Glutamine's protection against cellular injury is dependent on heat shock factor-1

Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. This study tested the hypothesis that GLN-mediated activation of the HSP pathway via heat shock factor-1 (HSF-1) is responsible for cellular protection. Wild-type HSF-1 (HSF-1^+/+) and knockout (HSF-1^–/–) mouse fibroblasts were used in all experiments. Cells were treated with GLN concentrations ranging from 0 to 16 mM and exposed to heat stress injury in a concurrent treatment model. Cell viability was assayed with phenazine methosulfate plus tetrazolium salt, HSP-70, HSP-25, and nuclear HSF-1 expression via Western blot analysis, and HSF-1/heat shock element (HSE) binding via EMSA. GLN significantly attenuated heat-stress induced cell death in HSF-1^+/+ cells in a dose-dependent manner; however, the survival benefit of GLN was lost in HSF-1^–/– cells. GLN led to a dose-dependent increase in HSP-70 and HSP-25 expression after heat stress. No inducible HSP expression was observed in HSF-1^–/– cells. GLN increased unphosphorylated HSF-1 in the nucleus before heat stress. This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation. knockout cells; amino acid; heat stress mechanism     

Glutamine's protection against sepsis and lung injury is dependent on heat shock protein 70 expression

Glutamine (GLN) has been shown to protect against inflammatory injury and illness in experimental and clinical settings. The mechanism of this protection is unknown; however, laboratory and clinical trial data have indicated a relationship between GLN-mediated protection and enhanced heat shock protein 70 (HSP70) expression. The aim of this study was to examine the hypothesis that GLN's beneficial effect on survival, tissue injury, and inflammatory response after inflammatory injury is dependent on HSP70 expression. Mice with a specific deletion of the HSP70 gene underwent cecal ligation and puncture (CLP)-induced sepsis and were treated with GLN (0.75 g/kg) or a saline placebo 1 h post-CLP. Lung tissue NF-kappaB activation, inflammatory cytokine response, and lung injury were assessed post-CLP. Survival was assessed for 5 days post-CLP. Our results indicate that GLN administration improved survival in Hsp70(+/+) mice vs. Hsp70(+/+) mice not receiving GLN; however, GLN exerted no survival benefit in Hsp70(-/-) mice. This was accompanied by a significant decrease in lung injury, attenuation of NF-kappaB activation, and proinflammatory cytokine expression in GLN-treated Hsp70(+/+) mice vs. Hsp70(+/+) mice not receiving GLN. In the Hsp70(-/-) mice, GLN's attenuation of lung injury, NF-kappaB activation, and proinflammatory cytokine expression was lost. These results confirm our hypothesis that HSP70 expression is required for GLN's effects on survival, tissue injury, and the inflammatory response after global inflammatory injury.     

Determinants of glutamine dependence and utilization by normal and tumor-derived breast cell lines

A continual supply of the amino acid glutamine (GLN) may be necessary for cancerous cell growth. GLN plays a central role in multiple metabolic pathways and has long been considered an essential component of tissue culture media. However, the GLN requirements of tumor cell lines and the factors that determine a cell's need for GLN have not been comprehensively studied. Also, it remains unclear how various metabolic pathways contribute to GLN consumption. In the present study, possible determinants of GLN metabolism were examined in seven breast cell lines, two derived from immortalized normal tissue and five of tumor origin. These cells exhibited different dependencies on media GLN concentration for growth and a wide range of GLN utilization rates. GLN uptake was facilitated by a single, common transporter functionally defined as System ASC. However, the affinities for GLN exhibited by this transporter differed appreciably between cell lines. Furthermore, the concentration at which media GLN became a limiting factor for cellular proliferation correlated with transporter affinity. The origin of the cell lines was not a determinant of GLN metabolism because immortalized cells of nontumor origin exhibited GLN dependence and utilization rates comparable to those of tumor-derived cells. The rates of CO₂ production from GLN were similar for each cell lines. Rates of GLN disappearance and glutamate appearance in media were strongly correlated, with 32–80% of media GLN converted to glutamate. Both rates were directly affected by media cystine concentration, suggesting that a large portion of glutamate efflux was coupled with cystine import through the amino acid transport system X These results demonstrated that cell growth is a function of GLN influx and suggest that GLN is used to supply glutamate and cystine, perhaps for glutathione synthesis. J. Cell. Physiol. 176:166–178, 1998. © 1998 Wiley-Liss, Inc.     

