We initiated this study to investigate the role of transcription factor NF-κB in the cochlea. NF-κB has been associated historically with inflammatory and immune responses, activating transcription of numerous genes involved in host defense. Very little is known about the role of NF-κB in the ear in general. Previously, NF-κB was found expressed in the murine cisplatin-treated cochlea, mostly in the stria vascularis and the spiral ligament, where it was proposed to activate transcription of inducible nitric-oxide synthase (iNOS) leading to increased production of NO and consequently to ear dysfunction (Watanabe et al. 2002
). Another study reported increased NF-κB activity in spiral ganglion neurites of aged mice (Inafuku et al. 2000
). NF-κB has also been tied to the inflammatory reactions of the middle ear epithelia during otitis media (Barrett et al. 2003
). Here we find that NF-κB/Rel family is expressed in the cochlea, and that at least one subunit, transcriptional activator RelA (p65), is present also in the nuclei of the hair cells and supporting cells of p5 rat organ of Corti (Fig. ). Nuclear localization of NF-κB in the organ of Corti was confirmed independently, in vivo
by immunohistochemistry (Fig. B) and in vitro
by immunoblotting of subcellular fractions (Fig. ). For the in vitro
biochemical studies, to minimize the possibility that changes in the localization and activity of RelA occur because of stress caused by the isolation procedure, explants were kept in culture for 72 h prior to further treatment.
Nuclear localization of RelA (p65) in hair cells and some supporting cells as well implies constitutive NF-κB activation but does not guarantee it per se (Fig. B). However, the morphology of hair cells as it was observed after treatment with the NF-κB inhibitor suggests that some form of NF-κB activity is required for hair cell survival. Disruption of orderly hair cell rows, degeneration, and loss of hair cells was observed within 24 h of treatment with NF-κB inhibitor (Fig. ). To ensure our observations were not due to reasons other than specific inhibition of NF-κB activity, control experiments were performed with an inactive mutated form of INH (INH-Mut). To determine whether cell death occurs by an apoptotic pathway following inhibition of NF-κB, caspase-3 activity assays were performed. Two different time points were assessed for the specific NF-κB inhibitor (INH). Comparison with a less specific NF-κB inhibitor parthenolide (PNT), which is also known to inhibit MAPK signaling, is provided. Assays showed 2- to 4.5-fold increase in caspase-3 activity in the organ of Corti after treatment with inhibitors, as compared to their inactive controls (Fig. ). The data obtained in these experiments strongly suggest that damage to hair cells occurs as a consequence of NF-κB inhibition and indicate that transcriptional activity of NF-κB is required for survival of neonatal cochlear hair cells in vitro even in resting conditions.
Because all our studies were performed with material isolated from p5 animals, we must leave open the possibility that expression patterns of NF-κB family may differ at distinct developmental stages. P5 animals were chosen for this study primarily because at this early neonatal stage, the otic capsule is not incorporated in dense bone, making isolation of material for biochemical studies plausible and relatively quick. It is worth noting that the rat cochlea is still immature in development 5 days after birth. While this article was in preparation, Jiang et al. (2005
) reported on the protective role of the NF-κB pathway in kanamycin-induced hair cell death in adult mice. Another very recent study conducted in the gerbil cochlea proposed a neuroprotective role of NF-κB through enhancing the survival of type II spiral ganglion neurons exposed to ouabain (i.e., G-strophanthidin), a poisonous plant alkaloid that blocks the sodium–potassium ATPase (Lang et al. 2005
In our experimental setting, inhibition of NF-κB activity resulted in rapid activation and execution of the apoptotic program, whereas hair cells seemed to suffer most. The findings are reminiscent of reports that NF-κB is constitutively activated in some neurons, and that inhibition of NF-κB transcriptional activity triggers neuronal apoptosis characterized by mitochondrial release of cytochrome c
, caspase-9 and -3 activation, and poly(ADP-ribose) polymerase- 1 cleavage. These events were reportedly preceded by selective reduction in Bcl-xL
, and A1/Bfl-1
transcription, i.e., NF-κB-dependent antiapoptotic B-cell CLL/lymphoma-2 (Bcl-2) family members (Chiarugi 2002
). In our study, we observed a decrease of gadd45β
mRNA and protein following inhibition of NF-κB in the organ of Corti (Fig. ). We were interested in this particular antiapoptotic NF-κB target gene because of reports of negative cross talk between the IKK/NF-κB and JNK pathways mediated by gadd45β (De Smaele et al. 2001
; Papa et al. 2004
; Tang et al. 2001
). c-Jun N-terminal kinase signaling is heavily implicated as one of the key mediators of auditory hair cell death, and our results open up an interesting possibility that NF-κB signaling could function in part to protect hair cells from JNK-mediated apoptosis by ensuring expression of GADD45β. Further research into the role of GADD45β in hair cells is needed to substantiate this hypothesis, however. It is also possible that NF-κB is involved in regulation of the cellular antioxidant program in the cochlea, as it has been proposed for neuronal cells and other cell types that harbor NF-κB in a constitutive activated state (Schütze et al. 1992
; Kaltschmidt et al. 1994
; Lezoualch'l et al. 1998
; Jang and Surh 2004
In summary, the results presented here are among the first to address the function in the cochlea of one of the most intensely studied eukaryotic transcription factors. Because NF-κB can be found in presumably a constitutively active state in the organ of Corti of p5 animals, and inhibition of its activity results in massive hair cell degeneration, it seems likely that NF-κB may participate in normal hair cell function.