Identification of short-lived long non-coding RNAs as surrogate indicators for chemical stress response

Abiotic and biotic stressors in human cells are often a result of sudden and/or frequent changes in environmental factors. The molecular response to stress involves elaborate modulation of gene expression and is of homeostatic, ecological, and evolutionary importance. Although attention has primarily focused on signaling pathways and protein networks, long non-coding RNAs (ncRNAs) are increasingly involved in the molecular mechanisms associated with responses to cellular stresses. We identified six novel short-lived long ncRNAs (MIR22HG, GABPB-AS1, LINC00152, IDI2-AS1, SNHG15, and FLJ33630) that responded to chemical stressors (cisplatin, cycloheximide, and mercury (II) oxide) in HeLa Tet-off cells. Our results indicate that short-lived long ncRNAs respond to general and specific chemical stressors. The expression levels of the short-lived long ncRNAs were elevated because of prolonged decay rates in response to chemical stressors and interruption of RNA degradation pathways. We propose that these long ncRNAs have the potential to be surrogate indicators of cellular stress responses.     

'Til cell death do us part: nitric oxide and mechanisms of hepatotoxicity

Like many juggernauts in biology, the elusive nature of nitric oxide (NO) sprints through the fields, sometimes the savior, at other times the scimitar. In the liver, which is the metabolic center of the organism, hepatocytes and immune cells trade blows using the reactive diatomic molecule NO to induce cellular damage under toxic conditions. In response, hepatocytes can utilize several mechanisms of NO to their protective advantage by prohibiting the activation of programmed cell death, a.k.a. apoptosis. The balance of these effects in this reactive milieu set the stage for the homeostatic response to cellular injury that determines whether hepatocytes will live, die, or regenerate. Insights that we and others have gained from the liver under pathologic conditions of stress can be applied to the understanding of cellular death mechanisms in other organs and tissues.     

BiP Availability Distinguishes States of Homeostasis and Stress in the Endoplasmic Reticulum of Living Cells

Accumulation of misfolded secretory proteins causes cellular stress and induces the endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). Although the UPR has been extensively studied, little is known about the molecular changes that distinguish the homeostatic and stressed ER. The increase in levels of misfolded proteins and formation of complexes with chaperones during ER stress are predicted to further crowd the already crowded ER lumen. Surprisingly, using live cell fluorescence microscopy and an inert ER reporter, we find the crowdedness of stressed ER, treated acutely with tunicamycin or DTT, either is comparable to homeostasis or significantly decreases in multiple cell types. In contrast, photobleaching experiments revealed a GFP-tagged variant of the ER chaperone BiP rapidly undergoes a reversible quantitative decrease in diffusion as misfolded proteins accumulate. BiP mobility is sensitive to exceptionally low levels of misfolded protein stressors and can detect intermediate states of BiP availability. Decreased BiP availability temporally correlates with UPR markers, but restoration of BiP availability correlates less well. Thus, BiP availability represents a novel and powerful tool for reporting global secretory protein misfolding levels and investigating the molecular events of ER stress in single cells, independent of traditional UPR markers.     

Heat shock protein expression in fish

Heat shock proteins (HSP) are a family of proteins expressed in response to a wide range of biotic and abiotic stressors. They are thus also referred to as stress proteins. Their extraordinarily high degree of identity at the amino acid sequence level and the fact that this cellular stress response has been described in nearly all organisms studied, make this group of proteins unique. We provide a brief historical overview of HSP research, as a background to summarizing what is known about HSP expression in fish. The expression of HSPs in fish has been described in cell lines, primary cultures of various cells, and in the tissues of whole organisms. Collectively, the data show that the expression of HSPs are affected in a wide variety of fish cells and tissues, in response both to biological stressors such as infectious pathogens, as well as to abiotic stressors such as heat and cold shock, and environmental contaminants. HSP research in fish is in its early stages and many studies are describing the expression of proteins in response to various stressors. Several studies have contributed to our understanding of the molecular nature and the molecular biology of HSPs in fish. Recent studies have shown a relationship between HSP expression and the generalized stress response in fish, but further research is needed to clarify the complex relationships between stress hormones and the cellular HSP response. In general, the HSP response seems to be related to the sensing of the stressor and the subsequent cellular effects which may adapt the cells to cope with the stressors. Consequently, such data may be of central importance in understanding the significance of HSP expression to the whole organism. We conclude with sections on laboratory methods used in HSP research and on potential applications of this knowledge in biomonitoring.     

