Stress-induced Protein Expression Research Articles

Derivatives of 3, 4, 5-Trimethoxycinnamic Acid Ameliorate Stress-Induced Anxiety in Mice and Rats.

Stress is an overwhelming problem associated with neuronal damage leading to anxiety and depression. The compound 3, 4, 5-trimethoxycinnamic acid (TMCA) has shown anti-stress effects; however, its derivatives remained unknown for their anxiolytic properties. Here, therefore, we investigated derivatives of TMCA (dTMCA) for their anxiolytic effects using immobilization and electric shock-induced stress in rats. Derivatives of TMCA ameliorated anxiety in mice and rats revealed by extended period of time spent in the open arms of elevated plus maze. Stress-mediated repression of tyrosine hydroxylase (TH) protein expression in the amygdala regions of rat brain and dopamine levels in the PC12 cells was restored by two selected derivatives (TMCA#5 and TMCA#9). Unlike TH expression, stress-induced protein expression of phospho-extracellular signal-regulated kinase (pERK) was unaffected by both derivatives in rats. Given the preferential inhibitory activity of dTMCA on dopamine and serotonin receptors, serotonergic road map of cellular signaling could be their target for anxiolytic effects. Thus, dTMCA would be promising agents to prevent neuronal damage associated with rampant stressful conditions.

Sargahydroquinoic acid (SHQA) suppresses cellular senescence through Akt/mTOR signaling pathway.

The effects of sargahydroquinoic acid (SHQA) on cellular senescence and the underlying mechanisms were investigated using human umbilical vascular endothelial cells (HUVECs). SHQA or DMSO was supplemented into the medium. Low dose of H2O2 was used to induce premature senescence. Replicative senescence was achieved by continuously culturing cells until they reached a plateau phase. Senescence biomarkers, including p53, p21, and p16 proteins, and SA-β-Gal activity were measured. Pretreatment of SHQA significantly suppressed the oxidative stress-induced protein expression of p53, p21, and p16, as well as the activity of SA-β-Gal. Additionally, SHQA also delayed the replicative senescence as indicated by an increased population doubling number, reduced protein expression of p53, p21, and p16, as well as a decreased SA-β-Gal activity. SHQA inhibited the phosphorylation of Akt, mTOR, and downstream targets of mTOR, such as p-S6K, which was elevated by premature senescence and replicative senescence. In the absence of senescence stimuli, SHQA also inhibited the Akt/mTOR signaling pathway and promoted autophagy. SHQA suppressed senescence induced by oxidative stress and replication through inhibiting the Akt/mTOR pathway. With the potential of acting as an Akt/mTOR inhibitor, SHQA might be useful for developing anti-ageing therapy.

The Influence of Copper Ions on Cellular Elements of the Coelomic Fluid of Starfish Asterias Rubens L.

This paper is devoted to studying the effect of copper ions on the number and ratio of various subpopulations and functional properties of cells in the coelomic fluid of the starfish Asterias rubens. The experiments were carried out at the White Sea Biological Station named after N.A. Pertsov of the Faculty of Biology of Moscow State University. Starfish were exposed for 96 h to copper chloride (II) in concentrations of Cu2 + ions 0.78, 1.95, and 3.91 μM in aquariums. The number of coelomocytes was significantly increased in starfish kept in aquariums with a maximum concentration of copper ions. The distribution of cell subpopulations was also changed. The proportion of small cells increased from 9 to 15.5%, that of agranulocytes increased from 61 to 75%, and that of granulocytes decreased from 30 to 8.5%. Expression of stress-induced proteins 70 (HSC70/HSP70 ) that was determined by Western blot analysis increased in starfish in all experimental aquariums. The viability of the isolated coelomocytes estimated by the absorption of neutral red (NR) dye increased in starfish in experimental aquariums with copper ion concentrations of 0.78 and 1.95 μM as compared with the control. However, at the concentration of 3.91 μM, NR absorption drastically decreased. Taken together, it was shown that A. rubens responded to the exposure to copper (II) ions by increasing the number of circulating coelomocytes and enhancing the proportion of phagocytes subpopulation. It presumably may serve as a compensatory mechanism in response to the toxic effect of copper (II).

