Heat Shock Transcription Factor 1 Is Required for Maintenance of ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 51, pp. 37285–37292, December 21, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Heat Shock Transcription Factor 1 Is Required for Maintenance of Ciliary Beating in Mice*□ S

Received for publication, June 4, 2007, and in revised form, October 23, 2007 Published, JBC Papers in Press, October 27, 2007, DOI 10.1074/jbc.M704562200

Eiichi Takaki‡, Mitsuaki Fujimoto‡, Takashi Nakahari§, Shigenobu Yonemura¶, Yoshihiko Miyata储, Naoki Hayashida‡, Kaoru Yamamoto‡, Richard B. Vallee**, Tsuyoshi Mikuriya‡ ‡‡, Kazuma Sugahara‡ ‡‡, Hiroshi Yamashita‡‡, Sachiye Inouye‡, and Akira Nakai‡1 From the Departments of ‡Biochemistry and Molecular Biology and ‡‡Otolaryngology, Yamaguchi University School of Medicine, Ube 755-8505, Japan, the §Department of Physiology, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan, the ¶Laboratory for Cellular Morphogenesis, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan, the 储Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan, and the **Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

Heat shock response is characterized by induction of a set of heat shock proteins (Hsps)2 and is a fundamental adoptive

* This work was supported in part by Grants-in-aid for Scientific Research and on Priority Areas-a Nuclear System of DECODE and Life of Proteins, from the Ministry of Education, Culture, Sports, Science and Technology, Japan, the Uehara Foundation, NOVARTIS Foundation, Nakatomi Foundation, Kao Foundation for Arts and Sciences, and the Yamaguchi University Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Movies S1 and S2. 1 To whom correspondence should be addressed. Tel.: 81-836-22-2214; Fax: 81-836-22-2315; E-mail: [email protected]. 2 The abbreviations used are: Hsp, heat shock protein; CBF, ciliary beat frequency; HSF, heat shock transcription factor; PBS, phosphate-buffered saline; GFP, green fluorescent protein; BSA, bovine serum albumin; DH2, dynein heavy chain 2; LIC3, dynein light intermediate chain 3.

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response in all organisms from bacteria to humans. This response is regulated mostly at the level of transcription by heat shock transcription factors that bind to the heat shock element in eukaryotes (1, 2). Among four HSF family members (HSF1– 4), HSF1 plays a key role in heat shock response in mammals, whereas HSF3 does so in avians (3, 4). This HSF-mediated induction of Hsps is required for acquisition of thermotolerance (5–7) and protection of cells from various pathophysiological conditions (8 –11). Inversely, HSF1 also regulates proapoptotic genes to decide on cell death or life in response to stress (12–14). In addition to the role in heat shock response, HSFs play critical functions in developmental processes such as gamatogenesis and neurogenesis (15–20), in maintenance of the sensory organs (21–24), and in immune response (25, 26). Although the precise mechanisms of how HSFs act in these physiological processes are still unclear, genetic evidence shows that HSFs regulate constitutive gene expression in unstressed cells and tissues (27, 28). Furthermore, it was revealed in sensory and immune cells that HSFs not only maintain protein homeostasis by regulating constitutive expression of Hsps, but are also involved in cell growth and differentiation by regulating expression of cytokines such as interleukin-6, fibroblast growth factors, and LIF (22, 24, 25). However, we do not know any client protein stabilized by Hsps that is under the control of HSFs in unstressed conditions. We previously showed abnormal nasal cavities in HSF1-null adult mice, which is associated with atrophy of the olfactory epithelium and sinusitis characterized by accumulation of mucus (24), and demonstrated that HSF1 is required to maintain olfactory neurogenesis. However, the mucus accumulation cannot be explained by this function of HSF1. Here, we examined the molecular mechanisms underlining mucus accumulation in the HSF1-null nasal cavity, and demonstrated that HSF1 is required to maintain ciliary beating in many organs, probably by regulating constitutive expression of Hsp90 that facilitates tubulin polymerization. This is the first demonstration that HSF1 plays a role in maintaining dynamic movement of a microstructure in cells.

MATERIALS AND METHODS Histopathology and Immunohistochemistry—HSF1-null (7) mice were maintained by crossing with ICR mice. Mice were JOURNAL OF BIOLOGICAL CHEMISTRY

