Transcription Of Heat Shock Proteins Research Articles

Effects of thermal stress on oxidative stress and antioxidant response, heat shock proteins expression profiles and histological changes in Marsupenaeus japonicus

Water temperature is a crucial ecological factor in aquatic environments. In the context of global warming, aquatic organisms are inevitably subject to thermal stress. The kuruma shrimp Marsupenaeus japonicus is an important commercial crustacean and has become one of the most important species in shrimp fisheries and aquaculture in China. However, M. japonicus showed relatively poor thermotolerance and frequently suffers mass mortalities during the summer months. The thermally sensitive properties of this species also make it a potential indicator species to reflect thermal alteration. Since this species raises both commercial and scientific interests, we investigated the effects of thermal stress on M. japonicus with special emphases on oxidative stress and antioxidant response, heat shock proteins expression profiles and histological changes. The results revealed that transcription of antioxidant enzymes (MjSeGpx and MjGSTMu) and heat shock proteins (Mjhsp60, Mjhsp70 and Mjhsp90) were all dramatically induced in both hepatopancreas and gill of M. japonicus under thermal stress with various expression patterns, and the up-regulated extent of the five genes exhibited a tendency of increasing with temperature. Several oxidative stress biomarkers, including MDA content, CAT activity, SOD activity and GST activity were assessed. The CAT activity was found to be more inducible than SOD and GST activities under thermal stress. In contrast to the higher basal antioxidant enzymes activities in the gill than that in the hepatopancreas, the hepatopancreas exhibited higher MDA content than that of gill. In consistent with the relatively higher MDA content, histological alterations appeared in the hepatopancreas of M. japonicus under thermal stress. This study provided comprehensive clues for uncovering the mechanisms underlying the thermal stress response and summer mortality syndrome of M. japonicus and evaluating the bioindicator potential of this species.

Rapid cold hardening protects against sublethal freezing injury in an Antarctic insect.

Rapid cold hardening (RCH) is a type of beneficial phenotypic plasticity that occurs on extremely short time scales (minutes to hours) to enhance insects' ability to cope with cold snaps and diurnal temperature fluctuations. RCH has a well-established role in extending lower lethal limits, but its ability to prevent sublethal cold injury has received less attention. The Antarctic midge, Belgica antarctica, is Antarctica's only endemic insect and has a well-studied RCH response that extends freeze tolerance in laboratory conditions. However, the discriminating temperatures used in previous studies of RCH are far below those ever experienced in the field. Here, we tested the hypothesis that RCH protects against non-lethal freezing injury. Larvae of B. antarctica were exposed to control (2°C), direct freezing (-9°C for 24 h) or RCH (-5°C for 2 h followed by -9°C for 24 h). All larvae survived both freezing treatments, but RCH larvae recovered more quickly from freezing stress and had a significantly higher metabolic rate during recovery. RCH larvae also sustained less damage to fat body and midgut tissue and had lower expression of two heat shock protein transcripts (hsp60 and hsp90), which is consistent with RCH protecting against protein denaturation. The protection afforded by RCH resulted in energy savings; directly frozen larvae experienced a significant depletion in glycogen energy stores that was not observed in RCH larvae. Together, these results provide strong evidence that RCH protects against a variety of sublethal freezing injuries and allows insects to rapidly fine-tune their performance in thermally variable environments.

