Protein kinase C micron plays an essential role in hypertonicity-induced heat shock protein 70 expression

Heat shock protein 70 (HSP70), which evidences important functions as a molecular chaperone and anti-apoptotic molecule, is substantially induced in cells exposed to a variety of stresses, including hypertonic stress, heavy metals, heat shock, and oxidative stress, and prevents cellular damage under these conditions. However, the molecular mechanism underlying the induction of HSP70 in response to hypertonicity has been characterized to a far lesser extent. In this study, we have investigated the cellular signaling pathway of HSP70 induction under hypertonic conditions. Initially, we applied a variety of kinase inhibitors to NIH3T3 cells that had been exposed to hypertonicity. The induction of HSP70 was suppressed specifically by treatment with protein kinase C (PKC) inhibitors (Go6976 and GF109203X). As hypertonicity dramatically increased the phosphorylation of PKC micron, we then evaluated the role of PKC micron in hypertonicity-induced HSP70 expression and cell viability. The depletion of PKC micron with siRNA or the inhibition of PKC micron activity with inhibitors resulted in a reduction in HSP70 induction and cell viability. Tonicity-responsive enhancer binding protein (TonEBP), a transcription factor for hypertonicity-induced HSP70 expression, was translocated rapidly into the nucleus and was modified gradually in the nucleus under hypertonic conditions. When we administered treatment with PKC inhibitors, the mobility shift of TonEBP was affected in the nucleus. However, PKC micron evidenced no subcellular co-localization with TonEBP during hypertonic exposure. From our results, we have concluded that PKC micron performs a critical function in hypertonicity-induced HSP70 induction, and finally cellular protection, via the indirect regulation of TonEBP modification.

CD36 signaling inhibits the translation of heat shock protein 70 induced by oxidized low density lipoprotein through activation of peroxisome proliferators-activated receptor gamma

Oxidized LDL (OxLDL), a causal factor in atherosclerosis, induces the expression of heat shock proteins (Hsp) in a variety of cells. In this study, we investigated the role of CD36, an OxLDL receptor, and peroxisome proliferator-activated receptor gamma (PPAR gamma) in OxLDL-induced Hsp70 expression. Overexpression of dominant-negative forms of CD36 or knockdown of CD36 by siRNA transfection increased OxLDL-induced Hsp70 protein expression in human monocytic U937 cells, suggesting that CD36 signaling inhibits Hsp70 expression. Similar results were obtained by the inhibition of PPAR gamma activity or knockdown of PPAR gamma expression. In contrast, overexpression of CD36, which is induced by treatment of MCF-7 cells with troglitazone, decreased Hsp70 protein expression induced by OxLDL. Interestingly, activation of PPAR gamma through a synthetic ligand, ciglitazone or troglitazone, decreased the expression levels of Hsp70 protein in OxLDL-treated U937 cells. However, major changes in Hsp70 mRNA levels were not observed. Cycloheximide studies demonstrate that troglitazone attenuates Hsp70 translation but not Hsp70 protein stability. PPAR gamma siRNA transfection reversed the inhibitory effects of troglitazone on Hsp70 translation. These results suggest that CD36 signaling may inhibit stress- induced gene expression by suppressing translation via activation of PPAR gamma in monocytes. These findings reveal a new molecular basis for the anti-inflammatory effects of PPAR gamma.

A study on the expression of HSP70 and iNOS after human brain contusion / 法医学杂志

OBJECTIVE@#To study the relationship between expression of HSP70, iNOS and traumatic brain contusion (TBI) in different posttraumatic intervals.@*METHODS@#35 samples of brain contusion were examined using immunohistochemecal staining to evaluate the expression of HSP70 and iNOS.@*RESULTS@#Maximal HSP70 expression was found at 0h after brain contusion. The intensity of HSP70 staining decreased remarkably to the minimum at 24h after TBI, then increased gradually. Expression of iNOS positive cells increased significantly and reached the maximum level 48h after TBI, then the expression decreased gradually from the 2nd day to the 11th day.@*CONCLUSION@#The changes of HSP70 and iNOS immunohistochemical staining can be used as a referential data for estimating time interval after human brain contusion.

