The human oesophageal squamous epithelium exhibits a novel type of heat shock protein response.

The human oesophageal epithelium is subject to damage from thermal stresses and low extracellular pH that can play a role in the cancer progression sequence, thus identifying a physiological model system that can be used to determine how stress responses control carcinogenesis. The classic heat shock protein HSP70 is not induced but rather is down-regulated after thermal injury to squamous epithelium ex vivo; this prompted a longer-term study to address the nature of the heat shock response in this cell type. An ex vivo epithelial culture system was subsequently used to identify three major proteins of 78, 70, and 58 kDa, whose steady-state levels are elevated after heat shock. Two of the three heat shock proteins were identified by mass spectrometric sequencing to be the calcium-calmodulin homologue transglutaminase-3 (78 kDa) and a recently cloned oesophageal-specific gene called C1orf10, which encodes a 53-kDa putative calcium binding protein we have named squamous epithelial heat shock protein 53 (SEP53). The 70-kDa heat shock protein (we have named SEP70) was not identifiable by mass spectrometry, but it was purified and studied immunochemically to demonstrate that it is distinct from HSP70 protein. Monoclonal antibodies to SEP70 protein were developed to indicate that: (a) SEP70 is induced by exposure of cultured cells to low pH or glucose starvation, under conditions where HSP70 protein was strikingly down-regulated; and (b) SEP70 protein exhibits variable expression in preneoplastic Barrett's epithelium under conditions where HSP70 protein is not expressed. These results indicate that human oesophageal squamous epithelium exhibits an atypical heat shock protein response, presumably due to the evolutionary adaptation of cells within this organ to survive in an unusual microenvironment exposed to chemical, thermal and acid reflux stresses.     

Induction of stress proteins in anoxic and hyperthermicSpodoptera frugiperda cells

In this study, we compare stress protein induction in anoxic and hyperthermicSpodoptera frugiperda cells. Anoxia transiently induces a cluster of heat shock proteins at 71 and 72 kDa. This is a subset of a larger group of stress proteins induced by heat shock. Several heat shock proteins reported in this study were previously undetected inS. frugiperda. With these additional proteins, the stress response of hyperthermicS. frugiperda closely resembles that ofDrosophila melanogaster. Prior investigations of stress protein induction during oxygen deprivation focused on mammalian cells. In sharp contrast to these cells, anoxicS. frugiperda cells neither induce glucose-regulated proteins nor suppress the heat shock family of 71/72 kDa proteins. These findings provide insight into the virtually unexplored area of stress protein induction in anoxic insect cells. In addition, they help to explain the effects of oxygen deprivation on heterologous protein yield from virally infected insect cells and to develop an oxygenregulated promoter for stably transformed insect cells.Abbreviations DO dissolved oxygen concentration - GRP's glucose-regulated proteins - HSP's heat shock proteins - ORP's oxygen-regulated proteins - PAGE polyacrylamide gel electrophoresis - Sf9 Spodoptera frugiperda cells     

The dual immunoregulatory roles of stress proteins

Stress proteins (SPs) from the heat shock and glucose-regulated protein families are abundant intracellular molecules that have powerful extracellular roles as immune modulators. Mammalian immune cells encounter both identical (self) SPs and non-identical SPs derived from invading pathogens. Although such extracellular SPs can function as powerful immunological adjuvants, SPs, including Hsp60 and Hsp70, can also attenuate inflammatory disease via apparent effects on immunoregulatory T cell populations. It therefore seems that the immunostimulatory and immunosuppressive potential of extracellular SPs depends on the context in which they are encountered by the cellular immune-response network. Conclusions regarding the immunobiology of these powerful immunomodulatory molecules must therefore take into account their dichotomous properties and their physiological role and importance must be interpreted in the context of the complex in vivo microenvironments in which these proteins exist.     

Inhibition of the heat shock response and synthesis of glucose-regulated proteins in Ca2+-deprived rat hepatoma cells

Rat hepatoma cells become refractory to the induction of heat shock proteins and highly resistant to severe hyperthermia when incubated in Ca2+-free medium. The Ca2+-depleted cells synthesize polypeptides identified as the glucose-regulated proteins, but these proteins do not appear to be directly involved in the inhibition of the heat shock response. The results suggest that a Ca2+-dependent metabolic process is involved in the generation of the heat shock signal and/or mediates a step in the subsequent cascade of events that leads to the induction of heat shock protein synthesis and cell death.     