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Heat shock transcription factors (HSFs) maintain protein homeostasis through regulating expression of heat shock proteins, especially in stressed conditions. In addition, HSFs are involved in cellular differentiation and development by regulating development-related genes, as well as heat shock genes. Here, we showed chronic sinusitis and mild hydrocephalus in postnatal HSF1-null mice, which are associated with impaired mucociliary clearance and cerebrospinal flow, respectively. Analysis of ciliary beating revealed that the amplitude of the beating was significantly reduced, and ciliary beat frequencies were lower in the respiratory epithelium, ependymal cells, oviduct, and trachea of HSF1-null mice than those of wild-type mice. Cilia possess a common axonema structure composed of microtubules of ␣- and ␤-tubulin. We found a marked reduction in ␣- and ciliary ␤iv-tubulin in the HSF1-null cilia, which is developmentally associated with reduced Hsp90 expression in HSF1-null mice. Treatment of the respiratory epithelium with geldanamycin resulted in rapid reduction of ciliary beating in a dose-dependent manner. Furthermore, Hsp90 was physically associated with ciliary ␤iv-tubulin, and Hsp90 stabilizes tubulin polymerization in vitro. These results indicate that HSF1 is required to maintain ciliary beating in postnatal mice, probably by regulating constitutive expression of Hsp90 that is important for tubulin polymerization.

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chamber, the volume of which was ⬃20 ␮l, and the rate of perfusion 200 ␮l/min (33, 34). The chamber was mounted on an differential interference contrast microscope (E600-FN, Nikon, Tokyo, Japan), which was connected to a high-speed digital video camera (FASTCAM 512PCI, Photoron, Tokyo). The sampling rate of the high-speed camera was 500 Hz. The ciliary beat frequency (CBF) of each tissue in a slice preparation was calculated from the time for 10 beating cycles (33, 34). Transmission Electron Microscopy—Mice were systemically anesthetized and transcardially perfused with a fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4). Blocks of bone containing paranasal sinuses and trachea were removed and soaked in the fixative for 16 h. Samples were then incubated for 10 days in a decalcification solution by changing the solution every day. Transmission electron microscopy was performed as described previously (22). Reverse Transcriptase-PCR Analysis—Total RNA was isolated from dissected tissues by using TRIzol reagent (Invitrogen). cDNAs were synthesized as described previously (24). Twenty-five to 35 cycles of PCR were performed using 1 ␮l from each reverse transcription product. Primers used for amplifying tubulin genes were: tuba-F, 5⬘-agaattccagaccaacctgg-3⬘; tuba-R, 5⬘-gtgttgctcagcatgcacac-3⬘; tubb3-F, 5⬘-attggcaacagcacg-3⬘; tubb3-R, 5⬘-tcacttgggcccctg-3⬘; tubb4-F; 5⬘-tcggagcagttcacc-3⬘; and tubb4-R, 5⬘-ttaagccacctcctct-3⬘. The primers tuba-F and tuba-R can amplify all ␣-tubulin genes (␣1 to ␣8) (Mouse Genome Informatics). The S16 ribosomal protein gene was amplified as a control (35). The amplified DNA was stained with ethidium bromide and photographed using an Epi-Light UV FA1100 (Aisin Cosmos R&D Co., Japan). Western Blot Analysis—Whole cell extracts from tissues were prepared and Western blot analysis was performed as described previously (24). Chromatin Immunoprecipitation—Mice were systemically anesthetized as described above. Tissues were dissected and immediately soaked in 1% formaldehyde in PBS at 37 °C for 10 min. Chromatin immunoprecipitation was performed using a chromatin immunoprecipitation assay kit (Upstate, New York) essentially according to the manufacturer’s instructions (22). Thirty-three cycles of PCR were performed to amplify a DNA fragment of the mouse Hsp90␣ (Hsp86) gene (⫺209 to ⫹56 from a transcription start site) containing a canonical heat shock element (36). Primers used to amplify chromatin immunoprecipitationenriched DNA were: Hsp90-CHIP-5, 5⬘-GCTGTGGAGGAGGGGCTTGCGTTCGTT-3⬘, and Hsp90-CHIP-3, 5⬘-GTGGCTGAATGAACACGCACGAGACGTGA-3⬘. Immunoprecipitation—HEK293 cells were plated in 100-mm dishes for 16 h, then transfected with an expression vector pEGFP2, pGFP2-␤iv-tubulin, or pGFP2-␤iii-tubulin (20 ␮g) by the calcium phosphate method as described previously (14). At 4 h after the transfection, cells were washed with PBS and incubated further for 44 h in normal medium. Cells were then washed with PBS, harvested, suspended in five packed cell pellet volumes of RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris (pH 7.5), 0.5 mM phenylmethylsulfonyl fluoride, 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin) for 10 min on ice. The supernatants were collected after centrifugation at 15,000 ⫻ g for 10 min, the 1 ␮l of preimVOLUME 282 • NUMBER 51 • DECEMBER 21, 2007


