Publications by authors named "Akira Nakai"

106 Publications

MED12 interacts with the heat-shock transcription factor HSF1 and recruits CDK8 to promote the heat-shock response in mammalian cells.

FEBS Lett 2021 May 31. Epub 2021 May 31.

Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan.

Activated and promoter-bound heat-shock transcription factor 1 (HSF1) induces RNA polymerase II recruitment upon heat shock, and this is facilitated by the core Mediator in Drosophila and yeast. Another Mediator module, CDK8 kinase module (CKM), consisting of four subunits including MED12 and CDK8, plays a negative or positive role in the regulation of transcription; however, its involvement in HSF1-mediated transcription remains unclear. We herein demonstrated that HSF1 interacted with MED12 and recruited MED12 and CDK8 to the HSP70 promoter during heat shock in mammalian cells. The kinase activity of CDK8 (and its paralog CDK19) promoted HSP70 expression partly by phosphorylating HSF1-S326 and maintained proteostasis capacity. These results indicate an important role for CKM in the protection of cells against proteotoxic stress.
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http://dx.doi.org/10.1002/1873-3468.14139DOI Listing
May 2021

Testicular localization of activating transcription factor 1 and its potential function during spermatogenesis.

Biol Reprod 2021 May 18. Epub 2021 May 18.

Department of Urology, School of Medicine, Yamaguchi University, Ube, Yamaguchi 755-8505, Japan.

Activating transcription factor 1 (ATF1), belonging to the CREB/ATF family of transcription factors, is highly expressed in the testes. However, its role in spermatogenesis has not yet been established. Here, we aimed to elucidate the impact of ATF1 in spermatogenesis by examining the expression pattern of ATF1 in mice and the effect of ATF1 knockdown in the mouse testes. We found that ATF1 is expressed in various organs, with very high levels in the testes. Immunohistochemical staining showed that ATF1 was localized in the nuclei of spermatogonia and co-localized with proliferating cell nuclear antigen. In ATF1-deficient mice, the seminiferous tubules of the testis contained cells at all developmental stages; however, the number of spermatocytes was decreased. Proliferating cell nuclear antigen expression was decreased and apoptotic cells were rare in the seminiferous tubules. These results indicate that ATF1 plays a role in male germ cell proliferation and sperm production.
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http://dx.doi.org/10.1093/biolre/ioab099DOI Listing
May 2021

Ferroptosis inducer erastin sensitizes NSCLC cells to celastrol through activation of the ROS-mitochondrial fission-mitophagy axis.

Mol Oncol 2021 Mar 6. Epub 2021 Mar 6.

Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, China.

Despite recent progress in non-small-cell lung cancer (NSCLC) treatment, treatment outcomes remain poor, mainly because of treatment resistance or toxicity. Erastin is a ferroptosis inducer that has shown promising cytotoxic effects in various types of cancers, including NSCLC. Celastrol is a triterpene extracted from the Tripterygium wilfordii that exhibits potential anticancer activity. However, the side effects of celastrol are severe and limit its clinical application. Combination therapy is a promising strategy to overcome the compensatory mechanisms and unwanted off-target effects. In the present study, we found that erastin synergized with celastrol to induce cell death at nontoxic concentrations. The combined treatment with celastrol and erastin significantly increased reactive oxygen species (ROS) generation, disrupted mitochondrial membrane potential, and promoted mitochondrial fission. Furthermore, cotreatment with erastin and celastrol initiated ATG5/ATG7-dependent autophagy, PINK1/Parkin-dependent mitophagy, and the expression of heat shock proteins (HSPs) in an HSF1-dependent manner. HSF1 knockdown further enhanced cell death in vitro and inhibited tumor growth in vivo. Our findings indicate that the combination of celastrol with erastin may represent a novel therapeutic regimen for patients with NSCLC and warrants further clinical evaluation.
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http://dx.doi.org/10.1002/1878-0261.12936DOI Listing
March 2021

Nuciferine protects against folic acid-induced acute kidney injury by inhibiting ferroptosis.

Br J Pharmacol 2021 Mar;178(5):1182-1199

Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, China.

Background And Purpose: Acute kidney injury is a common clinical problem with no definitive or specific treatment. Therefore, the molecular mechanisms of acute kidney injury must be fully understood to develop novel treatments. Nuciferine, a major bioactive compound isolated from the lotus leaf, possesses extensive pharmacological activities. Its effect on folic acid-induced acute kidney injury, however, remains unknown. Here, we aimed to clarify the pharmacological effects of nuciferine and its mechanisms of action in acute kidney injury.

Experimental Approach: The effects of nuciferine on folic acid-induced acute kidney injury in mice were investigated. HK-2 human proximal tubular epithelial cells and HEK293T HEK cells were used to evaluate the protective effect of nuciferine on RSL3-induced ferroptosis.

Key Results: Nuciferine treatment mitigated the pathological alterations, ameliorated inflammatory cell infiltration and improved kidney dysfunction in mice with folic acid-induced acute kidney injury. In HK-2 and HEK293T cells, nuciferine significantly prevented RSL3-induced ferroptotic cell death. Mechanistically, nuciferine significantly inhibited ferroptosis by preventing iron accumulation and lipid peroxidation in vitro and in vivo. Moreover, knockdown of glutathione (GSH) peroxidase 4 (GPX4) abolished the protective effect of nuciferine against ferroptosis.