Antagonistic effects of insulin and dexamethasone on glucose-regulated and heat shock protein synthesis

The present study extends our previous observation (Kasambalides and Lanks, J. Cell. Physiol., 114:93-98, 1983), that dexamethasone inhibits the alterations in heat shock protein (HSP) and glucose-regulated protein (GRP) synthesis caused by glucose deprivation. We now show that insulin, even in the presence of high extracellular glucose concentrations, will induce 95K and 82K GRP synthesis while suppressing 85K and 69K HSP synthesis. Heat shock of insulin-treated cultures causes induction of the 82K GRP rather than the 85K and 69K HSP's. All of the insulin effects are antagonized by dexamethasone. These data suggest that the changes in GRP and HSP synthesis induced by glucose deprivation and heat shock, respectively, may reflect the operation of a normal physiological mechanism that regulates glucose metabolism.     

Parameters for cellular viability and membrane function in chenopodium cells show a specific response of extracellular pH to heat shock with extreme Q10

The effect of brief heat shock on Chenopodium cells was investigated by measuring biochemical parameters for cellular vitality, membrane function and integrity: extracellular pH, release of osmotic compounds, phosphatase, protein and betalain, and cellular reduction of DCPIP and MTT. A threshold temperature was found at 45 degrees C, where release of osmotic compounds, protein and betalain, and reduction of DCPIP and MTT indicate loss of vitality. Extracellular pH and an alkaline phosphatase responded 10-20 degrees C below this threshold, suggesting that extracellular alkalinization, and probably the release of a phosphatase, are part of a specific cellular response to abiotic stress induced by heat shock. The extracellular proton concentration did not increase above 45 degrees C: this may indicate equilibration of gradients driving this process or an inactivation of cellular mechanisms responsible for extracellular alkalinization. The response of extracellular pH to heat shock in Chenopodium cell suspensions was fast, i.e., up to +1 pH in 5 min. Addition of the K+/H+ antiporter nigericin to Chenopodium cells caused an extracellular alkalinization similar to heat shock. The heat shock-induced extracellular alkalinization was characterized by Q10 values for distinct ranges of temperature (Q10 of 56 for 24-31 degrees C, 2.3 for 31-42 degrees C, and 1.0 for 42-50 degrees C). To the author's knowledge, the Q10 of 56 is the highest found up to now. These results suggest that extracellular protons are involved in temperature sensing and signalling in plant cells, probably via a channel-mediated pathway.     

Biochemical characterization of the mammalian stress proteins and identification of two stress proteins as glucose- and Ca2+-ionophore-regulated proteins

Biochemical properties of the heat shock or stress proteins of mammalian cells have been investigated using two-dimensional gel electrophoresis and immunological techniques. Of the major mammalian stress proteins (Mr = 72,000, 73,000, and 90,000) and minor stress proteins (Mr = 80,000, 100,000, and 110,000), the 80- and 90-kDa proteins were found to be phosphoproteins in all cell types examined. The 100-kDa protein was found to incorporate phosphate in only some cell types examined. In studies of the metabolic incorporation of mannose into the stress proteins, only the 100-kDa protein was found to be a glycoprotein. Two of the stress proteins, the 80- and 100-kDa species, were found to be identical with the proteins induced in cells grown in the absence of glucose (i.e. the "glucose-regulated proteins"). These same two proteins also were induced in cells treated with the calcium ionophore A23187. To begin examining the intracellular location of these multiregulated proteins, immunofluorescence microscopy studies were carried out using a monoclonal antibody against the 100-kDa stress protein. The antigen was localized primarily with the Golgi apparatus and less prominently with the plasma membrane and nucleus. Heat shock treatment resulted in an increased number of the cells exhibiting a nuclear location of 100 kDa.     

Proteomic analysis of cellular response to osmotic stress in thick ascending limb of Henle's loop (TALH) cells

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.     