A Periplasmic Antimicrobial Peptide-Binding Protein Is Required for Stress Survival in Vibrio cholerae.

Vibrio cholerae must sense and respond appropriately to stresses encountered in the aquatic environment and the human host. One stress encountered in both environments is exposure to antimicrobial peptides (AMPs), produced as a part of the innate immune response by all multicellular organisms. Previous transcriptomic analysis demonstrated that expression of Stress-inducible protein A (SipA) (VCA0732), a hypothetical protein, was highly induced by AMP exposure and was dependent on a specific uncharacterized two-component system. In order to better understand role of this protein in stress relief, we examined whether it shared any of the phenotypes reported for its homologs. SipA is required for survival in the presence of two other stressors, cadmium chloride and hydrogen peroxide, and it localizes to the bacterial periplasm, similar to its homologs. We also found that SipA physically interacts with OmpA. Importantly, we found that SipA binds AMPs in the bacterial periplasm. This suggests a model where SipA may act as a molecular chaperone, binding AMPs that enter the periplasm and delivering them to OmpA for removal from the cell. While El Tor V. cholerae strains lacking SipA do not show a survival defect in the presence of AMPs, we found that Classical sipA mutants are less able to survive in the presence of AMPs. This phenotype is likely masked in the El Tor background due to a functional lipid A modification system that increases AMP resistance in these strains. In summary, we have identified a protein that contributes to a novel mechanism of stress relief in V. cholerae.

HSP27: A Therapeutic Target in Myelofibrosis

▪Myelofibrosis (MF) is the most aggressive myeloproliferative neoplasms (MPN) with the highest degree of morbidity and mortality, including progressive bone marrow fibrosis resulting into bone marrow failure. JAK2 kinase inhibitors have been successfully used for a few years in MPN and more particularly for MF treatment. Despite their beneficial effects on spleen size and symptoms, JAK2 inhibitors induce low molecular and survival responses underscoring the urgent need for other therapeutic approaches. Recently, heat shock protein 90 (HSP90) - known to stabilize JAK2 - has been reported as a promising therapeutic target in MPN. However HSP90 inhibitors show toxicity and induce the expression of stress-inducible proteins like HSP70 and, most likely HSP27 as previously shown in other cancers. In addition, we and others have shown that HSP27, was strongly expressed in patients with idiopathic pulmonary, lung or kidney tubulointerstitial fibrosis, underlying a relevant role of HSP27 in fibrotic processes. Taking into account both the beneficial effects of HSP inhibitors in leukemia and in MPN, and the possible implication of HSP27 in fibrosis, we have evaluated in this work, the status of HSP27 in MF patient's samples and assess the effectiveness of an HSP27 oligonucleotide inhibitor called OGX-427.In this study, we first assessed the extracellular and intracellular level of HSPs from MF patients by ELISA, flow cytometry and by immunohistochemistry. We observed for the first time a specific increase in both intracellular and extracellular HSP27 in CD34+ circulating hematopoietic progenitor cells (n=9-16; P=0.0097) and in the sera of patients (n=24-27; P<0.0001) with MF compared with healthy donors, respectively. Moreover, we identified the presence of HSP27 in the bone marrow’s MF patients.We then investigated the in vivo impact of OGX-427, a specific inhibitor of HSP27, or an oligonucleotide control in a murine TPO-induced MF mouse model. The use of OGX-427 limited the progression of the disease in our MF mouse model (n=9). In particular, OGX-427 was associated with a marked reduction in both the spleen weight and size. Also, we noted a decrease of megakaryocyte hyperplasia in the bone marrow accompanied by a visible restoration of spleen structure and lymphoid white pulp territories with OGX-427.Taking altogether, our results support a key role of HSP27 in the pathophysiology in MF and highlight the potential therapeutic benefit of HSP27 inhibitors in this disorder. DisclosuresNo relevant conflicts of interest to declare.