Evaluation of physiological stress and nutritional deficiency related to cannibalism in early paralarvae of Patagonian red octopus Enteroctopus megalocyathus

Patagonian red octopus Enteroctopus megalocyathus is one of the two octopuses' species of great commercial importance in Chile. At the present time, the life cycle of this species in captivity has been closed at experimental level; however, high mortalities at the early paralarvae stage are a limiting factor for attaining sustainable production. Cannibalism has been identified as one of the main mortality causes at the early paralarvae rearing. Stress has been proposed as an important cause of cannibalism in cephalopods. Likewise, nutritional deficiency has been suggested as a potential cause to onset cannibalism. However, neither of both factors has been directly assessed in cephalopod paralarvae. In the present study, we aimed to elucidate if physiological stress due to a food shortage and/or to a high rearing density of paralarvae trigger cannibalism in E. megalocyathus. An experiment was carried out with newly hatched paralarvae, under conditions that trigger cannibalism with densities of 10 and 15 paralarvae L−1 combined with two measures of prey availability: 0 and 2 prey paralarva−1 day−1. Paralarvae alone in the aquarium fed ad libitum was used as a control to avoid cannibalism. Nutritional status was assessed as the total protein and energy content per paralarvae; and the 70 kD heat shock protein (HSP70) transcript level was used as a bioindicator of physiological stress, documented as a suitable stress biomarker for cephalopods. No differences were observed between the treatments and control in the absolute protein levels and energy content. There was a strong induction in HSP70 transcriptional level when high density and prey availability where combined, proving that there are only a few paralarvae rearing combinations that cause critical stress and trigger HSP70 transcription. In experimental conditions, nutritional status indicators showed that early paralarvae, with similar reserves of protein and energy, exhibited cannibalism. Results suggest that mortality as well as cannibalism is associated with competition for prey and with nutritional and energy imbalance due to food shortage that determines the expression of the related stress gene.

Inhibition of the ubiquitination of HSF1 by FBXW7 protects the intestine against ischemia-reperfusion injury.

Epithelial apoptosis is an important factor in intestinal ischemia-reperfusion (I/R) injury. Heat shock factor 1 (HSF1) is a classical stress response factor that directly regulates the transcription of heat shock proteins (HSPs) under stress conditions. Although HSPs are involved in protecting the intestine against I/R, the mechanism whereby HSF1 is regulated in I/R is poorly understood. Here, we show that the ubiquitin ligase FBXW7 targets HSF1 for ubiquitination and degradation in intestinal I/R. In this study, we found that FBXW7 expression was upregulated at the transcriptional level in intestinal mucosae subjected to I/R. In Caco-2 and IEC-6 cells subjected to hypoxia/reoxygenation (H/R), a high FBXW7 level led to excessive HSF1 ubiquitination and degradation. FBXW7 knockdown attenuated HSF1 ubiquitination and downregulation and accelerated HSPB1 and HSP70 expression. In addition, FBXW7 deletion alleviated the apoptosis of intestinal epithelial cells, as evidenced by decreased activation of caspase-3 and caspase-9. The results suggest that FBXW7 suppression protects against intestinal I/R, at least partly through the HSF1/HSP pathway. These findings indicate that FBXW7 may be a potential therapeutic target for inhibiting intestinal mucosa apoptosis during intestinal I/R.

Modulation of heat-shock response is associated with Di (2-ethylhexyl) phthalate (DEHP)-induced cardiotoxicity in quail (Coturnix japonica)

Di(2-ethylhexyl) phthalate (DEHP) is an omnipresent environmental pollutant with endocrine disrupting properties. As a plasticizer, DEHP can be leach from the plastic to transfer the external environment and thus enters the animal food chain, causing serious damage to the animal organs. The heat-shock response (HSR) comprising heat-shock protein (HSPs) and heat-shock transcription factor (HSFs) plays a pivotal role in various toxic stress conditions. For the sake of investigating the effects of DEHP exposure on cardiac toxicity and the regulation of HSR, male quail were fed the diet with 0, 250, 500 and 750 mg/kg DEHP by gavage administration for 45 days. Histopathological changes including cardiomyocyte swelling and muscle fiber dilatation were observed in the hearts exposed to DEHP. During the DEHP treatment, the mRNA expression of HSP60 and HSP70 were universally reduced, while the expression of other HSPs (HSP10, HSP25, HSP27, HSP40, HSP47, HSP90, HSP110) had different degrees of growth. In addition, the levels of HSF1, HSF2, and HSF3 were significantly increased. Given the facts above, DEHP exposure induced the toxic effects of quail heart. DEHP exposure did great harm to HSR via affecting the synthesis of HSFs to mediate the transcription of the HSPs. Ultimately, this study provided new evidence that DEHP-induced cardiotoxicity in quail was related to activation of HSR and playing a protective role.

