Dexamethasone treatment of tumor necrosis factor-alpha challenged organ of Corti explants activates nuclear factor kappa B signaling that induces changes in gene expression that favor hair cell survival

Abstract

The objective was to determine the role of nuclear factor kappa B (NFκB) in dexamethasone base (DXMb) protection of auditory hair cells from tumor necrosis factor-alpha (TNFα)-induced loss on gene expression and cell signaling levels. Organ of Corti (OC) explants from 3-day-old rats were cultured under one of the following conditions: (1) media only--no treatment; (2) media+TNFα; (3) media+TNFα+DXMb; (4) media+TNFα+DXMb+NFκB-Inhibitor (NFκB-I); or (5) media+TNFα+DXMb+NFκBI-Scrambled control (NFκBI-C). A total of 60 organ of Corti explants (OC) were stained with FITC-Phalloidin after 96 h in culture (conditions 1-5) for hair cell counts and imaging of surface characteristics. A total of 108 OC were used for gene expression studies (i.e. B-actin, Bax, Bcl-2, Bcl-xl, and TNFR1) after 0, 24, or 48 h in vitro (conditions 1-4). A total of 86 OC were cultured (conditions 1-3) for 48 h, 36 of which were used for phosphorylated NFκB (p-NFκB) ELISA studies and 50 for whole mount anti-p-NFκB immunostain experiments. TNFα+DXMb exposed cultures demonstrated significant upregulation in anti-apoptotic Bcl-2 and Bcl-xl genes and downregulation in pro-apoptotic Bax gene expression; DXMb treatment of TNFα explants also lowered the Bax/Bcl-2 ratio and inhibited TNFR1 upregulation. After inhibiting NFκB activity with NFκB-I, the gene expression profile following TNFα+DXMb treatment now mimics that of TNFα-challenged OC explants. The levels of p-NFκB and the degree of nuclear translocation are significantly greater in TNFα+DXMb exposed OC explants than observed in the TNFα and control groups in the middle+basal turns of OC explants. These findings were supported by the results of the hair cell counts and the imaging results obtained from the whole mount OC specimens. DXMb protects against TNFα-induced apoptosis of auditory hair cells in vitro via activation of NFκB signaling in hair cell nuclei, and regulation of the expression levels of anti- and pro-apoptotic genes and a pro-inflammatory gene.