Triggers of the HSP70 stress response: environmental responses and laboratory manipulation in an Antarctic marine invertebrate (Nacella concinna)

The Antarctic limpet, Nacella concinna, exhibits the classical heat shock response, with up-regulation of duplicated forms of the inducible heat shock protein 70 (HSP70) gene in response to experimental manipulation of seawater temperatures. However, this response only occurs in the laboratory at temperatures well in excess of any experienced in the field. Subsequent environmental sampling of inter-tidal animals also showed up-regulation of these genes, but at temperature thresholds much lower than those required to elicit a response in the laboratory. It was hypothesised that this was a reflection of the complexity of the stresses encountered in the inter-tidal region. Here, we describe a further series of experiments comprising both laboratory manipulation and environmental sampling of N. concinna. We investigate the expression of HSP70 gene family members (HSP70A, HSP70B, GRP78 and HSC70) in response to a further suite of environmental stressors: seasonal and experimental cold, freshwater, desiccation, chronic heat and periodic emersion. Lowered temperatures (−1.9°C and −1.6°C), generally produced a down-regulation of all HSP70 family members, with some up-regulation of HSC70 when emerging from the winter period and increasing sea temperatures. There was no significant response to freshwater immersion. In response to acute and chronic heat treatments plus simulated tidal cycles, the data showed a clear pattern. HSP70A showed a strong but very short-term response to heat whilst the duplicated HSP70B also showed heat to be a trigger, but had a more sustained response to complex stresses. GRP78 expression indicates that it was acting as a generalised stress response under the experimental conditions described here. HSC70 was the major chaperone invoked in response to long-term stresses of varying types. These results provide intriguing clues not only to the complexity of HSP70 gene expression in response to environmental change but also insights into the stress response of a non-model species.     

Lack of an HSP70 heat shock response in two Antarctic marine invertebrates

Members of the HSP70 gene family comprising the inducible (HSP70) genes and GRP78 (glucose-regulated protein 78 kDa) were identified in an Antarctic sea star (Odontaster validus) and an Antarctic gammarid (Paraceradocus gibber). These genes were surveyed for expression levels via Q-PCR after an acute 2-hour heat shock experiment in both animals and a time course assay in O. validus. No significant up-regulation was detected for any of the genes in either of the animals during the acute heat shock. The time course experiment in O. validus produced slightly different results with an initial down regulation in these genes at 2°C, but no significant up-regulation of the genes either at 2 or 6°C. Therefore, the classical heat shock response is absent in both species. The data is discussed in the context of the organisms’ thermal tolerance and the applicability of HSP70 to monitor thermal stress in Antarctic marine organisms.     

Antarctic marine molluscs do have an HSP70 heat shock response

The success of any organism depends not only on niche adaptation but also the ability to survive environmental perturbation from homeostasis, a situation generically described as stress. Although species-specific mechanisms to combat “stress” have been described, the production of heat shock proteins (HSPs), such as HSP70, is universally described across all taxa. Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) members, plus GRP78 (glucose-regulated protein, 78 kDa), a related HSP70 family member, were cloned using degenerate polymerase chain reaction (PCR) from two evolutionary divergent Antarctic marine molluscs (Laternula elliptica and Nacella concinna), a bivalve and a gastropod, respectively. The expression of the HSP70 family members was surveyed via quantitative PCR after an acute 2-h heat shock experiment. Both species demonstrated significant up-regulation of HSP70 gene expression in response to increased temperatures. However, the temperature level at which these responses were induced varied with the species (+6–8°C for L. elliptica and +8–10°C for N. concinna) compared to their natural environmental temperature). L. elliptica also showed tissue-specific expression of the genes under study. Previous work on Antarctic fish has shown that they lack the classical heat shock response, with the inducible form of HSP70 being permanently expressed with an expression not further induced under higher temperature regimes. This study shows that this is not the case for other Antarctic animals, with the two molluscs showing an inducible heat shock response, at a level probably set during their temperate evolutionary past.     