Expression profiles of 14 small heat shock protein (sHSP) transcripts during larval diapause and under thermal stress in the spruce budworm, Choristoneura fumiferana (L.).

Diapause is an important strategy for certain insect species to survive unfavorable environmental conditions, including low temperatures experienced when they overwinter in cold climate. Many studies have indicated that the increased expression of heat shock proteins during diapause improves the thermal tolerance of insects. However, the relationship between small heat shock proteins (sHSPs) and diapause is not clear or well-researched. In this study, we investigated the transcript levels of 14 sHSP genes in the spruce budworm, Choristoneura fumiferana, a major pest of spruce and fir in Canada, during pre-diapause, diapause, and post-diapause under normal rearing conditions and in response to a heat shock treatment. We found that sHSP expression profiles could be classified into five patterns under normal laboratory conditions: pattern I was upregulated only during pre-diapause, pattern II was upregulated only during diapause, pattern III was constantly expressed throughout diapause, pattern IV was upregulated in both pre-diapause and diapause, and pattern V was upregulated only during post-diapause. After heat shock, five different expression patterns were observed: pattern I responded weakly or not at all throughout diapause, pattern II responded weakly during the diapause stage but strongly at the onset of diapause and in the post-diapause period, pattern III was upregulated only during post-diapause, pattern IV was strongest during diapause, and pattern V was strongest only in early diapause. These complex expression profiles lead us to suggest that most of the sHSP genes are involved in the diapause process and that they may have multiple and important roles in different phases of this process.

Heat loss efficiency and HSPs gene expression of Nellore cows in tropical climate conditions.

Adaptation is a relevant characteristic to be understood in livestock animals in order to maintain and raise productivity. In Brazil, the Nellore beef cattle are widely disseminated and well-adapted breed that present good thermoregulatory characteristics for tropical environment conditions. Conversely, the physiological and cellular mechanisms required for thermoregulation and thermotolerance in this breed are still limited. The aim of this study was to comprehend the heat loss efficiency at the whole animal level and heat shock response at the cellular level of Nellore cows in tropical climate conditions. Healthy purebred Nellore cows were classified according to their capacity to lose body heat as Efficient or Inefficient based on vaginal temperature which was continuously monitored by data-loggers. Rectal, tail, and ocular temperatures, sweating rate, and respiratory frequency were collected to assess other thermoregulatory responses. Peripheral mononuclear cells were used for gene expression of heat shock proteins 60, 70, and 90 induced by in vitro heat treatments at 38, 40, and 42°C. In our findings, the Efficient cows presented higher sweating rates compared to Inefficient cows that presented higher rectal temperature with greater amplitude of vaginal temperature profile. Transcription of the HSP genes was stable at 38 and 40°C and decreased for all HSP genes at 42°C. In conclusion, the Nellore efficiency to lose heat was mainly associated with their sweating capacity and cellular thermotolerance confirmed by the maintenance of heat shock proteins transcripts under heat stress. Taken together, this knowledge contributes as a future key for genetic selection of adapted animals.

