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

The transcription factor FOXO3 is necessary to preserve cochlear hair cells. Growth factors, including TGF-ß, 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-ß 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-ß 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.