systemically anesthetized with ketamine (16 mg/kg, intraperitoneal) and xylazine (16 mg/kg, intraperitoneal), and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Tissues were removed and soaked in 4% paraformaldehyde for 12 h at 4 °C. After washing with PBS, the blocks were dehydrated, embedded in paraffin, and cut into sections 5-␮m thick. To prepare nasal sections, the fixed tissues were incubated in K-CX decalcification solution (Fujisawa, Osaka, Japan) for 24 h, and then neutralized in 5% Na2SO4 for 24 h. The sections were stained with hematoxylin and eosin. To identify mucus, sections were stained with periodic acid-Schiff and counterstained with hematoxylin. Immunostaining of the paraffin sections was performed as described previously (12). Antibodies used were antisera for mouse Hsp110 (␣Hsp110a), human Hsp90 (␣Hsp90c), human Hsp70 (␣Hsp70-1), human Hsp40 (␣Hsp40-1) (24, 29), mouse Hsp27 (␣mHsp27c), mouse TCP-1␣ (␣mTCP-1␣) (both were generated by immunizing rabbits with recombinant mouse Hsp27 or TCP-1␣), DH2 and LIC3 (30), or monoclonal antibodies for ␣-tubulin (fluorescein isothiocyanate-conjugated mouse IgG, F2168, Sigma) and ␤iv-tubulin (T7941, Sigma). Peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG was used as a second antibody. Signals were detected using a DAB substrate kit (Vector Laboratories, Inc.). Sections were counterstained with methyl green. To perform double staining, fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Cappel) and Alexa Fluor 568-conjugated anti-mouse IgG (Invitrogen) were used as second antibodies, and the sections were mounted in a Vectashield mounting medium with 4⬘,6diamidino-2-phenylindole (Vector Laboratories), and visualized using fluorescence microscopy (Axioplan 2, Zeiss). Estimation of Mucociliary Clearance—Analysis of the nasal mucociliary clearance was performed as described previously with some modifications (31). Pelican ink (5 ␮l) was injected into the nasal vestibules (a region 5 mm from the naris) using a micropipette with a fine catheter. The nose was divided into two through nasal septum, and the clearance of ink was examined. Assay of the Cerebrospinal Fluid Flow—The flow of cerebrospinal fluid was examined as described previously with modifications (32). The lateral wall of the lateral ventricle was dissected using a fine scalpel and forceps, and immediately soaked in a culture dish containing PBS at room temperature. For visualization of the flow, a small amount of pelican ink was placed on the surface of the lateral wall of the dissected ventricle. Movement of pelican ink was observed with a stereomicroscope (MZ6, Leica), and recorded with a digital camera and software (PowerShot S50, Canon). Analysis of Ciliary Movement—The nasal mucosa, trachea, oviducts, and inner surface of the lateral ventricle were removed from anesthetized mice, cut into small pieces (⬃5 mm square blocks), and suspended in control solution (121 mM NaCl, 4.5 mM KCl, 1 mM CaCl2, 1.5 mM NaHCO3, 1.5 mM NaHepes, 5 mM HHepes, and 5 mM glucose) at 4 °C. Each tissue block was cut into thin pieces by two adherent razor blades, and was placed on a coverslip precoated with Cell-Tak (Becton Dickinson Labware, Bedford, MA) to adhere slices firmly on the coverslip. The coverslip with slices was set in the perfusion


