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.

Nmi interacts with Hsp105ß and enhances the Hsp105ß-mediated Hsp70 expression.

The mammalian stress protein Hsp105α is expressed constitutively and is further induced under stress conditions, whereas the alternative spliced form, Hsp105ß is only expressed during mild heat shock. We previously reported that Hsp105α is localized mainly in the cytoplasm, whereas Hsp105ß is localized in the nucleus. Consistent with the different localization of these proteins, Hsp105ß but not Hsp105α induces the expression of the major stress protein Hsp70. We here identified N-myc and Stat interactor (Nmi), as an Hsp105ß-binding protein by yeast two-hybrid screening. Immunoprecipitation and pull-down assay showed that Nmi interacts with Hsp105ß in vivo and in vitro. Luciferase reporter gene assay and Western blotting showed that Nmi enhanced both the Hsp105ß-induced phosphorylation of Stat3 and the Hsp105ß-induced activation of the hsp70 promoter in a manner that is dependent on the Stat3-binding site, which results in an increase in Hsp70 protein levels. Most importantly, mild heat shock-induced Hsp70 expression, which is dependent on Hsp105ß, is suppressed by knockdown of endogenous Nmi. These results suggest that Nmi has a role as a positive regulator of Hsp105ß-mediated hsp70 gene expression along the Stat3 signaling pathway.

Increased expression of sorcin is associated with multidrug resistance in leukemia cells via up-regulation of MDR1 expression through cAMP response element-binding protein.

Sorcin, a 22 kDa Ca(2+) binding protein, was first identified in a vincristine-resistant Chinese hamster lung cell line, and was later demonstrated to be involved in the development of multidrug-resistance (MDR) phenotypes in a variety of human cancer cell lines. However, the exact role of sorcin in MDR cells is yet to be fully elucidated. Here we explored the role of sorcin in the development of MDR in leukemia cells, and revealed that the expression level of sorcin was directly correlated to the expression of MDR1/P-glycoprotein (P-gp). In addition, it was shown that sorcin induced the expression of MDR1/P-gp through a cAMP response element (CRE) between -716 and -709 bp of the mdr1/p-gp gene. Furthermore, overexpression of sorcin increased the phosphorylation of CREB1 and the binding of CREB1 to the CRE sequence of mdr1/p-gp promoter, and induced the expression of MDR1/P-gp. These findings suggested that sorcin induces MDR1/P-gp expression markedly through activation of the CREB pathway and is associated with the MDR phenotype. The new findings may be helpful for understanding the mechanisms of MDR in human cancer cells, prompting its further investigation as a molecular target to overcome MDR.

Naringenin inhibits the aggregation of expanded polyglutamine tract-containing protein through the induction of endoplasmic reticulum chaperone GRP78.

Polyglutamine (polyQ) diseases are inherited neurodegenerative diseases characterized by the aggregation of proteins containing expanded polyQ tract. Recently, we have shown that GRP78, the endoplasmic reticulum (ER) chaperone, was significantly decreased in the cells expressing enhanced green fluorescent protein with a pathological-length polyQ tract (EGFP-polyQ97), but not with a non-pathological-length polyQ tract (EGFP-polyQ24), and the expression levels of GRP78 were inversely related to the aggregation of EGFP-polyQ97. In this study, we performed the screening for compounds that modulate the GRP78 expression in herbal medicines, and found that naringenin, one of the major constitutions of Kanzo (Glycyrrhizae Radix), induced the expression of GRP78 in several mammalian cells. Furthermore, naringenin suppressed the protein aggregation caused by EGFP-polyQ97 in mammalian cells. These findings suggested that naringenin seemed to be a new inducer of GRP78 in mammalian cells, and may be a potential therapeutic agent for diseases caused by ER stress such as polyQ diseases.

Endoplasmic reticulum chaperone GRP78 suppresses the aggregation of proteins containing expanded polyglutamine tract.

