Alpha-lipoic Acid modulates heat shock factor-1 expression in streptozotocin-induced diabetic rat kidney

Increased oxidative stress and impaired heat shock protein (HSP) synthesis may contribute to diabetic nephropathy. The question of whether 8-week thiol antioxidant alpha-lipoic acid (LA) supplementation modulates HSP response and oxidative stress was studied in the kidney of streptozotocin-induced diabetic (SID) and nondiabetic rats. SID caused a histological mesangial expansion, tubular dilatation, and increased levels of transforming growth factor-beta (TGF-beta), a mediator of glomerulosclerosis. SID increased 4-hydroxynonenal (4-HNE) protein adduct formation, a marker of lipid peroxidation, and heme oxygenase-1 (HO-1), also a marker of oxidative stress. Moreover, SID increased the DNA-binding activity of heat shock factor-1 (HSF-1) and expression of heat shock protein 60 (HSP60). In contrast, LA supplementation partially reversed histological findings of glomerulosclerosis and decreased TGF-beta. LA also increased HSF-1 and decreased HO-1 protein expression, without affecting 4-HNE protein adduct levels. At the mRNA level, LA increased expression of HSF-1, HSP90, and glucose-regulated protein (GRP75) in both control and diabetic animals and HSP72 in SID rats. However, LA supplementation did not affect these HSPs at the protein level. These findings suggest that in addition to its antiglomerulosclerotic effects, LA can induce cytoprotective response in SID.     

Preconditioning of the liver for protecting the hepatic functional reserve

Ischemia reperfusion (I/R) injury of the liver is a major cause of post-surgical hepatic failure. The liver produced heat shock protein 72 (HSP72) 24 approximately 48 hours after heat shock preconditioning. We have found that these livers were provided with ischemic tolerance. The animal survival after I/R injury was improved, and the release of hepatic enzymes during reperfusion was significantly suppressed. As one of the mechanisms of this tolerance, the integrity of hepatic mitochondria was well maintained during I/R in the liver after heat shock preconditioning. In addition, the production of denatured proteins during I/R was significantly reduced in the heat shock group. This fact was well corroborated with the theoretical function of HSP72 as a molecular chaperon. Intra-vital microscopy showed that sinusoidal perfusion failure as well as leukocyte stagnation after ischemia were well suppressed. These beneficial effects of heat shock preconditioning on the hepatic microcirculation seemed to be related with the effective suppression of NF-kappaB activation and subsequent TNF-alpha production during I/R injury. We further studied the effects of geranyl-geranylacetone(GGA) in inducing HSP72 protein. Although GGA could not induce HSP72 by itself, GGA of 200mg/kg facilitated the production of HSP72 after heat shock preconditioning. With the preadministration of GGA, the heat stress to induce HSP72 (stress for preconditioning) could be minimized to one third. Although further investigation is necessary before the use of this strategy in the clinical setting of liver surgery, preconditioning and ischemic tolerance is a promising field of surgical research.     

Inhibition of NaCl-induced heat shock protein 72 expression renders MDCK cells susceptible to high urea concentrations

 Exposure of Madin-Darby canine kidney (MDCK) cells to elevated extracellular NaCl concentrations is associated with increased heat shock protein 72 (HSP72) expression and improved survival of these pretreated cells upon exposure to an additional 600 mM urea in the medium. To establish a causal relationship between HSP72 expression and cell protection against high urea concentrations, two approaches to inhibit NaCl-induced HSP72 synthesis prior to exposure to 600 mM urea were employed. First, the highly specific p38 kinase inhibitor SB203580 was added (100 μM) to the hypertonic medium (600 mosm/kg H₂O by NaCl addition, 2 days of exposure), which significantly reduced HSP72 mRNA abundance and HSP72 content. Survival of these cells after a 24-h urea treatment (600 mM) was markedly curtailed compared with appropriate controls. Second, a pcDNA3-based construct, containing 322 bases of the HSP72 open reading frame in antisense orientation and the geneticine resistance gene, was transfected into MDCK cells. Clones with strong inhibition of HSP72 synthesis and others which express the protein at normal levels (comparable to nontransfected MDCK cells) after heat shock treatment or hypertonic stress were established. When these transformants were subjected to hypertonic stress for 2 days prior to exposure to an additional 600 mM urea for 24 h, cell survival was significantly reduced in those clones in which HSP72 expression was strongly inhibited. These results provide further evidence for the protective function of HSP72 against high urea concentrations in renal epithelial cells. Received: 14 September 1998 / Received after revision and accepted: 16 November 1998     

Differential increase in the expression of heat shock protein family members during sleep deprivation and during sleep

