Increase In Hsp70 mRNA Levels Research Articles

Responses to elevated CO2 exposure in a freshwater mussel, Fusconaia flava.

Freshwater mussels are some of the most imperiled species in North America and are particularly susceptible to environmental change. One environmental disturbance that mussels may encounter that remains understudied is an increase in the partial pressure of CO2 (pCO2). The present study quantified the impacts of acute (6h) and chronic (up to 32days) exposures to elevated pCO2 on genes associated with shell formation (chitin synthase; cs) and the stress response (heat shock protein 70; hsp70) in Fusconaia flava. Oxygen consumption (MO2) was also assessed over the chronic CO2 exposure period. Although mussels exhibited an increase in cs following an acute exposure to elevated pCO2, long-term exposure resulted in a decrease in cs mRNA abundance, suggesting that mussels may invest less in shell formation during chronic exposure to elevated pCO2. In response to an acute elevation in pCO2, mussels increased hsp70 mRNA abundance in mantle and adductor muscle and a similar increase was observed in the gill and adductor muscle in response to a chronic elevation in pCO2. A chronic elevation in pCO2 also increased mussel MO2. This overall increase in hsp70 mRNA levels and MO2 in F. flava indicates that exposure to elevated pCO2 initiates activation of the general stress response and an increased energy demand. Together, the results of the present study suggest that freshwater mussels respond to elevated pCO2 by increasing processes necessary to 'deal with' the stressor and, over the long-term, may reduce their investment in non-essential processes such as shell growth.

Oxidative stress related to chlorpyrifos exposure in rainbow trout: Acute and medium term effects on genetic biomarkers

Organophosphates (OPs) are derivatives of phosphoric acid widely used in agriculture as pesticides. Chlorpyrifos (CPF) is an OP that is extremely toxic to aquatic organisms. Rainbow trout (Oncorhynchus mykiss) is considered as a sentinel model species for ecotoxicology assessment in freshwater ecosystems. An exposure study was carried out on rainbow trout to investigate genetic responses to CPF-induced oxidative stress by Real-Time PCR, and to determine the accumulation dynamics of CPF and toxic metabolite chlorpyrifos-oxon (CPF-ox) in edible parts, by HPLC–MS/MS. Among the genes considered to be related to oxidative stress, a significant increase in HSP70 mRNA levels was observed in liver samples up to 14days after CPF exposure (0.05mg/L). CPF concentrations in muscle samples reach mean values of 285.25ng/g within 96hours of exposure, while CPF-ox concentrations were always under the limit of quantification (LOQ) of the applied method. Our findings lead us to consider HSP70 as a suitable genetic marker in rainbow trout for acute and medium-term monitoring of CPF exposure, complementary to analytical determinations.

HSPA1A is an important regulator of the stability and function of ZNF198 and its oncogenic derivative, ZNF198–FGFR1

Mass spectroscopy analysis demonstrated that the HSPA1A protein is found in complex with the ZNF198 protein which is involved in a chromosome rearrangement with the FGFR1 gene in an atypical myeloproliferative disease. HSPA1A is a member of the HSP70 family of genes which has been shown to be inducible in a variety of circumstances. Exogenous expression of the ZNF198-FGFR1 fusion kinase gene as well as ZNF198 in a model cell system results in a large (>650-fold) increase in HSP70 mRNA levels. Using KNK437, a specific inhibitor of HSP70 transcription, we have demonstrated that an important function of HSPA1A is to stabilize the ZNF198 and ZNF198-FGFR1 proteins. In the absence of HSPA1A, specific functions of ZNF198-FGFR1 such as STAT3 phosphorylation is also lost. Treatment of cells with KNK437 in the presence of MG132, an inhibitor of proteasomal degradation of proteins, suggested that only the ZNF198-FGFR1 protein is subject to the proteasomal degradation pathway, while ZNF198 is not. These observations suggest an important role for HSPA1A in ZNF198 and ZNF198-FGFR1 mediated cellular function.

Peroxidative stress selectively down-regulates the neuronal stress response activated under conditions of endoplasmic reticulum dysfunction.

Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Here, we investigated the effect of peroxide exposure on the expression of genes coding for cytoplasmic and endoplasmic reticulum (ER) stress proteins. Primary neuronal cell cultures were exposed to H(2)O(2) for 6 h and mRNA levels of hsp70, grp78, grp94, gadd153 were evaluated by quantitative PCR. In addition, peroxide-induced changes in protein synthesis and cell viability were investigated. Peroxide treatment of cells triggered an almost 12-fold increase in hsp70 mRNA levels, but a significant decrease in grp78, grp94 and gadd153 mRNA levels. To establish whether peroxide exposure blocks the ER-resident stress response, cells were also exposed to thapsigargin (Tg, a specific inhibitor of ER Ca(2+)-ATPase) which has been shown to elicit the ER stress response. Tg exposure induced 7.2-fold, 3.6-fold and 8.8-fold increase in grp78, grp94 and gadd153 mRNA levels, respectively. However, after peroxide pre-exposure, the Tg-induced effect on grp78, grp94 and gadd153 mRNA levels was completely blocked. The results indicate that oxidative damage causes a selective down-regulation of the neuronal stress response activated under conditions of ER dysfunction. This down-regulation was only observed in cultures exposed to peroxide levels which induced severe suppression of protein synthesis and cell injury, implying a causative link between peroxide-induced down-regulation of ER stress response system and development of neuronal cell injury. These observations could have implications for our understanding of the mechanisms underlying neuronal cell injury in pathological states of the brain associated with oxidative damage, including Alzheimer's disease where the neuronal stress response activated under conditions of ER dysfunction has been shown to be down-regulated. Down-regulation of ER stress response may increase the sensitivity of neurones to an otherwise nonlethal form of stress.

