Silencing p27 reverses post-mitotic state of supporting cells in neonatal mouse cochleae

https://doi.org/10.1016/j.mcn.2009.08.011Get rights and content

Abstract

The post-natal cochlear mammalian epithelium have no capacity to proliferate in tissue, however, dissociated supporting cells exhibit the ability to divide and trans-differentiate into new hair cells in vitro, with this process found to be correlated with the downregulation of the cyclin-dependent kinase inhibitor p27kip1. Here we show that knockdown of p27kip1 with short hairpin RNA-expressing vectors results in the cell-cycle reentry of post-mitotic supporting cells in the post-natal mouse cochleae ex vivo. The p27kip1-knockdown cells incorporated BrdU, and then divided into two daughter cells. However, there was also activation of the apoptotic pathway in some supporting cells. These results indicate that the use of RNA interference to target p27kip1 is an effective strategy for inducing cell-cycle reentry in post-mitotic supporting cells in the post-natal mammalian cochleae, although additional manipulations of the supporting cells are required to achieve hair cell regeneration.

Introduction

Sensorineural hearing loss (SNHL) is one of the most common disabilities. Auditory hair cells in the cochlea are mechanoreceptors that play a crucial role in hearing. In mammals, if the hair cells are damaged or lost, the resulting SNHL is permanent. Therefore, one of the best approaches for improving hearing in mammals would be to find a way to induce hair cell regeneration.

The cochlear epithelium is composed of hair cells and supporting cells (SCs). In the avian auditory epithelium, SCs are able to reenter the cell cycle and proliferate in response to hair cell loss, which gives rise to both new hair cells and SCs (Corwin and Cotanche, 1988, Ryals and Rubel, 1988). In mice, progenitors of the cochlear epithelium stop dividing by embryonic day 14.5 (Ruben, 1967), with differentiation of the hair cells and SCs occurring after this terminal mitosis. Both hair cells and SCs maintain a post-mitotic state throughout life in adult mammals, and do not exhibit any spontaneous capacity to divide under normal conditions or in response to damage in vivo (Roberson and Rubel, 1994).

It has been determined that the cyclin-dependent kinase inhibitor (CKI), p27kip1, plays a crucial role in the entry of mammalian SCs into the G0 phase (Chen and Segil, 1999, Löwenheim et al., 1999). CKIs function by binding to and inhibiting the activity of the cyclin-dependent kinases that promote cell cycle progression and fulfill the cell cycle checkpoint functions (Sherr and Roberts, 1999). The p27kip1 acts as a negative regulator of the G1-S transition in the cell cycle (Harper, 2001). Studies on p27kip1-deficient mice have shown that when deleted, it results in the continued proliferation of SCs in postnatal mouse cochlear epithelia (Chen and Segil, 1999, Löwenheim et al., 1999). Recently, White et al. (2006) have reported that after dissociation, some of the mammalian SCs are able to recover their proliferative abilities. In addition, these dissociated SCs can also trans-differentiate into hair cells in vitro. These findings suggest that while these mammalian SCs might have a regenerative potential similar to avian auditory epithelia, this potential is suppressed within the tissue. This previous study also found that there was a correlation between the reduced expression levels of p27kip1 and the ability of the mammalian supporting cells to reenter the cell cycle in vitro. Overall, these previous results suggest that manipulation of the p27kip1 levels could be used therapeutically to stimulate the proliferation of mammalian SCs in tissue.

Discovery of gene inactivation by RNA interference (RNAi) has led to the development of a new targeted therapy for inner ear disease at the molecular level (Fire et al., 1998). RNAi is a two-stage intracellular process that converts the double-stranded RNA molecule precursors into functional small interfering RNAs. These small interfering RNAs are then incorporated into RNA-inducing silencing complexes. Subsequently, these duplexes unwind, with one strand used to target sequence-specific cleavage of the messenger RNAs that ultimately cause the knockdown of the expression of the targeted proteins (Elbashir et al., 2001).

Although studies on p27kip1-deficient mice have shown that its deletion results in continued SC proliferation in postnatal mouse cochlear epithelia (Chen and Segil, 1999, Löwenheim et al., 1999), the consequences of acute removal of p27kip1 from differentiated SCs have yet to be examined. Therefore, the aim of the current study was to examine the efficacy of RNAi in silencing p27kip1 in cochlear explant cultures from post-natal mice, and its potential for inducing mitosis in post-mitotic SCs in the cochlear epithelia after birth.

Section snippets

Co-transfection efficiency

In this study, the two different plasmids were simultaneously introduced into the cochlear explants in order to label cells in which short hairpin RNAs (shRNAs) were transfected. To examine the efficiency of co-transfection, we introduced two different plasmids, EGFP- and DsRed-expressing vectors, into five cochlear explants via electroporation. One day after the electroporation, EGFP+ cells were found in all explants, with the majority co-expressing DsRed (Fig. 1a, b). Immunostaining for p27

Animals

ICR mice (Japan SLC Inc., Hamamatsu, Japan) were maintained at the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Japan. Experimental protocols were approved by the Animal Research Committee of Kyoto University Graduate School of Medicine (MedKyo07062), and complied with the US National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals.

Explant culture

P3 ICR mice were deeply anesthetized with carbon dioxide and decapitated. The temporal bones were

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a Grant-in-Aid for Researches on Sensory and Communicative Disorders from the Ministry of Health, Labour and Welfare of Japan. We thank Tatsunori Sakamoto and Norio Yamamoto for critical review of this manuscript.

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