Increased expression of stress inducible protein 1 in glioma-associated microglia/macrophages

Factors released by glioma-associated microglia/macrophages (GAMs) play an important role in the growth and infiltration of tumors. We have previously demonstrated that the co-chaperone stress-inducible protein 1 (STI1) secreted by microglia promotes proliferation and migration of human glioblastoma (GBM) cell lines in vitro. In the present study, in order to investigate the role of STI1 in a physiological context, we used a glioma model to evaluate STI1 expression in vivo. Here, we demonstrate that STI1 expression in both the tumor and in the infiltrating GAMs and lymphocytes significantly increased with tumor progression. Interestingly, high expression of STI1 was observed in macrophages and lymphocytes that infiltrated brain tumors, whereas STI1 expression in the circulating blood monocytes and lymphocytes remained unchanged. Our results correlate, for the first time, the expression of STI1 and glioma progression, and suggest that STI1 expression in GAMs and infiltrating lymphocytes is modulated by the brain tumor microenvironment.

Proteomic analysis of copper stress responses in the roots of two rice (Oryza sativa L.) varieties differing in Cu tolerance

Background and aims Copper (Cu) is an essential micronutrient required for growth and development of plants. However, excess Cu is toxic to plants. To understand the mechanisms involved in copper stress response, a proteomic approach was used to investigate the differences in Cu stress-induced protein expression between a Cu-tolerant variety (B1139) and a Cu-sensitive one (B1195) of rice.

T-786C Polymorphism of the nos-3 Gene and the Endothelial Cell Response to Fluid Shear Stress—A Proteome Analysis

Endothelial dysfunction is a common denominator of cardiovascular disease. Central to endothelial dysfunction is a decrease in the bioavailability of nitric oxide (NO) synthesized by endothelial NO synthase (NOS-3). In vivo, the level of fluid shear stress (FSS) exerted by the flowing blood determines NOS-3 expression. However, in contrast to the -786T variant of the nos-3 gene, the -786C variant is not sensitive to shear stress. Consequently, cells homozygous for this variant have an inadequate capacity to synthesize NO. Therefore, we have compared shear stress-induced protein expression in human primary cultured endothelial cells with TT or CC genotype. Cells with the CC genotype exhibited a greatly reduced FSS-induced NOS-3 expression as well as a diminished NO synthesis capacity when compared to TT genotype cells. Proteome changes in response to FSS (30 dyn/cm(2) for 24 h) were monitored by 2D-gel electrophoresis/densitometry/mass spectrometry. Of a total of 14 FSS-sensitive proteins, 8 were identically expressed in all cells. Four proteins, all of them part of the NO-dependent endoplasmic reticulum-stress response, were up-regulated by FSS only in cells with TT genotype. In contrast, CC genotype cells responded to FSS with a unique increase in manganese-containing superoxide dismutase expression. These differences in protein expression may (i) reflect the low bioavailability of NO in cells homozygous for the -786C variant of the nos-3 gene and (ii) point to a mechanism by which this deficit is counterbalanced by protecting the less abundant NO from rapid degradation.

Expression of Major Histocompatibility Complex Class I Chain–Related Molecule A, NKG2D, and Transforming Growth Factor–β in the Liver of Humans with Alveolar Echinococcosis: New Actors in the Tolerance to Parasites?

Echinococcus multilocularis growth and persistent granuloma, which lead to the development of the severe parasitic disease alveolar echinococcosis (AE), might be caused by abnormal expression of stress-induced proteins, with subsequent abnormalities in T cell activation. Similar to its involvement in tumors, the NKG2D-major histocompatability complex class I chain-related molecules A and B (MICA/B) signaling system could be involved in host-parasite interactions; however, its involvement in helminthic diseases has never been studied. We studied MICA/B, NKG2D, and transforming growth factor-beta (TGF-beta) expression on liver sections and measured levels of soluble MICA in serum samples obtained from patients with progressive AE. Livers from healthy and cirrhotic subjects were studied as controls. Expression of MICA/B proteins was strongly enhanced in the hepatocytes and endothelial and bile duct cells; in the CD68+ cells of the periparasitic infiltrate, especially epithelioid and giant cells; and, also, in the metacestode germinal layer. Strong expression of MICA/B in the liver contrasted with low numbers of NK cells and lack of expression of NKG2D on the numerous CD8+ T lymphocytes of the periparasitic infiltrate, as well as with the absence of soluble MICA in serum. TGF-beta was strongly expressed by most of the infiltrating lymphocytes. Sustained expression of MICA/B molecules and TGF-beta might lead to modulation of NKG2D with subsequent inhibition of NKG2D-dependent cytotoxicity. Abnormalities of this signaling system could contribute to parasitic evasion of the host's immunity.

