Heat shock factor 1 ameliorates proteotoxicity in cooperation with the transcription factor NFAT
2010; Springer Nature; Volume: 29; Issue: 20 Linguagem: Inglês
10.1038/emboj.2010.225
ISSN1460-2075
AutoresNaoki Hayashida, Mitsuaki Fujimoto, Ke Tan, Ramachandran Prakasam, Toyohide Shinkawa, Liangping Li, Hitoshi Ichikawa, Ryosuke Takii, Akira Nakai,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle10 September 2010free access Heat shock factor 1 ameliorates proteotoxicity in cooperation with the transcription factor NFAT Naoki Hayashida Naoki Hayashida Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Mitsuaki Fujimoto Mitsuaki Fujimoto Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Ke Tan Ke Tan Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Ramachandran Prakasam Ramachandran Prakasam Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Toyohide Shinkawa Toyohide Shinkawa Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Liangping Li Liangping Li Max-Delbrück Center for Molecular Medicine, Berlin, Federal Republic of Germany Search for more papers by this author Hitoshi Ichikawa Hitoshi Ichikawa Genetics Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan Search for more papers by this author Ryosuke Takii Ryosuke Takii Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Akira Nakai Corresponding Author Akira Nakai Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Naoki Hayashida Naoki Hayashida Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Mitsuaki Fujimoto Mitsuaki Fujimoto Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Ke Tan Ke Tan Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Ramachandran Prakasam Ramachandran Prakasam Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Toyohide Shinkawa Toyohide Shinkawa Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Liangping Li Liangping Li Max-Delbrück Center for Molecular Medicine, Berlin, Federal Republic of Germany Search for more papers by this author Hitoshi Ichikawa Hitoshi Ichikawa Genetics Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan Search for more papers by this author Ryosuke Takii Ryosuke Takii Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Akira Nakai Corresponding Author Akira Nakai Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan Search for more papers by this author Author Information Naoki Hayashida1, Mitsuaki Fujimoto1, Ke Tan1, Ramachandran Prakasam1, Toyohide Shinkawa1, Liangping Li2, Hitoshi Ichikawa3, Ryosuke Takii1 and Akira Nakai 1 1Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Ube, Japan 2Max-Delbrück Center for Molecular Medicine, Berlin, Federal Republic of Germany 3Genetics Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan *Corresponding author. Department of Biochemistry and Molecular Biology, Yamaguchi University School of Medicine, Minami-Kogushi 1-1-1, Ube 755-8505, Japan. Tel.: +81 836 22 2214; Fax: +81 836 22 2315; E-mail: [email protected] The EMBO Journal (2010)29:3459-3469https://doi.org/10.1038/emboj.2010.225 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Heat shock transcription factor 1 (HSF1) is an important regulator of protein homeostasis (proteostasis) by controlling the expression of major heat shock proteins (Hsps) that facilitate protein folding. However, it is unclear whether other proteostasis pathways are mediated by HSF1. Here, we identified novel targets of HSF1 in mammalian cells, which suppress the aggregation of polyglutamine (polyQ) protein. Among them, we show that one of the nuclear factor of activated T cells (NFAT) proteins, NFATc2, significantly inhibits polyQ aggregation in cells and is required for HSF1-mediated suppression of polyQ aggregation. NFAT deficiency accelerated disease progression including aggregation of a mutant polyQ-huntingtin protein and shortening of lifespan in R6/2 Huntington's disease mice. Furthermore, we found that HSF1 and NFAT cooperatively induce the expression of the scaffold protein PDZK3 and αB-crystallin, which facilitate the degradation of polyQ protein. These results show the first mechanistic basis