Proteomic profiling of heat shock protein 70 family members as biomarkers for hepatitis C virus-related hepatocellular carcinoma

To identify proteins linked to the pathogenesis of hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV), we profiled protein expression levels in samples of HCC. To identify essential proteins, ten samples of HCV-related HCC were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. These experiments revealed increased levels of nine proteins in cancerous tissues compared to levels in corresponding noncancerous liver tissues. We focused on four members of the heat shock protein 70 family: 78 kDa glucose-regulated protein (GRP78), heat shock cognate 71 kDa protein (HSC70), 75 kDa glucose-regulated protein (GRP75), and heat shock 70 kDa protein 1 (HSP70.1). These results were confirmed by immunoblot analysis. In an additional 11 samples, the same expression patterns of these four proteins were observed. In total, 21 samples showed statistically significant up-regulation of GRP78, GRP75 and HSP70.1 in cancerous tissues. HSC70 showed a tendency toward overexpression. There has been no report describing overexpression of these four proteins simultaneously in HBV-related HCC as well as nonviral HCC. Our results suggest that these four proteins play important roles in the pathogenesis of HCV-related HCC and could be molecular targets for diagnosis and treatment of this disease.     

Isolation and analysis of ribonucleic acids from skeletal tissues

We report a method for the isolation of total cellular RNA from mineralized or cartilaginous tissues. The procedure accommodates the large amount of hydroxyapatite and high buoyant density proteoglycans present in skeletal tissue samples, as well as the low cell density characteristic of these tissues. The procedure can be reliably used for processing a large number of small (100-800 mg) tissue samples. Tissues are homogenized in guanidine hydrochloride solution, then centrifuged at low speed, and filtered to remove the nonsolubilized extracellular matrix proteins. Subsequent high speed density gradient centrifugation produces a high yield of RNA (0.2-0.6 micrograms RNA/mg tissue) which is precipitated in a low pH sodium acetate solution. RNA extracted by this method has been analyzed for the expression of various genes by Northern blotting. In addition to mRNAs of bone- and cartilage-specific proteins, messenger RNA for growth factors, proto-oncogenes, and heat shock proteins can be detected.     

The effect of extracellular Ca2+ and temperature on the induction of the heat-shock and glucose-regulated proteins in hamster fibroblasts

The expression of two stress-inducible protein families was examined in hamster fibroblast cells. These are the heat-shock and glucose-regulated proteins which have been shown to be highly inducible by heat and glucose-starvation, respectively. Our studies here demonstrate that the two sets of proteins can be induced simultaneously or separately. The enhanced synthesis of one set of proteins apparently does not affect the level of expression of the other set. We further show that pre-incubation of these fibroblast cells in calcium-free medium does not inhibit the synthesis of the 70 and 72-kilodalton heat-shock proteins at the elevated temperature. While extracellular calcium is apparently not involved in the activation of the heat-shock protein synthesis, its removal from the culture medium has a modest stimulative effect on the synthesis of the glucose-regulated proteins. Our results are consistent with the hypothesis that the expression of these two sets of proteins are regulated by separate control mechanisms.     

Role of Heat Shock Proteins in Diseases and Their Therapeutic Potential

Heat shock proteins are ubiquitously expressed intracellular proteins and act as molecular chaperones in processes like protein folding and protein trafficking between different intracellular compartments. They are induced during stress conditions like oxidative stress, nutritional deficiencies and radiation. They are released into extracellular compartment during necrosis. However, recent research findings highlights that, they are not solely present in cytoplasm, but also released into extracellular compartment during normal conditions and even in the absence of necrosis. When present in extracellular compartment, they have been shown to perform various functions like antigen presentation, intercellular signaling and induction of pro-inflammatory cytokines. Heat shock proteins represents as dominant microbial antigens during infection. The phylogenetic similarity between prokaryotic and eukaryotic heat shock proteins has led to proposition that, microbial heat shock proteins can induce self reactivity to host heat shock proteins and result in autoimmune diseases. The self-reactivity of heat shock proteins protects host against disease by controlling induction and release of pro-inflammatory cytokines. However, antibodies to self heat shock proteins haven been implicated in pathogenesis of autoimmune diseases like arthritis and atherosclerosis. Some heat shock proteins are potent inducers of innate and adaptive immunity. They activate dendritic cells and natural killer cells through toll-like receptors, CD14 and CD91. They play an important role in MHC-antigen processing and presentation. These immune effector functions of heat shock proteins are being exploited them as therapeutic agents as well as therapeutic targets for various infectious diseases and cancers.     

