HSP105 prevents depression-like behavior by increasing hippocampal brain-derived neurotrophic factor levels in mice.

Heat shock proteins (HSPs) are stress-induced chaperones that are involved in neurological disease. Although increasingly implicated in behavioral disorders, the mechanisms of HSP action, and the relevant functional pathways, are still unclear. We examined whether oral administration of geranylgeranylacetone (GGA), a known HSP inducer, produced an antidepressant effect in a social defeat stress model of depression in mice. We also investigated the possible molecular mechanisms involved, particularly focusing on hippocampal neurogenesis and neurotrophic factor expression. In stressed mice, hippocampal HSP105 expression decreased. However, administration of GGA increased HSP105 expression and improved depression-like behavior, induced hippocampal cell proliferation, and elevated brain-derived neurotrophic factor (BDNF) levels in mouse hippocampus. Co-treatment with GGA and the BDNF receptor inhibitor K252a suppressed the antidepressant effects of GGA. HSP105 knockdown decreased BDNF mRNA levels in HT22 hippocampal cell lines and hippocampal tissue and inhibited the GGA-mediated antidepressant effect. These observations suggest that GGA administration is a therapeutic candidate for depressive diseases by increasing hippocampal BDNF levels via HSP105 expression.

Expression and function of HSP110 family in mouse testis after vasectomy.

HSP110 functions to protect cells, tissues, and organs from noxious conditions. Vasectomy induces apoptosis in the testis; however, little is known about the reason leading to this outcome. The aim of the present study was to evaluate the expression and function of HSP110 in mouse testis after vasectomy. Following bilateral vasectomy, we used fluorescent Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to detect apoptosis, Western blotting and immunohistochemistry to examine HSP110 expression and localization. Serum antisperm antibody (AsAb) and testosterone were measured by Enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. Expression of endoplasmic reticulum stress (ERS) sensors and downstream signaling components was measured by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and the phosphorylation of eIF2α and JNK was detected by Western blotting. Vasectomy induced morphologic changes, increased apoptosis in the testis, increased serum AsAb, and decreased testosterone levels. After vasectomy, ORP150 mRNA level was increased first and then decreased, Bcl-2 was decreased, and the expression of HSPA4l, GRP78, GADD153, PERK, ATF6, IRE-1, XBP-1s, Bax, Bak, and caspases and the phosphorylation of eIF2α and JNK were increased. We present that an ER stress-mediated pathway is activated and involved in apoptosis in the testis after vasectomy. HSPA4l and ORP150 may play important roles in maintaining the normal structure and function of testis.

Nuclear heat shock protein 110 expression is associated with poor prognosis and chemotherapy resistance in gastric cancer.

Heat shock protein (HSP) expression is induced by the exposure to stress, such as fever, oxidative stress, chemical exposure, and irradiation. In cancer, HSP promotes the survival of malignant cells by inhibiting the induction of apoptosis. In colorectal cancer, a loss-of-function mutation of HSP110 (HSP110ΔE9) has been identified. HSP110ΔE9 inhibits the nuclear translocation of wild-type HSP110, which is important for its chaperone activity and anti-apoptotic effects. The patients carrying HSP110ΔE9 mutation exhibit high sensitivity to anticancer agents, such as oxaliplatin and 5-fluorouracil. There is still insufficient information about HSP110 localization, the clinicopathological significance of HSP110 expression, and its association with chemotherapy resistance in gastric cancer. Here, we found that high nuclear expression of HSP110 in gastric cancer tissues is associated with cancer progression, poor prognosis, and recurrence after adjuvant chemotherapy. In vitro results showed that HSP110 suppression increases the sensitivity to 5-fluorouracil and cisplatin of human gastric cancer cell lines. Our results suggest that nuclear HSP110 may be a new drug sensitivity marker for gastric cancer and a potential molecular therapeutic target for the treatment of gastric cancer patients with acquired anticancer drug resistance.

Yeast Two-Hybrid and One-Hybrid Screenings Identify Regulators of hsp70 Gene Expression.