for the observation that HSF1 has a much more profound effect on proteostasis than individual Hsp or combination of different Hsps, and suggest a new pathway for ameliorating protein-misfolding diseases. Introduction Cellular protein homeostasis or proteostasis involves controlling the concentration, conformation, binding interaction, and location of individual proteins, and is maintained by a network of pathways that influence protein synthesis, folding, translocation, assembly/disassembly, and degradation (Balch et al, 2008). Loss of cellular proteostasis results in many systemic and neurodegenerative disorders, termed protein-misfolding diseases or protein conformational diseases, caused by inherited misfolding-prone proteins such as mutant huntingtin, tau, and superoxide dismutase-1, or by metabolic/environmental stress-mediated misfolding of cellular proteins (Powers et al, 2009). Models have been established in yeast, Drosophila, and Caenorhabditis elegans that express misfolding proteins to identify regulators of proteostasis. These regulators include proteins that are involved in RNA metabolism, protein synthesis, protein degradation, ubiquitin-dependent protein catabolism, and lipid metabolism (Fernandez-Funez et al, 2000; Kazemi-Esfarjani and Benzer, 2000; Willingham et al, 2003; Nollen et al, 2004; Bilen and Bonini, 2007). Prominent among them are molecular chaperones and their stress-inducible response (Bukau et al, 2006; Ron and Walter, 2007; Morimoto, 2008). The heat shock response is characterized by the expression of a set of molecular chaperones termed heat shock proteins (Hsps) that facilitate the folding of proteins and maintain protein homeostasis (Parsell and Lindquist, 1993), and is regulated mainly at the transcriptional level by heat shock transcription factors (HSFs) (Wu, 1995). A single HSF, HSF1, regulates this response in yeast, Drosophila, and C. elegans, whereas four members of the HSF family (HSF1–4) have been characterized in vertebrates, and the expression of major Hsps is regulated mainly by HSF1 in mammals (Morimoto, 1998; Fujimoto et al, 2010). Gain of HSF1 function significantly ameliorates disease progression in C. elegans models of neurodegenerative disorders such as polyglutamine (polyQ) diseases (Hsu et al, 2003; Morley and Morimoto, 2004) and Alzheimer's disease (Cohen et al, 2006), whereas loss of HSF1 function accelerates it through the reduced expression of major Hsps. In mammals, this HSF1-mediated induction of Hsp expression is correlated with the acquisition of thermotolerance (McMillan et al, 1998) and the protection of cells from various pathological conditions such as neurodegenerative disorders (Fujimoto et al, 2005; Steele et al, 2008). However, HSF1 regulates the expression of not only major Hsps, products of classical heat shock genes, during heat shock, but also tremendous numbers of what are called non-classical heat shock genes in yeast, Drosophila, and mammalian cells (Hahn et al, 2004; Trinklein et al, 2004; Birch-Machin et al, 2005). Furthermore, HSF1 and other members of the HSF family are involved in the development and maintenance of neuronal, reproductive, and sensory tissues, which are associated with the expression of not only Hsps, but also development-related genes (Akerfelt et al, 2007; Nakai, 2009). Even though HSF1 regulates the expression of numerous genes, it is believed to maintain proteostasis by regulating the expression of major Hsps. Interestingly, the expression of classical heat shock genes is regulated by HSF3 in chicken cells (Nakai et al, 1995; Tanabe et al, 1998), whereas chicken HSF1 (cHSF1) is dispensable for their expression (Inouye et al, 2003). However, cHSF1 and mouse HSF3 can suppress polyQ aggregation without the expression of classical heat shock genes (Inouye et al, 2003; Fujimoto et al, 2010). Therefore, we hypothesized that HSFs might regulate non-classical heat shock genes involved in proteostasis. Here, we identified target genes of human HSF1 in HeLa cells using a DNA microarray, and examined whether they regulate