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RESULTS Mucus Accumulation in the * * * * * Nasal Cavity and Hydrocephalus in HSF1-null Mice—Previously, we 0 sec 3 sec 6 sec 9 sec 12 sec FIGURE 1. Mucus accumulation in the nasal cavity and hydrocephalus in HSF1-null mice. A, histological showed abnormal nasal cavity in examination of the paranasal sections of 6-week-old wild-type (⫹/⫹) (a) and HSF1-null (⫺/⫺) (d) mice. Periodic HSF1-null adult mice, which is assoacid-Schiff staining was performed. Arrows indicate mucus, which contains detached olfactory epithelial cells. ciated with atrophy of the olfactory Se, nasal septum; Tu, Turbinates. Bar, 1 mm. Mucociliary clearance was estimated (b, c, e, and f). Pelican ink was injected into the nasal vestibules (indicated as circles), and mucus transport of the ink was examined at 0 (b), 1 h epithelium and the accumulation of (c and e), and 3 h (f) after injection in wild-type (b and c) and HSF1-null (e and f) mice (n ⫽ 5; 5 individuals). Arrows mucus (Fig. 1A, a and d) (24). This indicate the turbinates. B, histological examination of the brain of 6-week-old mice by HE staining. Coronal revealed that HSF1 is required for sections are shown. LV, lateral ventricle; 3V, third ventricle; Aq, aqueduct; 4V, fourth ventricle. Bar, 1 mm. C, ependymal flow on the wall of the lateral ventricle. Pelican ink was placed onto the surface of the wall, and maintenance of olfactory neurogenits location was shown for 12 s after the injection (n ⫽ 5). Dashed red lines denote the lateral ventricle. Red arrows esis. However, the mucus accumuindicate normal direction of the flow. Asterisks indicate the site of wall adhesion. Bar, 1 mm. lation cannot be explained by this mune serum or antiserum for Hsp90 (␣hHsp90c) was added function of HSF1. Furthermore, the accumulation of mucus at 4 °C overnight, and mixed with 40 ␮l of protein A-Sepha- was not ameliorated in mice deficient for both HSF1 and HSF4, rose beads (1:1 suspension in PBS) (Amersham Biosciences) whereas the atrophied olfactory epithelium was partially by rotating at 4 °C for 1 h in the presence or absence of 1 ␮M restored in the same mice (24). We examined the mucociliary geldanamycin. The complexes were washed five times with clearance in the nasal cavity by monitoring movement of PelRIPA buffer, suspended in SDS sample buffer, and boiled for ican ink (31). The ink injected into the nasal vestibules was 3 min. The samples were loaded on SDS-PAGE, and trans- cleared after 1 h, and moved to the posterior turbinates (Fig. ferred onto nitrocellulose membranes. The membranes were 1A, b and c). In contrast, the ink was hardly removed from immunoblotted using a mouse monoclonal antibody for the vestibules of HSF1-null mice even after 3 h (Fig. 1A, e and GFP (GF200, Nacalai Tesque, Kyoto, Japan). Alternatively, f), indicating that the mucociliary clearance was severely immunoprecipitation was performed using antibody for impaired in HSF1-null mice. Therefore, HSF1 may have a GFP, and immunoblot analysis was performed using anti- direct role in mucociliary clearance in the nasal cavity. The whipping movement of the cilia generates moving force serum for Hsp90. In Vitro Tubulin Polymerization Assay—Hsp90 was purified of the mucus in the respiratory epithelium. As cilia are present from mouse L5178Y cells as described previously (37), and in many organs in the body, any dysfunction caused by genetic DECEMBER 21, 2007 • VOLUME 282 • NUMBER 51

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stored in a buffer containing 50 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, 10% glycerol at ⫺80 °C until use. A tubulin polymerization assay was performed according to the manufacturer’s instructions for a fluorescence-based tubulin polymerization assay kit (BK011, Cytoskeleton, Inc.). Briefly, a tubulin reaction mixture (50 ␮l) containing tubulin and a fluorescent reporter was mixed with bovine serum albumin (BSA, Fraction V, Nacalai Tesque), Hsp90 (5 ␮l), or paclitaxel that promotes microtubule polymerization and stability (38) and incubated at 37 °C until 40 min. Fluorescence emission at 460 nm (excitation wavelength is 360 nm) was measured by using a CytoFluor II Fluorescence Multi-well Plate Reader (PerSeptive Biosystems, Inc.). Statistical 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.

