Gene expression of heme oxygenase-1 during glial activation by lipopolysaccharide

The effect of bacterial lipopolysaccharide (LPS) on the expression of mRNA encoding heme oxygenase-1 (HO-1), which is the rate limiting enzyme in heme catabolism and is also known as heat shock protein 32 (HSP32), was examined in primarily cultured rat glial cells. Treatment of cells with LPS elicited an increase in HO-1 mRNA, accompanying down regulation of delta-aminolevulinate synthase, in a dose-dependent fashion. HO-1 mRNA increased markedly at 12 h after LPS treatment (10 microg/ml) and reached a maximum at 24 h. In contrast, HSP70, a major heat shock protein, slightly increased only at 6 h after LPS treatment and returned to the control level by 12 h. These results suggest that HSPs are induced under separate regulation during glial activation by LPS through oxidative stress, a part of which is likely mediated by intracellular free heme.     

Nitrosative stress, cellular stress response, and thiol homeostasis in patients with Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder with cognitive and memory decline, personality changes, and synapse loss. Increasing evidence indicates that factors such as oxidative and nitrosative stress, glutathione depletion, and impaired protein metabolism can interact in a vicious cycle, which is central to AD pathogenesis. In the present study, we demonstrate that brains of AD patients undergo oxidative changes classically associated with a strong induction of the so-called vitagenes, including the heat shock proteins (HSPs) heme oxygenase-1 (HO-1), HSP60, and HSP72, as well as thioredoxin reductase (TRXr). In inferior parietal brain of AD patients, a significant increase in the expression of HO-1 and TRXr was observed, whereas HO-2 expression was decreased, compared with controls. TRHr was not increased in AD cerebellum. Plasma GSH was decreased in AD patients, compared with the control group, and was associated with a significant increase in oxidative stress markers (i.e., GSSG, hydroxynonenal, protein carbonyl content, and nitrotyrosine). In AD lymphocytes, we observed an increased expression of inducible nitric oxide synthase, HO-1, Hsp72, HSP60, and TRXr. Our data support a role for nitrative stress in the pathogenesis of AD and indicate that the stress-responsive genes, such as HO-1 and TRXr, may represent important targets for novel cytoprotective strategies.     

Differential induction of brain heme oxygenase-1 and heat shock protein 70 mRNA in sepsis

We examined gene expression of heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1), which is the rate limiting enzyme in heme catabolism and is also known as heat shock protein 32 (HSP32), in the rat brain using a sepsis model induced by bacterial lipopolysaccharide (LPS). Intraperitoneal injection of LPS (10 mg/kg) to rats caused the elevation of body temperature and white blood cell (WBC) counts as well as marked elevation of serum interleukin-6 (IL-6) level, showing the typical pathological characteristics of sepsis. In this model, HO-1 mRNA increased at 6 h after LPS administration and continued to rise until 30 h. In contrast, HSP70 mRNA increased only between 3 h and 6 h after LPS administration, returning completely to the control level by 12 h. HO-1 mRNA was expressed predominantly in the cortex and the medulla oblongata, while HSP70 mRNA was expressed mainly in the striatum. HO-1 and HSP70 mRNA levels thus showed distinctive time courses and tissue distribution in the brain, suggesting that gene expression of these heat shock proteins (HSPs) is separately regulated.     

Redox regulation of heat shock protein expression by signaling involving nitric oxide and carbon monoxide: relevance to brain aging, neurodegenerative disorders, and longevity

Increased free radical generation and decreased efficiency of the reparative/degradative mechanisms both primarily contribute to age-related elevation in the level of oxidative stress and brain damage. Excess formation of reactive oxygen and nitrogen species can cause proteasomal dysfunction and protein overloading. The major neurodegenerative diseases are all associated with the presence of abnormal proteins. Different integrated responses exist in the brain to detect oxidative stress which is controlled by several genes termed vitagenes, including the heat shock protein (HSP) system. Of the various HSPs, heme oxygenase-I (HO-1), by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. The HO-1 gene is redox regulated and its expression is modulated by redox active compounds, including nutritional antioxidants. Given the broad cytoprotective properties of the heat shock response, there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. These findings have opened up new neuroprotective strategies, as molecules inducing this defense mechanism can be a therapeutic target to minimize the deleterious consequences associated with accumulation of conformationally aberrant proteins to oxidative stress, such as in neurodegenerative disorders and brain aging, with resulting prolongation of a healthy life span.     

