Potential roles of membrane fluidity and ceramide in hyperthermia and alcohol stimulation of TRAIL apoptosis

We recently reported that a mild heat shock induces a long lasting stimulation of TRAIL-induced apoptosis of leukemic T-lymphocytes and myeloid cell lines, but not normal T-lymphocytes, which correlates with an enhanced ability of TRAIL to recognize its receptors. As shown here, this phenomenon could be inhibited by the xanthogenate agent D609, a sphingomyelin/ceramide pathway inhibitor. A caspase-dependent and D609-sensitive two-fold increase in ceramide level was elicited by heat shock plus TRAIL combined treatment. One day after heat shock, a similar increase in ceramide was induced by TRAIL. Sphingolipids/ceramides are known to regulate membrane integrity, and heat shock increases membrane fluidity. In this regard, the heat shock plus TRAIL combined treatment resulted in a D609-sensitive membrane fluidization which was far more intense than that induced by heat shock only. We also report that membrane fluidizers, that mimic the effect of heat shock, such benzyl alcohol and ethanol, potently stimulated TRAIL-induced apoptosis. As heat shock, these alcohols increased, in a D609-sensitive manner, membrane fluidity in the presence of TRAIL, the recognition of TRAIL death receptors, and ceramide levels. These results suggest that stress agents that trigger ceramide production and an overall increase in membrane fluidity are stimulators of TRAIL apoptosis.     

Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii

A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat‐stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat‐induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat‐induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered.     

Changes in adsorption and permeability of mitoxantrone on plasma membrane of BCRP/MXR resistant cells

A selective analysis of adsorbed mitoxantrone (MTX) was performed by surface-enhanced Raman scattering (SERS) at the range of cellular membrane. Disruption of the membrane fluidity was carried out to appraise changes in membrane adsorption of MTX and drug uptake in sensitive (HCT-116 S) and resistant BCRP/MXR (HCT-116 R) cells. Based on spectral MTX modifications, micro-SERS spectroscopy discriminated clearly drug adsorption phenomena on plasma membrane from drug in solution. A 3-fold higher SERS intensity of MTX for HCT-116 R was observed concluding to a higher drug adsorption on resistant membrane. The increase of membrane fluidity with benzyl alcohol (BA) or chloroform (CF) resulted in a 3-fold decrease of MTX adsorption on HCT-116 R, exclusively. BA and CF improved intracellular accumulation of MTX (e.g., 823 and 191 pmol MTX/10(6) HCT-116 R incubated with or without BA). At 4 degrees C, drug accumulation measurements showed a decrease of MTX permeability in resistant membrane (42 pmol MTX/10(6) cells), restored with fluidizers (e.g., 342 pmol MTX/10(6) cells with BA). Fluorescence confocal microscopy involved an exclusive MTX emission around the plasma membrane of resistant cells whereas fluidizers increased the intracellular uptake of MTX in both cell lines at the same time with less drug emission around the plasma membrane. Changes of the membrane structure of resistant cells should modify both drug adsorption and membrane permeation.     

Rapid increases in inositol trisphosphate and intracellular Ca++ after heat shock

Heat shock (45 degrees C) caused a rapid (less than 1 min) release of inositol trisphosphate from the membranes of HA-1 CHO fibroblasts. The rise in inositol trisphosphate concentration was followed by an increase in intracellular free Ca++. In addition to the heat induced rise in intracellular free Ca++, we observed an increase in 45Ca++ influx following nonlethal heat shock (45 degrees C/10 min). The heat-induced increase in 45Ca++ influx was linearly related to membrane accumulation of phosphatidic acid, phosphoinositide metabolite that may be involved in Ca++ gating. These results suggest that the membrane may be the proximal target of heat shock; stimulation of rapid breakdown of polyphosphoinositides and subsequent increases in intracellular free Ca++ may provide a mechanistic insight into the pleiotropic effects of heat. In addition, the large increases in Ca++ influx could initiate a Ca++ dependent mechanism of thermal cell killing.     