Ciliary Dyskinesia in HSF1-null Mice whipping movement of the cilia was markedly impaired even in the tra+/+ cheae and oviducts (data not a b nasal shown), and CBF was decreased -/* from 14.38 ⫾ 0.90 and 10.90 ⫾ 0.96 +/+ Hz to 11.50 ⫾ 1.82 and 7.52 ⫾ 0.32 brain Hz in the HSF1-null tracheae and -/* +/+ -/15 oviducts, respectively (Fig. 2A). * +/+ These results indicate that HSF1 defic trachea d 10 ciency causes severe impairment of -/* ciliary beating in many organs. 5 +/+ As the cilium is composed of an oviduct axoneme structure that normally -/* 0 consists of nine peripheral microtu-/-/+/+ -/0 5 10 15 20 bule doublets arranged around two CBF (Hz) central microtubules (39), we next FIGURE 2. Ciliary dyskinesia in many organs of HSF1-null mice. A, estimation of CBF. Video differential examined the ultrastructure of the interference contrast images of the respiratory epithelium (nasal), lateral ventricle (brain), trachea, and oviduct of 6-week-old mice in a slice preparation were monitored. CBFs were estimated from frame images (n ⫽ 3). cilia by electron microscopy. In the Stars indicate p ⬍ 0.05. B, transmission electron microscopic analysis of cilia in the respiratory epithelium of HSF1-null respiratory epithelium, wild-type (⫹/⫹) (a) and HSF1-null (⫺/⫺) (b– d) mice. Cilia with eight microtubule doublets (b), with increased numbers of central microtubules (c), and with translocation of central microtubules (d) are shown. Bar, 100 nm. dynein arms and radial spokes were observed, but almost 10% of the cilia Percentages of cilia possessing abnormal structures were estimated (n ⫽ 3). A star indicates p ⬍ 0.05. possessed abnormal central micromutations of their components cause many phenotypes known tubules and microtubule doublets such as deletion or transpoas immotile ciliary syndrome or primary ciliary dyskinesia (39, sition (Fig. 2B). These abnormalities were characteristic of 40). Therefore, we examined the morphology of other organs some types of ciliary dyskinesia (42). Although it is unclear such as the tracheae, oviducts, and ventricles. Histological whether the abnormal structures in HSF1-null mice may be examination showed grossly normal morphology in the tra- responsible for ciliary dyskinesia, these results suggest that cheae and oviducts in 6-week-old HSF1-null mice. However, assembly or organization of microtubules might be impaired. Tubulin Expression Decreases in the Motile Cilia of HSF1we noticed hydrocephalus in HSF1-null 6-week-old mice (Fig. 1B). All of the ventricles, including the aqueduct and fourth null Mice—As HSF1-null mice have abnormal microtubule ventricle, were enlarged, indicating communicating hydro- ultrastructure, we examined expression of their major compocephalus. Hydrocephalus was observed even in 3-week-old nents such as tubulin, dynein, and chaperonin. Microtubules HSF1-null mice, but not in 2-week-old mice (data not shown). are formed by protofilaments of ␣/␤-tubulin heterodimers. Degeneration of neural cells was not found in the brains of Among five ␤-tubulin isotypes, ␤i- and ␤iv-tubulin are found in 6-week-old HSF1-null mice (data not shown) (20). One possible all axoneme structures (43, 44), and are required for ciliary reason for this phenotype may be impaired flow of cerebrospi- function and assembly (45). We found that the level of ␤ivnal fluid (41). Therefore, we examined the ependymal flow in tubulin in the cilia, as well as ciliary ␣-tubulin, was significantly isolated ventricles by dropping a small amount of Pelican ink decreased in the HSF1-null respiratory epithelium (Fig. 3A). onto the exposed surfaces of dissected walls of the lateral ven- Levels of ␤iv-tubulin were also reduced in cilia of the ventricles, tricles (Fig. 1C). The ink moved along the expected cerebrospi- trachea, and oviducts (Fig. 3B). Although the TCP-1 complex, nal fluid flow on the wall in wild-type mice, but stayed in the known as eukaryotic cytoplasmic chaperonin, directs folding of same place and hardly moved in HSF1-null mice. These results cytoskeletal proteins such as ␣/␤/␥-tubulin (46), expression of a indicate that the ependymal flow is impaired in HSF1-null mice, component, TCP-1␣, was constant in the cilia of HSF1-null and this is consistent with the notion of ciliary dysfunction in mice (Fig. 3A). Furthermore, levels of motor proteins such as both the respiratory epithelium and ependymal cells. dynein heavy chain 2 (DH2) and dynein light intermediate Ciliary Beating Is Severely Impaired in HSF1-null Mice—To chain 3 (LIC3), which are found in the cilia (30), were also conexamine whether or not ciliary movement is impaired in HSF1- stant in the HSF1-null respiratory epithelium. These data indinull mice, we monitored ciliary beating in the freshly isolated cate that ␣- and ␤iv-tubulin are specifically reduced in the cilia respiratory epithelium of the nasal cavity. The differential of the HSF1-null epithelium. interference contrast image showed beating cilia located on the To examine whether HSF1 directly regulates expression of ␣surface of respiratory cells, and each cilium movement was and ␤iv-tubulin genes, we analyzed expression of mRNA levels detected by video (supplementary Movies 1 and 2) (33, 34). In by using reverse transcriptase-PCR. mRNA levels of ␣- and ␤ivwild-type mice, the whipping movement of the cilia was tubulin in the HSF1-null respiratory epithelium were the same observed and CBF was 13.27 ⫾ 1.53 Hz (Fig. 2A). In marked as those in the wild-type epithelium (Fig. 3C), suggesting that contrast, the beating amplitude was significantly low, and CBF proteins of ␣- and ␤iv-tubulin may be unstable in the cilia of the reduced to 8.70 ⫾ 1.72 Hz in HSF1-null mice. Similarly, CBF in HSF1-null epithelium. the ventricles of wild-type mice was 18.65 ⫾ 2.54 Hz, whereas Reduction of Hsp90 in Affected Cilia of HSF1-null Mice—To that of HSF1-null mice was 8.60 ⫾ 1.82 Hz. Interestingly, the clarify the molecular mechanisms that connect HSF1 defi-