Patterns of heat-shock protein 70 biosynthesis following human traumatic brain injury.

Heat-shock protein 70 (hsp70) is activated upon cellular stress/injury and participates in the folding and intracellular transport of damaged proteins. The expression of hsp70 following CNS trauma has been speculated to be part of a cellular response which is involved in the repair of damaged proteins. In this study, we measured hsp70 mRNA and protein levels within human cerebral cortex subjected to traumatic brain injury. Specimens were obtained during routine neurosurgery for trauma and processed for Northern mRNA and Western protein analysis. The largest increase in hsp70 mRNA levels was detected in trauma tissue obtained 4-6 h following injury. By 24 h, hsp70 mRNA levels were similar to nontrauma comparison tissues. hsp70 protein expression exhibited its greatest increases at 12-20 h post-injury. Immunocytological techniques revealed hsp70 protein expression in cells with neuronal-like morphology at 12 h after injury. These results suggest a role for hsp70 in human cortex following TBI. Moreover, since the temporal induction pattern of hsp70 biosynthesis is similar to that reported in the rodent, our observations validate the importance of rodent brain injury models in providing useful information directly applicable to human brain injury.

Clonidine-Induced Heat-Shock Protein Expression in Rat Aorta.

BACKGROUND: Restraint-stress and administration of drugs that precipitate hypertension induce heat-shock protein (HSP) expression in the aorta. The exact mechanism supporting this hypertension-related HSP response is unclear because HSP induction is blocked by receptor-selective and nonselective antihypertensive agents. METHODS AND RESULTS: To identify mechanisms contributing to the pharmacological/physiological regulation of the HSP response in cardiovascular tissues, we administered clonidine to awake and freely moving animals to determine its effect on HSP expression in vivo. Inconsistent with previous work, we found that clonidine produced a dose-dependent and transient increase in HSP70 mRNA levels in the aorta. No other tissue examined displayed an HSP response after clonidine administration. Clonidine-induced HSP expression was not restricted to the HSP70 family; HSP89alpha, HSP89beta, and HSP60 were also induced. Interestingly, no heat-shock element-binding activity was observed after clonidine administration, suggesting that unusual transcriptional regulatory mechanisms mediate this response. Yohimbine and nifedipine blocked HSP70 mRNA expression, whereas isoproterenol, mecamylamine, and reserpine had no effect. CONCLUSIONS: The functional consequence of HSP expression in cardiovascular tissues may be to alter the responsiveness of cells in these tissues to subsequent drug or stress exposures, thereby implicating the HSP response as an important component of cardiovascular homeostasis. If so, treatment of mammalian organisms with drugs capable of inducting selective HSP expression in vascular tissue may alter the progression of cardiovascular disease processes.

Differential Expression of hsp70 Stress Proteins in Human Endothelial Cells Exposed to Heat Shock and Hydrogen Peroxide

The potential role of oxidative stress conditions in the induction of heat shock proteins was studied in human umbilical vein endothelial cells. We compared the effects of temperature (43 to 45 degrees C), exposure to hydrogen peroxide (H2O2) and oxygen metabolites generated by the enzyme system hypoxanthine-xanthine oxidase (O2- plus H2O2), as well as exposure to 95% O2, on the expression of the major 70-kD heat shock proteins (hsp70). Northern blot analysis indicated that: (1) heat shock induced a rapid and marked increase in hsp70 mRNA levels that reached a maximum during recovery from a 30-min exposure to 45 degrees C; (2) treatment with a 5-mM H2O2 bolus or 50 mU/ml xanthine oxidase also increased hsp70 mRNA levels but to a lesser extent than heat shock (about 10 and 25 times less, respectively); (3) no change was detected after a 5-day exposure to 95% O2. Nuclear run on transcription data and kinetics of mRNA decay in the presence of actinomycin D indicated that the observed increase in hsp70 mRNA levels in both heat-shocked and H2O2-treated cells was mainly due to a transcriptional induction. The kinetics of hsp70 synthesis correlated with the accumulation of hsp70 mRNA. Two-dimensional gel electrophoresis and immunologic analysis of these heat shock proteins revealed a series of at least five distinct hsp70 isoforms induced in heat-shocked cells, whereas only a specific subset of these proteins, mainly one acidic isoform, was induced in very low amounts in response to H2O2 treatment. These results clearly indicate that the endothelial cell responses to oxidative stress and heat shock differ in both qualitative and quantitative terms in respect to hsp70 induction. They also suggest that the intensity of this response to oxidative stress conditions may vary depending on the nature of the oxidative challenge.