proteostasis. We showed a novel HSF1-mediated proteostasis pathway, which is unrelated to the expression of major Hsps. Identification of the non-chaperone pathway explains why HSF1 has a great impact on protein homeostasis. Results Identification of HSF1-target genes that suppress polyQ aggregation To analyse target genes of human HSF1, we generated two independent HeLa clones (RDT1 and RDT2) expressing an actively mutated hHSF1 (hHSF1ΔRDT) (Fujimoto et al, 2005), as well as a HeLa clone expressing cHSF1 (HeLa/cHSF1). We first confirmed that protein and mRNA levels of major Hsps were increased in RDT1 and RDT2 cells compared with those in HeLa cells, whereas those were constant or even decreased in HeLa/cHSF1 cells (Supplementary Figure 1A and B). We then carried out a DNA microarray, and found 62 genes that showed a more than two-fold increase in both RDT1 and RDT2 cells (Supplementary Figure 1C; Supplementary Table 1). Among them, 29 genes were confirmed by RT–PCR to be increased in both RDT1 and RDT2 cells (Figure 1A; Supplementary Figure 1D). The expression of 18 genes (62%) was also increased in HeLa/cHSF1 cells (class a), whereas the expression of the other 11 genes (38%) was constant (class b) or decreased (class c). The 29 genes were temporarily induced in heat-shocked HeLa cells or in cells in which hHSF1ΔRDT was expressed under the control of tetracycline-responsive element, strongly suggesting that they are targets of hHSF1 (Supplementary Figure 2). The gene products included various kinds of proteins such as transcription factors and cytokines (Figure 1A). Figure 1.Identification of non-chaperone HSF1-target genes that suppress polyQ aggregation. (A) Summary of 29 novel HSF1-target genes, expression of which was increased in RDT1 and RDT2 lines. The expression of 18 genes (62%) was also increased in HeLa/cHSF1 cells (class a), whereas the expression of the other 11 genes (38%) was constant (class b) or decreased (class c). (B) Percentage of cells with polyQ inclusions in MEF cells co-infected with the retrovirus expressing each indicated gene and adenovirus expressing polyQ81-GFP. A retrovirus expressing an active hHSF1 (hHSF1ΔRDT) was used as a positive control. The averages of five experiments with the mean+s.d. are shown. (C) Soluble and insoluble fractions of polyQ81-GFP were analysed by western blotting (lower), and the insoluble polyQ81-GFP was quantified (upper). The averages of four experiments are shown. Error bars shows the mean+s.d. Statistical significance (P-value) was determined with an unpaired t-test. Download figure Download PowerPoint To search genes that regulate proteostasis, we established a procedure using MEF cells, in which each HSF1-target gene was expressed using a retrovirus and then a pathologic polyQ 81 fused to GFP (polyQ81-GFP) was expressed using an adenovirus (Fujimoto et al, 2005). We counted the number of cells with inclusion bodies at 24 and 36 h after the adenoviral infection, and found eight HSF1-target genes that inhibited aggregation at both time points similar to an active hHSF1 gene (Figure 1B; Supplementary Figure 3). One of these eight was a chaperone gene, αB-crystalline (CRYAB), but the rest were non-chaperone genes: transmembrane serine protease 3 (TMPRSS3), cysteine and glycine-rich protein 2 (CSRP2) encoding an actin-interacting and transcriptional co-activator, preferentially expressed antigen of melanoma (PRAME) encoding a transcriptional co-repressor, nuclear factor of activated T cell (NFATc2) encoding a transcription factor, prominin-2 (PROM2) encoding a membrane protein, dehydrogenase/reductase (SDR family) member 2 (DHRS2), and PDZ domain-containing 3 (PDZK3) encoding a scaffold protein, and were different from the genes that had a suppressive effect on polyQ aggregation in C. elegans and Drosophila, except for αB-crystalline (Nollen et al, 2004; Bilen and Bonini, 2007). These genes partially suppressed the accumulation of NP40-insoluble polyQ81-GFP (Figure 1C), and the reduction in mitochondrial activity (data not shown). The expression of these