Mitogen and lymphokine stimulation of heat shock proteins in T lymphocytes.

We have examined the effects of phytohemagglutinin (PHA) and the polypeptide growth factor interleukin 2 (IL-2) on the synthesis of the 70- and 90-kDa heat shock proteins (HSP70 and HSP90, respectively) in human T lymphocytes. Resting T cells (G0) stimulated with PHA responded with a generalized increase in protein synthesis that included HSP70. Gel blot analysis indicated that steady-state levels of HSP70 mRNA were not specifically modulated by PHA. Synthesis of HSP90 protein, however, peaked very rapidly following PHA stimulation and decreased sharply after 1 hr. When IL-2-dependent human T cells, synchronized by IL-2 deprivation, were treated with IL-2, synthesis of HSP70 mRNA was increased as much as 15-fold. HSP70 and HSP90 protein synthesis increased significantly upon IL-2 stimulation of human T lymphocytes. Two distinct members of the ancient family of heat shock genes, HSP70 and HSP90, are shown to be stimulated at the activation and progression stages of lymphocyte mitogenesis, which suggests that genetic mechanisms evolved from primitive stress/adaptation responses may be conserved in mammalian cellular activation.     

Expression of human HSP70 during the synthetic phase of the cell cycle.

Expression of the major heat shock and stress-induced protein, HSP70, is under complex regulatory control in human cells. In addition to being induced by physiological stress such as heat shock or transition metals, the HSP70 gene is induced by serum stimulation and immortalizing products of the adenovirus E1A 13S and polyoma large tumor antigen genes. Here we show that expression of the human HSP70 gene is tightly regulated during the cell cycle. Using selective mitotic detachment, a noninductive method to obtain synchronous populations of HeLa cells, we show that levels of HSP70 mRNA rapidly increase 10- to 15-fold upon entry into S phase and decline by late S and G2. A transient increase in HSP70 synthesis is detected during early S phase. The subcellular localization of HSP70 varies throughout the cell cycle; the protein is diffusely distributed in the nucleus and cytoplasm in G1, localized in the nucleus in S, and again diffusely distributed in G2 cells. We suggest that the temporal pattern of HSP70 expression during S phase, the nuclear localization, and activation by trans-acting immortalizing proteins indicate a role for HSP70 in the nucleus of replicating cells.     

Regulation of human heme oxygenase-1 gene expression under thermal stress

Heme oxygenase-1 is an essential enzyme in heme catabolism, and its human gene promoter contains a putative heat shock element (HHO-HSE). This study was designed to analyze the regulation of human heme oxygenase-1 gene expression under thermal stress. The amounts of heme oxygenase-1 protein were not increased by heat shock (incubation at 42 degrees C) in human alveolar macrophages and in a human erythroblastic cell line, YN-1-0-A, whereas heat shock protein 70 (HSP70) was noticeably induced. However, heat shock factor does bind in vitro to HHO-HSE and the synthetic HHO-HSE by itself is sufficient to confer the increase in the transient expression of a reporter gene upon heat shock. The deletion of the sequence, located downstream from HHO-HSE, resulted in the activation of a reporter gene by heat shock. These results suggest that HHO-HSE is potentially functional but is repressed in vivo. Interestingly, heat shock abolished the remarkable increase in the levels of heme oxygenase-1 mRNA in YN-1-0-A cells treated with hemin or cadmium, in which HSP70 mRNA was noticeably induced. Furthermore, transient expression assays showed that heat shock inhibits the cadmium-mediated activation of the heme oxygenase-1 promoter, whereas the HSP70 gene promoter was activated upon heat shock. Such regulation of heme oxygenase-1 under thermal stress may be of physiologic significance in erythroid cells.     

A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol

BACKGROUND/AIMS: A stress-inducible heat shock protein 70 (HSP70) is one of the best-known endogenous factors protecting cell injury under various pathological conditions. The aim of this study was to examine anti-apoptotic actions of a non-toxic HSP70 inducer, geranylgeranylacetone (GGA), on hepatocytes exposed to hydrogen peroxide (H2O2) or ethanol. METHODS: Primary cultures of rat hepatocytes were treated with different concentrations of GGA and exposed to 0.5 mM H202 or 100 mM ethanol. The heat shock response was assessed by measuring the activation of heat shock factor 1 (HSF1), HSP70 mRNA expression, and accumulations of HSP70, HSP90, and HSP27. Apoptosis was evaluated by DNA fragmentation. RESULTS: Pretreatment with 1 microM GGA for 2 h enhanced nuclear translocation and phosphorylation of HSF1, HSF1-DNA binding, HSP70 mRNA expression, and its accumulation, when the cells were exposed to H202 or ethanol. In association with this accelerated response, GGA suppressed the insult-induced activation of c-Jun N-terminal kinases, caspase 9, and caspase 3-like proteases, leading to significant inhibition of apoptosis. CONCLUSIONS: GGA exerted anti-apoptotic actions, at least in part, by priming hepatocytes for enhanced HSP70 induction. Our results suggest that GGA may have a potential benefit for the treatment of alcoholic and ischemia-reperfusion liver injuries.     

