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Active HSF1 Significantly Suppresses Polyglutamine Aggregate Formation in Cellular and Mouse Models

2005; Elsevier BV; Volume: 280; Issue: 41 Linguagem: Inglês

10.1074/jbc.m506288200

ISSN

1083-351X

Autores

Mitsuaki Fujimoto, Eiichi Takaki, Tetsuya Hayashi, Yasushi Kitaura, Yasunori Tanaka, Sachiye Inouye, Akira Nakai,

Tópico(s)

Muscle Physiology and Disorders

Resumo

Polyglutamine diseases are inherited neurodegenerative diseases characterized by misfolding and aggregation of proteins possessing expanded polyglutamine repeats. As overexpression of some heat shock protein (Hsp) suppresses polyglutamine aggregates and cell death, it is assumed that combined overexpression of Hsps will suppress that more effectively. Here, we examined the impact of active forms of heat shock transcription factor 1 (HSF1), which induces a set of Hsps, on polyglutamine inclusion formation and disease progression. We found that active HSF1 suppressed polyglutamine inclusion formation more significantly than any combination of Hsps in culture cells, possibly by regulating expression of unknown genes, as well as major Hsps. We crossed R6/2 Huntington disease mice with transgenic mice expressing an active HSF1 (HSF1Tg). Analysis of the skeletal muscle revealed that the polyglutamine inclusion formation and its weight loss were improved in R6/2/HSF1Tg mice. Unexpectedly, the life span of R6/2/HSF1Tg mice was significantly improved, although active HSF1 is not expressed in the brain. These results indicated that active HSF1 has a strong inhibitory effect on polyglutamine aggregate formation in vivo and in vitro. Polyglutamine diseases are inherited neurodegenerative diseases characterized by misfolding and aggregation of proteins possessing expanded polyglutamine repeats. As overexpression of some heat shock protein (Hsp) suppresses polyglutamine aggregates and cell death, it is assumed that combined overexpression of Hsps will suppress that more effectively. Here, we examined the impact of active forms of heat shock transcription factor 1 (HSF1), which induces a set of Hsps, on polyglutamine inclusion formation and disease progression. We found that active HSF1 suppressed polyglutamine inclusion formation more significantly than any combination of Hsps in culture cells, possibly by regulating expression of unknown genes, as well as major Hsps. We crossed R6/2 Huntington disease mice with transgenic mice expressing an active HSF1 (HSF1Tg). Analysis of the skeletal muscle revealed that the polyglutamine inclusion formation and its weight loss were improved in R6/2/HSF1Tg mice. Unexpectedly, the life span of R6/2/HSF1Tg mice was significantly improved, although active HSF1 is not expressed in the brain. These results indicated that active HSF1 has a strong inhibitory effect on polyglutamine aggregate formation in vivo and in vitro. Polyglutamine expansion is a major cause of inherited neurodegenerative diseases called polyglutamine diseases. Eight polyglutamine diseases have been identified, including Huntington disease, spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy (DRPLA), 2The abbreviations used are: DRPLA, dentatorubral pallidoluysian atrophy; CSA, cross-sectional area, Hsp, heat shock protein; HSF, heat shock transcription factor; hHSF1, human HSF1; cHSF1, chicken HSF1; HSE, heat shock elements; SCA, spinocerebellar ataxia; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; CMV, cytomegalovirus; pfu, plaque-forming unit. 2The abbreviations used are: DRPLA, dentatorubral pallidoluysian atrophy; CSA, cross-sectional area, Hsp, heat shock protein; HSF, heat shock transcription factor; hHSF1, human HSF1; cHSF1, chicken HSF1; HSE, heat shock elements; SCA, spinocerebellar ataxia; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; CMV, cytomegalovirus; pfu, plaque-forming unit. and five forms of spinocerebellar ataxia (SCAs). 