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,Akira Nakai

doi:10.1074/jbc.m704562200

Abstract

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 alpha- and beta-tubulin. We found a marked reduction in alpha- and ciliary betaiv-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 betaiv-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.

Highlights

Heat shock response is characterized by induction of a set of heat shock proteins (Hsps)2 and is a fundamental adoptive response in all organisms from bacteria to humans
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
This revealed that HSF1 is required for maintenance of olfactory neurogenesis

Summary

MATERIALS AND METHODS

Histopathology and Immunohistochemistry—HSF1-null [7] mice were maintained by crossing with ICR mice. 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 4Ј,6diamidino-2-phenylindole (Vector Laboratories), and visualized using fluorescence microscopy (Axioplan 2, Zeiss). 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 (ϳ5 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. Thirty-three cycles of PCR were performed to amplify a DNA fragment of the mouse Hsp90␣ (Hsp86) gene (Ϫ209 to ϩ56 from a transcription start site) containing a canonical heat shock element [36]. The supernatants were collected after centrifugation at 15,000 ϫ g for 10 min, the 1 ␮l of preim-

Tu Tu Se

Mucus Accumulation in the

The whipping movement of the cilia generates moving force