The mammalian stress protein Hsp105ß, which is specifically expressed during mild heat shock and localizes to the nucleus, induces the major stress protein Hsp70. In the present study, we performed yeast two-hybrid and one-hybrid screenings to identify the regulators of Hsp105ß-mediated hsp70 gene expression. Six and two proteins were detected as Hsp105ß- and hsp70 promoter-binding proteins, respectively. A luciferase reporter gene assay revealed that hsp70 promoter activation is enhanced by the transcriptional co-activator AF9 and splicing mediator SNRPE, but suppressed by the coiled-coil domain-containing protein CCDC127. Of these proteins, the knockdown of SNRPE suppressed the expression of Hsp70 irrespective of the presence of Hsp105ß, indicating that SNRPE essentially functions as a transcriptional activator of hsp70 gene expression. The overexpression of HSP70 in tumor cells has been associated with cell survival and drug resistance. We here identified novel regulators of Hsp70 expression in stress signaling and also provided important insights into Hsp70-targeted anti-cancer therapy. J. Cell. Biochem. 117: 2109-2117, 2016. © 2016 Wiley Periodicals, Inc.

Upregulation of heat shock proteins (HSPA12A, HSP90B1, HSPA4, HSPA5 and HSPA6) in tumour tissues is associated with poor outcomes from HBV-related early-stage hepatocellular carcinoma.

BACKGROUND: Heat shock proteins (HSPs) are overexpressed in human hepatocellular carcinoma (HCC) tissue and correlate with aggressiveness and prognosis of HCC. METHODS: Using the GSE14520 microarray expression profile from Gene Expression Omnibus, we compared HSP gene expression between tumour and non-tumour tissues and correlated this with outcomes in HCC patients. RESULTS: We analysed 220 hepatitis B virus (HBV)-related HCC patients and 25 HSPs in this study. With the exception of HSPA4L, HSPA12A and HSPB8, members of the HSP family, including HSPH1, HSPBP1, HSPA1A, HSPA1B, HSPA1L, HSPA2, HSPA4, HSPA5, HSPA8, HSPA9, HSPAA1, HSPAB1, HSPA14, HSPB11, HSPA13, HSP90B1 and HSPBAP1, were all overexpressed in tumour tissues (all P < 0.001). In contrast, HSPB6, HSPB7, HSPA6, HSPB2 and HSPB3 were upregulated in non-tumour tissues (all P < 0.001). Multivariate analysis showed that cirrhosis (HR = 5.282, 95% CI = 1.294-21.555, P = 0.02), Barcelona Clinic liver cancer (BCLC) staging (HR = 2.151, 95% CI = 1.682-2.750, P < 0.001), HSPA12A (HR = 1.042, 95% CI = 1.003-1.082, P = 0.033) and HSP90B1 (HR = 1.001, 95% CI = 1.000-1.001, P = 0.011) were negatively associated with survival of HBV-related HCC patients. Furthermore, advanced BCLC staging (HR = 1.797, 95% CI = 1.439-2.244, P < 0.001) was also associated with earlier recurrence of HCC. The high expression of HSPA4 (HR = 1.002, 95% CI = 1.000-1.004, P = 0.019), HSPA5 (HR = 1.0, 95% CI = 1.0-1.0, P = 0.046) and HSPA6 (HR = 1.008, 95% CI = 1.001-1.015, P = 0.021) was similarly associated with HCC recurrence. CONCLUSIONS: The expression of most HSPs was higher in tumour tissues than in non-tumour tissues. High BCLC staging scores, advanced cirrhosis and the overexpression of HSPA12A and HSP90B1 might be associated with poor survival from HCC, whereas high levels of HSPA4, HSPA5 and HSPA6 might be associated with earlier recurrence of HCC.

Identification of Hsf1 as a novel androgen receptor-regulated gene in mouse Sertoli cells.

Androgen signaling plays a crucial role in spermatogenesis, yet few downstream targets for this signaling pathway have been identified. In the current study, we found that the expression of heat-shock transcription factor 1 (Hsf1) was increased in the testes of Sertoli cell-selective androgen receptor knockout (S-AR(-/y) ) mice compared with wild-type mice by quantitative real-time PCR, and the expression of HSF1 in the S-AR(-/y) Sertoli cells was significantly increased, based on immunofluorescence analysis. In vitro cell-culture studies showed that testosterone repressed the expression of Hsf1 in TM4 cells, a mouse Sertoli cell line. Moreover, a luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay showed that testosterone repressed Hsf1 expression by facilitating the binding of androgen receptor to the Hsf1 promoter. Our experiments also demonstrated that testosterone-mediated inhibition of Hsf1 transcription down-regulated the expression of heat-shock proteins HSP105 and HSP60. Taken together, these results reveal that Hsf1 is a novel target of androgen receptor in mouse Sertoli cells, and testosterone and its receptor regulate the process of spermatogenesis partially by inhibiting Hsf1 expression.