Phosphorylated heat shock protein 27 represses growth of hepatocellular carcinoma via inhibition of extracellular signal-regulated kinase

Heat shock protein 27, one of the low molecular weight stress proteins, is recognized as a molecular chaperone; however, other functions have not yet been well established. Phosphorylated heat shock protein 27 levels inversely correlate with the progression of human hepatocellular carcinoma. This study shows that phosphorylated heat shock protein 27 interferes with cell growth of the hepatocellular carcinoma-derived HuH7 cells in the presence of the proinflammatory cytokine, tumor necrosis factor-alpha, via inhibition of the sustained activation of the extracellular signal-regulated kinase signal pathway. The activities of Raf/extracellular signal-regulated kinase and subsequent activator protein-1 transactivation and the induction levels of cyclin D1 were lower in HuH7 cells transfected with phosphorylated heat shock protein 27 than those with unphosphorylated heat shock protein 27. Moreover, phosphorylated heat shock protein 27 up-regulated the levels of p38 mitogen-activated protein kinase and mitogen-activated protein kinase phosphatase-1, an inhibitory protein of extracellular signal-regulated kinase. These results indicate that phosphorylated heat shock protein 27 might suppress the extracellular signal-regulated kinase activity in the hepatocellular carcinoma cells via two separate pathways in an inflammatory state. The extracellular signal-regulated kinase activity is inversely correlated with phosphorylated heat shock protein 27 at serine 15 and also in human hepatocellular carcinoma tissues in vivo. Because the extracellular signal-regulated kinase signal pathway is a major proliferation signal of hepatocellular carcinoma, activator protein-1 activation is an early event in hepatocarcinogenesis. These findings strongly suggest that the control of the phosphorylated heat shock protein 27 levels could be a new therapeutic strategy especially to counter the recurrence of hepatocellular carcinoma.     

The induction of stress-related proteins by lead

Differential inductive effects of lead on protein synthesis in rat fibroblasts and kidney epithelial cells were examined. The lead was administered as lead glutamate, a complex known to introduce lead into cells. Lead exposure induced the synthesis of three proteins which constitute two separate stress protein subgroups. Two of these proteins have been previously identified as the glucose-regulated proteins because their synthesis is induced by reagents which perturb glucose utilization. The third protein is inducible by several sulfhydryl-binding reagents including lead. This third protein has been compared with a protein, p32/6.3, of very similar size and isoelectric point, which has been associated with lead-induced intranuclear inclusion bodies. However, several features, including one-dimensional peptide maps, indicated that the third protein and p32/6.3 are not identical. The three lead-induced proteins are distinguished from the major group of stress proteins by their relative insensitivity to heat stress. Lead glutamate, on the other hand, induces neither the heat shock protein 70 nor metallothionein, both of which are strongly induced by several metals including cadmium, zinc, and mercury.     

In Vitro Heat Stability of Plasma Membrane Vesicles and Tonoplast Membrane Vesicles Isolated From the Hypocotyle of Phaseolus vulgaris

Microsome, plasma membrane vesicles and tonoplast membrane vesicles were isolated from the hypocotyles of Phaseolus vulgaris L. 85CT-49762, with very high heat tolerance potential. Comparing the H+-pump heat stability in vitro of the vesicles from the heat acclimated cells and the cells in which protein synthesis was inhibited by actidion during heat acclimation with that of normal cells, the authors found that heat acclimation could increase the heat stability of membrane vesicles, and that the heat shock proteins synthesized during heat acclimation were related to the effect. The authors further analysed the role of membrane peripheral proteins on H+-pump thermotolerance of membrane vesicles, and proved that heat shock protein HSP 70 and low molecular weight heat shock protein (LMW HSP) were able to protect H+-pump from heat destruction.     

