Heat Shock Protein 70 Protein Levels Research Articles

N-butyrate increases heat shock protein 70 through heat shock factor 1 and AMP-activated protein kinase pathways in human intestinal Caco-2 cells

Impaired integrity of the intestinal epithelium is a cause of intestinal and extraintestinal diseases. Heat shock protein 70 (HSP70), a cytoprotective protein, plays an important role in maintaining intestinal homeostasis. The intestinal expression of HSP70 is linked with the local microbiota. The present study investigated the molecular mechanisms underlying the upregulation of HSP70 by n-butyrate, a major metabolite of the intestinal microbiota in human intestinal Caco-2 cells. Treatment of Caco-2 cells with n-butyrate upregulated HSP70 protein and mRNA levels in a dose-dependent manner. Using luciferase reporter assay, it was found that n-butyrate enhanced the transcriptional activity of HSP70. These effects were sensitive to the inhibition of heat shock factor 1 (HSF1), a transcription factor, and AMP-activated protein kinase (AMPK). N-butyrate increased the phosphorylation (activity) of HSF1 and AMPK. Taken together, this study shows that n-butyrate is partly involved in the microbiota-dependent intestinal expression of HSP70, and the effect is exerted through the HSF1 and AMPK pathways.

Inducible Heat Shock Protein 70 Levels in Patients and the mdx Mouse Affirm Regulation during Skeletal Muscle Regeneration in Muscular Dystrophy.

Stress-inducible heat shock protein 70 (HSP70) is both a protective chaperone involved in protein homeostasis and an immune regulator. In both capacities, HSP70 has been implicated in muscle disorders, yet with fragmented and differing results. In this study we aimed to compare results obtained in the mouse model for the severest form of muscular dystrophy (MD) equivalent to Duchenne MD, termed the mdx mouse, with results obtained in human MD. Skeletal muscle and serum samples were obtained from 11 healthy controls, 11 fully characterized patients diagnosed with Becker MD and limb girdle MD (LGMD), and six muscle disease controls. In addition, muscle extracts were prepared from tibialis anterior of mdx and control mice at ages 4, 8 and 12 weeks. The HSP70 levels were quantified using RT-PCR, western blotting and protein arrays, and localized in muscle tissue sections using double immunofluorescence. We found selective and significant 2.2-fold upregulation of HSP70 protein in mdx tibialis muscle at the earliest disease phase only. In LGMD and Becker MD patients, HSP70 protein levels were not significantly different from those of healthy muscle and serum. HSP70 was localized to regenerating muscle fibers both in mouse and human MD skeletal muscle tissues. Toll-like receptor (TLR) 2 and TLR4 expression was moderately increased on the sarcolemma in MD muscle, yet protein levels were not significantly different from normal controls. HSP70 upregulation in MD appears disease stage-dependent, marking the phase of most active muscle regeneration in the mdx mouse. We postulate that well-timed supportive therapeutic interventions with HSP70 agonists could potentially improve muscle tissue's regenerative capacities in MD, attenuating loss of muscle mass while we await gene therapies to become more widely available.

Effect of water temperature on walleye pollock (Gadus chalcogrammus) embryos, larvae and juveniles: Survival, HSP70 expression, and physiological responses

This study investigated the survival rates, physiological responses, and the induction of heat shock protein 70 (HSP70) expression in size-specific walleye pollock (Gadus chalcogrammus) at different water temperatures. Four groups consisting of walleye pollocks in various lifecycle stages—embryos, larvae, small juveniles, and large juveniles—were used. Embryos had the highest hatching rate at 5 °C, which decreased as the water temperature increased. Larval survival rate was the highest at 5 °C. The median lethal temperatures (LT50) for small and large juveniles were 19.1 and 18.9 °C for 7 d, respectively. HSP70 was cloned using the pET21b + vector and expressed in Escherichia coli to analyze HSP70 expression in walleye pollock. Polyclonal antibodies were obtained from BALB/c mice after purification on a His-Trap HP column and used for protein analysis using enzyme-linked immunosorbent assay. HSP70 mRNA and proteins were expressed in similar patterns at both 5 °C and 8 °C, with a drastic increase in HSP70 expression induction at ≥11 °C. The gill tissues of small and large juveniles had the highest HSP70 mRNA and protein levels. Physiological responses of large juveniles' plasma showed that glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and glucose levels were highest at 14 °C, whereas total cholesterol and triglyceride levels were lowest. Plasma superoxide dismutase and cortisol levels were highest at 14 °C, whereas growth hormone and insulin-like growth factor-1 levels were highest at 8 °C. These results suggest that elevated water temperatures increased stress in the early lifecycle stages (embryos and larvae) of walleye pollock. HSP70 and stress-related indicator levels also increased with an increase in water temperature. Subsequently, the optimal water temperature for walleye pollock growth was determined to be below 8 °C.