Molecular mechanisms of glutamine action

Glutamine is the most abundant free amino acid in the body and is known to play a regulatory role in several cell specific processes including metabolism (e.g., oxidative fuel, gluconeogenic precursor, and lipogenic precursor), cell integrity (apoptosis, cell proliferation), protein synthesis, and degradation, contractile protein mass, redox potential, respiratory burst, insulin resistance, insulin secretion, and extracellular matrix (ECM) synthesis. Glutamine has been shown to regulate the expression of many genes related to metabolism, signal transduction, cell defense and repair, and to activate intracellular signaling pathways. Thus, the function of glutamine goes beyond that of a simple metabolic fuel or protein precursor as previously assumed. In this review, we have attempted to identify some of the common mechanisms underlying the regulation of glutamine dependent cellular functions.     

Glutamine deficiency in extracellular fluid exerts adverse effects on protein and amino acid metabolism in skeletal muscle of healthy,laparotomized, and septic rats

Characteristic feature of critical illness, such as trauma and sepsis, is muscle wasting associated with activated oxidation of branched-chain amino acids (valine, leucine, isoleucine) and enhanced release of glutamine (GLN) to the blood. GLN consumption in visceral tissues frequently exceeds its release from muscle resulting in GLN deficiency that may exert adverse effects on the course of the disease. In the present study, we investigated the effects of GLN depletion in extracellular fluid on GLN production and protein and amino acid metabolism in skeletal muscle of healthy, laparotomized, and septic rats. Cecal ligation and puncture (CLP) was used as a model of sepsis. After 24 h, soleus muscle (SOL, slow-twitch, red muscle) and extensor digitorum longus (EDL, fast-twitch, white muscle) were isolated and incubated in a medium containing 0.5 mM GLN or without GLN. L-[1-¹⁴C]leucine was used to estimate protein synthesis and leucine oxidation, 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. CLP increased GLN release from muscle, protein breakdown and leucine oxidation, and decreased protein synthesis. The effects were more pronounced in EDL. Alterations induced by laparotomy were similar to those observed in sepsis, but of a lower extent. GLN deficiency in medium enhanced GLN release and decreased intramuscular GLN concentration, decreased protein synthesis in muscles of intact and laparotomized rats, and enhanced leucine oxidation in SOL of intact and protein breakdown in SOL of laparotomized rats. It is concluded that (1) fast-twitch fibers are more sensitive to septic stimuli than slow-twitch fibers, (2) extracellular GLN deficiency may exert adverse effects on protein and amino acid metabolism in skeletal muscle, and (3) muscles of healthy and laparotomized animals are more sensitive to GLN deficiency than muscles of septic animals.     

Glutamine regulates Caco-2 cell tight junction proteins

Intestinal epithelial tight junction (TJ) barrier dysfunction may lead to inflammation and mucosal injury. Glutamine (GLN) plays a role in maintenance of intestinal barrier function in various animal models and critically ill humans. Recent evidence from intestinal cell monolayers indicates that GLN maintains transepithelial resistance and decreases permeability. The mechanisms of these effects remain undefined. We hypothesized that GLN affects proteins involved in the intercellular junctional complex. GLN availability was controlled in Caco-2 monolayers by addition to the medium and treatment with methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Expression of TJ proteins, claudin-1, occludin, and zonula occluden (ZO)-1 was measured by immunoblotting. Localization of TJ proteins was evaluated by immunofluorescence light microscopy. Structure of TJ was determined by transmission electron microscopy (TEM). Deprivation of GLN decreased claudin-1, occludin, and ZO-1 protein expression and caused a disappearance of perijunctional claudin-1 and a reduction of occludin but had no effect on ZO-1. TEM revealed that MSO-treated cells in the absence of GLN formed irregular junctional complexes between the apical lateral margins of adjoining cells. These findings indicate that TJ protein expression and cellular localization in Caco-2 cell monolayers rely on GLN. This mechanism may similarly relate to GLN-mediated modulation of intestinal barrier function in stressed animals and humans.     

