Tài liệu Báo cáo khoa học: Hsp105b upregulates hsp70 gene expression through signal transducer and activator of transcription-3 pdf

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Tài liệu Báo cáo khoa học: Hsp105b upregulates hsp70 gene expression through signal transducer and activator of transcription-3 pdf

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Hsp105b upregulates hsp70 gene expression through signal transducer and activator of transcription-3 Nobuyuki Yamagishi, Hajime Fujii, Youhei Saito and Takumi Hatayama Department of Biochemistry & Molecular Biology, Division of Biological Sciences, Kyoto Pharmaceutical University, Japan Keywords heat shock protein; Hsp70; Hsp105b; nuclear localization; STAT3 Correspondence T Hatayama, Department of Biochemistry & Molecular Biology, Division of Biological Sciences, Kyoto Pharmaceutical University, Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan Fax: +81 75 595 4758 Tel: +81 75 595 4653 E-mail: hatayama@mb.kyoto-phu.ac.jp (Received 14 May 2009, revised August 2009, accepted August 2009) doi:10.1111/j.1742-4658.2009.07311.x Hsp105a and Hsp105b are mammalian members of the Hsp105 ⁄ 110 family, a divergent subgroup of the Hsp70 family Hsp105a is expressed constitutively and induced by various forms of stress, whereas Hsp105b is an alternatively spliced form of Hsp105a that is expressed specifically during mild heat shock In a report, it was shown that Hsp105a and Hsp105b localize to the cytoplasm and of nucleus of cells, respectively, and that Hsp105b, but not Hsp105a, induces the expression of Hsp70 in mammalian cells Here, we examined the mechanism by which Hsp105b induces the expression of Hsp70 Using a series of deletion mutants of Hsp105b, it was revealed that the region between amino acids 642 and 662 of Hsp105b is necessary for the activation of the hsp70 promoter by Hsp105b 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 Hsp105b In addition, overexpression of Hsp105b 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 Hsp105b Furthermore, downregulation of Hsp105b reduced the expression of Hsp70 in heat-shocked cells On the basis of these findings, it is suggested that Hsp105b 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 Introduction Heat shock proteins are a set of highly conserved proteins produced in response to physiological and environmental stresses that serve to protect cells from stress-induced damage by preventing protein denaturation and ⁄ or repairing such damage [1] Mammalian heat shock proteins are classified into several families on the basis of their apparent molecular weight and function, such as Hsp105 ⁄ 110, Hsp90, Hsp70, Hsp60, Hsp40, and Hsp27 The Hsp70 family is the major and best-characterized group of heat shock proteins Several different species of Hsp70 family proteins are present in different compartments of eukaryotic cells, and play important roles as molecular chaperones that prevent the irreversible aggregation of denatured proteins Hsp70 family proteins also assist in the folding, assembly and translocation across the membrane of cellular proteins [2,3] Hsp70 family proteins are commonly composed of three functional domains: Abbreviations DOX, doxycycline; HSE, heat shock element; HSF, heat shock factor; INFa, interferon a; JAK, Janus kinase; NLS, nuclear localization signal; siRNA, small interfering RNA; STAT, signal transducer and activator of transcription 5870 FEBS Journal 276 (2009) 5870–5880 ª 2009 The Authors Journal compilation ª 2009 FEBS N Yamagishi et al the highly conserved N-terminal ATPase domain binds ADP ⁄ ATP, and can hydrolyze ATP; the central b-sheet domain directly binds peptide substrates; and the C-terminal a-helix domain regulates substrate binding [4–6] Hsp105a and Hsp105b are mammalian members of the Hsp105 ⁄ 110 family, a divergent subgroup of the Hsp70 family Hsp105a is expressed constitutively and induced by various forms of stress, whereas Hsp105b is an alternatively spliced form of Hsp105a that is expressed specifically during mild heat shock [7–9] These proteins suppress the aggregation of denatured proteins caused by heat shock in vitro, as does Hsp70 ⁄ Hsc70, but they have yet to be found to have refolding activity [10] In addition, Hsp105a and Hsp105b were suggested to function as a substitute for Hsp70 family proteins to suppress the aggregation of denatured proteins