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A. 2000; 97: 9701-9705Crossref PubMed Scopus (180) Google Scholar), which belong to diverse Hsp families, also suppress inclusion body formation and cell death. As one variety of Hsps acts coordinately to assist the folding of proteins (12Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2397) Google Scholar), it might be better to express a set of Hsps to inhibit aggregate formation (27Ross C.A. Poirier M.A. Nat. Med. 2004; 10: S10-S17Crossref PubMed Scopus (2350) Google Scholar, 28Landles C. Bates G.P. EMBO Rep. 2004; 5: 958-963Crossref PubMed Scopus (340) Google Scholar). The induction of Hsp expression on stress conditions such as heat shock is regulated by heat shock transcription factor 1 (HSF1), which binds to heat shock elements (HSE) located on the upstream region of all Hsp genes (29Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1509) Google Scholar). A pre-existing monomeric HSF1 is converted to a trimer that can bind to HSE when cells are heat-shocked. We previously generated an HSF1 mutant that up-regulates expression of a set of Hsps in the absence of stress in culture cells and generated transgenic mice expressing its mutant in tissues such as the heart and testes but not in the brain (30Nakai A. Suzuki M. Tanabe M. EMBO J. 2000; 19: 1545-1554Crossref PubMed Scopus (151) Google Scholar). This overexpression causes infertility in male mice but is not toxic to culture cells and to several other organs such as the heart. It was reported that overexpression of HSF1 could suppress inclusion formation of a polyglutamine protein in culture cells (31Rimoldi M. Servadio A. Zimarino V. Brain Res. Bull. 2001; 56: 353-362Crossref PubMed Scopus (25) Google Scholar). However, we do not know how strongly HSF1 can suppress aggregate formation and whether it can act in vivo in a mouse model. In fact, overexpression of Hsp70 has little effect on disease progression in some mouse models of polyglutamine disease (32Hansson O. Nylandsted J. Castilho R.F. Leist M. Jaattela M. Brundin P. Brain Res. 2003; 970: 47-57Crossref PubMed Scopus (97) Google Scholar, 33Hay D.G. Sathasivam K. Tobaben S. Stahl B. Marber M. Mestril R. Mahal A. Smith D.L. Woodman B. Bates G.P. Hum. Mol. Genet. 2004; 13: 1389-1405Crossref PubMed Scopus (274) Google Scholar, 34Helmlinger D. Bonnet J. Mandel J.L. Trottier Y. Devys D. J. Biol. Chem. 2004; 279: 55969-55977Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). By generating active HSF1 mutants, we examined the impact of active HSF1 on polyglutamine aggregate formation and disease progression by using a cellular model of inclusion formation of a polyglutamine protein derived from human DRPLA (35Onodera O. Burke J.R. Miller S.E. Hester S. Tsuji S. Roses A.D. Strittmatter W.J. Biochem. Biophys. Res. Commun. 1997; 238: 599-605Crossref PubMed Scopus (60) Google Scholar) and Huntington disease R6/2 model mice (36Mangiarini L. Sathasivam K. Seller M. Cozens B. Harper A. Hetherington C. Lawton M. Trottier Y. Lehrach H. Davies S.W. Bates G.P. Cell. 1996; 87: 493-506Abstract Full Text Full Text PDF PubMed Scopus (2536) Google Scholar). Our results clearly showed that active HSF1 inhibits inclusion body formation much more efficiently that any Hsp or any combination of Hsps. Furthermore, we showed that expression of an active HSF1 in nonneural tissues markedly prolonged the life span of Huntington disease R6/2 mice. Construction—A plasmid pHβ-hHSF1ΔRD was generated as described previously (30Nakai A. Suzuki M. Tanabe M. EMBO J. 2000; 19: 1545-1554Crossref PubMed Scopus (151) Google Scholar). A 1.3-kb HindIII fragment of pHβ-hHSF1ΔRD was inserted into a vector pcDNA3.1(+)(Invitrogen). Using this plasmid (pcDNA3.1/hHSF1ΔRD) as a template, leucine at amino acid 395 was substituted with glutamic acid using a QuikChange site-directed mutagenesis kit (Stratagene), generating a plasmid, pcDNA3.1/hHSF1ΔRDT. To confirm the mutation, sequencing reactions were performed with an ALFexpress AutoRead sequencing kit, and sequences were analyzed with an ALFexpress sequencer (Amersham Biosciences). cDNAs for FLAG-tagged hHSF1, hHSF1ΔRD, and hHSF1ΔRDT at the N terminus were created by PCR, and each cDNA was inserted into a pcDNA3.1(+) vector at HindIII and EcoRI sites. An hHsp70 expression vector, pcDNA3.1-hHsp70, was constructed by inserting a BamHI/XhoI fragment of pBK-CMV-HSP72 (a gift from Dr. H. Itoh, Akita University) (37Komatsuda A. Wakui H. Oyama Y. Imai H. Miura A.B. Itoh H. Tashima Y. Nephrol. Dial. Transplant. 