The abnormal level of HSP70 is related to Treg/Th17 imbalance in PCOS patients

BackgroundPolycystic ovary syndrome (PCOS) is a disease with chronic nonspecific low-grade inflammation. The imbalance of immune cells exists in PCOS. Several studies have found that heat shock protein 70 (HSP70) may be involved in the immunological pathogenesis of PCOS, but the relationship between HSP70 and Regulatory T cell (Treg)/T helper cell 17(Th17) ratio remains unclear. This study aims to explore the correlation between HSP70 and Treg/Th17 ratio and to provide evidence for the role of HSP70 in the immunological etiology of PCOS.ResultsThere was no significant difference in age and body mass index (BMI) between the two groups. The concentrations of basal estradiol (E2), basal follicle-stimulating hormone (FSH) did not show a significant difference between the two groups. The concentrations of basal luteinizing hormone (LH) (P < 0.01), testosterone (T) (P < 0.01), glucose (P < 0.001) and insulin (P < 0.001) in PCOS patients were significantly higher than those in the control group. The protein levels of HSP70 were significantly higher in serum in the PCOS group (P < 0.001). The percentage of Treg cells was significantly lower (P < 0.01), while the percentage of the Th17 cells of the PCOS group was significantly higher than that of the control group (P < 0.05). The ratio of Treg/Th17 in the PCOS group was significantly lower (P < 0.001). The concentrations of Interleukin (IL)-6, IL-17, and IL-23 were significantly higher, while the levels of IL-10 and Transforming growth factor-β (TGF-β) were significantly lower in the PCOS group (P < 0.001). Spearman rank correlation analysis showed a strong negative correlation of serum HSP70 levels with Treg/Th17 ratio, IL-10, and TGF-β levels. In contrast, HSP70 levels were significantly positively correlated with IL-6, IL-17, IL-23, LH, insulin, and glucose levels.ConclusionThe abnormal level of HSP70 is correlated with Treg/Th17 imbalance and corresponding cytokines, which indicates that HSP70 may play an important role in PCOS immunologic pathogenesis.

Effects of transport stress on pathological injury and expression of main heat shock proteins in the caprine stomach

BackgroundTransportation is necessary to introduce new breeds of goats to the farm and move the adult meat goat from the farm to the slaughterhouse. However, these actions may give rise to transport stress. Heat shock proteins (HSPs) are playing some important regulate roles during transport stress. The aim of this study was to evaluate the effects of transport stress on the pathological injury and HSPs expression in the stomach of goats. A total of three batches of Ganxi goats from western Jiangxi province were enrolled in this study. For each batch, twelve healthy adult male goats were randomly divided into three groups (four goats per batch and per group): Control group, stress group transported during 2 h and stress group transported during 6 h.ResultsOur results showed that the different degrees of stomach walls damage, with the change of expression levels of heat shock protein 27 (HSP27), heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90), occurred after goats transportation. In rumen, the mRNA and protein expressions of HSP27 and HSP70 were increased after transport stress, but not HSP90. In reticulum, all three HSPs mRNA and protein levels were upregulated after 2 h transport, but decreased after 6 h transport. In omasum, HSP27 and HSP70 mRNA and protein were increased after transport stress, however, HSP90 mRNA level only had a slightly enhancement after transport stress. In abomasum, HSP70 and HSP90 mRNA and protein levels were increased after transport stress, but HSP27 was decreased after transport stress.ConclusionsTaken together, these results revealed that the pathological changes in the gastric tissues and the stomach HSPs expression in goats are related to transport stress and duration. Moreover, this study also provides some new data to advocate reducing transport stress of goats and improving animal welfare.

Triptolide inhibits migration and proliferation of fibroblasts from ileocolonic anastomosis of patients with Crohn's disease via regulating the miR‑16‑1/HSP70 pathway.