in cells under severe stress, in which the cellular ATP level decreases markedly [11] In a preceding report, we showed that Hsp105a localizes in the cytoplasm of mammalian cells, whereas Hsp105b localizes in the nucleus [12] Furthermore, Hsp105b, but not Hsp105a, induces the expression of Hsp70 in mammalian cells [13] In this study, we showed that Hsp105b-mediated Hsp70 induction was regulated by signal transducer and activator of transcription (STAT)-3 but not heat shock factors (HSFs) Furthermore, Hsp105b seemed to upregulate Hsp70 expression in heat-shocked cells Results Mechanism of Hsp105b-induced Hsp70 expression As Hsp105b(D642–698), which lacked the region between amino acids 642 and 698, also failed to induce the luciferase activity, the region between amino acids 642 and 662 of Hsp105b seemed to be necessary for activation of the hsp70 promoter Furthermore, Hsp105bmNLS, in which several amino acids in the nuclear localization signal (NLS) sequence of Hsp105b were replaced, also failed to activate the hsp70 promoter, as described previously [13] We next examined the cellular localization of the deletion mutants of Hsp105b in COS-7 cells by indirect immunofluorescence, using antibody against myc-tag As shown in Fig 1C, Hsp105b localized to the nucleus of cells, but Hsp105bmNLS, which failed to activate the hsp70 promoter, localized to the cytoplasm of cells In addition, Hsp105b(1–756), which activates the hsp70 promoter like Hsp105b, localized mainly to the nucleus of cells, whereas Hsp105b(1–698), Hsp105b(1–677), and Hsp105b(1–662), which activate the hsp70 promoter to approximately 60% of the extent to which it is activated by Hsp105b, localized not only to the nucleus but also to the cytoplasm of cells However, Hsp105b(1–641), Hsp105b(1–591), and Hsp105b(D642– 698), which failed to activate the hsp70 promoter, also localized to the nucleus and cytoplasm Interestingly, Hsp105b(1–564) localized mainly to the nucleus of cells, like Hsp105b, although it failed to activate the hsp70 promoter These results suggest that the nuclear localization of Hsp105b was necessary but not sufficient for the Hsp105b-induced expression of the hsp70 gene Nuclear localization of Hsp105b is necessary but not sufficient for activation of the hsp70 promoter The promoter region between )206 and )187 bp of the hsp70 gene is essential for activation of the hsp70 promoter by Hsp105b In preceding reports, we have shown that Hsp105b, but not Hsp105a, localizes to the nucleus and induces the expression of Hsp70 in mammalian cells [12,13] To elucidate the mechanism by which Hsp105b induces the expression of Hsp70, a series of plasmids that expressed C-terminal deletion mutants of Hsp105b were cotransfected into COS-7 cells with the reporter plasmid pGL70()2616) (Fig 1A) As shown in Fig 1B, Hsp105b(1–756), lacking the C-terminal 58 amino acids, showed an approximately 10-fold induction in luciferase activity, similar to full-length Hsp105b Furthermore, Hsp105b(1–698), Hsp105b(1–677) and Hsp105b(1–662) induced approximately 60% of the luciferase activity as compared with the control However, Hsp105b(1–641), Hsp105b(1–591), and Hsp105b (1–564), lacking more than the C-terminal 173 amino acids of Hsp105b, failed to induce luciferase activity To identify the hsp70 promoter region that is necessary for the induction of Hsp70 by Hsp105b, a series of hsp70 5¢-promoter deletion-luciferase constructs were generated and transiently transfected into COS-7 cells with the Hsp105b expression plasmid (Fig 2A) As shown in Fig 2B, although Hsp105b enhanced the activity of hsp70 promoter deleted up to )218 bp, further deletion of the hsp70 promoter up to )194 bp abolished the responsiveness to Hsp105b Furthermore, we found two putative heat shock elements (HSEs) (5¢-NGAAN-3¢) and two putative STAT3-binding elements (5¢-CTGGRA-3¢) in the region between )206 and )187 bp of the hsp70 promoter through transcription factor database (TRANSFAC; http: ⁄ ⁄ www.biobaseinternational.com ⁄ pages ⁄ index) searches (Fig 2A) When the pGL70(–218)mut construct with five mutated bases within these elements were transiently FEBS Journal 276 (2009) 5870–5880 ª 2009 The Authors Journal compilation ª 2009 FEBS 5871 Mechanism of Hsp105b-induced Hsp70 expression A N Yamagishi et al B C 5872 FEBS Journal 276 (2009) 5870–5880 ª 2009 The Authors Journal compilation ª 2009 FEBS N Yamagishi