Polyglutamine (polyQ) diseases are inherited neurodegenerative diseases characterized by the aggregation of proteins containing expanded polyQ tract. It has been shown that expanded polyQ tract-containing proteins impair the functions of other cellular proteins. However, quantitative changes of cellular proteins in cells expressing expanded polyQ tract-containing proteins have not been performed. Here, we performed proteomic analysis of cells expressing expanded polyQ tract-containing proteins, and showed that GRP78, the endoplasmic reticulum (ER) chaperone, was significantly decreased in the cells expressing enhanced green fluorescent protein with a pathological-length polyQ tract (EGFP-polyQ97), but not with a non-pathological-length polyQ tract (EGFP-polyQ24). In addition, we revealed that down-regulation of GRP78 expression resulted in increase of the aggregation of EGFP-polyQ97. Conversely, the aggregation of EGFP-polyQ97 was suppressed by the overexpression of GRP78 in the cells. Furthermore, it seemed that the decreased GRP78 expression in the cells expressing EGFP-polyQ97 was due to the enhanced protein degradation of GRP78 through the ubiquitin-proteasome pathway. These findings indicated that GRP78, which has an inhibitory effect on the aggregation of proteins containing expanded polyQ tract, may be an effective target for the treatment of polyQ diseases.

Decreased expression of endoplasmic reticulum chaperone GRP78 in liver of diabetic mice.

To identify molecular targets associated with the development of diabetes, we analyzed the hepatic proteome of obese diabetic db/db mice using electrophoresis on a high-resolution two-dimensional gel combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. By comparison between non-diabetic db/+ and diabetic db/db mice, six proteins and one protein were significantly decreased and increased in the diabetic mice, respectively. Among these proteins, two of the decreased proteins are involved in endoplasmic reticulum (ER) stress-related unfolded protein response, GRP78 and protein disulfide isomerase A3, and it was revealed that the decreased GRP78 expression in the liver of diabetic db/db mice is due to the reduction of GRP78 protein synthesis rather than RNA transcription. In addition, we found that the treatment of human hepatocyte HepG2 cells with oleic acid decreased the expression of GRP78, and attenuated the activation of AKT by insulin stimulation. These results suggest that decreased GRP78 expression may induce resistance to insulin by inhibiting the AKT activation, and plays an important role in the development of type 2 diabetes.

Characterization of stress sensitivity and chaperone activity of Hsp105 in mammalian cells.

Hsp105 is a major mammalian heat shock protein that belongs to the Hsp105/110 family, a diverged subgroup of the Hsp70 family. Hsp105 not only protects the thermal aggregation of proteins, but also regulates the Hsc70 chaperone system in vitro. Recently, it has been shown that Hsp105/110 family members act as nucleotide exchange factors for cytosolic Hsp70s. However, the biological functions of Hsp105/110 family proteins still remain to be clarified. Here, we examined the function of Hsp105 in mammalian cells, and showed that the sensitivity to various stresses was enhanced in the Hsp105-deficient cells compared with that in control cells. In addition, we found that deficiency of Hsp105 impaired the refolding of heat-denatured luciferase in mammalian cells. In contrast, overexpression of Hsp105α enhanced the ability to recover heat-inactivated luciferase in mammalian cells. Thus, Hsp105 may play an important role in the refolding of denatured proteins and protection against stress-induced cell death in mammalian cells.

Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70.

Hsp105alpha and Hsp105beta are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105alpha is expressed constitutively in the cytoplasm of cells, while Hsp105beta, an alternatively spliced form of Hsp105alpha, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105beta but also Hsp105alpha accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105alpha and Hsp105beta with changes in the nuclear localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105alpha and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105alpha and Hsp105beta on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105alpha and Hsp105beta suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.

Hsp105beta upregulates hsp70 gene expression through signal transducer and activator of transcription-3.