Anastomotic fibrosis is highly likely to lead to reoperation in Crohn's disease (CD) patients. Triptolide (TPL) is considered to have anti-inflammatory and antifibrotic effects in a variety of autoimmune diseases, including CD. The present study aimed to investigate the effects of TPL on fibroblasts from strictured ileocolonic anastomosis of patients with CD and its underlying mechanism. Primary fibroblasts were obtained from strictured anastomosis tissue (SAT) samples and matched anastomosis-adjacent normal tissue (NT) samples which were collected from 10 CD patients who underwent reoperation because of anastomotic stricture. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure miR-16-1 and heat shock protein 70 (HSP70) levels. Western blotting was conducted to determine expression of HSP70, collagen I (Col-I), collagen III (Col-III) and α-smooth muscle actin (α-SMA) proteins. Agomir-16-1 and antagomir-16-1 were used to up and downregulate the expression of miR-16-1, respectively. Small interfering RNA (siRNA) was employed to inhibit the expression of HSP70. A wound healing assay was performed to measure the migration of fibroblasts. Cell proliferation was evaluated by MTT and 5-bromo-2-deoxyrudidine assays. Cell apoptosis was determined by caspase-3 activity and TUNEL assays. The results demonstrated that the levels of Col-I, Col-III and α-SMA were all significantly upregulated in SAT compared with NT. miR-16-1 levels in the SAT group were significantly compared with the NT group; conversely, the expression levels of HSP70 mRNA and protein in the SAT group were significantly lower compared with the NT group. Next, fibroblasts were treated with TPL to examine its effect on the miR-16-1/HSP70 pathway. The results demonstrated that the elevated expression of miR-16-1 in the SAT group was effectively inhibited by TPL treatment. Compared with the NT group, both the mRNA and protein levels of HSP70 were significantly downregulated in the SAT group cells, while TPL exhibited a strong promoting effect on HSP70 synthesis. Furthermore, upregulation of miR-16-1 reversed the effect of TPL on the miR-16-1/HSP70 pathway in fibroblasts from SAT. Overexpression of miR-16-1 significantly reversed the inhibitory effects of TPL treatment on migration, proliferation and extracellular matrix (ECM)-associated protein expression of fibroblasts from SAT. Finally, downregulation of miR-16-1 caused similar effects to the fibroblasts as the TPL treatment; however, the inhibitory effects on cell biological functions induced by antagomir-16-1 were all significantly reversed by HSP70 silencing. The present findings indicated that TPL may be a potential therapeutic option for postoperative anastomosis fibrosis of patients with CD. The miR-16-1/HSP70 pathway had a substantial role in the inhibitory effects of TPL on migration, proliferation and ECM synthesis rate of fibroblasts from strictured anastomosis tissues.

TGF-β and HSP70 profiles during transformation of yak hair follicles from the anagen to catagen stage.

Transforming growth factor-β (TGF-β) and heat shock protein 70 (HSP70) are important for the hair follicle (HF) cycle, but it is unclear whether they participate in HF regression in yak skin. In this study, we investigated the role of TGF-β, TGF-βRII, and HSP70 in the transition from anagen to catagen of HFs. The results showed that TGF-β2 transcription was significantly higher than that of TGF-β1 and TGF-β3 in the same periods. Meanwhile, the expressions of TGF-β2, TGF-βRII, and caspase-3 were higher in the catagen phase than that in mid-anagen, and some TGF-βRII-positive HF cells were terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive. Moreover, the HSP70 protein levels in mid-anagen were higher than those in late-anagen and catagen. These results suggested that TGF-β2 plays a major role in catagen induction in yak HFs, which might be achieved via TGF-βRII-mediated apoptosis in HF epithelial cells. In contrast, HSP70 might protect epithelial cells from apoptosis and ultimately inhibit HF regression. In conclusion, TGF-β2 has positive effects, whereas HSP70 has negative effects, on catagen induction.

Endurance training alters YKL40, PERM1, and HSP70 skeletal muscle protein contents in men with type 2 diabetes mellitus

ABSTRACTBackground: The fight against type 2 diabetes mellitus (T2DM) is tremendously challenging. This pilot study investigates whether endurance training (3 times per week for 3 months, moderate intensity) can change the skeletal muscle protein contents of chitinase-3-like protein-1 (YKL40), peroxisome proliferator-activated receptor y coactivator-1 and estrogen-related receptor-induced regulator in muscle-1 (PERM1) and heat-shock protein-70 (HSP70), which have been discussed as novel therapeutically relevant targets. Methods: Muscle biopsies were obtained from overweight/obese men with T2DM (n = 7, years = 63 ± 9) at T1 (6 weeks pre-training), T2 (1 week pre-training) and T3 (3 to 4 days post-training). The protein levels of YKL40, PERM1, and HSP70 were determined by immunohistochemistry. Results: YKL40, PERM1, and HSP70 were significantly upregulated following endurance training (T2–T3: +103%, +61%, +89%, p = 0.012, p = 0.010, p = 0.028). There was a fiber type-specific distribution of HSP70 with increased protein contents in type I fibers. A significant change in the fiber type distribution with an increase in type I fibers and a decrease in type II fibers was observed post-training. There were no significant differences for YKL40, PERM1, HSP70, or the fiber type distribution between T1 and T2. Conclusion: The training-induced upregulation of YKL40, PERM1, and HSP70 could help manage the diabetic disease and reduce its complications.