Non-invasive infra-red therapy (1072 nm) reduces ß-amyloid protein levels in the brain of an Alzheimer's disease mouse model, TASTPM.

BACKGROUND: Alzheimer's disease (AD) is the most prevalent neurodegenerative disease and common cause of dementias in the Western world. This study investigated the expression profile of heat-shock proteins (HSPs) involved in maintaining healthy neurons in the TASTPM AD mouse model, and whether chronic treatment with 1072 nm infra-red (IR1072) modified the expression profiles of HSPs and amyloidopathy in female TASTPM mice. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative immunoblotting and immunohistochemistry were used to examine the expression of proteins such as HSPs, phosphorylated tau (tau-P), amyloid precursor protein (APP), ß-amyloid1-40 (Aß), and Aß1-42. TASTPM mice at 3, 7 and 12 months were investigated as well as female TASTPM mice which had undergone a chronic, 5 month, IR1072 treatment. During the first 12 months of age, a critical period of AD progression, reduced HSP40 and HSP105 were observed. αB-crystallin, Aß1-42 and tau-P increased over this period, particularly between 3 and 7 months. Chronic IR1072 treatment of female TASTPM mice elicited significant increases in HSP60, 70 and 105 and phosphorylated-HSP27 (P-HSP27) (50-139%), together with a concomitant profound decrease in αB-crystallin, APP, tau-P, Aß1-40 and Aß1-42 (43-81%) protein levels at 7 months of age. Furthermore, IR1072 treatment elicited a modest, but significant, reduction in Aß1-42 plaques in the cerebral cortex. CONCLUSIONS/SIGNIFICANT FINDINGS: IR1072 treatment provides a novel non-invasive and safe way to upregulate a panel of stress response proteins in the brain, known to both reduce protein aggregation and neuronal apoptosis. This approach recently entered clinical trials for AD in the USA, and may provide a novel disease modifying therapy for a range of neuropathologies.

Maternal exercise reduces hyperthermia-induced apoptosis in developing mouse brain.

PURPOSE: Hyperthermia-induced apoptosis is mediated by mitochondrial pathway, and is temporally correlated with alterations in mitochondrial morphology in neuroepithelial cells. In addition, regular exercise up-regulates heat shock proteins (HSPs) that inhibit apoptosis. However, embryo-protective effects of maternal exercise against heat exposure during pregnancy have not been fully understood yet. MATERIALS AND METHODS: To investigate the role of maternal exercise in protecting embryos from hyperthermia, we measured apoptosis-related factors and HSPs in Hsp70 knockout mouse embryos. Pregnant mice were divided into control, exercise, hyperthermia-after-exercise, and hyperthermia groups. Where appropriate the swimming exercise was performed for 5-10 min/day from embryonic day (ED) 1 to ED 8, and hyperthermia (43°C, 5 min) was induced on ED 8. To characterise the effects of maternal exercise on apoptosis-related factors and HSPs, we performed western blotting and transmission electron microscopy. RESULTS: Caspase-9, -7, -3 and Bax were down-regulated in the hyperthermia-after-exercise group and Bcl-2, Hsp27 and Hsp110 were up-regulated. The number of apoptotic cells was markedly reduced in the hyperthermia-after-exercise group. CONCLUSIONS: Maternal exercise plays an important role in inhibiting apoptotic cell death in embryos against hyperthermic exposure during pregnancy.

The expression of heat shock proteins 27 and 105 in squamous cell carcinoma of the tongue and relationship with clinicopathological index.

INTRODUCTION: In oral cavity, the tongue is the most common site prone to development of squamous cell carcinoma (SCC). Considering malignant transformation as a cellular stress, the expression of heat shock proteins (HSPs) may be affected in this process. In this study we assessed the expression of HSP105 and HSP27 as two of the most interested stress proteins and investigated their relationship with grade and stage of the tongue SCC. MATERIAL AND METHODS: Fifty-six specimens including 31 early and 25 advanced tongue SCC were gathered. All specimens were graded histologically from I to III. Sixteen sections of normal oral mucosa were used as control group. The cellularity and intensity of HSP105 and HSP27 expression were studied immunohistochemically in both case and control groups. Results were expressed by histochemical score (HSCORE). RESULTS: Significant differences were observed between expression of HSPs and stage of the disease. From early to advanced stage, the expression of HSP105 and HSP27 increased and decreased, respectively. There was no relationship between histological grade of lesion and HSCORE of HSP105 expression (P=0.5), although, HSP27 expression had reverse relationship with the SCC histological grade. CONCLUSION: HSP27 and HSP105 may be indicated for prognostic purposes in evaluation of tongue SCC. HSP 27 may be used for more accurate microscopic grading of tongue SCC. Increased expression of HSP105 in advanced stage may lead to using this protein for immunotherapy of tongue SCC.