1999; 14: 1385-1390Crossref PubMed Scopus (51) Google Scholar) into the pcDNA3.1(+) vector. hHsp40 expression vector, pcDNA3.1-hHsp40, was constructed by inserting an ApaI/EcoRI fragment of phHsp40 (a gift from Dr. K. Ohtsuka, Chyubu University) (37Komatsuda A. Wakui H. Oyama Y. Imai H. Miura A.B. Itoh H. Tashima Y. Nephrol. Dial. Transplant. 1999; 14: 1385-1390Crossref PubMed Scopus (51) Google Scholar). mHsp110 cDNA (a gift from Dr. Hatayama, Kyoto Pharmaceutical University) (25Ishihara K. Yamagishi N. Saito Y. Adachi H. Kobayashi Y. Sobue G. Ohtsuka K. Hatayama T. J. Biol. Chem. 2003; 278: 25143-25150Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) was amplified by PCR with a 5′-primer containing a Kozak sequence (GCCACC) in front of a start codon and a 3′-primer and was inserted into the pcDNA3.1(+) vector at EcoRI and BamHI sites (pcDNA3.1-kozak-mHsp110). Cell Culture and Generation of HeLa Cells Stably Expressing hHSF1 or Hsp—HeLa and HEK293 cells were maintained at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. PC12 cells were maintained in DMEM containing 5% fetal bovine serum and 5% horse serum. Each expression plasmid was transfected into HeLa cells using a calcium phosphate method. At 4 h after transfection, the cells were treated for 3 min with 15% glycerol in an HBS buffer (140 mm NaCl, 25 mm HEPES, and 1.4 mm Na2HPO4). At 24 h after transfection, the cells were incubated in medium containing 1.5 mg/ml G418 disulfate (Nacalai Tesque, Kyoto, Japan). Clones expressing hHSF1, its mutant, or Hsp were selected by Western blot analysis as described below. HeLa cells stably expressing mHsp27 were generated previously (39Katoh Y. Fujimoto M. Nakamura K. Inouye S. Sugahara K. Izu H. Nakai A. FEBS Lett. 2004; 565: 28-32Crossref PubMed Scopus (24) Google Scholar). Western Blot Analysis—Cell extracts were prepared in lysis buffer containing 1.0% Nonidet P-40, 150 mm NaCl, 50 mm Tris-HCl (pH 8.0), 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 mm phenylmethylsulfonyl fluoride. Western blot analysis was performed as described previously (40Nakai A. Kawazoe Y. Tanabe M. Nagata K. Morimoto R.I. Mol. Cell. Biol. 1995; 15: 5168-5178Google Scholar). Antibodies used were antisera for HSF1 (αcHSF1x and αmHSF1g) (40Nakai A. Kawazoe Y. Tanabe M. Nagata K. Morimoto R.I. Mol. Cell. Biol. 1995; 15: 5168-5178Google Scholar, 41Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (183) Google Scholar), for Hsp110 (αHsp110a) (41Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (183) Google Scholar), for Hsp90 (αHsp90d) (42Katsuki K. Fujimoto M. Zhang X.Y. Izu H. Takaki E. Tanizawa Y. Inouye S. Nakai A. FEBS Lett. 2004; 571: 187-191Crossref PubMed Scopus (30) Google Scholar), for human Hsp27 (αhHsp27a, which was generated by immunizing rabbit with recombinant hHsp27), and for rat Hsp27 (a gift from Dr. K. Kato and H. Itoh, Aichi Human Service Center) (43Kato K. Ito H. Kamei K. Inaguma Y. Iwamoto I. Saga S. J. Biol. Chem. 1998; 273: 28346-28354Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) and mouse monoclonal antibodies for Hsp70 (W27, Santa Cruz Biotechnology), FLAG (M2, Sigma), GFP (GF200, Nacalai Tesque), and β-actin (AC-15, Sigma). To detect Hsp40, we generated antiserum αhHsp40a by immunizing rabbits with human Hsp40 (full-length) as described previously (41Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (183) Google Scholar). To analyze protein levels in the mouse tissues, tissues were homogenized in a Nonidet P-40 lysis buffer (150 mm NaCl, 1.0% Nonidet P-40, 50 mm Tris (pH 8.0), 