Early Developmental Decline in HSP Expression Affects Subsequent Response to Transient Heat Exposure

Importance of the stress response in acid-induced mucosal injury in opossum esophagus

Silencing of HSP70 and TRIAP1 Genes in Multiple Myeloma Cell Lines Induces Apoptosis through Apaf-1/Caspase 9 Pathway: New Potential Targets on the Way?

Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease.

Newborn animals are particularly sensitive to hypoxic stress. Oxygen is spared for sensitive tissues, including brain and heart. Scarce information is available concerning the molecular effects of hypoxia in the gastrointestinal tract (GIT). Moreover, stress protein expressions, including heat shock proteins (HSP), are still poorly documented in the GIT. Our objective was to determine the possible effect of hypoxia on HSP expression at birth. After western blotting, alphaB crystallin, HSP 27, and HSP 70 expressions were determined in newborn controls and piglets exposed to 1 or 4 h hypoxia (5% O2, 95% N2) allowed to recover from 1 to 68 h. Cytosol and nuclei were also separated and the extracts were tested for HSF1 and alphaB crystallin expressions. Surprisingly, alphaB crystallin was overexpressed in the stomach and colon in animals submitted to hypoxia, whereas HSP 27 and HSP 70 expression remained stable. Increases and return to basal levels in HSF1 and alphaB crystallin were simultaneously observed in the unique nuclear compartment. To our knowledge, the present study is the first to demonstrate the oxygen dependency of an HSP in the GIT, particularly in the colon in newborn piglets. The kinetics of alphaB crystallin overexpression after hypoxia parallels the activation of HSF1. This observation possibly indicates a correlation between this factor and alphaB crystallin after hypoxia. Taken together, the present results open the field of wide investigation about the specific response of this low-molecular-weight HSP and its possible involvement in pathological states in the GIT such as stomach and colon.

Heat shock (HS) activates mitogen-activated protein (MAP) kinases. Although prior exposure to nonlethal HS makes cells refractory to the lethal effect of a subsequent HS, it is unclear whether this also occurs in MAP kinase activation. This study was undertaken to evaluate the effect of a heat pretreatment on MAP kinase activation by a subsequent HS and to elucidate its possible mechanism. Preheating did not make BEAS-2B cells refractory to extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) activation by a second HS but accelerated their inactivation after HS. The rapid inactivation of ERK and JNK was dependent on de novo protein synthesis and associated with the up-regulation of heat shock protein 70 (HSP70). Moreover, the inhibition of phosphatase activity reversed this rapid inactivation. MAP kinase phosphatase-1 (MKP-1) expression was increased by HS, and the presence of its phosphorylated form (p-MKP-1) correlated with the observed rapid ERK and JNK inactivation. Blocking induction of p-MKP-1 with antisense MKP-1 oligonucleotides suppressed the rapid inactivation of ERK and JNK in preheated cells. HSP70 overexpression caused the early phosphorylation of MKP-1. Moreover, MKP-1 phosphorylation and the rapid inactivation of ERK were inhibited by blocking HSP70 induction in preheated cells. In addition, MKP-1 was insolubilized by HS, and HSP70 associated physically with MKP-1, suggesting that a chaperone effect of HSP70 might have caused the early phosphorylation of MKP-1. These results indicate that preheating accelerated MAP kinase inactivation after a second HS and that this is related to a HSP70-mediated increase in p-MKP-1.

Members of the yeast p24 family, including Emp24p and Erv25p, exist as heteromeric complexes that have been proposed to cycle between the endoplasmic reticulum (ER) and Golgi compartments. The specific functions and sites of action of p24 proteins are still unknown. Here we identified a human homolog of the yeast p24 family of proteins, named ERS25 (endoplasmic reticulum stress-response protein 25), and investigated its role in stress response. ERS25 is predicted to have an ER localization signal peptide, a GOLD (Golgi dynamics) domain, which is found in several eukaryotic Golgi and lipid-trafficking proteins, a coiled-coil region, and a transmembrane domain. We demonstrate that ERS25 is localized to the ER and is induced by ER-specific stress, heat shock, and oxidative stress. The selective induction of ERS25 by brefeldin A, but not tunicamycin, implicates the involvement of ERS25 in protein trafficking between the ER and the Golgi. Small interfering RNA-mediated inhibition of ERS25 results in a significant decrease in apoptosis as well as a reduction of reactive oxygen species induced by oxidative stress. Moreover, ERS25 depletion results in a significant increase in the levels of the ER chaperone HSP70 in response to heat-shock stress through increased levels of HSF-1. We also found that inhibition of ERS25 induction in response to heat shock enhanced the binding of HSP70 to Apaf-1, which is likely to interfere in stress-mediated apoptosis. Together, the data presented here demonstrate that ERS25 may play a critical role in regulation of heat-shock response and apoptosis.

