Cytoskeletal protein mRNA expression in the chick utricle after treatment in vitro with aminoglycoside antibiotics: effects of insulin, iron chelators and cyclic nucleotides

In birds, spontaneous recovery of the hair cells of the inner ear can occur after damage induced by aminoglycoside antibiotics. The factors that influence this recovery and the process of hair cell regeneration itself have until recently been investigated largely by morphological and histological methods. The aim of this work was to use a molecular biological approach to the analysis of hair cell regeneration by measuring the changes that occur in expression of mRNA for hair cell-specific cytoskeletal proteins fimbrin and class III beta-tubulin, along with that for beta-actin, in the utricle of chicks after hair cell damage both in vitro and in vivo. Utricles were removed from 1-day-old chicks and incubated with the aminoglycoside antibiotics gentamicin or neomycin (both 1 mM), or chicks were injected intraperitoneally with 100 mg/kg gentamicin or neomycin for 4 consecutive days. At the end of the treatment periods, total RNA was extracted from single utricles, reverse transcribed to cDNA and the cDNA amplified by PCR with primers for beta-actin, fimbrin and class III beta-tubulin. Co-amplification allowed quantitative comparison of mRNA between fimbrin, or class III beta-tubulin and beta-actin from the same utricle. Both aminoglycosides, either after 48 h in vitro or immediately after treatment in vivo, caused a significant decrease in the expression of fimbrin mRNA and class III beta-tubulin mRNA, relative to beta-actin mRNA, which itself increased. Light and electron microscopy confirmed that this corresponded to loss of, and damage to, hair cells. The relative expression of fimbrin and class III beta-tubulin mRNAs was partly restored almost to control levels 4 days after cessation of treatment in vivo and fully normalised by 4 weeks, by which time hair cells appeared normal. However, their relative expression remained depressed 4 days after removal of antibiotic in vitro. The iron chelator desferrioxamine (100 microM) in vitro prevented the aminoglycoside-induced reduction in relative expression of mRNA for both fimbrin and class III beta-tubulin. Neither insulin (5 microM) nor a combination of dibutyryl cyclic AMP (5 mM) and the phosphodiesterase inhibitor IBMX (0.5 mM) prevented the decrease in relative expression of the mRNAs for the hair cell-specific proteins, but both treatments allowed their partial recovery in vitro during the 4-day-period after removal of aminoglycoside. It is concluded that the cells of the sensory epithelium of the chick utricle subjected to aminoglycoside-induced damage undergo a process in which mRNA expression is switched away from the production of functional proteins and towards proteins necessary for structural re-organisation. The restoration of mRNA expression to a normal pattern is promoted by the growth factor insulin and by cyclic AMP.