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin) by Polytron (Kinematica, Inc., OH). After centrifugation at 15,000 × g for 10 min, supernatants were removed. Equal mounts of protein (100 μg) were loaded on SDS-PAGE and subjected to Western blot analysis. Gel Shift Assay—HEK293 cells were transfected with expression vectors for FLAG-tagged wild-type and mutant hHSF1 by using a calcium phosphate method, and cells were harvested after 24 h. Cells were frozen at -80 °C and then suspended in buffer C containing 20 mm HEPES (pH 7.9), 25% glycerol, 0.42 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A. After centrifugation at 100,000 × g for 5 min at 4 °C, the supernatants were frozen in liquid nitrogen and stored at -80 °C. Aliquots containing 10 μg of proteins were subjected to gel shift assay using a 32P-labeled ideal HSE-oligonucleotide as a probe (40Nakai A. Kawazoe Y. Tanabe M. Nagata K. Morimoto R.I. Mol. Cell. Biol. 1995; 15: 5168-5178Google Scholar). Ectopic Expression of GFP-Polyglutamine Protein and Estimation of Inclusion Formation—The expression vectors for Q19-GFP and Q81-GFP were kind gifts from Drs. W. J. Strittmatter, J. R. Burke, and Y. Nagai (Duke University Medical Center). In these plasmids, 19 or 81 CAG repeats of DRPLA cDNA were inserted into an expression vector, pEGFP-N1 (Clontech) (35Onodera O. Burke J.R. Miller S.E. Hester S. Tsuji S. Roses A.D. Strittmatter W.J. Biochem. Biophys. Res. Commun. 1997; 238: 599-605Crossref PubMed Scopus (60) Google Scholar). Each expression vector (2 μg) was mixed with DMEM containing nonessential amino acids (100 μl) and Plus reagent (20 μl) (Invitrogen) for 15 min. The mixture was combined with DMEM (50 μl) containing Lipofectin (0.5 μl) (Invitrogen) for 15 min and then added to a 35-mm dish containing 800 μl of DMEM. After the cells were incubated for 8 h, 2 ml of DMEM containing 15% fetal bovine serum was added to the dish. The medium was changed to DMEM containing 10% fetal bovine serum at 8 h after transfection. The formation of inclusion bodies was observed by detecting GFP fluorescence, and the cell morphology was examined by phase contrast under Axiovert 200 microscopy (Zeiss). The numbers of cells expressing GFP fusion protein and cells containing inclusion bodies were counted. To analyze solubility of Q81-GFP fusion protein in HeLa cells, cells were lysed on ice for 10 min in lysis buffer (400 μl) containing 40 mm HEPES (pH 7.5), 50 mm KCl, 1% Triton X-100, 2 mm dithiothreitol, 50 mm β-glycerophosphate, 5 mm EDTA, 5 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, and 5 μg/ml pepstatin (44Meriin A.B. Mabuchi K. Gabai V.L. Yaglom J.A. Kazantsev A. Sherman M.Y. J. Cell Biol. 2001; 153: 851-864Crossref PubMed Scopus (52) Google Scholar). After centrifugation at 15,000 rpm for 10 min, supernatants were removed. Pellets were suspended in 1× SDS sample buffer (400 μl) and were sonicated. Twenty microliters of the supernatant and pellet fractions were subjected to SDS-polyacrylamide electrophoresis and Western blot analysis. Generation of Adenoviruses Expressing an Active HSF1 and Hsps—An NheI/KpnI fragment of pcDNA3.1/hHSF1ΔRDT was inserted into pShuttle vector (Clontech). A viral DNA, pAd-hHSF1ΔRDT, was generated according to the manufacturer's instructions (Clontech). Ad-LacZ was similarly generated by using pShuttle-LacZ (Clontech). cDNA for mHsp110 was amplified by PCR using a 5′-primer containing NotI and Kozak (GCCACC) sequences and a 3′-primer containing EcoRV sequence and was inserted into a pShuttle-CMV vector (Stratagene) at NotI and EcoRV sites. cDNA for hHsp70 was amplified by PCR using a 5′-primer containing HindIII and Kozak sequences and a 3′-primer containing XhoI sequence and was inserted into the pShuttle-CMV vector at the same sites. A NotI/HindIII fragment of phHsp40 (38Ohtsuka K. Biochem. Biophys. Res. Commun. 