BackgroundChemotherapy resistance is an intractable problem in treating patients with epithelial ovarian cancer (EOC). Heat shock proteins (HSPs) act as apoptosis inhibitors and are highly conserved genetically. Most HSPs have strong cytoprotective effects, and their overexpression inhibits apoptosis. This has been demonstrated for HSP70. Heat shock protein 70 (HSP70) expression is abnormally upregulated in malignant cells. Furthermore, HSP70 can inhibit cell death and promote chemotherapeutic resistance. In our study, the relationship between the HSP70 gene and primary chemotherapy resistance and clinical outcome in patients with EOC was explored.MethodsQuantitative real-time polymerase chain (qRT-PCR) was applied to determine HSP70 messenger RNA (mRNA) levels, and immunohistochemistry assay was conducted to determine HSP70 protein level. HSP70 overexpression was assessed to clarify its role on chemotherapy resistance to cisplatin in SKOV3 cell lines.ResultsRT-qPCR assay indicated a strong relationship between HSP70 expression and chemotherapy resistance in patients with EOC. In cultured SKOV3 cells, overexpression of HSP70 inhibited cell sensitivity to cisplatin. Kaplan-Meier analysis demonstrated high HSP70 expression was associated with poor outcome of EOC patients. In multivariate models, high HSP70 expression independently predicted this poor outcome.ConclusionsHSP70 predicts the prognosis and response to chemotherapy in EOC patients.

Cancer cells with overexpression of heat shock protein 27 (HSP27) are resistant to chemotherapeutic drug doxorubicin (Dox). Paclitaxel (Pacl) was reported to suppress HSP27 expression in ovarian and uterine cancer cells. The purposes of this study were to investigate whether Pacl inhibits the expression of HSP27 in breast cancer cells, whether Pacl can sensitize breast cancer cells with HSP27 overexpression to Dox, and to define a more effective schedule for the combination of Dox with Pacl. The HSP27 high-expressing human breast cancer cell lines, MCF-7 and MDA-MB-435, and the HSP27 low-expressing cell line, MDA-MB-231, were used in this study. The level of HSP27, topoisomerase (Topo) IIalpha and beta expression were assessed by Western blotting. The cytotoxic activities of Dox, Pacl and combination of these two drugs were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and flow cytometric assays. Pacl (0.1 micromol/L) inhibited HSP27 expression by approximately 2-fold in MCF-7 and MDA-MB-435 cells, while up-regulating the level of topo IIalpha and beta. In contrast, expression of HSP27 in MDA-MB-231 did not change significantly following Pacl treatment. There were synergistic effects in both treatment sequences (Pacl-Dox and Dox-Pacl) when Pacl was combined with Dox. Compared with those treated with the Dox-Pacl sequence, the Pacl-Dox sequence had a stronger effect in cancer cells with HSP27 overexpression, as MCF-7 and MDA-MB-435 treated with the Pacl-Dox sequence had lower viabilities and a higher apoptotic rate. Paclitaxel significantly decreases the level of HSP27 in breast cancer cells overexpressing HSP27. In combination therapies, the Pacl-Dox sequence is more effective in clearing breast cancer cells with high HSP27 expression compared with the Dox-Pacl sequence.