et al transfected into COS-7 cells with the Hsp105b expression plasmid, the activation of the hsp70 promoter by Hsp105b was not observed (Fig 2B) On the other hand, after deletion of the sequence of the hsp70 promoter to )194 bp, responsiveness to heat shock was retained, and the heat inductivity of the hsp70 promoter was dependent on the length of the 5¢-flanking sequence of the hsp70 gene (Fig 2C) Thus, Hsp105b seemed to activate the hsp70 promoter by a mechanism different from the heat-induced activation of hsp70 promoter, and the region between )206 and )187 bp of the hsp70 promoter is necessary for Hsp105binduced expression of the hsp70 gene STAT3, but not HSF1, binds to the region between )206 and )187 bp of the hsp70 promoter Next, we investigated protein binding to the region between )206 and )187 bp of the hsp70 promoter, using a biotin-mediated oligonucleotide pull-down assay (Fig 3A) When the extracts from the cells overexpressing Hsp105b were incubated with a biotinylated oligonucleotide probe, GL70, containing the sequence from )212 to )185 bp of the hsp70 promoter, STAT3 was found to bind to the GL70 oligonucleotide but not the GL70mt oligonucleotide, which was mutated in the HSF1 ⁄ STAT3 consensus sequences On the other hand, HSF1 binding to GL70 or GL70mt was not observed To further examine whether STAT3 bound to the hsp70 promoter in vivo, chromatin immunoprecipitation analyses were performed using HeLa-tet ⁄ Hsp105b cells (Fig 3B) In the cells incubated with doxycycline (DOX), the chromatin containing the hsp70 promoter fragment was not coimmunoprecipitated with STAT3 antibody However, when Hsp105b was overexpressed, the binding of STAT3 to the region between )272 and )13 bp of the hsp70 promoter was observed using STAT3 antibody, whereas the hsp70 promoter fragment was not detected using normal rabbit IgG However, STAT3 did not bind to the region between )1860 and )1656 bp of the hsp70 promoter In addition, the Mechanism of Hsp105b-induced Hsp70 expression chromatin containing the hsp70 promoter fragment was pulled down by acetylated histone H4 antibody, regardless of Hsp105b overexpression, suggesting that Hsp105b did not affect the basal histone H4 acetylation of the hsp70 promoter These results suggested that STAT3 binds directly to the region between )206 and )187 bp of the hsp70 promoter in vitro and in vivo, resulting in the induction of Hsp70 expression STAT3 is required for the induction of hsp70 promoter activity by Hsp105b To further demonstrate that STAT3 plays essential roles in the activation of the hsp70 promoter by Hsp105b, STAT3 expression was downregulated by a small interfering RNA (siRNA) method In the experiment, we used human HEK293 cells in which STAT3 expression was effectively downregulated by the STAT3 siRNA, and monkey COS-7 cells in which it was not The transfection of STAT3 siRNA decreased STAT3 expression by up to approximately 25% in the cells, and the Hsp105b-mediated hsp70 promoter activation was significantly suppressed by the STAT3 siRNA but not by control siRNA (Fig 4) These results further suggested that STAT3 mediates Hsp105b-induced expression of the hsp70 gene STAT3 is activated by tyrosine phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding [14–16] We next examined whether Hsp105b stimulated the phosphorylation of STAT3 at Tyr705 using antibody against phosphoSTAT3 (Tyr705) When HeLa-tet ⁄ Hsp105b cells were treated with interferon a (INFa), an activator of the Janus kinase (JAK)–STAT pathway, STAT3 was phosphorylated at Tyr705 and translocated into the nucleus in cells incubated with or without DOX (Fig 5A,B) Furthermore, when Hsp105b was overexpressed in HeLa-tet ⁄ Hsp105b cells, STAT3 was phosphorylated and translocated into the nuclei without INFa treatment (Fig 5A,B), suggesting that Hsp105b stimulates the phosphorylation of STAT3 at Tyr705 and translocation into the nuclei of cells Fig The region between amino acids 642 and 662 of Hsp105b is necessary for activation of the hsp70 promoter by Hsp105b (A) Schematic representation of a series of C-terminal deletion mutants of Hsp105b The asterisk in the Hsp105bmNLS mutant indicates the location of the mutated NLS sequence (B) The expression plasmids for Hsp105b and its deletion mutants were cotransfected with the pGL70()2616) reporter construct into COS-7 cells, and luciferase activities were measured To