Conclusion And Implications: Nuciferine ameliorated renal injury in mice with acute kidney injury, perhaps by inhibiting the ferroptosis. Nuciferine may represent a novel treatment that improves recovery from acute kidney injury by targeting ferroptosis.
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http://dx.doi.org/10.1111/bph.15364DOI Listing
March 2021

HSF1 is required for induction of mitochondrial chaperones during the mitochondrial unfolded protein response.

FEBS Open Bio 2020 06 15;10(6):1135-1148. Epub 2020 May 15.

Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan.

The mitochondrial unfolded protein response (UPR ) is characterized by the transcriptional induction of mitochondrial chaperone and protease genes in response to impaired mitochondrial proteostasis and is regulated by ATF5 and CHOP in mammalian cells. However, the detailed mechanisms underlying the UPR are currently unclear. Here, we show that HSF1 is required for activation of mitochondrial chaperone genes, including HSP60, HSP10, and mtHSP70, in mouse embryonic fibroblasts during inhibition of matrix chaperone TRAP1, protease Lon, or electron transfer complex 1 activity. HSF1 bound constitutively to mitochondrial chaperone gene promoters, and we observed that its occupancy was remarkably enhanced at different levels during the UPR . Furthermore, HSF1 supported the maintenance of mitochondrial function under the same conditions. These results demonstrate that HSF1 is required for induction of mitochondrial chaperones during the UPR , and thus, it may be one of the guardians of mitochondrial function under conditions of impaired mitochondrial proteostasis.
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http://dx.doi.org/10.1002/2211-5463.12863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262932PMC
June 2020

The pericentromeric protein shugoshin 2 cooperates with HSF1 in heat shock response and RNA Pol II recruitment.

EMBO J 2019 12 28;38(24):e102566. Epub 2019 Oct 28.

Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan.

The recruitment of RNA polymerase II (Pol II) to core promoters is highly regulated during rapid induction of genes. In response to heat shock, heat shock transcription factor 1 (HSF1) is activated and occupies heat shock gene promoters. Promoter-bound HSF1 recruits general transcription factors and Mediator, which interact with Pol II, but stress-specific mechanisms of Pol II recruitment are unclear. Here, we show in comparative analyses of HSF1 paralogs and their mutants that HSF1 interacts with the pericentromeric adaptor protein shugoshin 2 (SGO2) during heat shock in mouse cells, in a manner dependent on inducible phosphorylation of HSF1 at serine 326, and recruits SGO2 to the HSP70 promoter. SGO2-mediated binding and recruitment of Pol II with a hypophosphorylated C-terminal domain promote expression of HSP70, implicating SGO2 as one of the coactivators that facilitate Pol II recruitment by HSF1. Furthermore, the HSF1-SGO2 complex supports cell survival and maintenance of proteostasis in heat shock conditions. These results exemplify a proteotoxic stress-specific mechanism of Pol II recruitment, which is triggered by phosphorylation of HSF1 during the heat shock response.
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http://dx.doi.org/10.15252/embj.2019102566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912021PMC
December 2019

HSF1 phosphorylation by cyclosporin A confers hyperthermia sensitivity through suppression of HSP expression.

Biochim Biophys Acta Gene Regul Mech 2019 08 3;1862(8):846-857. Epub 2019 May 3.

Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang, China. Electronic address:

Heat shock factor 1 (HSF1) is a transcription factor essential for tumorigenesis, and targeting HSF1 may be effective in combined therapeutics for cervical cancer. Cyclosporin A (CsA) is an immunosuppressant that has revolutionized organ transplantation. However, the roles and regulatory mechanisms by which CsA modulates HSP expression remain largely unknown. In this study, we found that CsA pretreatment prevented induction of HSPs during heat shock by enhancing the phosphorylation of Ser303 and Ser307 on HSF1 and thus inhibiting its transcriptional activity. Suppression of ERK1/2, GSK3β and CK2 activities attenuated CsA-induced down-regulation of HSP expression and up-regulation of HSF1 phosphorylation. CsA interfered with HSF1-SSBP1 complex formation and HSF1 nuclear translocation and recruitment to the HSP70 promoter. CsA clearly caused HeLa cell death during proteotoxic stress through reduced expression of HSPs. These results indicate that CsA suppresses HSP induction during heat shock by regulating the phosphorylation and nuclear translocation of HSF1. Our results provide a conceptual framework for the development of novel therapeutic strategies for cervical cancer through application of CsA during hyperthermia or chemotherapy.
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http://dx.doi.org/10.1016/j.bbagrm.2019.04.009DOI Listing
August 2019

Uric Acid-Induced Enhancements of Kv1.5 Protein Expression and Channel Activity via the Akt-HSF1-Hsp70 Pathway in HL-1 Atrial Myocytes.

Circ J 2019 03 20;83(4):718-726. Epub 2019 Feb 20.

Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Tottori University Graduate School of Medical Science.

Background: Intracellular uric acid is known to increase the protein level and channel current of atrial Kv1.5; however, mechanisms of the uric acid-induced enhancement of Kv1.5 expression remain unclear. Methods and Results: The effects of uric acid on mRNA and protein levels of Kv1.5, as well as those of Akt, HSF1 and Hsp70, in HL-1 cardiomyocytes were studied by using qRT-PCR and Western blotting. The uptake of uric acid was measured using radio-labeled uric acid. The Kv1.5-mediated channel current was also measured by using patch clamp techniques. Uric acid up-taken by HL-1 cells significantly increased the level of Kv1.5 proteins in a concentration-dependent manner, with this increase abolished by an uric acid transporter inhibitor. Uric acid slowed degradation of Kv1.5 proteins without altering its mRNA level. Uric acid enhanced phosphorylation of Akt and HSF1, and thereby increased both transcription and translation of Hsp70; these effects were abolished by a PI3K inhibitor. Hsp70 knockdown abolished the uric acid-induced increases of Kv1.5 proteins and channel currents.

Conclusions: Intracellular uric acid could stabilize Kv1.5 proteins through phosphorylation of Akt and HSF1 leading to enhanced expression of Hsp70.
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http://dx.doi.org/10.1253/circj.CJ-18-1088DOI Listing
March 2019

Poly(ADP-Ribose) Polymerase 1 Promotes the Human Heat Shock Response by Facilitating Heat Shock Transcription Factor 1 Binding to DNA.

Mol Cell Biol 2018 07 14;38(13). Epub 2018 Jun 14.

Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Minami-Kogushi, Ube, Japan

The heat shock response (HSR) is characterized by the rapid and robust induction of heat shock proteins (HSPs), including HSP70, in response to heat shock and is regulated by heat shock transcription factor 1 (HSF1) in mammalian cells. Poly(ADP-ribose) polymerase 1 (PARP1), which can form a complex with HSF1 through the scaffold protein PARP13, has been suggested to be involved in the HSR. However, its effects on and the regulatory mechanisms of the HSR are not well understood. Here we show that prior to heat shock, the HSF1-PARP13-PARP1 complex binds to the promoter. In response to heat shock, activated and auto-PARylated PARP1 dissociates from HSF1-PARP13 and is redistributed throughout the locus. Remarkably, chromatin in the promoter is initially PARylated at high levels and decondensed, whereas chromatin in the gene body is moderately PARylated afterwards. Activated HSF1 then binds to the promoter efficiently and promotes the HSR. Chromatin PARylation and HSF1 binding to the promoter are also facilitated by the phosphorylation-dependent dissociation of PARP13. Furthermore, the HSR and proteostasis capacity are reduced by pretreatment with genotoxic stresses, which disrupt the ternary complex. These results illuminate one of the priming mechanisms of the HSR that facilitates the binding of HSF1 to DNA during heat shock.
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http://dx.doi.org/10.1128/MCB.00051-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002698PMC
July 2018

Acute HSF1 depletion induces cellular senescence through the MDM2-p53-p21 pathway in human diploid fibroblasts.

J Cell Sci 2018 05 8;131(9). Epub 2018 May 8.

Laboratory of Molecular Genetics, The Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, Japan

Heat shock transcription factor 1 (HSF1) regulates the expression of a wide array of genes, controls the expression of heat shock proteins (HSPs) as well as cell growth. Although acute depletion of HSF1 induces cellular senescence, the underlying mechanisms are poorly understood. Here, we report that HSF1 depletion-induced senescence (HDIS) of human diploid fibroblasts (HDFs) was independent of HSP-mediated proteostasis but dependent on activation of the p53-p21 pathway, partly because of the increased expression of dehydrogenase/reductase 2 (DHRS2), a putative MDM2 inhibitor. We observed that HDIS occurred without decreased levels of major HSPs or increased proteotoxic stress in HDFs. Additionally, VER155008, an inhibitor of HSP70 family proteins, increased proteotoxicity and suppressed cell growth but failed to induce senescence. Importantly, we found that activation of the p53-p21 pathway resulting from reduced MDM2-dependent p53 degradation was required for HDIS. Furthermore, we provide evidence that increased DHRS2 expression contributes to p53 stabilization and HDIS. Collectively, our observations uncovered a molecular pathway in which HSF1 depletion-induced DHRS2 expression leads to activation of the MDM2-p53-p21 pathway required for HDIS.
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http://dx.doi.org/10.1242/jcs.210724DOI Listing
May 2018

The HSF1-PARP13-PARP1 complex facilitates DNA repair and promotes mammary tumorigenesis.

Nat Commun 2017 11 21;8(1):1638. Epub 2017 Nov 21.

Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Minami-Kogushi 1-1-1, Ube, 755-8505, Japan.