Expression of heat stress protein 70 mRNA in patients with chronic obstructive pulmonary disease and its significance / 华中科技大学学报（医学）（英德文版）

The effects of cigarette smoke extract (CSE) on the expression of heat stress protein 70 (Hsp70) in human bronchi smooth muscle cells were investigated in vitro, and the changes in Hsp70 mRNA in the patients with chronic obstructive pulmonary disease and their significance were explored. Human bronchi smooth muscle cells were cultured with CSE at the different concentrations. The expression of Hsp70 mRNA and Hsp70 was detected by reverse translation-polymerase chain reaction (RT-PCR) and Western blotting respectively. Levels of Hsp70 mRNA and Hsp70 in lymphocytes from 20 patients with COPD and 20 healthy smoking control subjects were measured by RT-PCR and Western blotting. The results showed the expression of both Hsp70 mRNA and Hsp70 was decreased conformably in human bronchi smooth muscle cells treated with CSE at certain concentration in vitro. The A values of the Hsp70 mRNA expression were 0.24 +/- 0.11 and 0. 42 +/- 0.13 respectively in COPD patients and healthy smoking controls with the difference being significant (P < 0.01). There was also significant difference in the A values of the Hsp70 expression between COPD patients and healthy smoking controls (20.9 +/- 9.9 vs 44.8 +/- 15.3, P < 0.01). The levels of Hsp70 mRNA had strongly positive correlation with Hsp70 protein (r = 0.85, P < 0.01). It was suggested that the expression of Hsp70 mRNA was in concordance with the expression of Hsp70, which could provide a basis on the study of Hsp70 gene regulation and Hsp70 gene in the development of COPD.

Mild heat stress at a young age in Drosophila melanogaster leads to increased Hsp70 synthesis after stress exposure later in life.

In a number of animal species it has been shown that exposure to low levels of stress at a young age has a positive effect on stress resistance later in life, and on longevity. The positive effects have been attributed to the activation of defence/cleaning systems (heat shock proteins (Hsps), antioxidases, DNA repair) or to effects of a changed metabolic rate, or both. We investigated the effect of mild stress exposures early in life on Hsp70 synthesis after a harder stress exposure later in life in five isofemale lines of Drosophila melanogaster. Female flies were either exposed to repeated bouts of mild heat stress (3 h at 34 degrees C) at a young age (days 2, 4 and 6 post-eclosion) or held under standard laboratory conditions. At 16 and 32 days of adult age, respectively, flies were exposed to a high-temperature treatment known to induce Hsp70 in the investigated species (1 h at 37 degrees C). Thereafter, the inducible Hsp70 levels were measured. Our data show a tendency towards increased Hsp70 synthesis with increased age for both 'mild stress' and 'no stress' flies. Moreover, the results show that flies exposed to mild stress at a young age synthesized more Hsp70 upon induction, compared to control flies, and that this difference was accentuated at 32 days compared to 16 days of age. Thus, bouts of mild heat stress at a young age impact on the physiological stress response system later in life. This may be caused by an increased ability to react to future stresses. Alternatively, the mild stress exposure at a young age may actually have caused cellular damages increasing the need for Hsp70 levels after stress exposure later in life. The importance of an Hsp70 upregulation (throughout life) in explaining the phenomenon of hormesis is discussed, together with alternative hypotheses, and suggestions for further studies.

Expression of HSP70 in peripheral lymphocytes of the patients with allergic rhinitis / 华中科技大学学报（医学）（英德文版）

The expression levels of heat shock protein 70 (HSP70) from peripheral lymphocytes of the patients with allergic rhinitis (AR) and the clinical implication were investigated. In the morning, 3 ml of fasting venous blood was taken out. The lymphocytes were isolated by using Ficoll-Hypaque and the expression of HSP70 in the lymphocytes was detected by using Western blot. In the AR patients the HSP70 level (41.49 +/- 15.77 integrated optical density, IOD) were significantly higher than that in the control group (23.89 +/- 10.13 IOD, P < 0.05). Western blot demonstrated that HSP70 bands in AR patients were more intensive than those in the control group. It was concluded that the elevated HSP70 level in peripheral lymphocytes of the AR patients might contribute to the development of AR.

Expression of heat shock protein 70 in freshwater prawn Macrobrachium malcolmsonii (H. Milne Edwards) following exposure to Hg and Cu.

Juveniles of freshwater prawn M. malcolmsonii were exposed to 1/6th concentration of LC50 of Hg and Cu for 48 hr. Sampling was done at 1 8, 12, 16, 24, 30, 36, 42 and 48 hr of exposure. Gill and hepatopancreas were dissected and subjected to one-dimensional electrophoresis. Western blotting was employed to determine the relative concentration of heat shock protein, hsp 70 (stress-70) in each sample. In the gill tissue of the prawn that had been exposed to Hg (0.024 mgHg 1(-1)), stress-70 was detected from the 1st hr till the 16th hr of exposure. But in the gills of Cu exposed prawn, synthesis of stress-70 appeared from the 1st hr till the 24th hr. Synthesis of hsp70 was not recorded after the 24th hr in the gills of exposed prawns. Synthesis of stress-70 was also found to be tissue-specific for both metals in this prawn. When the antibody probe raised against stress-70 was used, synthesis of stress-70 was not observed in hepatopancreas of prawns exposed to Hg or Cu, during the entire period of exposure of 48 hr. The expression of stress-70 in M. malcolmsonii following exposure to Hg and Cu is apparently only transient, and also a differential expression of stress-70 between gill and hepatopancreas was observed for both the metals.