Research papersDexamethasone protects auditory hair cells against TNFα-initiated apoptosis via activation of PI3K/Akt and NFκB signaling
Introduction
The pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) can play an important role in a variety of diverse cellular processes such as acute and chronic inflammation, septic shock, cellular proliferation, cell survival, and programmed cell death. One example is the observation that exposure of spiral ligament fibrocytes to TNFα can initiate secretion of chemokines and other inflammatory mediators which negatively impacts cochlear homeostasis and auditory function (Yoshida et al., 1999, Ichimiya et al., 2000, Ichimiya et al., 2003). Spiral ligament fibrocytes have been shown to release chemokines including TNFα when challenged with otitis media pathogens and are a likely source of TNFα in vivo (Moon et al., 2006). Other inner ear structures have also demonstrated TNFα production, including outer hair cells and supporting cells within the organ of Corti (So et al., 2007, Zou et al., 2005). Tumor necrosis factor receptor 1 (TNFR1) is the membrane bound receptor for TNFα and is expressed constitutively on most cell types, including those cells in the organ of Corti (Palladino et al., 2003, Dinh et al., 2008). Its activation can mediate endoplasmic stress-induced activation of c-Jun N-terminal kinases (JNKs). The three isoforms of JNK (i.e., JNK 1,2,3) are trauma activated members of the mitogen activated protein kinase (MAPK) family of signal molecules that have been associated with acoustic trauma, aminoglycoside ototoxicity, and electrode insertion trauma-induced hearing loss (Pirvola et al., 2000, Wang et al., 2003, Wang et al., 2007, Ylikoski et al., 2002, Zine and Van De Water, 2004, Eshraghi et al., 2007). Intrathecal delivery of TNFα induces meningitis in guinea pigs that results in a sensorineural hearing loss pattern similar to that which develops following pneumococcal-induced meningitis (Aminpour et al., 2005). A recent study that delivered TNFα directly into the scala tympani demonstrates that the presence of this inflammatory cytokine recruits inflammatory cells into the cochlea (Keithley et al., 2008). However, this study did not establish a direct link between the presence of this cytokine and production of hearing loss. Another animal study documented an increase in the expression levels of TNFα and its receptors (i.e., TNFR1 and -R2) within cochlear tissues of guinea pigs following exposure to transcranial vibration trauma suggesting that this inflammatory cytokine and its receptors play a role in the development of a post-trauma hearing loss (Zou et al., 2005). Due to the well established fact that TNFα is produced by the inner ear in times of stress, mediates inflammation, and causes cell death, this cytokine was chosen to model the inflammatory environment created by cochlear implant electrode insertion.
Treatment of TNFα-exposed spiral ligament cell cultures with dexamethasone (Dex) inhibits a TNFα-initiated inflammatory response (Maeda et al., 2005). Local delivery of glucocorticoids (e.g., Dex) into the scala tympani prevents either some or nearly all of the sensorineural hearing loss following exposure to: (1) electrode insertion trauma (EIT); (2) acoustic trauma; (3) aminoglycoside ototoxicity; (4) and an ischemia-reperfusion insult (Himeno et al., 2002, Takemura et al., 2004, Herr and Marzo, 2005, Tabuchi et al., 2005, Tabuchi et al., 2006, Eshraghi et al., 2007, Vivero et al., 2008). Glucocorticoids exert most of their action through the binding of the glucocorticoid receptors (GRs) (Tischner and Reichardt, 2007). GRs have been demonstrated throughout the organ of Corti, including the AHCs and supporting cells, of 3-day-old rat pups and adult rats (Zuo et al., 1995). Dex is a synthetic GR agonist that can inhibit TNFα-induced apoptosis in the MCF-7 cell line (Udo et al., 2001). Treatment of TNFα-challenged organ of Corti explants with Dex has also been shown to prevent death of auditory hair cells (AHCs), suppress TNFα-initiated up regulation of a pro-apoptotic member of this gene family (i.e., Bax), up regulate the expression levels of several anti-apoptotic members of the Bcl-2 family of genes (e.g., Bcl-xl), and down regulate TNFR1 gene expression (Dinh et al., 2008). The ability of