estimate the transfection efficiency of these plasmids, the levels of a series of C-terminal deletion mutants of Hsp105b and a-tubulin were determined by western blotting using antibodies against myc-tag and a-tubulin, respectively Relative activity is expressed as ratio to that of cells cotransfected with pGL70()2616) plasmid and pcDNA3.1(+)myc ⁄ His vector Values represent the means ± standard deviations of four independent experiments, and asterisks indicate significant differences (*P < 0.01, **P < 0.05) (C) The expression plasmids for Hsp105b and its deletion mutants were transfected into COS-7 cells At 48 h after transfection, cells were stained with Hoechst 33342 (blue), and the intracellular distribution of Hsp105b and its mutants was determined by indirect immunofluorescence microscopy using antibody against myc-tag (red) FEBS Journal 276 (2009) 5870–5880 ª 2009 The Authors Journal compilation ª 2009 FEBS 5873 Mechanism of Hsp105b-induced Hsp70 expression N Yamagishi et al A B C Fig The hsp70 promoter region between )218 and )194 bp is required for activation of the hsp70 promoter by Hsp105b (A) Schematic representation of 5¢-serial deletions of the hsp70 promoter reporter constructs (B) The expression plasmid for Hsp105b was cotransfected with the constructs in (A), and luciferase activities were measured (C) COS-7 cells were transfected with the constructs in (A) At 48 h after transfection, cells were treated at 45 °C for 10 min, and then maintained at 37 °C for h Luciferase activities were then measured Relative activities in (B) and (C) are expressed as ratios to that of cells transfected with pGL70()2616) plasmid and pcDNA3.1(+)myc ⁄ His vector Values represent the means ± standard deviations of three independent experiments, and the asterisks represent significant differences at P < 0.01 Downregulation of Hsp105 reduces the induction of Hsp70 expression during heat shock To elucidate the physiological role of Hsp105b-mediated regulation of Hsp70 expression, we examined 5874 whether Hsp105 affects Hsp70 expression during heat shock at 42 °C, at which temperature Hsp105b is induced As shown in Fig 6, when HeLa-tet ⁄ Hsp105b cells were treated at 42 °C for h, Hsp105b was induced at h, and the expression of Hsp70 increased FEBS Journal 276 (2009) 5870–5880 ª 2009 The Authors Journal compilation ª 2009 FEBS N Yamagishi et al Mechanism of Hsp105b-induced Hsp70 expression A A B B Fig STAT3, but not HSF1, binds to the region between )206 and )187 bp of the hsp70 promoter (A) HeLa-tet ⁄ Hsp105b cells were grown in medium without DOX for a period of 48 h Extracts from these cells were incubated with biotinylated GL70 or GL70mt oligonucleotides After unbound proteins were removed by washing, proteins bound to the oligonucleotides were eluted with excess biotin and detected by western blotting, using antibody against STAT3 or HSF1 The input represented 5% of the protein used in the pull-down assay (B) Chromatin immunoprecipitation analysis was performed with chromatin extracts from HeLatet ⁄ Hsp105b cells grown in medium with or without DOX for a period of 48 h DNAs from chromatins immunoprecipitated with an antibody against STAT3, acetylated histone H4 or normal rabbit IgG were amplified by PCR with two sets of specific primers for the hsp70 promoter The input represents 5% of the material used in the chromatin immunoprecipitation assays Similar results were obtained with two independent experiments gradually and markedly at h However, when Hsp105 expression was downregulated by Hsp105 siRNA, accumulation of Hsp70 at h was suppressed during heat shock at 42 °C Furthermore, the heatinduced Hsp70 was reduced by the downregulation of STAT3 (Fig 7) Thus, Hsp105b seemed to enhance the expression of Hsp70 during mild heat shock conditions Discussion We have shown that Hsp105b, but not Hsp105a, localizes to the nucleus and induces the expression of Fig STAT3 is required for activation of the hsp70 promoter by Hsp105b The expression plasmid for Hsp105b was cotransfected with pGL70()2616) plasmid and STAT3 or control siRNA into HEK293 cells (A) At 48 h after transfection, cells were harvested, and STAT3, Hsp105b and a-tubulin were detected by western blotting, using the respective antibodies (B) Luciferase activity was measured as hps70 promoter activity Relative activity is expressed as a ratio to that of cells cotransfected with