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Ciliary Dyskinesia in HSF1-null Mice and Hsp27 were localized both in the cilia and in the cytoplasm of the nasal respiratory epithelium (Fig. brain iv-tubulin 4A). Expression of Hsp110, Hsp70, and Hsp40 did not decrease in the trachea HSF1-null cilia. In marked contrast, Hsp90 and Hsp27 expression signif-tubulin icantly decreased in the HSF1-null oviduct cilia. Interestingly, Hsp27 expression disappeared both in the cilia DH2 and in the cytoplasm, whereas C Hsp90 expression disappeared brain nasal trachea lung especially in the cilia. Analysis of tub their expression in other ciliated tisLIC3 sues showed that Hsp27 was less tub 3 expressed in the tracheal epithetub 4 lium, oviduct epithelium, and 16s TCP-1 ependymal cells, whereas Hsp90 was highly expressed in all of the epithelia (Fig. 4B). Hsp90 expresFIGURE 3. Tubulin expression decreases in the cilia of HSF1-null mice. A, immunohistochemical examination of expression levels of major components in the cilia of the respiratory epithelium in 6-week-old wild-type sion markedly decreased in the (⫹/⫹) and HSF1-null (⫺/⫺) mice. The respiratory epithelium was stained with antibody specific for ␤iv-tubulin, HSF1-null cilia of the three tissues, ␣-tubulin, dynein heavy chain 2 (DH2), dynein light intermediate chain 3 (LIC3), or TCP-1␣, and counterstained with methyl green. Bar, 10 ␮m. B, immunohistochemical examination of ␤iv-tubulin in the cilia of the lateral as in the respiratory epithelium. ventricle (brain), trachea, and oviduct in 6-week-old mice. Bar, 10 ␮m. C, mRNA levels of tubulins. Reverse Furthermore, Hsp90 expression in transcriptase-PCR analysis of ␣-, ␤iii-, and ␤iv-tubulin, as well as S16 ribosomal protein was performed by using the respiratory cilia increased at 4 total RNAs isolated from the brain, nasal cavity, trachea, and lungs. weeks, but HSF1 deficiency caused decreased expression of both Hsp90 A B +/+ -/and ␤iv-tubulin at 4 weeks (Fig. 4C). -/+/+ -/+/+ HE Thus, decreased Hsp90 expression trachea Hsp110 in the HSF1-null respiratory epitheoviduct Hsp90 lium is temporally associated with reduction of ␤iv-tubulin expression. brain Hsp70 These results strongly suggest the Hsp40 nasal involvement of Hsp90 in the stabilHsp27 Hsp90 ity of ciliary tubulin. Hsp27 Although we showed decreased C D E -/+/+ -/+/+ expression of Hsp90 in HSF1-null t a l uc he 3W ciliated epithelial cells by using sa id ac na ov tr immunohistochemical analysis, we +/+ -/- +/+ -/- +/+ -/- +/+ -/nasal 4W HSF1 hardly detected any difference in trachea Hsp90 Hsp90 expression in wild-type and oviduct 3W -actin HSF1-null ciliated tissues by Westbrain ern blot analysis (Fig. 4D). This 4W result may be due to the small numFIGURE 4. Reduction of Hsp90 in the cilia of HSF1-null mice. A, immunohistochemical examination of major Hsps in the cilia of the nasal respiratory epithelium in 6-week-old wild-type (⫹/⫹) and HSF1-null (⫺/⫺) mice. bers of ciliated epithelial cells among Bar, 10 ␮m. B, immunohistochemical examination of Hsp90 and Hsp27 in the cilia of the tracheal epithelium, total cell numbers of each tissue. Nevoviduct epithelium, ependymal cells, as well as the nasal respiratory epithelium. C, immunohistochemical ertheless, chromatin immunoprecipiexamination of Hsp90 and ␤iv-tubulin in the nasal respiratory epithelium of 3- and 4-week-old mice. D, Western blot analysis of HSF1, Hsp90, and ␤-actin in the ciliated tissues. Whole tissue extracts isolated from the nasal tation analysis showed that HSF1 cavity, trachea, oviducts, and brain were subjected to Western blot analysis. E, chromatin immunoprecipita- bound to a promoter region of the tion-enriched DNAs from tissues of 6-week-old wild-type (⫹/⫹) and HSF1-null mice (⫺/⫺) using preimmune Hsp90␣ gene (mouse Hsp86 gene) (PI) and anti-HSF1 serum (␣-HSF1) as well as input DNA were amplified using primers specific for the Hsp90 in all four tissues (Fig. 4E). These promoter (⫺209 to ⫹56) by PCR analysis. results strongly suggest that HSF1 ciency with instability of ciliary tubulin, we examined expres- directly regulates Hsp90 expression in ciliated epithelial sion of Hsps, products of major HSF1-target genes. In addition cells. The Hsp90 Inhibitor Reduces Ciliary Beating—To examine to the TCP-1 complex, Hsps such as Hsp70, Hsp90, and Hsp40 are distributed in the cilia and are related to axonemal protein whether Hsp90 is required for ciliary beating, sliced tracheal turnover (47–50). Immunohistochemical study showed that tissues were soaked with a control solution, and then with a major cytoplasmic Hsps such as Hsp110, Hsp90, Hsp70, Hsp40, solution containing 1 to 100 nM geldanamycin, a specific inhib-

