HSP70 mRNA translation in chicken reticulocytes is regulated at the level of elongation

During heat shock of chicken reticulocytes the synthesis of a single heat shock protein, HSP70, increases greater than 10-fold, while the level of HSP70 mRNA increases less than 2-fold during the same period. Comparison of the in vivo levels of HSP70 and beta-globin synthesis with their mRNA abundance reveals that the translation of HSP70 mRNA is repressed in normal reticulocytes and is activated upon heat shock. In its translationally repressed state HSP70 mRNA is functionally associated with polysomes based on sedimentation analysis of polysomes from untreated or puromycin-treated cells and by analysis of in vitro "run-off" translation products using isolated polysomes. Treatment of control and heat shocked cells with the initiation inhibitor pactamycin reveals that elongation of the HSP70 nascent peptide is not completely arrested, but is slower in control cells. Furthermore, the inefficient translation of HSP70 mRNA in vivo is not due to the lack of an essential translation factor; HSP70 mRNA is efficiently translated in chicken reticulocyte translation extracts as well as in heterologous rabbit reticulocyte extracts. Our results reveal that a major control point for HSP70 synthesis in reticulocytes is the elongation rate of the HSP70 nascent peptide.     

HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise

Exercise causes heat shock (muscle temperatures of up to 45 degrees C, core temperatures of up to 44 degrees C) and oxidative stress (generation of O2- and H2O2), and exercise training promotes mitochondrial biogenesis (2-3-fold increases in muscle mitochondria). The concentrations of at least 15 possible heat shock or oxidative stress proteins (including one with a molecular weight of 70 kDa) were increased, in skeletal muscle, heart, and liver, by exercise. Soleus, plantaris, and extensor digitorum longus (EDL) muscles exhibited differential protein synthetic responses ([3H]leucine incorporation) to heat shock and oxidative stress in vitro but five proteins (particularly a 70 kDa protein and a 106 kDa protein) were common to both stresses. HSP70 mRNA levels were next analyzed by Northern transfer, using a [32P]-labeled HSP70 cDNA probe. HSP70 mRNA levels were increased, in skeletal and cardiac muscle, by exercise and by both heat shock and oxidative stress. Skeletal muscle HSP70 mRNA levels peaked 30-60 min following exercise, and appeared to decline slowly towards control levels by 6 h postexercise. Two distinct HSP70 mRNA species were observed in cardiac muscle; a 2.3 kb mRNA which returned to control levels within 2-3 h postexercise, and a 3.5 kb mRNA species which remained at elevated concentrations for some 6 h postexercise. The induction of HSP70 appears to be a physiological response to the heat shock and oxidative stress of exercise. Exercise hyperthermia may actually cause oxidative stress since we also found that muscle mitochondria undergo progressive uncoupling and increased O2- generation with increasing temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression, synthesis, and phosphorylation of HSP28 family during development and decay of thermotolerance in CHO plateau-phase cells.

We investigated a correlation between development of thermotolerance and expression, synthesis, or phosphorylation of HSP28 family in CHO plateau phase cells. After heating at 45.5 degrees C for 10 min, thermotolerance developed rapidly and reached its maximum 12-18 hr after heat shock. This acquired thermal resistance was maintained for 72 hr and then gradually decayed. In parallel, the levels of three 28 kDa heat shock proteins, HSP28a along with its two phosphorylated isoforms (HSP28b,c), increased and reached their maximum 24-48 hr after heat shock. The levels of these polypeptides, except HSP28c, remained elevated for 72 hr and then decreased. The level of HSP28 mRNA increased rapidly and reached its maximum 12 hr after heat shock. However, unlike thermotolerance and the levels of HSP28 family proteins, the level of HSP28 mRNA decreased rapidly within 72 hr. These results demonstrate a correlation between the amount of intracellular HSP28 family proteins and development and decay of thermotolerance.     