Activation of stress response by ionomycin in rat hepatoma cells

All living systems respond to a variety of stress conditions by inducing the synthesis of stress or heat shock proteins (HSPs), which transiently protect cells. HSP synthesis was preceded by an increase in intracellular free calcium concentration [(Ca(2+))i]. In this study, we show that Ca(2+) ionophore, ionomycin, induced an immediate increase in intracellular free Ca(2+) and examined how this increase affects heat shock response in rat hepatoma cell line H4II-E-C3. Results indicate that incubating H4II-E-C3 cells with 0.3 microM ionomycin at 37 degrees C for 15 min results in the induction of HSP 70 in both Ca(2+)-containing and Ca(2+)-free medium. Associated with this increase in free Ca(2+) is an in vivo change in membrane organization and activation of signaling molecules like ERKS and SAPKs/JNK. In Ca(2+) containing medium HSP 70 induction mediated by HSF-HSE interaction was faster upon ionomycin treatment as compared to heat shock. Our results show that ionomycin, at sub lethal concentration, increases intracellular free Ca(2+) concentration, activates SAPK/JNK and HSF-HSE interaction, and induces HSP 70 synthesis.     

Lipidomics reveals membrane lipid remodelling and release of potential lipid mediators during early stress responses in a murine melanoma cell line

Membranes are known to respond rapidly to various environmental perturbations by changing their composition and microdomain organization. In previous work we showed that a membrane fluidizer benzyl alcohol (BA) could mimic the effects of heat stress and enhance heat shock protein synthesis in different mammalian cells. Here we explore heat- and BA-induced stress further by characterizing stress-induced membrane lipid changes in mouse melanoma B16 cells. Lipidomic fingerprints revealed that membrane stress achieved either by heat or BA resulted in pronounced and highly specific alterations in lipid metabolism. The loss in polyenes with the concomitant increase in saturated lipid species was shown to be a consequence of the activation of phopholipases (mainly phopholipase A₂ and C). A phospholipase C–diacylglycerol lipase–monoacylglycerol lipase pathway was identified in B16 cells and contributed significantly to the production of several lipid mediators upon stress including the potent heat shock modulator, arachidonic acid. The accumulation of cholesterol, ceramide and saturated phosphoglyceride species with raft-forming properties observed upon both heat and BA treatments of B16 cells may explain the condensation of ordered plasma membrane domains previously detected by fluorescence microscopy and may serve as a signalling platform in stress responses or as a primary defence mechanism against the noxious effects of stresses.     

Heat stress stimulates inositol trisphosphate release and phosphorylation of phosphoinositides in CHO and Balb C 3T3 cells

Exposure of eukaryotic cells to elevated temperature leads to profound switches in cell metabolism and gene expression which may be involved in cellular homeostatic mechanisms. We have investigated the effect of heat shock (45 degrees C) on the metabolism of the phosphoinositides, a class of phospholipids involved in the function of Ca2+ -linked membrane receptors. Heat shock led to stimulation of phosphoinositide turnover in HA1-CHO and Balb C 3T3 cells, resulting in the rapid accumulation of inositol trisphosphate (IP3). Mitogenic and alpha 1 adrenergic stimulation, with serum or phenylephrine, led to similar increases in IP3. Heat shock also caused rapid increase in phosphorylation of polyphosphoinositides (PPI). Prolonged exposure to heat greater than 15 min at 45 degrees C led to progressive cellular toxicity which was associated with depletion of PPI. This decline in PPI concentration appeared to result from inhibition of PPI resynthesis. In this respect, heat may resemble some other types of cellular stresses in stimulating membrane phospholipases to deplete classes of membrane phospholipids. The induction of PPI turnover may, therefore, be involved in both pleiotropic responses to brief heat shock and toxicity resulting from prolonged thermal stress.     

Cold-shock regulation of the Arabidopsis TCH genes and the effects of modulating intracellular calcium levels.

The Arabidopsis TCH genes, which encode calmodulin-related proteins and a xyloglucan endotransglycosylase, are shown to be up-regulated in expression following cold shock. We investigated a possible role of fluctuations in intracellular calcium ion concentrations ([Ca2+]) in the cold-shock-induced TCH gene expression. Transgenic plants harboring the apoaequorin gene were generated to monitor [Ca2+]) and to test the necessity of cold-induced [Ca2+] increases for TCH expression. Cold-shock-induced [Ca2+] increases can be blocked by La3+ and Gd3+, putative plasma membrane Ca2+ channel blockers, and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an extracellular Ca2+ chelator. Cold-shock-induced expression of the TCH genes is inhibited by levels of La3+, Gd3+, and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, that have been shown to block [Ca2+] increases. These data support the hypotheses that (a) intracellular [Ca2+] increases following cold shock require extracellular Ca2+ and may derive from a Ca2+ influx mediated by plasmalemma Ca2+ channels, and (b) cold up-regulation of expression of at least a subset of the TCH genes requires an intracellular [Ca2+] increase. The inhibitors are also shown to have stimulus-independent effects on gene expression, providing strong evidence that these commonly used chemicals have more complex effects than generally reported.     