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Ciliary Dyskinesia in HSF1-null Mice not interact with GFP (data not shown). These results demonstrate direct binding of Hsp90 to ␤-tubulins. Hsp90 Promotes Tubulin Poly* merization—To examine whether * * * * * Hsp90 is involved in microtubule * * * bundling, we performed an in vitro * * tubulin polymerization assay. In this assay, polymerization is followed by fluorescence enhancement due to incorporation of a fluorescent reporter into microtubules Time (min) Time (min) as polymerization occurs. Tubulin polymerization reached a peak at 25 GA control +/+ -/min in the control tubulin reaction mixture, and reached a peak after only 5 min in a reaction mixture containing paclitaxel, a drug that binds to tubulin and promotes microtubule polymerization and stability (38) (Fig. 6C). Paclitaxel also elevated the peak level of tubulin polymerization. We purified mouse Hsp90 from culture cells (Fig. 6B), and examined the effects of Hsp90, as well as albumin. Although albumin had no effect on FIGURE 5. Hsp90 is required for ciliary beating. A, CBF of the tracheal epithelium of 6-week-old wild-type (left) tubulin polymerization, Hsp90 sigand HSF1-null (right) mice was estimated in the presence of geldanamycin (GA) at the indicated concentrations nificantly promoted tubulin polym(n ⫽ 3). CBF ratios (CBF/CBF0; CBF0 is CBF at 0 min) are shown. Stars indicate p ⬍ 0.05. B, localization of Hsp90 and ␤iv-tubulin in wild-type and HSF1-null nasal respiratory epithelium. Hsp90 and ␤iv-tubulin were double erization (Fig. 6C). Tubulin polymstained and counterstained with 4⬘,6-diamidino-2-phenylindole. Yellow bars indicate layers of the cilia. White erization reached a peak at 25 min, bar, 10 ␮m. C, effects of geldanamycin on localization of Hsp90 and ␤iv-tubulin in wild-type nasal respiratory and a marked peak level of tubulin epithelium. The slices were treated with 100 nM geldanamycin for 30 min, and then double stained as described polymerization in the presence of above. Bar, 10 ␮m. Hsp90 was similar to that in the itor of Hsp90, for 60 min. CBF ratios of the wild-type trachea presence of paclitaxel. This effect of Hsp90 was dose-dependdecreased after 100 nM geldanamycin treatment even at only 10 ent and was blocked by geldanamycin treatment (Fig. 6D). min after treatment (Fig. 5A). Consistent with reduced expres- These results indicate that Hsp90 facilitates microtubule sion of Hsp90 in the HSF1-null trachea, treatment with 1 nM polymerization. geldanamycin was enough to reduce CBF ratios of the HSF1null trachea, whereas treatment with 10 nM geldanamycin had DISCUSSION

A

HSF1

CBF ratio (CBF/CBF0)

HSF1

Hsp90

iv-tubulin

merge

merge

no effect on the CBF ratios of the wild-type trachea (Fig. 5A). These results indicate that Hsp90 is required for ciliary beating. We next examined localization and levels of ciliary tubulins in the presence of geldanamycin. Double staining showed that both Hsp90 and ␤iv-tubulin are expressed in a relatively distal part of the cilia (Fig. 5B). Expression of ␤iv-tubulin was reduced by geldanamycin treatment, and its localization was more restricted in the distal part of the cilia (Fig. 5C). These data suggest that Hsp90 is required for stability of ␤iv-tubulin. Hsp90 Interacts with Ciliary ␤iv-Tubulin—Our results suggest that Hsp90 directly interacts with ciliary tubulin. As a previous report showed that Hsp90 interacts with purified cytoplasmic microtubules (51), we generated expression vectors for ciliary ␤iv-tubulin and cytoplasmic ␤iii-tubulin fused to GFP (14). We found that Hsp90 interacted with both ciliary ␤ivtubulin and cytoplasmic ␤iii-tubulin, and this interaction was disrupted in the presence of geldanamycin (Fig. 6A). Hsp90 did


Epithelial cell cilia possess an axonemal architecture composed of a 9 ⫹ 2 arrangement of microtubules composed of ␣and ␤-tubulin, and beat rapidly. This beating moves the fluid across the epithelium, and plays a fundamental role in the mucociliary clearance, left-right patterning (39), generating ependymal flow (41), and neuronal cell migration (32). Reduced movement of ciliary beating causes primary ciliary dyskinesia, including Kartagener syndrome, characterized by situs inversus, bronchiectasis, and chronic sinusitis (40). Such dysmotility or immotility of the cilia results from genetic mutations of major components of the axonema such as dyneins (52–54), DNA polymerase ␭ (55), sperm-associated antigen 6 (56), and a thioredoxin family member (57). In this study, we demonstrated that HSF1 is required for maintenance of ciliary beating in various organs such as the nasal cavity, ventricles, trachea, and oviducts. Although a reduction in ciliary beating led by HSF1 deficiency did not cause clear phenotypes in all of the VOLUME 282 • NUMBER 51 • DECEMBER 21, 2007