热休克蛋白90在硫化氢抗化学性缺氧诱导心肌细胞氧化应激损伤中的作用

目的： 探讨热休克蛋白90（HSP90）在硫化氢（H2S）对抗化学性低氧模拟剂氯化钴（CoCl2）诱导H9c2心肌细胞氧化应激损伤中的作用。方法： 应用CoCl2处理H9c2心肌细胞,建立化学性缺氧损伤心肌细胞的实验模型。在CoCl2处理H9c2心肌细胞前30 min,把硫氢化钠（NaHS,H2S的供体）加入培养基中,作为预处理。应用高效液相色谱法（HPLC）检测细胞内ATP的含量;罗丹明123（Rh123）染色荧光显微镜照相检测线粒体膜电位（MMP）;超氧化物歧化酶（SOD）检测试剂盒检测SOD活性;免疫印迹法（Western blotting）检测血红素氧合酶-1（HO-1）的表达。结果： 600 μmol/L CoCl2明显地降低H9c2心肌细胞内SOD活性、ATP水平及MMP,并增加HO-1表达。400 μmol/L NaHS 预处理可显著地抑制CoCl2诱导的细胞毒性及氧化应激反应,使SOD活性、ATP水平及MMP提高,HO-1表达减少。热休克蛋白90抑制剂17-丙烯胺基-17去甲氧基格尔德霉素（17AAG）能明显地阻断H2S对CoCl2诱导的细胞毒性和氧化应激反应的抑制作用,使细胞内ATP水平及MMP降低,HO-1表达增多,但对SOD活性的影响不明显。结论： 热休克蛋白90可通过抑制化学性缺氧引起的氧化应激反应来介导H2S的心肌保护作用。     

Expression and localization of heat shock proteins in rat basophilic leukemia cells: differential modulation by degranulation,thermal or oxidative stress

BACKGROUND: Rat basophilic leukemia (RBL-2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell-dependent reactivity, we examined the adaptive responses of RBL-2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke. METHODS: HSP were determined by flow cytometry and immunocytochemistry. Degranulation was confirmed as the release of beta-hexosaminidase, determined spectrophotometrically, and by electron microscopy experiments. RESULTS: We found that RBL-2H3 cells respond to heat shock or oxidative injury by the synthesis of both the inducible 72 kDa HSP (Hsp70), and the oxidation-specific 32 kDa heme oxygenase (HO)-1. Heat shock induced mainly Hsp70 in a cell growth-dependent manner, whereas oxidative stress induced mainly HO-1 in a cell growth-independent manner. However, heat shock or oxidative stress had no significant effects on degranulation. CONCLUSION: Stress-mediated synthesis of HSP was not associated with RBL-2H3 degranulation and likewise, degranulation did not induce HSP.     

Effect of Vitamin C Supplements on Antioxidant Defence and Stress Proteins in Human Lymphocytes and Skeletal Muscle

Oxidative stress induces adaptations in the expression of protective enzymes and heat shock proteins (HSPs) in a variety of tissues. We have examined the possibility that supplementation of subjects with the nutritional antioxidant, vitamin C, influences the ability of lymphocytes to express protective enzymes and HSPs following exposure to an exogenous oxidant and the response of skeletal muscle to the physiological oxidative stress that occurs during exercise in vivo . Our hypothesis was that an elevation of tissue vitamin C content would reduce oxidant-induced expression of protective enzymes and HSP content. Lymphocytes from non-supplemented subjects responded to hydrogen peroxide with increased activity of superoxide dismutase (SOD) and catalase, and HSP60 and HSP70 content over 48 h. Vitamin C supplementation at a dose of 500 mg day⁻¹ for 8 weeks was found to increase the serum vitamin C concentration by ∼50 %. Lymphocytes from vitamin C-supplemented subjects had increased baseline SOD and catalase activities and an elevated HSP60 content. The SOD and catalase activities and the HSP60 and HSP70 content of lymphocytes from supplemented subjects did not increase significantly in response to hydrogen peroxide. In non-supplemented subjects, a single period of cycle ergometry was found to significantly increase the HSP70 content of the vastus lateralis. Following vitamin C supplementation, the HSP70 content of the muscle was increased at baseline with no further increase following exercise. We conclude that, in vitamin C-supplemented subjects, adaptive responses to oxidants are attenuated, but that this may reflect an increased baseline expression of potential protective systems against oxidative stress (SOD, catalase and HSPs).     

Increased expression of Leydig cell haem oxygenase-1 preserves spermatogenesis in varicocele

BACKGROUND: Oxidative stress is involved in the pathogenesis of testicular disorders. Haem oxygenase-1 (HO-1) plays an important cytoprotective role against oxidative stress. We investigated the presence of oxidative stress, represented by generation of 4-hydroxy-2-nonenal (4-HNE)-modified proteins, and expression of HO-1 in varicocele testes of human. METHODS: Thirty testicular biopsies from patients with left varicocele and 10 from patients with normal spermatogenesis were included. Generation of 4-HNE-modified proteins was examined as a marker of oxidative stress. Expression of HO-1 was assessed by western blotting and immunohistochemistry. The expression was compared with clinico-pathological parameters. RESULTS: Increased generation of 4-HNE-modified proteins was observed in varicocele testes. HO-1 expression was significantly correlated with varicocele grade (P < 0.01 in grade 2 and 3 compared to control) and expression of 4-HNE-modified proteins (r = 0.508, P < 0.01). The immunoreactivity was increased in Leydig cells in varicocele testes. There were significant correlations between age, total motile sperm count, Johnsen's mean score and HO-1:4-HNE-modified protein ratio (r = 0.206, 0.405 and 0.408, P = 0.027, 0.027 and 0.025 respectively). CONCLUSIONS: In testes with varicocele, there are increases in 4-HNE-modified proteins, suggesting that oxidative stress is present. Increased HO-1 expression, mainly in Leydig cells, is considered to protect the cells against oxidative stresses in varicocele testes.     