Poly(ADP-ribose) polymerase 1 (PARP1) is involved in DNA repair, chromatin structure, and transcription. However, the mechanisms that regulate PARP1 distribution on DNA are poorly understood. Here, we show that heat shock transcription factor 1 (HSF1) recruits PARP1 through the scaffold protein PARP13. In response to DNA damage, activated and auto-poly-ADP-ribosylated PARP1 dissociates from HSF1-PARP13, and redistributes to DNA lesions and DNA damage-inducible gene loci. Histone deacetylase 1 maintains PARP1 in the ternary complex by inactivating PARP1 through deacetylation. Blocking ternary complex formation impairs redistribution of PARP1 during DNA damage, which reduces gene expression and DNA repair. Furthermore, ternary complex formation and PARP1 redistribution protect cells from DNA damage by promoting DNA repair, and support growth of BRCA1-null mammary tumors, which are sensitive to PARP inhibitors. Our findings identify HSF1 as a regulator of genome integrity and define this function as a guarding mechanism for a specific type of mammary tumorigenesis.
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http://dx.doi.org/10.1038/s41467-017-01807-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696371PMC
November 2017

HSF1 and HSF3 cooperatively regulate the heat shock response in lizards.

PLoS One 2017 7;12(7):e0180776. Epub 2017 Jul 7.

Departments of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Minami-Kogushi, Ube, Japan.

Cells cope with temperature elevations, which cause protein misfolding, by expressing heat shock proteins (HSPs). This adaptive response is called the heat shock response (HSR), and it is regulated mainly by heat shock transcription factor (HSF). Among the four HSF family members in vertebrates, HSF1 is a master regulator of HSP expression during proteotoxic stress including heat shock in mammals, whereas HSF3 is required for the HSR in birds. To examine whether only one of the HSF family members possesses the potential to induce the HSR in vertebrate animals, we isolated cDNA clones encoding lizard and frog HSF genes. The reconstructed phylogenetic tree of vertebrate HSFs demonstrated that HSF3 in one species is unrelated with that in other species. We found that the DNA-binding activity of both HSF1 and HSF3 in lizard and frog cells was induced in response to heat shock. Unexpectedly, overexpression of lizard and frog HSF3 as well as HSF1 induced HSP70 expression in mouse cells during heat shock, indicating that the two factors have the potential to induce the HSR. Furthermore, knockdown of either HSF3 or HSF1 markedly reduced HSP70 induction in lizard cells and resistance to heat shock. These results demonstrated that HSF1 and HSF3 cooperatively regulate the HSR at least in lizards, and suggest complex mechanisms of the HSR in lizards as well as frogs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180776PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5501597PMC
October 2017

Role of Heat Shock Factor 1 in Conserving Cholesterol Transportation in Leydig Cell Steroidogenesis via Steroidogenic Acute Regulatory Protein.

Endocrinology 2017 08;158(8):2648-2658

Department of Urology, School of Medicine, Yamaguchi University, Ube, Yamaguchi 755-8505, Japan.

Testicular testosterone synthesis begins with cholesterol transport into mitochondria via steroidogenic acute regulatory (StAR) protein in Leydig cells. Acute heat stress is known to obstruct testicular steroidogenesis by transcriptional repression of StAR. In contrast, chronic heat stress such as cryptorchidism or varicocele generally does not affect testicular steroidogenesis, suggesting that Leydig cells adapt to heat stress and retain their steroid synthesis ability. However, the mechanisms of the stress response in steroid-producing cells are unclear. We examined the relationship between the heat stress response and heat shock factor 1 (HSF1), which protects cells from proteotoxic stress by inducing heat shock protein as a molecular chaperone. The influences of HSF1 deficiency on cholesterol transport by StAR and the expression of steroidogenic enzymes under chronic heat stress were studied in testes of HSF1-knockout (HSF1KO) mice with experimental cryptorchidism. StAR protein in wild-type-cryptorchid mice was transiently decreased after induction of cryptorchidism and then gradually returned to basal levels. In contrast, StAR protein in HSF1KO mice continued to decrease and failed to recover, resulting in impaired serum testosterone. StAR messenger RNA was not decreased with cryptorchidism, indicating that posttranslational modification of StAR, not its transcription, was obstructed in cryptorchidism. Other steroidogenic enzymes, including CYP11A1, 3β-HSD, and CYP17A1, were not decreased. Lipid droplets were increased in the cytosol of HSF1KO-cryptorchid mice, suggesting dysfunctional cholesterol transportation. These findings provide insight into the role of HSF1 in Leydig cell steroidogenesis, suggesting that it maintains cholesterol transport by recovering StAR under chronic heat stress.
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http://dx.doi.org/10.1210/en.2017-00132DOI Listing
August 2017

Biocompatible nanostructured solid adhesives for biological soft tissues.

Acta Biomater 2017 07 5;57:404-413. Epub 2017 May 5.

Department of Biomaterials, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. Electronic address:

Over the past few years, the development of novel adhesives for biological soft tissue adhesion has gained significant interest. Such adhesives should be non-toxic and biocompatible. In this study, we synthesized a novel solid adhesive using nanostructured hydroxyapatite (HAp) and evaluated its physical adhesion properties through in vitro testing with synthetic hydrogels and mouse soft tissues. The results revealed that HAp-nanoparticle dispersions and HAp-nanoparticle-assembled nanoporous plates showed efficient adhesion to hydrogels. Interestingly, the HAp plates showed different adhesive properties depending upon the shape of their nanoparticles. The HAp plate made up of 17nm-sized nanoparticles showed an adhesive strength 2.2times higher than that of the conventional fibrin glue for mouse skin tissues.