Hsp90

C

iv-tubulin

B

Ciliary Dyskinesia in HSF1-null Mice 5). Although it is unclear whether reduced tubulin levels in HSF1-null cilia are related with ciliary dysfunc4 3 4 3 4 3 4 3 200 : tion, Hsp90 does promote tubulin GFP 150 polymerization in vitro (Fig. 6). These GA - - - + + 100 results strongly suggest that HSF1GFP Input IP: P.I. 50 Hsp90 regulation may be required for contorol BSA 4 3 4 3 4 3 4 3 ciliary beating under physiological Hsp90 0 : CaCl2 conditions. Paclitaxel Hsp90 In chicken B lymphocytes, both GA - - - + + 0 10 20 30 40 HSF1 and HSF3 specifically up-regTime (min) ulate the expression of Hsp90␣ 250 under normal growth conditions. This regulation is required at mod200 erately high temperatures to stabilize the Cdc2 protein that promotes 97 150 cell cycle transition from the G2 66 100 phase to the mitosis phase, but is not required in unstressed conditions 50 45 BSA (27). Disruption of mouse HSF genes Hsp90 0 revealed tissue-specific regulation 0.39 (mg/ml) 0.23 0.39 Protein conc. 0 0.08 of basal expression of Hsps during + development. HSF1 is activated and GA FIGURE 6. Hsp90 interacts with ␤iv-tubulin and stabilizes tubulin polymerization. A, cell extracts were induces a set of Hsps during postnaprepared from 293 cells transfected with an expression vector for GFP-␤iv-tubulin (␤4) or GFP-␤iii-tubulin (␤3), tal development of olfactory neuroand immunoprecipitation was performed using preimmune (PI) serum, ␣Hsp90, or ␣GFP in the presence or genesis in mice (24). In the adult absence of 1 ␮M geldanamycin. Co-precipitation of Hsp90 or GFP-fused tubulins was analyzed by Western blotting using each antibody. B, purified mouse Hsp90 and BSA, as well as proteins for molecular mass stand- mouse heart, HSF1 is required for ards (kDa), were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. C, time-dependent tubulin basal expression of small Hsps that polymerization in the presence of Hsp90. A tubulin polymerization assay was performed for the indicated periods at 37 °C without or with Hsp90 (0.39 mg/ml), BSA (0.39 mg/ml), paclitaxel (3 ␮M), or CaCl2 (25 mM) as a protect against oxidative stress (28). negative control. Fluorescence was detected, and mean ⫾ S.D. are shown (n ⫽ 3). D, concentration-dependent In the lens of postnatal mice, HSF4 tubulin polymerization. Tubulin polymerization assay was performed for 20 min at 37 °C with the indicated regulates expression of Hsp25 and concentrations of Hsp90 or BSA. Geldanamycin (2 ␮M) was mixed in some reactions. Fluorescence was ␥-crystallin that has a role in protein detected, and mean ⫾ S.D. are shown (n ⫽ 3). stabilization under dehydrated condiciliated tissues, we found chronic sinusitis and mild hydroceph- tions (22, 23). However, we do not know any client protein of these molecular chaperones that explains the phenotypes of the mutant alus in HSF1-null mice. Previous reports showed that Hsp70, Hsp90, and TCP-1 mice. Here, we demonstrated that Hsp90 does promote tubulin (CCT) are associated with the axonema in cilia or flagella of polymerization by direct binding. We showed the first client provarious organisms such as chlamydomonas, sea urchins, tetra- tein whose polymerization could be regulated by HSF1-mediated hymena, and rabbits (47, 58, 59). Furthermore, the folding and expression of a molecular chaperone during development. assembly of tubulin is facilitated by Hsps such as Hsp70, Hsp90, and TCP-1 (46), and Hsp40, a component of the radial spoke Acknowledgments—We thank Dr. T. Nishida for the fluorescence complex (48, 49), is induced during cilium regeneration (60). multiwell plate reader and Dr. K. Yamaguchi for mouse maintenance. 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A


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Heat Shock Transcription Factor 1 Is Required for Maintenance of Ciliary Beating in Mice Eiichi Takaki, Mitsuaki Fujimoto, Takashi Nakahari, Shigenobu Yonemura, Yoshihiko Miyata, Naoki Hayashida, Kaoru Yamamoto, Richard B. Vallee, Tsuyoshi Mikuriya, Kazuma Sugahara, Hiroshi Yamashita, Sachiye Inouye and Akira Nakai J. Biol. Chem. 2007, 282:37285-37292. doi: 10.1074/jbc.M704562200 originally published online October 27, 2007

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