Hsp105alpha and Hsp105beta are mammalian members of the Hsp105/110 family, a divergent subgroup of the Hsp70 family. Hsp105alpha is expressed constitutively and induced by various forms of stress, whereas Hsp105beta is an alternatively spliced form of Hsp105alpha that is expressed specifically during mild heat shock. In a report, it was shown that Hsp105alpha and Hsp105beta localize to the cytoplasm and of nucleus of cells, respectively, and that Hsp105beta, but not Hsp105alpha, induces the expression of Hsp70 in mammalian cells. Here, we examined the mechanism by which Hsp105beta induces the expression of Hsp70. Using a series of deletion mutants of Hsp105beta, it was revealed that the region between amino acids 642 and 662 of Hsp105beta is necessary for the activation of the hsp70 promoter by Hsp105beta. Furthermore, it was shown that signal transducer and activator of transcription (STAT)-3 bound to the sequence of the hsp70 promoter between -206 and -187 bp, and that mutations of this sequence abrogated the activation of the hsp70 promoter by Hsp105beta. In addition, overexpression of Hsp105beta stimulated the phosphorylation of STAT3 at Tyr705 and its translocation to the nucleus. Downregulation of STAT3 expression resulted in reduction of the activation of the hsp70 promoter by Hsp105beta. Furthermore, downregulation of Hsp105beta reduced the expression of Hsp70 in heat-shocked cells. On the basis of these findings, it is suggested that Hsp105beta induces Hsp70 expression markedly through the STAT3 pathway in heat-shocked cells. This may represent the mechanism that connects the heat shock protein and STAT families for cell defense against deleterious stress.

Nuclear localization mechanism of Hsp105beta and its possible function in mammalian cells.

Hsp105alpha and Hsp105beta are mammalian stress proteins of the Hsp105/110 family. We have shown that Hsp105beta localizes to the nucleus, whereas Hsp105alpha localizes to the cytoplasm of mammalian cells. Hsp105alpha localizes in the cytoplasm, as the nuclear export signal (NES) activity rather than nuclear localization signal (NLS) activity dominates in Hsp105alpha, due to suppression of the NLS activity. In this study, we determined the mechanisms behind the nuclear localization of Hsp105beta, and revealed that the NES was suppressed by the N-terminal (amino acids 3-10) or C-terminal (amino acids 699-756) region of Hsp105beta, and the NLS activity rather than NES activity seemed to dominate in Hsp105beta. Furthermore, as Hsp105beta which localizes in the nucleus, functioned as an inducer of Hsp70 in mammalian cells, Hsp105 family proteins may play an important role in the protection of cells against deleterious stressor together with Hsp70.

Mammalian 105 kDa heat shock family proteins suppress hydrogen peroxide-induced apoptosis through a p38 MAPK-dependent mitochondrial pathway in HeLa cells.

Hsp105alpha and Hsp105beta are major heat shock proteins in mammalian cells that belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105alpha has opposite effects on stress-induced apoptosis depending on the cell type. However, it is not fully understood how Hsp105 regulates stress-induced apoptosis. In this study, we examined how Hsp105alpha and Hsp105beta regulate H2O2-induced apoptosis by using HeLa cells in which expression of Hsp105alpha or Hsp105beta was regulated using doxycycline. Overexpression of Hsp105alpha and Hsp105beta suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria in H2O2-treated cells. Furthermore, both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) were activated by treatment with H2O2, and the activation of both kinases was suppressed by overexpression of Hsp105alpha and Hsp105beta. However, H2O2-induced apoptosis was suppressed by treatment with a potent inhibitor of p38 MAPK, SB202190, but not a JNK inhibitor, SP600125. These findings suggest that Hsp105alpha and Hsp105beta suppress H2O2-induced apoptosis by suppression of p38 MAPK signaling, one of the essential pathways for apoptosis.

Targeted disruption of Hsp110/105 gene protects against ischemic stress.

BACKGROUND AND PURPOSE: Hsp110/105 belongs to the HSP110 heat shock protein family, which is a subgroup of the HSP70 family. In mammals, Hsp110/105 is constitutively expressed but exhibits particularly high levels in the brain. It has recently been shown that both Hsp110/105 and Hsp70 are elevated after cerebral ischemia. To study the physiological role of this protein in vivo, we generated hsp110/105 knockout (KO) mice and investigate the effect of reduced Hsp110/105 levels on focal cerebral ischemia. METHODS: hsp110/105 KO and wild-type mice were subjected to 30 minutes of transient middle cerebral artery occlusion followed by reperfusion for 24 hours. The infarct volume and neurological scores were measured and compared. The Hsp70 chaperone activity of thermally denatured firefly luciferase was measured in hsp110/105 KO embryonic fibroblasts. RESULTS: The infarct volume and neurological deficit scores were significantly (P<0.05) reduced in hsp110/105 KO mice compared with wild-type controls. In addition, hsp110/105 KO embryonic fibroblasts exhibited a dose-dependent suppression of Hsp70 chaperone activity by the presence of Hsp110/105. CONCLUSIONS: These results demonstrate that hsp110/105 KO mice are resistant to ischemic injury and that the protective effects of hsp110/105 deficiency in cerebral ischemia may partly be mediated by an increase in the chaperone activity of Hsp70.