Effects of temperature shifts on life parameters and expression of fatty acid synthesis and heat shock protein genes in temperate and Antarctic copepods Tigriopus japonicus and Tigriopus kingsejongensis

Global warming, increase of the atmospheric temperature leading to changes in climate, is a growing environmental concern for all organisms including marine organisms, and many efforts have been made to reveal the potential adverse effects on the systematics of aquatic organisms in response to the temperature changes. To examine the effects of temperature shifts on copepods in temperate and polar regions, we compared the life parameters and gene expression profiles of the de novo lipogenesis (DNL) pathway and heat shock protein (hsp) genes in the temperate copepod Tigriopus japonicus (T. japonicus) and the Antarctic copepod Tigriopus kingsejongensis (T. kingsejongensis). The median lethal temperature (LT50) and no observed effect level (NOEL) in the temperate copepod T. japonicus were determined to be 35.3 and 32 °C, respectively, in response to a temperature increase of 2 °C a day. In the Antarctic copepod T. kingsejongensis, the LT50 and NOEL were determined to be 24.8 and 12 °C, respectively. In addition, delayed developmental time and impaired fecundity were observed (P < 0.05) in response to temperature changes in T. japonicus. T. japonicus DNL pathway genes were down-regulated in response to high temperature, whereas T. kingsejongensis DNL pathway genes showed up-regulation in response to high temperature, indicating that these two Tigriopus species have different modes of action in response to temperature shifts. In both copepods, transcription of heat shock proteins (hsps) was mostly up-regulated in response to temperature shifts, but it showed moderate expression at 15 °C for T. japonicus and 4 °C for T. kingsejongensis. These findings indicate temperature shift-mediated species-specific modulations of the DNL pathway and hsps gene expression, leading to alteration of lipid synthesis and chaperoning with deleterious effects on the life parameters of these two copepods.

Kale supplementation up-regulates HSP70 and suppresses cognitive decline in a mouse model of accelerated senescence

Dietary interventions have been proposed as an effective way to prevent age-related senescence. Here, we investigated the ability of kale juice (KJ) dietary supplementation to delay cognitive decline in the senescence-accelerated mouse prone 8 (SAMP8) mouse. SAMP8 mice were fed a diet containing 0.8% (w/w) KJ or a control diet for 16 weeks, and cognitive performance was examined using the Morris water maze. KJ administration improved spatial and leaning memory as well as suppressed levels of serum 8-hydroxy-2′-deoxyguanosine and brain malondialdehyde with respect to the control group. Furthermore, the KJ-fed group showed a significant increase in the gene transcription and protein expression of heat shock protein 70 (HSP70). 1-Sinapoyl-2-feruloyl gentiobiose was identified as one of the active components of KJ affecting HSP70 expression. These findings suggest that dietary kale supplementation can suppress cognitive decline and age-related oxidative damage through the activation of HSP70 in SAMP8 mice.

STABILIZED1 as a heat stress-specific splicing factor in Arabidopsis thaliana

ABSTRACTTo overcome high temperature stress, plants have developed transcriptional cascades which express a large amount of chaperone proteins called heat shock proteins (HSPs). In our recent publication, we reported that STABILIZED1, as an U5-snRNP-interacting protein, is involved in the splicing of heat shock factor (HSF) and HSP transcripts during high temperature stress. This indicates that not only transcriptional regulation, but also post-transcriptional regulation by STA1, is essential for the full activation of HSF-HSP cascades and for thermotolerance. Here, we observed that the splicing of HSP transcripts was induced independent of STA1 at room temperature after heat acclimation, indicating that STA1 acts as a high temperature-specific splicing factor for the splicing of HSP transcripts. Our findings suggest the molecular mechanism for how HSF and HSP transcripts are spliced well under high temperature stress that blocks the splicing of overall transcripts.

Changes in activity and transcription of antioxidant enzymes and heat shock protein 90 in the water flea, Daphnia magna - exposed to mercury