Effects of in vitro heat shock on immune cells in diet-induced obese mice

Obesity has been associated with impaired immune responses and inflammation. The mechanisms underlying these immune disturbances in obesity are not yet clarified. This study investigated the effects of in vitro heat shock (HS) on immune cells from the point of view of thymocyte apoptosis and T-cell mitogen-stimulated splenocyte cytokine production as well as the heat shock protein 70 (HSP70) protein levels in diet-induced obese mice to explore a possible association between the disturbance of T cell immunity and HS response in obesity. Obese mice had increased apoptotic and necrotic thymocytes populations and increased splenocyte cytokine production of both proinflammatory and anti-inflammatory cytokines compared with lean mice. The in vitro HS at 42°C decreased the rate of live cells in thymocytes, and the degree of the decrease was larger in obese mice compared with lean mice. The in vitro HS increased the intracellular and extracellular HSP70 protein levels in thymocytes and splenocytes, while the effects of obesity on the HSP70 protein levels were not obvious. The in vitro HS prior to T cell mitogen stimulation decreased IFN-γ and IL-10 production by mitogen-stimulated splenocytes. This change in cytokine production due to HS was not affected by obesity. The obvious alteration of the HSP70 protein levels and association between cytokine production and the HS response in obesity were not found in this obesity model; however, our results indicate an association between the viability of thymocytes and an altered HS response in obesity and provide evidence that the increase in thymocyte apoptosis and acceleration of thymus involution in obesity could be, in part, due to the alteration of the HS response.

SIRT1 inhibits releases of HMGB1 and HSP70 from human umbilical vein endothelial cells caused by IL-6 and the serum from a preeclampsia patient and protects the cells from death.

Preeclampsia (PE), a pregnancy-specific disorder, is associated with inappropriate maternal inflammatory response, oxidative stress, and vascular endothelial cell dysfunction and damage. Releases of high mobility group box-1 (HMGB1) and heat-shock protein 70 (HSP70) into serum are considered to participate in the pathogenesis of PE. The deacetylase, sirtuin 1 (SIRT1), has protective effects against inflammation, apoptosis, and oxidative stress in various pathological conditions. We established a PE mouse model by injection of phosphatidylserine/dioleoyl-phosphatidycholine compounds, followed by measurement of the SIRT1 protein level in the placenta via Western blotting and the serum HMGB1 and HSP70 concentrations via enzyme-linked immunosorbent assay (ELISA). SIRT1 was down-regulated in the placenta of PE mice, in accompany with increased serum HMGB1 and HSP70 concentrations. We incubated human umbilical vein endothelial cells (HUVECs) with IL-6 and the serum from a PE patient individually to mimic status of vein endothelial cells in PE. Western blot and Immunofluorescent assays showed that HMGB1 and HSP70 protein levels were decreased in the cells, but they were increased in the cell medium based on ELISA. These suggested that HMGB1 and HSP70 were forced to be released from the cells. SIRT1 knockdown promoted the releases of HMGB1 and HSP70, whereas its over-expression inhibited the releases. Moreover, SIRT1 protected the cells from death. Collectively, SIRT1 inhibits the releases of HMGB1 and HSP70 from HUVECs caused by IL-6 and the serum from PE patient and protects the cells from death, thus SIRT1 is probably a potentially protective factor in placenta against PE.

Allicin protects traumatic spinal cord injury through regulating the HSP70/Akt/iNOS pathway in mice.

Allicin is a major component of garlic, extracted as an oily liquid. The present study was designed to investigate the beneficial effects of allicin on traumatic spinal cord injury (TSCI) in mice, and whether the effects are mediated via regulation of the heat shock protein 70 (HSP70), v-akt murine thymoma viral oncogene homolog 1 (Akt) and inducible nitric oxide synthase (iNOS) pathways. Adult BALB/c mice (30–40 g) received a laminectomy at the T9 vertebral level as a model of TSCI. In the present study, treatment of the TSCI mice with allicin significantly increased their Basso, Beattie and Bresnahan (BBB) scores (P<0.01) and reduced the spinal cord water content (P<0.01). This protective effect was associated with the inhibition of oxidative stress and inflammatory responses in TSCI mice. Western blot analysis demonstrated that allicin increased the protein levels of HSP70, increased the phosphorylation of Akt and reduced the iNOS protein expression levels in TSCI mice. Additionally, treatment with allicin significantly reduced the levels of ROS and enhanced the NADH levels in TSCI mice. Collectively, these data demonstrate that the effects of allicin on TSCI are mediated via regulation of the HSP70, Akt and iNOS pathways in mice.