1993; 197: 235-240Crossref PubMed Scopus (92) Google Scholar) was inserted into the pShuttle-CMV vector at the same sites. A NotI/NheI fragment of pcDNA3.1neo-mHsp27 (39Katoh Y. Fujimoto M. Nakamura K. Inouye S. Sugahara K. Izu H. Nakai A. FEBS Lett. 2004; 565: 28-32Crossref PubMed Scopus (24) Google Scholar) was inserted into the pShuttle-CMV vector. Viral DNAs containing cDNAs for Hsps were generated according to the manufacturer's instructions for the AdEasy adenoviral vector system (Stratagene). Viruses were infected into HEK293 cells, and the virus particles were enriched by CsCl gradient centrifugation and stored at -80 °C until use. Titers of virus stocks were 2-10 × 108 pfu/ml. Adenovirus Infection—HeLa cells plated in 60-mm dishes containing 4 ml of medium were infected with each adenovirus at a titer of 3 × 105 pfu/ml. More than 80% of HeLa cells were infected with Ad-LacZ at a titer of 2 × 105 pfu/ml judged by β-galactosidase staining. Abnormal morphology of HeLa cells was observed when they were infected with an adenovirus at a titer of 3 × 106 pfu/ml. At 24 h after infection of viruses, an expression vector for GFP-polyglutamine protein was transfected and maintained for 24-48 h. The numbers of cells expressing GFP fusion protein and cells containing inclusion bodies were counted, and these cells were harvested for the analysis of Hsps and GFP fusion protein expression by Western blot. To examine the effects of virus infection on the inclusion formation in PC12 cells, cells were infected with each adenovirus at a titer of 1.2 × 106 pfu/ml. Maintenance and Crossing of Transgenic Mice—A transgenic mouse line R6/2 (Jackson codes B6CBA-TgN(Hdexon1)62Gpb) was obtained from Jackson Laboratory (Bar Harbor, ME). To maintain the line, ovaries of R6/2 female mice were transplanted to (CBA × C57BL/6) F1 females, and ovarian-transplanted females were mated with (CBA × C57BL/6) F1 males. Genotyping was performed as described previously (36Mangiarini L. Sathasivam K. Seller M. Cozens B. Harper A. Hetherington C. Lawton M. Trottier Y. Lehrach H. Davies S.W. Bates G.P. Cell. 1996; 87: 493-506Abstract Full Text Full Text PDF PubMed Scopus (2536) Google Scholar). Transgenic mice expressing hHSF1ΔRD (HSF1ΔRD mice) (30Nakai A. Suzuki M. Tanabe M. EMBO J. 2000; 19: 1545-1554Crossref PubMed Scopus (151) Google Scholar) were maintained by crossing HSF1ΔRD females with (CBA × C57BL/6) F1 males. To generate double transgenic mice, HSF1ΔRD females were crossed with R6/2 males. Histopathology and Estimation of Polyglutamine Inclusion Formation in Tissues—Mice were sacrificed by cervical dislocation, and the quadriceps, heart, and brain were dissected and immediately frozen in isopentane and stored at -80 °C. Sections of 5-μm thickness were cut using a CM1900 cryostat (Leica). Immunohistochemistry was performed essentially as described previously (30Nakai A. Suzuki M. Tanabe M. EMBO J. 2000; 19: 1545-1554Crossref PubMed Scopus (151) Google Scholar). Antibodies used were goat IgG against Huntingtin (N-18, Santa Cruz Biotechnology) (1: 100 dilution in 3% dried milk) and fluorescein isothiocyanate-conjugated rabbit anti-goat IgG (Jackson) (1: 50 dilution in 3% dried milk). The sections were mounted in VECTASHIELD with 4′,6-diamidino-2-phe-nylindole (Vector Laboratories) and examined by Axioplan 2 microscopy (Zeiss). The numbers of Huntingtin-positive inclusions were counted in 500 nuclei, and the percentages of nuclei possessing the inclusions were calculated. All experimental protocols were reviewed by the Committee for Ethics on Animal Experiments of Yamaguchi University School of Medicine. To distinguish type I and type II fibers, histochemical reactions for myosin ATPase were performed at a series of pH (45Brooke M.H. Kaiser K.K. Arch. Neurol. 