Antioxidant compounds EGB-761 and BN-520 21 attenuate heat shock protein (HSP 72 kD) response, edema and cell changes following hyperthermic brain injury

Influence of the extract of Gingko biloba (EGB-761) and one of its constituent Gingkolide B (BN-52021) on hyperthermia induced cellular damage and heat shock protein (HSP 72kD) response was examined in a rat model. Rats subjected to 4h heat stress at 38 degrees C in a biological oxygen demand (BOD) incubator (relative humidity 50-55%, wind velocity 20-25cm/sec) resulted in profound edema and cell injury in many parts of the cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus and brain stem. Immunostaining of HSP 72 kD showed marked upregulation in the damaged and distorted neurons located within the edematous area. Pretreatment with EGB-761 (50mg/kg/day, p.o.) and BN-520 21 (2mg/kg, p.o.) per day for 5 days significantly reduced HSP expression and attenuated cell damage. Our results show that EGB-761 and its component Gingkolide B (BN-52021) has the capacity to reduce edema and cell injury following hyperthermia and this effect of the compound is somehow associated with a reduction in cellular stress response as evidenced with a reduction in HSP expression.     

HSP60, Bax, apoptosis and the heart

HSP60 has primarily been known as a mitochondrial protein that is important for folding key proteins after import into the mitochondria. It is now clear that a significant amount of HSP60 is also present in the extra-mitochondrial cytosol of many cells. In the heart, this cytosolic HSP60 complexes with Bax, Bak and Bcl-XL, but not with Bcl-2. Reduction in HSP60 expression precipitates apoptosis, but does not alter mitochondrial function. During hypoxia, HSP60 cellular distribution changes, with HSP60 leaving the cytosol, and translocating to the plasma membrane. Total cellular HSP60 does not change until 10 h of reoxygenation; however, release of cytochrome c from the mitochondria occurs prior to reoxygenation, coinciding with the redistribution of HSP60. The changes in HSP60, Bax and cytochrome c during hypoxia can be replicated by ATP depletion. HSP60 has also been shown to accelerate the cleavage of pro-caspase3. Thus, HSP60 has a complex role in apoptosis in the cell. Its binding to Bax under normal conditions suggests a key regulatory role in apoptosis.     

HSP70 and genomic stability

The 70 kDa heat shock proteins (HSP70s) were initially identified by their elevated expression following hyperthermic cell stress, however, these highly conserved proteins also protect critical cellular functions from a wider range of important environmental and physiological stresses. At least one result of HSP70 expression is inhibition of stress induced caspase activation as well as downstream events in the apoptotic cell death pathway. HSP70 have been reported upregulated in tumor cells, selective inhibition of such proteins might be valuable approach to treat cancer. A recent study revealed that cells with inactivated HSP70 displayed telomere instability and high frequency of spontaneous chromosomal aberrations, indicating a possible role for HSP70 proteins in the maintenance of genomic stability.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

HSP70 and Genomic Stability

The 70 kDa heat shock proteins (HSP70) were initially identified by their elevated expression following hyperthermic cell stress, however, these highly conserved proteins also protect critical cellular functions from a wider range of important environmental and physiological stresses. At least one result of HSP70 expression is inhibition of stress induced caspase activation as well as downstream events in the apoptotic cell death pathway. HSP70 have been reported upregulated in tumor cells, selective inhibition of such proteins might be valuable approach to treat cancer. A recent study revealed that cells with inactivated HSP70 displayed telomere instability and high frequency of spontaneous chromosomal aberrations, indicating a possible role for HSP70 proteins in the maintenance of genomic stability.     