Different localization of Hsp105 family proteins in mammalian cells.

Hsp105alpha and Hsp105beta of the HSP105 family are alternatively spliced products derived from an hsp 105 gene transcript. Hsp105alpha is constitutively expressed and also induced by various stress, whereas Hsp105beta, lacking 44 amino acids from Hsp105alpha, is specifically expressed during mild heat shock. Although Hsp105alpha is shown to localize in the cytoplasm of mammalian cells, cellular localization of Hsp105beta is not known. In this study, we showed that Hsp105beta localized in the nucleus of cells in contrast to cytoplasmic Hsp105alpha, suggesting that these proteins function in different cellular compartments of cells. Using deletion and substitution mutants of Hsp105alpha and Hsp105beta, we revealed that these proteins had a functional nuclear localization signal (NLS) and a nuclear export signal (NES). Furthermore, Hsp105alpha accumulated in the nucleus of cells when treated with leptomycin B, a specific inhibitor of NES-dependent nuclear export. siRNA for importin beta, an essential component for NLS-dependent nuclear transport, inhibited the nuclear localization of Hsp105beta. Furthermore, the 44 amino acids sequence found in Hsp105alpha but not in Hsp105beta suppressed the NLS activity. Thus, the different localization of Hsp105alpha and Hsp105beta is suggested to be due to the suppressed NLS activity in Hsp105alpha.

Heat-shock protein 105 interacts with and suppresses aggregation of mutant Cu/Zn superoxide dismutase: clues to a possible strategy for treating ALS.

A dominant mutation in the gene for copper-zinc superoxide dismutase (SOD1) is the most frequent cause of the inherited form of amyotrophic lateral sclerosis. Mutant SOD1 provokes progressive degeneration of motor neurons by an unidentified acquired toxicity. Exploiting both affinity purification and mass spectrometry, we identified a novel interaction between heat-shock protein 105 (Hsp105) and mutant SOD1. We detected this interaction both in spinal cord extracts of mutant SOD1(G93A) transgenic mice and in cultured neuroblastoma cells. Expression of Hsp105, which is found in mouse motor neurons, was depressed in the spinal cords of SOD1(G93A) mice as disease progressed, while levels of expression of two other heat-shock proteins, Hsp70 and Hsp27, were elevated. Moreover, Hsp105 suppressed the formation of mutant SOD1-containing aggregates in cultured cells. These results suggest that techniques that raise levels of Hsp105 might be promising tools for alleviation of the mutant SOD1 toxicity.

The phenylic hydroxyl group is essential for the induction of stress response by sodium salicylate.

We have shown that sodium salicylate (SA) activates the heat shock promoter and induces the expression of heat shock proteins (Hsps) with a concomitant increase in the thermotolerance of cells. To identify the functional groups of SA necessary for the induction of Hsps, we evaluated the effect of various derivatives of SA using a mammalian cell line containing a reporter gene downstream of an hsp105 promoter. Among the derivatives, the compounds in which the carboxyl group of SA was substituted activated the hsp105 promoter at 37 degrees C as SA did, but the compounds in which the hydroxyl group was substituted did not. Thus, the phenylic hydroxyl group but not the carboxyl group of SA seemed to be necessary for a stress-induced response. In addition, the orientation of two functional groups on the benzene ring of SA derivatives was also important for the induction of a response. Among these compounds, salicylalcohol which strongly induced the expression of Hsps suppressed the protein aggregation and apoptosis caused by an expanded polyglutamine tract in a cellular model of polyglutamine disease. These findings may aid in the development of novel effective Hsp-inducers.