Pre-existing inflammatory state compromises heat tolerance in rats exposed to heat stress

This study investigated the roles of endotoxemia and heat-induced tissue damage in the pathology of heat stroke. In groups of eight, male Wistar rats were treated with heat exposure only (HE), or heat exposure with turpentine (T+HE), dexamethasone (D+HE), and turpentine and dexamethasone combined (TD+HE). The rats remained sedated for 2 h after receiving the respective treatments, followed by heat exposure until the core temperature (T(c)) was 42 degrees C for 15 min; control rats received turpentine (T), dexamethasone (D), and turpentine and dexamethasone (TD) without heat stress. Blood samples were collected before treatment (baseline I), after 2 h of passive rest (baseline II), at T(c) 40 degrees C (T40), and 15 min after achieving T(c) 42 degrees C (T42). No rats died in the nonheat-stressed groups. Survival rate was lowest in the TD+HE rats (37.5%), followed by the HE (62.5%), T+HE (75%), and D+HE (100%) rats (P < 0.05). The duration of survival at T42 degrees C was shortest in the TD+HE rats (9.9 +/- 6.2 min) (P < 0.01), followed by the T+HE (11.3 +/- 6.1 min) and the HE (12.2 +/- 4 min) (P < 0.05) rats. The increase in plasma IL-6 concentrations was highest in the T+HE (352%) and HE (178%) rats (P < 0.05). D+HE treatment suppressed the increases in plasma aspartate transaminase, alanine aminotransferase, and IL-6 and LPS concentrations during severe heat stress. Heat stroke can be triggered by endotoxemia or heat-induced tissue damage, and preexisting inflammation compromises heat tolerance, whereas blocking endotoxemia increases heat tolerance.     

Thermal stress responses in Antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR

Living organisms have some common and unique strategies to response to thermal stress. However, the amount of data on thermal stress response of certain organism is still lacking, especially psychrophilic yeast from the extreme habitat. Therefore, it is not known whether psychrophilic yeast shares the common responses of other organisms when exposed to thermal stresses. In this work, the cold shock and heat shock responses in Antarctic psychrophilic yeast Glaciozyma antarctica PI12 which had an optimal growth temperature of 12 °C were determined. The expression levels of 14 thermal stress-related genes were measured using real-time quantitative PCR (qPCR) when the yeast cells were exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions. The expression profiles of the 14 genes at these three temperatures varied indicating that these genes had their specific roles to ensure the survival of the yeast. Under cold shock condition, the afp4 and fad genes were over-expressed possibly as a way for the G. antarctica PI12 to avoid ice crystallization in the cell and to maintain the membrane fluidity. Under the heat shock condition, hsp70 was significantly up-regulated possibly to ensure the proteins fold properly. Among the six oxidative stress-related genes, MnSOD and prx were up-regulated under cold shock and heat shock, respectively, possibly to reduce the negative effects caused by oxidative stress. Interestingly, it was found that the trehalase gene, nth1 that plays a role in degrading excess trehalose, was down-regulated under the heat shock condition possibly as an alternative way to accumulate trehalose in the cells to protecting them from being damaged.     

Purinergically induced membrane fluidization in ciliary cells: characterization and control by calcium and membrane potential.

To examine the role of membrane dynamics in transmembrane signal transduction, we studied changes in membrane fluidity in mucociliary tissues from frog palate and esophagus epithelia stimulated by extracellular ATP. Micromolar concentrations of ATP induced strong changes in fluorescence polarization, possibly indicating membrane fluidization. This effect was dosage dependent, reaching a maximum at 10-microM ATP. It was dependent on the presence of extracellular Ca2+ (or Mg2+), though it was insensitive to inhibitors of voltage-gated calcium channels. It was inhibited by thapsigargin and by ionomycin (at low extracellular Ca2+ concentration), both of which deplete Ca2+ stores. It was inhibited by the calcium-activated potassium channel inhibitors quinidine, charybdotoxin, and apamine and was reduced considerably by replacement of extracellular Na+ with K+. Hyperpolarization, or depolarization, of the mucociliary membrane induced membrane fluidization. The degree of membrane fluidization depended on the degree of hyperpolarization or depolarization of the ciliary membrane potential and was considerably lower than the effect induced by extracellular ATP. These results indicate that appreciable membrane fluidization induced by extracellular ATP depends both on an increase in intracellular Ca2+, mainly from its internal stores, and on hyperpolarization of the membrane. Calcium-dependent potassium channels couple the two effects. In light of recent results on the enhancement of ciliary beat frequency, it would appear that extracellular ATP-induced changes both in ciliary beat frequency and in membrane fluidity are triggered by similar signal transduction pathways.     