Differential expression of stress-inducible proteins in chronic hepatic iron overload

Introduction: Oxidative stress can trigger a cellular stress response characterized by induction of antioxidants, acute phase reactants (APRs) and heat shock proteins (HSPs), which are presumed to play a role in limiting tissue damage. In rodents, hepatic iron overload causes oxidative stress that results in upregulation of antioxidant defenses with minimal progressive liver injury. The aim of this study was to determine whether iron overload modulates expression of other stress-responsive proteins such as APRs and HSPs that may confer protection against iron-induced damage in rodent liver. Methods: Male rats received repeated injections of iron dextran or dextran alone over a 6-month period. Hepatic transcript levels for a panel of APRs and HSPs were quantitated by real-time PCR and protein expression was evaluated by Western blot and immunohistochemistry. Results: Hepatic iron concentrations were increased > 50-fold in the iron-loaded rats compared to controls. Iron loading resulted in striking increases in mRNAs for Hsp32 (heme oxygenase-1; 12-fold increase vs. controls) and metallothionein-1 and -2 (both increased ∼ 6-fold). Transcripts for α1-acid glycoprotein, the major rat APR, were increased ∼ 3-fold, while expression of other classical APRs was unaltered. Surprisingly, although mRNA levels for the HSPs were not altered by iron, the abundance of Hsp25, Hsp70 and Hsp90 proteins was uniformly reduced in the iron-loaded livers, as were levels of NAD(P)H:quinone oxidoreductase 1, an Hsp70 client protein. Conclusions: Chronic iron administration elicits a unique pattern of stress protein expression. These alterations may modulate hepatic responses to iron overload, as well as other injury processes.

Pharmacological inhibition of RhoA signaling prevents connective tissue growth factor induction in endothelial cells exposed to non-uniform shear stress

Shear stress changes play an important role in atheroma formation. This study focussed on atherogenic protein expression under non-uniform shear stress and the pharmacological modulation of shear-related endothelial dysfunction. Bifurcating flow-through cell culture slides were used to expose HUVECs to steady laminar or non-uniform shear stress for 18h at 10dyn/cm2. Protein expression was determined by immunofluorescence, and quantified using MetaVue software.Laminar shear stress resulted in cell alignment, reduced F-actin fibers, and significant induction of endothelial nitric oxide synthase expression. Under non-uniform shear stress at bifurcations, minor upregulation of adhesion molecules was observed. Connective tissue growth factor (CTGF) was significantly downregulated by laminar shear stress and induced in cells exposed to non-uniform shear stress. CTGF upregulation by non-uniform shear stress was RhoA-dependent, because it was almost completely inhibited in cells transfected with dominant negative RhoA-N19, and when cells were treated with 1μmol/L simvastatin during flow. Pre-incubation of HUVECs with inhibitors of Rho-associated kinase before exposure to flow significantly suppressed the CTGF induction in regions of non-uniform shear stress.In conclusion, non-uniform shear stress-dependent CTGF expression requires active RhoA and can be prevented pharmacologically. Interference with shear stress-induced protein expression may inhibit endothelial dysfunction in athero-prone vessel regions.

Expression of stress inducible protein 1 (Stip1) in the mouse testis

Phthalate esters are considered endocrine disruptors that interfere with the endocrine balance and development of the mammalian testis. Mono-2-ethylhexyl phthalate (MEHP), the active metabolite of the ubiquitously used plasticizer di-2-ethylhexyl phthalate (DEHP), acts upon Sertoli cells as initial target. By subtractive cDNA libraries we identified genes deregulated as response to MEHP in primary cultures of mouse Sertoli cells. The expression of mouse stress inducible protein 1 (Stip1) was detected as upregulated as a result of MEHP exposure. Stip1 is a cochaperone protein that is homologous to the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). To assess the presence and localization of Stip1 in mouse testis and its potential role in stress defense, we studied the expression pattern of the Stip1 protein by immunohistochemistry and of the mRNA by in situ hybridization. Both the protein and the mRNA of Stip1 were mainly found in the cytoplasm of all types of spermatogonia and spermatocytes up till zygotene, the expression decreased during late pachytene and was very weak in diplotene spermatocytes and round spermatids. Interestingly, this expression pattern resembled the pattern of stress sensitivity of spermatogenic cells in that the most sensitive cell types show the weakest expression of Stip1. This suggests an important role for Stip1 in the ability of germ cells to survive in stress conditions including high temperatures.