HSP70 heat shock proteins and environmental stress in Antarctic marine organisms: A mini-review

The ability to understand and predict the effects of environmental stress on biodiversity is becoming increasingly important in our changing environment. Antarctic marine species are some of the most stenothermal on the planet and many inhabit the waters off the Antarctic Peninsula which is one of the areas where there is rapid regional climate change. Therefore these animals are highly vulnerable to changing environmental temperatures and clearly we need to understand the complexities of their response, not just at the individual species level, but also the implications for the ecosystem as a whole. Heat shock proteins have a long history of use in studies of organism stress responses and have frequently been proposed as potential universal molecular biomarkers, especially for non-model species. In this mini-review, the heat shock response and heat shock proteins (specifically the HSP70 family) are examined in Antarctic marine species alongside their physiological capabilities and limits to answer a series of questions: do these animals have a heat shock response which includes the expression of HSP70 genes? What is the relationship between their heat shock response and physiological capabilities? Can HSP70 genes be used as molecular biomarkers for these species?     

The HSP70 heat shock response in the Antarctic fish <Emphasis Type="Italic">Harpagifer antarcticus</Emphasis>

Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) genes, plus GRP78 (Glucose-regulated protein 78 kDa) were surveyed for expression levels via Q-PCR after both an acute 2-h heat shock experiment and a time course assay in the Antarctic plunderfish Harpagifer antarcticus. In general, down regulation of all genes was observed during the course of the heat shock experiments. This thermally induced down regulation was particularly acute for the GRP78 gene, which at one time point was more than 100-fold down regulated. These results demonstrate the loss of the heat shock response in H. antarcticus, a basal member of the Notothenioidei. This finding is discussed with reference to the survival of Notothenioids during observed ocean warming and also the reorganisation of cellular protein mechanisms of species living in extreme environments.     

The 60-kDa Heat Shock Protein (HSP60) of the Sea Anemone <Emphasis Type="BoldItalic">Anemonia viridis:</Emphasis> A Potential Early Warning System for Environmental Changes

Expression of heat shock proteins (HSPs) is often correlated with adaptation to environmental stress. We examined the role of HSP60 (60 kDa) in acclimatization to thermal stress in the sea anemone Anemonia viridis. Using monoclonal antibodies, we identified HSP60 in sea anemones for the first time, and showed that its expression varied with changes in seawater temperature (SWT). Anemonia viridis displayed high levels of HSP60 when extreme temperatures prevailed in stressful habitats such as tidal pools. Specimens sampled from different temperature layers in the same tidal pool differed in their levels of HSP60. Specimens from subtidal zones exhibited a seasonal pattern of expression of HSP60, according to the seasonal SWT. The level of HSP60 was significantly higher in the summer (SWT, 31°C) than in other seasons throughout the year. This study suggests the use of HSP60 expression as a tool for stress detection in marine invertebrates. Received September 25, 2000; accepted February 26, 2001.     

Effects of rearing and induction temperature on the temporal dynamics of heat shock protein 70 expression in a butterfly

The temporal dynamics of heat shock protein 70 (HSP70) expression in response to longer‐term acclimation and rapid hardening in the butterfly Lycaena tityrus is investigated. After a 1‐h exposure to 1 °C or 37 °C, HSP70 is quickly up‐regulated within 1 h and down‐regulated within 2 h. The fast dynamic of HSP70 expression is in contrast to the patterns found in organisms inhabiting more stable thermal environments, and is interpreted as an adaptation to the large and rapid temperature variation experienced by flying ectotherms. HSP70 expression is higher in males than in females, as well as in animals reared at 27 °C than at 20 °C, although it is very similar across the high and low induction temperatures. Animals reared at the higher temperature, however, respond less strongly to high‐temperature stress.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein (HSP) genes (hsp70, hsc70, hsp90) were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus (Homoptera: Delphacidae), which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepi‐dopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock (40 °C) upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4°C did not change the expression levels of any hsp in either species.     

