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FOXO3/TGF-β signal-dependent ciliogenesis and cell functions during differentiation of temperature-sensitive mouse cochlear precursor hair cells

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Abstract

The transcription factor FOXO3 is necessary to preserve cochlear hair cells. Growth factors, including TGF-β, closely contribute to cochlear hair cell regeneration. In the present study, to investigate the roles of FOXO3 in the ciliogenesis and cell functions of cochlear hair cells, UB/OC-2 temperature-sensitive mouse cochlear precursor hair cells were treated with TGF-β receptor type 1 inhibitor EW-7197 or EGF receptor inhibitor AG-1478 after transfection with or without siRNA-FOXO3a. GeneChip analysis revealed that treatment with EW-7197 increased Foxo3 genes and decreased genes of Smads. During cell differentiation, treatment with EW-7197 or AG-1478 induced an increase in length of cilia-like structures that were positive for acetylated tubulin and inhibited cell migration. Treatment with EW-7197 also increased cell metabolism measured as mitochondrial basal respiration (oxygen consumption rate). The effects of EW-7197 were stronger than those of AG-1478. Knockdown of FOXO3 prevented the growth of cilia-like structures induced by EW-7197 or AG-1478 and induced cell migration under treatment with EW-7197. No change of the epithelial cell polarity molecule PAR3 was observed with any treatment. Treatment with the antimicrobial agent amikacin prevented the growth of cilia-like structures induced by EW-7197 and induced apoptosis. Pretreatment with the glucocorticoid dexamethasone inhibited the apoptosis induced by amikacin. This in vitro model of mouse cochlear hair cells suggests that FOXO3/TGF-β signaling plays a crucial role in ciliogenesis and cell functions during differentiation of cochlear hair cells. This model is useful for analysis of the mechanisms of hearing loss and to find therapeutic agents to prevent it.

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Acknowledgements

We thank Dr. Matthew Holley (The University of Sheffield, UK) for supplying the UB/OC-2 cell line.

Funding

This work was supported by JSPS KAKENHI grant numbers 19K07464, 20K18319, and 21K09610.

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Author notes

  1. Takuya Kakuki and Soshi Nishida have contributed equally to this work.

Authors and Affiliations

  1. Department of Otolaryngology, Sapporo Medical University School of Medicine, Sapporo, 060-8556, Japan

    Takuya Kakuki, Soshi Nishida, Kizuku Ohwada, Masaya Nakano & Kenichi Takano

  2. Department of Cell Science, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo, 060-8556, Japan

    Takayuki Kohno, Soshi Nishida, Takumi Konno, Kizuku Ohwada, Masaya Nakano, Mitsuki Tezuka & Takashi Kojima

  3. Department of Anatomy, Sapporo Medical University School of Medicine, Sapporo, 060-8556, Japan

    Shin Kikuchi

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  1. Takuya Kakuki
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  2. Takayuki Kohno
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  3. Soshi Nishida
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  4. Takumi Konno
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Correspondence to Takayuki Kohno or Takashi Kojima.

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418_2021_2068_MOESM1_ESM.tif

Supplementary file1 (TIF 2465 KB) (a) Immunocytochemistry for Ac-tubulin and γ-tubulin in UB/OC-2 cells treated with 100 μM amikacin after treatment with 10 μM AG-1478 at 39°C. Scale bar: 20 μm. (b) Western blotting for caspase 3, Ac-tubulin, and actin in 10 μM AG-1478 treated UB/OC-2 cells pretreated with 1 μM or 10 μM dexamethasone before treatment with 100 μM amikacin at 39°C

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Kakuki, T., Kohno, T., Nishida, S. et al. FOXO3/TGF-β signal-dependent ciliogenesis and cell functions during differentiation of temperature-sensitive mouse cochlear precursor hair cells. Histochem Cell Biol 157, 415–426 (2022). https://doi.org/10.1007/s00418-021-02068-8

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