B-chromosome effects on Hsp70 gene expression does not occur at transcriptional level in the grasshopper Eyprepocnemis plorans.

As intragenomic parasites, B chromosomes can elicit stress in the host genome, thus inducing a response for host adaptation to this kind of continuous parasitism. In the grasshopper Eyprepocnemis plorans, B-chromosome presence has been previously associated with a decrease in the amount of the heat-shock protein 70 (HSP70). To investigate whether this effect is already apparent at transcriptional level, we analyze the expression levels of the Hsp70 gene in gonads and somatic tissues of males and females with and without B chromosomes from two populations, where the predominant B chromosome variants (B2 and B24) exhibit different levels of parasitism, by means of quantitative real-time PCR (qPCR) on complementary DNA (cDNA). The results revealed the absence of significant differences for Hsp70 transcripts associated with B-chromosome presence in virtually all samples. This indicates that the decrease in HSP70 protein levels, formerly reported in this species, may not be a consequence of transcriptional down-regulation of Hsp70 genes, but the result of post-transcriptional regulation. These results will help to design future studies oriented to identifying factors modulating Hsp70 expression, and will also contribute to uncover the biological role of B chromosomes in eukaryotic genomes.

Association of Plasma Heat Shock Protein 70, Interleukin 6, and Creatine Kinase Concentrations in a Healthy, Young Adult Population.

Variations of baseline plasma concentrations of creatine kinase (CK), heat shock protein 70 (HSP70), and interleukin 6 (IL-6) have been reported. We report categorical associations which may influence these protein levels. Methods. Blood was harvested for DNA and plasma protein analysis from 567 adults. Mean protein levels of CK, HSP70, and IL-6 were compared by sex, ethnicity, genetic variants—CKMM Nco1 (rs1803285), HSPA1B +A1538G (rs1061581), and IL6 G-174C (rs1800795)—self-reported history of exercise, oral contraceptive use, and dietary supplement use. Results. SNP major allele frequencies for CKMM, HSPA1B, and IL6 were 70% A, 57% A, and 60%. Mean CK statistically differed by sex, ethnicity, oral contraceptives, and caffeine. Plasma HSP70 differed by caffeine and protein. Mean IL-6 concentration differed by sex, ethnicity, and genotype. Plasma IL-6 was significantly lower (29%) in males (1.92 ± 0.08 pg/mL) and higher (29%) among African Americans (2.85 ± 0.50 pg/mL) relative to the others. IL6 G-174C GG genotype (2.23 ± 0.14 pg/mL) was 19% greater than CG or CC genotypes. Conclusion. Differences in baseline CK and IL-6 plasma protein concentrations are associated with genetics, sex, ethnicity, and the use of oral contraceptives, caffeine, and protein supplements in this young and athletic population.

Effect of dietary manganese on antioxidant status and expression levels of heat-shock proteins and factors in tissues of laying broiler breeders under normal and high environmental temperatures.

To investigate the effect of Mn on antioxidant status and expression levels of heat-shock proteins/factors in tissues of laying broiler breeders subjected to heat challenge, we used a completely randomised design (n 6) with a factorial arrangement of 2 environmental temperatures (normal, 21 (sem 1)°C and high, 32 (sem 1)°C)×3 dietary Mn treatments (an Mn-unsupplemented basal diet (CON), or a basal diet supplemented with 120 mg Mn/kg diet as inorganic Mn sulphate (iMn) or organic Mn proteinate (oMn)). There were no interactions (P>0·10) between environmental temperature and dietary Mn in all of the measured indices. High temperature decreased (P<0·003) Mn content, and also tended (P=0·07) to decrease copper zinc superoxide dismutase (CuZnSOD) activity in the liver and heart. However, an increased manganese superoxide dismutase (MnSOD) activity (P<0·05) and a slight increase of malondialdehyde level (P=0·06) were detected in breast muscle. Up-regulated (P<0·05) expression levels of heat-shock factor 1 (HSF1) and HSF3 mRNA and heat-shock protein 70 (HSP70) mRNA and protein were found in all three tissues. Broiler breeders fed either iMn or oMn had higher tissue Mn content (P<0·0001), heart MnSOD and CuZnSOD activities (P<0·01) and breast muscle MnSOD protein levels (P<0·05), and lower (P<0·05) breast muscle HSP70 mRNA and protein levels than those fed CON. Broiler breeders fed oMn had higher (P<0·03) bone Mn content than those fed iMn. These results indicate that high temperature decreases Mn retention and increases HSP70 and HSF1, HSF3 expression levels in tissues of laying broiler breeders. Furthermore, dietary supplementation with Mn in either source may enhance heart antioxidant ability and inhibit the expression of HSP70 in breast muscle. Finally, the organic Mn appears to be more available than inorganic Mn for bone in laying broiler breeders regardless of environmental temperatures.