Phenotypic plasticity of HSP70 and HSP70 gene expression in the Pacific oyster (Crassostrea gigas): implications for thermal limits and induction of thermal tolerance

Pacific oysters, Crassostrea gigas, living at a range of tidal heights, routinely encounter large seasonal fluctuations in temperature. We demonstrate that the thermal limits of oysters are relatively plastic, and that these limits are correlated with changes in the expression of one family of heat-shock proteins (HSP70). Oysters were cultured in the intertidal zone, at two tidal heights, and monitored for changes in expression of cognate (HSC) and inducible (HSP) heat-shock proteins during the progression from spring through winter. We found that the "control" levels (i.e., prior to laboratory heat shock) of HSC77 and HSC72 are positively correlated with increases in ambient temperature and were significantly higher in August than in January. The elevated level of HSCs during the summer was associated with moderate, 2-3 degrees C, increases in the upper thermal limits for survival. We measured concomitant increases in the threshold temperatures (T(on)) required for induction of HSP70. Total hsp70 mRNA expression reflected the seasonal changes in the expression of inducible but not cognate members of the HSP70 family of proteins. A potential cost of increased T(on) in the summer is that there was no extension of the upper thermal limits for survival (i.e., induction of thermotolerance) after sublethal heat shock at temperatures that were sufficient to induce thermotolerance during the winter months.     

Effects of heat stress on ovarian development and the expression of HSP genes in mice

Heat stress reduces oocyte competence, thereby causing lower fertility in animals. Chronic and acute heat stresses cause extensive morphological damage in animals, but few reports have focused on the effects of chronic and acute heat stresses on ovarian function and heat shock protein (HSP) gene expression during ovarian injury. In this study, we subjected female mice to chronic and acute heat stresses; we then calculated the ovary index, examined ovary microstructure, and measured the expression of multiple HSP family genes. Chronic heat stress reduced whole-body and ovarian growth but had little effect on the ovarian index; acute heat stress did not alter whole-body or ovarian weight. Both chronic and acute heat stresses impaired ovary function by causing the dysfunction of granular cells. Small HSP genes increased rapidly after heat treatment, and members of the HSP40, HSP70, and HSP90 families were co-expressed to function in the regulation of the heat stress response. We suggest that the HSP chaperone machinery may regulate the response to heat stress in the mouse ovary.     

Effect of thermal stress on HSP70 expression in dermal fibroblast of zebu (Tharparkar) and crossbred (Karan-Fries) cattle

The present studies were conducted to investigate the difference response of dermal fibroblasts to heat stress in Tharparkar and Karan-Fries cattle. Skin is the most important environmental interface providing a protective envelope to animals. In skin, dermal fibroblasts are the most regular cell constituent of dermis that is crucial for temperature homeostasis. The study aimed to examine the reactive oxygen species (ROS) formation, cytotoxicity (%) and heat shock protein 70 (HSP70) genes expression in dermal fibroblast of Tharparkar and Karan-Fries cattle and to assess whether resistance of dermal fibroblast to heat stress is breed specific. Dermal fibroblasts from ear pinna of Tharparkar and Karan-Fries cattle were exposed at 25 °C, 37 °C, 40 °C and 44 °C for 3 h to measure the ROS, cytotoxicity (%) and HSP 70 (HSPA1A, HSPA2 and HSPA8) genes’ expression. The results showed that ROS formation at low temperature (25 °C) decreased in both breeds as compared to control (37 °C) and the differences were significant (P<0.0001). Heat stress at 40 °C did not increase ROS formation significantly in Tharparkar but increased significantly (P<0.001) in Karan-Fries cattle. The overall cytotoxicity (%) was also found to be significantly different (P<0.001) between Tharparkar and Karan-Fries cattle, and on exposure to different temperatures (P<0.001). The cytotoxicity (%) in dermal fibroblast cells of Karan-fries cows was more than Tharparkar. The expression studies indicated that all HSP70 genes (HSPA8, HSPA1A and HSPA2) were up-regulated at different temperatures in both breeds. In Tharparkar, the relative mRNA expression of HSPA8 gene was higher but HSPA1A and HSPA2 genes were low as compared to Karan-Fries cattle. At 40 and 44 °C, the relative expressions of inducible HSP 70 genes (HSPA1A and HSPA2) were higher in Karan-Fries than Tharparkar. In summary, dermal fibroblast resistance to heat shock differed between breeds. Dermal fibroblasts of Tharparkar were observed to be more heat tolerant than crossbred Karan-Fries cattle. The study concludes that zebu cattle (Tharparkar) dermal fibroblasts are more adapted to tropical climatic condition than crossbreed cattle (Karan-Fries). Differences exist in dermal fibroblasts of heat adapted and non-adapted cattle.