Although sleep is thought to be restorative from prior wakeful activities, it is not clear what is being restored. To determine whether the synthesis of macromolecules is increased in the cerebral cortex during sleep, we subjected C57BL/6 mice to 6 hours of sleep deprivation and then screened the expression of 1176 genes of known function by using cDNA arrays. The expression of the heat shock proteins (HSP), endoplasmic reticulum protein (ERp72) and glucose-regulated protein (GRp78), was among the genes whose expression was significantly elevated in the cortex during sleep deprivation, whereas GRp78 and GRp94 mRNAs were elevated in the cortex during recovery sleep after sleep deprivation, as confirmed by conventional and quantitative real-time polymerase chain reaction and/or Northern analyses. A systematic evaluation of the expression of six heat shock protein family members (ERP72, GRp78, GRp94, HSP27, HSP70-1, and HSP84) in seven brain regions revealed increased mRNA levels in cortex, basal forebrain, hypothalamus, cerebellum and medulla during sleep deprivation, whereas increased mRNA levels during recovery sleep were limited to the cortex and medulla. Immunohistochemical studies identified increased numbers of GRp78-, GRp94-, and ERp72-immunoreactive cells in the dorsal and lateral cortex during sleep deprivation but, during recovery sleep, elevated numbers of these cells were found only in the lateral cortex. In the medulla, increased numbers of GRp94-immunoreactive cells were observed in nucleus tractus solitarius, dorsal motor nucleus of the vagus and the rostroventrolateral medulla during recovery sleep. The widespread increase of heat shock protein family mRNAs in brain during sleep deprivation may be a neuroprotective response to prolonged wakefulness. In contrast, the relatively limited heat shock protein family mRNA expression during recovery sleep may be related to the role of heat shock proteins in protein biogenesis and thus to the restorative function of sleep.     

Hg2+ and Ni2+ alter induction of heat shock protein-72 in THP-1 human monocytes

The biological liabilities that result from the release of metal ions from biomedical alloys, particularly Ni(2+) and Hg(2+), continue to be a concern. Heat-shock proteins (HSP) are a class of molecular chaperones that may be induced under conditions of cellular stress, including oxidative stress. Our hypothesis was that because Hg(2+) and Ni(2+) alter other cellular stress responses such as glutathione levels and cytokine secretion, these metal ions may alter HSP induction in monocytes, which are key cells in the response of tissues to biomedical alloys. THP-1 monocytes were exposed to sublethal concentrations of Hg(2+) or Ni(2+) for 1 h with or without heat stress (43 degrees C), then allowed to recover at 37 degrees C for 2-6 h. HSP72 was measured using immunoblotting with phosphorimage quantification. Hg(2+) exposures of 2-10 micromol/L induced HSP72 without heat stress. With heat stress, HSP72 levels were altered by Hg(2+) versus heat stress alone. The response depended on the concentration of Hg(2+) and the recovery time. Hg(2+) at 10 micromol/L caused uniformly lower HSP72 levels. Ni(2+) exposures of 20-100 micromol/L did not induce HSP72 without heat stress, but significantly altered heat-induced HSP72 expression, with a significant increase in expression over heat alone at 40 and 100 micromol/L. Results from the current study support the hypothesis that these metal ions can, at concentrations relevant to those released from biomedical alloys, modulate HSP expression in human monocytes. The modulation of HSP expression indicates an early sign of cellular stress that may be important to the overall biological response to biomedical alloys containing and releasing these metal ions.     

Effects of heat stress and mechanical stretch on protein expression in cultured skeletal muscle cells

Effects of heat stress, mechanical stretching or a combination of both on the expression of heat shock proteins (HSPs) and total protein level were studied in a culture system. Rat skeletal muscle cells (L6) were cultured on flexible-bottomed culture plates. They were subjected to one of the four following conditions: (1) 97 h incubation at 37 °C, (2) 1 h incubation at 41 °C followed by 96 h incubation at 37 °C, (3) 1 h incubation at 37 °C followed by 96 h cyclic stretching (18% of initial length, 2-s stretch and 4-s release) at 37 °C or (4) 1 h incubation at 41 °C followed by 96 h cyclic stretching at 37 °C. The expression of HSP72 and HSP90 and total protein was determined in the crude homogenates, supernatant and pellets. Cellular protein concentrations in the homogenates and pellets were increased by heat stress and/or mechanical stress (stretch). A cumulative effect of the combination of heating and stretch on the protein concentration in the homogenates and in the pellets was noted. The expressions of HSP72 and HSP90 in the pellets were also increased by heat stress and/or stretch. However, HSP90 in the supernatant did not change following heat stress and/or stretch. The regulation of HSP72 and HSP90 expression in skeletal muscle cells may be closely related to total protein, the abundance of which is also stimulated by mechanical and heat stresses. These observations suggest strongly that heating and passive stretch of muscle may be useful as a means of increasing muscle mass, not only in athletes but also in patients during rehabilitation.     