Heat shock protein 27 expression in patients with chronic liver damage

The aim of this study was to evaluate a possible relationship between lymphomonocyte expression of heat shock proteins (HSP) 60/27 and plasma levels of pro-inflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and markers of antioxidant/oxidative status [glutathione (GSH), alpha glutathione-S-transferase activity (alpha GST), malonyldialdeyde (MDA), 4-hydroxinonenal (4-HNE), and S-nitrosothiols (S-NO)] in patients with chronic liver diseases. Entered into the study were 47 subjects: 10 healthy controls, 16 patients with HCV-related chronic hepatitis (CH), and 16 patients with HCV-related and 5 with alcohol-related liver cirrhosis (10 Child A and 11 Child B+C). HSP60 was clearly expressed only in 5% of patients and lowly in the control group. HSP27 was clearly expressed in 46.7% of CH and 71.4% of cirrhotic patients but was lowly present in healthy subjects. A significant difference was found between patients with a low expression of HSP27 (negative patients) and those with a high HSP27 expression (positive patients) of plasma levels both of antioxidants (GSH, p < 0.05), and of markers of enhanced production of free radicals and cytokines (alpha GST, TNF-alpha and IL-6, p < 0.05; MDA, 4-HNE and S-NO, p < 0.01) as well as for alcohol use and degree of liver impairment. The present data are the first showing that, particularly in conditions of enhanced oxidative stress, lymphomonocytes from liver disease patients present an increased expression of HSP27.     

Induction of heat shock protein 72 synthesis by endogenous tumor necrosis factor via enhancement of the heat shock element-binding activity of heat shock factor 1

Endogenous tumor necrosis factor (enTNF) acts as a resistance factor against cytotoxicity caused by heat by inducing manganous superoxide dismutase (MnSOD), thereby scavenging reactive oxygen free radicals. On the other hand, it is also well known that heat shock proteins (HSP) which are induced by heat stress behave as cytoprotective factor against this stress. However, the relationship of these two resistance factors is not elucidated yet. In the present study, we therefore proposed the possibility that enTNF enhances HSP72 expression. Heat-sensitive L-M (mouse tumorigenic fibroblast) cells, which normally do not express enTNF, were transfected with a nonsecretory-type human TNF-α expression vector to produce enTNF. Stable transfectants showed resistance to heat treatment and an increase of HSP72 expression. Conversely, when HeLa (human uterine cervical cancer) cells, which normally produce an appreciable amount of enTNF, were transfected with an antisense TNF-α mRNA expression vector to inhibit enTNF synthesis, their heat sensitivity was enhanced and HSP72 expression was reduced by half. Although enTNF caused no difference in the level of heat shock factor (HSF) 1 in these cells, enTNF expression correlated well with the binding activity of HSF-1 to a ³²P-labeled synthetic oligonucleotide containing the human heat shock element (HSE). These results indicate that enTNF participates not only in intrinsic resistance against heat via induction of MnSOD but also via enhancement of the HSE-binding activity of HSF 1 followed by augmentation of HSP72 expression.     

Expression of heat shock protein 47 is increased in remnant kidney and correlates with disease progression

Glomerulosclerosis is characterized by accumulation of the mesangial extracellular matrix, including type I and IV collagen. The processing for the collagens in the glomeruli may play a critical role for development of glomerulosclerosis. We examined the expression of heat shock protein 47 (HSP47), a collagen-binding molecular chaperone in the progresive glomerulosclerosis model. Subtotally nephrectomized rats, unlike sham-operated rats, developed focal and segmental glomerulosclerosis. Immunological staining demonstrated an increased expression of HSP47 which paralleled the expression of type I and IV collagen in the glomeruli of the nephrectomized rats as the glomerulosclerosis developed. The mRNA levels encoding type I and type IV collagen and HSP47 were increased 3.4 fold, 3.6 fold and 2.8 fold, respectively, at week 7 after nephrectomy. By in situ hybridization, the expression of HSP47 mRNA was determined to be localized to the glomeruli with segmental sclerosis. These results suggest that HSP47 may play a central role in the process of extracellular matrix accumulation during the development of glomerulosclerosis.