Mercury (Hg) has adverse effects in aquatic organisms but mechanisms underlying the effects of Hg in aquatic invertebrates are unclear. To investigate the oxidative stress response in the water flea Daphnia magna to Hg, intracellular reactive oxygen species (ROS) level, glutathione (GSH) content, and glutathione S-transferase (GST) and superoxide dismutase (SOD) activities were measured. Subsequently, transcriptional profiles of antioxidant enzyme-related genes (three GST isoforms, glutaredoxin, glutathione peroxidase and thioredoxin) and stress protein (HSP90) were examined at different developmental stages, concentrations and exposure times. The intracellular ROS level decreased at 48 h, whereas the total GSH content was greatly increased at 48 h. A significant increase in GST and SOD activities was also observed at 48 h. All the antioxidant-related genes were upregulated in juveniles more than neonates at 24 h exposure to Hg (0.08 μg/L), and their expression pattern was differently modulated according to the developmental stage at 48 h. The expression of HSP90 mRNA was observed at 24 h exposure to Hg in neonate (0.08-2 μg/L) and in juvenile (2 μg/L). These findings imply that these antioxidant enzymes and stress protein may be involved in cellular protection to Hg-induced toxicity. This study would be helpful in understanding the molecular mode of action of Hg in D. magna.

Targeted Deletion of Hsf1, 2, and 4 Genes in Mice.

Heat shock transcription factors (Hsfs) regulate transcription of heat shock proteins as well as other genes whose promoters contain heat shock elements (HSEs). There are at least five Hsfs in mammalian cells, Hsf1, Hsf2, Hsf3, Hsf4, and Hsfy (Wu, Annu Rev Cell Dev Biol 11:441-469, 1995; Morimoto, Genes Dev 12:3788-3796, 1998; Tessari et al., Mol Hum Repord 4:253-258, 2004; Fujimoto et al., Mol Biol Cell 21:106-116, 2010; Nakai et al., Mol Cell Biol 17:469-481, 1997; Sarge et al., Genes Dev 5:1902-1911, 1991). To understand the physiological roles of Hsf1, Hsf2, and Hsf4 in vivo, we generated knockout mouse lines for these factors (Zhang et al., J Cell Biochem 86:376-393, 2002; Wang et al., Genesis 36:48-61, 2003; Min et al., Genesis 40:205-217, 2004). Numbers of other laboratories have also generated Hsf1 (Xiao et al., EMBO J 18:5943-5952, 1999; Sugahara et al., Hear Res 182:88-96, 2003), Hsf2 (McMillan et al., Mol Cell Biol 22:8005-8014, 2002; Kallio et al., EMBO J 21:2591-2601, 2002), and Hsf4 (Fujimoto et al., EMBO J 23:4297-4306, 2004) knockout mouse models. In this chapter, we describe the design of the targeting vectors, the plasmids used, and the successful generation of mice lacking the individual genes. We also briefly describe what we have learned about the physiological functions of these genes in vivo.

Heat shock factor 4 regulates the expression of HSP25 and alpha B-crystallin by associating with DEXD/H-box RNA helicase UAP56.

Heat shock factor 4 controls the transcription of small heat shock proteins (e.g., HSP25, alpha B-cyrstallin, and r-crystallin), that play important roles in modulating lens proteostasis. However, the molecular mechanism underlying HSF4-mediated transcription is still unclear. Using yeast two hybrid, we found that HSF4 interacts with the ATP-dependent DEXD/H-box RNA helicase UAP56, and their interaction in lens epithelial cell line was further confirmed by GST-pull down assay. UAP56 is a vital regulator of pre-mRNA splicing and mature mRNA nuclear export. The immunofluorescence assay showed that HSF4 and UBA56 co-localize with each other in the nucleus of lens epithelial cells. Ectopic UAP56 upregulated HSF4-controlled HSP25 and alpha B-crystallin proteins expression, while knocking down UAP56 by shRNA reversed it. Moreover, UAP56 interacts with and facilitates the nuclear exportation of HSP25 and alpha B-crystallin mRNA without impacting their total mRNA expression level. In lens tissues, both UAP56 and HSF4 are expressed in the same nucleus of lens fiber cells, and their expression levels are simultaneously reduced with fiber cell maturation. Taken together, these data suggested that UAP56 is a novel regulator of HSF4 and might upregulate HSF4's downstream mRNA maturation and nuclear exportation.