glucocorticoids and the GR to provide anti-inflammatory and anti-apoptotic signals seem to be dependent on NFκB activation. Studies with spiral ganglion neurons demonstrate an inability of the glucocorticoid–GR complex to mediate survival signals in the presence of NFκB inhibition (Tahera et al., 2006a, Tahera et al., 2006b). Therefore, we predict a similar dependence on an active NFκB in AHCs for glucocorticoid otoprotection. Such a similarity in the cell signaling of spiral ganglion neurons and AHCs may be possible given their common embryological origin from the otic placode (Rubel and Fritzsch, 2002). This glucocorticoid reliance on active NFκB for otoprotection was hypothesized to involve the cell-signaling molecules PI3K and/or protein kinase B (i.e., Akt or PKB). The glucocorticoid–GR complex has been shown to activate both PI3K and PKB (Hafezi-Moghadam et al., 2002, Limbourg and Liao, 2003). PKB is capable of activating IKK, which leads to phosphorylation of IκB and subsequent NFκB activation. Such a pathway would result in augmented AHC survival if NFκB is producing pro-survival signals. Likewise, if NFκB is acting in a pro-survival manner and the hypothesized pathway is accurate, inhibition of PI3K and PKB should result in AHC death. Indeed, AHC toxicity secondary to gentamicin exposure has been shown to be augmented by the inhibition of PKB and PI3K (Chung et al., 2006).
The aim of the present study is to gain a better understanding of how Dex protects AHCs from an inflammatory environment (such as what may be found after cochlear implant electrode insertion). To accomplish this aim we use an in vitro model of inflammation consisting of 3-day-old (P-3) rat organ of Corti explants challenged at an ototoxic level of TNFα (2 μg/mL). The otoprotective action of Dex treatment (125 μg/mL) against TNFα-induced AHC loss was studied with a series of inhibitors of signal molecules that are thought to be downstream of Dex’s otoprotective activity. These inhibitors include mifepristone (Mif, glucocorticoid receptor antagonist), synthetic peptide NFκB inhibitor (NFκB-I), LY294002 (i.e., LY, PI3K inhibitor), and SH-6 (Akt/PKB inhibitor). To clarify the overall experimental plan of this study, the hypothesized signaling cascades in P-3 organ of Corti hair cells along with the inhibitors used and their proposed sites of action are shown in Fig. 1. In addition, to evaluate any AHC death caused by TNFα, the pancaspase inhibitor Boc-d-fmk was also used (although with TNFα only cultures).
Section snippets
Organ of Corti explants
All animals used in this study were treated in compliance with the published Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publications No. 80-23) revised 1996. The study was performed in accordance with the University of Miami, Internal Animal Care and Use Committee protocol # 08-099. Every effort was made to minimize the number of animals used in this study. Three-day-old (P-3) Sprague–Dawley rat pups (www.crivers.com) were placed in ice for 30
TNFα-induced HC death is dose dependent and displays a base to apex pattern of sensitivity
When TNFα was added to the culture medium of P-3 rat organ of Corti explants it induced both IHC and OHC losses that were dose dependent (Fig. 2A and B). This ototoxic effect of TNFα was most pronounced in the basal turn areas of the explants (basal turns vs. all turns data; compare Fig. 2B to A). For example, exposure to 2 μg/mL of TNFα resulted in a count of 39.3 ± 6.7 IHCs/415 μm when all turns were counted vs. 9.4 ± 6.2 IHCs/415 μm when only the basal turns were counted (p > 0.001).
TNFα-induced HC death is prevented by caspase inhibition
TNFα is known to
Discussion
Exposure of several cell types to the inflammation-related cytokine TNFα has been shown to induce apoptosis (Rath and Aggarwal, 1999, Udo et al., 2001). Specifically, TNFα has been associated with inner ear trauma, such as high levels of noise and vibration (Fujioka et al., 2006, Zou et al., 2005). In these settings, TNFα produced by spiral ligament fibrocytes in the lateral wall of the cochlea is pro-inflammatory and associated with apoptosis. Dinh et al. (2008) have demonstrated auditory hair
Acknowledgement
Supported by a grant from Advanced Bionics Corporation, Valencia CA to T.R.V.
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