Statement Of Significance: The present study indicates a new application of inorganic biomaterials (bioceramics) as a soft tissue adhesive. Organic adhesives such as fibrin glues or cyanoacrylate derivatives have been commonly used clinically. However, their limited biocompatibility and/or low adhesion strength are some drawbacks that impair their clinical application. In this study, we synthesized a novel solid adhesive with biocompatible and biodegradable HAp nanoparticles without the aid of organic molecules, and showed a rapid and strong adhesion of mouse soft tissues compared to conventional fibrin glues. Given the importance of wet adhesion in biomedicine and biotechnology applications, our results will help not only in developing an efficient approach to close incised soft tissues, but also in finding novel ways to integrate soft tissues with synthetic hydrogels (such as drug reservoirs).
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http://dx.doi.org/10.1016/j.actbio.2017.05.014DOI Listing
July 2017

Variations in brain defects result from cellular mosaicism in the activation of heat shock signalling.

Nat Commun 2017 05 2;8:15157. Epub 2017 May 2.

Center for Neuroscience Research, Children's National Medical Center, Washington, District of Columbia 20010, USA.

Repetitive prenatal exposure to identical or similar doses of harmful agents results in highly variable and unpredictable negative effects on fetal brain development ranging in severity from high to little or none. However, the molecular and cellular basis of this variability is not well understood. This study reports that exposure of mouse and human embryonic brain tissues to equal doses of harmful chemicals, such as ethanol, activates the primary stress response transcription factor heat shock factor 1 (Hsf1) in a highly variable and stochastic manner. While Hsf1 is essential for protecting the embryonic brain from environmental stress, excessive activation impairs critical developmental events such as neuronal migration. Our results suggest that mosaic activation of Hsf1 within the embryonic brain in response to prenatal environmental stress exposure may contribute to the resulting generation of phenotypic variations observed in complex congenital brain disorders.
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http://dx.doi.org/10.1038/ncomms15157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418582PMC
May 2017

Long-term melatonin treatment delays ovarian aging.

J Pineal Res 2017 Mar 23;62(2). Epub 2017 Jan 23.

Department of Obstetrics and Gynecology, Yamaguchi University Graduate School of Medicine, Ube, Japan.

Ovarian aging is characterized by gradual declines in oocyte quantity and quality. Melatonin is considered an anti-aging agent due to its cytoprotective actions as an antioxidant. This study examined whether long-term melatonin treatment would delay ovarian aging in mice. Female ICR mice (10 weeks old) were given melatonin-containing water (100 μg/mL; melatonin) or water only until 43 weeks of age. Their oocytes were recovered from the oviduct, and in vitro fertilization was performed. The ovaries were used for a histological analysis of the number of follicles. The mRNA expression of the aging-related sirtuin genes (SIRT1, SIRT3) and the autophagy-related gene (LC3) and the telomere length of the ovarian chromosomes were analyzed. Transcriptome changes in the ovaries were also characterized using microarray. The number of ovulated oocytes decreased with age; however, it was greater in melatonin-treated mice than that from control animals. The decreased fertilization rate and blastocyst rate during aging also were higher in the melatonin-treated mice than in the controls, as were the numbers of primordial, primary, and antral follicles. The mRNA expression of SIRT1 and LC3 and telomere length were enhanced due to melatonin treatment. Seventy-eight genes that were downregulated during aging and upregulated by melatonin were identified by a microarray analysis. Forty of these 78 genes were ribosome-related genes, and a free radical scavenging network was identified. The present results indicate that melatonin delays ovarian aging by multiple mechanisms including antioxidant action, maintaining telomeres, stimulating SIRT expression and ribosome function, and by reducing autophagy.
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http://dx.doi.org/10.1111/jpi.12381DOI Listing
March 2017

M3 Muscarinic Receptor Signaling Stabilizes a Novel Mutant Human Ether-a-Go-Go-Related Gene Channel Protein via Phosphorylation of Heat Shock Factor 1 in Transfected Cells.

Circ J 2016 Nov 1;80(12):2443-2452. Epub 2016 Nov 1.

Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science.

Background: Long QT syndrome 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Most of its mutations give rise to unstable hERG proteins degraded by the proteasome. Recently, carbachol was reported to stabilize the wild-type hERG-FLAG via activation of the muscarinic type 3 receptor (M3-mAChR). Its action on mutant hERG-FLAG, however, remains uninvestigated.Methods and Results:A novel mutant hERG-FLAG carried 2 mutations: an amino acid substitution G572S and an in-frame insertion D1037_V1038insGD. When expressed in HEK293 cells, this mutant hERG-FLAG was degraded by the proteasome and failed to be transported to the cell surface. Carbachol restored stability of the mutant hERG-FLAG and facilitated cell-surface expression. Carbachol activated PKC, augmented phosphorylation of heat shock factor 1 (HSF1) and enhanced expression of heat shock proteins (hsps), hsp70 and hsp90. Both a M3-mAChR antagonist, 4-DAMP, and a PKC inhibitor, bisindolylmaleimide, abolished carbachol-induced stabilization of the mutant hERG-FLAG.