Abstract TMEM-034: THE HEAT SHOCK TRANSCRIPTION FACTOR HSF1 INDUCES OVARIAN CANCER EPITHELIAL-MESENCHYMAL TRANSITION IN A 3D SPHEROID GROWTH MODEL

Abstract Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. These poor statistics are related to a lack of early symptoms and inadequate screening techniques. This results in the cancer going undetected until later stages when the tumor has metastasized through a process that requires the epithelial to mesenchymal transition (EMT). In lieu of traditional monolayer cell culture, EMT and cancer progression in general is best characterized through the use of 3D spheroid models. Compared to other cancers, ovarian cancer has very limited treatment options to date, with cisplatin and paclitaxel being the most common and efficient. Recently, a significant increase in heat shock factor 1 (HSF1) expression has been found in highly malignant ovarian cancer. HSF1 is the regulator of the heat shock response (HSR) and promotes the transcription of heat shock proteins (HSPs). Previous studies have shown that HSF1-induced chaperone activity may act to stabilize multiple oncogenes within many different cancer types. This leads to cancer proliferation, reduced apoptosis, and metastasis. In this study, we examine gene expression changes through microarray analysis in spheroid versus monolayer ovarian cancer cells treated with TGFβ to induce EMT. Interestingly, EMT and stress response pathways, including the heat shock response, were all significantly affected by 3D growth. HSF1 knockdown was utilized in both 2D and 3D cultures to further study this response. HSF1 knockdown is shown to inhibit ovarian cancer progression in both monolayer and spheroid model cells. Moreover, HSF1 knockdown in spheroid cells has a significantly greater impact on EMT markers than it does in 2D cultures. This indicates HSF1 may be a utilized as a target for chemotherapeutics in ovarian cancer patients and could potentially have implications in other such hormonal cancers. Citation Format: Trillitye Paullin, Chase Powell, Chris Menzie, Robert Hill, Christopher Martyniuk, and Sandy D. Westerheide. THE HEAT SHOCK TRANSCRIPTION FACTOR HSF1 INDUCES OVARIAN CANCER EPITHELIAL-MESENCHYMAL TRANSITION IN A 3D SPHEROID GROWTH MODEL [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-034.

Neurotoxic effects, molecular responses and oxidative stress biomarkers in Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) exposed to verapamil

Pharmaceutical drugs and their metabolites are detected in aquatic ecosystems and have been reported to cause ecotoxicological consequences to resident aquatic organisms. The study investigated the effects of acute and long-term exposure to verapamil on activities of acetylcholinesterase and antioxidant enzymes as well as mRNA expression of stress-related genes in brain and muscle tissues of Nile tilapia, Oreochromis niloticus. The 96h LC50 of verapamil to O. niloticus was 2.29mgL−1. Exposure to sub-lethal concentrations of verapamil (0.14, 0.29 and 0.57mgL−1) for period of 15, 30, 45 and 60days, led to inhibition of acetylcholinesterase activities in the brain and muscle of the fish. The activities of the oxidative enzymes such as the catalase, superoxide dismutase and glutathione peroxidase were also inhibited in both the tissues while there was an increase in the activities of glutathione–S-transferase and reduced glutathione in the muscle after 15 days at 0.29mgL−1. Lipid peroxidation and carbonyl protein showed elevated level, indicating a positive correlation with both time and concentration. The activities of energy-related biomarker (Na+-K+-ATPase) in both the tissues were significantly inhibited (p<0.05) compared with the control. Transcription of catalase (cat), superoxide dismutase (sod) and heat shock proteins 70 (hsp70) were up-regulated in both the tissues after the study period. Prolonged exposure to sub-lethal verapamil can result in oxidative stress, up-regulation of stress-related genes and neurotoxicity in O. niloticus.