BackgroundOverexpression of heat shock protein 70 (HSP70) has been observed in many types of cancer including gastric adenocarcinomas, although the exact role of HSP70 in carcinogenesis remains unclear.MethodsThe study analyzed a total of 458 radical gastrectomy specimens which were immunohistochemically stained with HSP70, p53, and Ki-67 antibodies.ResultsThe study determined that the expression of HSP70 was significantly increased in early gastric cancer (EGC) compared to advanced gastric cancer (p<0.001). The HSP70 expression was correlated with well-differentiated tumor type, intestinal type of Lauren classification and the lower pT and pN stage. Negative expression of Ki-67 and p53 expression was associated with poor prognosis. The study did not find any correlation between HSP70 and p53 expression. The study determined that HSP70 expression in the EGC subgroup was associated with a poor prognosis (p=0.009), as well as negative Ki-67 expression (p=0.006), but was not associated with p53. Based on multivariate analysis, HSP70 expression (p=0.024), negative expression of Ki-67, invasion depth and lymph node metastasis were determined to be independent prognostic markers.ConclusionsHSP70 is expressed in the early stages of gastric adenocarcinoma. In EGC, HSP70 is a poor independent prognostic marker and is correlated with a low proliferation index.

We previously showed that activation of the A1 adenosine receptor protected the kidney against ischemia-reperfusion injury by induction and phosphorylation of heat shock protein 27 (HSP27). Here, we used mice that overexpress human HSP27 (huHSP27) to determine if kidneys from these mice were protected against injury. Proximal tubule cells cultured from the transgenic mice had increased resistance to peroxide-induced necrosis compared to cells from wild-type mice. However, after renal ischemic injury, HSP27 transgenic mice had decreased renal function compared to wild-type mice, along with increased renal expression of mRNAs of pro-inflammatory cytokines (TNF-alpha, ICAM-1, MCP-1) and increased plasma and kidney keratinocyte-derived cytokine. Following ischemic injury, neutrophils infiltrated the kidneys earlier in the transgenic mice. Flow cytometric analysis of lymphocyte subsets showed that those isolated from the kidneys of transgenic mice had increased CD3(+), CD4(+), CD8(+), and NK1.1(+) cells 3 h after injury. When splenocytes or NK1.1(+) cells were isolated from transgenic mice and adoptively transferred into wild-type mice there was increased renal injury. Further, depletion of lymphocytes by splenectomy or neutralization of NK1.1(+) cells resulted in improved renal function in the transgenic mice following reperfusion. Our study shows that induction of HSP27 in renal tubular cells protects against necrosis in vitro, but its systemic increase counteracts this protection by exacerbating renal and systemic inflammation in vivo.

Although both the heat shock protein 70 (HSP70) and the activating transcription factor 5 (ATF5) have been shown to promote cell survival of transformed cells but not survival of non-transformed cells, the relationship of the two molecules is unknown. Here we show that HSP70 and ATF5 are concomitantly up-regulated upon transient but down-regulated over prolonged cellular stress and apoptotic stimulation in the rat C6 glioma and human U87 glioma cells. HSP70 interacts strongly with the N-terminal activation domain of ATF5, which is expected to be rigid and uniquely structured under physiological conditions because of extraordinary high concentration (over 25%) of proline residues. Binding of HSP70 to ATF5 is an ATP-driven process and requires functional ATPase on the nucleotide binding domain of the HSP70 molecule. Overexpression of HSP70 dramatically stabilizes the ATF5 protein, which is otherwise subject to rapid degradation, facilitated by both proteasome-dependent and caspase-dependent processes, whereas HSP70 depletion leads to acceleration of ATF5 degradation and transcription repression of Bcl-2 and Egr-1, which are downstream targets of ATF5 in C6 and U87 glioma cells. Our data reveal an essential role for HSP70 in maintaining high levels of ATF5 expression in glioma cells and support the conclusion that ATF5 is an important substrate protein of HSP70 that mediates HSP70-promoted cell survival in glioma cells.