Coping with stress: cellular relaxation techniques

Proteins damaged by stressors such as heat, oxidizing conditions or toxic agents are deleterious to cells and must be properly taken care of. Accordingly, misfolded proteins trigger a cellular stress response that aims to either repair defective polypeptides or eliminate faulty elements when salvage is not possible. This stress response provides time for additional stressor-specific pathways that adapt the cell to the changed environment if necessary. Recent studies have investigated how proteins that frustrate the folding machinery are recognized and cleared from the cell. Surprisingly, these clearance mechanisms are not restricted to the protein level. The stress response can also eliminate the mRNA of polypeptides that are refractory to folding.     

Stress, behaviour and reproductive performance in female cattle and pigs

Female farm animals are exposed to a great variety of environmental and management related stressors. As a consequence, their reproductive and maternal abilities may be compromised through mechanisms acting on the hypothalamic, pituitary, ovarian and uterine function. Responses to short- and long-term stressors may differ as short-term stressors often fail to affect reproduction or even may have stimulatory effects. Thus, the stress response induces diverse neuroendocrine reactions that can either increase or decrease the probability of an animal reproducing depending on the specific situation. The aim of the present review is to summarise the current knowledge on the stress concept and its implications on behaviour and reproductive performance in cows and female pigs as phenomena reported in laboratory animals are unable to explain all effects encountered in domesticated farm animals.     

Protein degradation and the stress response

Environmental stresses are manifold and so are the responses they elicit. This is particularly true for higher eukaryotes where various tissues and cell types are differentially affected by the insult. Type and scope of the stress response can therefore differ greatly among cell types. Given the importance of the ubiquitin proteasome system (UPS) for most cellular processes, it comes as no surprise that the UPR plays a pivotal role in counteracting the effects of stressors. Here we outline contributions of the UPS to stress sensing, signaling, and response pathways. We make no claim to comprehensiveness but choose selected examples to illustrate concepts and mechanisms by which protein modification with ubiquitin and proteasomal degradation of key regulators ensures cellular integrity during stress situations.     

Quantification of oxidative stress in live mouse embryonic fibroblasts by monitoring the responses of polyubiquitin genes

Stress-regulated polyubiquitin genes in mammals are expected to be upregulated under oxidative stress conditions. In order to assess gene regulation via the conventional method, the isolation of RNA molecules or the transfection of reporter constructs into cells is frequently required. If the stress response within cells can be monitored in a reversible manner with minimal manipulation, the study of the stress response pathways will become much easier. Herein, we have developed a simple fluorescence plate reader-based assay to monitor the stress responses of polyubiquitin genes in mouse embryonic fibroblasts, in which one allele of the ubiquitin-coding region of the polyubiquitin gene Ubb or Ubc was replaced by the eGFP-puro cassette, thereby placing GFP expression under the control of the endogenous polyubiquitin gene promoter. Using this simple assay, we established that both mammalian polyubiquitin genes are upregulated upon oxidative stress with slightly higher responses from the Ubb promoter. The principal advantage of this assay is that it allows for the monitoring of stress responses of polyubiquitin genes without disrupting cellular growth; this assay can therefore be applied repeatedly to the same cells. Furthermore, by calculating the increase in fluorescence deriving from newly synthesized GFP upon stress, which can be regarded as a bona fide polyubiquitin gene stress response, we were able to determine and directly compare the concentrations of various oxidative stressors that induce the similar cellular stress levels. Therefore, this simple assay may also be employed in the screening of potentially toxic reagents that induce the stress response pathways.     

The physiological response to anthropogenic stressors in marine elasmobranch fishes: a review with a focus on the secondary response

Elasmobranchs (sharks, rays, and skates) are currently facing substantial anthropogenic threats, which expose them to acute and chronic stressors that may exceed in severity and/or duration those typically imposed by natural events. To date, the number of directed studies on the response of elasmobranch fishes to acute and chronic stress are greatly exceeded by those related to teleosts. Of the limited number of studies conducted to date, most have centered on sharks; batoids are poorly represented. Like teleosts, sharks exhibit primary and secondary responses to stress that are manifested in their blood biochemistry. The former is characterized by immediate and profound increases in circulating catecholamines and corticosteroids, which are thought to mobilize energy reserves and maintain oxygen supply and osmotic balance. Mediated by these primary responses, the secondary effects of stress in elasmobranchs include hyperglycemia, acidemia resulting from metabolic and respiratory acidoses, and profound disturbances to ionic, osmotic, and fluid volume homeostasis. The nature and magnitude of these secondary effects are species-specific and may be tightly linked to metabolic scope and thermal physiology as well as the type and duration of the stressor. In fishes, acute and chronic stressors can incite a tertiary response, which involves physiological changes at the organismal level, thereby impacting growth rates, reproductive outputs or investments, and disease resistance. Virtually no studies to date have been conducted on the tertiary stress response in elasmobranchs. Given the diversity of elasmobranchs, additional studies that characterize the nature, magnitude, and consequences of physiological stress over a broad spectrum of stressors are essential for the development of conservation measures. Additional studies on the primary, secondary, and tertiary stress response in elasmobranchs are warranted, with particular emphasis on expanding the range of species and stressors examined. Future studies should move beyond simply studying the effects of known stressors and focus on the underlying physiological mechanisms. Such studies should include the coupling of stress indicators with quantifiable aspects of the stressor, which will allow researchers to test hypotheses on survivorship and, ultimately, derive models that effectively link physiology to mortality. Studies of this nature are essential for decision-making that will result in the effective management and conservation of these species.     