1970; 23: 369-379Crossref PubMed Scopus (1808) Google Scholar). The cross-sectional area (CSA) was estimated by using the NIH Image program. Electron Microscopy—For the transmission electron microscopic study, the specimens were fixed in 4% paraformaldehyde containing 0.25% glutaraldehyde and 4.5% sucrose, postfixed in 1% osmium tetroxide, dehydrated through passage in a series of graded ethanol, and embedded in Epon. Ultrathin sections obtained from the embedded blocks were stained with uranyl acetate and lead citrate and were examined with a Hitachi H-7000 electron microscope (46Hayashi T. James T.N. Buckingham D.C. Am. Heart J. 1995; 129: 946-959Crossref PubMed Scopus (25) Google Scholar). Stastical Analysis—Significant values were determined by analyzing data with the Mann-Whitney's U test using StatView version 4.5J for Macintosh (Abacus Concepts, Berkley, CA). A level of p < 0.05 was considered significant. Active Forms of HSF1—To examine the effects of HSF1-mediated gene activation in cells expressing the pathological length of polyglutamine proteins, it is necessary to introduce into cells an HSF1 mutant that has a stronger potential to activate target genes. HSF1 stays in an inactive monomer in the absence of stress and is activated through two steps: trimer formation and the acquisition of transcriptional activity. Previous studies showed the potential to activate heat shock genes of an HSF1 mutant, hHSF1ΔRD, lacking the regulatory domain that masks the activation domain (30Nakai A. Suzuki M. Tanabe M. EMBO J. 2000; 19: 1545-1554Crossref PubMed Scopus (151) Google Scholar, 31Rimoldi M. Servadio A. Zimarino V. Brain Res. Bull. 2001; 56: 353-362Crossref PubMed Scopus (25) Google Scholar, 47Xia W. Vilaboa N. Martin J.J. Mestril R. Guo Y. Voellmy R. Cell Stress Chaperones. 1999; 4: 8-18Crossref PubMed Google Scholar). However, a high expression level was required for hHSF1ΔRD to form a trimer that could bind to DNA (30Nakai A. Suzuki M. Tanabe M. EMBO J. 2000; 19: 1545-1554Crossref PubMed Scopus (151) Google Scholar). It was shown that a leucine zipper-like motif of HSF1 near the C terminus suppresses trimer formation (48Zuo J. Rungger D. Voellmy R. Mol. Cell. Biol. 1995; 15: 4319-4330Crossref PubMed Scopus (203) Google Scholar). Here, we substituted leucine at amino acid 395 in the suppression domain of the trimerization in hHSF1ΔRD with glutamic acid (hHSF1ΔRDT) (Fig. 1A). To compare protein levels of HSF1 mutants, a FLAG polypeptide was tagged at the N terminus of HSF1 and its mutants. These HSF1 mutants were transiently expressed in HEK293 cells, and the HSE binding activities were estimated. This clearly showed that hHSF1ΔRDT bound to HSE much more strongly than hHSF1ΔRD in HEK293 cells (Fig. 1B). To examine the potential to activate heat shock genes, we generated HeLa cells stably expressing similar levels of HSF1 mutants and estimated protein levels of Hsps. Hsp levels were increased in cells expressing both hHSF1ΔRD (HeLa/HSF1ΔRD cells) and hHSF1ΔRDT (HeLa/HSF1ΔRDT cells), but the levels were much higher in HeLa/HSF1ΔRDT cells (Fig. 1C). Growth rates and proportions of cells in each stage of the cell cycle in cells expressing an active HSF1 were similar to those in parental cells (data not shown) (30Nakai A. Suzuki M. Tanabe M. EMBO J. 2000; 19: 1545-1554Crossref PubMed Scopus (151) Google Scholar). An Active HSF1 Suppresses Polyglutamine Inclusions More Efficiently than Any Heat Shock Protein in Cells—To study the formation of inclusion bodies in culture cells, we transiently transfected polyglutamine-GFP fusion proteins into HeLa cells. When a nonpathologic length glutamine 19 from human DRPLA fused to GFP (Q19-GFP) was expressed under the control of a CMV promoter (35Onodera O. Burke J.R. Miller S.E. Hester S. Tsuji S. Roses A.D. Strittmatter W.J. Biochem. Biophys. Res. Commun. 1997; 238: 599-605Crossref PubMed Scopus (60) Google Scholar), diffuse GFP fluorescence was observed in both the cytoplasm and the nuclei of cells (data not shown). These cells, expressing Q19-GFP (30-40% of total cells), looked similar to cells that did not express the fusion protein by morphology (data not shown). In marked contrast, when a pathologic length polyglutamine 81 fused to GFP (Q81-GFP) was expressed, distinct inclusions were observed in about 25% of GFP-positive cells (data not shown). Phase contrast examinat

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