Heat shock and 5-azacytidine inhibit nitric oxide synthesis and tumor necrosis factor-alpha secretion in activated macrophages

To elucidate the role of stress response during macrophage activation, the effects of heat shock and the amino acid analog, 5-azacytidine on nitric oxide (NO) production, tumor necrosis factor-alpha (TNF-alpha) secretion, and heat shock protein (HSP) synthesis have been studied in murine peritoneal macrophages (C57BL/6). Heat shock (1 hr at 43 degrees C) or 5-azacytidine markedly inhibited the release of NO into the medium from interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-stimulated macrophages. Although heat shock significantly decreased TNF-alpha secretion only at the initiation stage of macrophage stimulation, 5-azacytidine treatment resulted in a more prolonged reduction in the secretion of TNF-alpha. When heat-shocked cells were stimulated with IFN-gamma plus LPS under normal culture conditions at 37 degrees C, the heat shock-induced inhibition of NO release reversed progressively with increasing recovery time. Although the total amount of cellular HSP72 measured by Western blot increased time-dependently over 7 hr, newly synthesized HSP72 measured by [35S]methionine incorporation was evident only after 1 and 3 hr of recovery time after heat shock treatment. At these time points, the lowest nitrite accumulation and TNF-alpha secretion into the medium was evident. It is concluded that signaling pathways related to newly synthesized HSP such as HSP72 are implicated in the down regulation of NO synthesis and TNF-alpha secretion in macrophages.     

Effects of quercetin and sunphenon on responses of cancer cells to heat shock damage.

Quercetin is a flavonoid well known to inhibit growth and heat shock protein (HSP) synthesis of cancer cells. However, sunphenon has been scarcely reported concerning effects on cancer cells. We compared the effects of sunphenon with those of quercetin on the human cholangio-cellular carcinoma cell line (HuCC-T1). Both flavonoids inhibited HuCC-T1 growth in a concentration-dependent manner without reduction of HSP70 and HSP90 expression before heat shock damage. The heat shock reduced the cell viability of the quercetin-treated HuCC-T1, but not that of the sunphenon-treated cells. This inhibitory effect of quercetin on tolerance to heat shock is thought to be due to marked suppression of HSP72. Sunphenon conversely increased HSP72 expression after heat shock. Although neither flavonoid altered HSP90 protein levels before and after heat shock, quercetin delayed the reorganization of filamentous actin (F-actin) during the recovery period after heat shock. Since HSP90 could preserve F-actin structure during stresses, quercetin might affect the interaction between HSP90 and F-actin without influencing HSP90 expression. In conclusion, quercetin would be more useful than sunphenon in combined therapy with hyperthermia for cancer.     

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 stress proteins HSP 72 & HSP 32 in response to endotoxemia

Pretreatment with heat decreases mortality and acute lung injury in the rat septic shock model, presumably by the production of heat shock proteins (HSP). However, endotoxin, a severe cell stresser, has not been shown to induce HSP 70. We investigated the effects of severe endotoxemia on the expression of specific protective stress proteins, including HSP 72 (inducible HSP 70), HSP 32 (heme oxygenase-1), and HSP 90. Fifteen rats received intravenously either 3 mg/kg of endotoxin (E. coli O127:B8 lipopolysaccharide, LPS) (n=9) or saline (n=6). Two hr later the spleen was removed and splenocytes were separated into three groups and analyzed for specific HSP by Western blot. In Group 1, both endotoxin-treated and saline-treated splenocytes were incubated for 3 hr at 37 degrees C. In Group 2, the splenocytes were washed twice, then heat shocked for 30 min at 42 degrees C and subsequently incubated for 2.5 hr at 37 degrees C. In Group 3, splenocytes were washed twice, then incubated for 3.0 hr at 37 degrees C. HSP 90 & HSP 70c (constitutive) were present in all groups. Consistent with observations by others, HSP 72 was not induced in Group 1. HSP 72 was induced in both the saline-treated and endotoxin-treated splenocytes after heating (Group 2). However, in the absence of heat stress, HSP 72 was present in endotoxin-treated but not in saline-treated splenocytes after incubation (Group 3). Conversely, HSP 32, while present in Group 1 splenocytes, was not detected in the endotoxin-treated splenocytes of Group 2 and Group 3, but was present in the saline-treated cells. In conclusion, endotoxemic shock results in induction of HSP 72 and depletion of HSP 32, but only after the cells have been washed and further incubated.     

Experimental pneumococcal meningitis causes central nervous system pathology without inducing the 72-kd heat shock protein.

We examined whether experimental pneumococcal meningitis induced the 72-kd heat shock protein (HSP72), a sensitive marker of neuronal stress in other models of central nervous system (CNS) injury. Brain injury was characterized by vasculitis, cerebritis, and abscess formation in the cortex of infected animals. The extent of these changes correlated with the size of the inoculum (P less than 0.003) and with pathophysiologic parameters of disease severity, i.e., cerebrospinal fluid (CSF) lactate (r = 0.61, P less than 0.0001) and CSF glucose concentrations (r = -0.55, P less than 0.0001). Despite the presence of numerous cortical regions having morphologic evidence of injury, HSP72 was not detected in most animals. When present, only rare neurons were HSP72 positive. Western blot analysis of brain samples confirmed the paucity of HSP72 induction. The lack of neuronal HSP72 expression in this model suggests that at least some of the events leading to neuronal injury in meningitis are unique, when compared with CNS diseases associated with HSP72 induction.