GDF15 as a central mediator for integrated stress response and a promising therapeutic molecule for metabolic disorders and NASH

BackgroundMitochondria is a key organelle for energy production and cellular adaptive response to intracellular and extracellular stresses. Mitochondrial stress can be evoked by various stimuli such as metabolic stressors or pathogen infection, which may lead to expression of ‘mitokines’ such as growth differentiation factor 15 (GDF15).Scope of reviewThis review summarizes the mechanism of GDF15 expression in response to organelle stress such as mitochondrial stress, and covers pathophysiological conditions or diseases that are associated with elevated GDF15 level. This review also illustrates the in vivo role of GDF15 expression in those stress conditions or diseases, and a potential of GDF15 as a therapeutic agent against metabolic disorders such as NASH.Major conclusionsMitochondrial unfolded protein response (UPRmt) is a critical process to recover from mitochondrial stress. UPRmt can induce expression of secretory proteins that can exert systemic effects (mitokines) as well as mitochondrial chaperons. GDF15 can have either protective or detrimental systemic effects in response to mitochondrial stresses, suggesting its role as a mitokine. Mounting evidence shows that GDF15 is also induced by stresses of organelles other than mitochondria such as endoplasmic reticulum (ER). GDF15 level is increased in serum or tissue of mice and human subjects with metabolic diseases such as obesity or NASH. GDF15 can modulate metabolic features of those diseases.General significanceGDF15 play a role as an integrated stress response (ISR) beyond mitochondrial stress response. GDF15 is involved in the pathogenesis of metabolic diseases such as NASH, and also could be a candidate for therapeutic agent against those diseases.     

Gadd45 in stress signaling

Gadd45 genes have been implicated in stress signaling in response to physiological or environmental stressors, which results in cell cycle arrest, DNA repair, cell survival and senescence, or apoptosis. Evidence accumulated implies that Gadd45 proteins function as stress sensors is mediated by a complex interplay of physical interactions with other cellular proteins that are implicated in cell cycle regulation and the response of cells to stress. These include PCNA, p21, cdc2/cyclinB1, and the p38 and JNK stress response kinases. What deterministic factors dictate whether Gadd45 and partner proteins function in either cell survival or apoptosis remains to be determined. An attractive working model to consider is that the extent of cellular/DNA damage, in a given cell type, dictates the association of different Gadd45 proteins with particular partner proteins, which determines the outcome.     

Minimal peroxide exposure of neuronal cells induces multifaceted adaptive responses

Oxidative exposure of cells occurs naturally and may be associated with cellular damage and dysfunction. Protracted low level oxidative exposure can induce accumulated cell disruption, affecting multiple cellular functions. Accumulated oxidative exposure has also been proposed as one of the potential hallmarks of the physiological/pathophysiological aging process. We investigated the multifactorial effects of long-term minimal peroxide exposure upon SH-SY5Y neural cells to understand how they respond to the continued presence of oxidative stressors. We show that minimal protracted oxidative stresses induce complex molecular and physiological alterations in cell functionality. Upon chronic exposure to minimal doses of hydrogen peroxide, SH-SY5Y cells displayed a multifactorial response to the stressor. To fully appreciate the peroxide-mediated cellular effects, we assessed these adaptive effects at the genomic, proteomic and cellular signal processing level. Combined analyses of these multiple levels of investigation revealed a complex cellular adaptive response to the protracted peroxide exposure. This adaptive response involved changes in cytoskeletal structure, energy metabolic shifts towards glycolysis and selective alterations in transmembrane receptor activity. Our analyses of the global responses to chronic stressor exposure, at multiple biological levels, revealed a viable neural phenotype in-part reminiscent of aged or damaged neural tissue. Our paradigm indicates how cellular physiology can subtly change in different contexts and potentially aid the appreciation of stress response adaptations.     