Cell-specific expression of heat shock proteins in chicken reticulocytes and lymphocytes

We have found that chicken reticulocytes respond to elevated temperatures by the induction of only one heat shock protein, HSP70, whereas lymphocytes induce the synthesis of all four heat shock proteins (89,000 mol wt, HSP89; 70,000 mol wt, HSP70; 23,000 mol wt, HSP23; and 22,000 mol wt, HSP22). The synthesis of HSP70 in lymphocytes was rapidly induced by small increases in temperature (2 degrees-3 degrees C) and blocked by preincubation with actinomycin D. Proteins normally translated at control temperatures in reticulocytes or lymphocytes were not efficiently translated after incubation at elevated temperatures. The preferential translation of mRNAs that encode the heat shock proteins paralleled a block in the translation of other cellular proteins. This effect was most prominently observed in reticulocytes where heat shock almost completely repressed alpha- and beta-globin synthesis. HSP70 is one of the major nonglobin proteins in chicken reticulocytes, present in the non-heat-shocked cell at approximately 3 X 10(6) molecules per cell. We compared HSP70 from normal and heat-shocked reticulocytes by two-dimensional gel electrophoresis and by digestion with Staphylococcus aureus V8 protease and found no detectable differences to suggest that the P70 in the normal cell is different from the heat shock-induced protein, HSP70. P70 separated by isoelectric focusing gel electrophoresis into two major protein spots, an acidic P70A (apparent pl = 5.95) and a basic P70B (apparent pl = 6.2). We observed a tissue-specific expression of P70A and P70B in lymphocytes and reticulocytes. In lymphocytes, P70A is the major 70,000-mol-wt protein synthesized at normal temperatures whereas only P70B is synthesized at normal temperatures in reticulocytes. Following incubation at elevated temperatures, the synthesis of both HSP70A and HSP70B was rapidly induced in lymphocytes, but synthesis of only HSP70B was induced in reticulocytes.     

BiP, HSP70, NDK and PDI in wheat endosperm. II. Effects of high temperature on protein and mRNA accumulation

The effects of high temperature on accumulation of the 70‐kDa heat shock protein (HSP70) and nucleoside diphosphate kinase (NDK) as well as two other proteins that have roles in the biosynthesis of storage proteins were examined during grain development. An HSP70 homolog and a 17‐kDa NDK were co‐purified from wheat endosperm, their identity verified, and a cDNA for an HSP70 expressed in endosperm was isolated. Wheat plants ( Triticum aestivum , cvs Butte and Vulcan) were heat shocked at 40°C or exposed to maximum daily temperatures of 37 or 40°C during early or mid‐grain fill. Antibodies and cDNA probes for BiP, HSP70, NDK and PDI were used to examine the effect of high temperatures on the accumulation of protein and mRNA in the endosperm. HSP70 mRNA levels increased substantially when plants were exposed to heat shock or to a 1‐day gradual increase to 40°C. The effects of a 5‐day heat treatment on mRNA levels were more complicated and depended on the developmental stage of the grain. A treatment that began at 7 days post‐anthesis (DPA) decreased the level of mRNA for HSP70, BiP, PDI and NDK, whereas a treatment that began at 14 DPA slightly increased mRNA levels. The same treatments increased the accumulation of HSP70 but did not affect BiP, PDI, or NDK protein levels. This is the first detailed report on the effects of heat on mRNA and protein levels for HSP70 in a developing seed storage tissue.     

The Drosophila hsp70 message is rapidly degraded at normal temperatures and stabilized by heat shock

When heat-shocked Drosophila cells are returned to normal temperatures, heat-shock protein (HSP) synthesis is repressed and normal protein synthesis is restored. The repression of HSP70 synthesis is accompanied by the selective degradation of its mRNA. We have engineered cells to produce a modified hsp70 mRNA that behaves exactly as the wild-type message. That is, it is stable during heat shock but degraded during recovery when protein synthesis returns to normal. When this message, placed under the control of the metallothionein promoter, is induced at normal temperatures it is rapidly degraded, with a half life of 15-30 min. Apparently, the hsp70 message is inherently unstable. During heat-shock, degradation of the message is suspended; during recovery degradation is restored.     

Is the major Drosophila heat shock protein present in cells that have not been heat shocked?

When eukaryotic cells are exposed to elevated temperatures they respond by vigorously synthesizing a small group of proteins called the heat shock proteins. An essential element in defining the role of these proteins is determining whether they are unique to a stressed state or are also found in healthy, rapidly growing cells at normal temperatures. To date, there have been conflicting reports concerning the major heat-induced protein of Drosophila cells, HSP 70. We report the development of monoclonal antibodies specific for this protein. These antibodies were used to assay HSP 70 in cells incubated under different culture conditions. The protein was detectable in cells maintained at normal temperatures, but only when immunological techniques were pushed to the limits of their sensitivity. To test for the possibility that these cells contain a reservoir of protein in a cryptic antigenic state (i.e., waiting posttranslational modification for use at high temperature), we treated cells with cycloheximide or actinomycin D immediately before heat shock. HSP 70 was not detected in these cells. Finally, we tested for the presence of a reservoir of inactive messages by using a high stringency hybridization of 32P- labeled cloned gene sequences to electrophoretically separated RNAs. Although HSP 70 mRNA was detectable in rapidly growing cells, it was present at less than 1/1,000th the level achieved after induction.     