TISSUE- AND DOSE-DEPENDENT ALTERATION OF STRESS-INDUCIBLE PROTEINS BY β2-ADRENOCEPTOR AGONIST, SALBUTAMOL, IN RATS

The effects of selective beta(2)-adrenoceptor agonists on proinflammatory cytokines and the expression of stress-inducible proteins have not yet been clarified. We investigated the effect of a higher dose (60 mg/kg intravenously) of salbutamol, a selective beta(2)-adrenoceptor agonist, on the induction of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha in plasma and the expression of protein and mRNA of metallothioein-1 (MT-1), heme oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) in heart, lung, liver and spleen in rats. The plasma IL-6 concentration was significantly increased after administration with a maximum increase at 3 hr in a dose-dependent manner, but IL-1beta and TNF-alpha concentrations were not changed. MT-1 mRNA increased in heart, lung and liver, but not in spleen, and MT-1 protein increased in endocardium, fibroblasts of lung and periportal regions in liver. HO-1 mRNA was not changed in lung, decreased at 3 hr in liver and spleen, and increased at 6 hr in liver. Contrary to liver, HO-1 mRNA in the heart increased at 3 hr and decreased at 6 hr. HO-1 protein increased in cardiomyocytes and centrilobular regions in the liver. iNOS mRNA increased in lung, liver and spleen, but decreased in the heart, and iNOS protein increased in alveolar type II cells and hepatocytes, and decreased in necrotic cardiomyocytes. In contrast, a lower dose (6 mg/kg intravenously) of salbutamol suppressed lipopolysaccharide-induced HO-1 and iNOS mRNA. We conclude that salbutamol tissue- and dose-dependently alters the expression of stress-inducible proteins.

Photoreceptor degeneration and loss of retinal function in the C57BL/6-C2J mouse.

The C57BL/6-c(2J) (c2J) mouse strain has been shown in earlier studies to be highly resistant to light damage. Subsequent studies related this resistance to an amino acid substitution (leu450met) in a pigment epithelial enzyme (RPE65), which slowed the rate of rhodopsin regeneration. The present study was conducted to examine patterns of photoreceptor death, electrophysiological function (the ERG) and trophic factor expression over the life of the C57BL/6-c(2J) retina. Observations were made on two C57BL/6J-c(2J) substrains, one albino (Tyr/Tyr) and one pigmented (Tyr/(+)), and two nondegenerative strains, one albino (BALB/cJ) and one pigmented (C57BL/6J). Mice were raised in dim cyclic light (12 hours at 5 lux, 12 hours in the dark), and a developmental series of retinas of each strain was taken between postnatal day (P)4 and (P365(+)). Retinas were examined for cell death by using the TUNEL technique, stress-induced protein expression (FGF-2 and GFAP), and measures of retinal thickness. The dark-adapted ERG was recorded in dark-adapted conditions in early adulthood (13-15 weeks) and late adulthood (>1 year). In both C57BL/6-c(2J) substrains, the retina showed marked degenerative features when compared with two control strains, BALB/cJ (leucine at codon 450 in RPE65) and C57BL/6J (methionine). During development and into young adulthood, photoreceptor death rates were abnormally high, levels of two stress-inducible proteins (FGF-2 and GFAP) were abnormally high, and the ERG (electroretinogram) was significantly reduced in amplitude (<50% of values in BALB/cJ or C57BL/6J). The rate of photoreceptor death remained abnormally high into young adulthood (2-3 months) but decreased to control levels by 1 year. Accordingly, the thickness of the outer nuclear layer and the ERG were stable over the same period. Results suggest that a still-unidentified stress increases photoreceptor death in the C57BL/6-c(2J) retina during the critical period of photoreceptor development and into young adulthood, upregulates stress-inducible factors, and markedly limits the amplitude of the ERG. These degenerative changes do not continue after early adulthood, the retina remaining stable in structure and function into late adulthood. The degenerative changes were apparent in both albino and pigmented C57BL/6-c(2J) substrains. Their genetic cause remains unknown.

