative, pro-inflammatory, apoptotic and necrotic endpoints. Cellular oxidative stress was characterised by measurement of the intracellular glutathione ratio, intracellular ROS production and activation of the Nrf2-controlled Anti-oxidative Response Elements (ARE). The inflammatory response of the cells was determined through quantification of secreted cytokines, IL-1a, IL-6 and IL-8. Apoptotic and necrotic responses were characterised by measurements of Caspase 3/7 activity and live-cell protease activity. Stably transfected luciferase reporter cell lines were utilised to quantify the transcriptional control of anti-oxidant and inflammatory pathways. All cell stress response endpoints were activated by exposure to positive controls or CSEaq. The observed concentration-dependent lowering of the glutathione ratio and increase in intracellular ROS generation corresponded with an increase in Nrf2 transcriptional activation of the ARE. It has been demonstrated that this model was able to distinguish between CSEaq from the two different reference cigarettes. We propose that this model may be suitably sensitive for biological comparisons of cigarettes against different nicotine delivery products.

The Novel Hydroxylamine Derivative NG-094 Suppresses Polyglutamine Protein Toxicity in Caenorhabditis elegans

Aggregation-prone polyglutamine (polyQ) expansion proteins cause several neurodegenerative disorders, including Huntington disease. The pharmacological activation of cellular stress responses could be a new strategy to combat protein conformational diseases. Hydroxylamine derivatives act as co-inducers of heat-shock proteins (HSPs) and can enhance HSP expression in diseased cells, without significant adverse effects. Here, we used Caenorhabditis elegans expressing polyQ expansions with 35 glutamines fused to the yellow fluorescent protein (Q35-YFP) in body wall muscle cells as a model system to investigate the effects of treatment with a novel hydroxylamine derivative, NG-094, on the progression of polyQ diseases. NG-094 significantly ameliorated polyQ-mediated animal paralysis, reduced the number of Q35-YFP aggregates and delayed polyQ-dependent acceleration of aging. Micromolar concentrations of NG-094 in animal tissues with only marginal effects on the nematode fitness sufficed to confer protection against polyQ proteotoxicity, even when the drug was administered after disease onset. NG-094 did not reduce insulin/insulin-like growth factor 1-like signaling, but conferred cytoprotection by a mechanism involving the heat-shock transcription factor HSF-1 that potentiated the expression of stress-inducible HSPs. NG-094 is thus a promising candidate for tests on mammalian models of polyQ and other protein conformational diseases.

Secretory pathway stress responses as possible mechanisms of disease involving Golgi Ca2+ pump dysfunction

In mammalian tissues, uptake of Ca(2+) and Mn(2+) by Golgi membranes is mediated by the secretory pathway Ca(2+) -ATPases, SPCA1 and SPCA2, encoded by the ATP2C1 and ATP2C2 genes. Loss of one copy of the ATP2C1 gene, which causes SPCA1 haploinsufficiency, leads to squamous cell tumors of keratinized epithelia in mice and to Hailey-Hailey disease, an acantholytic skin disease, in humans. Although the disease phenotypes resulting from SPCA1 haploinsufficiency in mice and humans are quite different, each species-specific phenotype is remarkably similar to those arising as a result of null mutations in one copy of the ATP2A2 gene, encoding SERCA2, the endoplasmic reticulum (ER) Ca(2+) pump. SERCA2 haploinsufficiency, like SPCA1 haploinsufficiency, causes squamous cell tumors in mice and Darier's disease, also an acantholytic skin disease, in humans. The phenotypic similarities between SPCA1 and SERCA2 haploinsufficiency in the two species, and the general functions of the two pumps in consecutive compartments of the secretory pathway, suggest that the underlying disease mechanisms are similar. In this review, we discuss evidence supporting the view that chronic Golgi stress and/or ER stress resulting from Ca(2+) pump haploinsufficiencies leads to activation of cellular stress responses in keratinocytes, with the predominance of proapoptotic pathways (although not necessarily apoptosis itself) leading to acantholytic skin disease in humans and the predominance of prosurvival pathways leading to tumors in mice.

Regulation of the hepatocellular response to endoplasmic reticulum stress by the transcription factor c-Jun/AP-1

Accumulation of viral or misfolded proteins in the endoplasmic reticulum (ER) and subsequent activation of cellular stress responses is an important step in the pathogenesis of inflammatory and metabolic liver disease. The transcription factor c-Jun/AP-1 regulates hepatocyte survival, proliferation and liver tumorigenesis, however, its functions during the ER stress response are incompletely understood and were addressed here using primary hepatocytes and livers from hepatocyte-specific c-Jun knockout mice (k.o.). ER stress was induced in vitro pharmacologically by Thapsigargin and Tunicamycin, respectively, and resulted in a robust induction of c-Jun expression. Interestingly, expression of various ER stress-related genes such as the pro-apoptotic transcription factor Gadd153, the ER resident chaperone Bip or splicing (i.e. activation) of the transcription factor XBP-1 was increased in k.o. hepatocytes. Moreover, signs of increased hepatocyte damage and cell death such as cytoplasmic vacuolization and ER swelling as determined by electron microscopy, cleavage of caspases 3, 7 and 12 and increased expression of the chemokines CXCL1 and CXCL2 were apparent in hepatocytes lacking c-Jun. Recent data suggest that autophagy, a conserved process of self-degradation of organelles, promotes cell survival during acute ER stress and is regulated by the Jun kinase JNK. Chemical inhibition of JNK or autophagy in ER-stressed wild-type hepatocytes also resulted in cytoplasmic vacuolization reminiscent of the phenotype observed in k.o. cells, in which autophagosome numbers were consistently reduced. Moreover, increased hepatic expression of ER stress related genes, cytoplasmic vacuolization and hepatocyte death were also apparent upon induction of ER stress in k.o. mice in vivo. These data suggest that c-Jun protects hepatocytes against excessive activation of the ER stress response and subsequent cell death, possibly by interacting with the autophagy machinery.