Hypertonic stress induces rapid and widespread protein damage in C. elegans

Proteostasis is defined as the homeostatic mechanisms that maintain the function of all cytoplasmic proteins. We recently demonstrated that the capacity of the proteostasis network is a critical factor that defines the limits of cellular and organismal survival in hypertonic environments. The current studies were performed to determine the extent of protein damage induced by cellular water loss. Using worm strains expressing fluorescently tagged foreign and endogenous proteins and proteins with temperature-sensitive point mutations, we demonstrate that hypertonic stress causes aggregation and misfolding of diverse proteins in multiple cell types. Protein damage is rapid. Aggregation of a polyglutamine yellow fluorescent protein reporter is observable with <1 h of hypertonic stress, and aggregate volume doubles approximately every 10 min. Aggregate formation is irreversible and occurs after as little as 10 min of exposure to hypertonic conditions. To determine whether endogenous proteins are aggregated by hypertonic stress, we quantified the relative amount of total cellular protein present in detergent-insoluble extracts. Exposure for 4 h to 400 mM or 500 mM NaCl induced a 55-120% increase in endogenous protein aggregation. Inhibition of insulin signaling or acclimation to mild hypertonic stress increased survival under extreme hypertonic conditions and prevented aggregation of endogenous proteins. Our results demonstrate that hypertonic stress causes widespread and dramatic protein damage and that cells have a significant capacity to remodel the network of proteins that function to maintain proteostasis. These findings have important implications for understanding how cells cope with hypertonic stress and other protein-damaging stressors.     

Effects of husbandry and management systems on physiology and behaviour of farmed and laboratory rabbits

The major issues regarding the welfare of both farmed and laboratory rabbits are reviewed, according to husbandry and management systems. The main stressors that can affect welfare and homeostatic responses in rabbits are also reviewed. An overview of the most widespread housing systems for both farmed and laboratory rabbits is presented. The main problems related to housing and management are identified, in particular those related to individual and group housing, space requirements and group size, as well as human-animal interaction. The effects of psychological and physical stressors on physiology and behaviour are illustrated through examples in various rearing conditions. Psychological stressors include social stress and fear, while physical stressors include environmental variables such as housing system and climatic factors, i.e. heat. Welfare indicators are identified that can be monitored to determine the effects of individual and environmental variables on the animals' possible coping strategies. Physiological indicators include the neuro-endocrine and psycho-neuro-immuno-endocrine measurements, while behavioural indicators include the behavioural repertoire and responses to behavioural tests. Some possible ways to enhance welfare are indicated, such as enrichment of the environment and improved handling procedures.     

Long-lasting, sex- and age-specific effects of social stressors on corticosterone responses to restraint and on locomotor responses to psychostimulants in rats

Many neural systems are undergoing marked development over adolescence, which may heighten an animal's vulnerability to stressors. One consequence may be altered sensitivity to drugs of abuse. We previously reported that social stressors in adolescence increased behavioral sensitization to nicotine in adulthood in female, but not male, rats. Here we examined whether social stressors in adolescence alter the functioning of the hypothalamic-pituitary-adrenal (HPA) axis by examining corticosterone release in response to restraint in adulthood. To further assess effects of social stressors on behavioral sensitivity to psychostimulants, we examined locomotor activity in response to nicotine and to amphetamine. In a second set of experiments, we investigated whether the same procedure of social stressors administered in adulthood produces effects similar to that observed when administered in adolescence. Rats underwent daily 1 h isolation followed by pairing with a new cage mate on either postnatal days 33-48 (pubertal stress: PS) or days 65-80 (adult stress: AS). Three weeks later rats tested for either: (a) corticosterone levels were measured in response to restraint, or (b) locomotor sensitization to nicotine (0.25 mg/kg; 5 days) followed by an amphetamine challenge (0.5 mg/kg) 24 h later. Effects of social stressors were evident only in females. PS females had increased locomotor activity to amphetamine compared to controls, and AS females had increased corticosterone release compared to controls. No effect of the social stressors was found in males at either age except for reduced weight gain during the stress procedure. Thus, females are more susceptible to the enduring effects of these moderate social stressors than are males. However, in terms of behavioral sensitivity to drugs of abuse, females may be more susceptible to stressors during adolescence than adulthood, although the reverse appears to be true for HPA function.