Conclusions: M3-mAChR activation leads to enhancement of hsp expression via PKC-dependent phosphorylation of HSF1, thereby stabilizing the mutant hERG-FLAG protein. Thus, M3-mAChR activators may have a therapeutic value for patients with LQT2. (Circ J 2016; 80: 2443-2452).
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http://dx.doi.org/10.1253/circj.CJ-16-0712DOI Listing
November 2016

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1.

J Arrhythm 2016 Oct 25;32(5):433-440. Epub 2015 Nov 25.

Division of Cardiovascular Medicine, Department of Molecular Medicine and Therapeutics, Faculty of Medicine, Tottori University, Japan.

Background: The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS).

Methods: We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping.

Results: When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone.

Conclusions: A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG.
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http://dx.doi.org/10.1016/j.joa.2015.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063263PMC
October 2016

A study of hearing function and histopathologic changes in the cochlea of the type 2 diabetes model Tsumura Suzuki obese diabetes mouse.

Acta Otolaryngol 2016 Nov 16;136(11):1097-1106. Epub 2016 Jun 16.

a Department of Otolaryngology , Yamaguchi University Graduate School of Medicine , Ube City , Yamaguchi Prefecture , Japan.

Objectives: This study used Tsumura Suzuki Obese Diabetes (TSOD) mice as a spontaneous type 2 diabetes model and Tsumura Suzuki Non-obesity (TSNO) mice as controls to investigate factors involved in the onset of hearing impairment.

Method: Body weight, blood glucose levels, and auditory brainstem responses (ABRs) were measured. The cochleae were excised and evaluated histopathologically.

Results: The TSOD mice showed significant hyperglycemia at 2-7 months and severe obesity at 5-10 months; significantly elevated ABR thresholds at 8-10 months; and the capillary lumens in the cochlea stria vascularis were narrower in the TSOD mice than in the TSNO mice. At 17 months, India ink vascular staining of the TSOD mice's cochleae revealed decreased capillary density in the stria vascularis. The vascular area of capillaries in the stria vascularis and the vascular area were significantly smaller in TSOD mice. Histopathological analysis showed vessel wall thickening in the modiolus and narrowed capillaries in the stria vascularis, suggesting reduced blood flow to the inner ear.

Conclusion: The diabetes mice model used in our study showed early age-associated hearing loss, and histopathology showed findings of vessel wall thickening in the modiolus, narrowing of capillaries in the stria vascularis, and chronically reduced blood flow in the cochlea.
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http://dx.doi.org/10.1080/00016489.2016.1195012DOI Listing
November 2016

Anti-TNF-α Agent Infliximab and Splenectomy Are Protective Against Renal Ischemia-Reperfusion Injury.

Transplantation 2016 Aug;100(8):1675-82

1 Department of Urology, Graduate School of Medicine, Yamaguchi University, Yamaguchi, Japan.2 Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Yamaguchi University, Yamaguchi, Japan.

Background: Renal ischemia-reperfusion (I/R) injury is associated with delayed graft function and results in poor long-term graft survival. We previously showed that splenectomy (SPLN) protects the kidney from I/R injury and reduces serum TNF-α levels. Herein, we further investigated the effects of SPLN on inflammatory responses and tissue injury in renal I/R by examining the expression of major inflammatory cytokines and heat shock protein 70 (HSP70). Because it was shown previously that the anti-TNF-α agent infliximab (IFX) attenuated renal I/R injury, we also investigated whether IFX administration mimics the effects of SPLN.

Methods: The left renal pedicles of adult male Wistar rats were clamped for 45 minutes and then reperfused for 24 hours; right nephrectomy and SPLN were performed immediately. A separate cohort was administered IFX 1 hour before surgery in lieu of SPLN.

Results: Serum creatinine and blood urea nitrogen levels were markedly elevated by I/R injury; these increases were significantly reversed by IFX. Furthermore, IFX inhibited the induction of inflammatory cytokines and HSP70 during renal I/R injury. Time-dependent profiles revealed that the expression of inflammatory cytokines was elevated immediately after I/R, whereas levels of HSP70, serum creatinine, and blood urea nitrogen began to rise 3 hours postreperfusion. Macrophages/monocytes were significantly increased in I/R-injured kidneys, but not in those administered IFX. The outcomes of SPLN mirrored those of IFX administration.

Conclusions: Splenectomy and TNF-α inhibition both protect the kidney from I/R injury by reducing the accumulation of renal macrophages/monocytes and induction of major inflammatory cytokines.
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http://dx.doi.org/10.1097/TP.0000000000001222DOI Listing
August 2016

[Regulation of proteostasis capacity in the mitochondria].

Authors:
Ke Tan Akira Nakai

Seikagaku 2015 Dec;87(6):758-61

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December 2015

Molecular basis of HSF regulation.