Exposure to ELF magnetic fields modulate redox related protein expression in mouse macrophages.

The interaction of extremely low frequency (ELF) magnetic fields (MF) with cells can induce alterations in various cell physiological processes. Here, we present evidence that exposure of mouse macrophages to 50 Hz, 1.0 mT MF lead to immune cell activation seen as increased production of reactive oxygen species (ROS), and also to modulation on the expression level of important proteins acting in redox regulatory processes and thus explaining the noted changes in ROS levels seen after exposure. The MF exposure caused slight and transient decreases after short term exposures (2h or less) of clathrin, adaptin, PI3-kinase, protein kinase B (PKB) and PP2A, whereas longer exposures had no effect. The levels of the NAD(P)H oxidase subunit gp91phox oscillated between increased and normal levels compared to controls. The stress proteins Hsp70 and Hsp110 exhibited increased levels at certain time points, but not generally. The effects of MF on protein levels are different from the effects exerted by 12-O-tetradecanolyphobol-13-acetate (TPA) or LPS, although all three factors cause increases in ROS release. This suggests that ELF MF interacts with other cellular constituents than these chemicals, although induced pathways at least partially converge.

Expression of heat shock protein 105 and 70 in malignant melanoma and benign melanocytic nevi.

BACKGROUND: Heat shock proteins (HSPs) restore immature proteins or denatured proteins, thus protecting cells. Also, the expression of some HSPs is elevated substantially in malignant tumors, but the expression of HSPs in association with melanoma has yet to be studied. Therefore, we examined the expression patterns of HSP 70 and 105 in melanoma, benign melanocytic nevi and normal human skin. METHODS: Two specimens of malignant melanoma, two of benign melanocytic nevi and six of normal human skin were analyzed using Western blot analysis for expression of HSP 70 and 105. In another set, 16 specimens of malignant melanoma, 24 of benign melanocytic nevi and eight of normal human skin were analyzed for the expression of HSP 105 using immunohistochemical studies. RESULTS: The Western blot analysis showed that HSP 70 was overexpressed in all three types. But, the HSP 105 was hardly expressed in normal human skin and benign melanocytic nevi. However, in malignant melanoma, the HSP 105 was overexpressed, and immunohistochemical examination of HSP 105 showed a result similar to that of Western blot analysis. CONCLUSIONS: In our study, HSP 105 is thought to be a more relevant tumor-associated antigen in malignant melanoma than is HSP 70.

Significant overexpression of Hsp110 gene during colorectal cancer progression.

Colorectal cancer (CRC) is one of the most frequent malignant diseases in the world. Metastatic spread of the cancer to the lymph nodes is a crucial factor for progression and therapeutic management of the disease. We analysed gene expression profiles of CRC patiens by low-density cancer-focused oligonucleotide microarrays to identify new predictive markers of the extent of the disease and for better understanding of CRC progression. Relative expression levels of 440 genes known to be involved in cancer biology were obtained by low-density oligonucleotide microarrays from 20 tumor samples. Statistical analysis of gene expression data identified 3 genes (HSP110, HYOU1 and TCTP) significantly up-regulated in primary tumors of patients who developed lymph node metastasis. We have shown, for the first time, that up-regulation HSP110 and HYOU1 expression is associated with lymph node involvement in CRC. We validated the differences in HSP110 expression in an independent group of 30 patients of all clinical stages by real-time PCR. We identified significant up-regulation of HSP110 expression in colorectal tumors compared to adjacent non-tumoral tissue (p<0.0003). We observed significant differences of HSP110 gene expression between metastatic and localized disease (p=0.031) and negative trend of HSP110 gene expression and overall survival of CRC patients. We suggest that HSP110 gene is a promising molecular predictor in CRC.

Examination of the expression of the heat shock protein gene, hsp110, in Xenopus laevis cultured cells and embryos.