The Effect of Acute Exercise on Heat Stress Response and Autophagy Induction in Humans: Meta‐Analysis

Eukaryotes employ heat stress response and autophagy to manage protein homeostasis. Autophagy is responsible for degradation of dysfunctional cellular constituents. On the other hand, heat shock proteins modulate protein folding and repair. Recent studies have shown that inhibition of heat shock factor 1 and upregulation of heat shock protein 70 (HSP70) activates and inhibits autophagy, respectively. In the present studies, we examined how these systems cooperate to maintain protein homeostasis during and after acute exercise in humans. A meta-analysis of studies that investigated the effects of acute exercise on autophagy, HSP70, protein synthesis, and protein breakdown in humans was performed. 53 publications with 566 participants were identified. Linear and nonlinear regression analyses were performed to describe relationships between time since initiation of exercise and expression of HSP70, autophagy activation, protein synthesis and breakdown. Our model shows that LC3 protein expression (a marker of autophagy) activation was increased about 14 h from the start of exercise, whereas HSP70 protein levels were elevated at about 105 h after exercise initiation. After initial increase (first 8 hours), protein synthesis gradually declined. Our model also showed a small increase in protein breakdown at about 24 h after the start of exercise and then steadily decreasing. We conclude that heat stress response and autophagy are linked and cooperate to maintain protein homeostasis during and after one bout of exercise in humans.

Contrasting cellular stress responses of Baikalian and Palearctic amphipods upon exposure to humic substances: environmental implications

The species-rich, endemic amphipod fauna of Lake Baikal does not overlap with the common Palearctic fauna; however, the underlying mechanisms for this are poorly understood. Considering that Palearctic lakes have a higher relative input of natural organic compounds with a dominance of humic substances (HSs) than Lake Baikal, we addressed the question whether HSs are candidate factors that affect the different species compositions in these water bodies. We hypothesized that interspecies differences in stress defense might reveal that Baikalian amphipods are inferior to Palearctic amphipods in dealing with HS-mediated stress. In this study, two key mechanisms of general stress response were examined: heat-shock protein 70 (HSP70) and multixenobiotic resistance-associated transporters (ABCB1). The results of quantitative polymerase chain reaction (qPCR) showed that the basal levels (in 3-day acclimated animals) of hsp70 and abcb1 transcripts were lower in Baikalian species (Eulimnogammarus cyaneus, Eulimnogammarus verrucosus, Eulimnogammarus vittatus-the most typical littoral species) than in the Palearctic amphipod (Gammarus lacustris-the only Palearctic species distributed in the Baikalian region). In the amphipods, the stress response was induced using HSs at 10 mg L(-1) dissolved organic carbon, which was higher than in sampling sites of the studied species, but well within the range (3-10 mg L(-1)) in the surrounding water bodies populated by G. lacustris. The results of qPCR and western blotting (n = 5) showed that HS exposure led to increased hsp70/abcb1 transcripts and HSP70 protein levels in G. lacustris, whereas these transcript levels remained constant or decreased in the Baikalian species. The decreased level of stress transcripts is probably not able to confer an effective tolerance to Baikalian species against further environmental stressors in conditions with elevated HS levels. Thus, our results suggest a greater robustness of Palearctic amphipods and a higher sensitivity of Baikalian amphipods to HS challenge, which might prevent most endemic species from migrating to habitats outside Lake Baikal.