Molecular and functional characterization of the stress-induced protein (SIP) gene and its two transcripts generated by alternative splicing. SIP induced by stress and promotes cell death.

We have used a quantitative fluorescent cDNA microarray hybridization approach to identify pancreatic genes induced by the cellular stress promoted by acute pancreatitis in the mouse. We report the cloning and characterization of one of them that encodes the stress-induced proteins (SIP). The mouse SIP gene is organized into five exons and expands over approximately 20 kilobase pairs. Exon 4 (38 base pairs) is alternatively spliced to generate two transcripts. Northern blot and in situ hybridization showed that both SIP mRNAs are rapidly and strongly induced in acinar cells of the pancreas with acute pancreatitis. They are also constitutively expressed in several other tissues, although with different ratios. They encode proteins of 18 and 27 kDa (SIP(18) and SIP(27)). SIP(27) is identical to the thymus-expressed acidic protein (TEAP) protein, formerly described as a thymus-specific protein. Expression of the SIP(18) and SIP(27)/EGFP or V5 fusion proteins showed that both are nuclear factors. We monitored SIP expression in NIH3T3 cells submitted to various stress agents. UV stress, base damaging, mutagenic stress, ethanol, heat shock, and oxidative stress induced the concomitant expression of SIP(18) and SIP(27) mRNAs. Finally, transient transfection of SIP(18) and SIP(27) expression plasmids induced death by apoptosis in COS7 cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining. In conclusion, the SIP gene is an important element of cellular stress response. It is expressed in many tissues and induced by a variety of stress agents affecting many cellular pathways. SIP generates, by alternative splicing, two nuclear proteins that can promote cell death by apoptosis.

Hypothermia during reperfusion after asphyxial cardiac arrest improves functional recovery and selectively alters stress-induced protein expression.

This study examined whether prolonged hypothermia induced 1 hour after resuscitation from asphyxial cardiac arrest would improve neurologic outcome and alter levels of stress-related proteins in rats. Rats were resuscitated from 8 minutes of asphyxia resulting in cardiac arrest. Brain temperature was regulated after resuscitation in three groups: normothermia (36.8 degrees C x 24 hours), immediate hypothermia (33 degrees C x 24 hours, beginning immediately after resuscitation), and delayed hypothermia (33 degrees C x 24 hours, beginning 60 minutes after resuscitation). Mortality and neurobehavioral deficits were improved in immediate and delayed hypothermia rats relative to normothermia rats. Furthermore, both immediate and delayed hypothermia improved neuronal survival in the CA1 region of the hippocampus assessed at 14 days. In normothermia rats, the 70-kDa heat shock protein (Hsp70) and 40-kDa heat shock protein (Hsp40) were increased within 12 hours after resuscitation in the hippocampus. Delayed hypothermia attenuated the increase in Hsp70 levels in the hippocampus but did not affect Hsp70 induction in the cerebellum. Hippocampal expression of Hsp40 was not affected by hypothermia. These data indicate that prolonged hypothermia during later reperfusion improves neurologic outcome after experimental global ischemia and is associated with selective changes in the pattern of stress-induced protein expression.

Diversity in protein synthesis and viability of Helicobacter pylori coccoid forms in response to various stimuli.

The viability of the coccoid forms of Helicobacter pylori was evaluated by assessing protein synthesis. Metabolic labeling studies showed the synthesis of proteins and the specific protein profiles of H. pylori coccoids produced under various conditions. Harsh conditions such as aerobiosis and starvation (lack of horse serum) in the culture did not affect the synthesis of proteins in the coccoids. Lowering of the pH to that of gastric secretions induced expression of several proteins in the coccoids. However, the coccoids produced under prolonged microaerobic conditions exhibited a profile of acid stress-induced protein expression different from that induced by aerobiosis or starvation. Our data suggest that coccoid H. pylori exhibits diversity in viability following exposure to different stresses and that the response to acid stress of coccoid H. pylori could be involved in infection of the host stomach.

血流刺激により内皮に発現される急性反応蛋白

血流刺激により内皮に発現される急性反応蛋白