Eukaryotic organisms respond to various stresses with the synthesis of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is part of the HSP70/DnaK superfamily. In this study, we have examined, for the first time, the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. Sequence analysis revealed that the protein encoded by the hsp110 cDNA exhibited 74% identity with its counterparts in mammals and only 27-29% with members of the Xenopus HSP70 family. Hsp110 mRNA and/or protein was detected constitutively in A6 kidney epithelial cells and was inducible by heat shock, sodium arsenite, and cadmium chloride. However, treatment with ethanol or copper sulfate had no detectable effect on hsp110 mRNA levels. Similar results were obtained for hsp70 mRNA except that it was inducible with ethanol. In Xenopus embryos, hsp110 mRNA was present constitutively during development. Heat shock-inducible accumulation of hsp110 mRNA occurred only after the midblastula stage. Whole mount in situ hybridization analysis revealed that hsp110 mRNA accumulation in control and heat shocked embryos was enriched in selected tissues. These studies demonstrate that Xenopus hsp110 gene expression is constitutive and stress inducible in cultured cells and developmentally- and tissue specifically-regulated during early embryogenesis.

Correlation of Hsp110 expression with caspase-3 and -9 during apoptosis induced by in vivo embryonic exposition to retinoic acid or irradiation in early mouse craniofacial development.

OBJECTIVE: To analyze the expression and role of three proteins (HSP110, caspase-3 and caspase-9) during craniofacial development. DESIGN: Seven pregnant C57Bl/6J mice received, by force-feeding at gestation day 9 (E9), 80 mg/kg of all-trans retinoic acid mixed to sesame oil. Seven pregnant NMRI mice received two grays irradiation at the same gestation day. Control mice of both strains (seven mice for each strain) were not submitted to any treatment. Embryos were obtained at various stages after exposition (3, 6, 12 and 24 h), fixed, dehydrated and embedded. Coronal sections (5 microm) were made. Slide staining occurred alternatively using anti-Hsp110, anti-caspase-3 and anti-caspase-9 immunohistochemistry. RESULTS: Expression of HSP110, caspase-3 and caspase-9 was found in cells of well-known locations of programmed cell death. After retinoic acid exposure, expressions were increased especially in neural crest cells of mandibular and hyoid arches. Quantification of positive cells shows that caspase-9 and Hsp110 were expressed before caspase-3. After irradiation, the expression of the three proteins quickly increased with a maximum 3 h after irradiation. For all three models of apoptosis (physiological, retinoic-induced and irradiation-induced) HSP110 positive cells were more numerous than caspase-3 positive cells. Caspase-3 positive cells were more numerous than caspase-9 positive cells especially in mesectodermal irradiation-induced apoptotic cells. CONCLUSION: The findings show a potential function of HSP110 in apoptosis during embryo development. Caspase-3-expressing cells are more numerous than cells expressing caspase-9, especially irradiation-induced apoptotic neural crest cells. This suggests that other caspases, still to be identified, may activate caspase-3 in this model.

DNA vaccination of HSP105 leads to tumor rejection of colorectal cancer and melanoma in mice through activation of both CD4 T cells and CD8 T cells.

We report that HSP105, identified by serological identification of antigens by recombinant expression cloning (SEREX), is overexpressed in a variety of human cancers, including colorectal, pancreatic, thyroid, esophageal, and breast carcinoma, but is not expressed in normal tissues except for the testis. The amino acid sequences and expression patterns of HSP105 are very similar in humans and mice. In this study, we set up a preclinical study to investigate the usefulness of a DNA vaccine producing mouse HSP105 whole protein for cancer immunotherapy in vivo using BALB/c and C57BL/6 mice, Colon26, a syngeneic endogenously HSP105-expressing colorectal cancer cell line, and B16.F10, a melanoma cell line. The DNA vaccine was used to stimulate HSP105-specific T-cell responses. Fifty percent of mice immunized with the HSP105 DNA vaccine completely suppressed the growth of subcutaneous Colon26 or B16.F10 cells accompanied by massive infiltration of both CD4+ T cells and CD8+ T cells into tumors. In cell transfer or depletion experiments we proved that both CD4+ T cells and CD8+ T cells induced by these vaccines play critical roles in the activation of antitumor immunity. Evidence of autoimmune reactions was not present in surviving mice that had rejected tumor cell challenges. We found that HSP105 was highly immunogenic in mice and that the HSP105 DNA vaccination induced antitumor immunity without causing autoimmunity. Therefore, HSP105 is an ideal tumor antigen that could be useful for immunotherapy or the prevention of various human tumors that overexpress HSP105, including colorectal cancer and melanoma.