genes was also increased in HeLa/cHSF1 cells, except that of DHRS2 (Supplementary Figure 1D). The results showed for the first time that non-classical heat shock genes activated by HSF1 can prevent polyQ from forming aggregates. It is noticeable that overexpression of an active HSF1 gene suppressed polyQ aggregation more than that of any HSF1-target gene. Increased aggregation in HSF1-null cells is reversed by overexpression of HSF1-target genes As a deficiency of HSF1 greatly accelerates the polyQ aggregation in C. elegans (Hsu et al, 2003; Morley and Morimoto, 2004) and mouse cells (Homma et al, 2007), we analysed the contribution of the newly identified HSF1 targets in HSF1-null cells. We first confirmed in our MEF assay the increase in the number of cells forming polyQ inclusions and the level of NP40-insoluble polyQ protein in HSF1-null cells (Figure 2A). Furthermore, the level of ubiquitylated cellular protein was markedly increased in HSF1-null cells (Figure 2A; Supplementary Figure 4A and B), indicating disruption of the ubiquitin-proteasome system (Bennett et al, 2007). In the HSF1-null cells, levels of major Hsps such as Hsp110, Hsp90, Hsp70, Hsp40, and Hsp27 are the same as those in wild-type cells (Supplementary Figure 4C). In contrast, the expression of four of the newly identified genes, NFATc2, PDZK3, CSRP2, and CRYAB, was decreased in HSF1-null cells (Figure 2B). Therefore, we re-expressed the genes in HSF1-null cells, and found a significant reduction in the number of cells with polyQ inclusions, the amount of insoluble polyQ protein, and the accumulation of NP40-insoluble ubiquitylated cellular proteins, similar to the overexpression of Hsp70-1 and Hsp27 genes (Figure 2C). These results suggested that the impaired proteostasis in HSF1-null MEF cells is at least in part because of the down-regulation of the four HSF1 targets. Figure 2.Overexpression of target genes improves aggregate formation in HSF1-null cells. (A) Percentage of cells having polyQ inclusions (left), accumulation of insoluble polyQ protein (middle), and accumulation of insoluble ubiquitylated protein in WT and HSF1-null MEF cells. Error bars show the mean+s.d. (B) mRNA levels of novel HSF1-target genes in HSF1-null MEF cells relative to the levels in WT cells. The averages of three experiments are shown. Error bars show the mean+s.d. (C) Re-expression of NFATc2, PDZK3, CSRP2, or CRYAB in HSF1-null MEF cells restored HSF1 deficiency, such as overexpression of Hsp70-1, Hsp27, or three Hsps containing Hsp70-1, Hsp27, and Hsp40 (Hsp mix). Percentage of cells with polyQ inclusions (left), accumulation of insoluble polyQ protein (middle), and accumulation of insoluble ubiquitylated protein (right) are shown. The averages of three experiments are shown. Error bars show the mean +s.d. Statistical significance (P-value) was determined with an unpaired t-test. Download figure Download PowerPoint NFATc2 is required for HSF1-mediated suppression of polyQ aggregation Among the four targets of HSF1, we focused on the transcription factor NFATc2 (NFAT1), a member of the NFAT family (Hogan et al, 2003), as it strongly suppressed polyQ aggregation and its expression greatly depended on HSF1 in MEF cells (Figure 2B and C). The expression of NFATc2 was decreased in HSF1-null cells, and heat shock treatment markedly increased the amount of NFATc2 in wild type, but not in HSF1-null cells (Figure 3A). Overexpression of hHSF1 restored the expression of NFATc2 mRNA in the HSF1-null cells, whereas overexpression of hHSF1 mutants, hHSF1R176P and hHSF1ΔAB that cannot form a trimer (Inouye et al, 2007), did not because of an inability to bind the promoter (Figure 3B; Supplementary Figure 5A). The mouse NFATc2 gene contains two alternative 5′ exons, IA and IB (Vihma et al, 2008). Both exons were transcribed in the cerebral cortex, whereas only exon IA was transcribed in MEF cells (Supplementary Figure 5B). The expression of the exon IA transcript was induced during heat shock and was decreased in HSF1-null MEF cells and in the HSF1-null cerebral cortex (Supplementary Figure 5C). We performed a reporter analysis of each upstream sequence, the P1 (2.0 kb) or P2 (3.0 kb) promoter, in HEK293 cells, and found that the reporter activity of the P1 promoter increased two-fold after heat shock such as that of the Hsp70 promoter, whereas the reporter activity of the P2 promoter was unchanged (Figure 3C). Deletion of region 6 (−1500 to −2000) or mutation of the HSE3 sequence in the region 6 abolished both the constitutive and the heat-induced reporter activity of the P1 promoter. A ChIP analysis showed that HSF1 binds to region 6 in both unstressed and heat-shocked cells (Supplementary Figure 5D). These results indicate that a trimeric HSF1 binds to region 6 through HSE3 and activates the NFATc2 gene under normal and heat shock conditions. Figure 3.NFATc2 has a major function in HSF1-mediated suppression of polyQ aggregation. (A) Western blot of NFATc2 protein in WT and HSF1-null MEF cells. Cells were heat shocked at 42°C for 30 min and recovered for 9 h. (B) RT–PCR analysis of NFATc2 mRNA levels in WT and HSF1-null MEF cells expressing GFP, hHSF1, or hHSF1 mutants (hHSF1R176P and hHSF1ΔAB) that cannot form trimers. (C) Reporter analysis of the mouse NFATc2 promoter under normal and heat-stressed conditions. The promoter was divided into six regions (region 1–6). HSE2 and HSE3 in region 6 were mutated in pP1-luc-m1 and pP1-luc-m2, respectively. Error bars show the mean+s.d. The averages of three experiments are shown with P-values. (D) Percentage of cells with polyQ inclusions (left), accumulation of insoluble polyQ protein (middle), and that of insoluble ubiquitylated protein (right) in WT and NFATc2-null MEF cells. Error bars show the mean+s.d. The averages of three experiments are shown. (E) Percentage of cells with polyQ inclusions in WT, NFATc2-null, and HSF1-null MEF cells infected with Ad-hHSF1ΔRDT or Ad-NFATc2 (1.0, 0.1, and 0.01 × 106 pfu/ml) (upper). Levels of HSF1 and NFATc2 proteins are shown by western blotting (middle). The accumulation of insoluble polyQ protein, accumulation of insoluble ubiquitylated protein, and expression of Hsp70 and β-actin are also shown (lower). Error bars show the mean + s.d. The averages of three experiments are shown. (F) Accumulation of insoluble ubiquitylated protein in WT, NFATc2-null, and HSF1-null MEF cells expressing GFP (cont.), hHSF1ΔRDT, or NFATc2. Error bars show the mean +s.d. The averages of three experiments are shown. Statistical significance (P-value) was determined with an unpaired t-test through Figure 3. Download figure Download PowerPoint We next examined polyQ aggregation in NFATc2-null cells and found increased numbers of cells with polyQ inclusions and increased amounts of NP40-insoluble polyQ81-GFP in the cells (Figure 3D). Furthermore, the accumulation of insoluble ubiquitylated cellular proteins was significantly increased in these cells. Considering that NFATc2 is a downstream target of HSF1, we examined whether NFATc2 has a critical function in HSF1-mediated proteostasis or not. Overexpression of NFATc2 in HSF1-null cells efficiently reduced the number of cells with polyQ inclusions, associated with a decrease in the amount of NP40-insoluble polyQ81-GFP (Figure 3E). In marked contrast, overexpression of hHSF1ΔRDT in NFATc2-null cells suppressed the polyQ aggregation only partially, although the expression of Hsp70 was well induced. Similarly, overexpression of NFATc2 in HSF1-null cells significantly suppressed the accumulation of insoluble ubiquitylated proteins, whereas overexpression of hHSF1ΔRDT in NFATc2-null cells suppressed it only partially (Figure 3F). These results clearly show that NFATc2 is required for HSF1-mediated suppression of polyQ aggregation. Shortening of lifespan in both HSF1-null and NFATc2-null Huntington's disease mice To investigate the impact of HSF1 and NFATc2 on polyglutamine disease in vivo, we used a model of Huntington's disease, the R6/2 mice (Mangiarini et al, 1996), which was transgenic for the human huntingtin gene carrying fewer CAG repeats (95–97-fold). A single nuclear