Decreased levels of heat shock proteins in gut epithelial cells after exposure to plant lectins

BACKGROUND: The enterocytes of the intestinal epithelium are regularly exposed to potentially harmful substances of dietary origin, such as lectins. Expression of heat shock proteins (HSPs) by this epithelium may be part of a protective mechanism developed by intestinal epithelial cells to deal with noxious components in the intestinal lumen. AIM: To investigate if the lectins PHA, a lectin from kidney beans (Phaseolus vulgaris) and WGA, a lectin from wheat germ (Triticum aestivum) could modify the heat shock response in gut epithelial cells and to establish the extent of this effect. METHODS: Jejunal tissue sections from PHA and WGA fed rats were screened for expression of HSP70, HSP72, and HSP90 using monoclonal antibodies. Differentiated Caco-2 cells, the in vitro counterpart of villus enterocytes, were exposed to 100 microg/ml of PHA-E(4) or WGA for 48 hours and investigated for changes in DNA and protein synthesis by double labelling with [2-(14)C]thymidine and L-[methyl-(3)H]methionine. The relative concentrations of HSP60, HSP70, HSP72, and HSP90 and binding protein (BiP) in these cells exposed to lectins were analysed by polyacrylamide gel electrophoresis and immunoblotting. To establish if lectin exposed differentiated Caco-2 cells were still capable of producing a heat shock response, these cells received a heat shock (40 degrees C, 41 degrees C, and 42 degrees C) for one hour and were allowed to recover for six hours at 37 degrees C. During heat shock and recovery periods, lectin exposure was continued. RESULTS: Constitutive levels of HSPs were measured in the intestinal cells of lactalbumin fed (control) rats, as may be expected from the function of this tissue. However, in PHA and WGA fed rats a marked decline in the binding of antibodies against several HSPs to the intestinal epithelium was found. These results were confirmed by in vitro experiments using differentiated Caco-2 cells exposed to PHA-E(4) and WGA. However, after exposure to lectins, these cells were still capable of heat induced heat shock protein synthesis, and total protein synthesis was not impaired indicating specific inhibition of HSP synthesis in non-stressed cells. CONCLUSIONS: We conclude that PHA and WGA decrease levels of stress proteins in rat gut and enterocyte-like Caco-2 cells, leaving these cells less well protected against the potentially harmful content of the gut lumen.     

Kruppel样因子4过表达对C2C12肌原细胞中热休克蛋白25表达的影响

目的观察Kruppel样因子4过表达对热休克蛋白25表达的影响。方法运用稳定转染pcDNA3.1/myc-His(-)和Kruppel样因子4-pcDNA3.1/myc-His(-)两种细胞株,采用逆转录聚合酶链反应观察Kruppel样因子4在生理状态和热休克反应时对热休克蛋白25 mRNA表达的影响;采用Western blot观察Kruppel样因子4在生理状态和热休克反应时对热休克蛋白25蛋白表达的影响。结果在正常状态下,两种细胞热休克蛋白25 mRNA及蛋白表达水平均较低。生理状态下,热休克恢复1 h及3 h Kruppel样因子4过表达细胞株热休克蛋白25 mRNA表达水平明显高于转空载体细胞;至热休克恢复6 h后两组细胞热休克蛋白25 mRNA表达无明显差异。在生理状态及热休克恢复不同时间后,Kruppel样因子4过表达细胞热休克蛋白25蛋白表达水平均较空载体组明显增加。结论在生理状态下和热休克反应中,Kruppel样因子4过表达均能够促进C2C12肌原细胞中热休克蛋白25高表达。     

Contrasting effects of triiodothyronine on heat shock protein 27 induction and vascular endothelial growth factor synthesis stimulated by TGF-beta in osteoblasts

In osteoblast-like MC3T3-E1 cells, we recently reported that transforming growth factor-beta (TGF-beta) stimulates the induction of heat shock protein 27 (HSP27). In the present study, we investigated the effects of triiodothyronine (T(3)) on the TGF-beta-stimulated induction of HSP27 and synthesis of vascular endothelial growth factor (VEGF) in these cells. T(3) by itself had little effect on the level of HSP27, however, it significantly reduced the TGF-beta-stimulated HSP27 accumulation in a dose-dependent manner in the range between 1 pM and 100 nM. The TGF-beta-stimulated increase in the level of mRNA for HSP27 was also attenuated by T(3). On the other hand, T(3), which alone stimulated the release of VEGF, more than additively stimulated the TGF-beta-induced VEGF release. T(3) enhanced the TGF-beta-induced increase in the levels of mRNA for VEGF. These results strongly suggest that T(3) has contrasting effects on HSP27 induction and VEGF synthesis induced by TGF-beta in osteoblasts.