Enzyme‐Treated Asparagus Extract Promotes Expression of Heat Shock Protein and Exerts Antistress Effects

A novel enzyme-treated asparagus extract (ETAS) has been developed as a functional material produced from asparagus stem. Studies were conducted to determine the effect of ETAS on heat shock protein 70 (HSP70) expression and alleviation of stress. HeLa cells were treated with ETAS, and HSP70 mRNA and protein levels were measured using a reverse transcription-polymerase chain reaction (RT-PCR) assay and an enzyme-linked immunosorbent assay (ELISA), respectively. ETAS showed significant increases in HSP70 mRNA at more than 0.125 mg/mL and the protein at more than 1.0 mg/mL. The antistress effect was evaluated in a murine sleep-deprivation model. A sleep-deprivation stress load resulted in elevation of blood corticosterone and lipid peroxide concentrations, while supplementation with ETAS at 200 and 1000 mg/kg body weight was associated with significantly reduced levels of both stress markers, which were in the normal range. The HSP70 protein expression level in mice subjected to sleep-deprivation stress and supplemented with ETAS was significantly enhanced in stomach, liver, and kidney, compared to ETAS-untreated mice. A preliminary and small-sized human study was conducted among healthy volunteers consuming up to 150 mg/d of ETAS daily for 7 d. The mRNA expression of HSP70 in peripheral leukocytes was significantly elevated at intakes of 100 or 150 mg/d, compared to their baseline levels. Since HSP70 is known to be a stress-related protein and its induction leads to cytoprotection, the present results suggest that ETAS might exert antistress effects under stressful conditions, resulting from enhancement of HSP70 expression.

Molecular Mechanisms Of Inhibition Of Ribosomal Biogenesis and Translational Flux By The Selective Inhibitor Of Nuclear Export (SINE) XPO1/CRM1 Antagonist KPT-185 In Mantle Cell Lymphoma

Exportin 1 (XPO1/CRM1) mediates transport of a number of cargo molecules including transcription factors and ribosomal subunits from the nucleus to cytoplasm. XPO1 is critical for cancer cell survival and proliferation, and we reported that high XPO1 expression correlates with poor prognosis in AML (Kojima, Blood, 2013). Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma that frequently shows chemoresistance. The overexpression of cyclin D1 due to the specific translocation t(l1 ;14)(q13;q32) in MCL cells is believed to be associated with oncogenesis, and additional genetic events such as mutation/overexpression of p53 are adverse prognostic indicators. Since a number of signaling pathways are dysregulated in MCL, novel strategies aimed at restoring multiple anti-oncogenic pathways, are of considerable interest. We have previously reported anti-proliferative effects of the small molecule SINE XPO1 antagonist KPT-185 in MCL cells, in which KPT-185 abrogates MCL-related cyclin Dl overexpression and upregulates pro-apoptotic PUMA in a p53-independent manner (Tabe, ASH. 2012).In this study, we identified pro-survival pathways involved in XPO1-dependent nuclear export in MCL cells, using the isobaric tags for relative and absolute quantification (iTRAQ) with two-dimensional-liquid chromatography-tandem mass spectrometry. Two MCL cell lines with known p53 status and sensitivities to KPT-185 were analyzed; wt-p53 Zl38 (IC50 35 nM, ED50 62 nM) and mt-p53 Jeko-1 (IC50 103 nM, ED50 618 nM). iTRAQ proteomics identified a total of 2,255 unique proteins in Zl38 and of 2,179 in Jeko-1 cells (KPT-185 of 50 nM for Zl38 and 100 nM for Jeko-1, 18 h), including 75 proteins (62 downregulated and 13 upregulated proteins) consistently altered after KPT-185 treatment in both cells lines. Notably, 81% of the downregulated proteins (50/62) were ribosomal proteins, and iTRAQ further detected the significant repressions of EIF4A1/PIM2 (eukaryotic translation initiation factor 4A1) and EEF2 (eukaryotic elongation factor 2), suggesting that KPT-185 inhibited the XPO1-dependent nuclear export of ribosomal subunits, which led to a defect of ribosomal biogenesis. Very recently, the coordination between the net translational activity of ribosomal biogenesis and the transcriptional regulation via the multifaceted transcription factor HSF1 (heat shock factor 1) has been reported (Santagata, Science, 2013) and HSF1 was identified as a prime transducer that regulates a transcriptional network of genes driving heat-shock proteins, protein synthesis, and energy metabolism. In our study, iTRAQ consistently detected the KPT-185 induced decreased protein levels of HSF1 target HSP70 (Heat shock protein 70), FASN (Fatty acid synthase), phospho-HSP90 (Heat shock protein 90) and EEF1A1 (Eukaryotic translation elongation factor 1 alpha 1), and increased levels of phospho-HNRNPD (Heterogeneous nuclear ribonucleoprotein D, a nucleic acid binding protein which contributes pre-mRNA processing in nucleus). These results indicate that XPO1 may also be affecting transcriptional processes critical for cellular metabolism and survival. Translation initiation factor EIF4Al/PlM2 is known to be associated with an aggressive clinical course in B-cell lymphomas (Gomez-Abad, Blood. 2011), and downregulation of PIM1 kinase via ribosomal protein deficiency induces cell-cycle inhibitor p27KIP (Morishita, Cancer Res. 2008) and inhibits oncogenic transcription factor c-Myc (Iadevaia, Oncogene. 2010). Of note, iTRAQ detected the KPT-185 induced depletion of ribosomal proteins RPS19 and RPL11, which interact with PIM1 kinase and c-Myc, respectively. We confirmed KPT-185 induced downregulation of PIM1 and c-Myc and upregulation of p27KIP by Western blot. KPT-185 further reduced phospho-S6K, a substrate of mTORC1 and a major negative regulatory axis of autophagy, and induced a shift from LC3-I to LC3-II, suggesting that CRM1 inhibition by KPT-185 causes autophagy through suppression of mTOR signaling.In summary, this is the first investigation of XPO1 inhibition in MCL cells using the iTRAQ proteomics approach. The results suggest that XPO1 inhibition targets ribosomal biogenesis, in addition to its nuclear retention of numerous client proteins including p53. This finding elucidates a novel mechanism and target of KPT-185 and warrants further investigations. Disclosures:Andreeff:Karyopharm Therapeutics: Research Funding.