Arctigenin from Fructus Arctii is a novel suppressor of heat shock response in mammalian cells.

Because heat shock proteins (Hsps) are involved in protecting cells and in the pathophysiology of diseases such as inflammation, cancer, and neurodegenerative disorders, the use of regulators of the expression of Hsps in mammalian cells seems to be useful as a potential therapeutic modality. To identify compounds that modulate the response to heat shock, we analyzed several natural products using a mammalian cell line containing an hsp promoterregulated reporter gene. In this study, we found that an extract from Fructus Arctii markedly suppressed the expression of Hsp induced by heat shock. A component of the extract arctigenin, but not the component arctiin, suppressed the response at the level of the activation of heat shock transcription factor, the induction of mRNA, and the synthesis and accumulation of Hsp. Furthermore, arctigenin inhibited the acquisition of thermotolerance in mammalian cells, including cancer cells. Thus, arctigenin seemed to be a new suppressive regulator of heat shock response in mammalian cells, and may be useful for hyperthermia cancer therapy.

Synthetic small interfering RNA targeting heat shock protein 105 induces apoptosis of various cancer cells both in vitro and in vivo.

We previously reported that heat shock protein 105 (HSP105), identified by serological analysis of a recombinant cDNA expression library (SEREX) using serum from a pancreatic cancer patient, was overexpressed in various human tumors and in the testis of adult men by immunohistochemical analysis. In the present study, to elucidate the biological function of the HSP105 protein in cancer cells, we first established NIH3T3 cells overexpressing murine HSP105 (NIH3T3-HSP105). The NIH3T3-HSP105 cells acquired resistance to apoptosis induced by heat shock or doxorubicin. The small interfering RNA (siRNA)-mediated suppression of HSP105 protein expression induced apoptosis in human cancer cells but not in fibroblasts. By a combination of siRNA introduction and doxorubicin or heat shock treatment, apoptosis was induced synergistically in a human colon cancer cell line, HCT116. In vivo, siRNA inoculation into the human gastric cancer cell line KATO-3 established in the flank of an NOD SCID mouse suppressed the tumor growth. This siRNA-induced apoptosis was mediated through caspases, but not the p53 tumor suppressor protein, even though the HSP105 protein was bound to wild-type p53 protein in HCT116 cells. These findings suggest that the constitutive overexpression of HSP105 in cancer cells is involved in malignant transformation by protecting tumor cells from apoptosis. HSP105 may thus be a novel target molecule for cancer therapy and a treatment regimen using synthetic siRNA to suppress the expression of HSP105 protein may provide a new strategy for cancer therapy.

Hsp105 family proteins suppress staurosporine-induced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells.

Hsp105 (Hsp105alpha and Hsp105beta), major heat shock proteins in mammalian cells, belong to a subgroup of the HSP70 family, HSP105/110. Previously, we have shown that Hsp105alpha has completely different effects on stress-induced apoptosis depending on cell type. However, the molecular mechanisms by which Hsp105alpha regulates stress-induced apoptosis are not fully understood. Here, we established HeLa cells that overexpress either Hsp105alpha or Hsp105beta by removing doxycycline and examined how Hsp105 modifies staurosporine (STS)-induced apoptosis in HeLa cells. Apoptotic features such as the externalization of phosphatidylserine on the plasma membrane and nuclear morphological changes were induced by the treatment with STS, and the STS-induced apoptosis was suppressed by overexpression of Hsp105alpha or Hsp105beta. In addition, we found that overexpression of Hsp105alpha or Hsp105beta suppressed the activation of caspase-3 and caspase-9 by preventing the release of cytochrome c from mitochondria. Furthermore, the translocation of Bax to mitochondria, which results in the release of cytochrome c from the mitochondria, was also suppressed by the overexpression of Hsp105alpha or Hsp105beta. Thus, it is suggested that Hsp105 suppresses the stress-induced apoptosis at its initial step, the translocation of Bax to mitochondria in HeLa cells.

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.