Shh Initiates Cochlear Hair Cell Regeneration Through Down-regulation Of PRb

[Objects] Hair cell loss caused by ototoxic chemicals and noise exposure is a major reason for senserineural hearing loss. But unlike invertebrate animals, hair cells in mammalian inner ear cannot be regenerated intrinsically. Thus, hearing loss is permanent and there is no effective way for treatment. Triggering cochlear hair cell regeneration in situ is one of the ideal approaches for restoring hearing that is lost as a result of hair cell damage. Current work focuses on the identification of pathway that can be explored for hair cell regeneration by cell cycle re-entry.[Methods] In our study, we use in vitro neonatal cochlea explant culture to investigate the effect and mechanism of Shh on hair cell regeneration in neonatal mammalian inner ear. We studied cell cycle re-entry in adult mouse inner ear by conditionally deleting floxed Rbl gene using Cre mediated strategy and evaluated proliferation by BrdU incorporation with specific hair cell and supporting cell markers.[Results]1). In rat neonatal cochlea explant, Shh can trigger hair cell and supporting cell proliferation after hair cell damage, shown by BrdU incorporation. Further, proliferating supporting cells transdifferentiate into hair cells, shown by hair cells that are labelled with progenitor cell markers Pax2and Sox2.2). RT-PCR study results show that, by culturing neonatal cochlea with Shh, the Hh pathway is activated, shown by up-regulation of several downstream factors. The regulation of cell proliferation and differentiation is achieved by Hh signaling pathway activation.3). By activating Hh pathway, the fuction of pRb is suppressed. Shh suppresses Rb function by reducing Rbl expression and by phosphorylation of pRb, resulting in the upregulation of cyclin B1, cyclin D2, cyclin D3and CDK1and causing cell cycle re-entry.4). By deleting Rbl in adult inner ear culture, hair cell and supporting cells can both go back to cell cycle in cochlea and utricle.[Conclusion]1).Shh can trigger supporting cell and hair cell proliferation in neonatal rat cochlea after hair cell damage.2). In in vitro environment, Hh signaling pathway can be activated by treating with Shh, shown by increased expression of Shh pathway genes.3). The effect of Shh on promoting inner ear proliferation is mediated through suppression of pRb function, which is a key factor in the maintenance post mitotic cells in the inner ear.4). Deletion of Rbl in adult cochlea together with Fgf signaling is sufficient to induce cell cycle re-entry in both hair cells and supporting cells.