A Rhodiola Rosea Root Extract Protects Skeletal Muscle Cells Against Chemically Induced Oxidative Stress by Modulating Heat Shock Protein 70 (HSP70) Expression

Rhodiola rosea is a perennial plant in the Crassulaceae family, recently postulated to exert its adaptogenic functions partially by modulating the expression of molecular factors such as heat shock proteins (HSP). The aim of this study was to analyze the efficacy of a Rhodiola rosea extract (Rhodiolife) in protecting murine skeletal muscle cells (C2 C12 myotubes) from chemically induced oxidative stress and to establish whether modulation of HSP70 expression is observed. C2 C12 cells treated with Rhodiolife did not experience any loss of viability (p > 0.05) at concentrations of 1-100 µg/mL for up to 24 h. In control cultures, viability decreased 25% following exposure to 2 mM H2 O2 (1 h). However, no significant decrease in viability in cells pre-treated with extract at concentrations as low as 1 µg/mL was observed. HSP70 mRNA levels were up-regulated two-fold in cell cultures treated with Rhodiolife (10 µg/mL), and expression was further enhanced by exposure to H2 O2 (six-fold, p < 0.05). HSP70 protein levels were maintained in pre-treated cell cultures compared to controls but was significantly lower (-50%) in cells lacking treatment exposed to H2 O2 . The present results indicate that Rhodiolife protects C2 C12 myotubes against peroxide-induced oxidative stress through the modulation of the molecular chaperone HSP70.

Anti-hypoxia effect of adenovirus-mediated expression of heat shock protein 70 (HSP70) on primary cultured neurons

Heat shock protein 70 (HSP70) has attracted great attention recently in hypoxia injury because of its close link to the recovery after hypoxic-ischemic damage in organs. However, the cellular mechanism underlying its protective roles remains unclear. In this study, we developed a recombinant adenovirus containing HSP70-GFP (vAd-HSP70-GFP) and studied the effect of virus-mediated expression of exogenous HSP70 gene on neurons in response to hypoxia-reoxygenation injury. Virus-mediated expression of HSP70 was detected as early as 24 hr and lasted until 10 days after infection. Neurons with 48 hr vAd-HSP70-GFP infection were exposed to 0, 0.5, 1, 2, 3, or 4 hr hypoxia followed by 1 hr reoxygenation. The mRNA and protein levels of HSP70 in neurons exposed to different lengths of hypoxia were compared by using RT-PCR and Western blotting (WB). The 1-hr hypoxia exposure showed the most significant increases in the HSP70 mRNA and protein level compared with other exposure durations. MTT assay showed that HSP70 overexpression significantly increased the neuronal viability, accompanied by decreased lactate dehydrogenase (LDH) activity in the culture medium after hypoxia-reoxygenation. Neurons with vAd-HSP70-GFP exhibited increased levels of mitochondrial cytochrome C (Cyt-C) and decreased levels of cytoplasmic Cyt-C compared with vAd-GFP-infected cells. These results suggest a neuroprotective role of exogenous HSP70 against hypoxia-reoxygenation injury, possibly via preventing initiation of mitochondrial apoptosis.