Heat-Induced Proteasomic Degradation of HSF1 in Serum-Starved Human Fibroblasts Aging in Vitro

The exposure of human fibroblasts (HF) aging in vitro to heat shock resulted in an attenuated expression of the heat shock-inducible HSP70. When late passage cells were cultured in the continuous presence of serum, we observed a reduced accumulation of the cytoplasmic polyadenylated HSP70 mRNA. The levels of HSF1 activation and nuclear HSP70 mRNA were comparable to those of early passage cells (M. A. Bonelli et al., Exp. Cell Res. 252, 20-32, 1999). When late passage cells were serum-starved overnight, we observed a reduced activation of HSF1 and a decreased level of HSP70 mRNA during heat shock. However, at 37 degrees C the levels of HSF1 differed little between late passage HF and early passage cells, irrespective of the presence of serum. Interestingly, during heat shock a marked decrease in the level and, consequently, in the binding activity of HSF1 was noted only in serum-starved, late passage HF. The decrease in the level of HSF1 was counteracted by back addition of serum to the cells during heat shock. Addition of the specific proteasome inhibitor MG132 blocked a decrease in HSF1 during heat shock, maintaining levels observed in late passage cells and HSF1 activity comparable to that of early passage HF. The recovery of the level and activity of HSF1 observed in late passage HF incubated in the presence of MG132 suggests that heat shock unmasks a latent proteasome activity responsible for HSF1 degradation.     

The Effect of Manganese-induced Cytotoxicity on mRNA Expressions of HSP27, HSP40, HSP60, HSP70 and HSP90 in Chicken Spleen Lymphocytes in Vitro

The purpose of this study was to investigate the effect of manganese (Mn)-induced cytotoxicity on heat shock proteins in chicken spleen lymphocytes. Lymphocytes were cultured in medium in the absence and presence of MnCl₂ (2?×?10^?4, 4?×?10^?4, 6?×?10^?4, 8?×?10^?4, 10?×?10^?4, and 12?×?10^?4 mmol/L) for 12, 24, 36, and 48 h in vitro. Then, the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 were examined by real-time quantitative PCR. The results showed that the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 in all treatment groups at all time points, except mRNA levels of HSP27 at 48 h, had the same tendency. As manganese concentration increased, the mRNA expression of the heat shock proteins first increased and then decreased. In other words, we demonstrated that the mRNA expression of the heat shock proteins was induced at lower concentrations of manganese and was inhibited at higher concentrations. Mn had a dosage-dependent effect on HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression in chicken spleen lymphocytes in vitro.     

Reduction of HSP70 and HSC70 Heat Shock mRNA Induction by Pentobarbital After Transient Global Ischemia in Gerbil Brain

Abstract: The effect of pentobarbital on the induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brains was investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brains, HSP70 mRNA was scarcely present, whereas HSC70 mRNA was present in most cell populations. After a 5-min occlusion of bilateral common carotid arteries, HSP70 and HSC70 mRNAs were induced together in several cells and were especially dense in hippocampal dentate granule cells at 3 h, but the strong hybridization of the mRNAs continued only in hippocampal CA1 cells by 2 days. At 7 days after the ischemia, CA1 neuronal cell death was apparent, and the HSP70 mRNA disappeared and HSC70 mRNA content returned to the sham level, except for in the CA1 cells. Pretreatment with pentobarbital (40 mg/kg, i.p.) greatly reduced or inhibited the induction of HSP70 and HSC70 mRNAs at both early (3-h) and late (2-day) phases after ischemia. The drug also prevented CA1 cell death at 7 days along with the maintenance of expression of HSC70 mRNA at the sham control level. Hypothermic effects of pentobarbital were noted at 30 and 60 min after the reperfusion, whereas at 2 h there was no statistical significance between the control and drug-treated groups. The great reduction of HSP70 and HSC70 mRNA induction at both early and late phases after ischemia suggests that pentobarbital reduces intra- and/or postischemic stress and may protect CA1 cells from ischemic damage. These effects of the drug may be mainly due to its specific action rather than its hypothermic effects.