A role of heat shock protein 27 (HSP27) as a potential biomarker has been reported in various tumour entities, but comprehensive studies in pancreatic cancer are lacking. Applying tissue microarray (TMA) analysis, we correlated HSP27 protein expression status with clinicopathologic parameters in pancreatic ductal adenocarcinoma specimens from 86 patients. Complementary, we established HSP27 overexpression and RNA-interference models to assess the impact of HSP27 on chemo- and radiosensitivity directly in pancreatic cancer cells. In the TMA study, HSP27 expression was found in 49% of tumour samples. Applying univariate analyses, a significant correlation was found between HSP27 expression and survival. In the multivariate Cox-regression model, HSP27 expression emerged as an independent prognostic factor. HSP27 expression also correlated inversely with nuclear p53 accumulation, indicating either protein interactions between HSP27 and p53 or TP53 mutation-dependent HSP27-regulation in pancreatic cancer. In the sensitivity studies, HSP27 overexpression rendered HSP27 low-expressing PL5 pancreatic cancer cells more susceptible towards treatment with gemcitabine. Vice versa, HSP27 protein depletion in HSP27 high-expressing AsPC-1 cells caused increased gemcitabine resistance. Importantly, HSP27 expression was inducible in pancreatic cancer cell lines as well as primary cells. Taken together, our study suggests a role for HSP27 as a prognostic and predictive marker in pancreatic cancer. Assessment of HSP27 expression could thus facilitate the identification of specific patient subpopulations that might benefit from individualized treatment options. Additional studies need to clarify whether modulation of HSP27 expression could represent an attractive concept to support the incorporation of hyperthermia in clinical treatment protocols for pancreatic cancer.

1. The relative expression of heat shock protein (HSP) genes (HSP60, HSP70 and HSP90) was performed using quantitative real-time PCR on tissue from the heart ventricles to investigate the effect of 3,5,3′-l-triiodothyronine (T3)-induced pulmonary hypertension in broiler chickens.2. The ratio of the right ventricle to total ventricle (index of pulmonary hypertension) was increased in the treated groups at 12 and 42 d of age compared to controls but was significant only at 42 d.3. The HSP genes were expressed in the right and left ventricles of control but T3-treated broilers at 12 and 42 d of age. The relative amounts of HSP60 and HSP90 gene expression in the right ventricle of treated groups were significantly increased at 12 d and decreased at 42 d of age compared to controls.4. Variations of HSP60 and HSP90 mRNAs in the left ventricle were not significant. The relative amount of HSP70 mRNA expression in the right and left ventricles of treated groups was significantly decreased at 42 d of age compared to controls. HSP70 mRNA expression did not differ between the right and left ventricles at 12 d of age.5. It is concluded that gene expression of HSPs (i.e. HSP60 and HSP90) was upregulated in the heart of chickens developing pulmonary hypertension syndrome, probably to delay the pathological process of disease. The right ventricle from hearts of pulmonary hypertensive chickens showed considerable reductions of HSP60, HSP70 and HSP90, which is evidence of a loss of compensatory responsiveness in dilated heart.

Desert birds must cope with occasional and unpredictable heat waves, which are slowly becoming more frequent with climate change. Different orders of birds have different physiological and behavioural capacities that may aid survival during a heat wave. To date, the expression of genes related to heat exposure have not been studied across different bird orders. We hypothesised that acutely exposing native Australian birds whose natural habitat include arid environments to a high temperature (45 °C), similar to during a heat wave, would result in the upregulation of genes with protective effects against cell damage (BCL-2, VEGFA and heat shock proteins) and inflammation (interleukins), as well as the downregulation of genes involved in the coagulation pathway (fibrinogen). We used eight each of captive-bred Budgerigars (Melopsittacus undulatus), Zebra Finches (Taeniopygia guttata) and Diamond Doves (Geopelia cuneata). Four birds of each species were exposed to a temperature that was within the zone of thermal neutrality (35 °C), while the other four birds were exposed to a higher temperature (45 °C). The mRNA expression of selected genes were then measured using high-throughput qPCR platform (Fluidigm®, BioMark™). The results supported the hypothesis that acute exposure to a high temperature would result in the upregulation of heat shock protein (HSP) genes, but there was no significant upregulation of other genes with protective effects against cell damage nor genes associated with inflammation. The results also do not support the hypothesis that acute heat exposure would result in downregulation of the genes involved in the coagulation pathway in these birds. Among all the tissues that were analysed, the gastrointestinal tissue had the highest number of upregulated HSP genes, possibly indicating that this tissue requires the most protection to continue functioning. Diamond Dove organs also had the highest number of HSP genes upregulated, possibly a reflection of their ability to better protect their cells at high temperatures.

Cloning and expression analysis of a HSP70 gene from Pacific abalone ( Haliotis discus hannai)