control siRNA, pGL70()2616) plasmid, and pcDNA3.1(+)myc ⁄ His vector Values represent the means ± standard deviations of four independent experiments, and the asterisks indicate significant differences at P < 0.01 Hsp70 in mammalian cells [12,13] Here, we examined the mechanism by which Hsp105b induces the expression of Hsp70, and showed that Hsp105b induces the expression of Hsp70 through the transactivation of STAT3 in mammalian cells Indeed, it was found that STAT3 bound to the sequence between )206 and )187 bp of the hsp70 promoter, and mutation of the conserved motif for STAT3 binding in this region abrogated activation of the hsp70 promoter by Hsp105b Furthermore, downregulation of STAT3 expression resulted in reduced Hsp105b-induced expression of the hsp70 gene FEBS Journal 276 (2009) 5870–5880 ª 2009 The Authors Journal compilation ª 2009 FEBS 5875 Mechanism of Hsp105b-induced Hsp70 expression N Yamagishi et al A A B B C Fig Activation of STAT3 and nuclear translocation by Hsp105b HeLa-tet ⁄ Hsp105b cells were incubated with or without DOX for 48 h, and were treated without or with 100 ngỈmL)1 INFa for 10 (A) Phosphorylated STAT3 (Tyr705) (p-STAT3), STAT3, Hsp105, Hsp70 and a-tubulin were analyzed by western blotting, using the respective antibodies (B) These cells were stained with Hoechst 33342 (blue), and the intracellular distribution of phosphorylated STAT3 was determined by indirect immunofluorescence microscopy using antibody against phospho-STAT3 (red) The major regulators of hsp gene expression are HSFs, which form a multimember protein family in vertebrates [17] Each HSF seems to have special functions in response to distinct stimuli under normal or stressed conditions HSF1 is a typical transcription factor, and is responsive to various stresses, such as heat shock [18] Under nonstress conditions, HSF1 exists in an inactive form as a monomer; in response to stress, it forms homotrimers that bind to the HSEs in the 5876 Fig Hsp105b upregulates the expression of Hsp70 during mild heat shock (A) Hsp105 or control siRNA was transfected into HeLa-tet ⁄ Hsp105b cells grown in medium with DOX At 48 h after transfection, the cells were treated at 42 °C for h, and the expression of Hsp105, Hsp70 and a-tubulin was analyzed by western blotting, using the respective antibodies (B, C) The density of bands was quantified by densitometry, and was corrected with the density of a-tubulin as loading control Relative levels of Hsp70 (B) and Hsp105b (C) are represented as ratios of respective levels in the cells heat-shocked at 42 °C for h with transfection of control siRNA Values represent the means ± standard deviations of three independent experiments, and the asterisks indicate significant differences at P < 0.01 promoter regions of hsp genes [19] In addition, an inflammatory cytokine, interleukin-6, induces the expression of Hsp70 and Hsp90b via STAT-like binding FEBS Journal 276 (2009) 5870–5880 ª 2009 The Authors Journal compilation ª 2009 FEBS N Yamagishi et al A B Fig Requirement of STAT3 for the induction of Hsp70 during mild heat shock (A) STAT3 or control siRNA was transfected into HeLa-tet ⁄ Hsp105b cells grown in medium with DOX At 48 h after transfection, the cells were treated at 42 °C for h, and the expression of STAT3, Hsp70 and a-tubulin was analyzed by western blotting, using the respective antibodies (B) The density of bands was quantified by densitometry, and was corrected with the density of a-tubulin as loading control Relative levels of Hsp70 are represented as ratios of respective levels in the cells heat-shocked at 42 °C for h with transfection of control siRNA Values represent the means ± standard deviations of three independent experiments, and the asterisks indicate significant differences at P < 0.01 sites that are located close to the HSEs in the hsp70 and hsp90b promoters The activation of hsp promoters is mediated through the STAT3 signaling pathway [20] STAT1 also enhances the activation of hsp70 and hsp90 promoters [21] As we showed, in this study, that STAT3 directly bound to the region between )206 and )187 bp of the hsp70 promoter and enhanced the expression of the hsp70 gene in the cells overexpressing Hsp105b, STAT family proteins seem to play an important role in the regulation of hsp gene expression STAT family proteins are present in a latent, monomeric form in the cytoplasm, and