polyQ-huntingtin aggregate per cell was observed in the striatum at 38 weeks (Figure 4I and J), whereas several small aggregates were observed in the nucleus at 8 weeks (Figure 4A, B, E, and F). Remarkably, numbers of cells having the aggregates in the striatum were increased and each aggregate in both the nucleus and cytoplasm was more evident in HSF1-null and NFATc2-null R6/2 mice at 8 weeks than in wild-type R6/2 mice (Figure 4C, D, G, H, and K; Supplementary Figure 6A). Furthermore, the accumulation of polyQ-huntingtin protein (Supplementary Figure 6B) and the formation of its highly insoluble aggregates examined by filter trap assay (Figure 4L) were increased in the HSF1-null and NFATc2-null brain compared with that in the wild-type R6/2 brain, although deficiency of HSF1 or NFATc2 did not affect mRNA level of polyQ-huntingtin (Supplementary Figure 6C). PolyQ-huntingtin aggregates did not appear in other tissues such as the skeletal muscle at 38 weeks (data not shown) (Fujimoto et al, 2005). Figure 4.Shortening of lifespan in HSF1-null and NFATc2-null Huntington's disease mice. PolyQ-huntingtin protein aggregates were detected by immunohistochemistry using a goat anti-huntingtin antibody in the striatum of 8-week-old R6/2 mice (95–97 CAG repeats) with or without HSF1 (CBA background) (A–D) and in that of 8-week-old R6/2 mice with or without NFATc2 (BALB/c background) (E–H). Typical inclusions in R6/2 mice at 38 weeks old are shown (I, J). Boxed regions are magnified in the insets. Bar, 50 μm. (K) Percentage of cells with inclusions in (A–H). The averages of three mice are shown with P-values calculated using an unpaired t-test. Error bars show the mean +s.d. (L) Filter trap assay of polyQ-huntingtin protein in the brain of HSF1-null and NFATc2-null R6/2 mice. SDS-insoluble aggregates were trapped on a cellulose acetate membrane, and immunoblotting was performed using a goat anti-huntingtin antibody (left). The intensity of the signals was quantified, and the averages of three mice in each genotype are shown with P-values using an unpaired t-test (right). Error bars show the mean+s.d. (M) The lifespan of R6/2 mice with or without HSF1 or NFATc2. Download figure Download PowerPoint We also examined lifespan. First, we generated HSF1-null R6/2 (CAG95–97) mice with the C57BL/6 background, but found that they died quite early (within 6 weeks), whereas 80% of control R6/2 (CAG95–97) mice lived for up to 40 weeks (data not shown). Therefore, we generated HSF1-null R6/2 mice against the CBA background and NFATc2-null R6/2 mice against the BALB/c background. The R6/2 mice with the CBA or BALB/c background lived for up to 40 weeks, whereas the HSF1-null R6/2 mice with the CBA background and NFATc2-null R6/2 mice with the BALB/c background died in 25 and 13 weeks, respectively (Figure 4M). In addition, both HSF1-null and NFATc2-null R6/2 mice showed neurological symptoms much earlier than R6/2 mice. In fact, clasping appeared at 8–11 weeks in HSF1-null R6/2 mice (n=5) and at 5–8 weeks in NFATc2-null R6/2 mice (n=10), whereas it appeared at 27 weeks in HSF1-hetero-R6/2 mice (n=10) and at 32 weeks in NFATc2-hetero-R6/2 mice (n=12). Taken together, both HSF1 deficiency and NFATc2 deficiency caused accelerated polyQ protein expression and aggregation in the brain, and resulted in a marked shortening of lifespan. Both HSF1 and NFATc2 are required for marked activation of PDZK3 and CRYAB As HSF1 deficiency and NFATc2 deficiency showed similar phenotypes in cultured cells and in mice, we examined whether HSF1 and NFATc2 target the same genes. Using HeLa cells expressing NFATc2-HA under the control of a tetracycline-responsive promoter, we found that the expression of PDZK3 and CRYAB, which are targets of HSF1, was induced in response to elevated levels of NFATc2 (Figure 5A). Conversely, expression of the two genes as well as CSRP2 was decreased in NFATc2-null MEF cells (Figure 5B). ChIP assays revealed that HSF1 and NFATc2 directly bind to region 2 (−298 to −179) and region 3 (−500 to −294) of the PDZK3 promoters, respectively (Figure 5C; Supplementary Figure 7). Similarly, HSF1 and NFATc2 directly bind to region 2 (−297 to −91) and region 1 (−93 to −69) of the CRYAB promoters, respectively (Figure 5D; Supplementary Figure 7). Heat shock induced binding of not only HSF1, but also NFATc2 to both promoters. HSF1 or NFATc2 binding to the promoters was not affected by the other factor under control and heat shock conditions. These results showed that HSF1 and NFATc2 independently bind to the PDZK3 and CRYAB genes before and after heat shock. Figure 5.Both HSF1 and NFATc2 are required for marked activation of PDZK3 and CRYAB. (A) RT–PCR of novel HSF1-target genes in HeLa cells expressing tetracycline-off NFATc2-HA. Expression levels relative to those in control cells are shown. (B) RT–PCR of novel HSF1-target genes in NFATc2-null MEF cells. Expression levels are shown as in (A). The averages of three experiments are shown with P-values using an unpaired t-test. Error bars show the mean + s.d. in (A) and (B). (C) In vivo binding of HSF1 and NFATc2 in the PDZK3 promoter. ChIP of control and heat-shocked (42°C, 15 min) WT, HSF1-null (left), and NFATc2-null (right) MEF cells was performed using a preimmune serum (p.i.), an antiserum for HSF1, or that for NFATc2. DNA fragments of three regions (R1, R2, R3) were amplified by PCR using primers listed in Supplementary Table 4. (D) In vivo binding of HSF1 and NFATc2 in the CRYAB promoter. ChIP assay was performed as above. DNA fragments of two regions (R1, R2) were amplified by PCR. (E) RT–PCR of PDZK3 and CRYAB mRNA in wild-type, HSF1-null, and NFATc2-null MEF cells overexpressing hHSF1ΔRDT, NFATc2, or both (upper). Levels of HSF1 and NFATc2 proteins are shown (lower). Error bars show the mean +s.d. Download figure Download PowerPoint Remarkably, the level of PDZK3 mRNA in NFATc2-null cells overexpressing only an active hHSF1 was similar to that in wild-type cells, whereas it was significantly increased in the same cells overexpressing both an active hHSF1 and NFATc2 (Figure 5E). Although the expression of CRYAB mRNA was slightly induced by overexpressing an active hHSF1 in NFATc2-null cells, it was markedly induced by overexpression of both factors. The cooperative effect on the expression of PDZK3 and CRYAB mRNA was also observed when we overexpressed an active hHSF1 and/or NFATc2 in HSF1-null cells. These results suggested that both HSF1 and NFATc2 may be required for efficient expression of PDZK3 and CRYAB. We next examined the expression of target genes in mice. The expression of PDZK3 and CRYAB mRNAs was decreased in both HSF1-null and NFATc2-null mice in the cerebral cortex, heart, skeletal muscle, and spleen (Supplementary Figure 8A–E). The expression of NFATc2 mRNA was also decreased in various tissues of HSF1-null mice, though not in the skeletal muscle. Furthermore, the expression of NFATc2 and PDZK3 mRNA was increased in the skeletal muscle of HSF1 transgenic mice (Supplementary Figure 8F) (Fujimoto et al, 2005). Thus, the expression of these genes is under the control of HSF1 in vivo in mice. PDZK3 and CRYAB promote degradation of polyQ protein We next examined whether the overexpression of PDZK3 and CRYAB can reverse the accelerated aggregation in NFATc2-null cells and found that it greatly inhibited polyQ aggregation, similar to the overexpression of NFATc2 (Figure 6A). Furthermore, the knockdown of PDZK3 mRNA resulted in a marked increase in polyQ aggregates and that of CRYAB mRNA a moderate increase (Figure 6B). These results clearly indicated that both HSF1 and NFATc2 suppress polyQ aggregation in part by up-regulating the expression of PDZK3 and CRYAB genes. Figure 6.PDZK3 and CRYAB suppress polyQ aggregation by promoting degradation of polyQ protein. (A) Re-expression of NFATc2, PDZK3, and CRYAB in NFATc2-null MEF cells reversed the effects of NFATc2 deficiency, more than that of hHSF1ΔRDT. At 48 h after infection with a retrovirus expressing each indicated gene, NFATc2-null cells were infected with Ad-polyQ-GFP for 24 h. The perc
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