Increasing Melanoma Cell Death Using Inhibitors of Protein Disulfide Isomerases to Abrogate Survival Responses to Endoplasmic Reticulum Stress

Exploiting vulnerabilities in the intracellular signaling pathways of tumor cells is a key strategy for the development of new drugs. The activation of cellular stress responses mediated by the endoplasmic reticulum (ER) allows cancer cells to survive outside their normal environment. Many proteins that protect cells against ER stress are active as protein disulfide isomerases (PDI) and the aim of this study was to test the hypothesis that apoptosis in response to ER stress can be increased by inhibiting PDI activity. We show that the novel chemotherapeutic drugs fenretinide and velcade induce ER stress-mediated apoptosis in melanoma cells. Both stress response and apoptosis were enhanced by the PDI inhibitor bacitracin. Overexpression of the main cellular PDI, procollagen-proline, 2-oxoglutarate-4-dioxygenase beta subunit (P4HB), resulted in increased PDI activity and abrogated the apoptosis-enhancing effect of bacitracin. In contrast, overexpression of a mutant P4HB lacking PDI activity did not increase cellular PDI activity or block the effects of bacitracin. These results show that inhibition of PDI activity increases apoptosis in response to agents which induce ER stress and suggest that the development of potent, small-molecule PDI inhibitors has significant potential as a powerful tool for enhancing the efficacy of chemotherapy in melanoma.

Cadmium-induced Activation of Stress Signaling Pathways, Disruption of Ubiquitin-dependent Protein Degradation and Apoptosis in Primary Rat Sertoli Cell-Gonocyte Cocultures

Cadmium (Cd) is a ubiquitous environmental pollutant that has been associated with male reproductive toxicity in both humans and animal models. The underlying mechanism of this response, however, is still uncharacterized. To address this issue, we employed a recently developed and optimized three-dimensional primary Sertoli cell-gonocyte coculture system and examined the time- and dose-dependent effects of Cd on morphological alterations, cell viability, activation of stress signaling pathway proteins, and the disruption of the ubiquitin proteasome system (UPS). Our results demonstrated that Cd exposure lead to time- and dose-dependent morphological changes that are associated with the induction of apoptosis. In response to Cd, we also saw a disruption of the UPS as evaluated through the accumulation of high-molecular weight polyubiquitinated proteins (HMW-polyUb) as well as alterations in proteasome activity. Robust activation of cellular stress response, measured through the increased phosphorylation of stress-activated protein kinase/c-jun N-terminal kinase and p38, paralleled the accumulation of HMW-polyUb. In addition, p53, a key regulatory protein, was upregulated and underwent increased ubiquitination in response to Cd. To further characterize the role of the UPS in Cd cellular response, we compared the above changes with two classic proteasomal inhibitors, lactacystin, and MG132. The stress response and the accumulation of HWM-polyUb induced by Cd were consistent with the response seen with MG132 but not with lactacystin. In addition, Cd treatment resulted in a dose- and time-dependent effect on proteasome activity, but the overall Cd-induced proteasomal inhibition was unique as compared to MG132 and lactacystin. Taken together, our studies further characterize Cd-induced in vitro testicular toxicity and highlight the potential role of the UPS in this response.

The TORrid affairs of viruses: effects of mammalian DNA viruses on the PI3K–Akt–mTOR signalling pathway

The successful replication of mammalian DNA viruses requires that they gain control of key cellular signalling pathways that affect broad aspects of cellular macromolecular synthesis, metabolism, growth and survival. The phosphatidylinositol 3'-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) pathway is one such pathway. Mammalian DNA viruses have evolved various mechanisms to activate this pathway to obtain the benefits of Akt activation, including the maintenance of translation through the activation of mTOR. In addition, viruses must overcome the inhibition of this pathway that results from the activation of cellular stress responses during viral infection. This Review will discuss the range of mechanisms that mammalian DNA viruses use to activate this pathway, as well as the multiple mechanisms these viruses have evolved to circumvent inhibitory stress signalling.