are activated by specific tyrosine phosphorylation by JAK family mem- Mechanism of Hsp105b-induced Hsp70 expression bers The phosphorylation of STATs leads to homodimerization or heterodimerization, and the STATs then migrate to the nuclei of cells to activate target gene expression [14–16] We showed here that Hsp105b stimulated the phosphorylation of STAT3 at Tyr705 and its translocation into the nucleus Hsp105b seems not to activate JAKs directly, as JAKs localize mainly into the cytoplasm As Hsp105a, which localizes in the cytoplasm of cells, did not enhance the expression of Hsp70, Hsp105b may activate unknown factor(s) in the nucleus, which stimulate the phosphorylation of STAT3 by JAKs Further studies are required to elucidate the precise mechanisms by which Hsp105b activates STAT3 HSP105 ⁄ 110 family proteins are suggested to prevent the aggregation of denatured proteins caused by heat shock in vitro, as does Hsp70 ⁄ Hsc70 [10,22,23] More recently, mammalian Hsp105 ⁄ 110 and the yeast homologs Sse1p ⁄ 2p have been shown to act as efficient nucleotide exchange factors for Hsp70 and its orthologs in Saccharomyces cerevisiae, Ssa1p and Ssb1p, respectively, and enhance Hsp70-mediated chaperone activity [24–26] However, although HSP105 family proteins are important components of the Hsp70 chaperone machinery, excess Hsp110 seems to have a negative effect on Hsp70-mediated chaperone activity, owing to it accelerating substrate cycling to such an extent that the reaction becomes unproductive for folding [25] Hsp105b-induced expression of Hsp70 may be important for Hsp70-mediated protein refolding by the correction of the ratio between Hsp105 and Hsp70 chaperones in the cells Furthermore, as Hsp105b is necessary for the marked expression of Hsp70 under mild heat shock conditions, Hsp105b seems to play an important role in protection against deleterious stressors by Hsp70 Although further study will be required to clarify the mechanisms by which Hsp105b induces the activation of STAT3, the present findings may provide clues to the cellular function of HSP105 family proteins in the chaperone network of mammalian cells Experimental procedures Antibodies The following antibodies were used for western blotting, immunofluorescence, and gel shift experiments: Hsp105, rabbit anti-(human Hsp105 IgG) [27]; Hsp70, mouse anti(human Hsp70 IgG), which only reacted with inducible Hsp70 (clone C92F3A-5; Stressgen, Ann Arbor, MI, USA); myc tag, mouse anti-myc IgG (Invitrogen, Carlsbad, CA, USA); HSF1, rabbit anti-(human HSF1 IgG) (Stressgen); STAT3, rabbit anti-(mouse STAT3 IgG) (K-15; Santa Cruz FEBS Journal 276 (2009) 5870–5880 ª 2009 The Authors Journal compilation ª 2009 FEBS 5877 Mechanism of Hsp105b-induced Hsp70 expression N Yamagishi et al Biotechnology, Santa Cruz, CA, USA); phosphorylated STAT3, rabbit anti-[phospho-STAT3 (Tyr705) IgG] (D3A7; Cell Signaling Technology, Danvers, MA, USA); a-tubulin, mouse anti-a-tubulin IgG (clone DM1A; Sigma, Saint Louis, MI, USA) Table Primers used for construction of 5¢-serial deletions of hsp70 promoter reporter constructs Plasmid Orientation Sequence (5¢- to 3¢) pGL70()1194) Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense GGAGGTGGAGCAATTAGCCG GGGTTATGTTAGCTCAGTTACAGTA CTTTCCCCAAGTGCTCCTCCTA GGGTTATGTTAGCTCAGTTACAGTA TGGACGCGCGTAACCCGCAC GGGTTATGTTAGCTCAGTTACAGTA GCGCTGAAGCGCAGGCGGTCA GGGTTATGTTAGCTCAGTTACAGTA TGTCCCCTCCAGTGAATCCCAGA GGGTTATGTTAGCTCAGTTACAGTA ACTCTGGAGAGTTCTGAGCAG GGGTTATGTTAGCTCAGTTACAGTA pGL70()800) Cells African green monkey kidney COS-7 cells and human embryonic kidney HEK293 cells were obtained from the RIKEN Bioresource Center Cell Bank (Tsukuba, Japan) HeLa-tet ⁄ Hsp105a and HeLa-tet ⁄ Hsp105b cells, which express either mouse Hsp105a or Hsp105b by removing DOX from the culture medium, have been described previously [28] These cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37 °C with 95% air and 5% CO2 pGL70()298) pGL70()218) pGL70()194) peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG, and the antibody–antigen complexes were detected using a western blot luminol reagent (Santa Cruz Biotechnology) Plasmids The expression plasmids for mouse Hsp105a, Hsp105b and a mutant with substitutions in the NLS or a series of C-terminal deletion mutants of Hsp105b in mammalian cells have been described previously [12,13,29] The construction of the reporter