Authors:
Akira Nakai

Nat Struct Mol Biol 2016 Feb;23(2):93-5

Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan.

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http://dx.doi.org/10.1038/nsmb.3165DOI Listing
February 2016

Retinoic acid has the potential to suppress endometriosis development.

J Ovarian Res 2015 Jul 31;8:49. Epub 2015 Jul 31.

Department of Obstetrics and Gynecology, Yamaguchi University Graduate School of Medicine, Minamikogushi 1-1-1, Ube, 755-8505, Japan.

Background: Despite endometriosis is common estrogen dependent disease afflicting women in reproductive age, the pathogenesis has not been fully elucidated. Retinoic acid has various functions in cells as biologic modulator, and aberrant retinoid metabolism seems to be involved in the lesions of endometriosis. In order to evaluate the potential of all-trans retinoic acid (ATRA) for therapeutic treatment, a transcriptome analysis and estradiol measurements in cultured endometriotic cells and tissues were conducted.

Methods: The mRNA expression levels in ATRA-treated endometriotic stromal cells (ESC) isolated from ovarian endometrial cysts (OEC) were investigated. Estradiol production in OEC tissues was also investigated.

Results: In the isolated ESC culture supplemented with ATRA for four days, total RNA was extracted followed by a transcriptome analysis using GeneChip. Forty-nine genes were upregulated and four genes were down-regulated by the ATRA treatment. Many upregulated genes were associated with the negative regulation of cellular proliferation. In addition, ATRA treatment decreased the mRNA expression of 17-beta-dehydrogenase 2 (HSD17B2) which converts estradiol into estrone in a dose-dependent manner, and the ELISA measurements indicated that estradiol production in the OEC tissue was inhibited by ATRA treatment.

Conclusions: Retinoic acid has the potential to suppress endometriosis development.
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http://dx.doi.org/10.1186/s13048-015-0179-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521362PMC
July 2015

Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

Eur J Immunol 2015 Jul 28;45(7):2028-41. Epub 2015 Apr 28.

Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan.

Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease, and disease activity is associated with serum IFN-α level. Plasmacytoid dendritic cells (pDCs) sense microbial as well as self-nucleic acids by TLRs 7 and 9 and produce a large amount of IFN-α. Here, we show that heat shock protein 90 (Hsp90) associates with and delivers TLR7/9 from the ER to early endosomes for ligand recognition. Inhibition of Hsp90 by various approaches including the use of Hsp90 inhibitor, a geldanamycin derivative, suppressed the Hsp90 association with TLR7/9, which resulted in inhibition of IFN-α production, leading to improvement of SLE symptoms in mice. Notably, we observed that serum Hsp90 is clearly increased in patients with active SLE compared with that in patients with inactive disease. Furthermore, we demonstrated that serum Hsp90 detected in SLE patients binds to self-DNA and/or anti-DNA Ab, thus leading to stimulation of pDCs to produce IFN-α. Our data demonstrate that Hsp90 plays a crucial role in the pathogenesis of SLE and that an Hsp90 inhibitor will therefore provide a new therapeutic approach to SLE and other nucleic acid-related autoimmune diseases.
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http://dx.doi.org/10.1002/eji.201445293DOI Listing
July 2015

Analysis of the heat shock factor complex in mammalian HSP70 promoter.

Methods Mol Biol 2015 ;1292:53-65

Departments of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Minami-Kogushi 1-1-1, Ube, 755-8505, Japan.

The heat shock response is characterized by the induction of heat shock proteins (HSPs) and is one of prominent mechanisms that regulate proteostasis capacity in the cell. In mammals, heat shock factor 1 (HSF1) regulates the expression of HSPs transcriptionally in both unstressed and stressed cells. Recent reports show that the HSF1-RPA complex constitutively gains access to nucleosomal DNA in part by recruiting a histone chaperone and a chromatin-remodeling component. Here, we describe the strategies to substitute endogenous HSF1 with ectopically expressed HSF1 or its mutant and to detect the occupancy of HSF1 transcription complex including RPA in vivo on two heat shock response elements located close together in the human or mouse HSP70 promoters by chromatin immunoprecipitation assay with high sensitivity and specificity.
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http://dx.doi.org/10.1007/978-1-4939-2522-3_4DOI Listing
December 2015

Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity.

Nat Commun 2015 Mar 12;6:6580. Epub 2015 Mar 12.

Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Minami-Kogushi 1-1-1, Ube 755-8505, Japan.

Heat-shock response is an adaptive response to proteotoxic stresses including heat shock, and is regulated by heat-shock factor 1 (HSF1) in mammals. Proteotoxic stresses challenge all subcellular compartments including the mitochondria. Therefore, there must be close connections between mitochondrial signals and the activity of HSF1. Here, we show that heat shock triggers nuclear translocation of mitochondrial SSBP1, which is involved in replication of mitochondrial DNA, in a manner dependent on the mitochondrial permeability transition pore ANT-VDAC1 complex and direct interaction with HSF1. HSF1 recruits SSBP1 to the promoters of genes encoding cytoplasmic/nuclear and mitochondrial chaperones. HSF1-SSBP1 complex then enhances their induction by facilitating the recruitment of a chromatin-remodelling factor BRG1, and supports cell survival and the maintenance of mitochondrial membrane potential against proteotoxic stresses. These results suggest that the nuclear translocation of mitochondrial SSBP1 is required for the regulation of cytoplasmic/nuclear and mitochondrial proteostasis against proteotoxic stresses.
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http://dx.doi.org/10.1038/ncomms7580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4558571PMC
March 2015

Fatty acid-binding protein 7 regulates function of caveolae in astrocytes through expression of caveolin-1.