Alpha-adrenergically mediated changes in membrane lipid fluidity and Ca2/ binding in isolated rat liver plasma membranes

Noradrenaline (0.1-5 microM, in the presence of 5 microM propranolol to block beta-receptors), ATP (100 microM) and angiotensin II (0.1 microM), which are thought to increase cytosolic Ca2+ concentration by mobilizing Ca2+ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the alpha-antagonists phenoxybenzamine (50 microM) and phentolamine (1 microM). The response to a maximal dose of noradrenaline (5 microM) and that to ATP (100 microM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 microM) along with the foreign amphiphile Na+-oleate (1-30 microM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg2+, reducing free Ca2+ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca2+ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 microM) and angiotensin II (0.1 microM) also promoted a small release of 45Ca2+ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the alpha-antagonist phentolamine (5 microM). It is proposed that noradrenaline, via alpha-adrenergic receptors and other Ca2+ -mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca2+ bound to phospholipids, removing thus the mechanical constraints brought about by this ion.     

The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

The heat shock response (HSR) is a highly conserved molecular response to various types of stresses, including heat shock, during which heat-shock proteins (Hsps) are produced to prevent and repair damages in labile proteins and membranes. In cells, protein unfolding in the cytoplasm is thought to directly enable the activation of the heat shock factor 1 (HSF-1), however, recent work supports the activation of the HSR via an increase in the fluidity of specific membrane domains, leading to activation of heat-shock genes. Our findings support the existence of a plasma membrane-dependent mechanism of HSF-1 activation in animal cells, which is initiated by a membrane-associated transient receptor potential vanilloid receptor (TRPV). We found in various non-cancerous and cancerous mammalian epithelial cells that the TRPV1 agonists, capsaicin and resiniferatoxin (RTX), upregulated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70 and Hsp90 respectively, while the TRPV1 antagonists, capsazepine and AMG-9810, attenuated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70, Hsp90, respectively. Capsaicin was also shown to activate HSF-1. These findings suggest that heat-sensing and signaling in mammalian cells is dependent on TRPV channels in the plasma membrane. Thus, TRPV channels may be important drug targets to inhibit or restore the cellular stress response in diseases with defective cellular proteins, such as cancer, inflammation and aging.     

A role for the thermal environment in defining co-stimulation requirements for CD4+ T cell activation

Maintenance of normal core body temperature is vigorously defended by long conserved, neurovascular homeostatic mechanisms that assist in heat dissipation during prolonged, heat generating exercise or exposure to warm environments. Moreover, during febrile episodes, body temperature can be significantly elevated for at least several hours at a time. Thus, as blood cells circulate throughout the body, physiologically relevant variations in surrounding tissue temperature can occur; moreover, shifts in core temperature occur during daily circadian cycles. This study has addressed the fundamental question of whether the threshold of stimulation needed to activate lymphocytes is influenced by temperature increases associated with physiologically relevant increases in temperature. We report that the need for co-stimulation of CD4+ T cells via CD28 ligation for the production of IL-2 is significantly reduced when cells are exposed to fever-range temperature. Moreover, even in the presence of sufficient CD28 ligation, provision of extra heat further increases IL-2 production. Additional in vivo and in vitro data (using both thermal and chemical modulation of membrane fluidity) support the hypothesis that the mechanism by which temperature modulates co-stimulation is linked to increases in membrane fluidity and membrane macromolecular clustering in the plasma membrane. Thermally-regulated changes in plasma membrane organization in response to physiological increases in temperature may assist in the geographical control of lymphocyte activation, i.e., stimulating activation in lymph nodes rather than in cooler surface regions, and further, may temporarily and reversibly enable CD4+ T cells to become more quickly and easily activated during times of infection during fever.     

热休克转录因子1与机体耐热性的相关研究

热休克转录因子1(HSF1)能够启动各种热休克蛋白基因的诱导表达,这对防止机体免受热应激损伤具有重要的意义。从HSF1的结构、功能及活化过程等几个方面阐述了HSF1的生理特征及其与机体耐热性能之间的关系。