plasmid pGL70()2616), which contains the firefly luciferase cDNA driven by a human hsp70 promoter sequence from )2616 to +150, has been described previously [13] A series of 5¢-deletions of the human hsp70 promoter sequence were generated by self-ligation of the DNA sequence made by PCR, with pGL70()2616) as the template DNA and a specific set of primers (Table 1) The substitution construct of pGL70()218) was made using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA), according to the manufacturer’s instructions The following oligonucleotides were used as primers for mutagenesis: 5¢-CAG AAC TCT CCA GAG TCT GAT GAG ATC TAC TGG AGG GGA CAG GGT T-3¢ and 5¢-AAC CCT GTC CCC TCC AGT AGA TCT CAT CAG ACT CTG GAG AGT TCT G-3¢ (substituted nucleotides underlined) Western blotting Cells (5 · 105 cells per 35 mm diameter dish) were lysed with 0.1% SDS and boiled for Aliquots (20 lg of protein) of cell extracts in SDS sample buffer (62.5 mm Tris ⁄ HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.00125% bromophenol blue) were subjected to SDS ⁄ PAGE, and then transferred to nitrocellulose membranes by electrotransfer The membranes were blocked with 5% skimmed milk in Tris-buffered saline (20 mm Tris ⁄ HCl, pH 7.6, 137 mm NaCl) containing 0.1% Tween20, and incubated with the indicated primary antibodies Then, the membranes were incubated with horseradish 5878 pGL70()405) Measurement of hsp70 promoter activity For the transfection of reporter constructs, COS-7 cells in 24-well plates (7 · 104 cells per well) were grown and washed twice with Opti-MEM The cells were then transfected with 1.5 lg of reporter plasmid and 0.75 lg of the expression vector for Hsp105b or its deletion constructs by lipofection, using DMRIE-C reagent according to the manufacturer’s instructions (Invitrogen) At 48 h after transfection, the cells were lysed with the cell culture lysis reagent (Promega, Madison, WI, USA), and aliquots of the cell extracts were subjected to measurement of luciferase activity, as described previously [11] The protein concentration of the cell lysates was also determined, to normalize for protein content For the knockdown of STAT3 expression, 10 pmol of the STAT3 siRNA (Santa Cruz Biotechnology) or RISC-free control siRNA (Dharmacon, Chicago, IL, USA) was cotransfected into HEK293 cells with 1.5 lg of reporter plasmid and 0.75 lg of the expression vector for Hsp105b, using Lipofectamine 2000, according to the manufacturer’s instructions (Invitrogen) Indirect immunofluorescence Cells grown on collagenized coverslips (2 · 104 cellsỈcm)2 in 24-well plates) were fixed with 4% paraformaldehyde for 30 at room temperature and permeabilized with icecold methanol for 10 After being washed twice with NaCl ⁄ Pi, the cells were incubated with blocking solution (NaCl ⁄ Pi containing 5% BSA) at 37 °C for h Then, the cells were incubated with mouse anti-myc Ig (1 : 100) or rabbit anti-phospho-STAT3 (Tyr705) IgG for 1–2 h at 37 °C After multiple washes with NaCl ⁄ Pi, the cells were FEBS Journal 276 (2009) 5870–5880 ª 2009 The Authors Journal compilation ª 2009 FEBS N Yamagishi et al incubated with rhodamine-conjugated anti-mouse IgG or rhodamine-conjugated anti-rabbit IgG (1 : 50; Molecular Probes, Eugene, OR, USA) for 1–2 h at 37 °C, and stained with 10 lm Hoechst 33342 for 10 in the dark After multiple washes with NaCl ⁄ Pi, the cells were observed using a confocal laser scanning microscope (LSM410; Zeiss, Jena, Germany) Biotin-mediated oligonucleotide pull-down assay Whole cell extracts were prepared by lysing cells with extraction buffer as described previously [30] Two milligrams of whole cell extract was incubated with 0.5 nmol of biotinylated double-stranded oligonucleotide (GL70, 5¢-biotin-CTC CAG TGA ATC CCA GAA GAC TCT GGA G-3¢; GL70mt, 5¢-biotin-CTC CAG TAG ATC TCA AGA GAC TCT GGA G-3¢) in binding buffer [50 mm Tris ⁄ HCl, pH 7.8, 100 mm NaCl, 1.5 mm MgCl2, mm EDTA, 0.1% NP-40, 10% (v ⁄ v) glycerol, 0.5 mgỈmL)1 salmon sperm DNA] at °C for 30 The samples were precleared with CL-4B Sepharose at °C for 30 min, and the remaining DNA was precipitated with 30 lL of a 50% slurry of Ultralink streptavidin gel (Pierce, Rockford, IL, USA) at °C for h Bound fractions were washed five times with binding buffer, and DNA-bound proteins were eluted with binding buffer containing mgỈmL)1 biotin; this was followed by SDS ⁄ PAGE and western blotting