Higher dietary protein increases growth performance, anti-oxidative enzymes activity and transcription of heat shock protein 70 in the juvenile sea urchin (Strongylocentrotus intermedius) under a heat stress

This study was conducted to investigate the effect of dietary protein concentration (12%, 18%, 24%, 30% and 36%) on the growth performance, activity of anti-oxidative enzymes and heat shock protein 70 (HSP70) transcription in the sea urchin (Strongylocentrotus intermedius) under a heat stress. After 112 days of feeding trial the sea urchins were heat stressed (26 °C) and the coelomic fluid and intestine sampled at time 0 and 15 min, 2 h and 6 h. The results showed that an increase in dietary protein (12%–24%), significantly increased (p < 0.05) the sea urchin weight gain rate (WGR). As dietary protein increased (from 18% to 36%), the gonadosomatic index (GI) of juvenile sea urchins also significantly increased (p < 0.05) from 18.0% to 22.6%. Superoxide dismutase (SOD) activity increased with dietary protein increase (12%–30%) and the enzyme activity was significantly higher (p < 0.05) in the coelomic fluid of sea urchins that were fed with 30% protein diets when compared to 12% and 36% protein diets at all time points after the heat stress. Catalase (CAT) activity showed a similar tendency with the increase in dietary protein concentration at time 0 and 15 min after the heat stress (p < 0.05). Transcription of HSP70 in the intestine also showed a similar trend to SOD and was highest in the animals that were fed with 30% protein diets (p < 0.05). Our results suggest that 24% protein diets could meet the requirements for growth performance but a 30% protein diet resulted in improved gonad development and anti-heat stress capacity in this sea urchin species.

Enhanced stress responses and metabolic adjustments linked to diapause and onset of migration in the large milkweed bug Oncopeltus fasciatus

AbstractIn response to short‐day photoperiods in the autumn, the large milkweed bug Oncopeltus fasciatus Dallas enters a reproductive diapause and migrates south to avoid the adverse environmental conditions and food shortages that prevail in the winter. Milkweed bugs are one of only a few temperate insects that undergo long distance migration during diapause, making them a good model for investigating trade‐offs associated with migratory diapause. Although enhanced stress tolerance is typical of diapause, it is unclear whether this aspect of diapause would be retained in a species that migrates to more favourable conditions. The present study tests (i) whether diapause enhances thermal tolerance; (ii) whether food shortage, which is required for maximal expression of diapause, influences thermal tolerance during diapause; and (iii) whether the potential changes in stress tolerance are associated with upregulated heat shock protein expression or metabolic adjustments (including cryoprotectant synthesis), or both. Both cold tolerance at −10 °C and heat tolerance at 43 °C are significantly higher in diapausing O. fasciatus, whereas food restriction has no further effect on thermal tolerance. None of the heat shock protein transcripts measured are significantly upregulated in response to diapause, and the experiments also fail to detect any cryoprotectant accumulation during diapause. Thus, although heat shock proteins and cryoprotectants are common mechanisms for enhancing thermal tolerance in many diapausing insects, the results of the present study suggest that alternative mechanisms are responsible in milkweed bugs.

Cytosolic calcium transients are a determinant of contraction-induced HSP72 transcription in single skeletal muscle fibers.

The intrinsic activating factors that induce transcription of heat shock protein 72 (HSP72) in skeletal muscle following exercise remain unclear. We hypothesized that the cytosolic Ca(2+) transient that occurs with depolarization is a determinant. We utilized intact, single skeletal muscle fibers from Xenopus laevis to test the role of the cytosolic Ca(2+) transient and several other exercise-related factors (fatigue, hypoxia, AMP kinase, and cross-bridge cycling) on the activation of HSP72 transcription. HSP72 and HSP60 mRNA levels were assessed with real-time quantitative PCR; cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was assessed with fura-2. Both fatiguing and nonfatiguing contractions resulted in a significant increase in HSP72 mRNA. As expected, peak [Ca(2+)]cyt remained tightly coupled with peak developed tension in contracting fibers. Pretreatment with N-benzyl-p-toluene sulfonamide (BTS) resulted in depressed peak developed tension with stimulation, while peak [Ca(2+)]cyt remained largely unchanged from control values. Despite excitation-contraction uncoupling, BTS-treated fibers displayed a significant increase in HSP72 mRNA. Treatment of fibers with hypoxia (Po2: <3 mmHg) or AMP kinase activation had no effect on HSP72 mRNA levels. These results suggest that the intermittent cytosolic Ca(2+) transient that occurs with skeletal muscle depolarization provides a sufficient activating stimulus for HSP72 transcription. Metabolic or mechanical factors associated with fatigue development and cross-bridge cycling likely play a more limited role.