Glia 2015 May 19;63(5):780-94. Epub 2015 Jan 19.

Department of Organ Anatomy, Yamaguchi University Graduate School of Medicine, Ube, Japan.

Fatty acid-binding proteins (FABPs) bind and solubilize long-chain fatty acids, controlling intracellular lipid dynamics. FABP7 is expressed by astrocytes in the developing brain, and suggested to be involved in the control of astrocyte lipid homeostasis. In this study, we sought to examine the role of FABP7 in astrocytes, focusing on plasma membrane lipid raft function, which is important for receptor-mediated signal transduction in response to extracellular stimuli. In FABP7-knockout (KO) astrocytes, the ligand-dependent accumulation of Toll-like receptor 4 (TLR4) and glial cell-line-derived neurotrophic factor receptor alpha 1 into lipid raft was decreased, and the activation of mitogen-activated protein kinases and nuclear factor-κB was impaired after lipopolysaccharide (LPS) stimulation when compared with wild-type astrocytes. In addition, the expression of caveolin-1, not cavin-1, 2, 3, caveolin-2, and flotillin-1, was found to be decreased at the protein and transcriptional levels. FABP7 re-expression in FABP7-KO astrocytes rescued the decreased level of caveolin-1. Furthermore, caveolin-1-transfection into FABP7-KO astrocytes significantly increased TLR4 recruitment into lipid raft and tumor necrosis factor-α production after LPS stimulation. Taken together, these data suggest that FABP7 controls lipid raft function through the regulation of caveolin-1 expression and is involved in the response of astrocytes to the external stimuli. GLIA 2015;63:780-794.
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http://dx.doi.org/10.1002/glia.22784DOI Listing
May 2015

ATF1 modulates the heat shock response by regulating the stress-inducible heat shock factor 1 transcription complex.

Mol Cell Biol 2015 Jan 13;35(1):11-25. Epub 2014 Oct 13.

Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Minami-Kogushi, Ube, Japan

The heat shock response is an evolutionally conserved adaptive response to high temperatures that controls proteostasis capacity and is regulated mainly by an ancient heat shock factor (HSF). However, the regulation of target genes by the stress-inducible HSF1 transcription complex has not yet been examined in detail in mammalian cells. In the present study, we demonstrated that HSF1 interacted with members of the ATF1/CREB family involved in metabolic homeostasis and recruited them on the HSP70 promoter in response to heat shock. The HSF1 transcription complex, including the chromatin-remodeling factor BRG1 and lysine acetyltransferases p300 and CREB-binding protein (CBP), was formed in a manner that was dependent on the phosphorylation of ATF1. ATF1-BRG1 promoted the establishment of an active chromatin state and HSP70 expression during heat shock, whereas ATF1-p300/CBP accelerated the shutdown of HSF1 DNA-binding activity during recovery from acute stress, possibly through the acetylation of HSF1. Furthermore, ATF1 markedly affected the resistance to heat shock. These results revealed the unanticipated complexity of the primitive heat shock response mechanism, which is connected to metabolic adaptation.
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http://dx.doi.org/10.1128/MCB.00754-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4295370PMC
January 2015

Heat shock factor 1 is required for migration and invasion of human melanoma in vitro and in vivo.

Cancer Lett 2014 Nov 3;354(2):329-35. Epub 2014 Sep 3.

Department of Dermatology, Yamaguchi University Graduate School of Medicine, Ube, Japan.

Heat shock factor 1 (HSF1) is a major transactivator of the heat shock response. Recent studies have demonstrated that HSF1 is involved in tumor initiation, maintenance, and progression by regulating the expression of heat shock proteins (HSPs) and other molecular targets. Furthermore, HSF1 was identified as a potent proinvasion oncogene in human melanomas. However, the biological functions of HSF1 in human melanoma remain poorly understood. To determine the functional role of HSF1 in melanoma, we used short hairpin RNA (shRNA) to silence HSF1 in human melanoma cell lines and investigated its effect on cell migration and invasive ability in vitro. We found that HSF1 knockdown led to a marked reduction in migration and invasive ability, and these functions were restored by overexpression of wild-type HSF1. To confirm the in vitro results, we performed subcutaneous xenograft experiments in athymic nude mice. We found that HSF1 was required for melanoma invasion and metastasis, as well as tumorigenic potential in vivo. Overall, these results show that HSF1 is indispensable for melanoma progression and metastasis, and suggests that HSF1 could be a promising therapeutic target for melanoma.
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http://dx.doi.org/10.1016/j.canlet.2014.08.029DOI Listing
November 2014