Knockdown of mouse VCAM-1 by vector-based siRNA

Graft rejection is critically dependent on the recruitment of leukocytes via adhesion molecules on the endothelium, and inhibition of these interactions can prolong graft survival. We have therefore developed an approach using siRNA to inhibit the expression of VCAM-1 in endothelial cells. We transfected siRNA constructs into murine corneal and vascular endothelium and looked at expression of VCAM-1 and other surface molecules by flow cytometry. Adhesion assays (both static and under flow) were used to determine the effect of VCAM-1 inhibition. The activation of cellular stress responses was assessed by RT-PCR. Constructs encoding siRNA can block expression of VCAM-1 in both corneal and vascular endothelial cells (in the latter case after cytokine stimulation). Inhibition of VCAM-1 expression reduced the ability of T cells to adhere to endothelium. However, there were non-specific effects of siRNA expression, including upregulation of (Programmed Death Ligand 1) PDL1 and decreased cell growth. Analysis of stress pathways showed that the endothelial cells transfected with siRNA had upregulated molecules associated with cell stress. While these data are supportive of a potential therapeutic role for siRNA constructs in blocking the expression of adhesion molecules, they also highlight potential non-specific effects of siRNA that must be carefully considered in any application of this technology.

Odorants as cell-type specific activators of a heat shock response in the rat olfactory mucosa.

Heat shock, or stress, proteins (HSPs) are induced in response to conditions that cause protein denaturation. Activation of cellular stress responses as a protective and survival mechanism is often associated with chemical exposure. One interface between the body and the external environment and chemical or biological agents therein is the olfactory epithelium (OE). To determine whether environmental odorants affect OE HSP expression, rats were exposed to a variety of odorants added to the cage bedding. Odorant exposure led to transient, selective induction of HSP70, HSC70, HSP25, and ubiquitin immunoreactivities (IRs) in supporting cells and subepithelial Bowman's gland acinar cells, two OE non-neuronal cell populations involved with inhalant biotransformation, detoxification, and maintenance of overall OE integrity. Responses exhibited odor specificity and dose dependency. HSP70 and HSC70 IRs occurred throughout the apical region of supporting cells; ubiquitin IR was confined to a supranuclear cone-shaped region. Electron microscopic examination confirmed these observations and, additionally, revealed odor-induced formation of dense vesicular arrays in the cone-like regions. HSP25 IR occurred throughout the entire supporting cell cytoplasm. In contrast to classical stress responses, in which the entire array of stress proteins is induced, no increases in HSP40 and HSP90 IRs were observed. Extended exposure to higher odorant doses caused prolonged activation of the same HSP subset in the non-neuronal cells and severe morphological damage in both supporting cells and olfactory receptor neurons (ORNs), suggesting that non-neuronal cytoprotective stress response mechanisms had been overwhelmed and could no longer adequately maintain OE integrity. Significantly, ORNs showed no stress responses in any of our studies. These findings suggest a novel role for these HSPs in olfaction and, in turn, possible involvement in other normal neurophysiological processes.

Transgenic nematodes as biomonitors of microwave-induced stress

Transgenic nematodes ( Caenorhabditis elegans strain PC72), carrying a stress-inducible reporter gene ( Escherichia coli β-galactosidase) under the control of a C. elegans hsp16 heat-shock promoter, have been used to monitor toxicant responses both in water and soil. Because these transgenic nematodes respond both to heat and toxic chemicals by synthesising an easily detectable reporter product, they afford a useful preliminary screen for stress responses (whether thermal or non-thermal) induced by microwave radiation or other electromagnetic fields. We have used a transverse electromagnetic (TEM) cell fed from one end by a source and terminated at the other end by a matched load. Most studies were conducted using a frequency of 750 MHz, at a nominal power setting of 27 dBm. The TEM cell was held in an incubator at 25°C inside a shielded room; corresponding controls were shielded and placed in the same 25°C incubator; additional baseline controls were held at 15°C (worm growth temperature). Stress responses were measured in terms of β-galactosidase (reporter) induction above control levels. The time-course of response to continuous microwave radiation showed significant differences from 25°C controls both at 2 and 16 h, but not at 4 or 8 h. Using a 5×5 multiwell plate array exposed for 2 h, the 25 microwaved samples showed highly significant responses compared with a similar control array. The wells most strongly affected were those in the rows closest to the source, whereas the most distant row did not rise above control levels, suggesting a shadow effect. These differential responses are difficult to reconcile with general heating effects, although localised power absorption affords a possible explanation. Experiments in which the frequency and/or power settings were varied suggested a greater response at 21 than at 27 dBm, both at 750 and 300 MHz, although extremely variable responses were observed at 24 dBm and 750 MHz. Thus, lower power levels tended, if anything, to induce larger responses (with the above-mentioned exception), which is opposite to the trend anticipated for any simple heating effect. These results are reproducible and data acquisition is both rapid and simple. The evidence accrued to date suggests that microwave radiation causes measurable stress to transgenic nematodes, presumably reflecting increased levels of protein damage within cells (the common signal thought to trigger hsp gene induction). The response levels observed are comparable to those observed with moderate concentrations (ppm) of metal ions such as Zn 2+ and Cu 2+. We conclude that this approach deserves further and more detailed investigation, but that it has already demonstrated clear biological effects of microwave radiation in terms of the activation of cellular stress responses ( hsp gene induction).