Role of reactive oxygen species in injury-induced insulin resistance

Acute insulin resistance is common after injury, infection, and critical illness. To investigate the role of reactive oxygen species (ROS) in critical illness diabetes, we measured hepatic ROS, which rapidly increased in mouse liver. Overexpression of superoxide dismutase 2, which decreased mitochondrial ROS levels, protected mice from the development of acute hepatic insulin resistance. Insulin-induced intracellular signaling was dramatically decreased, and cellular stress signaling was rapidly increased after injury, resulting in the hyperglycemia of critical illness diabetes. Insulin-induced intracellular signaling, activation of stress (c-Jun N-terminal kinase) signaling, and glucose metabolism were all normalized by superoxide dismutase 2 overexpression or by pretreatment with antioxidants. Thus, ROS play an important role in the development of acute hepatic insulin resistance and activation of stress signaling after injury.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes.     

Adaptive responses to the stress induced by hyperthermia or hydrogen peroxide in human fibroblasts

Perturbation of oxidant/antioxidant cellular balance, induced by cellular metabolism and by exogenous sources, causes deleterious effects to proteins, lipids, and nucleic acids, leading to a condition named "oxidative stress" that is involved in several diseases, such as cancer, ischemia-reperfusion injury, and neurodegenerative disorders. Among the exogenous agents, both H(2)O(2) and hyperthermia have been implicated in oxidative stress promotion linked with the activation of apoptotic or necrotic mechanisms of cell death. The goal of this work was to better understand the involvement of some stress-related proteins in adaptive responses mounted by human fibroblasts versus the oxidative stress differently induced by 42 degrees C hyperthermia or H(2)O(2.) The research was developed, switching off inducible nitric oxide synthase (iNOS) expression through antisense oligonucleotide transfection by studying the possible coregulation in the expression of HSP32 (also named HO-1), HSP70, and iNOS and their involvement in the induction of DNA damage. Several biochemical parameters, such as cell viability (MTT assay), cell membrane integrity (lactate dehydrogenase release), reactive oxygen species formation, glutathione levels, immunocytochemistry analysis of iNOS, HSP70, and HO-1 levels, genomic DNA fragmentation (HALO/COMET assay), and transmembrane mitochondrial potential (deltaPsi) were examined. Cells were collected immediately at the end of the stress-inducing treatment. The results, confirming the pleiotropic function of i-NOS, indicate that: (i). HO-1/HSP32, HSP70, and iNOS are finely tuned in their expression to contribute all together, in human fibroblasts, in ameliorating the resistance to oxidative stress damage; (ii). ROS exposure, at least in hyperthermia, in human fibroblasts contributes to growth arrest more than to apoptosis activation; and (iii). mitochondrial dysfunction, in presence of iNOS inhibition seems to be clearly involved in apoptotic cell death of human fibroblasts after H(2)O(2) treatment, but not after hyperthermia.     

Coniferyl Aldehyde Attenuates Radiation Enteropathy by Inhibiting Cell Death and Promoting Endothelial Cell Function

Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function.     

Identification of BC005512 as a DNA damage responsive murine endogenous retrovirus of GLN family involved in cell growth regulation

Genotoxicity assessment is of great significance in drug safety evaluation, and microarray is a useful tool widely used to identify genotoxic stress responsive genes. In the present work, by using oligonucleotide microarray in an in vivo model, we identified an unknown gene BC005512 (abbreviated as BC, official full name: cDNA sequence BC005512), whose expression in mouse liver was specifically induced by seven well-known genotoxins (GTXs), but not by non-genotoxins (NGTXs). Bioinformatics revealed that BC was a member of the GLN family of murine endogenous retrovirus (ERV). However, the relationship to genotoxicity and the cellular function of GLN are largely unknown. Using NIH/3T3 cells as an in vitro model system and quantitative real-time PCR, BC expression was specifically induced by another seven GTXs, covering diverse genotoxicity mechanisms. Additionally, dose-response and linear regression analysis showed that expression level of BC in NIH/3T3 cells strongly correlated with DNA damage, measured using the alkaline comet assay,. While in p53 deficient L5178Y cells, GTXs could not induce BC expression. Further functional studies using RNA interference revealed that down-regulation of BC expression induced G1/S phase arrest, inhibited cell proliferation and thus suppressed cell growth in NIH/3T3 cells. Together, our results provide the first evidence that BC005512, a member from GLN family of murine ERV, was responsive to DNA damage and involved in cell growth regulation. These findings could be of great value in genotoxicity predictions and contribute to a deeper understanding of GLN biological functions.