Heat shock response of germinating embryos of wheat : effects of imbibition time and seed vigor

Seeds frequently face a hostile environment during early germination. In order to determine whether seeds have evolved unique mechanisms to deal with such environments, a survey of the heat shock response in isolated embryos of wheat (Triticum aestivum L.) was undertaken. Embryos simultaneously heat shocked and labeled following several different periods of prior imbibition up to 12 hours synthesized many groups of heat shock proteins (hsps) typical of other plant and animal systems. Also, five developmentally dependent hsps, present only in treatments imbibed less than 6 hours prior to heat shock, were detected. These proteins have relative molecular masses of 14, 40, 46, 58, and 60 kilodaltons. One of the developmentally dependent hsps is among the most highly labeled hsps found in early imbibed embryos. The possibility that this protein is the E_m protein is discussed. The hypothesis that the capacity for hsp synthesis is affected by seed vigor was also tested. The heat shock responses of embryos from two high and two low vigor seed lots were compared using one- and two-dimensional electrophoresis of labelled protein extracts. The results indicate that both of the low vigor lots tested had weaker heat shock responses than their high vigor counterparts overall. Not all hsps were relatively less abundant in low vigor embryos. The developmentally dependent hsps showed little relationship to vigor. Some of the developmentally dependent hsps were actually made in greater amounts, relative to other proteins, in the low vigor seed lots. The results presented here demonstrate that imbibing embryos are capable of expressing an enhanced heat shock response, and that this response is related to seed vigor.     

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium.

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.0 to 5.0. Two-dimensional gel electrophoretic analysis of proteins labeled with H2(35)SO4 for 20 min during the shift showed that in E. coli, 13 polypeptides were elevated 1.5- to 4-fold, whereas in S. typhimurium, 19 polypeptides were increased 2- to 14-fold over the pH 7.0 control. Upon long-term growth at pH 5.0, almost double the number of polypeptides were elevated twofold or more in S. typhimurium compared with E. coli. In E. coli, there was no apparent induction of heat shock proteins upon growth at pH 5.0 in SMM. However, growth of E. coli in a complex broth to pH 5.0, or subsequent growth of fresh E. coli cells in the filtrate from this culture, showed that a subset of five polypeptides is uniquely induced by low pH. Two of these polypeptides, D60.5, the inducible lysyl-tRNA synthetase, and C62.5, are known heat shock proteins. Measurements of the internal pH (pHi) and growth rates of both organisms were made during growth in SMM at pH 7.0, pH 5.0, and upon the pH shift. The data show that the pHi of E. coli decreases more severely than that of S. typhimurium at an external pH of 5.0; the growth rate of E. coli is about one-half that of S. typhimurium at this pH, whereas the two organisms have the same growth rate at pH 7.0. The two-dimensional gel, growth, and pHi experiments collectively suggest that, at least in SMM, S. typhimurium is more adaptive to low-pH stress than is E. coli.     

Heat shock proteins in health and disease: therapeutic targets or therapeutic agents?

For many years, heat shock or stress proteins have been regarded as intracellular molecules that have a range of housekeeping and cytoprotective functions, only being released into the extracellular environment in pathological situations such as necrotic cell death. However, evidence is now accumulating to indicate that, under certain circumstances, these proteins can be released from cells in the absence of cellular necrosis, and that extracellular heat shock proteins have a range of immunoregulatory activities. The capacity of heat shock proteins to induce pro-inflammatory responses, together with the phylogenetic similarity between prokaryotic and eukaryotic heat shock proteins, has led to the proposition that these proteins provide a link between infection and autoimmune disease. Indeed, both elevated levels of antibodies to heat shock proteins and an enhanced immune reactivity to heat shock proteins have been noted in a variety of pathogenic disease states. However, further evaluation of heat shock protein reactivity in autoimmune disease and after transplantation has shown that, rather than promoting disease, reactivity to self-heat shock proteins can downregulate the disease process. It might be that self-reactivity to heat shock proteins is a physiological response that regulates the development and progression of pro-inflammatory immunity to these ubiquitously expressed molecules. The evolving evidence that heat shock proteins are present in the extracellular environment, that reactivity to heat shock proteins does not necessarily reflect adverse, pro-inflammatory responses and that the promotion of reactivity to self-heat shock proteins can downregulate pathogenic processes all suggest a potential role for heat shock proteins as therapeutic agents, rather than as therapeutic targets.