Abstract A41: The heat shock transcription factor HSF1 induces ovarian cancer epithelial-mesenchymal transition in a 3-D spheroid growth model.

Abstract Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. These poor statistics are related to a lack of early symptoms and inadequate screening techniques. This results in the cancer going undetected until later stages when the tumor has metastasized through a process that requires the epithelial to mesenchymal transition (EMT). In lieu of traditional monolayer cell culture, EMT and cancer progression in general is best characterized through the use of 3D spheroid models. Compared to other cancers, ovarian cancer has very limited treatment options to date, with cisplatin and paclitaxel being the most common and efficient. Recently, a significant increase in heat shock factor 1 (HSF1) expression has been found in highly malignant ovarian cancer. HSF1 is the regulator of the heat shock response (HSR) and promotes the transcription of heat shock proteins (HSPs). Previous studies have shown that HSF1-induced chaperone activity may act to stabilize multiple oncogenes within many different cancer types. This leads to cancer proliferation, reduced apoptosis, and metastasis. In this study, we examine gene expression changes through microarray analysis in spheroid versus monolayer ovarian cancer cells treated with TGFβ to induce EMT. Interestingly, EMT and stress response pathways, including the heat shock response, were all significantly affected by 3D growth. HSF1 knockdown was utilized in both 2D and 3D cultures to further study this response. HSF1 knockdown is shown to inhibit ovarian cancer progression in both monolayer and spheroid model cells. Moreover, HSF1 knockdown in spheroid cells has a significantly greater impact on EMT markers than it does in 2D cultures. This indicates HSF1 may be a utilized as a target for chemotherapeutics in ovarian cancer patients and could potentially have implications in other such hormonal cancers. Citation Format: Trillitye Paullin, Chase Powell, Menzie Christopher, Robert Hill, Christopher Martyniuk, Sandy Westerheide. The heat shock transcription factor HSF1 induces ovarian cancer epithelial-mesenchymal transition in a 3-D spheroid growth model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A41.

Interaction of Heat Shock Protein Cpn10 with the Cyclin E/Cdk2 Substrate Nuclear Protein Ataxia-Telangiectasia (NPAT) Is Involved in Regulating Histone Transcription

Precise modulation of histone gene transcription is critical for cell cycle progression. As a direct substrate of Cyclin E/CDK2, nuclear protein ataxia-telangiectasia (NPAT) is a crucial factor in regulating histone transcription and cell cycle progression. Here we identified that Cpn10/HSPE, a 10-kDa heat shock protein, is a novel interacting partner of NPAT. A pool of Cpn10 is colocalized with NPAT foci during G1 and S phases in nuclei. Gain- and loss-of-function experiments unraveled an essential role of Cpn10 in histone transcription. A conserved DLFD motif within Cpn10 was critical for targeting NPAT and modulating histone transcription. More importantly, knockdown of Cpn10 disrupted the focus formation of both NPAT and FADD-like interleukin-1β-converting enzyme-associated huge protein without affecting Coilin-positive Cajal bodies. Finally, Cpn10 is important for S phase progression and cell proliferation. Taken together, our finding revealed a novel role of Cpn10 in the spatial regulation of NPAT signaling and disclosed a previously unappreciated link between the heat shock protein and histone transcription regulation.