Chromatin immunoprecipitation HeLa-tet ⁄ Hsp105b cells were grown in medium with or without DOX for a period of 48 h and subjected to chromatin immunoprecipitation analysis Cells (5 · 107 cells) were cross-linked with a final concentration of 1% formaldehyde for at room temperature, and this was followed by quenching with a final concentration of 0.25 m glycine The cell pellets were then collected by centrifugation (450 g, °C, min) and washed three times with lysis buffer (10 mm Tris ⁄ HCl, pH 7.5, 10 mm NaCl, mm MgCl2, 0.5% NP-40) The lysates were then sonicated on ice to generate chromatin fragments with an average DNA length of 500 bp Immunoprecipitation was performed after preclearing with salmon sperm DNA saturated protein A–agarose beads at °C overnight using anti-mouse STAT3 IgG (K-15; Santa Cruz Biotechnology), acetylated histone H4 serum (positive control; #06-866; Upstate Biotechnology, Lake Placid, NY, USA), or normal rabbit IgG (negative control) The immunocomplexes were washed three times in buffer A (50 mm Hepes ⁄ KOH, pH 7.5, mm EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) containing 150 mm NaCl, twice in buffer A containing 500 mm NaCl, twice in buffer B (10 mm Tris ⁄ HCl, pH 8.0, 250 mm LiCl, mm EDTA, 0.5% NP-40, 0.1% sodium deoxycholate), and finally once in TE buffer (10 mm Tris ⁄ HCl, pH 8.0, mm EDTA) The immunoprecipitated chromatin was Mechanism of Hsp105b-induced Hsp70 expression then eluted with elution buffer (50 mm Tris ⁄ HCl, pH 7.5, 10 mm EDTA, 1% SDS), and DNA–protein cross-links were removed by incubating with a final concentration of mgỈmL)1 pronase at 65 °C for h Then, DNA was purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) Immunoprecipitated DNA and input samples obtained prior to immunoprecipitation were analyzed by PCR (35 cycles of 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s), using a set of specific primers for the human hsp70 gene between )272 and )13 (forward, 5¢-CCA TGG AGA CCA ACA CCC T-3¢; reverse, 5¢-CCC TGG GCT TTT ATA AGT CGT-3¢), or the human hsp70 gene between )1860 and )656 (forward, 5¢-TCT ATC TCT CGA TGG ATA CAG A-3¢; reverse, 5¢-AGG ACA GTA GAA TTA GGT CAC T-3¢) Knockdown of Hsp105a and Hsp105b The double-stranded RNA targeting Hsp105 (Dharmacon; 5¢-GCA AAU CAC UCA UGC AAA CUU-3¢) was resuspended to make a 20 lm solution, following the manufacturer’s instructions siRNA transfections were carried out using siLentFect reagent, according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA) At 72 h after transfection, cells were harvested for western blotting Statistical analysis The significance of differences was assessed using an unpaired Student’s t-test A probability level (P) of less than 0.05 was considered to be statistically significant Acknowledgements This study was supported in part by a Grant-in-Aid for 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Hatayama T & Yukioka M (1988) Common antigenicity of mouse 42 degrees C-specific heat-shock protein with mouse HSP 105 J Biochem 103, 81–85 Yamagishi N, Ishihara K, Saito Y & Hatayama T (2006) Hsp105 family proteins suppress staurosporineinduced apoptosis by inhibiting the translocation of Bax to mitochondria in HeLa cells Exp Cell Res 312, 3215– 3223 Yamagishi N, Saito Y, Ishihara K & Hatayama T (2002) Enhancement of oxidative stress-induced apoptosis by Hsp105alpha in mouse embryonal F9 cells Eur J Biochem 269, 4143–4151 Ishihara K, Horiguchi K, Yamagishi N, Saito Y & Hatayama T (2003) Identification of sodium salicylate as an hsp inducer using a simple screening system for stress response modulators in mammalian cells Eur J Biochem 270, 3461–3468 FEBS Journal 276 (2009) 5870–5880 ª 2009 The Authors Journal compilation ª 2009 FEBS ... localization of Hsp105b was necessary but not sufficient for the Hsp105b- induced expression of the hsp70 gene Nuclear localization of Hsp105b is necessary but not sufficient for activation of the hsp70. .. the heat-induced activation of hsp70 promoter, and the region between )206 and )187 bp of the hsp70 promoter is necessary for Hsp105binduced expression of the hsp70 gene STAT3, but not HSF1, binds... at P < 0.01 Hsp70 in mammalian cells [12,13] Here, we examined the mechanism by which Hsp105b induces the expression of Hsp70, and showed that Hsp105b induces the expression of Hsp70 through the

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